Agronomy Research

Established in 2003 by the Faculty of Agronomy, Estonian Agricultural University

Aims and Scope:

Agronomy Research is a peer-reviewed international Journal intended for publication of broad-
spectrum original articles, reviews and short communications on actual problems of modern
biosystems engineering incl. crop and animal science, genetics, economics, farm- and
production engineering, environmental aspects, agro-ecology, renewable energy and bioenergy
etc. in the temperate regions of the world.

Copyright:
Copyright 2009 by Estonian University of Life Sciences, Estonian Research Institute of
Agriculture, Latvia University of Agriculture, Aleksandras Stulginskis University, Lithuanian
Institute of Agriculture and Lithuanian Institute of Horticulture. No part of this publication may
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Agriculture and Lithuanian Institute of Horticulture.

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ISSN 1406-894X
 CONTENTS
VII FOOD SCIENCE AND TECHNOLOGY .….…………………………………… 693
M. Ahokas, A.-L. Välimaa, T. Lötjönen, A. Kankaala, S. Taskila and E. Virtanen
Resource assessment for potato biorefinery: Side stream potential in Northern
Ostrobothnia ....…………………………………………………………………………… 695
D. Baranenko, V. Kolodyaznaya and Y. Broyko
Effect of cold treatment on the amino acid composition of veal .………………………… 705
N. Iakovchenko and L. Silantjeva
Vegetable ingredients in soft cheese made from concentrated skim milk
by ultrafiltration ...………………………………………………………………………… 717
L. Kuznetsova, L. Zabodalova and D. Baranenko
On the potential of lupin protein concentrate made by enzymatic hydrolysis of
carbohydrates in dairy-like applications ......……………………………………………… 727
T.Michlová, Š. Horníčková, H. Dragounová and A. Hejtmánková
Quantitation of vitamins A and E in raw sheep milk during lactation period ….………… 737
U. Moor, P. Põldma, T. Tõnutare, A. Moor and M. Starast
The effect of modified atmosphere storage on the postharvest quality of the
raspberry ‘Polka’ .....……………………………………………………………………… 745
H. Mootse, A. Pisponen, S. Pajumägi, A. Polikarpus, V.Tatar, A. Sats and
V. Poikalainen
Investigation of Casein Micelle Particle Size Distribution in Raw Milk of Estonian
Holstein Dairy Cows ...…………………………………………………………………… 753
L. Nadtochii and A. Koryagina
Fermented sauces for child nutrition from age three ...…………………………………… 759
O. Orlova and U. Nasonova
The unique characteristics of milky-wax ripe walnuts and their usage ….…………….… 769
A. Pastukhov and H. Dogan
Studying of mixing speed and temperature impacts on rheological properties of
wheat flour dough using Mixolab …....…………………………………………………… 779
A. Pisponen, S. Pajumägi, H. Mootse, A. Sats, V. Poikalainen and A. Karus
Effect of cooling rates and low crystallization temperatures on morphology of lactose
crystals obtained from Ricotta cheese whey .………………………………………...…… 787
A. Põldvere, L. Lepasalu, A. Tänavots, J. Olt, U. Sannik, A. Sats, R. Saar,
R. Martinson and V. Poikalainen
An alternative method for meat shear energy estimation during ageing .………………… 793
A. Sats, H. Mootse, S. Pajumägi, A. Pisponen, V. Tatar and V. Poikalainen
Estimation of Particle Size Distribution in Bovine Colostrum Whey by Dynamic
Light Scattering (DLS) Method …………………………..……………………………… 801

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A. Sats, H. Mootse, L. Lepasalu and V. Poikalainen
Use of Delvotest T for Quantitative Estimation of β-lactam Antibiotic Residues
in Waste Milk and for Evaluation of Thermal Treatment Efficiency –
a Methodical Pilot Study .....……………………………………………………………… 807
E. Suchkova, B. Shershenkov and D. Baranenko
Effect of ultrasonic treatment on metabolic activity of Propionibacterium shermanii,
cultivated in nutrient medium based on milk whey .……………………………………… 813
R. Vokk, E. Tedersoo, T. Lõugas, K. Valgma and J. Rosend
Comparative study on anti-oxidant activity of garlic grown in different regions ..………. 821
L. Zabodalova, T. Ishchenko, N. Skvortcova, D. Baranenko and V. Chernjavskij
Liposomal beta-carotene as a functional additive in dairy products .…………………….. 825

VIII ERGONOMICS ..………………………………………………………………… 835

P. Kic and L. Růžek

Microbiological environment in special rooms of university campus …………………… 837

H. Kalkis, Z. Roja and V. Kalkis

Physical Load Analysis in Hotel Cleaning Work ………………………………………… 843

S.N. Kalnins, J. Gusca, S. Valtere, R. Vanaga and D. Blumberga

Transition to low carbon society. Evaluation methodology ……………………………… 851

T. Koppel and P. Tint

Reducing exposure to extremely low frequency electromagnetic fields from portable
computers ………………………………………………………………………………… 863

Ü. Kristjuhan
Ergonomics is slowing down ageing and postponing ageing related diseases …………… 875

Ü. Kristjuhan
What is priority in the problem of ageing? .……………………………………………… 881

V. Nídlová and J. Hart

The impact of light conditions on identifying facial features …………………………… 889

K. Reinhold, S. Kalle and J. Paju

Exposure to high or low frequency noise at workplaces: differences between
assessment, health complaints and implementation of adequate personal protective
equipment ………………………………………………………………………………… 895

M. Reinvee and K. Jansen

Utilisation of tactile sensors in ergonomic assessment of hand–handle
interface: a review ………………………………………………………………………… 907

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T. Sirge, J. Ereline, T. Kums, H. Gapeyeva and M. Pääsuke
Musculoskeletal symptoms, and perceived fatigue and work characteristics in
supermarket cashiers ……………………………………………………………………… 915

A. Traumann, M. Kritsevskaja, P. Tint and D. Klauson

Air quality as an important indicator for ergonomic offices and school premises .……… 925

IX ENGINEERING DESIGN AND MODELLING,

AUTOMATION TECHNOLOGY,
ENVIRONMENTAL IMPACT …………………………………………………… 935
V. Bulgakov, S. Pilipaka, V. Adamchuk and J. Olt
Theory of motion of a material point along a plane curve with a constant pressure and
velocity ....………………………………………………………………………………… 937
J. Hart, V. Nídlová and M. Přikry
Reliability of detection of sources of infrared radiation in security alarm and
distress signal systems .…………………………………………………………………… 949
V. Osadcuks, A. Pecka, A. Lojans and A. Kakitis
Experimental research of proximity sensors for application in mobile robotics in
greenhouse environment ..………………………………………………………………… 955
L. Laivina, J. Pubule and M. Rosa
A multi-factor approach to evaluate environmental impact statements ..………………… 967

X REVIEWS ..………………………………………………………………………… 977

A. Beloborodko, M. Rosa, F. Romagnoli and D. Blumberga
Overview of the waste-to-energy sector in Latvia: driving forces for
a cluster creation .………………………………………………………………………… 979
M. Kiviste and R. Lindberg
The feasibility of phase change materials in building structures for saving heating energy
in the Nordic climate …….……………………………………………………………… 989
M. Repele, A.Paturska, K. Valters and G. Bazbauers
Life cycle assessment of bio-methane supply system based on natural
gas infrastructure ....……………………………………………………………………… 999

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VII FOOD SCIENCE AND
TECHNOLOGY

693
694
 Agronomy Research 12(3), 695–704, 2014

Resource assessment for potato biorefinery: Side stream

potential in Northern Ostrobothnia

M. Ahokas1,3,*, A.-L. Välimaa1, T. Lötjönen2, A. Kankaala1, S. Taskila3

and E. Virtanen1
1
MTT Agrifood Research Finland, Biotechnology and Food Research, P.O. Box 413,
FI90014 University of Oulu, Oulu, Finland
2
MTT Agrifood Research Finland, Plant Production Research, Tutkimusasemantie 15,
FI92440 Ruukki, Finland
3
University of Oulu, Chemical Process Engineering, P.O. Box 4300, FI90014
University of Oulu, Finland; *Correspondence: [email protected]

Abstract. Potato industry side-streams consist of a significant amount of the original biomass.
However, tightened demands of EU legislation together with the costs of side stream processing
have forced potato industry towards more efficient use of the raw material. For this purpose, we
have examined the possibility to recover main fractions from potato side streams, such as
proteins, fibers and starch, and utilize them in a manner of biorefinery concept. The aim of the
present research was to evaluate the potential for a potato biorefinery based on biomasses
available at area of Northern Ostrobothnia, Finland. Study shows, that there is enough side-
streams available to build a concept, which produces more value added products, like fibers and
proteins. In this report, the main conclusions of the research are presented together with state-
of-art on potato waste water processing technologies and current applications of their products.

Key words: side streams, potato, biorefinery, starch, fiber, protein, resource assessment.

INTRODUCTION

Potato production can be conducted in a variety of different conditions, which

makes it a commonly cultivated crop across the world, also grown in all EU countries.
Global annual production of potato is over 350 million tons from which EU countries
produce nearly 55 million tons (FAO, statistics from 2012). Finnish annual production
is approximately 500,000 t corresponding to harvested area of 20,700 ha.
The geographical scope of the table potato market is rather wide; competition
between production areas is enabled due to tolerance of potatoes to carriage and thus
low transportation costs. The rising demand for potato products in Central and Eastern
Europe, in Asia and South America also offers new business opportunity for European
potato producers. Potatoes used for processing have lower unit value, which leads to
increased transportation costs compared to table potatoes. Thus the processing plants
are located close to the cultivation areas.
The main center of production for Finnish potatoes is in Ostrobothnia, where the
conditions are most suitable for the purpose. The present research focuses on area of

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Northern Ostrobothnia, which extends across Finland from the Gulf of Bothnia coast to
the Russian border and has an area of 37,000 km² and population of about 380,000
people (7.2% of the Finnish population). The harvest area of potato in the region is
approximately 3,800 ha accounting to 114.9 million tons (Tike, 2013). The majority of
harvest is used as seed potato or as food (table potato or food industry). Starch potato is
not cultivated in large extend.
Potato cultivation and processing creates significant amounts of side streams that
are not included in the main products. These are especially produced during processing
of potato, i.e. peeling, packaging and washing, while harvesting residues comprise
smaller but still remarkable share of non-used biomass. The amount of produced side
streams varies from process to process; in some peeling processes up to half of the
biomass ends up to side stream (Peusa & Piilo, 2006). Further processing of side
streams is costly and thus affects economy of industry. It is also notable that the
biological burden of these side streams is relatively high. For instance, biological
oxygen consumption of potato peel mass can be 10,000 mg L – 1 (Peusa & Piilo, 2006)
while that of municipal waste is only approximately 200 mg L – 1 (Riekkinen, 2007).
Additional pressure comes from EU directives regarding end-of waste criteria for
biodegradable waste subject to biological treatment (compost/digestate) in accordance
with Article 6 of Directive 2008/98/EC of the European Parliament and of the Waste
Framework Directive (JRC 2014). Thus, it is obvious that the valorization of potato
industry side streams would be beneficial for both environment and industry.
Data on produced side streams in the research area were collected by interviewing
of industry, supermarkets and potato farmers. Based on the survey, preliminary
feasibility evaluation was prepared for potato biorefinery concept around main
compounds in the side streams. By definition biorefining means sustainable processing
of biomass into spectrum of marketable products and energy. The basis of this research
was the assumption that biorefinery is the most feasible manner for optimal use of the
produced side streams. The preliminary biorefinery concept was thus build based on
the known composition of potato, the composition of processing side streams and the
estimation of resource potential and location. In our further research we intend to
develop necessary separation and purification processes for these compounds, and to
integrate the developed processes as potato biorefinery concept.

MATERIALS AND METHODS

An assessment of potato industry side streams in Northern Ostrobothnia was

conducted by direct industry survey. The survey was conducted during
January–February 2012. The target group included 28 packing plants (seed and food
potatoes), 7 potato processing plants (peeling plants, refining plants), and 11 large
retail stores (supermarkets). The target companies were asked to describe the amount
of produced side streams from potato production, processing and distribution, and the
current use of these streams. The companies were also asked the current costs of
processing the side streams and to evaluate, which price they would sell side stream
potatoes. In total the survey included 15 questions.
Resource estimates were made of the quantities of materials produced and the
potential for their use in biorefinery. Quantities of materials were estimated by
summing the answers from industry survey. Based on known compositions of potato
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industry side streams, the initial biorefinery plan included separation and upgrading of
starch, fibers, proteins and solid residues.
The main compound in potato is water, forming approximately 63–87% w/w
(Storey, 2007, Burlingame et al., 2009). The other main compounds are starch
(9.1– 22.6%) and other carbohydrates (mainly glucose, fructose and saccharose); and
fibers, from which 0.87–1.22% are soluble and 0.41–2.53% insoluble. Potato bulb
contains small amounts of proteins (0.85–4.2%), fat (0.05–0.51%), trace elements and
vitamins (6.5–34 mg 100 g-1 of vitamin C). In addition, potato contains toxic thermo
tolerant glycoalkaloids 0.071–175 mg 100 g-1 (Burlingame et al., 2009).
Potato starch is located in starch granules composed of amylose and amylopectin
(Storey 2007). Starch concentration depends on species, cultivation conditions and
season; in spring potato it is 10% and in starch potato 21% (Kangas et al., 2007). The
concentration of starch is highest in peels and smallest in the middle of potato in which
it can be even totally absent (Karlsson & Eliasson, 2003; Virtanen, et al., 2005). Native
potato starch is most suitable as filler and ‘binding agent’ and thus its properties have
been changed to allow wider spectrum of applications, first with chemical means
(Kraak, 1993) and later also biologically (Jobling, 2004). Modified starches are used
e.g. in building and textile industries (Lyckeby, 2012). Starch production creates potato
fruit juice (PFJ) and potato pulp as side streams, both of which contain proteins and
fibers. These side streams could be utilized better which would eventually also
improve the economy of industry.
Potato fiber includes other carbohydrates than starch, i.e. cellulose, hemicellulose,
pectic substances and pentosan. Those are present in tuber cell walls and intracellular
structures, and they comprise approximately 2.3% w/w of potato (Storey, 2007). Main
polysaccharides in fibers are cellulose (10–12%), pectin (0.7–1.5%) and hemicellulose
(1%). Pectic substances are mainly protopectin (70%), soluble pectin (10%) and pectic
acid (13.3%). Potato hemicellulose is composed of glucuronic acid, xylose,
galacturonic acid and arabinose (Kadam et al., 1991). Additionally starch resistant to
small intestine digestion is present.
Potato contains 6.9–46.3 g protein per kg of wet weight, the concentration being
highest in peel layer (Karlsson & Eliasson, 2003). The quality of potato protein
depends on amino acid composition. The quality is measured using biological value,
which describes the proportion of nitrogen in protein available for human consumption.
If all of the nitrogen can be utilized in human body the biological value is 100, which is
the reference value from egg white. Potato protein has biological value of 90–100
which means excellent composition of amino acids for human nutrition. It contains
nearly all essential amino acids for human with only exceptions of methionine and
cysteine (Storey, 2007). In addition, potato waste may include also high-valued protein
fractions that could be utilized commercially (Schieber & Saldaña, 2009).
Minerals comprise 1.1% of potato tuber weight (Storey, 2007). According to
research by MTT (Ahokas et al., 2012) dry matter mean value is 22.3%. The average
concentrations of phosphorus, potassium, calcium, sulfur, sodium, magnesium, copper,
mangane, zinc, iron and boron of five different cultivars has been studied in field tests
during 2005–2011 (Table 1).

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Table 1. Minerals in potato tubers (Ahokas et al., 2012)
Element P K Ca S Na Mg Cu Mn Zn Fe B
g kg-1
1.9 21.7 0.3 1.2 1.5 0.0 0.0046 0.0076 0.0113 0.0339 0.0065

The majority of potato industry side streams are formed during peeling, cutting
and packaging. Peeling of potatoes produces washing waters that include peel residues
and PFJ. In wet peeling 25–50% of the raw material ends up into residues. Its solid
content is 10–15%, which includes also some earth. Therefore, it is not usable as
animal feed. Dry peeling produces 50–100% less side streams than wet peeling. The
produced side streams include earthy water (produced during washing), peel mass
(pure potato), starch and PFJ. Peel mass (potato pulp) resulting from the industrial
starch processing is highly viscous and contains 16–17% by weight of dry matter of
which 30–35% starch and 60–65% non-starch polysaccharide material (NSP) (Mayer
et al., 2008). Additionally minerals are included (K : 20 g kg-1, C : < 0.8 g kg-1,
P : 1.4 g kg -1, Mg : 0.85 g kg-1). Peel mass may also be stored for some days due to
natural lactic acid fermentation; however, it is advisable to use it fresh (Peusa & Piilo,
2006). The energy content of peel mass is 13.6 MJ Kg-1 (Peusa & Piilo, 2006).

Table 2. The yield estimate of value added products from potato residue feedstock.
Yield
Product Available products in market
kg t-1
Recovered wastewater 2,000 Process Water,
Ethanol 331 Several potable vodka brands
Potato protein 102 Tubermine®, PRO GO™,
Potato protein isolate 0.63 Slendestra™, SUPRX™, Gly-Sea-Max™, Solathin™
Potato fiber 384 Vitacel™, PENFibe®RO, Pofiber (Semper), Potex
1
Izmirlioglu & Demirci (2012) (33g L-1 ethanol from waste potato mash); 2Approximate value
based on Karup Kartoffelmelfabrik (2007); 3US Patent 6414124 2002 (900 mg kg-1 potato);
4
Mayer et al. (2012) (24% by weight of the pulp dry matter)

The amount of produced PFJ is approximately 70% the whole peel mass. It is
easily spoiling and difficult to handle and its biological oxygen consumption is rather
high. PFJ contains 23.5% solids which is formed by starch (17.4%), proteins (2.5%),
fibers (1.8%), soluble carbohydrates (0.5%) and minerals (1%) (Bergthaller et al.,
1999). Nutrients are present as follows: N : 0.33% (from which half is soluble),
P : 0.045%, K : 0.47%, Mg : 0.03% and Ca : 0.002% (Riekkinen, 2007). PFJ may be
utilized as fertilize in farming according to respective legislation (Peusa & Piilo, 2006).
Cutting processes produce classification and cutting residues. In addition, spoiled,
under – or over dimensioned and incorrectly shaped potatoes are discarded to waste
during packaging (Peusa & Piilo, 2006). The amount and solid content of side streams
vary between processes.
The preliminary biorefinery concept was designed as a basis for the resource
assessment and estimation of biorefining potential in the region. This concept is based
on literature review on potato side streams, composition of potato and existing
technologies for separation and purification of main components starch, fibers and

698
proteins. To calculate material balance, certain yield values were chosen based on the
literature review. These yield values are presented in Table 2.

RESULTS AND DISCUSSION

The response rate of industry survey was 72%, which was considered sufficient to
assessment of resource potentials. The results of industry survey are summarized in
Table 3.

Table 3. The results of industry survey. Standard error of the mean in parentheses
Answers Treatment capasity Sorting waste Potato residue
Product
(n) T a-1 t a-1 (in total) t a-1
Seed potato
14 450–9,000 (1,513) 25–250 (44) 1,600
packing
Food potato
8 1,500–10,000 (955) 250–1,000 (95) 3,700
packing
Potato peeling 4 180–3,500 (706) 90–480 (80) 1,100
Markets 7 8–90 (10) 220

Packaging plants classify the potatoes in order to remove spoiled, under - or over
dimensioned and incorrectly shaped potatoes. A minor part of harvest is usually left
unsold e.g. due to excess supply. The annual processing volumes at seed potato
packaging plants and food potato packaging plants were 450–9,000 t a-1 and 1,500–
10,000 t a-1, respectively (Table 3). Potato processing steps and produced side streams
at a typical seed and/or food potato packaging plant are presented in Fig. 1.

Figure 1. Process steps and produced side streams at a typical seed or food potato packaging
plant. Solid line – primary use of side stream, dashed line – secondary use of side stream.

The amount of unsold potatoes at seed and food potato plants are 3–15% and
10–17% from total raw material, respectively. The difference is due to possibility of
seed potato packaging plants to direct unsold potatoes to be used as food. In total, side

699
streams from potato packaging plants count to approximately 5,000 t a-1, from which
approximately 85% is further utilized at food production (2nd class potatoes). Thus,
only 15% of packaging plant side streams, accounting to 1,100 t a-1, can be included in
the total side stream potential of the region. This residue is currently composted and
used either as fertilizer or animal feed. However, companies are willing to sell their
side streams if the income from it would be higher than current prices for 2 nd class
potatoes, and the transportation of biomass would be arranged without extra cost.
Based on our estimation, the price should be approximately 13 euro t-1 without
transportation cost.
Approximately half of the interviewed packaging plants are able to wash the
potatoes before packaging. Washing waters contain mostly earth and they are
processed by sedimentation and sand filters. The recovery of biomass compounds from
these waters can be considered unprofitable (Lehto et al., 2007).
Based on visual evaluation of potato farmers, approximately 5% of the harvest is
left to fields. The majority of this residue is formed by under dimensioned potatoes.
The exact amount of the residues is likely to depend on several factors, such as earth
humidity and specifications of harvest equipment. The farmers recognized possibilities
for improved harvest e.g. via minor equipment adjustments; however, this choice is not
attractive unless the price for 2nd class potatoes increases. In regional context the
harvest residues may be up to 6,000 t a-1 accounting a significant amount of biomass.
The major potato processing plants in the region are chip production plant in
Pyhäntä and multiproduct plant in Vihanti. In these processes 27–54% of the raw
material ends up to side streams which accounts to 16,100 t a-1. Vihanti plant share of
this is 15,000 t a-1, from which the majority is used in animal feed. This side stream is
composed of classification and peeling residues of 1st class potatoes, and peeling
residues of 2nd class potatoes that are used for potato flake production.
Other processing plants, mostly operating as peeling and cutting units, direct their
peeling biomasses either as feed or composting. The estimated total side stream
potential from peeling plants is 1,100 t a-1. The process steps of potato processing
plants are presented in Fig. 2.
Peeling plants that employ grinding method produce large amounts of starch
containing water and PFJ. According to survey this side stream can be of similar size
as the solid peeling residue. The surveyed companies used sedimentation, filtration and
centrifugation for separation of solids. Centrifuges were generally considered effective
but also too expensive for small companies.
Answers were received from 7 out of 11 surveyed retail stores. The annual
amount of unsold potatoes, vegetables and fruits was 8–90 t a-1 in each store. The
majority of this residue consisted of fruits and vegetables. Thus, the potential of potato
side stream from the retail stores is rather low. Furthermore, retail stores suggested that
the side streams should be removed in short sequences, several times a week, which
together with small amounts of produced side streams would lead to relatively high
logistic cost. Currently these residues are mostly composted which also generates costs
to the companies. Small portion of the residues ends up as animal feed.
In terms of feedstock potential the most feasible location for the biorefinery
would be in the proximity of Vihanti plant. The biorefinery would operate using side
streams of potato flake production and peeling residues which results to minimal
effects on material balances in plant itself. This would reduce the use of side streams
700
for animal feed production. However, if bioethanol is produced in the biorefinery, also
2nd-class potatoes could be applied for this purpose.

Figure 2. Process steps and produced side streams at potato processing plants. Solid line –
primary use of side stream, dashed line – secondary use of side stream.

Based on rough estimations, the capacity of biorefinery could be approximately

20,000 t a-1 biomass feedstock for its operation. According to the present results
17,000 t a-1 could be acquired within the studied region with relatively small
arrangements. The acquisition of retail store side streams could add to regional side
stream potential if it could be feasibly arranged. The potential could be further
increased if the use of harvest residues could be arranged in a feasible manner.
Considering maximal logistic cost of 10 euro t-1, feasible range for feedstock
transportation would be approximately 130 km (Paappanen et al., 2008). The
respective zone of supply would include the whole region of Northern Ostrobothnia
and areas of Central Ostrobothnia. It can be assumed that sufficient amount of
feedstock is available within this area. However, it is notable that the availability of
side streams depends on both annual harvest and potato demand, and therefore the
annual feedstock demand of 20,000 t may not be available at all times.

701
Figure 3. Estimation of material flow for potato biorefinery.

Material flow was estimated using the values from literature presented in Table 2.
Assuming a feedstock of 20,000 t a-1 the potato biorefinery would produce 660 t a-1
ethanol, 760 t a-1 potato fiber, 200 t a-1 potato protein and 12 t a-1 protein isolates Also
40,000 tons of waste water can be recycled. Estimation of material flow is presented in
Fig. 3.

CONCLUSION

1. The total potato side stream in the studied region would be about 17,200 t a-1. The
biggest side stream (15,000 t a-1) comes from Vihanti potato product plant. At the
present, main part (85%) of packing plants side stream is transported to Vihanti
plant for potato flake production.
2. Only 15% of packing plants side stream (1,100 t) and the peels (1,100 t) can be
added to Vihanti plant side stream, when estimating total side stream of the region.
3. Based on rough estimations biorefinery would require approximately 20,000 t a-1
biomass feedstock for its operation. According to the present results 17,000 t a-1
could be acquired within the studied region with relatively small arrangements.
4. The acquisition of retail store side streams could add to regional side stream
potential if it could be feasibly arranged.
5. The potential could be further increased if the use of harvest residues could be
arranged in a feasible manner.
6. In terms of biorefinery operations, significant role is played by the feedstock
availability.
7. It is important to obtain low-cost but high yield chemical recovery process.
8. According to presented biorefinery concept, potato protein conversion, including
ethanol recovery process and fiber production to high value products seems to be
the most beneficial technical solution.

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ACKNOWLEDGEMENTS. This research has been financed by European Regional
Development Fund, Council of Oulu Region in projects A31611 and A32182.

REFERENCES

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 Agronomy Research 12(3), 705–716, 2014

Effect of cold treatment on the amino acid composition of veal

D. Baranenko*, V. Kolodyaznaya and Y. Broyko

Institute of refrigeration and biotechnologies, ITMO University, 191002, Lomonosova

Street 9, Saint-Petersburg, Russia; *Correspondence: [email protected]

Abstract. Veal is a promising raw material for use in the daily diet, as well as for production of
functional and dietary foods. However the effect of cold treatment on the amino acid
composition of veal has not been sufficiently studied. The aim of this study was the amino acid
composition analysis of veal subjected to various variants of cold treatment.
The selected material under research was muscle tissue of hip parts from calves, grown in the
Leningrad Region, Russia and aged no more than 3 months. Cooling to 4 ± 1°C and rapid
freezing to the temperature of minus 18°C at the cooling air temperatures of minus 24°C and
minus 35°C were used as variants of cold treatment. Amino acid composition analyses were
carried out using precolumn derivatization with phenylisothiocyanate and reversed-phase
gradient HPLC on the Shimadzu 20-AD chromatograph with spectrophotometric detection at
254 nm.
The results show the effect of cold treatment on the content of free amino acids and total amino
acid composition of veal. In many respects changes in amino acid composition are concerned
with moisture losses during the refrigerating treatment. The dependence between the change in
amino acid content and the structure of its side chain group type is shown. Amino acid score for
essential amino acids was calculated and conclusions about changes in biological value of veal
protein were made.
The obtained data can be used in biological value calculation of the multi-component products
and food rations with veal subjected to refrigerating treatment.

Key words: cooling, freezing, HPLC, protein, meat.

INTRODUCTION

The main sources of protein for humans are products of animal origin and some
legumes. Protein biological value depends strongly on the type of raw material, and it
determines formation of the daily diet and reasoning of the consumption norms for
animal and vegetable proteins. The physiological average daily requirements in
proteins are systematically studied and analyzed in the decisions of the FAO/WHO and
national organizations of different countries, including the Russian Federation (FAO
1970; FAO/WHO 1991, 2007, 2011). However the effect of cold treatment on the
amino acid composition of meat has not been sufficiently studied.
Because of its chemical composition, veal is a promising raw material for use in
the daily diet, as well as for production of functional and dietary foods. Some of these
products are characterized by high protein content, at that the share of complete
proteins should be at least 60% of the total protein content. Furthermore, the internal
organs of calves are used to obtain a large number of biologically active substances, it

705
is necessary to store and rationally process the rest of the carcass. In this regard, a
study of veal is of particular interest.
In chicken muscle proteins after freezing at -30°C and frozen storage at -5°C for
10 weeks, the changes occurring in myofibrillar proteins as a result of frozen storage
were indicated by the loss of -SH groups, ATPase activity, solubility, and water-
holding capacity (Khan et al., 1968). Also the results show that rapid freezing
preserves the integrity of muscle proteins to a greater extent than slow freezing (Khan
& Berg, 1967). Freezing of bovine muscle has a denaturating effect on myofibrillar
proteins; the lower the freezing rate the greater the loss (Wagner & Anon, 1985).
Disulfide bond formation with the concomitant decrease in sulfhydryl group was
found in all species of some tropical fish during frozen storage (Benjakul et al., 2003).
It was found that the denaturation of proteins during freezing is closely related to
surface-induced denaturation (Chang et al., 1996). The review was made to describe
the various stages of freezing of freeze-dried therapeutic proteins and examine the
consequences of the various stresses developing during freezing on protein stability;
however, the study of changes of amino acid composition was not carried out
(Bhatnagar et al., 2007). Decrease in the content of each of sixteen amino acids was
found in Lactobacillus bulgaricus after freeze-drying (Mitić, 1976).
For other food products effect of freezing to -25°C on amino acid composition
was studied for selected species of edible mushroom (Bernaś & Jaworska, 2012). After
freezing at temperature of cooling air of -35°C and frozen storage mushrooms
contained significantly higher levels (3–118% on average) of leucine, lysine,
methionine, and phenylalanine, but lower levels (8–61% on average) of cysteine,
histidine, isoleucine, tyrosine and valine, compared with canned products.
Nutritional composition of veal is studied in several works, including the amino
acid composition of preruminant calves and special fed veal ribeyes (Williams, 1978;
Riss et al., 1983; Williams, 2007). However, information about the changes in amino
acid composition of veal depending on conditions of cold treatment could not be found.
The aim of this study was the amino acid composition analysis of veal subjected
to different variants of cold treatment.

MATERIALS AND METHODS

The selected material under research was muscle tissue of hip parts from calves,
grown in the Leningrad Region, Russia and aged no more than 3 months. The meat
was placed in a refrigerator at a temperature of 4 ± 1°C in 2 h after slaughter. After
three days of refrigerated storage, the samples with a thickness of 2.0 ± 0.3 cm and a
weight of 200 ± 5 g were isolated from the musculi biceps femoris et gluteus
superficialis and subjected to rapid freezing to -18°C. Freezing at the temperatures of
the cooling air of -24°C and -35°C were used as variants of cold treatment. Storage of
frozen meat at -18°C held no longer than 24 h. Prior to analysis, each sample was
placed in an individual plastic container and defrosted for 4 h at 5°C under natural
convection.
Standard samples of amino acids (Sigma), phenylisothiocyanate (Sigma),
acetonitrile (HPLC-grade, J.T.Baker) were used for the research. Isopropyl alcohol
(HPLC-grade), sodium acetate (puriss.), hydrochloric acid (puriss.), sodium hydroxide

706
(puriss. spec.) were from Vekton, Russia. Water for analysis was produced with a
Milli-Q purification system (Millipore) from twice-distilled water.
Amino acid composition analyses were carried out using precolumn derivatization
with phenylisothiocyanate and reversed-phase gradient HPLC. The method is a
combination and modification of methods described in Heinrikson & Meredith (1984),
Gunawan et al. (1990), Fierabracci et al. (1991), González-Castro et al. (1997). The
used method is certified by All-Russian Research Institute of Metrology and has a
number of M-02-902-142-07.
Analyses of amino acids derivatives were performed by liquid chromatograph
Shimadzu LC-20 Prominence with spectrophotometric detector (254 nm); column with
a reverse phase C18 (250 x 4.6 mm, 5 µm, manufactured by Supelco) and a
corresponding precolumn; mobile phase – a mixture of 6 mM sodium acetate, pH = 5.5
(component A), 1% isopropyl alcohol solution in acetonitrile (component B), and
6 mM sodium acetate pH = 4.05 (component C). Chromatographic analyses were
carried out in a gradient mode. Mobile phase flow rate was 1.2 ml min-1.
Standard samples of amino acids were dissolved in 1 M hydrochloric acid
solution. Aliquots of standard solution of 15, 25, 50, 100 and 150 µl were placed in
five test-tubes. Hydrochloric acid was removed from aliquots by drying on a water bath
at 60°C in a stream of air through the capillary by suction created by an electric
vacuum pump. Sodium hydroxide solution (0.10 ml of 0.15 M) was added to the dried
amino acids, mixture was stirred, and then 0.35 ml of phenylisothiocyanate in
isopropyl alcohol and 0.05 ml of water were added. The solution was thoroughly
stirred and left for 20 min at room temperature, then evaporated to dryness at a
temperature of 60°C. The dry residue was dissolved in 1 ml of water. The resulting
solution was subjected to chromatographic analysis.
Sample preparation for determining the total contents of amino acids included the
acid hydrolysis with 6 M hydrochloric acid at 110°C for 16–18 h. Samples (~ 0.2 g)
were placed in vials, that were filled with a solution of hydrochloric acid and
hermetically sealed. Before sealing the vapor phase was purged for 3 min with nitrogen
to prevent oxidation of amino acids by atmospheric oxygen. After cooling, the
hydrolyzates were filtered; aliquots (0.2–0.3 ml) were placed in test-tubes and
evaporated to dryness on a water bath at 60°C in a stream of air similar to the standard
solutions. Sodium hydroxide solution (0.10 ml of 0.15 M) was added to the dried
aliquots, mixture was stirred, and then 0.35 ml of phenylisothiocyanate in isopropyl
alcohol and 0.05 ml of water were added. The solution was also thoroughly stirred and
left for 20 min at room temperature, then evaporated to dryness at a temperature of
60°C. The dry residue was dissolved in 1 ml of water. The resulting solution was
subjected to chromatographic analysis.
For the determination of free amino acids, the meat samples were homogenized;
sample (5 g) was placed in a glass cup, filled with 50 ml of ethyl alcohol, mixed with a
magnetic stirrer, and small amount of fluid was filtered through a membrane filter.
Aliquots (0.2 ml) were collected, placed in vials and evaporated to dryness on a water
bath at 60°C in a stream of air. Phenylisothiocyanate derivatisation was then
performed; samples were dried and then dissolved in 1 ml of water similar to the
standard solutions and the solutions after acid hydrolysis. The obtained samples were
subjected to chromatographic analysis.

707
 All experiments were performed with at least three replicates; data was processed
by methods of mathematical statistics at theoretical frequency 0.95.

RESULTS AND DISCUSSION

Changes in free amino acids after freezing

Free essential and non-essential amino acids content change after veal freezing
process is shown in Table 1. As can be seen from Table 1 the total amount of these
acids is increased regardless of the freezing temperature. However, more free essential
amino acids are accumulated in the meat frozen at a temperature of -24°C (1.92 times)
than at -35°C (1.31 times).

Table 1. The contents of free amino acids in veal

Mass fraction of free amino acids in meat,
mg g(protein)-1
Amino acid after freezing at t, °С
before freezing
-24 -35
Essential
Histidine 1.47 ± 0.06 2.2 ± 0.2 1.15 ± 0.08
Isoleucine 0.17 ± 0.01 0.3 ± 0.03 0.25 ± 0.02
Leucine 0.31 ± 0.02 0.64 ± 0.04 0.44 ± 0.04
Lysine 0.18 ± 0.01 0.76 ± 0.08 0.63 ± 0.05
Methionine 0.021 ± 0.001 0.042 ± 0.004 0.038 ± 0.002
Cysteine + Cystine trace amounts trace amounts trace amounts
Phenylalanine 0.2 ± 0.02 0.31 ± 0.02 0.24 ± 0.02
Tyrosine 0.19 ± 0.01 0.39 ± 0.03 0.29 ± 0.02
Threonine 0.29 ± 0.02 0.77 ± 0.05 0.59 ± 0.05
Tryptophan trace amounts trace amounts trace amounts
Valine 0.24 ± 0.02 0.49 ± 0.03 0.4 ± 0.03
Total: 3.071 5.902 4.028
Non-essential
Alanine 1.11 ± 0.08 1.7 ± 0.1 2.4 ± 0.2
Arginine 11.0 ± 0.8 15.2 ± 0.9 11.7 ± 0.6
Serine 0.34 ± 0.02 0.60 ± 0.04 0.41 ± 0.04
Aspartic acid & Asparagine 2.6 ± 0.2 1.9 ± 0.2 1.54 ± 0.08
Glutamic acid & Glutamine 2.4 ± 0.1 1.41 ± 0.08 1.2 ± 0.1
Glycine 0.61 ± 0.06 0.74 ± 0.05 0.76 ± 0.06
Proline 0.17 ± 0.01 0.24 ± 0.02 0.19 ± 0.01
Hydroxyproline 0.037 ± 0.003 0.042 ± 0.003 0.032 ± 0.002
Total: 18.267 21.832 18.232
In total: 21.338 27.734 22.26

708
 The content of free non-essential amino acids changed in a lesser degree. Thus,
their content in veal, frozen at -24°C increased 1.20 times and at -35°C remained
almost unchanged.
Analysis of the data presented in Table 2 shows that the change in the amount of
free amino acids after freezing depends not only on the temperature of the process, but
also on the structure of their side chain group type. Content of certain amino acids is
reduced. Thus, meat freezing at -24°C and -35°C reduced the amount of aspartic acid
and asparagine 1.37 and 1.69 times, glutamic acid and glutamine 1.70 and 2.00 times,
respectively. Possibly, this change is due to the fact that the polar amino acid with
anionic radicals – aspartic acid and its monoamide glutamine have clearly marked
hydrophilic properties. Reactions of these acids deamination with formation of
nitrogen-free compounds – glutaric and succinic acids could take place during phase
transformation of water into ice and freezing. The histidine content decrease in 1.28
times during veal freezing at -35°C was also noted, which can probably be explained
by the reaction of decarboxylation and the formation of nitrogenous compound
histamine.
However, the content of most free essential and non-essential amino acids has
increased in meat frozen at -24°C and at -35°C. It should be noted that the greatest
changes take part in the polar amino acids with hydrophilic nonionogenic and cationic
radicals (Table 2). Thus, the amount of polar amino acids with hydroxyl nonionogenic
radicals increased 2.15 and 1.57 times in meat frozen at -24°C and -35°C, respectively.
The biggest changes from the polar amino acids with cationic radicals underwent
lysine, its content significantly increased 4.22 and 3.50 times in meat, frozen at -24°C
and -35°C. The amount of arginine in meat frozen at -35°C mostly unchanged.
The freezing temperature has no significant effect on the content of non-polar
amino acids with hydrophobic side chains. Thus, the amount of amino acids having
aliphatic hydrocarbon radicals increases 1.57 and 1.69 times in meat frozen at -24°C
and -35°C respectively. The exceptions are the amino acids alanine, leucine and
isoleucine. The amount of alanine increased 1.53 and 2.16 times, valine – 2.04 and
1.67 times, isoleucine 1.76 and 1.47 times in meat frozen at -24°C and -35°C,
respectively.
The amount of phenylalanine is increased 1.55 and 1.20 times, methionine – 2.00
and 1.81 times in meat frozen at -24°C and -35°C respectively.
Accumulation (content increase) of free amino acids during meat freezing
obviously is due to the proteolysis of muscle and connective tissue proteins. It is
known that proteins ability to be attacked by proteolytic enzymes is greatly enhanced
during freezing (Lawrie, 1968).
Meat protein denaturation takes place during a freezing process, it is
accompanied by conformational changes in a structure of a protein molecule. Since
denaturation ruptures large number of weak hydrogen and then hydrophobic and ionic
bonds, it should be assumed that cryodenaturation destroys mainly secondary structure
of proteins. This structure is formed by an interaction between functional groups of
amino acids by hydrogen bonds between the oxygen atoms and amino groups nitrogen
atoms.

709
Table 2. Changes in the free amino acids contents in veal after freezing depending on the
polarity of radicals
Mass fraction of free amino acids in meat,
Functional mg g(protein)-1
Amino acid
group after freezing at t, °С
before freezing
-24 -35
Nonpolar amino acids with hydrophobic side chains
Alanine 1.11 ± 0.08 1.7 ± 0.1 2.4 ± 0.2
Glycine 0.61 ± 0.06 0.74 ± 0.05 0.76 ± 0.06
Valine Aliphatic 0.24 ± 0.02 0.49 ± 0.03 0.4 ± 0.03
Leucine hydrocarbon 0.31 ± 0.02 0.64 ± 0.04 0.44 ± 0.04
Isoleucine R-groups 0.17 ± 0.01 0.3 ± 0.03 0.25 ± 0.02
Proline 0.17 ± 0.01 0.24 ± 0.02 0.19 ± 0.01
Hydroxyproline 0.037 ± 0.003 0.042 ± 0.003 0.032 ± 0.002
Total: 2.65 4.15 4.47
Phenylalanine Aromatic, 0.2 ± 0.02 0.31 ± 0.02 0.24 ± 0.02
heterocyclic
Tryptophan hydrocarbon trace amounts trace amounts trace amounts
R-groups
Total: 0.20 0.31 0.24
Methionine -SH 0.021 ± 0.001 0.042 ± 0.004 0.038 ± 0.002
Total: 0.021 0.042 0.038
In total: 2.87 4.50 4.75
Polar amino acids with hydrophilic non-ionogenic radicals
Tyrosine 0.19 ± 0.01 0.39 ± 0.03 0.29 ± 0.02
Serine -OH 0.34 ± 0.02 0.60 ± 0.04 0.41 ± 0.04
Threonine 0.29 ± 0.02 0.77 ± 0.05 0.59 ± 0.05
Cysteine + Cystine -SH trace amounts trace amounts trace amounts
Total: 0.82 1.76 1.29
Polar amino acids with anionic radicals
Aspartic acid & Asparagine 2.6 ± 0.2 1.9 ± 0.2 1.54 ± 0.08
-COOH
Glutamic acid & Glutamine 2.4 ± 0.1 1.41 ± 0.08 1.2 ± 0.1
Total: 5.0 3.31 2.74
Polar amino acids with cationic radicals
Histidine 1.47 ± 0.06 2.2 ± 0.2 1.15 ± 0.08
Lysine 0.18 ± 0.01 0.76 ± 0.08 0.63 ± 0.05
Arginine 11.0 ± 0.8 15.2 ± 0.9 11.7 ± 0.6
Total: 12.65 18.16 13.48

710
 Hydrogen bonds are formed between uncharged hydrophilic groups
(-OH, -CO-NH2, SH-groups) and any other hydrophilic groups. Secondary structure of
proteins has regular structures of two types: α-helix or β-structure. α-helical structure is
formed by a great amount of hydrogen bonds and it is among the most stable
conformation of the peptide backbone corresponding to the free energy minimum.
As a result of α-helix formation the polypeptide chain is shortened, but during
cryodenaturation due to rupture of hydrogen bonds, the polypeptide chain elongates
and becomes more accessible to the action of enzymes, as evidenced by the increase in
free amino acids content (Table 1, 2).
In contrast to α-helices, breaking of hydrogen bonds that form β-structure by lots
of hydrogen bonds between peptide groups linear regions of a single polypeptide chain
or between different polypeptide chains does not cause elongation of the latter.
Consequently the action of enzymes in β-structure of the denatured protein molecules
will be hindered.
It should be noted that a possible cryodenaturation result is violation of the
tertiary structure of proteins formed by hydrophobic, ionic and hydrogen bonds, but not
covalent ones. Hydrophobic interactions occur between hydrophobic amino acid
radicals, as well as Van der Waals forces between the closely spaced to each other
atoms. As a result, hydrophobic core is formed inside of the protein globule. In the
denatured protein hydrophobic radicals that in the native molecule structure are hidden
within a hydrophobic core appear on the surface. In absence of strong repulsive charge
molecules associate with each other by hydrophobic bonds that result in decreased
proteins solubility. In addition, the compact dense spatial structure of the native protein
after cryodenaturation is considerably increased in size and becomes also easily
accessible to the action of enzymes.
Hydrophilic groups of amino acid radicals are also involved in the formation of
the protein molecules tertiary structure. They tend to form hydrogen bonds with water,
and therefore, they are mainly located on the surface of the protein molecule.
All hydrophilic amino acid group radicals trapped within the hydrophobic core
interact with each other through ionic and hydrogen bonding. Ionic bonds occur
generally between charged (anionic) carboxyl groups of aspartic and glutamic acids
and the positively charged (cationic) groups of lysine, arginine or histidine.

Changes in proteins amino acids after freezing

The total amount of essential and nonessential amino acids and amount of each
amino acid of veal protein are reduced during the freezing process regardless of
temperature. However, such changes depend considerably on the structural
characteristics of amino acids, chemical structure of their radicals and their solubility
in water as well as on the freezing temperature.
Thus, the amount of essential amino acids of veal frozen at temperatures of -24°C
and -35°C decreased by 10% and 7%, non-essential – by 17% and 9%, respectively
(Table 3).

711
Table 3. Changes of essential and non-essential amino acids contents in veal proteins after
freezing
Mass fraction of amino acids in meat,
mg g(protein)-1
Amino acid after freezing at t, °С
before freezing
-24 -35
Essential
Histidine 17.7 ± 1.8 13.2 ± 0.9 14.6 ± 1.3
Isoleucine 49 ± 5 40 ± 4 43.4 ± 3.1
Leucine 48 ± 4 46.6 ± 2.2 47 ± 4
Lysine 102 ± 7 91 ± 10 90 ± 5
Methionine 40 ± 4 36.4 ± 3.0 36.7 ± 3.2
Cysteine + Cystine 21.0 ± 2.0 20.4 ± 0.9 20.8 ± 1.9
Phenylalanine 44.3 ± 3.9 40 ± 4 43.1 ± 3.5
Tyrosine 32.8 ± 1.4 30.6 ± 3.1 31.4 ± 1.7
Threonine 33.3 ± 1.9 24.6 ± 2.2 28.7 ± 2.8
Tryptophan 22.4 ± 2.4 19.7 ± 1.2 21.0 ± 1.7
Valine 67 ± 4 65 ± 5 66 ± 6
Total: 477.5 427.5 442.7
Non-essential
Alanine 32.3 ± 2.6 28.7 ± 1.6 30.4 ± 2.8
Arginine 52 ± 5 38.5 ± 1.6 43.4 ± 2.7
Serine 16.2 ± 1.0 13.5 ± 1.1 14.2 ± 1.4
Aspartic acid & Asparagine 47.4 ± 3.8 36.5 ± 1.8 39 ± 4
Glutamic acid & Glutamine 39.1 ± 3.5 28.7 ± 2.8 32.6 ± 1.5
Glycine 72 ± 7 68.3 ± 3.7 70.4 ± 3.8
Proline 26.1 ± 1.8 22.5 ± 1.4 24.6 ± 1.6
Hydroxyproline 62 ± 4 50.6 ± 3.7 60 ± 5
Total: 347.1 287.3 314.6
In total: 824.6 714.8 757.3

Table 4 shows that the freezing process and its temperature have least effect on
changes of amino acids with a nonpolar (hydrophobic) side chain group type compared
to other studied amino acids. Their content decreases in veal frozen at -24°C and -35°C
by 10% and 4%, respectively.
It is known that non-polar radicals having aliphatic hydrocarbon chains (radicals
of alanine, valine, leucine, isoleucine, proline and methionine) and aromatic rings
(radicals of phenylalanine and tryptophan) tend to each other or to other hydrophobic
molecules in water, reducing their contact surface with water (Baynes & Dominiczak,
2009). So it can be assumed that freezing of moisture has no significant effect on deep
proteolysis of proteins associated with the cleavage of amino acids with hydrophobic
radicals.

712
Table 4. Changes in the amino acids contents in veal after freezing depending on the polarity of
radicals
Mass fraction of amino acids in meat,
Functional mg g(protein)-1
Amino acid
group before after freezing at t, °С
freezing -24 -35
Nonpolar amino acids with hydrophobic side chains
Alanine 32.3 ± 2.6 28.7 ± 1.6 30.4 ± 2.8
Glycine 72 ± 7 68.3 ± 3.7 70.4 ± 3.8
Valine Aliphatic 67 ± 4 65 ± 5 66 ± 6
Leucine hydrocarbon 48 ± 4 46.6 ± 2.2 47 ± 4
Isoleucine R-groups 49 ± 5 40 ± 4 43.4 ± 3.1
Proline 26.1 ± 1.8 22.5 ± 1.4 24.6 ± 1.6
Hydroxyproline 62 ± 4 50.6 ± 3.7 60 ± 5
Total: 356.4 321.7 341.8
Phenylalanine Aromatic, 44.3 ± 3.9 40 ± 4 43.1 ± 3.5
heterocyclic
Tryptophan hydrocarbon 22.4 ± 2.4 19.7 ± 1.2 21.0 ± 1.7
R-groups
Total: 66.7 59.7 64.1
Methionine -SH 40 ± 4 36.4 ± 3.0 36.7 ± 3.2
Total: 40 36.4 36.7
In total: 463.1 417.8 442.6
Polar amino acids with hydrophilic non-ionogenic radicals
Tyrosine 32.8 ± 1.4 30.6 ± 3.1 31.4 ± 1.7
Serine -OH 16.2 ± 1.0 13.5 ± 1.1 14.2 ± 1.4
Threonine 33.3 ± 1.9 24.6 ± 2.2 28.7 ± 2.8
Cysteine + Cystine -SH 21.0 ± 2.0 20.4 ± 0.9 20.8 ± 1.9
Total: 103.3 89.1 95.1
Polar amino acids with anionic radicals
Aspartic acid & Asparagine 47.4 ± 3.8 36.5 ± 1.8 39 ± 4
-COOH
Glutamic acid & Glutamine 39.1 ± 3.5 28.7 ± 2.8 32.6 ± 1.5
Total: 86.5 65.2 71.6
Polar amino acids with cationic radicals
Histidine 17.7 ± 1.8 13.2 ± 0.9 14.6 ± 1.3
Lysine 102 ± 7 91 ± 10 90 ± 5
Arginine 52 ± 5 38.5 ± 1.6 43.4 ± 2.7
Total: 171.7 142.7 148.0

713
 Amino acids with uncharged hydrophilic radicals undergo more changes than
ones with nonpolar radicals. Radicals of these acids better dissolve in water, since they
are composed of polar functional groups (hydroxyl in serine, threonine and tyrosine,
thiol in cysteine) that form hydrogen bonds with water. In this regard, water freezing
and proteins denaturation followed by a break first of all of weak hydrogen bonds lead
to a decrease of these acids amounts during freezing.
Amount of amino acids with polar non-ionogenic radicals is reduced by 14% and
8% in veal proteins during freezing at temperatures of -24°C and -35°C, respectively.
Significant content changes were noted for amino acids with polar anionic and
cationic radicals. Amounts of amino acids with negatively charged polar radicals –
aspartic acid and asparagine, glutamic acid and glutamine, after veal freezing at -24°C
are reduced by 23% and 27%, after freezing at -35°C – by 18% and 17%, respectively.
The total content of amino acids with polar positively charged cations (histidine,
lysine, arginine) in veal frozen at temperatures of -24°C and -35°C decreased by 17%
and 14%, respectively.
The contents of amino acids such as tyrosine, leucine, valine and cysteine do not
change at studied veal freezing temperatures, obviously due to their low solubility in
water. In this regard, water freezing does not affect their structural changes.
Amino acid score of indispensable amino acids was calculated to estimate the
changes in the biological value of veal after freezing (Table 5). It was achieved by a
comparison of the content of the amino acid in the protein with its content in the
requirement pattern (Eq 1) (FAO/WHO, 2007).

(1)

Table 5. Changes in amino acid score of veal proteins after freezing

Adult requirements, Amino acid score
Amino acid mg g(protein)-1 after freezing at t, °С
before freezing
(FAO/WHO, 2007) -24 -35
Histidine 15 1.18 0.88 0.97
Isoleucine 30 1.63 1.33 1.45
Leucine 59 0.81 0.79 0.80
Lysine 45 2.27 2.02 2.00
Methionine 16 2.50 2.28 2.29
Cysteine 6 3.50 3.40 3.47
Phenylalanine +
38 2.03 1.86 1.96
tyrosine
Threonine 23 1.45 1.07 1.25
Tryptophan 6 3.73 3.28 3.50
Valine 39 1.72 1.67 1.69

714
 CONCLUSIONS

The content of most free amino acids has increased in veal after freezing and
defrosting. The greatest changes took part in the free polar amino acids with
hydrophilic nonionogenic and cationic radicals. The amount of free amino acids with
hydroxyl nonionogenic radicals increased 2.15 and 1.57 times after freezing at -24°C
and -35°C, respectively. The content of free amino acids with cationic radicals
increased 1.44 times in veal, frozen at -24°C. Accumulation of free amino acids during
meat freezing is associated with the proteolysis of muscle and connective tissue
proteins.
Freezing temperature has a weak effect on the total content of amino acids with
hydrophobic (nonpolar) radicals in veal. Their content decreases by 10% and 4% after
freezing at -24°C and -35°C, respectively. Significant changes are established for
amino acids with polar anionic and cationic radicals during veal freezing at -24°C. The
content of amino acids with polar anionic radicals decreased by 25%, with cationic
radicals – by 17%. This can probably be explained by the reaction of decarboxylation
and the formation of nitrogenous compounds.
It was found that the veal freezing process at temperatures -24°C and -35°C
reduces amino acid score of all essential amino acids, although to varying degrees.
Median amino acid score decrease of 10% occurred after freezing at -24°C (minimum
decrease of 2% and maximum of 26%). After freezing at -35°C median amino acid
score decrease was 7% (minimum–1%, maximum–18%). In cooled veal the only
limiting amino acid was leucine with amino acid score of 0.81. There were two
limiting amino acids in veal after freezing – leucine and histidine. Their scores after
freezing at -24°C were 0.79 and 0.88, at -35°C – 0.80 and 0.97, respectively.
It is recommended to freeze veal rapidly at -35°C and below to slow the
proteolysis and save contents of amino acids with polar nonionic, anionic and cationic
radicals.

ACKNOWLEDGEMENTS. This work was partially financially supported by the government

of the Russian Federation, Grant 074-U01.

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Baynes, J. & Dominiczak, M. H. 2009. Medical biochemistry. Elsevier Health Sciences, 712 pp.
Benjakul, S., Visessanguan, W., Thongkaew, C. & Tanaka, M. 2003. Comparative study on
physicochemical changes of muscle proteins from some tropical fish during frozen
storage. Food Res. Int. 36(8), 787–795.
Bernaś, E. & Jaworska, G. 2012. Effect of preservation method on amino acid content in
selected species of edible mushroom. LWT-Food Sc. and Tech. 48(2), 242–247.
Bhatnagar, B.S., Bogner, R.H. & Pikal, M.J. 2007. Protein stability during freezing: separation
of stresses and mechanisms of protein stabilization. Pharm. Dev and Tech. 12(5),
505–523.
Chang, B.S., Kendrick, B.S. & Carpenter, J.F. 1996. Surface‐induced denaturation of proteins
during freezing and its inhibition by surfactants. J. of Pharm. Sc. 85(12), 1325–1330.
FAO. 1970. Amino-Acid content of foods and biological data on proteins. FAO food and
nutrition series. Rome, Italy: FAO.

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FAO/WHO. 1991. Protein Quality Evaluation: Report of the Joint FAO/WHO Expert
Consultation. FAO food and nutrition paper 51.
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WHO/FAO/UNU expert consultation. Geneva, Switzerland: World Health Organization,
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nutrition paper 92.
Fierabracci, V., Masiello, P., Novelli, M. & Bergamini, E. 1991. Application of amino acid
analysis by high-performance liquid chromatography with phenyl isothiocyanate
derivatization to the rapid determination of free amino acids in biological samples. J. of
Chrom. B: Biomed. Sc. and App. 570(2), 285–291.
González-Castro, M.J., López-Hernández, J., Simal-Lozano, J. & Oruna-Concha, M.J. 1997.
Determination of amino acids in green beans by derivatization with phenylisothiocianate
and high-performance liquid chromatography with ultraviolet detection. J. of Chrom. Sc.
35(4), 181–185.
Gunawan, S., Walton, N.Y. & Treiman, D.M. 1990. High-performance liquid chromatographic
determination of selected amino acids in rat brain by precolumn derivatization with
phenylisothiocyanate. J. of Chrom. A, 503, 177–187.
Heinrikson, R.L. & Meredith, S.C. 1984. Amino acid analysis by reverse-phase high-
performance liquid chromatography: precolumn derivatization with phenylisothiocyanate.
An. Biochem. 136(1), 65–74.
Khan, A.W. & Berg, L. 1967. Biochemical and quality changes occurring during freezing of
poultry meat. Journ. of Food Sc. 32(2), 148–150.
Khan, A.W., Davidkova, E. & Berg, L. 1968. On cryodenaturation of chicken myofibrillar
proteins. Cryobiol. 4(4), 184–188.
Lawrie, R.A. 1968. Chemical changes in meat due to processing — A review. J. of the Sc. of
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Mitić, S. 1976. Transformation of amino acid composition in bacterial cells of Lactobacillus
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Wagner, J.R. & Anon, M.C. 1985. Effect of freezing rate on the denaturation of myofibrillar
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Williams, P. 2007. Nutritional composition of red meat. Nutr. & Diet. 64(s4), S113–S119.

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 Agronomy Research 12(3), 717–726, 2014

Vegetable ingredients in soft cheese made from concentrated

skim milk by ultrafiltration
N. Iakovchenko* and L. Silantjeva

Institute of Refrigeration and Biotechnologies, ITMO University, Lomonosova

Street 9, 191002, Saint-Petersburg, Russia; *Correspondence: [email protected]

Abstract. The objective of this research was to develop the technology and composition of soft
cheese made from concentrated skimmed milk by ultrafiltration with a low concentration factor.
UF enables reduction in the quantities of starter, rennet, colorants, and cheese making costs per
vat. The use of UF milk appeal to lactose intolerant consumers because of the low lactose levels
in the product.
Ultrafiltration was carried out using polyethersulfone membranes. Jerusalem potato and carrot
were chosen as vegetable ingredients. Since the soft cheese was made from skimmed milk
retentate, there was a necessity to compensate the lack of fat. In this research the impact of
orange fiber on organoleptic qualities was determined. For imparting mouthcoating, creaminess,
and providing for an even meltaway effect in the mouth CITRI–FI 100 FG (pure orange fiber),
CITRI–FI 200 FG (orange fiber with guar gum), CITRI–FI 300 FG (orange fiber with xanthan
gum) were chosen. Application of CITRI–FI 200 FG and CITRI–FI 300 FG enables the
production of high quality curd only up to certain concentrations, which do not have a
significant impact on the organoleptic qualities of soft cheese. The use of CITRI–FI 100 FG
enables to get firm curd and can at the same time create organoleptic properties of a fat-
containing product in the finished soft cheese.
Thus, on the basis of data CITRI–FI 100 FG is recommended to use for manufacturing soft
cheese where mouthfeel is needed. Optimal concentrations of CITRI–FI 100 FG were
determined.

Key words: Ultrafiltration, Jerusalem potato, soft cheese, vegetable ingredients, orange fiber.

INTRODUCTION

Due to the present unfavourable environmental and economic situation, the

dietary patterns of the population are undergoing a significant change towards an
imbalance of major components in diet (Bogatyrev, 1985; Bobylin et al., 2000). Thus,
the main problem in the field of nutrition is to create a range of products promoting
improvement of human health in daily consumption. Our research tries to solve this
problem by creating products with both dairy and non-dairy components. These
components help to improve food and the biological values of the products.
Nowadays, with increasing concern about consumer health, the aim is to reduce fat
and calorie consumption. Low fat, low calorie foods, which look and taste like their fatty
counterparts higher in calories, have been gaining in popularity. Thus, it is topical to
concentrate on the development of food products, which are nutritious and contain
reduced levels of calories and fat.

717
 By analyzing international experience, it was determined that increasing
production and expanding the range of soft cheeses could be a good idea. Along with
the high biological value, this type of cheese can be sold without ripening. Despite the
perspective in the manufacture of soft cheese, most of the whey proteins are lost in the
whey.
Ultrafiltration has been used in cheese making since about 1971 (Maubois &
Mocquot, 1971). Eventually the use of ultrafiltration has gained significance in cheese
making industry. Ultrafiltration enables the concentration, separation, and recovery of
individual milk components (Salhab, 1998). It provides complete retention of whey
proteins and improvement of the traditional technological process of manufacturing
cheese (Lelievre & Lawrence, 1988).
Low concentration factor (CF) of skimmed milk permits the use of traditional
equipment for cheese making and the only investment expenses are the purchase of an
ultrafiltration plant and putting it in service. Ultrafiltration can help minimize seasonal
variations in milk composition, which, in turn, can help standardize rennet coagulation
time, gel strength, and cheese yield (Lucey, 2000).
Based on the analysis of information data, Jerusalem potato and carrot purée have
been chosen as the vegetable ingredient. Jerusalem potato is a source of inulin and
oligofructose. Inulin and oligofructose improve immunity as well as calcium
absorption, reduce cholesterol in the blood and even reduce the risk of colon cancer
(Davidovich, 1957; Perkovets, 2007). Jerusalem artichoke has an ability of not
accumulating heavy metals (Reshetnik et al., 1997).
Carrot is used in cases of anemia, scurvy, rheumatism, visual disturbances,
cardiovascular diseases, and loss of strength. Carrot has hematopoietic, bactericidal, anti-
inflammatory, choleretic, diuretic, laxative, analgesic, and anti-sclerotic effect
(Novichikhina, 2003).
Orange fiber is a natural fiber, derived from the cell tissues of dried orange pulp
without using chemical reagents and only by mechanical treatment. It has a positive effect
on the physiological processes of the human organism: purifies from slags, reduces
cholesterol, eliminates heavy metals and improves the functioning of the gastrointestinal
tract (Gubina, 2010).
Based on the foregoing, the objective of this research was to develop the
composition of soft cheese made from concentrated skimmed milk by ultrafiltration, using
vegetable components.

MATERIAL AND METHODS

Preparation of retentate
Whole cow milk was obtained from a local market. Fat was removed by mechanical
separation that resulted in skimmed milk (8.65% non-fat solids, 3.19% total protein,
0.09% fat) and cream. Before concentration the skimmed milk was heated to 65°C and
held at that temperature during 25 s. The skimmed milk was concentrated by using
laboratory scale set Vivaflow 50. (‘Vivascience’, Sartorius group) (Fig. 1).
Ultrafiltration was carried out using polyethersulfone membranes (molecular weight cut-
off of 30 kDa, pump flow 200–400 ml min-1), until a retentate with CF 2 was obtained.
The temperature during the process was 50°С. Polyethersulfone membranes are preferred

718
for their low fouling characteristics, broad pH range, and durability. They also exhibit no
hydrophobic or hydrophilic interactions.

Figure 1. Vivaflow 50 set up.

Starter culture: BC–Uglich–No 4 (Lactococcus lactis subsp. lactis, Lactococcus

lactis subsp. cremoris, Lactococcus lactis subsp. diacetylactis, Leuconostoc lactis).
Ferment: Calf rennet was obtained from Moscow rennet factory.
Jerusalem potato: Powder of Jerusalem potato was obtained from Seliger.
Carrot purée: Sterilized carrot purée was obtained from a local store.
CITRI-FI: Orange fiber was obtained from Fiberstar Inc., USA.
Non–fat solids, total protein, and fat of skimmed milk were measured using milk
analyzer ‘Klever–2’.
To determine the concentrations of Jerusalem potato suitable for cheese
manufacturing, retentate was divided into several parts. Each part (1 l.) with added
Jerusalem potato was heated at 74 ± 2°C for 20–25 s. and cooled to 29°C. The doses of
Jerusalem potato ranged from 1% to 5% of retentate weight with the increment of 1%.
The control sample contained no vegetable component. Starter culture prepared by
inoculating sterilized skimmed milk with culture was added in the amount of 1%.
Lactococcus lactis subsp. lactis is used as an active acidifier. Lactococcus lactis subsp.
cremoris, Lactococcus lactis subsp. diacetylactis, Leuconostoc lactis are very
important for the flavour of the cheese. During the fermentation of starter culture,
lactic acid accumulates, which causes the pH–value to decrease, resulting in better
whey drainage. The mixtures were coagulated with calf rennet and left to curdle. After
coagulation the curds were subjected to organoleptic assessment. Jerusalem potato was
chosen because of its medicinal properties, specific pleasant taste qualities, and medical
recommendation for consumption. But adding this component resulted in cheese with
unacceptable colour for consumers. Carrot purée was decided to add for correction.
Investigations for determining the concentrations of sterilized carrot purée were
carried out according to the above procedure with only one addition, so that carrot purée
was added after the pasteurization of the mixture. The concentrations of Jerusalem potato
were 2% and 3%. The concentrations of sterilized carrot purée ranged from 1% to 9%
with the increment 1%.

719
 Despite the fact that inulin and oligofructose in Jerusalem potato have the capacity of
being used as fat replacers with excellent mouthfeel characteristics and provide better
organoleptic properties, their concentrations in the product are insufficient to get soft
cheese with the appearance, taste, consistency, and texture of fat–containing cheese.
To maintain a desirable creamy mouthfeel characteristic of the product, orange fiber
was chosen. This fiber is a powder of light cream colour with a neutral taste and smell. As
fiber does not require pre-hydration, it was added in the mixture in different
concentrations together with the other dry ingredient before heat treatment. The following
additives recommended by the manufacturer were chosen: CITRI–FI 100 FG (pure
orange fiber), CITRI–FI200 FG (orange fiber with guar gum), and CITRI–FI 300 FG
(orange fiber with xanthan gum). To determine the possible use of the additive, samples of
soft cheeses were obtained with additive dose ranging from 0.05% to 0.4% of mixture
weight with the increment of 0.05%. After complete coagulation, the curds were cut into
cubes, which were settled for 10 min. The curds were stirred carefully during the next 20
min and settled for 15 min. After that, the whey was drained and curds were transferred
into perforated moulds for draining and pressed under the force of gravity. The samples
of cheese were turned upside down three times during the first 5 h of draining and
pressing. The pressing temperature was 16–18°C. Then the cheeses were soaked in
concentrated brine solution (18–22%) for salting for an appropriate period of time
depending on the size of the cheese sample.
Organoleptic assessment
Samples were evaluated for organoleptic properties by a taste panel of the 11 staff
members and students from Milk Technology and Food Biotechnology Department. The
participants were selected and trained in accordance with the ISO 8586-1 standard
(1993). Requirements for the work of the group of assessors were according to ISO 8589
standard (2007).

Organoleptic evaluation of cheese curds

The aim of the cheese curds sensory evaluation was to determine the acceptable
concentrations of vegetable additive for the manufacturing of soft cheese. The quality of
the cheese curds was evaluated for appearance (colour, colour homogeneity),
consistency, and texture (hardness, and flavour (odour and taste), using a 7–point scale
(Pereira et al., 2011). The participants were asked to assess a number of specific
attributes (Tables 1 and 2). The cheese curds were randomly coded with three-digit
numbers.

Table 1. Description of organoleptic attributes for several sensory features, used by selected
assessors to assess cheese curds with various concentrations of Jerusalem potato
Organoleptic Continuous scale
Sensory features
attribute 0 7
Appearance Colour White Dark beige
Intensity of
Jerusalem potato
Taste and odour taste and odour Not detected Pronounced
Creaminess Not detectable Intensive
Aftertaste Not intensive Intensive
Consistency and texture Firmness Weak, very soft Very firm

720
Table 2. Description of organoleptic attributes for several sensory features, used to assess cheese
curds with concentrations of Jerusalem potato (2% and 3%) and various concentrations of
sterilized carrot purée
Continuous scale
Sensory features Organoleptic attribute
0 7
Appearance Colour Gray beige Creamy
Colour homogeneity Heterogeneous Homogeneous
Intensity of Jerusalem
Taste and odour potato taste and odour Slight Pronounced
Creaminess Detectable Intensive
Aftertaste Slight Very intensive
Consistency and texture Firmness Weak, very soft Very firm

Cheese organoleptic evaluation

The aim was to compare more than two samples; therefore, the method of
ordering by preference was applied. The samples were coded with three-digit random
numbers. A cheese sample was divided into various portions and equilibrated at room
temperature. Its overall acceptability was evaluated, using a five–point hedonic scale
(from 1 = I disliked very much to 5 = I liked very much). The samples were presented to
the group in randomized order after salting. For assessment were chosen samples with
CITRI–FI concentrations that allowed to obtain firm cheese curds with a sharp, clean
split which were suitable for manufacturing cheeses with more or less improved
organoleptic quality.

pH measurement
pH values were measured using pH–meter (pH 4–10 with a glass combination
electrode).

Moisture measurement
Moisture content was determined by drying method using moisture determining
device ‘ELEX – 7’.

RESULTS AND DISCUSSIONS

The results of the sensory assessment of cheese curds quality are given in
Figs. 2–4.
It can be seen (Fig. 2) that appearance, taste and odour, consistency, and texture
of cheese curds were affected by the concentrations of Jerusalem potato.
The control cheese curd was a little firmer than the cheese curds with increasing
concentration of Jerusalem potato. The colour range of the control sample and the
samples with Jerusalem potato addition varied significantly. The concentration increase
up to 3% led to insignificant decrease in curd firmness. The increase in concentrations
of vegetable additive more than 3% led to a gradual decrease in organoleptic
characteristics. The consistency of cheese curds became weaker, porous and even
deliquescent. The taste and flavour of Jerusalem potato were strongly pronounced. All
that makes these cheese curds the least acceptable for manufacturing cheese.

721
Figure 2. Spider plot of sensory attributes (lowest = 0; highest = 7) of cheese curds samples
with various concentrations of Jerusalem potato, where: control sample, 1%,
2%, 3%, 4%, 5%.

Thus, the most acceptable concentrations of Jerusalem potato for cheese

manufacturing are in the range from 2% to 3%. Further concentration increase leads to
deterioration in the consistency of the curds, which complicates obtaining soft cheeses and
makes them unacceptable for the consumer.
The data obtained from the profiles (Figs. 3, 4) showed that with the increase in
carrot purée concentration and constant Jerusalem potato concentration, the cheese
curd appearance became better. The samples gradually obtained a creamy colour. At
the same time, an increase in carrot purée concentration resulted in a decrease in the
consistency quality of the curd. When comparing the samples, it was found out that the
higher the concentration of carrot purée, the lower the intensity of Jerusalem potato
taste and odour, creaminess, and aftertaste in the cheese curd samples.
Based on the data, the recommended concentration of Jerusalem potato is no more
than 2%, while for sterilized carrot purée the concentration should be no more than 8%.
The optimal concentration of carrot purée is 8%, as this concentration improves colour
and positively influences the taste of the product. With the increase in concentration of
Jerusalem potato more than 2% and sterilized carrot purée more than 8%, deterioration in
the quality of the curd occurred.
Research on laboratory scale has established relationships between concentrations of
orange fiber and organoleptic characteristics of the soft cheese. The increase in the
amount of CITRI–FI 300 FG has a negative effect on the quality of the curds. Acceptable
curds may be obtained if the amount of the additive does not exceed 0.05 % of the weight
of the mixture. But this amount has no positive effect on the organoleptic characteristics
of the final product. With increasing dosage of CITRI–FI 300 FG, curds became mushy

722
and with a ragged split, making the further process of obtaining a product with acceptable
organoleptic characteristics complicated.

Figure 3. Descriptive sensitive profiles for samples with 3% of Jerusalem potato and various
concentrations of carrot purée, where: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%.

Figure 4. Descriptive sensitive profiles for samples with 2% of Jerusalem potato and various
concentrations of carrot purée, where: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%.

723
 The application of CITRI–FI 200 FG to improve the organoleptic characteristics of
cheese revealed that an increase in the amount of additive has a negative impact on the
taste and consistency of the obtained curds, and hence on the organoleptic characteristics
of the product. The concentration of the additive more than 0.25 % of the weight of the
mixture resulted in porous consistency of the curds, which further resulted in large losses
of solids in the whey. With increasing dosage of CITRI–FI 200 FG, the finished product
obtains an off-flavour. The amount of the additive lower than 0.25 % did not lead to
significant improvement in the taste characteristics of the final product.
The application of CITRI–FI 100 FG at an approximate level of 0.05% to 0.4%
enables to obtain curds with a sharp, clean split. The finished product has gained creamy
mouth feel characteristics and an acceptable texture. Based on the sensory characteristics
of the soft cheese, the addition of CITRI–FI 100 FG to the cheeses at levels 0.25 % or
0.3% of mixture weight was preferred. Further increase does not lead to significant
improvement in the quality of the finished product.
For providing a soft cheese with the texture, smoothness, and organoleptic properties
of a fatty product, the possibility of using CITRI–FI was investigated. The positive data
were obtained. The researches on the effect of CITRI–FI concentrations on pH value of
the soft cheeses were carried out. The data are presented in Fig. 5.

CITRI–FI 100 FG, CITRI–FI 200 FG, CITRI–FI 300 FG

Figure 5. Effect of CITRI–FI concentrations on pH value of the soft cheeses.

The overall quality of cheese samples (Fig. 6) with the addition of CITRI–FI 100
(0.25%) was more acceptable for the panelists than control samples without CITRI–FI
and with concentrations of CITRI–FI 300F G (0.05%) and CITRI–FI 200 FG.

724
 5
4
3
2
1
0
overall acceptibility

Figure 6. Overall quality of the products investigated, where: control sample, CITRI–FI
300FG, CITRI–FI 200FG, CITRI–FI 100FG.

CONCLUSIONS

1. In the course of the production of soft cheese from ultrafiltrated skimmed milk,
Jerusalem potato and carrot purée can be used as vegetable ingredients with dosage 2%
and 8%, respectively.
2. In order to produce soft cheese with the texture, smoothness and organoleptic
properties of a fatty product, but containing no substantial fat, it is recommended to use
CITRI–FI 100 FG at a level 0.25%.
3. The increase in CITRI-FI concentrations was stated to reduce the pH value of the
soft cheese inconsiderably.

ACKNOLWLEDGEMENTS. This work was partially financially supported by the government

of the Russian Federation, Grant 074-U01. The author, Iakovchenko N., is very grateful to the
family for moral and financial support. The author wish to acknowledge Zyryanova A., Rykov
S., and all those persons who have supported me.

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manufacture of combined soft cheeses. International scientific-practical conference
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99–100 (in Russian).
Bogatyrev, G.N. 1985. Designing of Food. Chemistry and Life. 12, 3–9 (in Russian).
Davidovich, S.S. 1957. Jerusalem potato. State Publishing House of Agricultural Literature,
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Gubina, I. 2010. ‘CITRI-FI’ – a new component of a healthy diet. Milk Processing 3, 51.
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of assessors, Part 1. Selected assessors.
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 Agronomy Research 12(3), 727–736, 2014

On the potential of lupin protein concentrate made by

enzymatic hydrolysis of carbohydrates in dairy-like applications
L. Kuznetsova*, L. Zabodalova and D. Baranenko

Institute of Refrigeration and Biotechnologies, ITMO University, Lomonosova

Street 9, 191002, Saint-Petersburg, Russia;
*Correspondence: [email protected]

Abstract. The aim of this research was to study the parameters of obtaining lupin concentrates
by enzymatic hydrolysis of wholegrain lupin flour and application of these concentrates in the
technology of high-protein dairy-like products. The following enzymes preparations were used:
Celluclast BG, Cellulaza 100, Pentopan Mono BG and α-amylase. The usage of Cellulaza 100
and Pentopan Mono BG showed the highest protein amounts in the lupin concentrates under the
test conditions. Three factors were studied to take effect on crude protein content in the product:
temperature in the range 50–60°C; cellulase dosage between 0.54–1.62 units g-1; ratio water:
flour – 10 : 1, 15 : 1, 20 : 1. Optimum hydrolysis conditions for Cellulaza 100 were temperature
of 55°C; ratio water: flour of 15 : 1; cellulase dosage of 1.08 units g-1. Crude protein content in
the final product increased on 12% compared with the original flour and on 8–9% compared to
the lupin concentrate obtained without enzymes. Hydrolysis by multienzymatic compositions
was tried as an alternative way of increasing the efficiency of the process. However hydrolysis
by multienzymatic compositions was not yet found so efficient as hydrolysis by pure Cellulaza
100. The lupin protein concentrate was dispersed in water and mixed with skimmed milk to have
total product protein content about 5%. The mixture was fermented by yogurt starter culture;
consumer properties of final products were investigated. Fermented products supplement the diet
with vegetable proteins, fats, carbohydrates and fiber, which have high biological value.

Key words: lupin protein concentrates, lupin whey, multienzymatic compositions, fermented
products, analogues of dairy products.

INTRODUCTION

Limitation of resources of food, caused by both environmental and demographic

factors, makes the scientists and manufacturers looking for the new ways to meet the
needs in essential food nutrients. The main interest in lupin for foods is related to its
high content of protein which is considered as a good source of lysine
(El-Adawy et al., 2001). Typically, lupin seeds have a crude protein content of
31–42%, which is higher than the content of most other grain legumes (Pollard et al.,
2002). Lupin already has many human consumption applications, such as bread
making, pasta products, sausage substitutes, egg and milk replacers (Xu & Mohamed,
2003; Xu et al., 2006). Sweet lupin diet helps lowering whey cholesterol level (Chango
et al., 1998). Whole lupin flour-enriched foods as well as isolated lupin protein and
fiber fractions appear to have a cardioprotective action (Belski, 2012).
Yet the application of lupin products in food is limited, largely due to their
‘green’ and ‘bean-like’ flavor and antinutrirional substances. Fermentation of lupin

727
protein extracts using several lactic acid bacteria was conducted to reduce off-flavour
formation in stored samples (Schindler et al., 2011). The main antinutritional
substances in lupin are various alkaloids of the quinolizidine group. Hexane-defatted
flakes of lupin (Lupinus rautabilis) were extracted under various conditions with
alcohols to remove alkaloids (Blaicher et al., 1981). Production of protein isolates can
overcome this problem too. Alkaloids are water-soluble and would be removed during
preparation of the isolates (Lqari et al., 2001). The usage of some organic solvents
improves lupin protein isolates quality (Bader et al., 2011).
Protein concentrates are considered to have greater than 50% protein and they are
usually native flour (dehulled kernels) from which the carbohydrates (free sugars and
oligosaccharides) and other soluble materials have been removed. Australian scientists
have proposed the method of obtaining the protein preparation from lupin grains,
which includes selection, treatment and fractionation of grains, their fragmentation,
extraction of the protein, precipitation and drying (Sipsas, 2003). Muranyi et al. (2013)
have studied two important techniques of protein isolation: the alkaline extraction with
subsequent isoelectric precipitation and the salt-induced extraction followed by dilutive
precipitation.
Plant proteins isolates and concentrates are used in the production of meat
products, analogues of dairy products and combined foods. Lupin seeds and lupin seed
protein isolates were used in the manufacture of fermented sausages (Papavergou et al.,
1999). Soybeans are used as raw material to obtain soy yoghurt. It is hypoallergenic, so
such products can be used in the diet of people suffering from milk protein intolerance
(Osman & Razig, 2010; Vij et al., 2011). Soy-coconut yogurts were also studied
(Kolapo & Olubamiwa, 2012). The milk-like product from Lupinus сampestris was
obtained by using an alkaline thermal treatment (Jimenez-Martınez et al., 2003).
Usage of enzymes is an alternative removal antinutritional substances method.
Microbial hydrolytic enzymes are able to destroy many antinutritional components in
plant raw material, which perform linking and protective function, such as phytin,
cellulose, hemicellulose and lignin, and others (Ferket & Middleton, 1998). The
improvement of carbohydrate extractability due to hydrolysis of polysaccharides
allows producing a final product with higher protein content.
The most widespread, commercial enzyme products currently available for
biomass hydrolysis are produced by submerged fermentation of the saprophytic
mesophilic fungus Trichoderma reesei (Olsson & Ahring, 2007). The commercial
enzyme preparation ‘Celluclast’ is a multiactive carbohydase for degradation of
cellulose, cellobiose and higher polymers of glucose that could be used for improving
malt quality (Grujic, 1998). Usage of Celluclast 1.5L for pectin extraction increases the
pectin yield (Yuliarti et al., 2011). The degradation of biomaterial by cellulase is
accompanied by the release of substrates for the action of other enzymes, particularly
for xylanase and mannanase. The glyukuronoksilan and mannan are rapidly
decomposed in a system with cellulase (Viikari et al., 1994). The application of
arabinofuranosidase with xylanase leads to complete removal of xylan (Makkonen &
Nakas, 2005).
The aim of this research was to study the parameters of obtaining lupin
concentrates by enzymatic hydrolysis of wholegrain lupin flour and application of
these concentrates in the technology of high-protein dairy-like products.

728
 MATERIALS AND METHODS

Materials
Lupinus angustifolius wholegrain flour was provided by the All-Russian Scientific
Research Institute of Lupin, Bryansk, with a crude protein content of 46%, crude fat –
7.1%, fiber – 4.0%.
Selection of enzyme preparations was carried out on the basis of carbohydrate
composition of lupin seeds, which includes fiber, small share of starch (about 4%),
hemicelluloses and pectin (in total 10%) (Kupcov & Takunov, 2006).
The following enzymes preparations were used:
 Celluclast BG – cellulase preparation made by submerged fermentation of the
selected strain of fungus Trichoderma reesei, containing 3,500 endoglucanase
units gram-1. Preparation was provided by Novozymes, Denmark;
 Cellulaza 100 – cytolytic complex enzyme preparation derived from a mixed
culture of fungi Aspergillus foetidus and Trichoderma viride, containing 540
сellulase units gram-1, Sibbiofarm, Russia;
 Pentopan Mono BG – xylanase preparation from fungi Aspergillus oryzae,
containing 2,500 fungal xylanase units gram-1, Novozymes, Denmark;
 α-amylase, containing 950 fungal amylase units gram-1. Preparation was provided
by State scientific institution All-Russia Scientific Research Institute of Fats of
Russian Academy of Agricultural Sciences, Russia.

The yogurt starter culture of Streptococcus salivarius ssp. thermophilus and

Lactobacillus delbrueckii ssp. bulgaricus were provided by the State scientific
institution All-Russia Scientific Research Institute of Fats of Russian Academy of
Agricultural Sciences, Russia.

Preparation of the lupin concentrate

The used method is close to the one for obtaining the soy concentrate in the acidic
medium (Sair, 1959). The full-fat lupin flour was diluted with water in a ratio of 1 : 10.
The resulting solution was adjusted to pH 4.5 by adding 5% HCl to achieve protein
isoelectric point. The following process was carried out in a thermostatic vessel with a
magnetic stirrer (Khalil et al., 2006). Different variants of enzyme preparations were
added after the suspension reached a temperature of 50ºC. The mixture was maintained
for 40 min.
The mechanical separation of phases was performed by centrifugation at 4000  g
for 30 min. The sediment obtained after centrifugation was the final product (the lupin
concentrate). The sediment contained protein and fat fractions as the flour used had a
fat content of 7.1%.
The sediment protein content was analyzed after centrifugation. Total content of
protein, water-soluble carbohydrates and their component composition in the lupin
whey (supernatant) were analyzed.

Preparation of the fermented products

For making the non-dairy vegetable yoghurt analog lupin protein concentrate was
dissolved in water to a dry matter content of 10%. The dispersion was neutralized with
alkali to pH 6.8–6.9, homogenized at 60–65ºC, pasteurized at 92ºC for 3 min and
729
cooled to a temperature of fermentation. Sucrose was added in an amount of 1% to
increase the quantity of nutrients for a starter culture. Fermentation was carried out to
achieve the required pH values of 3.8–4.2.
The combined dairy vegetable product was made by adding skim milk instead of
sucrose to the pasteurized dispersion in ratio of dairy and lupin proteins 50 : 50. Total
protein content of the mixture was about 5%. Fermentation was held to pH 4.4–4.5.
For all products the yogurt starter culture was used in an amount of 5% of the
system mass. The temperature of fermentation was 42 ± 2ºC.

Measurements
Water content in the lupin protein concentrate was determined by the gravimetric
method (AOAC, 1998). The content of crude protein was determined by Kjeldahl
method on automated analyzer Kjeltec Auto (Tecator, Sweden) according to standard
protocol of manufacturer. Crude protein content was estimated using a conversion
factor 6.25 from total nitrogen.
The content of crude fat was determined by the Soxhlet method on automated
analyzer SER 148 (VELP Scientifica, Italy) according to standard protocol of
manufacturer. The ceramic fiber filter method was used to determine the crude fiber
(AOAC, 1980).
Analysis of the total content of water-soluble carbohydrates was conducted by
Bertrand method (Bertrand & Thomas, 1910). Changes in pH were measured with
Orion 920A pH-meter (Russia).
Component analysis of mono- and disaccharides was conducted by HPLC
‘Stayer’ (Akvilon, Russia) with refractometric detector, the column ‘Luna NH2 5µ’,
(Phenomenex, USA). The mobile phase consisted of acetonitrile and water in volume
ratio of 77 : 23. ‘Stayer’ HPLC system with spectrophotometric detector and column
‘Luna C18’ (Phenomenex, USA) was used for analysis of the organic acids in whey,
which was obtained by centrifugation of the fermented products samples at 4,000  g
for 30 min. Solution of 0.1% orthophosphoric acid in distilled water was used as a
mobile phase.

Statistical evaluation of the data

All experiments were performed with at least three replicates; data was processed
by methods of mathematical statistics at theoretical frequency 0.95. Statistical
processing of data was carried out using computer programs Microsoft Office Excel
2010 and Mathcad 15.0.

RESULTS AND DISCUSSION

Effect of the enzyme preparations on the lupin concentrate

Enzymes with different substrate specificities were tried for the hydrolysis of lupin
flour polysaccharides. Bioconversion efficiency was evaluated by the content of crude
protein in the lupin concentrate and in the lupin whey (Table 1). The data was compared
with the results for the negative control sample (the lupin concentrate obtained without
enzymes).
Part of the water-soluble protein fraction is transferred to the whey during the
lupin concentrate making; despite the system pH value of 4.5 is near the isoelectric

730
point. On average lupin concentrate loses 19% of protein with the lupin whey
according to the mass balance. The usage of Cellulaza 100 and xylanase preparation
leads to the highest protein amounts in the lupin concentrates under the test conditions.
Cellulaza 100 was used for further optimization of the hydrolysis conditions.

Table 1. Crude protein and water content in the products of the lupin concentrate making, %
The name of the Concentrate protein, Whey protein,
Concentrate water
enzyme on a dry basis on a dry basis
Control sample 50.4 ± 1.3 75.2 ± 1.1 28.0 ± 1.0
Cellulaza 100 53.3 ± 1.1 77.9 ± 0.5 34.0 ± 1.5
Celluclast BG 50.0 ± 1.1 79.2 ± 0.6 30.4 ± 1.0
Pentopan Mono 53.0 ± 1.2 78.6 ± 0.9 34.1 ± 1.4
α-amylase 48.2 ± 1.1 71.6 ± 0.4 29.4 ± 1.2

Optimization of the hydrolysis conditions

Optimum hydrolysis conditions were
necessary for increasing the efficiency of
the process. Three factors were studied to
take effect on crude protein content in the
product (Y, % to dry substance):
temperature in the range of 50–60°C (Z1);
cellulase dosage of 0.54–1.62 u g-1 (Z2);
ratio water: flour – 10 : 1, 15 : 1, 20 : 1
(Z3). The mixture was exposed to the
hydrolysis for 40 min.
Optimal process parameters were
obtained by means of rotatable plan of the
second order and regression equation
coefficients were found. The resulting
response surfaces have the form of an
elliptic paraboloid (Fig. 1).
The significance of the regression
equation coefficients was determined by the
Student's criterion. The adequacy of the
regression equation was estimated by the
Fisher test.
Optimum hydrolysis conditions for
Cellulaza 100 were temperature of 55°C;
ratio water: flour of 15 : 1; cellulase dosage
of 1.08 units g-1. Crude protein content in
the final product under these conditions was
59.3 ± 1.1% on a dry basis. Crude protein
content increased on 12–13% compared Figure 1. Dependence of crude protein
with the initial flour and on 8–9% compared content in the lupin concentrate on
to the lupin concentrate obtained without temperature, ratio water: flour and cellulase
enzymes. dosage.

731
 Hydrolysis by multienzymatic compositions
Hydrolysis by multienzymatic compositions was tried as an alternative way of
increasing the efficiency of the process. The degradation of non-starchy
polysaccharides was conducted in the presence of Cellulaza 100 or Celluclast BG. An
attempt was made to improve Cellulaza 100 hydrolysis efficiency with help of
α-amylase. The synergetic effect is known between xylanase and cellulase. These
enzymes act on cellulose, xylan and other hemicelluloses of lupin flour (Jeffries,
1996). So there was an attempt to raise the protein yield of the concentrates made with
Celluclast BG with help of the xylanase preparation Pentopan Mono.
The compositions of the ferments included cellulases in the optimal dosage of
1.08 u g-1 and other enzymes in recommended or higher dosages. The ratio of enzymes
in the compositions was calculated according to their declared activity (Table 2).
Hydrolysis was carried out in the previously found optimal conditions for
Cellulaza 100.

Table 2. Effect of multienzymatic compositions on the content of crude protein, fat and fiber in
the lupin concentrates
Content in % on a dry basis
No Multienzymatic composition
crude protein crude fat crude fiber
1 1.08 ± 0.02 u g-1 ‘Cellulaza 100’ &
54.9 ± 1.4 10 ± 1 5±1
0.7 ± 0.2 u g-1 α-amylase
2 1.08 ± 0.02 u g-1 ‘Celluclast BG’ &
56.0 ± 1.1 10 ± 1 5±1
5 ± 1 u g-1 ‘Pentopan Mono’
1.08 ± 0.02 u g-1 ‘Celluclast BG’ &
3 51.9 ± 1.3 9±1 5±1
25 ± 1 u g-1 ‘Pentopan Mono’

About 20% of cellulose was subjected to bioconversion to soluble carbohydrates

according to the material balance. Multienzymatic composition based on cellulase
1.08 ± 0.02 u g-1 and xylanase 5 ± 1 u g-1 showed highest protein yeild results under the
used conditions. However hydrolysis by multienzymatic compositions was not yet
found so efficient as hydrolysis by pure Cellulaza 100. Moreover, increased content of
Pentopan Mono resulted in reduced crude protein content in the lupin concentrate of the
third sample. This may be due to the inhibition of the action of one enzyme preparation
by hydrolysis products of other one. This issue requires further study.

Transition of carbohydrate and protein fractions in the lupin whey

The lupin whey of samples after hydrolysis with 1.08 ± 0.02 u g-1 ‘Celluclast BG’ &
5 ± 1 u g-1 ‘Pentopan Mono’ was analyzed as they showed highest protein yeild results
under the used conditions for multienzimatic compositions. The resulting lupin whey
contains some extractive substances from lupin seeds (organic acids, soluble
carbohydrates and vitamins, other biologically active substances), molecular nitrogen
compounds (amino acids, peptides, albumin fraction of proteins) and lipids, which were
released from the initial substrate in the process of hydrolytic destruction of cellular
structures (Table 3). The initial lupin flour and the resulting lupin whey had a dry
substance content of 90% and 2%, respectively.

732
Table 3. Crude protein, mono- and disaccharides percentage of the initial lupin flour and
resulting lupin whey
% to dry substance
flour whey
Crude protein 46.3 ± 1.1 30.0 ± 1.4
Mono- and disaccharides 3.4 ± 0.5 25.0 ± 0.3

The balance of material is shown in equation 1:

Мflour ·Sflour ·Cflour = Мwhey ·Swhey ·Cwhey, or 100·0,90·0,034 → 1280·0,02·0,25 (1)

where: M – mass of the product (g); S – solids (dry substance), share units; C –
carbohydrates, share units.

Total content of mono- and disaccharides in the lupin whey according to the
material balance has increased in 2 times in comparison with their content in the initial
flour.
Mass fraction of total sugar (to invert sugar) in the whey was 0.5% (25% to dry
substance). Non-starchy polysaccharides hydrolysis increases the concentration of
soluble disaccharides (in particular, sucrose) and monosaccharides in the whey up to
17% and 3% to dry substance, respectively. High performance liquid chromatography
of the lupin whey showed that the ratio sucrose: glucose: fructose is 10 : 1 : 1.
Transition of protein fraction to the whey was 18% of the amount contained in the
flour. That corresponds to the data obtained for the samples hydrolysed with
Cellulaza 100.
Thus, 20% of the hydrolyzed cellulose and some quantity of the hydrolyzed xylan
increase yields of mono- and disaccharides in the whey twice but accompanied by the
protein loss of 18%.

Application of the lupin concentrates in dairy-like products

Three fermented products were studied: the non-dairy vegetable yoghurt analog,
the combined dairy vegetable product and the control product based on a skim cow
milk. All received clots had homogeneous consistency with minor release of whey,
sour-sweet taste and fruity smell. The usage of vegetable raw material in the combined
product leads to dynamic reduction of pH in the first 6 h of fermentation comparing to
the control product (Fig. 2). This gives the possibility to reduce the fermentation time.
The initial pH value of the lupin dispersion was 1.2 units lower than in the control
sample. Buffer capacity of this dispersion was lower compared to the skim milk because
of its plant origin.
Composition of organic acids in the whey obtained by centrifugation of the
products samples characterizes the biochemical process of fermentation (Table 4).
Increase in the acidity of the combined product in comparison with the control
product mainly depends on the malic acid production. This is due to the formation of
by-products of homofermentative lactic acid fermentation, in particular malic acid.
Organic acids are produced in varying degrees in the process of fermentation and
storage of yoghurts (Fernandez-Garcia & McGregor, 1994). The course of lactic acid

733
fermentation can be different depending on environmental conditions. Malic, propionic
and some other organic acids are formed on the Embden-Meyerhof-Parnas pathway.

Figure 2. Dependence of active acidity on the fermentation time and the product base.

Table 4. Composition of organic acids in the whey samples

Content of lactic acid, Content of malic acid, The amount of lactic and
Whey sample g dm-3 g dm-3 malic acids, g dm-3
Vegetable analog 3.93 ± 0.19 0.17 ± 0.03 4.1
Combined product 6.7 ± 1.3 1.9 ± 0.4 8.6
Control product 6.9 ± 1.2 – 6.9

Table 5. Macronutrients and energy value of the fermented products

Vegetable yoghurt
Parameter Combined product Control product
analog
Protein, % 4.8 ± 0.2 4.8 ± 0.2 5.0
Fat, % 0.8 ± 0.1 0.6 ± 0.2 1.5
Fiber, % 0.4 ± 0.1 0.2 ± 0.1 –
Energy value, kcal (per 100 g) 61.3 67.4 75.1

The fermented products supplement the diet with vegetable proteins, fats,
carbohydrates and fiber, which are necessary for the proper functioning of the
gastrointestinal tract and have high biological value (Table 5).
The vegetable yoghurt analog and the combined product can be classified as low-
calorie dietary foods. Fiber in the composition of these products would have a positive
effect on intestinal motility. Sensory analysis of the products with hydrolyzed lupine
flour revealed no ‘green’ and ‘bean-like’ flavors.

734
 CONCLUSIONS

Enzymatic treatment of the lupin flour is an effective method for increasing the
protein content in the concentrate production. Hydrolysis of the lupin flour with
Cellulaza 100 increased crude protein content in the lupin concentrate on 12–13%
compared with the initial flour and on 8–9% compared to the lupin concentrate obtained
without enzymes. This led to сrude protein content in the concentrate of 59.3 ± 1.1% on
a dry basis. Average protein loss from moving in the whey amounted 18–19%.
Improving efficiency of hydrolysis using a combination of enzyme preparations still not
yielded the expected results, and requires further study.
The formation of malic acid during lactic acid fermentation of products containing
lupin concentrates gives the possibility to reduce the fermentation time. The received
products have homogeneous consistency, sour-sweet taste and fruity smell without
negative flavor. They can be classified as low-calorie dietary foods with energy value
less than 70 kcal per 100 g.
The proposed ways of making fermented products allow reducing the cost of
vegetable yoghurt analogue in 1.7 times, the combined product in 1.3 times compared
with the traditional product in Russia. Implementation of these products can help
diversifying production due to the necessity of compensation of technological risks and
of using new food sources.

ACKNOWLEDGEMENTS. This work was partially financially supported by the government of

the Russian Federation, Grant 074-U01.

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of lupin proteins produced by ultrafiltration-diafiltration. J Am. Oil Chem. Soc.
83(6), 553–558.
Yuliarti, O., Matia-Merino, L., Goh, K.K., Mawson, J.A. & Brennan, C.S. 2011. Effect of
Celluclast 1.5L on the Physicochemical Characterization of Gold Kiwifruit Pectin. Int. J.
Mol. Sci.12, 6407–6417.

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 Agronomy Research 12(3), 737–744, 2014

Quantitation of vitamins A and E in raw sheep milk during

lactation period
T. Michlová1, Š. Horníčková1, H. Dragounová2 and A. Hejtmánková
1
Department of Chemistry, Faculty of Agrobiology, Food and Natural Resources,
Czech University of Life Sciences Prague, the Czech Republic
2
Dairy Research Institute Ltd, Prague, the Czech Republic

Abstract. In this article, the influence of breed and lactation stage on vitamin A and E content
in raw sheep milk was studied. The milk of the East Friesian, Romanov and Lacaune sheep
breeds was included in the study. The samples were taken once a month throughout lactation.
The total average content of vitamin A in raw milk of all sheep breeds during lactation was
0.76 ± 0.19 mg kg-1 of milk and the total average content of vitamin E was 2.86 ± 0.99 mg kg1
of milk. The content of vitamin A and E during lactation varied in respect to the breed type. The
highest average content of vitamin A and E was detected in the Romanov sheep:1.01 ± 0.19
mg kg-1 of milk for vitamin A and 4.26 ± 1.90 mg kg-1 for vitamin E. The lowest average value
of vitamin A was found in the milk of the East Friesian sheep (0.56 ± 0.10 mg kg-1). This milk
showed also the lowest content of vitamin E 2.11 ± 0.53 mg kg-1. The highest content of
vitamins was found in summer and at the end of lactation, which is in September, and the
lowest values appeared in early lactation, which is in April.

Key words: Lactation, sheep milk, sheep breed, vitamin A, vitamin E.

INTRODUCTION

The production of sheep milk has been increasing in the Czech Republic in recent
years. People are becoming more and more aware of the milk and dairy products of
these small ruminants. Due to the increasingly expanding network of farmers’ markets,
it is much easier to get hold of these products. The East Friesian and Romanov sheep
are the most widely bred breeds in the Czech Republic. The Lacaune sheep have
gained popularity only recently in the Czech Republic. Sheep milk is much easier to
digest than cow milk. Its fat is dispersed in smaller fat beads (Jandal, 1996). In
addition, due to the better digestibility of sheep milk proteins, sheep milk is an
important constituent of the diet of people suffering from allergies. Milk contains
relatively low amounts of vitamin A and E; however, due to its frequent consumption
in various forms, it represents an important dietary resource, taking into account the
higher average fat content in sheep milk (7%) (Raynal-Ljutovac et al., 2008). Thus, a
higher expression of lipophilic vitamins in sheep milk when compared to cow and goat
milk is to be expected.

737
 Vitamin A plays a role in the biochemical pathways related to visual perception,
affects the growth, differentiation and maturation of gametes, and is important for fetal
development, growth, and bone development (Debier et al., 2005). It plays a role in the
synthesis of proteins, nucleic acids, and lipoproteins. Vitamin A is also an effective
antioxidant. Vitamin deficiency is associated with vision disturbances (night
blindness), inhibition of growth and deformities of bone and reproductive organs. High
doses of vitamin A result in increased hepatic reserve. In pregnant women, it may
have teratogenic effects (Miller et al., 1998). The recommended daily dose for an adult
is in the range of 0.8 mg (2,600 IU) – 1 mg (3,300 IU) and for children from 0.4 to
0.6 mg (1,300–2,000 UI) (Capito and Calleja, 2006).
Vitamin E is a very important antioxidant. It has an important function in
protecting the body against free oxygen radicals, which can lead to DNA damage. It is
also a factor that slows down the ageing of the body and plays a role in the prevention
of cardiovascular diseases and cancer (Eitenmiller & Junsoo, 2004). Vitamin E is
present in food, being dissolved in fats, and is released and subsequently absorbed
during their cleavage in the intestine. The recommended daily dose of vitamin E is
from 10 to 15 mg for adults, this value is around from 5 to 8 mg for children (Monsen,
2000). Vitamin E deficiency is often associated with disorders of fat absorption or
distribution or cystic fibrosis (Pekmezci, 2011).
The content of vitamins in raw milk is influenced by many factors. These include
animal species, breed, stage of lactation, and individual health status. According to
Zervas & Tsiplakou (2011), another important aspect is the nutrition of the animal and
the specific character of farming.
There are not many studies, which focus on the investigation of vitamin content in
raw sheep milk in the Czech Republic. The aim of our study was to determine the
content of fat-soluble vitamins A and E in the milk of sheep from private farms in the
Czech Republic and to assess the effect of lactation period on the content of these
vitamins.

MATERIALS AND METHODS

Experimental material
Pooled milk samples of the East Friesian sheep (a herd of 380 head), the
Romanov sheep (a herd of 130 head), and Lacaune sheep (a herd of 85 head) were
collected throughout the lactation from April to September on private farms once a
month, in cooperation with the Dairy Research Institute. The milk samples were taken
first 20 days after parturition.
The ration of all breeds was based on full-day pasture ad libitum, hay, and silage.
Mineral licks have been used in all cases as a dietary supplement: MILLAPFOS,
BIOSAXON for the Romanov sheep, RUMIHERB, NATURMIX for the East Friesian
sheep, and SANO for the Lacaune sheep. A mixture of pressed grains was also
supplemented.

738
 The chemicals. Determination of vitamins
For the preparation of the analytical samples, we used the following standards and
chemicals: DL-α-tocopherol, 98.2% (CALBIOCHEM, Canada), tocopherol set
(CALBIOCHEM, Canada), retinol, > 99% (Sigma-Aldrich, Germany), pyrocatechol,
> 99.5% (Sigma-Aldrich, Germany), potassium hydroxide, min. 85% (Lachema, Czech
Republic), methanol, p.a., content 99.5% (Lachner, Czech Republic), hexane, clean
min. 95.0%, Penta, Czech Republic, methanol, super gradient, content min. 99.9%
(Lachner, Czech Republic), and treated distilled water (Milipore, France).
Measurement of vitamin E and A content in milk samples
Vitamin E (or the individual tocopherols (T) and tocotrienols (TKT)) and vitamin
A were determined by high performance liquid chromatography with fluorescence and
spectrophotometric detection, respectively.
Approx. 1 g of homogenized sample was weighed in a plastic tube with a lid. 200
ml of methanol pyrocatechol (0.2 g ml-1) was added, then 5 ml 1M KOH, and the
mixture was vortexed for 20 seconds. Subsequently, the sample was saponified for 10
minutes on ultrasound. Then the mixture was vortexed again for 20 seconds. Then, 5
ml of hexane and 1 ml of distilled water were added to the mixture. The mixture was
vortexed for 1 minute. Subsequently, 3 ml were taken from the upper hexane layer and
evaporated on a rotary evaporator until dry. The residue was dissolved in 0.5 ml of
methanol and an aliquot was transferred through a nylon filter into a 1 ml Eppendorf
tube, which was placed in the freezer (-20ºC) for 30 minutes. Subsequently, the sample
was centrifuged for 2 minutes (by 14.4 rpm) and drained off into a dark vial. For the
analytic extension we used the chromatographic system Ultimate 3,000 (Dionex,
USA), consisting of a quaternary high-pressure pump, an autosampler, a column
thermostat, a fluorescence detector and a diode array detector. We employed an
analytical column with a precolumn packed with Develosil 5µm RPAQUEOUS
(250 × 4.6 mm); Develosil 5µm C30 UG-100A (10 × 4 mm) (Phenomenex, USA),
which allows the separation of all forms of tocopherols and tocotrienols (Fig. 1).

Figure 1. Chromatogram of vitamin E (compared to the standard): 2 – standard, 1 – sample.

739
A mixture of MeOH: deionized water (97 : 3, v/v) was used as the mobile phase; flow
rate of 1 ml per minute. Column temperature was 30°C and 10 ml sample injection was
applied. For the detection of tocol wavelengths we selected: excitation 292 nm and
emission at 330 nm. Vitamin A was determined by spectrophotometric detection at
λ = 325 nm (Fig. 2).

Figure 2. Chromatogram of vitamin A (compared to the standard): 2 – standard, 1 – sample.

The content of analytes in the samples was evaluated by external calibration.

Calibration curve of all tocols and vitamin A was linear in the range from 0.05 to
10 µg ml-1. The detection limit for each tocol, expressed as a ratio of three times the
value of the signal-to-noise ratio, was as follows: δ-tocotrienols and δ-tocopherol
0.01 µg ml-1, β-tocotrienol, γ-tocotrienol, β-tocopherol and γ-tocopherol 0.025 µ ml-1,
α-tocotrienol and α-tocopherol 0.05 µ ml-1, vitamin A 0.025 µ ml-1. The results were
processed with Chromeleon and MS Excel. The results were expressed as m kg-1 of
milk. Statistical analysis was done in Statistica Version 9. The measured values were
processed by the analysis of variance method (ANOVA), using post-hoc Tukey’s tests
for more detailed evaluation.

RESULTS AND DISCUSSION

The content of vitamin A and E was detected in raw milk of 3 sheep breeds – the
East Friesian, the Romanov and the Lacaune. The average values of vitamin A and E in
the milk samples of individual breeds are presented in Table 1. The total average
content of vitamin A in raw milk of all sheep breeds during lactation was
0.7–0.1 m kg-1 of milk and the total average content of vitamin E was 2.8–0.9 m kg-1.
The correlation between the content of vitamin A and vitamin E was found (R2 = 0.62,
P < 0.05).

740
 The results show significant differences in the content of vitamins A and E in
relation to the breed of sheep. The highest (P < 0.05) average content of both, vitamins
A and E, was found in the milk of the Romanov sheep (1.01 ± 0.19 mg kg-1 and
4.26 mg kg-1 ± 1.90, respectively) when compared with two other breeds. The average
content of vitamin E in the milk of the Lacaune sheep vs. milk of the East Friesian
breed was not statistically different while the difference between these two breeds was
found for vitamin A in milk (Table 1.) Raynal-Ljutovac et al (2008) found the content
of vitamin E in sheep milk 1.1 mg kg-1. The content of vitamin A is according to this
author 0.8 mg kg-1. Park et al (2007) gives the value of vitamin A content 0.44 mg kg-1
(146 UI). Thus, values of vitamin A content determined in this study correspond to
values published in Czech and foreign studies, while the content of vitamin E was
higher than most of the published values related to sheep milk.

Table 1. The average content and standard deviation of vitamins A and E in sheep milk
East Friesian Lacaune Romanov
Vitamin A 0.56 ± 0.10a 0.70 ± 0.11b 1.01 ± 0.21c
Vitamin E 2.11 ± 0.53a 2.21 ± 0.84a 4.26 ± 1.91b
The values in the same line marked with different letters (a–d) differ significantly (P ≤ 0.05)

The amount of vitamins in sheep milk varies during lactation. The lowest
measured value of vitamin E (0.15 mg kg-1) among all the breeds was found in the milk
of the Romanov sheep in April. The highest value was found in September
(6.70 mg kg-1) in the milk of the Romanov sheep, too. The content of vitamin E in the
milk of the Lacaune sheep and the East Friesian sheep had a similar time-course to the
milk of the Romanov sheep but the values were lower through all the lactation stages.
The lowest content of vitamin E among all the breeds was found at the beginning of the
lactation period, which is in April. The average value was 0.59 ± 0.39 mg kg-1. The
highest average value of vitamin E among all the breeds was recorded at the end of
lactation, which is in September (4.4 ± mg kg-1). In other months, the content of
vitamin E was not statistically different from each other. It appears that the content of
vitamin E in raw sheep milk rises from April to September, and thus increases during
lactation (Fig. 3).

741
 8

Content of vitamin E (mg.kg-1)

7
6
5
4
3 East Friesian
2 Lacaune
1
Romanov
0

Month

Figure 3. Content of vitamin E during lactation.

The values of vitamin A during lactation are recorded in Fig. 4. The lowest value
of vitamin A (mg kg-1) between all the monitored breeds was found in the milk of the
East Friesian sheep in August. The highest content 1.25 mg kg-1 was recorded in
September in the milk of the Romanov sheep. The values of vitamin A had a very
similar time-course to that of vitamin E.

1,40
Content of vitamin A (mg.kg-1)

1,20

1,00

0,80

0,60 East Friesian

0,40 Lacaune
0,20 Romanov

0,00

Month

Figure 4. Content of vitamin A during lactation.

742
 The content of vitamin A in milk increases from the beginning of the lactation
period till June, then decreases slightly and takes the highest values in September.
Statistically highest average content of vitamin A 0.93 mg kg-1 was determined in
September, the lowest average content 0.58 ± 0.08 mg kg-1 in April. The values
measured in May, June and July were not statistically different.
It can be presumed that fluctuations in the above mentioned vitamin levels during
lactation are associated with warm weather in the summer months and also with
changes in the fat content of sheep milk during lactation.
The content of both vitamins in sheep milk experimentally determined in this
study was higher than the content of both vitamins in cow milk given in literature. The
difference is more striking for vitamin E than for vitamin A (Table 2).

Table 2. Comparison of sheep and cow milk (average content)

Sheep milk Cow milk
Vitamin A 0.76 ± 0.19 mg kg-1 0.30–1.00 mg kg-1*
Vitamin E 2.86 ± 0.99 mg kg-1 0.20–1.20 mg kg-1*
*Fox & McSweeney (1998)

CONCLUSION

There were found significant differences in the content of vitamin A and E

between three sheep breeds. The highest content of both, vitamins A and E, was found
in the milk of the Romanov sheep. The milk content of both vitamins differed in
respect to the lactation period as well. The highest content was found in September and
the lowest in April. A strong correlation was found between the average content of the
two examined vitamins over the entire monitored period. Sheep milk contains a higher
amount of both vitamins compared to cow milk.

ACKNOWLEDGEMENTS. This work was supported by a grant MSMT 2B08072 and an

institutional support of the Ministry of Agriculture of the Czech Republic No. RO0511.

REFERENCES

Capita R. & Caleja C.A. (2006). Evaluation of vitamin and mineral intakes and impact of snack
foods on Spanish adults. Nutr Res. 26, 255–265.
Debier, C., Pottier, J., Gofee, C.H. & Larondelle, Y. (2005). Present knowledge and unexpected
behaviours of vitamins A and E in colostrum and milk. Livest Prod Sci. 98, 135–147.
Eitenmiller, R. & Junsoo, L. 2004. Vitamin E – Food Chemistry, Composition and Analysis.
Food Science and technology, Marcel Dekker, New York, 530 pp.
Fox, P.F. & Mcsweeney, P.L.H. 1998. Dairy Chemistry and Biochemistry. Blackie Academic &
Professional, an imprint of Chapman & Hall, London. 487 pp.
Jandal, J.P. (1996). Comparative aspects of goat and sheep milk. Small Ruminant Res.
22, 177–185.
Miller, R.K., Hendrikx A.G., Mills, J.L., Hummler, H. & Wiegand, U.W. (1998). Perinconcepti
onal vitamin A use: How much is teratogenic? Reprod Toxicol. 12, 75–88.

743
Monsen, E.R. (2000). Dietary Reference Intakes for The Antioxidant Nutrients: Vitamin C,
Vitamin E, Selenium, and Carotenoids. J Am Diet Assoc. 6, 637–640.
Öste, R., Jägerstad, M., Anderson, I. 1997. Vitamins in milk and milk products. In Fox, P.F.
(ed.): Advanced Dairy Chemistry, Vol. 3 Lactose, water, salts and vitamins. 2nd ed.,
Chapman & Hall, London. 536 pp.
Park, Y.W., Juáres, M., Ramos, M. & Haenlein, G.F.V. (2007). Physico-
chemical characteristics of goat and sheep milk. Small Ruminant Res. 68, 88–113.
Pekmezci, J. (2011): Vitamin E & Immunity. Vitamins and Hormones. 86, 179–215.
Raynal-Ljutovac, K., Lagriffoul, G., Paccard, P., Guillet, I. & Chilliard, Y. (2008). Composition
of goat and sheep milk products: An update. Small Ruminant Res. 79, 57–72.
Zervas, G. & Tsiplakou, E. (2011). The effect of feeding system on the characteristic of product
of small ruminants. Small Ruminant Res. 101, 140–149.

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 Agronomy Research 12(3), 745–752, 2014

The effect of modified atmosphere storage on the postharvest

quality of the raspberry ‘Polka’
U. Moor1,*, P. Põldma1, T. Tõnutare1, A. Moor2 and M. Starast1
1
Institute of Agricultural and Environmental Sciences, Estonian University of Life
Sciences, Kreutzwaldi 1, EE5104 Tartu, Estonia;
*Correspondence: [email protected]
2
Institute of Economics and Social Sciences, Estonian University of Life Sciences,
Kreutzwaldi 1, EE5104 Tartu, Estonia

Abstract. The aim of the experiment was to determine the effect of a passive modified
atmosphere package (MAP) (30 μm LDPE bag (Estiko, Estonia), the Xtend® raspberry bag
(Stepac, Israel) and an active MAP (30 μm LDPE bag, flushed with a gas mixture containing
10% O2 and 15% CO2) on the postharvest quality of the raspberry ’Polka’. Raspberries stored in
macroperforated punnets (normal atmosphere – NA) served as the control. The raspberries were
initially stored for 3 days at 1.6°C, then a half of the bags were moved into simulated retail
conditions (6ºC) and a half remained at 1.6°C for another 24 hours.
The raspberry weight, O2 and CO2 content of the packages were measured daily. The fruit dry
matter (DM) and soluble solids content (SSC), titratable acidity (TA), ascorbic acid content
(AAC), and total anthocyanins (ACY) were determined at harvest and the total antioxidant
activity (TAA) and rotting (weight of rotten berries) were determined after storage. Neither the
active nor passive MAP suppressed rotting significantly. The raspberries stored at 1.6°C, had
the best quality in passively modified LDPE bags, since the fruit had higher SSC and TA and
lower ACY content compared to the control. After the simulated retail conditions, the Xtend ®
bags turned out to be the most suitable, since the fruit had the lowest ACY content (the fruit did
not become too dark), but the highest TAA.

Key words: Rubus idaeus, soluble solids, titratable acids, ascorbic acid, anthocyanins, total
antioxidant activity.

INTRODUCTION

Growing primocane raspberry cultivars is a good opportunity for berry growers in

Estonia and other Nordic countries to prolong the fresh raspberry season. Also,
pesticide need is reduced in primocane raspberries, since the oviposition period of the
dominant pest, the raspberry beetle (Byturus tomentosus De Geer), does not coincide
with the flowering period of the primocane cultivars (Vetek & Penzes, 2008). Also,
frost damage during the winter does not affect the yield of primocane raspberries as
much as it does summer fruiting raspberry cultivars.
The main risk in Estonia for cultivating primocane raspberry is the cold climate,
since early autumn frosts and low temperatures in September may hinder raspberry
ripening. The Polish primocane raspberry ‘Polka’ has been introduced as a self-
supporting, semi vigorous, early autumn bearing, and high yielding cultivar (Danek,

745
2002). The first commercial plantations with the raspberry ‘Polka’ have been
established in Estonia. Besides the local market, Finland is the main destination for
Estonian berries and the producers are looking for cultivars, which would stand long
distance transportation. Danek (2002) has stated that the fresh fruit of ‘Polka’ are firm
and cohesive with tight skin. This statement makes it possible to hypothesize that this
cultivar could have good storage potential. However, to our knowledge, postharvest
experiment results with ‘Polka’ raspberries have not been published yet.
The aim of the current research was to determine the effect of normal and
modified atmosphere storage on the postharvest quality of the raspberry ’Polka’,
considering both the external quality and the nutritional value of the fruit.

MATERIALS AND METHODS

The ‘Polka’ raspberries were grown in a commercial plantation in South Estonia (NL
58º15’33’’; EL 26º35’33’’), where brown pseudopodzolic soils dominate. The
plantation was 3 years old and was established using black polyethylene mulch and drip
irrigation. The plant to plant spacing was 0.5 m and the distance between the rows was 3
m. Weather conditions in 2008 were not favourable for raspberries, since the summer
was very rainy. The fruit development and ripening period in August was extremely
wet, since 216 mm rained, which was almost three times more than the average (79
mm).
The raspberries were harvested at commercial maturity on September 14. The
fruit with uniform size and colour and free from defects were picked directly into 250g
macroperforated plastic punnets, transported to the university, cooled down to 1.6°C
and packed as follows:
1) 250g macroperforated plastic punnets covered with a lid (control);
2) Xtend® raspberry bag (Stepac, Israel);
3) passive modified 30 μm low density polyethylene (LDPE) bag (Estiko,
Estonia);
4) actively modified 30 μm LDPE bag, flushed with a gas mixture containing 10%
O2 and 15% CO2).

One treatment consisted of six replicate bags (four punnets in one bag). The
raspberries were stored for 4 days: on the 3rd day, a half of the bags were moved into
simulated retail conditions (6ºC) and a half remained at 1.6°C. The storage room
relative humidity ranged from 96 to 98%.
The raspberries were weighed and the O2 and CO2 contents measured from the
packages every day with a hand-held gas analyser OXYBABY V (WITT-Gasetechnik
GmbH & Co KG, Germany). The fruit dry matter (DM) and soluble solids content
(SSC), titratable acidity (TA), ascorbic acid content (AAC), and total anthocyanins
(ACY) were determined at harvest and at the end of storage. Total antioxidant activity
(TAA) and rotting (weight of berries with rots) were determined after storage. A fruit
was considered rotten if even one of the drupelets was infected.
For determination of the AAC, ACY and TAA, ten randomly chosen fruit from
each treatment were weighed into a titration vessel separately for each analysis and
extraction solution was added immediately to avoid breakdown of the easily oxidised
compounds in the air. For the AAC determination, the fruit were crushed quickly with

746
a homogenizator and titrated with dichlorophenolindophenol; also, voltamperometric
indication was used (method M569/570 (www.mt.com)). TA was determined by
titration to pH 8.2 with 0.1 NaOH. The Titrator Mettler Toledo DL50 with the
autosampler Rondolino was used for titration of the AAC and TA. The SSC (%) was
measured using the digital refractometer PAL-1 (ATAGO CO., Ltd., Japan). The
content of total anthocyanins was estimated by a pH differential method (Cheng &
Breen, 1991). Total anthocyanins were calculated as cyanidin-3-glycoside, one of the
major anthocyanins in raspberries (Boyles & Wrolstad, 1993). The TAA was
determined using the 1.1-diphenyl-2-picrylhydrazyl (DPPH) discoloration assay
described by Brand-Williams et al. (1995) with some modifications. The results of the
TAA are reported as Trolox equivalents (TE) per 100 gram of fresh fruit weight.
Significant differences between treatments and the effect of retail and cool-store
temperature were tested by a two-way analysis of variance. In figures and tables, the
mean values followed by the same letter are not significantly different at P ≤ 0.05.

RESULTS AND DISCUSSION

Gas composition in the MA packages

The O2 content in both passively modified packages decreased rapidly during the
first 24 hours, reaching 15.7% in the Xtend® film and 14.9% in the LDPE film
(Fig. 1A). During the next two days, the decrease was very little and the final content
of O2 in the Xtend® film was 15.1% and in the LDPE film 13.1%. In the actively
modified LDPE package, the O2 content increased slightly, being 12.4% after 24 hours
and 11.9% at the end of cool storage.
The CO2 content in the passively modified packages increased to 5.1% in the
LDPE film and to 6.1% in the Xtend® film during the first 24 hours (Fig. 1B). The
final CO2 content in the LDPE film was 5.9% and in the Xtend® film 7.3%. In the
actively modified LDPE package, the CO2 content decreased rapidly, being 8.7% after
24 hours. At the end of cool storage, the CO2 content in the active MAP was 5.9%,
which was exactly the same as in the passive LDPE package.

Figure 1. Changes in the O2 (A) and CO2 (B) concentrations (% ± SD) in different modified
atmosphere packages of ‘Polka’ raspberries stored at +1.6 °C for 3 days.

747
 Fruit spoilage
The losses caused by rots were extensive in the present study, ranging from 9 to 23%
(Table 1). The reason was obviously the extremely rainy weather during the fruit
ripening period. The effect of the MA conditions on rotting in our trial was not
significant. The mean effect of storage treatment showed that the Xtend® film decreased
rotting compared to both LDPE films, but not compared to the control. Several authors
have reported that high CO2 concentrations (10, 20 and 30%) suppress rotting of
raspberries (Agar & Streif, 1996; Haffner et al., 2002). In our trial, the CO2
concentration in the passive MAP ranged from 5 to 7% and in the active MAP the CO2
concentration also decreased below 10% after 24 hours. The mentioned CO2
concentrations were probably too low to suppress rotting.

Table 1. The effect of different MA packages on fruit rotting, the SSC and TA of the raspberry
‘Polka’ after 4 days of storage in coolstore (+1.6 °C) and after 3 days in coolstore +24 hours in
simulated retail conditions (+6ºC)
Control Xtend® Passively Actively Mean
modified modified
LDPE LDPE
Rotting weight, %
Coolstore 16a 9a 16a 16a 14B
Retail 16a 13a 23a 22a 18A
Mean 16AB 11B 20A 20A
Soluble solids,%
Coolstore 8.6b 9.3ab 9.7a 9.4ab 9.3A
Retail 9.2a 8.2b 8.7ab 9.1ab 8.8B
Mean 8.9A 8.7A 9.2A 9.2A
Titratable acids, %
Coolstore 1.54b 1.66a 1.68a 1.53b 1.60A
Retail 1.50b 1.57a 1.48b 1.50b 1.51B
Mean 1.52B 1.61A 1.58A 1.51B

Weight loss, dry matter and taste-related parameters

The average fruit DM content at harvest was 13.7 ± 0.4%. By the end of storage,
it ranged from 12.7 to 13.3%. The effect of storage treatment on the DM content was
not significant.
The weight loss of the control treatment ranged from 1 to 2% and in the MA
packages from 0.1 to 0.2%. Thus, weight loss was not a limiting factor for berry
quality during the experiment, since the loss according to Kenny (1975) needs to be at
least 8% to cause a quality reduction. Haffner et al. (2002) also found in a trial with 5
raspberry cultivars that postharvest treatments did not affect fruit weight loss and dry
matter content.
The initial SSC of the raspberries was 10.8 ± 0.4%, which is in agreement with
the previously reported values for ‘Polka’ of 10.7–11.3% (Grajkowski & Ochmian,
2007). After storage, the SSC of the raspberries ranged from 8.6 to 9.7% (Table 1). In
cool storage, the raspberries from the passive LDPE had retained the highest value of
soluble solids. In retail conditions, the SSC was the highest in control fruit and the
lowest in the Xtend® package. The average effect of storage temperature showed that
24 hours in retail conditions had decreased the SSC significantly.

748
 At harvest, the raspberry TA was 1.84 ± 0.03% and after storage it ranged from
1.48 to 1.68% (Table 1). Grajkowski & Ochmian (2007) have reported the raspberry
‘Polka’ mean TA to be 1.75%, which is similar to our findings. After storage in cool
conditions, the fruit TA was significantly higher in the passively modified LDPE and
the Xtend® packages. In retail conditions, the TA content was higher in the Xtend®
film. 24 hours in retail conditions had decreased the TA significantly.
Relatively high sugars and acids are required for good flavour of the fruit. The
passively modified LDPE film maintained the highest SSC and TA in cool conditions,
but not in retail conditions.

Bioactive compounds and total antioxidant activity

The AAC of the raspberry fruit before storage averaged 36 ± 3 mg 100 g FW-1.
Haffner et al. (2002) have studied the L-ascorbic acid content of 5 raspberry cultivars
in Norway and the mean for all cultivars at the time of harvest was 23.2 mg in 100 g of
berries. These results confirm that ‘Polka’ is a valuable cultivar with high ascorbic acid
content. After 4 days of cool storage, the raspberry AAC had decreased and ranged
from 22 to 27 mg 100 g FW-1 (Fig. 2). The AAC in the Xtend® film was lower
compared to the LDPE films and the control after cool storage. As an average of the
experiment, 24 hours in retail conditions did not cause a significant decrease in the
AAC and no significant differences in the AAC between packages were found in retail
conditions.

Figure 2. The effect of different MA packages on the ascorbic acid content of the raspberry
‘Polka’ after 4 days of storage in coolstore (+1.6°C) and after 3 days in coolstore +24 hours in
simulated retail conditions (+6ºC).

The initial ACY content in the raspberries was 40 ± 3 mg 100 g FW-1. Haffner et
al. (2002) have found that the mean ACY level of 5 raspberry cultivars in Norway was
40 mg 100 g-1 fresh weight. After 4 days of cool storage, the fruit ACY content ranged
from 34 to 43 mg 100 g FW-1 (Fig. 3). After cool storage, the raspberries held in the
actively and passively modified LDPE packages had significantly lower ACY contents

749
compared to the control. Thus, in the MA packages, the ACY synthesis was slowed
down. Haffner et al. (2002) also found that compared to the raspberries stored in
normal atmosphere, the ACY content of the raspberries from controlled atmosphere
conditions was significantly lower. Among other phenolic compounds, anthocyanins
are derived from the phenylpropanoid pathway, which is activated by the enzyme
phenylalanine ammonia-lyase (PAL). It has been demonstrated with table grapes
(Romero et al., 2008) that CO2-treated fruit have lower PAL activity and consequently
also lower ACY content.
In retail conditions, the ACY content in both LDPE films had increased to the
similar level with the control and the fruit from the Xtend® film had the lowest ACY
content. The average effect of the last diurnal temperature was significant: as expected,
warmer temperature increased the ACY content, which was not a favourable result in
the particular cultivar. Darkening of the attractive red colour is considered to be one of
the characteristics of quality loss in raspberries (Haffner et al., 2002). The fruit of the
raspberry ‘Polka’ are of dark colour compared to some other cultivars. According to
the experience of the growers, when selling ‘Glen Ample’ and ‘Polka’ fruit side by
side, consumers prefer ‘Glen Ample’, which is of lighter colour. The dark colour of
‘Polka’ fruit is associated with overripeness. There are very few published studies
identifying the sensory properties important to the consumer acceptance of raspberries.
Villamor et al. (2013) have found that high colour intensity and green aroma were
associated with negative drivers of liking of fresh raspberries.
Haffner et al. (2002) have stated that cultivars with a lighter red colour with less
blue will keep a better colour after harvest compared to darker and more bluish
cultivars.

Figure 3. The effect of different MA packages on the anthocyanin content of the raspberry
‘Polka’ after 4 days of storage in coolstore (+1.6°C) and after 3 days in coolstore +24 hours in
simulated retail conditions (+6ºC).

The TAA of the raspberries ranged from 95 to 154 TE mg 100 g FW-1 (Fig. 4). In
cool conditions, the raspberries from the passively modified LDPE had the highest
TAA, followed by the actively modified LDPE bag, the Xtend® bag and finally the

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control, where the raspberry TAA was 32% lower than in the first mentioned
treatment. In retail conditions, the situation had changed and the highest TAA was
determined in the raspberries stored in the Xtend® package. Both LDPE bags had
decreased raspberry TAA. The average effect of storage temperature was significant:
24 hours in retail conditions had significantly decreased the TAA. It has been reported
that cyanidin-based anthocyanins with a o- dihydroxy moiety have the greatest
antioxidant activity among anthocyanins (Rice-Evans et al., 1996). Therefore it was
expected that in treatments where the ACY concentration was high, the TAA would
also be high. For an unknown reason, negative correlation between the ACY and TAA
was found. Schotsmans et al. (2007) carried out a controlled atmosphere storage
experiment with rabbiteye blueberries and found that the antioxidant activity and total
phenolic content were positively correlated when the berries were stored in a regular
atmosphere, but negatively correlated after storage in a controlled atmosphere. The
mechanisms for reversed correlation remain unclear.

Figure 4. The effect of different MA packages on the total antioxidant activity of the raspberry
‘Polka’ after 4 days of storage in coolstore (+1.6 °C) and after 3 days in coolstore +24 hours in
simulated retail conditions (+6ºC).

CONCLUSIONS

The modified atmosphere packages used in the current experiment did not
suppress Botrytis rot probably due to the insufficient content of carbon dioxide (less
than 10%). It would be worth trying other materials less permeable to oxygen and
carbon dioxide in order to increase the CO2 content to a sufficient level for suppressing
microbial activity. However, the effect of MA on the fruit taste-related parameters and
the antioxidant properties indicates the possibility to improve the consumer
acceptability of ‘Polka’ raspberries by using modified atmosphere. The raspberries
stored at 1.6°C, had the best quality in passively modified LDPE bags, since the fruit
had higher SSC and TA and lower ACY content compared to the control, meaning that
these fruits might have a more intensive taste and lighter red colour compared to
others. After simulated retail conditions, the Xtend® bags turned out to be the most

751
suitable, since the fruit had the lowest ACY content (the fruit did not become too dark),
but the highest antioxidant activity.

ACKNOWLEDGEMENTS. The current research was supported by the Estonian

Science Foundation Grant No. 7515 and the Estonian Ministry of Agriculture.

REFERENCES

Agar, I.T. & Streif, J. 1996. Effect of high CO2 and controlled atmosphere (CA) storage on the
fruit quality of raspberries. Gartenbauwissenschaft 61, 261–267.
Kenny, A. 1975. Handling strawberries and raspberries for fresh market II. Precooling. Farm
and Food Res. 6(3), 64–66.
Danek, J. 2002. ‘Polka’ and ‘Pokusa’ – new primocane fruiting raspberry cultivars from Poland.
Acta Hort. 585, 197–198.
Boyles, M.J. & Wrolstad, R.E. 1993. Anthocyanin composition of red raspberry juice:
influences of cultivar, processing, and environmental factors. J. Food Sci. 58, 1135–1141.
Brand-Williams, W., Cuvelier, M.E. & Berset, C. 1995. Use of a free radical method to evaluate
antioxidant activity. Food Science and Technol. 28, 25–30.
Cheng, G.W. & Breen, P.J. 1991. Activity of phenylalanine ammonialyase (PAL) and
concentrations of anthocyanins and phenolics in developing strawberry fruit. J. Am. Soc.
Hortic. Sci. 116, 865–868.
Grajkowski, J. & Ochmian, I. 2007. Influence of three biostimulants on yielding and fruit
quality of three primocane raspberry cultivars. Acta Scientarium Polonorum Hortorum
Cultus 6(2), 29–36.
Haffner, K., Rosenfeld, H.J., Skrede, G. & Wang, L. 2002. Quality of red raspberry Rubus
idaeus L. cultivars after storage in controlled and normal atmospheres. Postharvest Biol.
& Technol. 24, 279–289.
Rice-Evans, C.A., Miller, N.J. & Paganga, G. 1996. Structure- antioxidant activity relationships
of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933
Romero, I., Sanchez-Ballesta, M.T., Maldonado, R., Escribano, M.I. & Merodio, C. 2008.
Anthocyanin, antioxidant activity and stress-induced gene expression in high CO2-treated
table grapes stored at low temperature. J. Plant Phys. 165, 522—530.
Schotsmans, W., Molan, A. & MacKay, B. 2007. Controlled atmosphere storage of rabbiteye
blueberries enhances postharvest quality aspects. Postharvest Biol. & Technol. 44, 277–
285.
Vetek, G. & Penzes, B. 2008. The possibilities of organic raspberry production – setting a
Hungarian example. In Dimza, I. et al. (eds.). Proceedings of the International Scientific
Conference, Sustainable Fruit Growing: from Plant to Product; 2008 May 28–31;
Jurmala – Dobele, Latvia. Latvia State Institute of Fruit-Growing, pp. 233–242.
Villamor, R.R., Daniels, C.H., Moore, P.P. & Ross, C.F. 2013. Preference mapping of frozen
and fresh raspberries. J. Food Sci. 78, 911–919.

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 Agronomy Research 12(3), 753–758, 2014

Investigation of Casein Micelle Particle Size Distribution in Raw

Milk of Estonian Holstein Dairy Cows
H. Mootse*, A. Pisponen, S. Pajumägi, A. Polikarpus, V.Tatar, A. Sats and
V. Poikalainen

Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life

Sciences, Kreutzwaldi 56/5, EE51014 Tartu, Estonia;
*Correspondence: [email protected]

Abstract. The particle size of milk influences its microstructure and defines many properties of
dairy products such as colloidal stability, texture etc. Differences in particle size can
significantly affect milk processing especially when membrane technology is used.
Aim of this investigation was to estimate casein micelle size in the raw milk of Estonian
Holstein dairy cows and its variability concerning individual animals.
Milk samples were collected during 12 months with the interval of 25–35 days. DLS analyses
were performed using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK).
Average mode of casein micelle particles size in raw milk of 44 cows was 171.13 nm with the
variation range 70.1 nm and its distribution resembled a normal one. Casein micelles size mode
of individual cows varied in a wide range from 148.5 (with variation range 18.2) to 194.1 (with
variation range 27.6) nm which may be caused by differences in physiological and health status,
stage of lactation and other factors concerning milk production.

Key words: casein micelle, dynamic light scattering, particle size, particle size distribution.

INTRODUCTION

Particle size (PS) and particle size distribution (PSD) gives valuable information
about colloidal systems, among others milk and dairy products also (Beliciu & Moraru,
2009). Commonly used techniques for the analysis of PS and PSD are dynamic light
scattering (DLS), nanoparticle tracking analysis (NTA), scanning electron microscopy
(SEM), size exclusion chromatography (SEC), cell electrophoresis, analytical
ultracentrifugation (AUC) etc. Various analytical methodologies may give different
results (Anema et al., 2005; Thu Tran Le et al., 2008; Dejan, 2010; Raza et al., 2011).
Of the mentioned techniques, DLS is the most user-friendly and it gives relatively
accurate and consistent results of protein samples which can be obtained in short
period of time (Vasco et al., 2010). Main challenges to estimate casein micelle (CM)
PSD by DLS, is the fact that measurements need to be performed with considerably
pure solution and at low concentration of sample (Alexander & Dalgleish, 2006). For
reliable estimation of CM PSD, 10 to 1 μg milk must be diluted in one millilitre of
solution (Beliciu & Moraru, 2009). Casein micelle consists of four different protein
fractions (αS1-, αS2-, β-, κ-casein). Each CM is covered with a layer of water molecules
which affects the hydrodynamic diameter of micelle.

753
 In former studies CM PSD had been mainly estimated in raw bulk milk, skimmed
milk, reconstituted skimmed milk, lactose-free milk and pasteurized milk
(Martin et al., 2007; Tran Le et al., 2008; Liu et al., 2013).
There is few published research which deal with CM in raw milk of individual
cows, for example Bijl et al. (2014) investigated how milk chemical composition
influenced casein micelle size of individual cows and de Kruif & Huppertz (2012)
investigated how lactation stage affects CM PSD. The aim of current study was to
approve suitability of dynamic light scattering (DLS) method for estimation of casein
micelle PSD in raw milk and investigation of its variability of individual Estonian
Holstein dairy cows during one year period.

MATERIALS AND METHODS

Raw milk samples of 44 Estonian Holstein dairy cows were collected at the
Experimental Farm of Estonian University of Life Sciences (EMÜ) from January 2013
to December 2013, with the interval of 25 to 35 days. After milking samples were
cooled down and stored at 5oC, all analyses where made in the same day.
For stable results, samples were diluted at refrigerator temperature (5°C) just
before the measurement using RPMI 1640 (PAA Laboratories GmbH, Pasching,
Austria) as diluting media to concentration 1μg/ml, and filtered before measurement
using a 0.45 μm, Ø 15mm Premium Syringe Filters (Agilent Technologies, Santa
Clara, California). This procedure was necessary to remove larger particles such as fat,
dust etc. 1500 μl of each sample was inserted into a single-use disposable sizing
cuvette DTS0012 (Sarstedt REF 67.754, Sarstedt AG&Co, Nümbrecht, Germany).
DLS analyses were performed using a Zetasizer Nano ZS analyzer (Fig. 1). The
particle size estimations were made at fixed 173° backscattered angle using the default
‘protein analysis mode’ with automatic duration and four consequent measurements
from a sample without delay. Automatic attenuation selection was switched off and
number six was inserted as value for attenuator.

Figure 1. Typical example of CM PSD and Malvern Zetasizer Nano ZS analyser.

754
 Measurement temperature was chosen 20°C, because this is a temperature
commonly used in DLS measurements studies (Beliciu & Moraru, 2009). Data
collection and first elaboration of these was carried out by Zetasizer software 7.01. The
mode of PSD (mean intensity size of hydrodynamic diameter), average of total PSD
(harmonic intensity of averaged particle diameter or Z-Average diameter) and all other
data were exported to Microsoft Excel for further analyses. Typical example of CM
PSD and measurement equipment are presented in Fig. 2.

RESULTS AND DISCUSSION

From obtained data only mean intensity of different variables of size was used.
This index corresponds to the mode of CM PSD curve and represents the most
essential information in it. Average of CM PSD modes of 328 samples was 171.13 nm
with variation range 70.1 nm and it had standard deviation (SD) of 14.06. Histogram of
these modes resembles normal distribution and covers the range 135–210 nm (Fig. 2).
More than half of the modes (58.5%) covered the range 155–175 nm. Tails of this
modes distribution were represented by four (1.2%) samples in range from
135–140 nm and six (1.5%) samples in the range 200–210 nm. Former studies about
CM PSD in bulk (and treated) milk by different methods showed up quite the same
variability in average mode of CM PSD: 150–200 nm (Table 1). Some variation in
results can be explained by different measurement methodologies used for preparation
of probes and by differences in milk itself (bulk milk of different production system,
cows breed etc.).
80 72
70 63
57
n 60
48
50 43
40
30
20 14 13
8 6
10 4
0

Size (nm)

Figure 2. Histogram of CM PSD modes in raw milk probes of Estonian Holstein dairy cows.

Studies of Beliciu & Moraru (2009) and Liu et al. (2010) are in agreement with
results of our investigation. They found that CM PSD average in treated bulk skim
milk varies from 176.3 to 178.8 nm. This result is only somewhat bigger than average
mode of CM PSD in our study. Overall accordance of above mentioned studies with
our investigation suggests that DLS method is suitable for estimation of CM PSD in
raw milk of cows too.
To investigate the role of individual animals on mode of CM PSD, all data were
rearranged into groups by cow number. Of all animals under this study (n = 39) milk of

755
six cows has been analysed 3–5 times and milk of 33 cows 6–11 times during the total
investigation period.

Table 1. Summary of former studies about the CM size in milk

Author Method Mode of CM size, nm
Tran et al., 2008 NTA 192
Martin et al., 2007 Cell electrophoresis 185
Tran et al., 2008 DLS 186
DLS at 20°C 177
Liu et al., 2010
DLS at 40°C 200
DLS at 20°C 176
Beliciu and Moraru, 2009
DLS at 50°C 194
De Kruif and Huppertz, 2012 DLS 154–230
Raza Hussain et al., 2011 DLS (solvent NaCl) 150

Differences in number of analyses were caused by changes in lactation stage

mainly.Individual dairy cows’ average mode of CM PSD in descending order is
presented in Fig. 3 and numerical values of modes and their variations are given in
Table 2.

Figure 3. Individual dairy cows average mode of CM PSD in descending order.

Average mode of CM PSD in this study varied in range from 148.5 nm to

194.06 nm (group average of 39 cows was 170.95 nm, with variation range of
27.40 nm and SD of 19.4). This data is similar to the results obtained in analyses about
the total herd (44 cows/328 samples).
Largest average mode of CM PSD in this dataset was 194.06 nm, with 9.1 nm
variation and the smallest one had PSD of 148.5 nm with 27.6 nm variation. In Fig. 4
casein micelle PSD variability of five individual cows are presented. Cow No. 1 and
cow No. 39 average mode of CM PSD have extreme values (min and max), three other
cows (10, 18, 32) have average mode closer to mean of the herd. Average mode value
of CM PSD and its variation of different cows seem to be independent from each other.
It was confirmed by statistical analyses – they showed up only a very slight positive
correlation (0.195). Also increasing number of samples does not make this relation

756
better. For example, by 10 cows of 11 which had 10 CM PSD estimations each,
variation mean value (19.4 nm) of the mode was exceeded. The only reasonable
explanation to that may be found in cows’ individuality.

Figure 4. Casein micelle PSD variability of individual cows 1, 10, 18, 32 and 39.

Table 2. Average modes of CM PSD in milk of individual cows in descending order, their
variations and standard deviations
Cow identi- Variation SD of Cow identi- Variation SD of
fication Mode of range of CM CM Count fication Mode of range of CM CM Count of
number CM PSD PSD PSD of samples number CM PSD PSD PSD samples
1 194.1 18.2 12.9 3 21 170.6 15.8 11.2 9
2 188.3 25.5 18.0 6 22 169.3 31.7 22.4 10
3 186.2 25.5 18.0 9 23 167.2 15.1 10.7 9
4 184.9 57.0 40.3 9 24 167.0 15.1 10.7 7
5 182.2 25.5 18.0 10 25 166.4 23.2 16.4 9
6 181.7 42.5 30.1 10 26 165.6 37.0 26.2 10
7 180.9 24.3 17.2 7 27 165.4 38.8 27.4 11
8 180.8 33.2 23.5 9 28 164.3 37.0 26.2 7
9 180.5 25.5 18.0 10 29 163.8 23.2 16.4 9
10 179.7 70.1 49.6 9 30 162.1 15.1 10.7 9
11 179.6 16.6 11.7 6 31 161.4 22.2 15.7 3
12 178.7 24.3 17.2 9 32 160.7 7.4 5.2 5
13 178.2 42.5 30.1 10 33 159.5 28.9 20.4 10
14 177.3 33.2 23.5 10 34 158.7 30.3 21.4 10
15 176.0 24.3 17.2 9 35 158.6 22.2 15.7 10
16 175.8 31.7 22.4 8 36 155.4 7.1 5.0 8
17 173.2 15.8 11.2 9 37 154.6 43.4 30.7 10
18 173.1 15.8 11.2 5 38 152.0 27.6 19.5 3
19 172.7 15.8 11.2 7 39 148.5 27.6 195 7
20 171.9 31.7 22.4 3 Average 170.9 27.4 19.4 8.1

Also the absence of tight correlation between CM PSD average mode and its
variation refers to influence of certain factors connected to cows’ individuality
(changes in physiological status, disease incidences, stages of lactation, etc). All these
aspects should be topics of further investigations.

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 CONCLUSION

For the first time, studies of casein micelle PSD variability in milk samples of
individual Estonian Holstein dairy cows have been carried out by DLS measurements
during one year period. The main results of this study can be summarized as follows:
1) Average mean intensity (mode) of CM PSD in raw milk of Estonian Holstein dairy
cows was 171.13 nm and its variation (range 135–210 nm) resembled statistically
normal distribution. 2) Weak correlation between CM PSD average mode and its
variation in milk samples of individual cows may refer to the possible influence of
cows’ physiological status, disease incidences and stages of lactation etc. which will be
studied in further research.

REFERENCES

Alexander, M. & Dalgleish, D.G. 2006. Dynamic light scattering techniques and their
applications in food science. Food Biophysics 1, 2–13.
Anema, S.G., Lowe, E.K. & Stockmann, R. 2005. Particle size changes and casein
solubilisation in high-pressure-treated skim milk. Food Hydroc. 19, 257–267.
Beliciu, C.M. & Moraru, C.I. 2009. Effect of solvent and temperature on the size distribution of
casein micelles measured by dynamic light scattering. J. Dairy Sci.92, 1829–1839.
Bijl, E., de Vries, R., van Valenberg, H., Huppertz, T. & van Hooijdonk, T. 2014. Factors
influencing casein micelle size in milk of individual cows: Genetic variants and
glycosylation of k-casein. Int. Dairy J. 34, 135–141.
Dejan, A. 2010. Dynamic light scattering and application to proteins in solutions. University of
Ljubljana Faculty of Mathematics and Physics, Department of Physics, Ljubljana, 19 pp.
Kruif de C.G. (Kees) & Huppertz, T. 2012. Casein Micelles: Size Distribution in Milks from
Individual Cows. J. Agric. Food Chem. 60, 4649−4655.
Liu, D.Z., Weeks, M.G., Dunstan, D. E. & Martin, G.J.O. 2013. Temperature-dependent
dynamics of bovine casein micelles in the range 10–40°C. Food Chem. 141, 4081–4086.
Martin, G.J.O., Williams, R.P.W. & Dunstan, D.E. 2007. Comparison of Casein Micelles in
Raw and Reconstituted Skim Milk. J. Dairy Sci. 90, 4543–4551.
Raza, H., Claire, G. & Joël, S. 2011. Revealing Casein Micelle Dispersion under Various
Ranges of Nacl: Evolution of articles Size and Structure. Eng. Tech. 51, 972–982.
Tran, Le T., Saveyn, P., Hoa, H.D. & Van der Meeren, P. 2008. Determination of heat-induced
effects on the particle size distribution of casein micelles by dynamic light scattering and
nanoparticle tracking analysis. Int. Dairy J.18, 1090–1096.
Vasco, F., Andrea, H. & Wim, J. 2010. Critical Evaluation of Nanoparticle Tracking Analysis
(NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates.
Pharm. Res. 27(5), 796–810.

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 Agronomy Research 12(3), 759–768, 2014

Fermented sauces for child nutrition from age three

L. Nadtochii* and A. Koryagina

ITMO University, Lomonosova Street 9, 191002 Saint-Petersburg, Russia;

*Correspondence: [email protected]

Abstract. One of the directions in modern food science is the development of diets for children
of different age groups based on their physiological needs and psycho-emotional activity. One
of the approaches to solving this problem is functional product design. Such products are not
medicaments, but help to prevent the diseases and ageing processes of the human organism.
The purpose of our research was to develop fermented sauce compositions based on milk and
plant ingredients for child nutrition.
Mass fractions of pumpkin and banana purée, berry syrup and stabilizing additives were
experimentally determined.

Key words: Fermented sauces, child nutrition, fruit and vegetable purées, berry syrups, corn
starch.

INTRODUCTION

The creation and launching of functional food products is one of the directions of
the human nutrition program initiated by the UN. Even in the developed countries there
is a deficit in biologically active substances, macronutrients and micronutrients, and
ballast substances in the diet. Functional food products which are not medications help
to prevent diseases and ageing. 65% of the modern market of functional food products
consists of dairy products (Zakharova & Mazeeva, 2010).
One of the priorities in the field of healthy eating is the existence of a varied
range of products for functional purposes. Vegetable raw materials with a wide range
of biologically active substances (vitamins, minerals, dietary fiber, antioxidants, etc.)
are currently widely used. The presence of these ingredients improves many
physiological processes in the body and improves the immune system. One of the most
accessible ways to create products with such qualities is the development of technology
for the production of functional foods with a complex mineral composition. The
creation of multicomponent products is mainly aimed at the regulation of amino acids,
lipids, carbohydrates, minerals and vitamins in human diet. (Tikhomirovа, 2009).
Children and elderly people have become an increasingly important segment of the
consumers and such products are aimed specifically at these two groups
(Murray et al., 2001).
The present study focuses on the development of safe functional foods intended
for children. A balanced diet is one of the conditions for the normal development of
resilience in children, helping to cope with exposure and infection. Baby food plays a
significant role in ensuring children access to balanced products, the production of

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which is possible only in the conditions of specialized modern enterprises. The most
important are the products included in the daily diet. A special group of them is sauces.
Due to the presence of extractive, aromatic and flavoring substances stimulating the
secretion of digestive glands, sauces are a good source of key components in food
(Tarasova & Taghiyev, 2009).
At present, various types of sauces and soups are extremely popular in the
consumer market because of the significant expansion of the average consumer basket.
However, not all products in this category, available now in the market, are ideal for
the children. The abundance of these products is unacceptable for children because of
their content of additives, stabilizers, preservatives, and artificial dyes. That is why the
study is so important in the current situation in the food market, as regards children of
pre-school and school age. According to the Institute of Nutrition of Russian Academy
of Medical Sciences, a broad-scale monitoring of the nutritional status of school-age
children has shown a shortage of high-grade proteins (30–70%), fat (10–40%), and
vitamins (40–70%). The vast majority of pupils (about 80%) are eating at school
cafeterias and buffets during school hours. Due to deficient school meals, according to
the I.М. Sechenov Moscow Medical Academy, 37% of children regularly experience
hunger and 39% from time to time. Because of considerable mental stress at school, the
well-being of pupils depends on regular meals and a high-calorie diet. Childhood and
adolescence is characterized by relatively high energy consumption. Thus, the energy
consumption of children 7–10 years of age constitutes about 80 kcal kg-1 of body
weight, 13–16 year old adolescents 50–65 kcal kg-1, and adults 45 kcal kg-1.
The recommended consumption rates of basic nutrients for children and
adolescents by age and gender according to the Institute of Nutrition and the sanitary
standard 2.4.5.2409-08 ‘Нygienic requirements for catering pupils in general
educational institutions, institutions of primary and secondary professional education’
are shown in Table 1.

Тable 1. Recommended consumption rates of basic nutrients for children and adolescents
according to age
Quantity, per day
Poultry nutrition
7–11 years 11–18 years
Рrotein, % of calories 14 13
Fat, % of calories 30 30
NLC, % of calories 10 10
Sugar, % of calories 10 10
Calcium, mg 1,100 1,200
Vitamin C, mg 80 100
Energy value, kcal 2,400 2,800

Feeding children of school and pre-school age is an important task. In fact, it has
become increasingly difficult to work out a balanced and safe diet for the children due
to the abundance of the latest developments of food industry filling the store shelves.
Not all of these innovations are suitable for children, but the children prefer such
products because of their pleasant organoleptic properties. However, there is a way out.
The new products can be both useful and valuable in terms of nutritional value, but at
the same time tasty foods with health benefits that babies could drink every day. The

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younger generation is fond of sweet dairy desserts. Therefore, one solution to the
shortage of dairy products in the diet of children of pre-school and school age may be
the development of new functional milk based products, which could easily be
combined with their taste preferences. Sauce could be ideal as such a product, as it is
both fashionable and popular.

МATERIALS AND METHODS

A study on the development of composition and technology of fermented sauces

for three year old infants was conducted in the laboratory of technology of milk and
food biotechnology at Institute of Refrigeration and Biotechnology of Saint Petersburg
National Research University of Information Technologies, Mechanics and Optics.
Тhe objects of research were:
 skimmed milk powder with a fat content of 1.25%;
 homogenized mashed pumpkin ‘Umnitsa’ for early childhood (Ivanovo plant of
baby food);
 homogenized banana purée for early childhood (Ivanovo plant of baby food);
 blueberries, sugar free syrup ‘Huckleberry’ (‘Petrodiet’);
 viburnum berry syrup (‘Vifiteh’);
 corn starch premium sort (‘Starch-center’);
 sourdough of pure cultures of viscous Streptococcus thermophilus.

Milk powder was used in the experiment with the aim of eliminating differences
in physical and chemical indicators of raw milk. However, at the implementation of
fermented sauce into production, it is recommended to use raw milk as a base.
Raw materials for the manufacturing of fermented sauces must meet the
microbiological requirements presented in Table 2. The milk used for manufacturing
baby food products should not be below the first grade. Manufactured fillers comply
with microbiological criteria, as do products for babies from 6–10 months.

Тable 2. Acceptable levels of bacteria and somatic cells in raw milk

Weight of the product, which is Max content of
KMAFAnM
Product milk, grade not allowed somatic cells
Pathogens,
including
Coliform bacteria salmonella
Supreme 1*105 – 25 4*105
the first 5*105 – 25 1*106
the second 4*106 – 25 1*106

Integrated study design adopted these methodologies:

1. Determination of active acidity;
2. Determination of dry matter and mass fraction;
3. Definition of syneresis properties.

761
 Determination of active acidity
Potentiometric method is based on the measuring of voltage between two
electrodes (electrode measurement and comparison). Electrodes couple before the
measurement and should be placed in a glass with a solution of hydrochloric acid
molar concentration of 0.1 mol/dm3 in intervals between measurements. Electrodes are
washed by distilled water before tests and sediment particles must be removed through
filter paper.
We poured a 40 ± 5 cm3 sample of milk or liquid product in a glass with a
capacity of 50 cm3 at the temperature 20 ± 2°С and dipped in the lead pair. After that
we took the readings.
The measurement of pH in each sample is repeated twice, each time removing the
electrodes from the sample and immersing them into the product.
The immersion depth of the electrode pair into the glass during the trial shall be
not be less than 30 mm and that of combined electrodes no less than 16 mm. It is not
allowed to touch the electrodes or the glass of the temperature compensator bottom and
sides.
After the measurement of each sample of milk or dairy products, an electrode
couple should be rinsed with distilled water at the temperature 30–40°С and the
sediment particles in the distilled water must be removed through filter paper.
The final results of the measurement of active acidity in milk and dairy products
are obtained by taking the arithmetic mean of the results of two parallel determinations.

Determination of moisture and dry matter content in sauces

It is necessary to put a doubled cheesecloth in an aluminum weighing bottle, then
to exsiccate the content with an open lid at 105°С for 20–30 min, after that to cool it in
the desiccator for 20–30 min by closing the lid, and then to weigh the content.
3 ml of the investigated product should be taken into a prepared aluminum
weighing bottle pipette, distributed at regular intervals over the entire surface of the
doubled cheesecloth and weighed after closing the lid. Then open aluminum container
and lid in the oven at 105°С for 60 min, after which the aluminum container is closed,
cooled and weighed. Drying and weighing continue during 20–30 min until the
difference in mass between two successive weighings is no more than 0.001 g.

Definition of syneresis properties

The study of syneresis properties of clots was conducted as follows: a 10 ml test
sample was placed into a measuring tube and centrifuged for 30 min, noting every
5 min the precipitated serum volume. By results of the research the rate of sample
syneresis was found out and the ability of serum clotting was concluded.

Research organization
In the course of this study, we carried out experiments on pumpkin and banana
puree and syrup percentage, as well as on the doses of stabilizer for a product with high
organoleptic properties and a texture typical of this type of products. The control
sample of sauce No 1 was prepared on the basis of raw milk without the addition of
plant fillers and was produced from banana purée; the control sample of sauce No 2
was prepared from pumpkin purée.

762
 All samples of sauces were made on the basis of skimmed milk, reduced to a
mass fraction of solids 16%, fermented together with pure cultures of lactic acid of
viscous Streptococcus thermophilus. Before developing, all the samples were subjected
to intensive mixing in a blade mixer at the speed of 800 RPM to prevent stratification
of the foundations and filler. Further samples were put in a thermostat at the
temperature 44 ± 2°C and fermented for 4–6 hours. The finished product was sent
ripening to the holding room at 4 ± 2°С.

RESULTS AND DISCUSSION

The dynamics of decreasing the acidity of sauce samples during the process of
fermentation was studied.
In the first stage of the research the effect of doses of applied plant fillers on the
fermentation of samples was investigated. Fig. 1 shows the use of different doses of
pumpkin purée added in the amount of 15, 30, 45% of the mass of the initial mixture.
Fig. 1 shows that the fermentation all samples lasted for approximately six hours.
It should be noted that the change in acidity was faster in the control sample, as
skimmed milk is a suitable environment for the development of starter microflora. The
graph also shows the reduction in active acidity depending on the doses of filler: the
higher the dose, the slower pH decreases. This may be due to the presence of large
quantities of organic acids in samples with high filler content, which impede the rapid
multiplication of the microflora of the leaven. However, adding a filler up to 30% of
the amount had a slight effect on the fermentation of the mixture, which is why further
investigation used a dose of about 30% for making a plant component.
7
6,5 control
6 sample with
5,5 15% puree
sample with
pH

5 30% puree

4,5
0 2 4 6 8
t, min
Figure 1. Сhange in the active acidity of samples with different doses of added pumpkin purée.

Banana and pumpkin purée were selected as plant components. Fig. 2 shows the
variation in the active acidity of the selected samples in comparison with the control
sauce.
The initial values of the active acidity of the samples with fillers are lower than in
the control sample. But the development of the starter microflora occurs rapidly in the
control sample as noted previously. However, after 6 hours of fermentation, samples
reach similar values of active acidity. This allows establishing the necessary time for
the fermentation of all samples, which is 6 hours.
The study should also figure out how to affect the process of fermentation by
adding viburnum syrup as a sweet component of the recipe. Fig. 3 shows the effect of

763
syrup (in an amount of 9%) on the fermentation process of the sample with pumpkin
purée.
Fig. 3 shows that the decrease in active acidity is faster in the sample without
syrup. It is obvious that a high content of sugar and organic acid does not make the
microflora of the starter culture evolve quickly and seamlessly in the sample with
syrup, and as a result, the fermentation occurs at a slower speed compared to the
sample without syrup. It should be noted that samples obtained in both the first and
second experiments had a good flavour and taste but the sauces lacked the required
consistency. At the end of the fermentation the samples had the sleek, glossy clot, but a
heterogeneous structure: when mixing, there was a small separation of the serum that
affected the consistency of the product.
7
6,5
control
6
Sample No1
5,5
Sample No2
5
рН

4,5
4
0 2 4 t, min 6 8

Figure 2. Сhange in the active acidity of samples with different fillers.

6 Sample
pH

No2 (with
5 syrup)

4
0 2 4t, min 6 8

Figure 3. Сhange in the active acidity of samples with addition of syrup and without syrup.

7
Control with the
6,5 stabilizer
6
5,5 Sauce No2 with
pH

5 the stabilizer
4,5 Control without
4 the stabilizer
0 5 10
t, min

Figure 4. Change in active acidity of samples with and without stabilizer.

764
 Consequently, it appears to be necessary to add a stabilizer in the manufacturing
process of the fermented sauces. Fig. 4 presents diagrams of decrease in active acidity
during fermentation of samples (control sauce and sample No2) with different fillers
with and without a stabilizer. The stabilizer was added according to the
recommendations in the amount of 2.7%.
On comparing these graphs we can say that the presence of the stabilizer does not
affect the fermentation of the samples significantly. However, with the introduction of
the stabilizer, we are reaching the desired consistency and structure of samples. Thus,
the inclusion of a stabilizer in the formulation of sauces is appropriate.

Study on syneresis properties of samples.

Syneresis properties are an important quality indicator in the production of
fermented products. It is required that the allocation of serum during centrifugation be
minimal (V, %).
As a result of this experiment, the following data presented in
Fig. 5 were obtained.

80
Сontrol with the
70 stabilizer
60 Control without the
stabilizer
50 Sauce No1 with the
V,%

40 stabilizer
30 Sause No1 without
the stabilizer
20 Sauce No2 with the
10 stabilizer
0 Sause No2 without
0 10 20 30 40 the stabilizer
t, min
Figure 5. Syneresis properties of sauce samples with and without stabilizer.

Fig. 5 shows the syneresis properties of test samples with and without stabilizer.
The graph shows the apparent stabilizing effect of structure with stabilizer: samples
with the stabilizer separated the serum significantly less than samples without the
stabilizer.
Samples No1 and 2 without the stabilizer are more resilient than the control sauce
without the stabilizer. This may be due to the stabilizing quality of dietary fiber, which
is part of banana and pumpkin purée.

Composition and properties of developed sauces

While working out the sour sauce recipe, it was decided to use 34–37% of plant
component ingredients of the total weight of the finished product. Such a filler dose
provides the necessary organoleptic qualities and functionality without violating the
regulations for child nutrition products.

765
 In order to obtain the desired consistency of the product of this type and the
stabilization of the system, it was decided to introduce corn starch in the amount of
2.7% of the total weight of the sauce as a stabilizer.
Recipes for cooking sauces No 1 and No 2 for 1,000 kg of product are presented
in Tables 3, 4.
Table 3. Recipe of sour sauce No 1 with banana purée for 1,000 kg of product (excluding
losses)
Сomponent Weight, kg
Water 515.0
Banana purée 283.0
Skimmed milk powder 69.0
Blueberry syrup 61.0
Sourdough (viscous thermopile Streptococcus) 45.0
Stabilizer (corn starch) 27.0

Table 4. Recipe of sour sauce No 2 with pumpkin purée for 1,000 kg of product (excluding
losses)
Сomponent Weight, kg
Water 496.0
Pumpkin purée 273.0
Skimmed milk powder 68.0
Viburnum syrup 91.0
Sourdough (viscous thermopile Streptococcus) 45.0
Stabilizer (corn starch) 27.0

The moisture content of the dry matter is an important characteristic of the

finished product. The data obtained are summarized in Table 5.
Table 5. Moisture content of dry matter in sauce samples
Samples Dry matter content, % Moisture content, %
Control sample 14.9 ± 0.2 85.1 ± 0.2
Sauce No 1 17.1 ± 0.2 82.9 ± 0.2
Sauce No 2 14.2 ± 0.2 85.8 ± 0.2

The present study also examined the organoleptic parameters of the samples.
Children have been documented to have different taste thresholds than adults
(Glanville et al., 1964; Hermel et al., 1970). It is apparent that children have different
dietary habits and preferences than adults. Whether this is a result of differences in
perception per se, familiarity, a learned behaviour or a combination of these, is not yet
clear. It may therefore be desirable to train children for descriptive profiling or
investigate these implications more thoroughly (Moskowitz, 1994;
Chen & Resurreccion, 1996; Baxter et al., 1998).
As it was not possible to teach children sensory analysis in the present study,
organoleptic evaluation of the received sauce samples was carried out by a group of 8
people from the academic staff working at the Department of Milk Technology and
Food Biotechnology. The panelists were trained in accordance with the
ISO 8586-1: 1993.

766
 Preparation and use of samples for sensory analysis was carried out according to
the recommendations (Seon-Suk et al., 2012). The evaluation was conducted for each
indicator on a five-point system. Samples were served with a glass of water after they
were left at the room temperature for 10 min. Samples were presented at the same time
in each session. Sensory evaluation of samples in each analysis was carried out 2 times.
The control points of organoleptic evaluation were taste, colour, and consistency.
Results of the evaluation are summarized in Tables 6 and 7 and displayed on the chart.

Table 6. Results of organoleptic evaluation of fermented sauces with different fillers

Points
Samples
Sour milk taste Taste filler Colour Consistency
Sample No 1 4.25 4.88 4.13 4.75
Sample No 2 4.38 4.88 4.88 4.88

Sample No 2 got the highest score in organoleptic evaluation of the best consumer
properties; however, sample No 1 received high praise too, particularly for its sour
milk taste, taste filler and consistency, losing only in colour. The latter is not
substantial enough to discard the sample from further research. The samples must meet
the requirements shown in Table 7.

Тable 7. Organoleptic characteristics of fermented sauces with different fillers

Sauce with mashed banana Sauce with pumpkin purée
Organoleptic characteristics
and blueberry syrup and cranberry syrup
Sour milk, sweet, with a Sour milk, sweet, with a
Taste and flavor strong banana taste and pronounced taste of pumpkin
flavour of blueberries and cranberry flavour
Consistency Homogeneous, viscous Homogeneous, viscous
Appearance Homogenous mass Homogenous mass
Colour Beigish-grey Bright orange

Тable 8. Microbiological safety indicators for sauces

The microorganisms identified Sauces for children's and dietary nutrition
Spore mesophilic aerobic and Meet the requirements of the industrial sterility. In the case
facultative anaerobic-team of determining the number of these micro-organisms, it
В.subtilis. should be no more than 11 cells in 1 g (cm3) of the product
Do not meet the requirements of the industrial sterility
Mesophilic Clostridium
detected in 10 g product (cm3)

Fermented sauces must meet the requirements ‘Hygienic requirements for safety
and nutritional value of foods. Public health regulations 2.3.2.1078-01’ shown in
Table 8.

СONCLUSION

The study was carried out to develop the composition and technology of
fermented sauces for children from age three. Based on the results of the research we
can make the following conclusions:

767
 1) The possibility of using filling materials of plant origin for the production of
fermented sauces was proved: it is possible to use banana and pumpkin purée and berry
syrup for this purpose in an amount of 34–37% of the total weight of the finished
product. It also provides a perspective for enlarging the assortment of fermented sauces
by introducing other combinations of fruit or vegetable purées and berry syrups;
2) The viscous thermopilus culture of Streptococcus (Streptococcus thermophilus)
promotes the clot formation of moderately viscous consistency with a pleasant sweet
taste of fermented sauces;
3) The use of corn starch as stabilizer allows to achieve the desired consistency
and structure of sauces: samples with added starch separated almost 2 times less whey
than samples without stabilizers;
4) The requirements for fermented sauces with various fillings were identified.
The organoleptic characteristics: taste of fermented milk with a pronounced taste of
fillers, aroma with a pronounced flavour of fillers; consistency is homogeneous,
viscous.

ACKNOWLEDGEMENTS. This work was partially financially supported by the government

of the Russian Federation, Grant 074-U01.

REFERENCES

Baxter, I.A. & Jack, F.R. & Schro der, M.J.A. 1998. The use of the repertory grid method to
elicit perceptual data from primary school children. Food Qual. Prefer. 9(2), 73–80.
Chen, A.W. & Resurreccion, A.V. (1996). Age appropriate hedonic scales to measure food
preferences of young children. J. Sens. Stud. 11, 141–163.
Dobrynina, E.S. & Lomovskij, O.I. 2010. Development of new formulations of sauces and
dressings functional purpose. Food industry. 54–55.
ISO 8586-1: 1993 (Sensory analysis. General guidance for the selection, training and
monitoring of assessors, Part 1. Selected assessors.
Glanville, E.V., Kaplan, A.R. & Fischer, R. 1964. Age, sex and taste sensitivity. J. Gerontol.
34, 834–840.
Hermel, J., Schonwetter, S.V. & Samueloff, S. 1970. Taste sensation and age in man. Journal of
Oral Medicine. 25, 39–42.
Moskowitz, H.R. 1994. Children versus adults. Food concepts & products. CT: Food &
Nutrition Press. 293–331.
Murray, J.M., Delahunty, C.M. & Baxter, I.A. 2001. Descriptive sensory analysis: past, present
and future. Food Res. Int. 34: 461–471.
Seon-Suk J., Seung-Joo L., Palanivel G. & Hae-Soo K. 2012. Comparative Study of Flavor,
Texture, and Sensory in Cream Cheese and Cholesterol-removed Cream Cheese. Food
Sci. Biotechnol. 21(1), 159–165.
Tarasova, L.I. & Taghiyev, T.G. 2009. Sauces and mayonnaises-is there a difference. Oil and
fat industry. 7. (In Russian).
Tikhomirovа, N.A. 2009. Current state and prospects of development of functional food
products. The dairy industry, 5–6. (In Russian).
Tikhomirovа, N.А. 2009. Healthy eating for students. Тhe dairy industry.78–79. (In Russian).
Tikhomirovа, N.A. & Churakov, M.M. 2007. Functional fermented sauces. The dairy industry.
3, 59–60. (In Russian).
Zakharova, L.M. & Mazeeva, I.A. 2010. Technology of new functional foods milk based.
Storage and processing of agricultural raw materials. 63. (In Russian).
.

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 Agronomy Research 12(3), 769–778, 2014

The unique characteristics of milky-wax ripe walnuts

and their usage
O. Orlova* and U. Nasonova

Institute of Refrigeration and Biotechnologies, ITMO University, Lomonosova

Street 9, 191002 Saint-Petersburg, Russia; *Correspondence: [email protected]

Abstract. The objective of the research is to obtain biologically valuable and safe food
products, which have functional qualities and extended shelf life by using the antimicrobial
properties of walnuts and walnut leaves of milky-wax ripeness. A study was conducted on the
possibility of using walnuts of milky-wax ripeness (walnut materials) in the production
technology of functional foodstuffs (cheese products, beverages, cheese, desserts, and bakery
products). The study identified types and possible amounts of walnut additives into the
developed products. Various additives were obtained during the research, such as extracts,
tinctures, dry powders, and capsules. The most unique specific substance in raw nut is juglone.
Juglone is a natural antibiotic that inhibits more than 100 kinds of pathogenic microorganisms.
The developed products with the addition of walnuts and walnut leaves of milky-wax ripeness
contain high amounts of vitamins (especially C, E, A, and group B) and minerals (iodine and
manganese). For this reason, walnut contains the natural antibiotic juglone, which increases the
shelf life of products up to 21 days without the use of stabilizers or preservatives. Because of
the multifunctional properties of raw nut, more specifically the antibacterial, fungicidal and
preservative properties of juglone, the additives ensure a sustainable microbiological state of the
products. Juglone allows sparing technological heat treatment of raw milk (40 ± 2°C) that
reduces energy costs for the operation and the equipment. Various population groups can
successfully use the designed products as functional food; what is more, they can be used for
medical purposes.

Key words: Walnut, fermented products, dairy products, juglone, preservative, milky-wax
ripeness.

INTRODUCTION

Walnut is a unique vegetable raw material, all parts of which can be used by
humans. Already the ancient Greeks and Romans mentioned walnut. Theophrastus,
‘the father of botany’, was one of the first to describe walnut. This plant is mentioned
in the writings of Cicero, Pliny, Virgil, and Hippocrates (Derzhavina, 2000). Michuryn
called walnut a tree, which is like an industrial complex, as absolutely all its parts are
used by humans: ripe and unripe fruit, shells and partitions, green pericarp and leaves,
bark, wood, and roots (Richter & Yadrov, 1985).
Curative and preventive properties of walnut have long been used by people in the
areas of its growth – Moldova, Northern Caucasus, Romania, Tibet, Greece, Japan,
China, France, etc. (Richter & Yadrov, 1985). The composition of milky-wax ripe raw
walnuts includes essential oils, organic acids, alkaloids, glycosides, saponins,

769
coumarins, carotenoids, water-soluble vitamins, volatile, phenolic compounds, tannins,
and trace elements (Yenikeeva, 2008). These natural complexes induce the therapeutic
use of milky-wax ripe walnuts as supplements. The chemical composition of all parts
of the walnut depends on the type, location, and environmental growing conditions.
All parts of walnut contain vitamins C, A, E, and group B, organic acids,
minerals, and tannins. The unripe fruit of walnut contains up to 3–5% of vitamin C.
The maximum content of vitamin C in unripe walnuts is observed at the beginning of
endocarp solidification (Pilipenko & Orlova, 2009).
The main groups of minor bioactive components of food plants (quinones and
hydroquinones) include juglone, which occurs in walnuts of milky-wax ripeness.
Juglone (5-hydroxy-1,4-naphthoquinone) is a natural antibiotic with high bactericidal
qualities. The German chemists Vogel and Reischauer first isolated it in 1856 from the
green husk of walnut (Yenikeeva, 2008). Juglone inhibits the activity of
phosphatidylinositol-3-kinase, which indicates its anticarcinogenic properties without
marked toxicity inherent to other cytostatics (Babich & Stern, 1993). Juglone has high
antimicrobial impact on both gram-positive bacteria (Staphylococcus aureus and
Streptococcus mutans), and gram-negative microorganisms (Esherichia coli and
Pseudomonas aeruginosa), as well as on pathogenic yeast organisms (Candida
albicans) (Polonik et al., 2004; Babula et al., 2005).
The aims of our work were to develop curd products using milky-wax ripe
walnut, evaluate their chemical composition (fatty acids and amino acids), and monitor
changes in sensory, physicochemical and microbiological properties of samples during
storage.

MATERIALS AND METHODS

Materials
Non-fat curd, cream, starter for curd on pure cultures of lactic streptococci,
calcium chloride, rennet powder, additives of fruit of walnut of milky-wax ripeness.

Additive preparation
For the production of supplements, we used different varieties of milky-wax ripe
walnuts grown in the environmentally friendly Tsimlyanskii district of Rostov region
(Russia). Harvesting of walnuts took place from 12 to 14 July in 2011 (average
temperature was 28 ± 2°C) during fruit formation while they are rich in biologically
active medicinal substances.
Honey was used as a preservative for the chopped nuts and as a sweetener. We
used flower honey, which meets the requirements of the Russian standards (Russian
consumer oversight agency, 2001).
Walnut additive technology consists of the following stages: receiving and sorting
walnuts; washing walnuts and peeling them mechanically with a carborundum;
chopping walnuts with a cutting-pulping machine, mixing chopped walnuts with honey
heated to 38 ± 2°C, packaging and corking; storing at temperature 6 ± 2°C. The
resulting additive is made of crushed milky-wax ripe walnuts
(seed + partition + endocarp) mixed with honey; the color of the additives
is homogenous and depends on the type of honey - from pale straw to dark brown; it
tastes of honey and has a pronounced walnut flavour.
770
 The preparation of curd products
Pasteurized cow cream (fat 25%) and non-fat curd were used for curd production.
The cream should be homogenized (we used homogenizator Twin Panda Niro Soavi –
100 liters per hour) at temperature 60–70°С and pressure (8.0–10.0) MPa, then
pasteurized at temperature 92 ± 2°C and incubated from 2 to 8 seconds, and then
cooled to a temperature not exceeding 80°C (Orlova, 2007). The prepared curd and
cream mixture was vacuumized and heated in a universal homogenizing module, after
which it was stirred for 30 seconds at 1,500 rev min-1. When the temperature reached
40 ± 2°C, the vacuum was turned off. Then we brought the supplement made of milky-
wax ripe walnuts and honey. The additive was weighed, heated while mixing in the
digester till 38 ± 2°C, after which it was added to the prepared curd and cream mixture.
For removing air bubbles and distributing the additives uniformly, we started the stirrer
and vacuum for 30 seconds. After that the product was packaged in polystyrene cups
with a capacity of 200 grams. The packaged product was kept at irregular temperature
for 2–3 h to prevent the formation of condensate, after which it was sent to
the refrigerating chamber for cooling down till temperature 4 ± 2°C and the formation
of structure for 6–8 h. The product should be stored at temperature 4 ± 2°C.
Manufacturing technology of curd products with the additive (15%) based on
milky-wax ripeness walnuts is shown in Fig. 1.

The stage of manufacturing Parameters and indices

Acceptance of raw materials Monitoring of sensory,

physicochemical and
additive curd cream
microbiological properties

Preparing a mixture of curd and

T = 4 ± 2°C
cream

T = 40 ± 2°C
Thermal treatment of a mixture
duration - 30 s

Introduction of additives of milky-

T = 40 ± 2°C
wax ripeness walnuts and honey,
the mixing time - 30-40 s
mixing

Packaging and labeling T = 40 ± 2°C

Aftercooling and structure T = 6 ± 2°C

formation duration - 6-8 hours

T = 4 ± 2°C
Storage
duration - 14 days

Figure 1. Curd products manufacturing technology.

771
 Sensory evaluation
The taste, aroma, colour, and texture of all curd product samples were evaluated
by a trained panel of 12 members using a five-point score system (5 excellent, 1
unacceptable).
Twelve panelists (eight women, four men; age 22–38 years) familiar with sensory
evaluation techniques and regular consumers of curd products estimated the sensory
properties of the samples.

Analysis
Juglone content of the walnut additives was determined by capillary
electrophoresis using KAPEL 105 (sample injection 450 mbar*s, voltage 20 kV,
temperature 20°C, detection 254 nm).
Amino acids, fatty acids, fat, solids, carbohydrates and titratable acidity were
determined according to AOAC (1998).

Experimental design of shelf-life

Samples stored at 4 ± 2°C were evaluated on days 1, 7, 14, and 21 taking into
account the factor of safety. The shelf-life of the developed products was evaluated
according to MUK 4.2.1847–04 ‘Control methods. Biological and microbiological.
Sanitary-epidemiological assessment of terms of consumption and suitability for food
storage. Methodology.’ (Russian consumer oversight agency, 2004).

Microbiological counts
The amount of S. Aureus, coliforms, yeasts and molds, and pathogens was
determined by the methods described by Neusely et al., 2012.

RESULTS AND DISCUSSION

We have identified the possible amounts of walnut additives used for the
developed products.
We have found out the dependence of the estimated organoleptic characteristics
of the final product on the amount of contributed additive, which is expressed by the
following equation 1:

Y = (28.026 - 0.962 x)/(1 - 0.065 x + 0.0013 x2) (1)

Approximating function, which expresses the dependence of the organoleptic

characteristics on the amount of additive and its deviation from the experimental
values, is presented in Fig. 2.
The samples with 5, 10, 15% of additive had the most acceptable sensory
properties. When we added more supplements, the taste of the final product was too
sweet, and the consistency too liquid. The samples with the addition of less than 5%
were not sweet and the taste of the additives was almost imperceptible. The resulting
products had a smooth consistency without whey separation, a fermented taste of nuts
and honey, and the colour varied from light cream to cream.

772
 S = 1.2 20 36 7 07
r = 0 .96 64 12 2 0 Residuals
25 3
45. 2.1

75
42.
7
1.0
25
40.
Y Axis (units)

Y Axis (units)
75 0.0
0
37.

25
35.
7
-1.0
75
32.

25 3
30. 0.1 3.9 7.7 11.5 15.3 19.1 22.9 -2.1 0.1 5.8 11.5 17.2 22.9

X Axis (units)

Figure 2. Approximation between organoleptic characteristics and the percentage of additives:

X- the percentage of additive components, Y – organoleptic characteristics, points.

Table 1 shows the results of a study of the chemical composition of the samples
of curd products. The maximum value of solids was observed in the sample with 15%
additive, being 36.08%. The quantity of juglone had maximum value in the sample
with 15% additive (Pilipenko & Orlova, 2009).

Table 1. Chemical composition of test samples

Mass fraction of dry Mass fraction Fat mass Mass fraction of Mass fraction
Sample
substances,% of protein,% fraction,% carbohydrates,% of juglone,%
Base 36.03 14.23 10.0 11.8 0
5% 32.43 16.26 10.0 6.17 0.24
10% 34.23 15.30 10.0 8.93 0.47
15% 36.08 14.39 10.0 11.69 0.71

In order to characterize the biological value of the investigated products, the

composition of amino acids was determined and the amino-acid score was calculated.
The results are presented in Table 2. The results of the study of biological values
indicate that the proteins of the new types of curd products are characterized by a
complete set of essential amino acids. It was found out that the additive has increased
the content of essential amino acids, which were especially limited in the base sample.
The sample with 15% of additive had the greatest number of them. It can be explained
by the fact that walnut protein has increased the content of lysine (up to 12.4 g/100 g of
protein), methionine (5.6 g/100 g of protein), and tryptophan (to 3.4 g/100 g protein).
The fat of the product consisted of a mixture of milk and vegetable fats. The
lipids of milk fat have 70.0% of saturated fatty acids, and about 4.0% of
polyunsaturated fatty acids. The lipids of milky-wax ripe walnuts consist of 84.5% of
unsaturated fatty acids, the content of linoleic and α-linolenic acid can be up to 56.0%
and 11.0% respectively.

773
Table 2. Contents of essential amino acids in test samples
Base 5% additive 10% additive 15% additive
Name of amino
X*, X*, X*, X*,
acids А*, % А*, % А*, % А*, %
mg g-1 mg g-1 mg g-1 mg g-1
Valine 50.28 100.56 55.91 111.82 57.31 114.62 58.64 117.28
Isoleucine 48.10 120.25 54.93 137.33 57.31 143.28 59.64 149.10
Leucine 79.69 113.84 93.51 133.59 98.53 140.76 103.51 147.87
Lysine 63.77 115.95 73.67 133.95 76.77 139.58 79.82 145.13
Methionine +
cystine 27.97 79.91 30.55 87.29 33.13 94.66 35.71 102.03
Threonine 36.46 91.15 50.28 125.70 60.42 151.05 70.52 176.30
Tryptophan 9.83 98.30 10.97 109.70 12.11 121.10 13.25 132.50
Phenylalanine +
82.83 138.05 101.43 169.05 111.36 185.60 121.24 202.07
tyrosine
X is amino acid content in 1g protein product; А* is amino-acid score of the amino acid.
*

The results of determining the fatty acid composition of curd products are
presented in Table 3. PUFA ratio ω6, ω3 is 9.886 : 1, and recommended by the Institute
of Nutrition, the ratio is 10 : 1 for a healthy person.

Table 3. Fatty acid composition of products

Fatty acid content in the test samples, g
Name of acid 5% 10% 15%
Base additive additive additive
Sum of saturated acids 7.047 6.526 6.142 5.563
Butyric 0.211 0.198 0.185 0.149
Capric 0.254 0.217 0.198 0.162
Myristic 1.384 1.354 1.347 1.331
Palmitic 3.722 3.526 3.282 3.213
Stearic 0.946 0.938 0.721 0.583
Arachic 0.090 0.070 0.056 0.049
Unidentified 0.440 0.223 0.353 0.276
Amount of unsaturated acids 2.953 3.474 3.858 4.437
Myristoleic 0.113 0.075 0.053 0.060
Palmitoleic 0.248 0.252 0.211 0.228
Oleic 2.090 2.141 2.159 2.214
Linoleic (ω6) 0.189 0.698 1.124 1.523
α-linolenic (ω3) 0.081 0.121 0.147 0.168
Arachidonic (ω6) 0.153 0.144 0.141 0.139
Unidentified 0.078 0.043 0.023 0.105
ω6/,ω3 4.275 7.020 8.687 9.886

The results of organoleptic evaluation of the curd products during storage are
presented in Fig. 3. The best results on the first day of storage have samples No 1 and
No 4, since by all indications they have received the highest score – 20 points.
Sample No 4 got the highest score after 21 days of storage. There was no change
in the appearance of the sample; the surface remained smooth and glossy with evenly
distributed patches of additive. The consistency of sample No 4 was the same as that of

774
the fresh sample: rather viscous, thick, and slightly fluffy. The colour was cream and
uniform throughout the mass, a little more intense than that of the fresh sample. The
taste and smell of sample No 4 on the 21st day of storage were weaker, having a less
pronounced taste of nuts and honey.

1 day 7 day 14 day 21 day

20 20 20
Organoleptic assessment,scores

20 18 18 18
17 17 17 19,5
15 15 17
15 13
10
10
6
5

0
base 5% 10% 15%
Figure 3. Organoleptic assessment during the storage of different percentages of additive
components.

On the 21st day of storage, the control sample had a too acid taste and yeast
flavour. Separation of serum and unpleasant odour were observed. We concluded that
the product was not suitable for consumption.
The change in titratable acidity is shown in Fig. 4.

140
Titratable acidity, 0T

135 control sample

130 without additive
125
120 sample with 5% of
115 additive
110
105 sample with 10% of
100 additive
95
sample with 15% of
90
additive
1 day 7 day 14 day 21 day

Figure 4. Change in titratable acidity of curd products during the storage.

775
 In the control sample without the additive, quite a rapid growth of it can be
observed at the beginning of the storage. After 14 days of storage, titratable acidity of
the control sample did not meet the requirements of Russian legislation (Rosstandart,
2008). Introduction of supplement to the curd product allowed halting the growth of
acidity in the product, especially in the sample with 15% of the additive. It is obvious
that the preservative effect of juglone slows the formation of acids. It is known that
juglone has properties, which partially inhibit the growth of lactic acid bacteria even in
low concentrations (Babula et al., 2005).
We have investigated microbial indicators of samples in accordance with Russian
legislation (Rosstandart, 2008). The results of microbiological tests (on day 14) are
shown in Table 4.
In the studied samples of curd products molds, coliform bacteria in 0.001 g of
product and pathogens including salmonella in 25 g were not detected. Colony-forming
units (CFU) Staph. aureus in 1 g were not detected. The amount of yeast in the control
sample did not meet the requirements of Russian legislation. Samples No 1, 2 and 3 are
within the expected microbiological levels for this type of product and present no food
safety concern (Rosstandart, 2008).

Table 4. The result of microbiological tests

Sample No 1
Product weight Sample No1 Sample No 2 Sample No 1
Name of sample with
(g, cm3), which is control sample sample with 5% sample with
indicator 10% of
not allowed without additive of additive 15% of additive
additive
CGB (coliforms),
0,001 not detected not detected not detected not detected
in 0.01 cm3
S. aureus, in 0.1g 0,1 not detected not detected not detected not detected
Pathogenic,
including 25 not detected not detected not detected not detected
salmonella
Yeast, CFU/g 100 110 45 22 20
Molds, CFU/g 50 not detected not detected not detected not detected

We assume that the reduction in yeast amount is related to the increase in additive
percentage. It may be explained by the presence of naphthoquinone juglone.

CONCLUSION

Milky-wax ripe walnut is a unique product. Its amazing properties have been used
since ancient times. The use of the unique characteristics of milky-wax ripe walnut
(especially juglone) enables to develop fortified foods, increase the shelf life of
products due to their content of naftachinon – juglone in walnuts. These studies have
shown only a small part of the results of introducing walnut based additives into curd
products.
Introduction of the additives allows obtaining a cream-colored product with a
pronounced walnut and honey taste and odour and a smooth and pleasant consistency.
The additive of milky-wax ripe walnut slows acid formation.

776
 In contrast to the control sample, the samples with the additive displayed an
increase in:
 The amount of essential amino acids, thus improving the biological value of the
final product;
 The ratio of ω6/ω3 from 4.275 to 9.886.
The results of monitoring changes in sensory, physicochemical and
microbiological properties of samples during storage allow us to recommend the shelf
life of 14 days (4 ± 2°С) for curd products with additive.

ACKNOWLEDGEMENTS. This work was partially financially supported by the government

of the Russian Federation, Grant 074-U01.

REFERENCES

AOAC. 1998. Official Method of Analysis. Association of Official Analytical Chemists (16th
Edn), Washington DC, USA.
Al-Kadamany, E., Khattar, M., Haddad, T. & Toufeili, I. 2003. Estimation of shelf-life of
concentrated yogurt by monitoring selected microbiological and physicochemical changes
during storage. Lebensm.-Wiss.U.-Technol. 36, 407–414.
Babich, H. & Stern, A. 1993. In vitro cytotoxicities of 1,4-naphthoquinone and hydroxylated
1,4-naphthoquinones to replicating cells. J Appl Toxicol. 13(5), 353–358.
Babula, P., Mikelovab, R., Potesilb, D., Adamb, V., Kizekb, R., Haveld, L. & Sladkya ,Z. 2005.
Simultaneous determination of 1,4-naphtoquinone, lawsone, juglone and plumbagin by
liquid chromatography with UV detection. Biomed. Papers V, 25–28 (in Russian).
Derzhavina, N.A. 2000. Healing walnut. Respeks, St. Petersburg, 64pp. (in Russian).
Ji, Y., Qua, Z. & Zoua, X. 2009. Juglone-induced apoptosis in human gastric cancer SGC-7901
cells via the mitochondrial pathway. Exp. Toxicol. Pathol. 63(1–2), 69–78.
Karsheva, M., Paskov, V., Tropcheva, R., Georgieva, R. & Danova, S. 2013. Physicochemical
parameters and rheological properties of yoghurts during the storage. J. Chem. Technol.
Metall. 48, 483–488.
Liu, L., Li, W., Koike, K., Zhang, S. & Nikaido, T. 2004. New alpha-tetralonyl glucosides from
the fruit of Juglans mandshurica. Chem Pharm Bull, 52(5), 566–569.
Monks, T.J., Hanzlik, R.P., Cohen, G.M., Ross, D. & Graham, D.G. 1992. Quinone chemistry
and toxicity. Toxicol Appl Pharmacol. 112(1), 2–16.
Neusely, S., Taniwaki, M.H., Junqueira, V.C., Silveira, N., Nascimento, M. & Gomes, R.A.
2012. Microbiological Examination Methods of Food and Water: A Laboratory Manual,
484 pp.
Orlova, O.U. 2007. Walnuts milk-wax maturity in cheese products. Storage and processing of
agricultural 9, 40–42. (in Russian).
Pilipenko, T.V. & Orlova, O.U. 2009. Development of formulations of foods using raw walnut
milky-wax ripeness. In Baranenko A.V.(ed.): Collection of materials 3rd International
Scientific and Technical Conference Low-Temperature and Food Technologies in XXI
century. SPbGUNiPTS St. Petersburg. pp. 290–291. (in Russian).
Polonik, S. Prokofiev, N. Agafonova, I. & Uvarov, N. 2004.The antitumor and immuno
stimulatory activity O- and S-atsetilglikozidov 5-hydroxy-1,4-naphthoquinone (juglane).
Pharmaceutical Chemistry Journal 8, 15–17.(in Russian).
Richter, A.A. & Yadrov, A.A. 1985.Walnut.Agropromizdat,Moscow, 215 pp.(in Russian).
Rosstandart, 2008. Federal Law No 88 on ‘Technical Regulations for Milk and Milk Products’
Moscow, 124 pp.(in Russian).

777
Russian consumer oversight agency, 2004. MUK 4.2.1847-04 ‘Control methods. Biology and
microbiological. Sanitary-epidemiological assessment of terms reasons consumption and
fitness for storage of food. Methodology’, 16 pp.
Russian consumer oversight agency, 2001. Hygienic requirements for safety and nutrition value
of food products. Sanitary and epidemiological rules and regulations. SanPin 2.3.2.1078-
01, 180 pp.
Segura-Aguilar, J., Jonsson, K., Tidefelt, U. & Paul, C. 1992. The cytotoxic effects of 5-OH-1,
4-naphthoquinone and 5,8-diOH-1,4-naphthoquinone on doxorubicin-resis- tant human
leukemia cells (HL-60). Leuk Res. 16(6–7), 631–637.
Yenikeeva, R.A. 2008. Study on Farmakognostichesky study and standardization of raw
materials and preparations of walnut(Juglansregia L.).Autoabstract, Moscow, 21 pp.

778
 Agronomy Research 12(3), 779–786, 2014

Studying of mixing speed and temperature impacts on

rheological properties of wheat flour dough using Mixolab
A. Pastukhov1,* and H. Dogan2
1
ITMO University, Institute of Refrigeration and Biotechnologies, 191002,
Lomonosova Street. 9, office 2111 Saint-Petersburg, Russia;
*Correspondence: [email protected]
2
Kansas State University, Department of Grain Science and Industry, 201
Shellenberger Hall, Manhattan, KS 66506, KS, USA

Abstract. Wheat flour dough is highly non-Newtonian, time-dependent, strain-dependent and

viscoelastic. These rheological properties are very sensitive to temperature, water content and
composition. Dough mixing is one of the most important ways to characterize the quality of
wheat flours. Proper dough development is affected by mixing intensity (mixing speed) and
work imparted to the dough. The objective of this research was to study impact of mixing speed
and temperature on thermomechanical properties of breadmaking quality wheat flours using
Mixolab. Analysis was carried out at the constant water absorption (98% db) using standard
Chopin+ protocol, which consisted of a heating/cooling cycle after a certain mixing time at
constant mixing speed (60–120 rpm). Effect of temperature at 80 rpm, 100 rpm, 120 rpm, and
effect of mixing speed at 30C, 40C, 50C were also studied. Strong relationships were
observed between the mixing speed (rpm) and the Mixolab parameters (dough consistency
during mixing (C1), mixing stability, protein weakening (C2), starch gelatinization (C3),
amylase activity (C4) and starch gelling (C5).
Mixing temperature was observed to have higher impact on dough consistency and stability
than mixing speed. Softening effect of temperature was more significant at low mixing speeds.

Key words: mixing behavior, pasting, torque, dough consistency.

INTRODUCTION

The bread-making process consists of the three main steps. Those are mixing,
fermentation and baking. The mixing process is the crucial operation in bakery product
production by which the wheat flour, water, and additional ingredients are changed
through the mechanical energy flow to coherent dough. (Gras et al., 2000; Zheng et al.,
2000; Wilson et al., 2001). Dough mixing is one of the most important ways to
characterize the quality of wheat flour samples. The dough development is a dynamic
process where the viscoelastic properties are continuously changing. Therefore, dough
properties are strongly influenced by the way of their mixing. For achieving the proper
dough development, two basic requirements must be satisfied. The imparted mixing
energy or work input must be higher than the critical limit of energy needed for gluten
formation, and the mixing intensity must be above the critical level for the dough
development (Kilborne & Tipples, 1972). These requirements vary with the flour

779
properties and the type of mixer used (Frazier et al., 1975; Oliver & Allen, 1992). For
this reason, decisions with respect to adequacy of dough mixing are still partly based
on operator experience. Recently, a number of analytical methods have been
investigated to monitor dough development based on physical or chemical description
of dough properties. The most popular in-line process measurements, based on changes
in dough physical properties, are that of mixing torque or power consumption of the
mixer.
At laboratory scale (using analytical methods after dough sampling), dough
development has been largely investigated by microscopy and chemical analysis. In
industry, a wide variety of mixing geometries and speeds are used for dough
development. The way the dough is mixed has a major impact on the rheological
properties due to the time- and strain-dependent nature of dough. The farinograph and
mixograph are two common devices for assessing flour properties during mixing in lab
scale. Both mixers provide empirical measurements related to the torque and work
input required to produce optimally mixed dough, despite dissimilar geometries and
mixing actions. A new generation of analytical equipment is represented by Chopin
(Tripette et Renaud, Paris, France). This apparatus measures and plots in real time the
torque (in N·m) produced by passage of the dough between the two mixing arms, thus
allowing the study of mixing and pasting behavior of the wheat flour dough systems.
Mixolab could play a key role in ensuring flour performance matches customers’
expectation in finished product (Gedrovica & Karklina, 2011). The quality of wheat-
hemp composites prepared with different amounts of hemp flour (5, 10, 15 and 20%)
was characterised by the mixolab rheological test by Hruskova et al. The most precise
distinguishing of samples was observed during the mixing and starch retrogradation
phases of the test. Correlation analysis confirmed proper relationships between
mixolab and rheological parameters related both to protein properties (C1, C2, C1–C2
vs. farinograph and extensigraph ones) and starch or starch gel properties (C3, C4, C5
vs. amylograph ones) ( Hruskova et al., 2013). There were several research studies of
thermomechanical properties of different types of wheat, such as Indian and Chines
(Dhaka & Khatkar, 2013) and (Chen et al., 2013).The objective of this research was to
study impact of mixing speed on thermomechanical properties of Hard Red Spring
flours using Mixolab. Hard Red Spring wheat flour stands out as the aristocrat of wheat
for baking bread, bagels and hard rolls. It has the highest protein content of all U.S.
wheats (usually 13–16%) which, in turn, corresponds with greater gluten content in
dough. Understanding the effects of mixing speed variation on gluten strength and also
starch gelatinization, amylase activity and starch gelling of Hard Red Spring wheat
flour is important when creating the distinct structural and textural characteristics that
consumers desire in baked products.

780
 MATERIALS AND METHODS

This study was done in the laboratory of Department of grain Science and
Industry at Kansas State University.
One batch of Hard Red Spring wheat (1.4% ash, 13.2% protein, 16% moisture,
98% water absorbtion) was used for the experiments in this study during 5 consecutive
days.
A standard Mixolab curves (Fig. 1) were used to determine a set of parameters
listed in Table 1. C1 and C2 are related to protein quality, whereas C3, C4 and C5 are
related to the starch characteristics. Fig.1 shows the results of experiment No 3
(Table 2). Correlations between mixing speed and the mixolab parameters and also the
correlations between initial bowl temperature and mixolab parameters were
investigated.

Figure 1. Mixolab Chopin+ protocol curve. Experiment No 3 (80 rpm and 30C); where: α, β,
and γ are the indicators of protein weakening, starching speed and enzymatic degradation.
Zone 1: Dough Development – at constant temperature, the start of the test determines the water
absorption capacity of the flours and measures the characteristics of dough during mixing
(stability, elasticity, absorbed power); Zone 2: Protein reduction (α) – when dough temperature
increases, consistency decreases. The intensity of this decrease depends on protein quality;
Zone 3: Starch gelatinisation (β) – as from a certain temperature, the phenomena linked to
starch gelatinisation become dominant and an increase in consistency is then observed. The
intensity of this increase depends on the quality of the starch and, in some cases, on the
additives; Zone 4: Amylase activity (γ) – The value of consistency at the end of the plateau
depends considerably on the endogenous or added amylasic activity. The greater the decrease in
consistency, the greater the amylasic activity.

781
 The Mixolab is a recording dough mixer used to measure the rheological
properties of doughs subject to the dual stress of mixing and temperature changes. It
measures the torque (in N·m) produced by the dough between two mixing blades. The
test is based on the preparation of a constant dough sample weight hydrated to obtain a
target consistency during the first test phase. In the ‘Chopin+’ protocol, the dough
weight is 75 grams and the target consistency is 1.1 N·m (± 0.05 Nm).
Mixolab analysis were carried out at the constant water absorption (98% db)
using standard ‘Chopin+’ protocol, which consisted of a heating/cooling cycle after a
certain mixing time at constant mixing speed (60–120 rpm). Required amount of flour
for analysis was calculated by Mixolab software according to input values of flour
mixtures moisture as well as water absorption. The total mass of flour and distilled
water placed into bowl was 75 g. Initial bowl temperature for each experiment is
shown in Table 2.

Table 1. Mixolab parameters

Point Description
C1 Maximum consistancy obtained in the first 8 min
(water absorbtion)
C2 Protein weakening as a function of mechanical
work and temperature
C3 Starch gelatinisation
C4 Hot gel stability
C5 Starch retrogradation in the cooling phase
Slope α – slope of curve between end of Protein weakning speed under the effect of heat
period at 30°C and C2
Slope β – Slope of curve between C2 and Starch gelatinisation speed
C3
Slope γ – Slope of curve between C3 and C4 Enzyme degradation speed

Table 2. Experiment composition (target torque for C1 – 1.1 N·m)

Experiment Number of Mixing speed, Initial bowl and 1-st step
experiment rpm temperature, °C
1 60 30
2 70 30
3 80 30
Speed effect
4 90 30
study
5 100 30
6 110 30
7 120 30
8 80 40
9 80 50
Temperature and
10 100 40
speed effects
11 100 50
study
12 120 40
13 120 50

782
 After dough mixing stage (8 minutes) samples temperature increase with the
speed 4°C min-1 during 15 minutes; at this point, there was a holding period for 7
minutes at 90°C, followed by a temperature decrease with the speed 4°C per min
during 10 minutes; then the mixture reached 50°C and hold at this temperature for 5
minutes. Total analysis time was 45 min. The mixing speed during the entire assay
from very beginning until the end was 60, 70, 80, 90, 100, 110 and 120 rpm,
respectively to the experiment. 5 replicates were carried out for each type of
experiments.

RESULTS AND DISCUSSION

Initial testing has focused on mixing flour-water dough to peak development at

varying speeds (Fig. 2). Work input to reach peak torque was determined and
compared (Pastukhov & Dogan, 2010).

Figure 2. Mixolab curves obtained at varying mixing speeds (60–120 rpm) using ‘Chopin+’
protocol (Speed effect study).

Strong relationships were observed between the mixing speed (rpm) and the
following Mixolab parameters: (Fig. 3) a) time needed to reach point C1; b) torque in
point C1; c) torque in point C2; d) C1–C2 difference indicating progressive protein
weakening; e) torque in point C3; f) torque in point C4; g) C3–C4 difference indicating
starch stabilization; h) α; i) total work done (sum of the torques during the experiment).

783
 It is known from (Sabovics et al., 2011) that decrease of triticale flour proportion
in blend during mixing with constant speed results to increasing of the dough stability
and does not change dough properties substantially. Changing the mixing speed we
discovered that dough consistency increased while the stability decreased with
increasing mixing speed (Fig. 2, Fig. 3). The higher the mixing speed the faster the
achievement of point C1 takes place and the higher the torque in this point (Fig. 3 a, b).
The same situation with torque in points C2, C3, C4, but the time needed to reach these
points are increasing with increasing of mixing speed. C2–C1 difference increased
indicating progressive weakening in dough network at elevated mechanical energy
input and temperature. Maximum viscosity (point C3) increased possibly due to quick
rupture of starch granules leading to lower pasting temperatures and to higher paste
consistency. C3–C4 difference – fall in viscosity (stability when hot) is decreasing
when mixing speed is increased. Value of α slope increases monotonically with
increasing of mixing speed, indicating the protein weakening (Fig. 3 h).

Figure 3. Correlations between mixing speed and Mixolab parameters.

Fig. 4 shows the effect of temperature at 80 rpm (a), 100 rpm (b), 120 rpm (c),
and effect of mixing speed at 30C (d), 40C (e), 50C (f) observed in the second set of
experiments

784
Figure 4. Effect of temperature at a – 80 rpm, b – 100 rpm, c – 120 rpm, and effect of mixing
speed at d – 30C, e – 40C, f – 50C.

CONCLUSIONS

Results indicated that the speed at which dough is deformed during mixing can
cause it to develop differently.
Dough development time decreased significantly with gradual increase in mixing
speed.
Stability of gluten network dropped sharply as mixing speed increased as
indicated by C1–C2 and α (slope of the descending curve) values.
Increase in mixing speed resulted in increased higher dough consistency
independent from the mixing temperature. Mixing temperature was observed to have
higher impact on dough consistency and stability than mixing speed. Softening effect
of temperature was more significant at low mixing speeds. The present study showed
that Mixolab has ability to easily model different speed variations and results of these
experiments indicate that the speed at which dough is deformed during mixing can
785
cause it to develop differently. However, further work is required for modeling more
complicated mechanical motion of mixing arms as we can meet in real mixers. It can
be concluded that Mixolab is a suitable instrument for progressive work in scientific
laboratories and industrial bakeries.

ACKNOWLEDGEMENTS. This study was financially supported by Fulbright scholarship.

REFERENCES

Chen, F., Li, H., Li, X., Dong, Z., Zuo, A., Shang, X. & Cui, D. 2013. Alveograph and Mixolab
parameters associated with Puroindoline-D1 genes in Chinese winter wheats. J. Sci. Food
Agric. 93(10), 2541–2548.
Dhaka, V. & Khatkar, B.S. 2013. Mixolab thermomechanical characteristics of dough and bread
making quality of Indian wheat varieties. Qual. Assur. Saf. Crop. 5(4), 311–323.
Gedrovica, I. & Karklina, D. 2011. Influence of Jerusalem artichoke powder on dough
rheological properties. Proceedings of the 6th Baltic Conference on Food Science and
Technology ‘Innovations for Food Science and Production’ FOODBALT-2011, Jelgava,
May 5–6, pp.7–12.
Gras, P.W., Carpenter, H.C. & Andersen, R.S. 2000. Modelling the developmental rheology of
wheat flour dough using extension tests. J. Cereal Sci. 31, 1–13.
Hruskova, M., Svec, I. & Jurinova, I. 2013. Changes in Baking Quality of Composite
Wheat/Hemp Flour Detected by Means of Mixolab. Cereal Res. Commun. 41(1),
150–159.
Kilborne, R.H. & Tipples, K.H. 1972. Factors affecting mechanical dough development. I.
Effect of mixing intensity and work input. Cereal Chem. 49, 48–53.
Olivier, J.R. & Allen, H.M. 1992. The prediction of breadmaking performance using the
farinograph and extensograph. J. Cereal Sci. 15, 79–89.
Pastukhov, A.S. & Dogan, H. 2010. Effect of kneading speed on thermo mechanical properties
of flour doughs. Kansas State University, Manhattan, KS, U.S.A. Cereal Foods World,
55: A63
Sabovics, M., Straumite, E. & Galoburda R. 2011. Assessment of the rheological properties of
flour using the mixolab. Proceedings of the 6th Baltic Conference on Food Science and
Technology ‘Innovations for Food Science and Production’ FOODBALT-2011, Jelgava,
May 5–6, pp.33–38.
Wilson, A.J., Morgenstern, M.P. & Kavale, S. 2001. Mixing response of a variable speed 125 g
laboratory scale mechanical dough development mixer. J. Cereal Sci. 34, 151–158.
Zheng, H., Morgenstern, M.P., Campanella, O.H. & Larsen, N.G. 2000. Rheological properties
of dough during mechanical dough development. J. Cereal Sci. 32, 293–306.

786
 Agronomy Research 12(3), 787–792, 2014

Effect of cooling rates and low crystallization

temperatures on morphology of lactose crystals obtained
from Ricotta cheese whey

A. Pisponen*, S. Pajumägi, H. Mootse, A. Sats, V. Poikalainen and A. Karus

Department of Food Science and Technology, Estonian University of Life Sciences,

Kreutzwaldi 56/5, EE51014 Tartu, Estonia; *Correspondence: [email protected]

Abstract. In the traditional process of lactose production from cheese whey crystallization
temperature is reached by slow cooling. Lactose morphology obtained in this way has been well
investigated. The objective of this work was to study morphological parameters of lactose
crystals during crystallization at low temperatures, using rapid and extra rapid cooling. Ricotta
whey was chosen for analysis because this raw material has been poorly investigated as a
source of lactose production. Three temperatures (2, 6 and 12 C) were used for crystallization
of lactose. Rapid (0.5°C min-1) and extra rapid cooling (3°C min-1) rates were used to achieve
these temperatures. Dimensions of crystals were examined with optical stereo-, and scanning
electron microscopes. Pure lactose solution was used as a reference during the study. The
results of the study showed the impact of some Ricotta whey components on the crystals
morphology and size. These components reduce crystals size, and linked with low
crystallization temperature, modify the shape of crystals faces. Extra rapid cooling of Ricotta
whey contributes to the growth of needle-like crystals more than the rapid one. In a pure lactose
solution no needle-shaped crystals were observed.

Key words: lactose, Ricotta cheese whey, crystallization, cooling rate.

INTRODUCTION

Industrial crystallization of lactose from cheese whey is a slow process based on

cooling of deproteinated and concentrated whey. First of all, a concentrate is cooled
rapidly to 30°C. After that it is seeded and cooled rather slowly, at about 1 to 3°C h -1.
Crystallization temperature of 20°C is normally recommended. (Tan, 2010) As early as
in 1930s, Whritter & Could (1931) proposed such scheme for lactose production
because they found that lactose crystallization was faster at 30°C than at any lower
temperature. Crystallization of lactose at low temperatures has been well studied over
many decades in connection with ice-cream investigations (Nickerson, 1954, 1956;
Livney et al., 1995). The phenomenon of cooling rate in general has also been
investigated and used for manufacturing of lactose (Paterson, 2010). No information
was found in the literature concerning the effects of low crystallization temperatures
and the cooling rate on the morphological parameters of lactose crystals. The objective
of this work was to study morphological parameters of lactose crystals during
crystallization at low temperatures, using rapid and extra rapid cooling.

787
 MATERIALS AND METHODS

Ricotta cheese whey was used for this study because of additional
deproteinization process carried out during Ricotta production. It was provided by a
local cheese factory, OÜ Põltsamaa Juustutööstus, Estonia. The lactose, fat and protein
contents were measured at the Milk Analysis Laboratory of the Estonian Animal
Recording Centre using an automated infrared milk analyzer (CombiFoss FT+,
Foss Electric, Denmark). The ash content was estimated using the International IDF
Standard 42B : 1990; pH was measured by the METTLER TOLEDO pH-meter
(Mettler-Toledo International Inc., Switzerland). The dry matter content was measured
using METTLER TOLEDO RH83 moisture analyzer (Mettler-Toledo International
Inc., Switzerland).
Ricotta whey was heated to 90°C and then 0.4% w/w of lactic acid (Sigma-
Aldrich Chemie GmbH, Germany) was added for precipitation of protein residues,
which were removed by filtering through common paper filter. Concentration of
Ricotta cheese whey was carried out by water evaporation at low temperature (60°C)
during approximately 3.5 h. Concentration factor of 10 was achieved by this treatment.
It corresponds to total solids concentration of ca 50% w/w (Pisponen et al., 2013).
Concentrated Ricotta whey was poured into test tubes, by 10 ml into each one.
Crystallization was carried out at the temperatures of 2, 6 and 12°C during 24 hours
without agitation in an incubator Panasonic MIR-154-PE (Japan). Rapid (0.5°C min-1)
and extra rapid cooling (3°C min-1) rates were used to achieve these temperatures.
Extra rapid cooling was carried out using ultra low temperature freezer Panasonic
MDF-C8V1 (Japan). The pure lactose solution with concentration 50% w/w was used
as reference solution and was subjected to the same stages, in order to study the impact
of impurities on crystallization process. Thus, there were two investigated parameters
for each sample: cooling rate and temperature of crystallization. Crystals were washed
with 10% w/w spirit solution and distilled water to clean crystals from molasses.
The shape and the size of crystals were examined using a microscope
Nikon SMZ 1000 (Nikon Corporation, Japan), equipped with the digital camera
Nikon DS-U2/L2 USB (Nikon Corporation, Japan). The height of crystals was
estimated using the software NIS-Elements D 3.1 (Nikon Corporation, Japan). Used
measurement technique has been described in our earlier study in Pisponen et al.
(2013). At least 40 crystals were examined for each data set. In order to avoid errors,
only single, clearly distinguishable crystals were inspected. A scanning electron
microscope Leo 1430VP (LEO Electron Microscopy Ltd, England) was used for
studying the surface of crystals.

RESULTS AND DISCUSSION

The content of dry matter, fat, protein and lactose in Ricotta cheese whey are
given in Table 1. Five measurements were considered for each data set. Data values
obtained in this study were lower than those of the normal cheese whey (Jelen, 2003).
The average values of crystals heights are shown in Table 2. Crystals obtained
from pure lactose solution in most cases were larger than crystals obtained from
Ricotta cheese whey. This difference can be explained by presence of protein residues
in Ricotta cheese whey. Modler & Lefkovitch (1986) and Miumoni et al. (2005)
788
demonstrated in their research with cheese whey that protein lowered the final size of
crystals. Minerals, contained in Ricotta cheese whey, can also lower the final size of
crystals. Guu & Zall (1991) found, that potassium, sodium and calcium can retard or
inhibit lactose crystallization from cheese whey. General appearance of crystals is
shown in Fig. 1.

Table 1. Content of dry matter, fat, protein, lactose, ash content (%w/w) and pH in native
Ricotta cheese whey
Dry matter Fat Protein Lactose pH Ash content
Average 4.9 0.16 0.44 3.85 5.67 0.51
Standard deviation 0.23 0.01 0.02 0.07 0.08 0.08

Table 2. The average, ± standard deviation, minimum and maximum values (within brackets)
of crystals height (µm) obtained from different solutions under different crystallization
temperatures and cooling rates
Temperature,°C 2 6 12
Extra Extra
Rate of cooling Rapid Rapid Rapid Extra rapid
rapid rapid
Ricotta cheese 155 ± 20 78 ± 26 182 ± 76 133 ± 73 179 ± 59 123 ± 53
whey (128–196) (41–127) (93–388) (39–384) (80–351) (62–337)
Pure lactose 166 ± 77 128 ± 52 165 ± 66 192 ± 72 158 ± 68 220 ± 65
solution (77–450) (39–288) (46–302) (94–385) (55–387) (111–372)

Rapid cooling does not affect the size of crystals obtained from pure lactose
solution; they remained almost identical at different crystallization temperatures. When
using extra rapid cooling, the difference in crystals sizes became obvious. The height
of crystals, obtained from pure lactose solution, increased along with rising of
crystallization temperatures. There was no such a clear dependence in size of crystals,
obtained from Ricotta cheese whey; the shortest crystals appeared at 2°C and the
highest at 6°C. Difference in sizes between crystals, obtained at 6 and 12°C, was
considerably smaller than between 2 and 6°C. This indicates to a certain optimum
between 2 and 12°C. A large amount of needle-shape crystals was obtained at 6 and
12°C during crystallization from Ricotta cheese whey at extra rapid cooling (Fig. 1).
Needle-shaped crystals are formed when the super saturation of material in solution is
very high (Hartel, 2001). Extra rapid cooling increases the content of crystalline
material in solution and promotes the appearance of a host in crystallization centers
(Paterson, 2010). In our experiment, extra rapid cooling could promote occurrence of
even more crystallization centers, which lead to appearance of extra small crystals.
There were no needle-shaped crystals observed in a pure lactose solution (Fig 1),
which leads to the conclusion that impurities have bigger impact on the crystal form
than crystallization conditions. Crystals obtained in the research were generally smaller
than crystals obtained in our earlier study at the same concentration factor, but at a
slower cooling rate (Pisponen et al., 2013).

789
Figure 1. Lactose crystals forms depending on crystallization temperature and cooling rate:
solution: I–Ricotta cheese whey II–pure lactose solution: a–rapid cooling and b–extra rapid
cooling.

The SEM photos show that faces of some crystals, grown in Ricotta cheese whey,
were modified (Fig. 2 b). According to Hartel`s (2001) theory crystals grow
imperfectly, if their growth speed is too rapid. Molecules do not have time to attach the
right place on the crystal face (in the crystal lattice). They rapidly incorporate at
random position, making crystal surface rough. In our case crystals were growing very
rapidly due to a sharp drop in temperature. But crystals, grown at the same conditions
in pure lactose solution, had smooth faces (Fig. 2 a).
It can be supposed, that not only growth speed can affect the shape of crystals.
Macromolecules from Ricotta cheese whey such as proteins may adsorb on the crystal
surface and influence the manner by which molecules attach to the crystals lattice
(Hartel & Shastry, 1991). Riboflavin can also adsorb on the growing crystal and
modify its shape (Holsinger, 1988). Lifran et al. (2007) found, that riboflavin makes
the tip of the crystal irregular, which can be observed on crystals, obtained from
Ricotta cheese whey (Fig. 2 b).

790
Figure 2. SEM images of lactose growing in a – pure supersaturated lactose solution, b –
Ricotta cheese whey.

According to Cabrera-Vermilyea (1958) model, impurities adsorbed on the crystal

face prevent the smooth growth of a new layer. Crystal of lactose grows in a spiral
(Dincer et al., 2009); therefore layers grow one upon another. If the lower layer`s
growth is stopped by immobile impurity, the upper layer grows to it, until it completely
covers impurity and move farther (Hartel, 2001). Any kind of particles adsorbed on a
crystal face, or rough and pitted surface, can potentially serve as nucleation centers.
Furthermore, a rapid rate of cooling generally leads to nucleation at a lower
temperature, rather than the slow cooling. (Hartel, 2001) In our case, these effects
could trigger nucleation on the crystal surface and increase its roughness. However, it
contradicts the theory that under conditions of rapid growth the additives cannot adsorb
on the surface because of competition with the molecules of crystallizing material
(Holsinger, 1988).
It remains unclear why (010) faces (base of crystal) were well-developed when
other faces were rough (Fig. 2 b). Inasmuch as (010) face becomes the fastest growing
face (Dincer et al., 2009), it can be assumed that it starts to form until the
supersaturation becomes critical and impurities from solution accumulates in the
boundary layer of the crystal later on (Hartel & Shastry, 1991).

CONCLUSIONS

This study presents influence of rapid and extra rapid cooling rate and
crystallization at low temperatures on the morphological parameters of lactose crystals.
Experiments show that temperature of crystallization and cooling rate impact mainly
the size of lactose crystals. A strong dependence between crystallization temperature,
cooling rate and crystals sizes was observed. Morphology of crystals was affected by
impurities from solution more than crystallization conditions. There was some amount
of irregular and needle-shaped crystals, obtained from Ricotta cheese whey. In a pure
lactose solution no needle-shaped crystals were observed. The impact of impurities on
certain faces of crystal requires an additional research.

791
 REFERENCES

Cabrera, N. & Vermilyea, D.A. 1958. The Growth of Crystal from Solution. In Doremus, R.H.,
Roberts, B.W. & Turnbull, D. (eds.): Growth and Perfection of Crystals. Chapman &
Hall, London, pp. 393–410.
Dincer, T.D., Ogden, M.I. & Parkinson, G.M. 2009. Crystal growth mechanisms of the (010)
face of alpha-lactose monohydrate crystals. J. Crys. Gro. 311, 2427–2432.
Guu, M.Y.K. & Zall, R.R. 1991. Lactose crystallization: Effects of minerals and seeding.
Process Biochem. 26, 167–172.
Hartel, R.W. 2001. Crystallization in Foods, Aspen Publishers, Inc, Gaithersburg, 325 pp.
Hartel, R.W. & Shastry, A.V. 1991. Sugar crystallization in food products. Crit. Rev. Food Sci.
& Nutr. 1(1), 49–112.
Holsinger, V.H. 1988. Lactose. In Jennes, R., Marth, E.H., Wong, N.P., Keeney, M. (eds):
Fundamentals of Dairy Chemistry, 3rd edn, Springer, New York, pp. 279–343.
Jelen, P. 2003. Whey: Composition, properties, processing and uses. In Roginski, H. (ed):
Encyclopedia of Dairy Science. Academic Press, Waltham, pp. 2653–2661.
Lifran, E.V., Vu, T.T.L., Durham, R.J., Hourigan, J.A. & Sleigh, R.W. 2007. Crystallization
kinetics of lactose in the presence of lactose phosphate. Powder Tech. 179, 43–54.
Livney, Y.D., Donhowe, D.P. & Hartel, R.W. 1995. Influence of temperature on crystallization
of lactose in ice-cream. Int. J. Food Sci. Tech. 30, 311–320.
Miumoni, A., Schuck, P. & Bouhallab, S. 2005. Kinetics of lactose crystallization and crystal
size as monitored by refractometry and laser light scattering: effect of proteins. Dairy Sci.
Tech 85, 253–260.
Modler, H.W. & Lefkovitch, L.P. 1986. Influence of pH, casein and whey protein denaturation
on the composition, crystal size nad yield of lactose from condensed whey. J. Dairy Sci.
69, 684–697.
Nickerson, T.A. 1956. Lactose crystallization in ice cream II. Factors affecting rate and
quantity. J. Dairy Sci. 39(10), 1342–1350.
Nickerson, T.A. 1954. Lactose crystallization in ice cream I. Control of crystal size by seeding.
J. Dairy Sci. 37(9), 1099–1105.
Paterson, AH.J. 2010. Production and Uses of Lactose. In McSweeney, P.L.H. & Fox, P.F.
(eds): Advanced in Dairy Chemistry Volume 3: Lactose, Water, Salts and Minor
Constituents, 3rd edn, Springer, New York, pp. 105–120.
Pisponen, A., Pajumägi, S., Mootse, H., Karus, A. & Poikalainen, V. 2013. The lactose from
Ricotta cheese whey: the effect of pH and concentration on the size and morphology of
lactose crystals. Dairy Sci. & Technol. 93, 477–486.
Tan, R. 2010. Manufacture of Sweetened Condenced Milk and the Significance of Lactose
Therein. In McSweeney, P.L.H., Fox, P.F. (eds): Advanced in Dairy Chemistry Volume 3:
Lactose, Water, Salts and Minor Constituents, 3rd edn, Springer, New York, pp. 36–57.
Whritten, E.O. & Gould, S.P. 1931. Speed of crystallization of lactose, galactose, glucose and
sucrose from pure solution. Ind. Eng. Chem. 23(6), 670–673.

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 Agronomy Research 12(3), 793–800, 2014

An alternative method for meat shear energy

estimation during ageing
A. Põldvere1, L. Lepasalu1, A. Tänavots1, J. Olt1, U. Sannik1,2, A. Sats1,
R. Saar1, R. Martinson3 and V. Poikalainen1,*
1
Estonian University of Life Sciences, Kreutzwaldi 1, EE51014 Tartu, Estonia;
*Correspondence: [email protected]
2
Competence Center of Food and Fermentation Technologies, Akadeemia tee 15,
EE12618 Tallinn, Estonia
3
AS Rakvere Meat Processing Plant, Roodevälja, Sõmeru, EE44207 Lääne-Viru
County, Estonia

Abstract. The aim of this research was to study an alternative method (gravitational impulse
method) for meat shear energy estimation. A falling shear blade (guillotine) with constant
potential energy was used. Shear energy was determined as the difference between the initial
potential energy of the blade and the residual energy measured via the impulse provided by the
shear blade during collision with the force plate on the bottom of the device. The shear energy
measured by the gravitational impulse method was compared to the data received by the texture
analyser TMS PRO based on the Warner-Bratzler method. The meat shear parameters of de-
boned beef striploin samples were measured after 2, 7, 14, 21, 28 and 35 days of ageing at 0–
2°C. The results of both methods (gravitational impulse method and Warner-Bratzler method)
indicated similar trends of shear energy lessening during beef ageing. Based on the preliminary
results, it can be concluded that the gravitational impulse method is suitable for evaluation of
meat texture during ageing as well. The main advantage of this method is its simplicity and
the low price of the device.

Key words: Warner-Bratzler share force test, gravitational impulse method, beef shear energy.

Abbreviation key: MLD – Musculus longissimus dorsi, WB – Warner-Bratzler, GIM –

gravitational impulse method.

INTRODUCTION

Beef texture is tough, thus it must be aged before consumption to improve the
meat tenderness (Field et al., 1971; Jennings et al., 1978; Mottram, 1998). Textural
parameters are very important during meat processing and regarding the sensory
characteristics of meat products. Special texture analyzers are produced for quantitative
evaluation of meat texture, which measure force dynamics at constant cutting speed.
Such texture analyzers based on the Warner-Bratzler (WB) methodology and with
determined characteristics are mainly used in scientific investigations (Wheeler et al.,
1999; Hopkins et al., 2011). Application of these devices in meat industry is not
common due to their operational complexity and considerably high price.

793
 The aim of this work was development and evaluation of an alternative device
based on the gravitational impulse method (GIM) for estimation of beef shear energy
during ageing. Shear energy of aged steak samples was measured by the gravitational
impulse method (GIM) and with the TMS PRO equipment based on the Warner-
Bratzler method (Tommy et al., 2013) during the study.

MATERIALS AND METHODS

An experimental device using the gravitational impulse method (GIM) for

estimation of meat shear energy was developed at the Department of Food Science and
Technology of the Estonian University of Life Sciences. The GIM device contains a
blade, a cutting table and a force plate with a transducer to record impulses (Fig 1). To
carry out measurements, a meat sample is placed on the cutting table between the shear
blade and force plate, where the sample is penetrated by the free fall of the blade. After
shredding the sample, the blade falls onto the force plate and its transducer generates
an impulse, which is recorded by a measurement controller. The weight of the blade is
1.10 kg and its initial height from the force plate is 460 mm. The shear energy can be
determined as a difference between the initial potential energy of the blade and the
residual energy generating an impulse on the force plate. The softer the structure of the
meat, the stronger the strike to the force transducer and vice versa.

Figure 1. The test device to determine the shear energy of meat by using GIM method (1 –
blade, 2 – force transducer, 3 – blade leading rods, 4 – slidebearing, 5 – cutting table).

Physics of the GIM device based on energy balance. If the blade is lifted to a
certain altitude (h), it holds the potential energy (EP).
While dropping the blade from that height, the potential energy (EP) turns into
kinetic energy (EK), the end value of which corresponds to the size of the impulse (i)
generated by the force plate transducer 1 (Fig. 2). This can be estimated by calculation
of the surface area under the graph of force dynamics. The meat sample on cutting
table will consume a part of that energy (shear energy EL), and the force transducer will
record a lower result accordingly.

794
Figure 2. Example of the force dynamics recorded by the force plate transducer of the GIM
device during a measurement cycle.

Experiments were carried out at the Department of Food Science and Technology
(Estonian University of Life Sciences) in 2013. Three deboned M. longissimus dorsi
muscles (MLD) removed from beef carcasses two days after slaughtering were used for
the experiments. Each muscle was cut into six samples with the weight of about 300 g
each and aged in vacuum packages at 0–2°C for 2, 7, 14, 21, 38 and 35 days.
Hanzelková et al. (2011) have suggested that for improvement of beef tenderness it
should be aged for at least 14 days. The total ageing time in our investigation was
chosen to cover all spans reported in the literature on different meat ageing studies
(Koohmaraie et al., 1995; Sañudos et al., 2004; Vieira et al., 2007; Muchenje et al.,
2008). Preparation of probes for analyses was carried out according to the
methodology of WB (Savell et al., 2013). Probes were obtained from the meat samples
by using a hollow drill with the inner diameter of 20 mm. Up to ten probes were drilled
off each of the MLD segments along the muscle fibres (Fig. 3). The shear force
dynamics of both raw and thermally treated samples of aged beef were registered by
the WB and GIM methods. Later, total shear energy was calculated from force
dynamics. Probes were sheared from the middle perpendicularly to the muscle fibres in
six separate trials. For thermal treatment, meat samples were heated in a water bath
until the inner temperature reached 72–76°C.

Figure 3. Samples of meat taken by hollow drill.

795
 The texture analyzer TMS PRO with a 1000 N force transducer and blade
movement speed of 500 mm min-1 was used for the WB method. Total shear energy
consumption during cutting the probe was estimated by calculating the surface area
under the force dynamic curve (Fig. 4). The blades in both (WB and GIM) devices had
similar configurations with a 60° V-shaped incision and thickness of 1.016 mm).

Figure 4. Force dynamics during the Warner-Bratzler test and determination of shear energy by
calculating the surface area under the curve (Luno et al., 1999).

To assess the effect of ageing on the shear energy of samples, a one-way analysis
of variance was performed with the spreadsheet program Excel 2010. For
determination of statistical difference between the devices, student's t-test was used.
The mean differences in shear energy between ageing days were evaluated by the
statistical package R (R Core Team, 2013).

RESULTS AND DISCUSSION

The data of the WB method showed lower shear energy consumption when
cutting raw meat samples compared to thermally treated ones (P < 0.001). The latter
consumed 0.62 J more energy in comparison with raw meat (P = 0.001) (Fig. 5). The
parameters of raw and thermally treated meat samples did not differ significantly
(P = 0.880) when using the GIM method. It can be concluded that the GIM method is
not suitable for determining the differences between boiled and raw meat.
Estimation of shear energy is especially important in the course of aging, because
a decrease in it characterizes the enzymatic processes in the muscle, which have an
effect on the muscle fibres where myofibrillar proteins degrade and meat becomes
softer (Koohmaraie et al., 1995). The shear energy consumption measured by the WB
method in our experiments decreased significantly (P < 0.001) during ageing (Fig. 6a).
The shear energy of thermally treated meat (compared to raw meat) was 0.8 J higher at
the beginning of ageing (day 2), however, at the end of ageing (day 35), the difference
had decreased to 0.35 J.

796
Figure 5. The shear energy consumption of raw and thermally treated meat in comparison with
the WB and the GIM methods.

The shear energy consumed by the GIM method for cutting raw meat samples
decreased even more during ageing (Fig. 6b), with a little bit lower statistical reliability
(P = 0.028).

Figure 6. Changes in shear energy consumption by raw and thermally treated meat during
ageing estimated with WB (a) and GIM (b) devices.

Different studies about the effect of beef ageing have produced somewhat
different results. Roncalés et al. (1995) found that increasing MLD muscle ageing time
changed the texture of the meat – meat tenderised throughout the whole ageing period.
Huff & Parrish (1993) and Sañudos et al. (2004) concluded that 21-day ageing of MLD
muscles decreased the shear force evenly in time. Our experimental data confirmed this
statement. Vieira et al. (2007) and Muchenje et al. (2008) obtained different results –
the conclusion of the first study was that the WB shear force value of MLD muscle
decreased (P < 0.1) between 14 and 28 ageing days, and the second study showed that
MLD muscle already reached the final tenderness by the second day of ageing and the
WB shear force did not reduce significantly later (up to the 21st ageing day).
Accioli et al. (1995) found that the WB shear force values differed significantly
between the 9th and 16th days of ageing, however, there were no significant differences
between the 16th and 23rd day.

797
 In our investigation, the average shear energy of raw and thermally treated meat
samples decreased during ageing by using both (WB and GIM) meat shearing methods
(Tables 1, 2, Figs 7, 8).

Table 1. The effect of ageing on raw meat shear energy consumption by using the WB and
GIM methods (a, b, c, and d – the means within each effect with one letter in common do not
differ significantly, P > 0.05)
Energy consumption, J Ageing, days
2 7 14 21 28 35
WB method 1.05a 0.89ab 0.82abc 0.68bc 0.58c 0.57c
GIM method 3.15a 2.15ab 1.87ab 1.87ab 1.56ab 0.87b

Table 2. The effect of ageing on boiled meat shear energy consumption by using the WB and
GIM methods (a, b, c, and d – the means within each effect with one letter in common do not
differ significantly, P > 0.05)
Energy consumption, J Ageing, days
2 7 14 21 28 35
WB method 1.85a 1.56ab 1.68ab 1.17bc 1.25bc 0.92c
GIM method 2.37a 2.31a 2.07a 1.85a 1.71a 1.16a

The effect of ageing on the shear energy of raw meat is illustrated on Fig. 8. The
statistical significance of this effect was < 0.001 in the case of the WB method and
0.02 in the case of the GIM method. The shear energy values obtained by the GIM
method were larger compared to the WB method throughout the total ageing period.
Shearing energy decreased noticeably faster at the beginning of the ageing period in
comparison with the end of the period. On the second day of ageing, the shear energy
consumption by the GIM was 2.1 J (3.15–1.05 J) higher than that of the WB. At the
end of the ageing period (35 days), this difference had decreased to 0.3 J. These data
indicate the much greater sensitivity of the GIM method in the first week of ageing,
compared to the WB method. Therefore, development of a new express device for
evaluation of the initial stage of meat ageing would be reasonable.

Figure 7. Changes in raw meat shear energy consumption measured by WB and GIM devices
during the beef ageing period.

798
 Thermally treated and raw meat showed similar trends in the changes of shear
energy consumption for both (WB and GIS) methods (Table 2, Fig. 8) with differences
in statistical significance. Shearing of thermally treated meat with the WB method
showed a significant decrease in shear energy consumption during ageing days
(P < 0.001), but the GIM method did not show a significant difference (P = 0.38).

Figure 8. Changes in thermally treated meat shear energy consumption measured by WB and
GIM devices during the ageing period.

CONCLUSIONS

1. The effect of ageing on the structure of meat can be determined both by the
classical (WB) and the gravitational impulse method (GIM).
2. The GIM method is more sensitive in determining the tenderness of aged raw
meat compared to the WB method. The advantage of the gravitational method is its
simplicity.
3. On the base of the GIM method, development of a new texture analyzer for
meat industry would be reasonable.

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 Agronomy Research 12(3), 801–806, 2014

Estimation of Particle Size Distribution in Bovine Colostrum

Whey by Dynamic Light Scattering (DLS) Method
A. Sats*, H. Mootse, S. Pajumägi, A. Pisponen, V. Tatar and V. Poikalainen

Estonian University of Life Sciences, Institute of Veterinary Medicine and Animal

Sciences, Department of Food Science and Technology, Kreutzwaldi 56/5, EE51014
Tartu, Estonia; *Correspondence: [email protected]

Abstract. Colostrum whey consist bioactive compounds in considerable concentration. For

isolation of these compounds the particle size has crucial importance. The aim of this work was
to study possibilities of using dynamic light scattering method – DLS (Malvern Zetasizer Nano
ZS) for colostrum whey particle size distribution estimation. The first and second milking
colostrum samples were skimmed by centrifugal separation and casein of these was
enzymatically coagulated by rennet (chymosin). Obtained whey was diluted (1:200) by distilled
water and filtered (cut-off 0.45 μm) to get probes for estimation of particle size. Particle size
distribution in colostrum whey probes had maximally three peaks and polydispersity indices
from 0.157 to 0.541. Prevailing peak of the distribution was found at size from 144 to 210 nm,
which apparently corresponds to hydrodynamic diameter of immunoglobulin IgG1.

Key words: Milk, Colostrum, Whey, Particle size distribution, immunoglobulin.

INTRODUCTION

Colostrum is a complex fluid rich in nutrients and is also characterised by its high
level of bioactive (e.g. antimicrobial) components, like immunoglobulins (Ig),
especially IgG1, growth factors, especially insulin-like growth factor-1 (IGF-1),
transforming growth factor beta-2 (TGF-b2) and growth hormone (GH) as well as
lactoferrin, lysozyme and lactoperoxidase (table 1). Colostrum whey contains a
significant amount of those bioactive compounds (Pakkanen & Aalto, 1997; Elfstrand
et al., 2002; Marnila & Korhonen, 2002; Kehoe et al., 2007), which can be used in
livestock husbandry, in food and feed supplements, in medical products, etc.
Concentration of proteins and/or bioactive components in bovine colostrum and milk
are shown in Table 1.
The pharmaceutical and biotechnological industries have recently shown interest
in bovine colostrum as a source of growth factors and other specific bioactive
components. Also, a multitude of health products and foods made from various
colostrum fractions have been launched on the market. Fractionation of colostrum
components (immunoglobulins, lactoferrin, glycomacropeptides, etc.) from colostral
whey may result in profitable returns and hence, more research into effects of fractions
or individual components compared to whole colostrum is being undertaken. (Tripathi
& Vashishtha, 2006)

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Table 1. Concentration of proteins and/or bioactive components in bovine colostrum and milk.
Data adapted from: Pakkanen & Aalto 1997; Marnila & Korhonen, 2002; Elfstrand et al., 2002;
Kehoe et al., 2007
Protein and Growth Factors Colostrum Milk
Casein 26 g l-1 29 g l-1
-1
α-lactalbumin 2gl 1.4 g l-1
-1
β-lactoglobulin 8gl 3.3 g l-1
-1
IgG1 48 k–87 g l 0.7 g l-1
-1
IgG2 1.6–2.9 g l 0.05 g l-1
-1
IgM 3.7–6.1 g l 0.05 g l-1
-1
IgA 3.2–6.2 g l 0.1 g l-1
-1
Serum Albumin 1.2 g l 0.4 g l-1
-1
Lactoferrin 1.0–2.0 g l 0.1 g l-1
-1
Lactoperoxydase 30 mg l 20 mg l-1
-1
TGF-b2 20–40 mg l 1–2 μg l-1
-1
IGF-1 0.1–2 mg l 25 μg l-1
-1
Lysozyme 0.1–0.7 mg l 0.1–0.3 mg l-1
Values are depending on postpartum timeline and cow´s individuality. Data represent range.

To extract bioactive compounds from colostrum membrane filtration may be used

(Elfstrand et al., 2002; Venkiteshwaran et al., 2008). Based on the particle size
distribution it is possible to select optimum separation technologies and also evaluate
the protein composition of colostrum whey. Although there are some references
concerning skimmed milk (Beliciu & Moraru, 2009) and whey (Giroux et al., 2009)
particle size distribution (PSD) by dynamic light scattering (DLS) method, there is no
such data about PSD in bovine colostrum available. An important factor in use of
separation technologies and of DLS is the knowledge about possible polydispersity
which is caused by considerable differences in particle size. In application of DLS
method for investigation of particle size it is essential to prevent:
1. the presence of large particles in the sample
2. excessively high concentration of nanoparticles in the sample
3. denaturation or aggregation of particles due to pH or some other co-factor as
temperature, concentration etc. (Dalgleish & Hallett, 1995; Alexander & Dalgleish,
2006; Beliciu & Moraru, 2009).
The aim of current research was to evaluate the suitability of DLS method for the
estimation of particle size distribution and its polydispersity in colostrum whey.

MATERIALS AND METHODS

First and second milking colostrum (1.5 l) was collected and frozen at the EULS
Märja experimental cowshed. Five first and seven second milking samples of
colostrum were under investigation. To ensure efficient separation of fat frozen
colostrum was warmed up to separation temperature 55°C in water bath. Fat separation
process was conducted by separator Armfield FT15 (10,000 rpm). Casein was
precipitated at 35°C using rennet (Formatase 2200 TL) which is derived from a
fermentation process of the fungus Rhizomucor miehei and does not affect whey
proteins. In order to secure large particles (casein-dust, fat, etc.) free and transparent
solution, colostrum whey was diluted (1 : 200) and filtered by syringe filter (cut-off

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0.45 µm). Since whey proteins are more stable close to neutral pH, distilled water (pH
6.8–7.2) was used as diluting solution for reduction of particles concentration in
current research. Isoelectric points of whey proteins are presented in Table 2 (Pouliot
& Gauthier, 2006). Dilution of whey was carried out before filtering to prevent
clogging of the filter. The effect of pH and dilution environment on colostrum whey
particle size distribution was not studied because this needs a detailed investigation.

Table 2. Isoelectric point of whey proteins and growth factors present in milk (Pouliot &
Gauthier, 2006)
Milk protein IgG1 IgG2 BSA βLg αLa GF LF LP
Isoelectrical point, pH 6.5 8 4.7 5 4.7 4.7–9.5 9 10
BSA – serumalbumin, βLg – β-lactoglobulin, αLa – α-lactalbumin, GF – Growth factors, LF –
Lactoferrin, LP – Lactoperoxydase.

For estimation of colostrum whey particle size distribution Malvern Zetasizer

Nano-ZS analyser which is based on dynamic light scattering (DLS) method was used.
This device measures the time dependent fluctuations in the scattering intensity of light
to determine the translational diffusion coefficient, and subsequently the hydrodynamic
diameter by the Stokes-Einstein equation. Each measurement consisted of 3 subsequent
individual runs of which the average result was calculated. Measurements were
conducted at 22.0ºC and the light scattering was detected at 173 degrees. The detection
range of device is from 0.1 nm to 10 μm. The data obtained by the Malvern Zetasizer
Nano-ZS analyser was exported into Microsoft Excel for further analyses.

RESULTS AND DISCUSSION

The particles size distribution (PSD) of diluted and filtered colostrum whey
(DFCW) was found to be in wide range and can therefore be described as multimodal
dispersion (indicates polydispersity). Polydispersity in PSD is reflected clearly by
existence of three peaks (Fig. 1).

Pea
k1

Pea Pea
k3 k2

Figure 1. Example of particles size distribution (PSD) graph with three peaks of diluted and
filtered first milking colostrum whey (cow No. 550).

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 PSD is usually described by overall average or cumulant average (z-average) of
particle diameter (size). In case of polydisperse solutions interpretation of PSD results
using z-average might be insufficient. To describe the polydispersity of the PSD in
DFCW z-average, mean intensity size and area intensity percentage of each peak and
polydispersity index (PDI) were calculated (Table 3).

Table 3. Results of particle size distribution in colostrum whey estimated by DSL method. (PDI
– polydispersity index, MI – mean intensity which corresponds to mode of the peak, AI – area
intensity which corresponds to partial area under the peak of the total distribution)
Cow No/ z-average, Peak 1 Peak 2 Peak 3 Peak 1 Peak 1 Peak 1
milking nm PDI MI, nm MI, nm MI, nm AI, % AI, % AI, %
528/ I 94.5 0.541 177.7 45.1 11.6 80.1 9.1 7.5
624/ I 106.5 0.533 209.6 51.8 12.6 80.3 13.5 6.3
550/ I 122.8 0.458 211.0 61.1 14.0 96.1 3.9 0.0
70/ I 132.2 0.226 167.7 30.9 0.0 99.2 0.8 0.0
439/ I 113.5 0.310 173.9 39.8 16.7 92.8 3.7 3.5
Average of
1st milking 113.9 0.414 188.0 45.7 11.0 89.7 6.20 3.46
St.dev. 13.0 0.13 18.5 10.3 5.75 8.0 4.53 3.11
550/ II 148.3 0.290 200.5 63.4 0.0 97.5 2.5 0.0
92/ II 132.3 0.192 155.6 0.0 0.0 100.0 0.0 0.0
44/ II 105.1 0.311 144.1 13.7 0.0 96.1 3,9 0.0
60/ II 185.5 0.157 199.8 0.0 0.0 100.0 0.0 0.0
17/ II 129.7 0.239 168.9 0.0 0.0 100.0 0.0 0.0
70/ II 138.0 0.271 184.1 50.48 0.0 97.4 2.6 0.0
76/ II 135.0 0.328 196.9 43.86 0.0 90.1 9.9 0.0
Average of 2nd
milking 139.1 0.255 178.6 24.50 0.0 97.3 2.7 0.0
St.dev. 22.51 0.06 21.07 25.29 0.0 3.28 3.28 0.0
Average of
1st and 2nd
milking 126.5 0.335 183.3 35.1 5.50 93.5 4.5 1.7
St.dev. 22.82 0.12 20.57 22.95 6.56 6.86 4.22 2.63

PSD of first milking probes had 2–3 peaks with average mean intensity sizes
188.0, 45.7 and 11.0 nm for peaks 1, 2 and 3 (may also be absent) respectively. Mean
intensity and area intensity varied between 173.9–209.6 nm and 80.1–99.2% for peaks
1 and between 30.9–61.1 nm and 0.8–13.5% for peak 2. PSD of second milking
colostrum probes had 1–2 peaks with average mean intensity sizes of 178.6 nm for
peak 1 and 24.5 nm for peak 2. Mean intensity and area intensity of the dominating
peak 1 varied between 144.1–200.5 nm and 90.1–100.0% respectively.
Polydispersity index (PDI) indicates how homogenous the probes appeared to be,
at least from a light scattering perspective. PDI larger than 0.2 indicates that the simple
cumulant fitting is not a complete representation and that more than a single species are
present (Nobbmann, 2007). It is acknowledged that polydispersity affects the DLS
measurement results. Although it is also clear that the mean intensity results of the
dominating peak are less affected and the role of smallest particles to it may be
neglected. The second milking colostrum probes showed clearly more homogenous
PSD results having only two peaks maximally and lower mean PDI (0.255) compared
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to first milking colostrum probes (mean PDI = 0.414). This can be explained by rapid
postpartum changes in colostrum composition (Elfstrand et al., 2002). According to
published investigations about the content of colostrum and normal milk protein
compounds (Table 1), and the fact that casein and fat fractions were removed from
probes, it can be assumed that dominating peak of PSD in our experiments represented
major colloidal compound of colostrum whey – immunoglobulin IgG1.
Because of wide variance in polydispesity index values (PDI = 0.157–0.541) and
considerably large mean size of dominating particles certain aggregation of those in
DFCW probes can be assumed also. There are several studies about the influence of
various factors including storage conditions and processing variables such as heat, pH
and pressure on stability of bovine IgG (Elfstrand et al., 2002; Godden et al., 2006;
Mcmartin et al., 2006; Indyk et al., 2007). Still, from the point of view of PSD
estimation by DLS-method the question about possible aggregation of IgG remains to
be answered.
Further on more profound investigations about sample treatment impact (dilution
media, pH, heat treatment, refrigerated preservation, etc.) towards PSD of bovine
colostrum whey proteins are required. The certain aggregation degree of whey proteins
(especially IgG1) by regulation of temperature and pH could even enhance efficiency
of filtration or other extraction technology (bigger particles ensure more effective
separation). However, it is essential to preserve the bioactivity of protein during this
kind of treatment.

CONCLUSIONS

This study showed that DLS method is applicable for evaluation of particle size
distribution and polydispersity of the colostrum whey proteins. Based on the cited
knowledge about content of colostrum protein compounds and the fact that casein and
fat fractions were removed, it can be assumed that highest mean intensity of PSD in
our study represents major colloidal compound of colostrum whey – immunoglobulin
IgG1. Further research about the effect of pH and dilution environment in the phase of
sample preparation and possible aggregation of IgG on colostrum whey particle size
estimation by DSL-method is needed.

ACKNOWLEDGEMENTS. We would like to thank the staff and management at the EULS
(Estonian University of Life Sciences) experimental dairy farm for kindly providing bovine
colostrum for this study.

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Elfstrand, L., Lindmark-Mansson, H., Paulsson, M., Nyberg, L. & Akesson, B. 2002.
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Indyk, H.E., Williams, J.W. & Patel, H.A. 2007. Analysis of denaturation of bovine IgG by heat
and high pressure using an optical biosensor. Int. Dairy J. 18, 359–366.
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Marnila, P. & Korhonen, H. 2002. Colostrum. Encyclopedia of Dairy Sciences. 1, 471–477.
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Fetrow, J., Wells, S. & Chester-Jones, H. 2006. Heat treatment of bovine colostrum. I:
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Nobbmann, U., Connah, M., Fish, B., Varley, P., Gee, C., Mulot, S., Chen, J., Zhou, L., Lu, Y.,
Sheng, F., Yi, J. & Harding, S.E. 2007. Dynamic light scattering as a relative tool for
assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol.
Genet. Eng. Rev. 24, 117–28.
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Colostrum. Int. Dairy J. 7, 285–297.
Pouliot, Y. & Gauthier, S.F. 2006. Review. Milk growth factors as health products: Some
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Tripathi, V. & Vashishtha, B. 2006. Bioactive Compounds of Colostrum and Its Application.
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Venkiteshwaran, A., Heider, P., Teysseyre, L. & Belfort, G. 2008. Selective precipitation-
assisted recovery of immunoglobulins from bovine serum using controlled-fouling
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 Agronomy Research 12(3), 807–812, 2014

Use of Delvotest T for Quantitative Estimation of β-lactam

Antibiotic Residues in Waste Milk and for Evaluation of
Thermal Treatment Efficiency – a Methodical Pilot Study
A. Sats*, H. Mootse, L. Lepasalu and V. Poikalainen

Estonian University of Life Sciences, Institute of Veterinary Medicine and Animal

Sciences, Department of Food Sciences, Kreutzwaldi 56/5, EE51014 Tartu, Estonia;
*Correspondence: [email protected]

Abstract. The aim of this work was to study possibilities of using microbiological broad-
spectrum inhibitor test (Delvotest T) for express estimation of antibiotic residues in bovine milk.
For quantitative estimation the waste-milk samples were stepwise diluted (dilution array) by
antibiotic-free milk until negative test result was achieved. Another objective was to evaluate
heat treatment efficiency of waste-milk in order to degrade antibiotic residues in it. Heat
treatment (in water bath) at 90°C was chosen according to references in literature. In order to
study certain drug residue the waste-milk samples were collected at the next milking after
medical treatment. Two different drugs were investigated: Carepen and Norocillin. Preliminary
results indicated that the average concentration of antibiotic residue (active substance of drugs)
exceeded European MRL (Maximum Residue Limit) 4,100 (± 2,408) and 13.0 (± 5.7) times
respectively. The average heat treatment duration at 90°C (until negative test result of
Delvotest T) was 15.0 (± 5.0) and 7.8 (± 1.5) hours respectively.

Key words: Waste-milk, Antibiotic residues, Delvotest T, Heat treatment.

INTRODUCTION

It is widely known that improper use of antibiotics may lead to residues in milk,
especially when withdrawal times are not respected. These residues can be dangerous
for human health. They may cause allergic reactions, antibiotic resistance of pathogens
etc. Antibiotic residues can also create a technological problem for industry production
concerning bacterial fermentation processes in dairy products (Packham et al., 2001).
According to Statistics Estonia (www.stat.ee, 2014), the production of raw milk in
2012 was 721,200 tons. In addition to that, there is an estimated 13–19 thousand tons
of raw milk containing antibiotic residue (AR). This milk is also known as waste-milk
(WM), which is still an unused resource. For the use of that resource inactivation or
degradation of AR is to be carried out. To control these processes estimation of
antibiotic concentration in milk is of crucial importance.
Nowadays there are several receptor-based lateral flow assay tests employed
routinely at the farm level and in the dairy industry because they are fast and simple to
use. Microbiological test kits based on the inhibition of Geobacillus
stearothermophilus are most frequently used for the screening analysis of milk in farms
and dairy industries. According to Reybroeck and Ooghe (2012) the Delvotest T is a

807
new version of Delvotest SP-NT with an improved detection capability. It detects β-
lactam (and many others) antibiotics efficiently in respect of actual Maximum Residue
Limit (MRL) legislation (Commission Regulation (EU) No. 37/2010 and
amendments). The important advantage of microbiological methods is that false
negative samples can be almost excluded. Although it must be acknowledged that false
positive results could occur if other than antibiotic inhibitors are present. Disadvantage
of this method is that threshold level (MRL) of antibiotic in milk could be estimated
only. For concentration studies expensive and sophisticated methods basing on
chromatography should be used.
Thermal treatment is likely one possibility for antibiotics degradation in milk.
There are several studies concerning thermal degradation of β-lactam antibiotics from a
pharmaceutical aspect in aqueous solutions under different storage conditions, at
different pH, etc. Also there are some studies about the effects of temperature on β-
lactam antibiotics in foodstuffs (Moats, 1999; Hanway et al., 2005; Zorraquino et al.,
2008; Roca et al., 2010, 2011, 2012).
Roca et al. (2011) investigated the effect of heating on the stability of β-lactams
(penicillins and cephalosporins) in skim milk (skim milk powder was reconstituted to
10%). The results indicated that the conventional milk processing techniques causes
minor losses in the concentration of these antibiotics and therefore do not prevent these
antimicrobial substances from reaching consumers. Although it was clearly shown that
milk sterilization at 120°C for 20 min had significant impact towards stability of β-
lactams. Zorraquino et al. (2008) used a bioassay based method on the inhibition of
Geobacillus stearothermophilus var. calidolactis. They studied three industrial heat
treatments regimes. Of those classic sterilization (120°C for 20 min) showed
convincingly highest level of heat inactivation of over 65% for penicillins although
penicillin G concentrations in fortified milk samples were used only three times over
MRL. However it can be concluded that duration of thermal treatment is a substantial
factor concerning inactivation of β-lactam antibiotics.
Researches dealing with quantity of antibiotic residue in milk have been
conducted mostly by spiking/fortifying milk (skim milk or commercial UHT milk)
with antibiotic and the analysis of drug concentration performed by sophisticated
laboratory equipment such as liquid chromatography etc. The aim of this work was to
study actually treated cow’s raw milk and to conduct a pilot experiment for
investigation of a simple alternative for estimation of the quantity of (β-lactam)
antibiotic residues in milk by Delvotest T. Additional goal was to scan thermal
degradation of such milk.

MATERIALS AND METHODS

The samples of raw milk for this study were collected from treated cow´s in Põlva
and Hummuli farms and cooled down. Two veterinary drugs Carepen and Norocillin
were under investigation in current study. Both drugs contained the same active
substance (PBP – procaine benzylpenicillin) but concentrations and route of
administration were different (Table 1). Carepen was used intramammary in case of
mastitis and Norocillin intramusculary in case of leg disease. The samples (1.5 l per
cow) were collected during next milking after the treatment (assuming that the milk

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contains maximal possible concentration of antibiotic residue then). Experiments were
conducted on the following day after collection of the samples.

Table 1. Description of drugs studied in current work, EU maximum residue limit (MRL) and
detection limit (DL) of Delvotest T
Drug AS in AS DL of
Route of admin. drug admin. MRL Delvotest T
Drug AS* administration (ml) (mg ml-1) (ml) (µg kg-1) (µg kg-1)
Carepen PBP** Intramammary 10 60 600 4 4
Norocillin PBP** Intramusculary 40 300 1,200 4 4
*AS is active substance
**PBP is procaine benzylpenicillin

Quantitative estimation of antibiotic by Delvotest T

The Delvotest T is a microbiological broadspectrum inhibitor test. To screen milk
on the presence of antimicrobials it uses Geobacillus stearothermophilus var.
calidolactis as a test organism. Microplates were incubated (floating on the water
surface) in a waterbath at 64°C. After 3 h incubation, the color change of the pH
indicator in the agar of the wells (yellow–negative, purple–positive) was recorded. For
quantitative AR (antibiotic residue) estimation the waste-milk was diluted by
antibiotic-free milk in order to achieve an array of different concentrations (dilution
factors) of WM (Fig. 1 a). The 2.5% fat content commercial milk used as dilution
environment was purchased from ordinary public store and tested by Delvotest T (no
positive test results were found in current study).

Table 2. Example of dilution arrays used for quantitive estimation of antibiotic residue (AR).
Bold represents AR below MRL (Maximum Residue Limit)
Veterinary Drug Carepen
1st dilution array 500 1,000 3,000 5,000 7,000 9,000
Delvotest T results pos pos neg neg neg neg
2nd dilution array to refine
results 1,000* 1,500 2,000 2,500 3,000*
Delvotest T results pos neg neg neg neg
Veterinary Drug Norocillin
1st dilution array 10 30 50 100 150 200
Delvotest T results pos neg neg neg neg neg
2nd dilution array to refine
results 10* 15 20 25 30*
Delvotest T results pos neg neg neg neg
* – dilution was made as control to ensure reliability of results

The dilution factor at which negative test result occurred in 1st dilution array was
refined by 2nd dilution array and considered as detection limit (DL) of Delvotest T
witch conveniently equals to MRL. Example of dilution arrays for quantitive
estimation of antibiotic residue (AR) in case of Norocillin and Carepen are shown in
Table 2. Negative results were taken into account only in case of clear colour change.

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 Heat treatment
For the heat treatment 50 ml sealed sample container was placed in thermostatic
bath at 90°C. The 100 µl probes for detection of antibiotic residue (AR) were taken
from the container hourly. Heat treatment was considered sufficient, and degradation
of AR completed at a probe with negative test result (Fig. 1b). Objective was to
establish the thermal degradation duration (TDD) at which negative test occurred
(AR < MRL).
Roca et al (2011) found that the half-life of benzylpenicillin (PBP) at 90°C is 52
min. In current study this value was taken for bases and the predicted duration of
thermal degradation (Table 3) was calculated by equations 1, 2, where PT is predicted
time, t1/2 is half-life of benzylpenicillin at 90°C (according to Roca et al (2011), 52
min), n1/2 is count of t1/2 (half-life of PBP), AR is estimated antibiotic residue and MRL
is maximum residue limit of AR.

PT= n1/2t1/2 (1)

n1/2= (logAR-logMRL)/log2 (2)

Figure 1. Delvotest T results: a) estimation of concentration (dilution factors: 500–2,000) b)

influence of thermal treatment duration (4–11 hours). Arrow points to the lowest dilution factor
(1,500) and thermal treatment duration (10 hours), at which negative test occurred
(AR < MRL). Originally colour photos are represented here in grayscale mode.

RESULTS AND DISCUSSION

Table 3 presents estimated quantity of antibiotic residue (AR) in raw waste-milk

and thermal degradation time of veterinary drugs (Carepen and Norocillin) AR. The
difference between average AR concentrations of two drugs was remarkable
(315 times). Significantly higher AR concentrations of Carepen can be explained by
route of administration (intramammary or intramusculary). In addition to that the
composition of drug (excipient) might have some impact to the results. Suggested 1st
dilution arrays for quantity estimation of Norocillin and Carepen AR are presented in
Table 2.
High standard deviation values concerning negative Delvotest T results (DF neg
in Table 3), computational concentrations of AR (CC of AR) and thermal degradation
duration (TDD) can be explained by differences in waste-milk quantity (WMQ) of
particular milking. Negative correlation between AR concentration and WMQ also
confirms that. High standard deviation value of TDD compared to PT of TDD can be

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explained by cow´s individuality (milk production level, composition differences, etc.)
which influences quantity of AR.
Initially just slight difference (in view of AR concentration) concerning the
duration of thermal degradation of different WM samples as well as different drugs can
be simply explained by exponential character of half-life degradation.
Remarkable correlation between computational concentration of AR and thermal
degradation time supports the assumption that quantitative estimation of AR could be
carried out by using Delvotest T on an array of diluted milk samples.

Table 3. Results of quantitative estimation and duration of thermal degradation of antibiotic

residue in waste-milk
CC of AR, TDD till PT of TDD*,
Cow No Drug DF neg µg kg-1 neg, h h WMQ, kg
4,859 Carepen 1,500 6,000 10 9.2 16
5,364 Carepen 7,500 30,000 23 11.2 13
5,894 Carepen 3,500 14,000 19 10.8 12
6,805 Carepen 5,500 22,000 15 11.3 11
6,737 Carepen 2,500 10,000 14 9.8 12
Average 4,100 16,400 15.0 10.24 12.8
St dev 2,408 9,633 5.0 0.79 1.92
Concentration correlation 0.82 0.84 -0.45
439 Norocillin 5 20 6 2.0 13
123 Norocillin 15 60 7 3.4 6
6,790 Norocillin 20 80 10 3.8 9
5,979 Norocillin 15 60 8 3.4 10
5,286 Norocillin 10 40 8 2.9 16
Average 13.0 52.0 7.8 3.1 10.8
St dev 5.70 22.80 1.48 0.69 3.83
Concentration correlation 0.83 0.80 -0.65
*
– Based on the half-life of benzylpenicillin at 90°C (Roca et al., 2011)
DF neg – Dilution factors of negative Delvotest T results
CC of AR – Computational concentration (CC) of AR based on DL=MRL (4 µg kg-1)
TDD till neg – Thermal treatment (90ᵒC) duration till neg. test result occurred
PT of TDD – Predicted time of thermal degradation duration (TDD)
WMQ – waste-milk quantity

Table 3 also presents predicted time (PT) of thermal degradation duration (TDD)
based on the half-life of benzylpenicillin at 90°C described by Roca`s research group
and estimated AR concentration results from our study. Compared to PT of TDD, the
TDD of Carepen and Norocillin AR was significantly longer. This can be explained by
the fact that the results were expressed only in case of clear colour change. So
considering that, the actual concentration of AR might have been lower and/or the
duration of AR degradation therefore shorter. It also might be caused by the
composition of drug (excipient), route of administration, cow´s individuality, etc.
Further studies are needed to work out determined procedures for implementing
proposed method in practice and for evaluation of it.

811
 CONCLUSIONS

For quantitative estimation of antibiotic residue in milk expensive and

sophisticated laboratory equipment such as liquid chromatography etc. is normally
used. The current pilot study demonstrates simple potential alternative for express
quantitative estimation of antibiotic residues in bovine milk by applying Delvotest T to
an array of diluted milk samples. This method is probably applicable in case of
receptor-based lateral flow assay tests (testkits) also, which are employed routinely at
the farm level and in the dairy industry because they are fast and simple to use.
Preliminary conclusions of current study are: 1) proposed express method for
quantitative estimation of AR in waste-milk and in heat treated waste-milk by
Delvotest T is applicable; 2) the heat treatment study confirmed that procaine
benzylpenicillin (PBP) in waste-milk can be degraded by long-term thermal treatment
at 90°C.

ACKNOWLEDGEMENT. This work supports the investigation project ‘Technological

solutions for utilization of waste-milk and possibilities for reducing of its amount’. We are
thankful to partners of the project and staffs of the Põlva and Hummuli dairy farms.

REFERENCES

European Council. 2010. Council Regulation 37/2010 of the European Communities.

Pharmacologically active substances and their classification regarding maximum residue
limits in foodstuffs of animal origin. Off. J. Eur. Union. L15, 1b–72.
Hanway, W.H., Hansen, A.P., Anderson, K.L., Lyman, R.L. & Rushing, J.E. 2005. Inactivation
of Penicillin G in Milk Using Hydrogen Peroxide, J. Dairy Sci. 88, 466–469.
Moats, W.A. (1999). The effect of processing on veterinary residues in foods. Advances in
experimental medicine and biology Volume: 459, 233–241.
Packham, W., Broome, M.C., Limsowtin, G.K.Y. & Roginski. H. 2001. Limitations of standard
antibiotic screening assays when applied to milk for cheesemaking. Aust. J. of Dairy
Technol. 56(1), 15–18.
Reybroeck, W. & Ooghe, S. 2012. Validation report of the Delvotest T. January 6th 2012, 1–13.
Roca, M., Castillo, M., Marti, P., Althaus, R.L. & Molina, M.P. 2010, Effect of Heating on the
Stability of Quinolones in Milk J. Agric. Food Chem. 58, 5427–5431.
Roca, M., Villegas, L., Kortabitarte, M.L., Althaus, R.L. & Molina, M.P. 2011. Effect of heat
treatments on stability of β-lactams in milk, J. Dairy Sci. 94, 1155–1164.
Roca, M., Althaus, R.L. & Molina, M.P. 2012. Thermodynamic analysis of the thermal stability
of sulphonamides in milk using liquid chromatography tandem mass spectrometry
detection. Food Chem. 136(2), 376–383.
Statistics Estonia. http://pub.stat.ee/px-web.2001/Database/Majandus/13Pellumajandus/
06Pellumajandussaaduste_tootmine/02Loomakasvatussaaduste_tootmine/02Loomakasvat
ussaaduste_tootmine.asp. Accessed 10.1.2014.
Zorraquino, M.A., Roca, M., Fernández, N., Molina, M.P. & Althaus, R.L. 2008. Heat
inactivation of beta-lactam antibiotics in milk. J. Food Prot. 71, 1193–1198.

812
 Agronomy Research 12(3), 813–820, 2014

Effect of ultrasonic treatment on metabolic activity of

Propionibacterium shermanii, cultivated in nutrient medium
based on milk whey
E. Suchkova, B. Shershenkov* and D. Baranenko

Institute of Refrigeration and Biotechnologies, ITMO University, Lomonosova 9,

Saint-Petersburg, Russia; *Correspondence: [email protected]

Abstract. Utilization of milk whey still remains one of the most serious problems of the
modern world dairy industry. Because of high biological value of whey it can be used as
nutrient media in techniques of direct microbiological synthesis of complex high-molecular
substances, for example the B12 vitamin, which participate in various biocatalytic reactions in
organism providing normal functioning of the brain, nervous and hematogenic systems.
The main industrial producer of B12 vitamin is Propionibacterium shermanii species, which can
use lactose as main carbon source and can develop directly in milk whey. However, common
ways of vitamin B12 microbiological production can’t be applied on the food plants and there
must be used alternative safe methods, such as ultrasonic treatment of the cell culture, which
can carry out the direct modulation of metabolic activity of bacteria for increasing of a yield of
B12 vitamin.
For the definition of ultrasonic processing influence on metabolism of Propionibacterium
freudenreichii ssp. shermanii industrial strain it was cultivated in the nutrient media based on
milk whey and treated by low intensity ultrasound at a frequency of 20 kHz within 10 and 20
min each 24 h. Received results allow to suggest that ultrasonic modulation of propionic
bacteria metabolic activity can be used for an intensification of B 12 vitamin biotechnological
obtaining and manufacturing of fermented food products based on milk whey and enriched with
B12 vitamin.

Key words: ultrasound, Propionibacterium shermanii, B12, cobalamine, milk whey.

INTRODUCTION

The one of the most important vitamins for the human health is the B12 vitamin
which involves several kinds of specific forms of cobalt-containing compounds, known
as cobalamins. The main industrial form of vitamin is cyanocobalamin due to its high
stability. Cobalamines serve as cofactors for various important enzymes participating
in maintenance of normal DNA synthesis, regeneration of methionine and other
biocatalytic reactions providing normal functioning of the brain and nervous system,
blood formation, etc. Deficiency of these compounds in diet can lead to different
neurological diseases, vascular damage, thrombosis and even to serious form of anemia
called Addison’s pernicious anemia (Green & Miller, 2010).
Naturally they are synthesized only by microorganisms and contain only in food
of an animal origin. The lack of these compounds leading to various diseases often can
be observed in the diet through various gastric disorders, such as chronic gastritis that
813
lead to malabsorption of vitamin, or inadequate dietary intake, for example in
vegetarian diet. Deficiency of B12 can be corrected by introduction special food
products enriched with high amounts of vitamin which can provide adequate B12 intake
even through a passive diffusion mechanism that allows assimilate only of 1–2% of
vitamin containing in food. That type of food products also can be used as medical
food for people who can’t absorb vitamin B12 via the normal physiologic route. The
recommended dietary allowance of B12 for males and females is 2.4 μg per day and
higher, so such products should contain at least 240 μg kg-1 of vitamin.
However nowadays B12 vitamin is one of the most expensive vitamins and it is
used mostly as medical agent for injections and due to its high cost it can’t be used as
simple food additive.
Vitamin B12 is produced exclusively by biosynthetic way. The main industrial
producer of B12 vitamin is Propionibacterium freudenreichii ssp. shermanii which was
recently used mainly as a ripening culture in Swiss-type cheeses. These
microorganisms synthesize exclusively biologically active forms of vitamin and able to
use a variety of carbon substrates, including lactose and lactic acid, that allows them to
grow in milk whey (Falentin et al., 2010).
Milk whey contains from 4.2 to 7.5% of solids, most of which are disaccharide
lactose (3.5–5.2%), a large number of organic and inorganic compounds containing
essential chemical elements such as potassium, calcium, magnesium, sodium and the
phosphorus in natural proportions, water- and fat-soluble vitamins from milk such as
A, B1, B6, С, E, and nitrogenous substances like whey proteins and free amino acids.
All that allow considering whey as a valuable nutrient media for propionic
bacteria. The highest vitamin B12 yield for Propionibacterium freudenreichii cultivated
on milk whey was 15 μg mL-1 (Berry & Bullerman, 1966) and that shows high
application potential for enriching fermented products with natural vitamins.
Profitable production of various functional products with the use of milk whey
enriched with B12 vitamin based on propionic fermentation can be arranged
economically on the dairy factories. This can partially solve the problem of profitable
utilization of milk whey which still remains one of the most serious problems of the
modern world dairy industry.
Traditional ways of vitamin B12 microbiological production demand long-time
multistage process of synthesis of the precursor and the vitamin itself, high-
temperature processing for vitamin releasing from the cells, and also using of
dangerous substances, as cyanides and the phenol for stabilization and extraction of
vitamin, application of which at the food factories is inadmissible due to their high
toxicity. Economically effective alternative to the common techniques of vitamin
obtaining and stabilization can be ultrasonic treatment of the Propionibacterium cell
culture developing directly in food product for the purpose of B12 synthesis
intensification and soft destruction of bacteria cells without product overheating.
Low-frequency ultrasound was proved itself as a powerful and economic tool for
intensification of biochemical reactions by means of increasing an exchange between
cells and a nutrient medium (Christi, 2003), reducing duration of enzymatic hydrolysis
and maintaining activity of some enzymes during cavitation (Rokhina et al., 2009).
For example ultrasound treatment with 20 kHz frequency which is widely used in
food industry ultrasonic equipment increases growth rate and overall biomass of the
cyanobacteriae cells (Francko et al., 1994). Treatment on this frequency is also used for
814
stimulation of anaerobic digestion processes in wastewater treatment plants
(Dewil et al., 2006). Therefore low intensity ultrasound treatment of liquid medium
which cause acceleration and initiation of chemical reactions in the sonicated medium
can change the metabolic activity of bacteria cells (Akopyan & Ershov, 2005). By
means of ultrasonic treatment it is possible to carry out the direct modulation of
metabolic activity of bacteria for B12 vitamin yield increase without increasing in
duration of production process using no special devices (Suchkova & Shershenkov,
2013).

MATERIALS AND METHODS

For the definition of ultrasonic processing influence on industrial strain of

Propionibacterium freudenreichii ssp. shermanii metabolism bacteria cells were
cultivated during 96 h in the nutrient media based on milk whey with concentration of
lactose of 5% and adding of 0.002% CoCl2·6H2O and 0.001% MgSO4 according to
Zalashko (1990). The strain used for vitamin B12 production was industrial strain
Propionibacterium freudenreichii I-63, which is a gram-positive, non-spore forming,
rod-shape, and aerotolerant anaerobic bacterium producing vitamin B12 intracellularly.
The major problem in vitamin B12 production using Propionibacterium is the
growth inhibition of the cell due to the accumulation of inhibitory metabolites such as
propionic acid and acetic acid. Control of pH at approximately 7.0 is critical for
reaching high vitamin B12 yields (Ye et al., 1996). Therefore рН was kept at level
about 6.9 by means of periodic neutralization of the nutrient media during the
cultivation by calcium carbonate.
Cell culture was treated by low intensity ultrasound at operating frequency of
typical ultrasonic homogenizer 20 kHz within 10 and 20 min each 24 h.
First several cultivation cycles were performed in anaerobic conditions during all
96 h. Since oxygen is required for the biosynthesis of the lower ligand of active form
of vitamin B12, 5, 6-dimethylbenzimidazole, main product of this cultivation was
vitamin analogs called corrinoids, which lack of nucleotide.
Next cultivation cycles were performed with continuous aeration of fermented
media by means of sparging by sterile air during the second stage of cultivation after
48 h of anaerobic cultivation.
Measurement of titratable acidity of milk whey by 0.1N NaOH solution for
definition of amount of organic acids synthesized by bacteria as primary metabolites
was performed for research of metabolic activity of bacteria during the cultivation.
Results are presented and compared in degrees of Turner (Table 1). Comparative
photometric definition of corrinoid compounds concentration in the milk whey after
cultivation was also made.

RESULTS AND DISCUSSION

It was found out that increase in duration of low intensity ultrasound treatment
cause about 1–4% increase of total titratable acidity in all 20 minute treated samples
and it depends upon duration of ultrasound treatment.

815
Table 1. Average total titratable acidity of fermented milk whey probes in various treatment
conditions
Cultivation Duration of treatment, min
time, hours 0 10 20
ºТ % ºТ % ºТ %
0 3.1 100.0 3.1 100.0 3.1 100.0
24 40.6 100.0 41.6 102.5 39.5 97.3
48 74.9 100.0 74.9 100.0 74.9 100.0
72 108.9 100.0 111.0 101.9 113.0 103.8
96 140.7 100.0 140.4 99.8 146.4 104.0

That points on acceleration of fermentation processes and bacteria activity

increase. Also changes in morphology of the bacteria cells subjected to ultrasound
treatment and reduction of their sizes in comparison with untreated culture were
observed. Average linear size of cells without treatment was 1.0–1.5 μm; linear size of
treated cells in both experiments was 0.5–0.8 μm (Fig. 1).

Figure 1. Microscope slides of fermented whey samples after anaerobic cultivation (untreated
culture; 10 min treatment; 20 min treatment respectively).

Photometric analysis of fermented media was made after completion of

fermentation process, thermal destruction of cells and filtration of the media. Received
results are the same both for anaerobic cultivation and mixed anaerobic-aerobic
cultivation cycles (Figs 2, 3).
During cultivation propionic bacteria use Co (II) from solution and turn it into
corrin ring of cobalamines. Formed corrinoids have light absorption maximum which
varies from 351 to 375 nm for various forms (Dolgov et al., 2004). Due to this fact it is
possible to estimate their concentration in the fermented media and reveal the tendency
of ultrasound impact on bacteria metabolic activity.

816
Figure 2. Absorption spectrum of fermented whey samples received after 96 h of anaerobic
cultivation (1 is for untreated culture; 2 is for 10 min treatment; 3 is for 20 min treatment).

Figure 3. Absorption spectrum of fermented whey samples after 48 h of anaerobic and 48 h of

aerobic cultivation (1 is for untreated culture; 2 is for 10 min treatment; 3 is for 20 min
treatment).

817
 For comprehensive analysis of corrinoid concentration graphical method based on
additivity principle was used. For that purpose was measured difference between levels
of 351–375 nm optical density peaks of averaged absorption spectra, received by
comparing spectra of multiple probes, and overall optical density of probe averaged
spectra without peak (Fig. 4). Results are presented and compared in relative
photometrical units (Table 2).

Figure 4. Graphical method for comparation of corrinoid concentration on fragments of

absorption spectra of fermented whey samples.

Table 2. Comprehensive analysis of corrinoid concentration in fermented milk whey probes in

various treatment conditions
With aeration Without aeration
Treatment duration, min
0 10 20 0 10 20
Absorption maximum, nm 361 359 362 362 360 361
Overall optical density 1.717 2.032 2.211 1.601 1.786 1.860
Optical density without peak 1.585 1.817 1.967 1.455 1.630 1.697
Optical density difference 0.132 0.215 0.244 0.146 0.156 0.163
Content of corrinoids,
13.2 21.5 24.4 14.6 15.6 16.3
photometrical units (D·100)
Relative content of corrinoids, % 100.0 162.9 184.8 100.0 106.8 111.6

Results show that formation of corrinoids is minimal without treatment and

increase with increasing duration of ultrasound treatment from 10 to 20 min
respectively.
Overall affect of the low-intensity ultrasound on metabolism of
Propionibacterium can be noticed by comparing relations of left and right sides of
different spectra and needs further investigation.

818
 CONCLUSION

Received results show that ultrasonic modulation of a metabolism of propionic

bacteria is the perspective direction for an intensification of B12 vitamin and its analogs
obtaining and it can be also used in production of the various fermented food products
enriched with B12 vitamin.
Received enriched milk whey can be used as an additive for production of the
various dairy products enriched with the vitamin. Also there is an interest in the
probiotic activity of Propionibacterium freudenreichii which can produce bifidogenic
compound 1,4-dihydroxy-2-naphthoic acid and stimulates growth of bifidobacteria
(Hugenshmidt et al., 2010). This effect can be also used in various fermented dairy
products.
Enriched whey can be used for production of other enriched non-dairy like bakery
products and confectionery for the purpose of ensuring the intake of daily requirement
of B12 vitamin.

ACKNOWLEDGEMENTS. This work was partially financially supported by Government of

Russian Federation, Grant 074-U01.

REFERENCES

Akopyan, B.V. & Ershov, Yu. A. 2005. Basics of ultrasound interaction with biological
objects. MGTU, Moscow, 224 pp. (in Russian).
Berry, E.C. & Bullerman, L.B. 1966. Use of cheese whey for vitamin B12 production. II.
Cobalt precursor and aeration levels. Appl. Microbiol. 3, 356–357.
Dewil, R., Baeyens, J. & Goutvrind R. 2006. Ultrasonic treatment of waste activated sludge.
Environ. Prog. 25, 121–128.
Dolgov, V.V., Ovanesov, E.N. & Schetnikovich, K.A. 2004. Photometric analysis in laboratory
practice. Russian medical academy of post-graduate education, Moscow, 142 pp.
(in Russian).
Falentin, H., Deutsch, S.-M., Jan, G., Loux, V., Thierry, A., Parayre, S., Millard, M.-B.,
Dherbecourt, J., Cousin, F., Jardin, J., Siguier, P., Couloux, A., Barbe, V., Vacherie, B.,
Winker, P., Gibrat, J.-F., Gaillardin, C. & Lortal, S. 2010. The Complete Genome of
Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and
Probiotic Applications. PLoS ONE 5, 1–12.
Francko, D.A., Al-Hamdani, S. & Joo, G.-J. 1994. Enhancement of nitrogen fixation in
Anabaena flos-aquae (Cyanobacteria) via low-dose ultrasonic treatment. J. Appl. Phycol.
6, 455–458.
Green, R. & Miller, J. 2010. Handbook of Vitamins, 4th edition. 13. Vitamin B12. CRC Press,
413–458, 608 pp.
Hugenschmidt, S., Miescher-Schwenninger, S., Gnehm, N. & Lacroix, C. 2010. Screening of a
natural biodiversity of lactic and propionic acid bacteria for folate and vitamin B12
production in supplemented whey permeate. Int. Dairy J. 20, 852–857.
Rokhina, E.V., Lens, P. & Virkutyte, J. 2009. Low-frequency ultrasound in biotechnology: state
of the art. Trend Biotech.27, 298–306.
Suchkova, E.P. & Shershenkov, B.S. 2013. Ultrasound modulation of Propionibacterium
freudenreichii subsp. shermanii metabolic activity for production enriched by B12 vitamin

819
 food products. ESJ ‘The processes and devices of food industry’, 2. (in Russian, English
abstr.) http://processes.open-mechanics.com/articles/844.pdf
Ye, K., Shijo, M., Jin, S. & Shimizu, K. 1996. Efficient production of vitamin B12 from
propionic acid bacteria under periodic variation of dissolved oxygen concentration.
J. Ferment. Bioeng. 82, 484–491.
Zalashko, M.V. 1990. Biotechnology of milk whey utilisation. Agropromizdat, Moscow, 192 pp.
(in Russian).

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 Agronomy Research 12(3), 821–824, 2014

Comparative study on anti-oxidant activity of garlic grown in

different regions
R. Vokk*, E. Tedersoo, T. Lõugas, K. Valgma and J. Rosend

Department of Food Processing, Tallinn University of Technology,

Ehitajate tee 5, Tallinn, EE19086, Estonia; *Correspondence: [email protected]

Abstract. Currently reliance on natural products such as garlic and other vegetables is gaining
popularity to combat various physiological threats including oxidative stress, cardiovascular
complexities, cancer insurgence, and immune dysfunction. Garlic (Allium sativum L.) holds a
unique position in history, belongs as a natural ingredient to different cuisines and was
recognized for its therapeutic potential. Extensive research work has been carried out on the
health promoting properties of garlic, often referred to its sulfur containing metabolites i.e.
allicin and its derivatives. The aim of the present study was to compare garlic originated from
different parts of the world (Chinese, Spanish, Lithuanian etc) on the basis of their anti-oxidant
activity to evaluate their potential for different applications. As a result distinctive differences
have been found in anti-oxidant activity of different garlic varieties. Garlic grown in Estonia
possessed the highest antioxidant activity among the raw varieties. Black garlic has remarkable
higher anti-oxidant content in comparison with other garlic samples.

Key words: anti-oxidant content, garlic, black garlic.

INTRODUCTION

Garlic (Allium sativum L.) is one of the bulbous rooty vegetables, which has been
used for many centuries both as a flavouring agent and as a home remedy for the
treatment of different health disorders. This vegetable is still mostly valued in different
parts of the world due to its content of various flavouring components mostly presented
by sulfur-containing chemical compounds. It has been shown that garlic aroma
character is determined by the presence of di-2-propenyl disulfide (Gorinstein et al.,
2005). Fortunately, garlic is also amongst a number of herbs having strong anti-
microbial properties (Vokk, 1996). In recent decades application of garlic in
phytotherapy has been widely investigated (Omar et al., 2007; Gorinstein et al., 2010).
It is well known that aging garlic at a constant temperature and humidity without
adding any additives to a raw material, increases polyphenol compound presence.
Thus, aged black garlic should exert stronger anti-oxidant effect compared to raw
garlic, without decreasing the original effectiveness of the garlic. Gorinstein et al
(2005) have shown that the bioactive compounds, electrophoretic patterns, and anti-
oxidant potential of fresh Polish, Ukrainian and Israeli garlic samples are comparable
but garlic samples subjected to heating at 100°C during 20 minutes preserve their
bioactive compounds and anti-oxidant potential.

821
 Even for the recommendations given by the physicians who wish to offer a patient
herbal option for anti-microbial and/or anti-oxidative agents having several options to
choose from.

MATERIALS AND METHODS

Physico-chemical and anti-oxidative measurements were performed to study

garlic varieties grown in different climate conditions to compare their anti-oxidant
activity.
The garlic samples were harvested in 2012–2013, the garlic bulbs were clean,
white or greyish and well cured, the cloves were firm to the touch. The Estonian garlic
was purchased from the producer Kadastiku in South Estonia. Other samples were
purchased on the local market.
Black garlic (in the present work was used processed Spain garlic harvested in
2012) was also included into the study and the material was produced in 2013 by FS
Baltic. Black garlic was produced by method of aging garlic, which in an embodiment
involved the following procedures: classifying the garlic according to its condition and
pre-treating it to achieve clean appearance; sealing the garlic in a vinyl pack by 10 kg
and storing it in a tray; putting said tray in an aging device for a black garlic, applying
steam and heat for 1–3 h while maintaining a temperature of 80–100°C inside the
aging device, and treating by steam under a high temperature and high humidity
condition; main aging by applying steam and heat to the treated garlic for 198 h while
maintaining a temperature of 72–78°C inside the aging device; after-aging by applying
steam and heat to the garlic, which had undergone main aging process, for 35 h while
maintaining 60–69°C temperature inside the aging device; drying the after-aged garlic
for 51 h while maintaining a temperature of 50–58°C inside the aging device; and low
temperature after-aging by cooling obtained dried garlic at low temperature for 168 h
while maintaining 0–5°C temperature inside the aging device. Provided here the
method of aging black garlic characterized by drawing optimum time and condition
without taking the storage condition, size, etc of the garlic (US Patent No
US20110129580).
Sample preparation was performed as follows: 20 g of garlic was pounded with a
pestle in a mortar until no garlic structure was recognizable any more (during a
minute). To 0.5 g of disrupted material 20 ml of water (for the water soluble anti-
oxidant measurements or 20 ml of methanol for lipid soluble anti-oxidant
measurements) was added, shaked vigorously on Fortex and centrifuged 2 minutes at
13,200 rpm. 1 ml of supernatant was taken from both samples to perform anti-oxidant
measurements. All the measurements of anti-oxidant activities were perfomed with the
aim on Photochem, Analytik Jena. Preparation of the calibration curve was performed
as described in the standard kit protocol ACL and ACW using volumes as displayed in
the schemes in Manual (Photochem, Manual).
According to the principle of the ACW and ACL measurement method the free
radicals were produced by optical excitation of a photosensitizer substance. Those
radicals were partially eliminated from the sample by reaction with the anti-oxidants
presented in the sample. In the measuring cell the remaining radicals caused the
detector substance to luminescence and thereby the anti-oxidant capacity of the sample
is determined. The anti-oxidative capacity of the sample was quantified by comparison
822
with the standard (constructed calibration curve with ascorbic acid or Trolox).

At least four parallels were performed. All samples needed dilution and the
dilution rate was taken into account for final calculations. The results were given as
ascorbic acid equivalent for water soluble anti-oxidants and Trolox equivalent for lipid
soluble anti-oxidants in all investigated samples.

RESULTS AND DISCUSSION

In all garlic samples the moisture content has been determined by Halogen
Moisture Analyzer and the results are given in Tabel 1.

Tabel 1. Moisture content of different garlic varieteis

Origin and year of harvesting Moisture content, %
Estonian 2012 57.8
Chinese 2012 67.61
Spanish2012 37.66
Lithuanian 2013 63.17
Polish 2013 59.48
Chinese 2012 in a set 54.35
Black garlic 2013 45.37

Moisture content of different garlic samples varies significantly in the range from
37.66 to 67.61%. Moisture content was taken into account for further calculations of
anti-oxidant acitivity.
In Tabel 2 all the calculations concerning the water soluble anti-oxidants and lipid
soluble anti-oxidants are given per 1 g of dry weight of the product and also per 1 g of
the product itself.

Tabel 2. Anti-oxidant activity of garlic samples (ACW – water soluble anti-oxidants and
ACL – lipid soluble anti-oxidants)
Sample origin ACW expressed as ascorbic ACL expressed as Trolox
acid equivalent
µmol 1g dry µmol 1g-1
-1
µmol 1g-1 dry µmol 1g-1
weight product weight product
Estonian 2012 524.07 221.16 3.35 1.41
Chinese 2012 85.02 27.54 4.32 1.40
Spanish2012 14.05 8.76 2.40 1.50
Lithuanian2013 30.46 11.22 8.34 3.07
Polish 2013 16.94 6.86 11.32 4.59
Chinese 2012, in a set 84.25 38.46 6.51 2.97
Black garlic 2013 1,082.92 591.60 7.96 4.35

As a result distinctive differences have been found in anti-oxidant activity of

different garlic varieties. Over 90 times higher anti-oxidant content in Spain garlic has
been observed after aging process. Lipid soluble anti-oxidant content had less

823
remarkable differences. Would be interesting to produce black garlic from Estonian
garlic expressing very high anti-oxidant content in a raw material. Over the past
decades the application of herbs and other natural products has gained popularity, mint,
garlic, pumpkin and beetroot amongst them. Garlic is known as a potent cardiovascular
agent (Capasso, 2013). Therefore, garlic is a proper supplement in anti-atherosclerotic
diets (Gorinstein et al., 2005). Latest investigations have given information about a
possible role of black garlic in treatment of type 2 diabetes mellitus (Lee, 2009).

CONCLUSIONS

Garlic has been studied as one of the vegetables possessing comparatively high
anti-oxidative activity. However the garlic bioactive value differs depending on the
region, as it has been shown in the present investigation. It should be interesting to
estimate different cultivars grown in the same region under the similar climatic
conditions. Black garlic has very high potential as the source of anti-oxidants and it
could be explained by changes in the chemical composition during the aging process.

REFERENCES

Capasso, A. 2013. Anti-oxidant action and therapeutic efficacy of Allium sativum L. Molecules,
18(1), 690–700.
Gorinstein, S., Drziewiecki, J., Leontowicz, H., Leontowitcz, M., Naiman, K., Jastrzebski, Z.,
Zachwieja, Z., Barton, H., Shtabsky, B., Katrich, E. & Trakhtenberg, S. 2005. Comparison
of the bioactive compounds and anti-oxidants potentials of fresh and cooked Polish,
Ukrainian, and Israeli garlic. J. Agric. Food. Chem., 53, 2726–2732.
Gorinstein, S., Leontowicz, H., Leontowicz, M., Jastrzebski, Z., Najman, K., Tashma, Z.,
Katrich, E., Heo, B., Cho, J., Park, Y. & Trakhtenberg, S. 2010. The influence of raw and
processed garlic and onions on plasma classical and non-classical atherosclerosis indices:
investigations in vitro and in vivo. Phytother Res, 24, 706–714.
Lee, Y., Gweon, O., Seo, Y., Im, J., Kang, M., Kim, M. & Kim, J. 2009. Anti-oxidant effect of
garlic and aged black garlic in animal model of type 2 diabetes mellitus. Nutr. Res. Pract.,
3(2), 156–161.
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US Patent US201110129, 2009, published in 2011.

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 Agronomy Research 12(3), 825–834, 2014

Liposomal beta-carotene as a functional additive in dairy

products
L. Zabodalova1*, T. Ishchenko1, N. Skvortcova1, D. Baranenko1 and
V. Chernjavskij2
1
The Institute of Refrigeration and Biotechnologies, ITMO University, 191002
Lomonosova Street 9, Saint-Petersburg, Russia;
*Correspondence: [email protected]
2
Research Institute of Influenza, 197376, prof. Popova str., 15/17, Saint-Petersburg;
Russia

Abstract. Encapsulation is of growing use in food technologies for various bioactive

compounds. The study addresses the aspect of possible improved physiological activity that
encapsulated form can enable. Liposomes obtained by dehydration/rehydration method were
used as carriers of beta carotene. The liposomal additive was used for fortification of low-fat
milk drink. Formation, structure and stability of the liposomes were controlled in the product
matrix by transmission electron microscopy. Fraction-dispersed composition and aggregate
stability of liposomal particles were investigated by means of dynamic light scattering.
Liposomal beta-carotene was found to maintain stability during storage at 4 ± 2ºС within 15
days. Hexobarbital sleeping time test performed on mice fed with the product showed enhanced
hepatostimulation effect achieved by the liposomal additive compared to free beta carotene.
Liposomal beta-carotene is suggested to be used for development of functional health-
promoting dairy products.

Key words: liposomes, beta-carotene, bioactive compounds, encapsulation, hepatostimulation

activity.

INTRODUCTION

Dietary supplements are bioactive substances of natural origin for nutritional

balance normalization and health promotion. Dietary supplements are the most
efficient way of micronutrients deficiency elimination if their dosage corresponds to
physiological requirements (Gichev & Gichev, 2009). Many dietary supplements
contain substances of adaptogenic and restorative action, which stimulate body
defense, enhance general resistance and vitality, physical and mental performance and
reduce negative environmental effects and stress. Dietary supplements are the
ingredients that provide health benefits beyond nutritional value, enhance physiological
performance and prevent diseases (Paliyath et al., 2011).
Beta-carotene is a hydrocarbon with molecular formula C40H56 containing 11
conjugated double bonds. It is a biologically active substance playing important role in
vital functions (pro-vitamin A). Beta-carotene is also an antioxidant involved in free
radical defense. Fortification of food products is the most rational way of ensuring a

825
daily intake of β-carotene. However, beta-carotene is sensitive to environmental factors
and rapidly loses activity being exposed to high temperature, light or oxygen
(Krichkovskaya et al., 2001).
Combination of instability and hydrophobicity of crystal beta-carotene hampers
its direct application in food processes. Development of acceptable β-carotene forms is
required for introduction to food compositions. Encapsulation enables carotenoids
stability optimization, dispersion in aqueous media and bioavailability improvement.
The methods of microencapsulation of bioactives are used in medicamental
compositions. Both natural substances (lipids, proteins, polysaccharides, etc.) and
synthetic polymers (nylon, polyethylenimine, polylysine) are utilized as a wall material
(Zuidam & Nedovic, 2010; Abbas, 2012).
Microencapsulation technology combines various physical and chemical methods
including coacervation, precipitation, melt injection, fluidized bed coating, extrusion,
vapor condensation, cross-linked polymers, polycondensation and polymerization. The
method choice is mainly determined by properties of encapsulated ingredient (Sotnikov
et al., 1999). For instance, the most complete encapsulation of flavorings can be
achieved by spray drying. In this method encapsulation composition includes water-
dispersed wall material, active ingredient, antioxidants and emulsifier (Merritt, 1981;
Rich & Reineccius, 1988).
Preparation of water-dispersed forms of hydrophobic beta-carotene using
microencapsulation includes several stages. Beta-carotene is dissolved in lipid fraction
with emulsifiers and solvent at elevated temperature and pressure. Aqueous solutions
of protectant colloid (gelatin or polymer) and softener (sugar) are prepared separately.
The components are mixed and cooled down by pressure relieve. This results in
formation of microparticles, namely, oil drops containing dissolved beta-carotene
coated in protective shell. Fine powder is obtained after drying; it can be dispersed in
water (Antoshkiw et al., 1982; Agapova et al., 1994).
Solubilization of beta-carotene can be achieved by using Tween 80
(polyoxyethylene sorbitan monooleate). Authors propose to use the solubilizate both
for fatty and aqueous food systems (Sotnikov, 1995). Microemulsion ‘Vetoron’
produced in Russia includes alpha-tocopherol acetate, ascorbic acid and other
bioactives in different proportions. There are successful results of using ‘Vetoron’ for
medical applications (Sorokina & Lokshina, 2010). The possibility of using ‘Vetoron’
for fondant manufacture is currently investigated (Davidovich, 2009).
It should be noted that microencapsulates contain, as a rule, 1.5–10% of beta-
carotene, up to 10% of stabilizer and antioxidants and 80–88.5% of non-ionogenic
surfactants. In other words, they are mainly surfactants with beta-carotene addition and
their combined biological effect requires special investigation (Shih, 2004). A way to
obtain aqueous dispersion of beta-carotene at concentration of 80% requires
application of extensive amounts of chlororganic solvents (dichlorethane, ethyl
chloride, chlorophorm) or toluene (Bocharov, 2000). This makes application of this
technique in food industry impossible (Zuidam & Nedovic, 2010).
Beta-carotene oil-in-water emulsions are prepared in few stages. At first stage β-
carotene solution in non-polar solvent is added to vegetable oil at high temperature.
This is followed by emulsification in aqueous phase containing stabilizer (Zuidam &
Nedovic, 2010). Oil forms of beta-carotene increase its absorbtion in vitro and in vivo.

826
However, their use is limited and not applicable for products with reduced or zero fat
(Berketova et al., 1995).
Liposomal encapsulation is a process that has been widely employed in medicine,
pharmacology, cosmetology and genetic engineering (Lipowsky & Sackmann, 1995;
Jesorka & Orwar, 2008; Mozafari et al., 2008). Liposomes are one of the most well
studied supramolecular structures. There are researches on the diversity of lipid
vesicles in terms of structure (size, number of layers), trapping capacity and
localization in supramolecular structure (Gregoriadis, 2006). Liposomes can be objects
of many applications because of independent biological activity of intact liposomes
and enhanced efficacy of encapsulated ingredients (Bolshunova et al., 2010; Umnova,
2010). At present, liposomes are studied as an independent object in colloidal
chemistry. They are considered as a target carrier of chemicals to cells, tissues and
organs and as a model for biological membranes studies. However, their structure and
properties still require investigation.
One of the promising liposomes applications is encapsulation of bioactive
compounds and design of functional additives for food products. In the review by
Gresler et al. (2008) liposomal technologies are considered as one of prospective lines
in food industry. Benefits provided by embedding of active ingredients into liposomes
include following: improved bioavailability of encapsulated ingredients; protection
from environmental factors, e.g. oxygen, acids, processing temperature; ability to turn
hydrophobic compounds into a water-dispersed form for better delivery; controlled
release of an active ingredient (Mozafari et al., 2008).
The liposomes production processes known from literature could be adapted for
the production of carotenoid-loaded liposomes as described by Ax (2003). In order to
obtain the desired liposome size distribution, the dispersion is subjected to high-
pressure homogenization, for example, by a microfluidizer.
The aim of this study was to show the possibility of designing a functional
additive with beta-carotene in liposomal form and its usage for producing a dairy
product. The additive should enhance biological activity and, therefore, improve health
benefits of a product for daily consumption.

MATERIALS AND METHODS

Soya lecithin powder LeciPRO 90C (Orison Chemicals Ltd., China) was used as a
liposomal wall material. Active ingredient for liposome encapsulation was crystalline
beta-carotene (DSM Nutritional Products AG, Switzerland). Oil solution of
α-tocopherol acetate 300 mg ml-1 (GalenoPharm, Russia) was used for antioxidant
protection. Bioactive additive ‘Vetoron’ (Akvion, Russia) containing 20 mg ml-1 of
beta-carotene and 8 mg ml-1 of vitamin E in water solution was used for preparation of
non-liposomal carotene.
Carotene-loaded liposomes were prepared by dehydration/rehydration technique
followed by mechanical homogenization (Weissig, 2010; Zabodalova et al., 2011).
Soya lecithin was dissolved in approximately ten-fold n-hexane (Komponent-Reaktiv,
Russia). Beta-carotene was dissolved in the minimal amount of chloroform
(approximately 1 ml per 50 mg beta-carotene) and added to lipid solution to ensure the
level of active ingredient being 0.5% (w/w) of lecithin. Antioxidant protection was
achieved by addition of α-tocopherol in form of oil solution at the amount of 0.2%
827
(w/w) of lipid to the mixture. The blend was evaporated by the rotary evaporator
EV311-V Plus (LabTech Inc., Italy) until lipid film was obtained at the flask bottom.
Ethanol 50% solution was introduced to the dry film for better emulsification in the
amount equal to soya lecithin weight, thoroughly stirred and the resulting mixture was
left overnight. Next day the mixture was diluted with distilled water to ensure the lipid
level of 10% (v/v). This mixture was an object of study as a water suspension of
liposomes. The mixture was homogenized using mechanical mixer Biomix LE-402
(LaborMIM, Hungary) at the stirring rate of 15,000 rpm.
Empty liposomes were prepared in the same manner excluding the step of beta-
carotene solution addition.
Reconstituted skim milk was used as a dairy product for fortification with
liposomal beta-carotene. The skim milk powder contained 1% of fat (w/w), 36% of
protein (w/w), 52% of lactose (w/w), 6% of ash (w/w) and 5% of water (w/w); it was
obtained from the Bellakt Company (the Republic of Belarus). The skim milk powder
was dissolved in distilled water (9 g skim milk powder per 100 ml distilled water) at
40–45°C. The mixture was left for 40 min for protein hydration and then pasteurized
for 20−25 s at 76 ± 2°С. Liposomal additive was introduced into refrigerated milk with
temperature of 4 ± 2°С in form of water suspension with 10% of lipid (v/v) and
thoroughly stirred. Liposomal beta-carotene was added in the amount of 40% of daily
adult requirement (5 mg) in beta-carotene established by the national health authorities
(Rospotrebnadzor, 2009; Tutelyan, 2009) if consumed with 1 portion of the product
(200 ml). Thus, the final levels of soya lecithin and beta-carotene in the product were
0.2 g and 1 mg per 100 ml of product, respectively.
Formation and internal structure of the liposomes were studied using electronic
microscope JEM-100C (JEOL, USA). Two methods were used: negative contrast and
ultra-thin slices technique. The first method was used as a preliminary to determine
whether the desired structures were formed. Negatively stained samples were
examined on support films with grids. The sample was mixed with a contrast solution.
After 1 min of incubation, it was applied to the grid, the excess solution was drained,
and the preparation was examined immediately (Lichtenberg et al., 1981; Podgorny &
Ovchinnikov, 2002). Ultra-thin slices technique is more informative as it allows not
only to determine the size, but also to obtain information about the internal structure.
Ultra-thin slices were embedded in epoxy resin Araldite (Fluka, Switzerland) (Sora,
2007).
Fraction-dispersed composition and aggregate stability of liposomal particles
were investigated by means of dynamic light scattering using spectrometer LKS-03
(Intoks, Russia). The method is based on scattered light intensity shift resulting from
molecular movement in liquid. The technique provides information on particles size
distribution. The percentage of diffused light corresponding to each fraction is related
to surface area of diffusing particles which gives an indirect estimate of fraction share
(Chernjavskij et al., 1985; Torchilin & Weissig, 2003). Since the dairy product matrix
is too complex, fraction-dispersed composition of carotene-loaded liposomes was
studied in the water suspension.
Titratable acidity and pH were controlled in the dairy product during storage to
detect the effect of liposomal additive on milk stability. Titratable acidity was
controlled according as follows: 10 ml of the product were mixed with 20 ml of
distilled water and 3 drops of 1% phenolphthalein in a flask. The mixture was titrated
828
with 0.1 M NaOH until the defined pink color appeared. Titratable acidity was
expressed as the volume of NaOH multiplied by ten (°T).
Beta-carotene concentrations in the dairy product and in the water suspension of
liposomes during storage were determined using spectrophotometric method on
UV-2,600 spectrophotometer (Shimadzu, Japan). The samples were diluted with
distilled water to have 1% of fat (v/v). Beta-carotene was extracted from 1 ml aliquots
of the dairy product or the water suspension using 2 ml of chloroform. Then 0.7 ml of
the extract was collected and 3 ml of chloroform were added. The absorbance of
carotenoid was measured in 1 cm cuvettes at a wavelength of 451 nm. Concentration
was determined using standard curve (Dawson et al., 1989).
Biological activity of the dairy products was investigated using hexobarbital
sleeping time test on white outbred mice (Dearing et al., 2006). Hexobarbital is a drug
used for general anesthesia. It is degraded by microsomal enzymes in the liver that
results in awakening. The test was performed on five groups with 10 animals in each
(males, weight of 18–20 g). The dairy product samples were injected intragastrically in
the dose of 0.2 ml per animal daily during 5 days. The last injection was made 30 min
before hexobarbital injection. Hexobarbital was injected to all groups intraperitoneally
in the dose of 60 mg kg-1. The sleeping time was determined in seconds.
All measurements were performed at least in triplicate. Statistical analysis was
performed using Microsoft Office Excel tools at theoretical frequency of 0.95.

RESULTS AND DISCUSSION

The liposomes characteristics

Fraction-dispersion composition and aggregative stability of liposomal dispersion
in water and dairy product matrix were studied using transmission electron
microscopy. Electron microscopy of the dairy product with carotene-loaded liposomes
on the first day of storage proved the presence of continuous hollow spherical
structures (Fig. 1). Unilamellar hollow vesicles and several multi-layered hollow
structures can be distinguished clearly. The dark circles are probably slices of lipid
drops with phospholipid and protein coatings (Gorbatova et al., 2011).

Figure 1. Electron microscopy image of ultra-thin slice of beta-carotene loaded liposomes in

the dairy product matrix on the first day of storage.

829
 The stability of lammelar structures of liposomes was studied in the dairy product
during 10 days of refrigerated storage at 4 ± 2°С (Fig. 2). No structure disruption or
aggregation of lipid vesicles were observed, however monolamellar liposomes
dominate in the field of vision. Lipid drops tend to aggregate. This indicates the
stability of liposomal additive in the dairy product during refrigerated storage for the
time of observation.

Figure 2. Electron microscopy image of ultra-thin slice of beta-carotene loaded liposomes in

the dairy product matrix after 10 days of storage at 4 ± 2°С.

It should be noted that homogenization transfer of multi-layered liposomes to

mono-layered ones and reduction of their mean diameter also occurs after mechanical
stirring or sonication (Weissig, 2010).
Liposomes of different size and characteristics can be obtained depending on a
preparation technique. The size is important in terms of liposomes application.
Although the ultra-thin slices images can give a brief estimate of size distribution, it
cannot be considered reliable as during sample preparation certain deformations of
shape and structure occur.
More objective data on fraction-dispersed composition was obtained by means of
dynamic light scattering (Table 1). It should be noted that the actual mean diameter is
lower than the mean hydrodynamic diameter as measured by this method.

Table 1. Mean hydrodynamic diameter (d, nm) and percent of scattered light (%) of carotene-
loaded liposomes in water suspension during storage at 4 ± 2ºС
Storage duration, days
Method 0 7 27
d, nm % d, nm % d, nm %
1,100 12 − − − −
without homogenization 430 60 530 27 350 85
126 28 220 63 115 11
17,500 30 21,000 40 22,000 12
730 33 525 40 500 45
with homogenization
260 33 170 19 190 42
80 3 − − − −

830
 It is established that most of the particles in the solution have a size less than
1 μm. Different stereometry of lipids from the raw material may cause erratic
distribution of formed lamellar particles size. Changes in particles size in the
unhomogenized sample were not observed during the period of study (27 days at
4 ± 2ºС). It can be said that in this sample liposomes have stable sizes of about 0.5 μm
or less. First fraction with the size of 1.1 μm could be formed by united particles which
are subsequently separated.
The fraction with sizes of 17–22 μm was established in the water suspension
liposomes samples subjected to the homogenization. Since there were no particles of
this size in the original solution before homogenization, it can be assumed that this
fraction is represented by a group of smaller particles joined together. This supposition
is supported by this fraction part reduction from 30% to 12% in 27 days, which may be
associated with the detachment of liposomes from aggregates formed during
homogenization. Large fraction formation indicates the necessity of selecting another
conditions or equipment for homogenization.

The products stability during storage

Stability of beta-carotene in liposome form during storage at 4 ± 2ºС was
determined in two samples: the fortified dairy product and the water suspension of
liposomes. Oxidized beta-carotene does not absorb at a wavelength of 451 nm. The
oxidation rate of beta-carotene and the residual amount of non-oxidized beta-carotene
in liposomes can be studied by the concentration in an extract from liposomes. The
concentrations of beta-carotene in the chloroform extracts from the product and the
suspension did not change during the observation period of 0–15 days and were
4.7 ± 0.5 µmol l-1 and 3.3 ± 0.7 µmol l-1, respectively. This means that the liposomal
beta-carotene is not exposed to oxidation.
The stability of the dairy product was evaluated by controlling titratable acidity
and pH in reconstituted skim milk, in the product fortified with liposomal beta-
carotene and in water suspension of liposomes. Both reconstituted skim milk with and
without liposomal beta-carotene had pH of 6.75 ± 0.13 and titratable acidity of
18.9 ± 1.1°T for 0–13 days of storage at 4 ± 2ºС. Water suspension of liposomal
additive had pH of 7.04 ± 0.09 for the same period and conditions of storage. Thus the
liposomal additive did not affect pH and titratable acidity and did not lead to earlier
spoilage of the product compared to the control sample.

Bioactivity of liposomal beta-carotene

Liposomal additive has a wide range of bioactivity due to its composition. The
effect of the fortified dairy product consumption on the liver function of mice was
chosen to study its bioactivity. The hexobarbital sleeping time test was performed
using three samples: reconstituted skim milk, reconstituted skim milk fortified with
liposomal beta-carotene and reconstituted skim milk fortified with water-soluble beta-
carotene (‘Vetoron’). The two latter samples contained equal amounts of beta-carotene
ensuring 40% of daily requirement of an adult ‒ 1 mg%. The first control group
received no treatment; the other control group received distilled water. The results are
given in Table 2.

831
Table 2. The effect of the different products on hexobarbital sleeping time of mice
Hexobarbital sleeping time
Group
min % of control
Control (intact animals) 40 ± 5 100
Distilled water 41 ± 4 100
Skim milk 39 ± 3 98
Skim milk + ’Vetoron’ 30 ± 5 75
Skim milk + liposomal beta-carotene 24 ± 4 60

The data indicates decrease in hexobarbital sleeping time of animals treated with
the samples fortified with beta-carotene. The sample with liposomal beta-carotene is
more efficient than the one with water-soluble beta-carotene.
It should be noted that the lipids in liposomal wall bear not only structural
function. Phospholipids also possess bioactivity and employed in the drugs stimulation
of a liver function. An activation of liver detoxication function due to the functional
additive intake was shown in the hexobarbital sleeping time test. This may indicate the
ability of liposomes to penetrate hepatocytes and the effect of the functional additive
on the liver detoxification rate (Shleikin et al., 2006). Further study is required to
determine which components of the liposomal additive, jointly or separately, and in
what form affect the liver function.
Liposomal beta-carotene has two active components: beta-carotene itself and
lecithin as the wall material. Both contribute to hepatostimulation activity of the
additive. However, it is not the only bioactive property. The additive can give a wide
range of health benefits including anti-inflammatory, immunostimulatory activities,
etc.
The results are in accordance with studies of other authors. Liposomal form
doubles bactericidal and bacteriostatic activity of the phytochemical compositions as
shown in the bioactivity comparison of their native and liposomal forms (Umnova,
2010). It was shown in the same research, that liposomal form of bioactives increases
their membrane-stimulating properties by 40% and antioxidant activity by 25%.
Liposomal form of Bergenia crassifolia leaves extract had a three times high
immunomodulatory effect under cold stress as native extract (Bolshunova et al., 2010).
Stimulating effect of the empty liposomes was also noted. Researches confirm
liposomal form to be promising for phytocompositions and encapsulation of bioactives.

CONCLUSIONS

The novel functional additive for dairy products is proposed. The method used for
liposome preparation is low-cost, simple and enables the production of unilamellar
vesicles with the mean diameter below 1 μm. The stability of liposomes in dairy
product matrix during storage is confirmed by transmission electron microscopy. The
liposomal beta-carotene is not exposed to oxidation during storage for 15 days at
4 ± 2ºС.
The fortified low-fat milk drink was prepared with addition of liposomal beta-
carotene to pasteurized reconstituted skim milk. The intake of 200 ml of the fortified
product can supply 40% of daily requirement in beta-carotene. The liposomal additive

832
did not lead to earlier spoilage of the product as the product had pH of 6.75 ± 0.13 and
titratable acidity of 18.9 ± 1.1°T for 0–13 days of storage at 4 ± 2ºС.
The activation of liver detoxication function due to the fortified dairy product
intake was shown in the hexobarbital sleeping time test on mice. The contribution of
each component of the liposomes in this bioactive property requires further study as
well as other possible health benefits of the functional additive.

ACKNOWLEDGEMENTS. This work was partially financially supported by the government

of the Russian Federation, Grant 074-U01.

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