3.
1 Procurement of Milk:
Standardized milk (fat- 4.5% and SNF- 8.5%) was procured from the Department of
Livestock Products Technology, Lala Lajpat Rai University of Veterinary and Animal Sciences
for the preparation of Paneer and the Whey separated out was used for the preparation of the
product to carry out research study.
3.2 Analysis of Milk:
The milk obtained was subject to testing to ensure it met the necessary fat and SNF
standards. The testing procedures used for this purpose are as follows:
3.2.1. Fat
The milk was being analyzed for fat by using Gerber method as per FSSAI, Manual of
Method of Analysis of Foods, 2016. Milk was mixed with sulphuric acid and iso-amyl alcohol in
a special Gerber tube, permitting dissolution of the protein and release of fat. The tubes were
centrifuged and the fat rising into the calibrated part of the tube was measured as a percentage of
the fat content of the milk sample.
3.2.2. SNF
The sample of milk was mixed well and then poured into a dry cylinder that allowed the
lactometer to float without touching the sides. The lactometer was then placed into the cylinder
and the reading was taken as soon as it became stationary. The corrected lactometer reading
(CLR) was noted at 27˚C.
SNF % (Solids Not Fat) was calculated as
CLR\4 + 0.25xF + 0.6, where F was the Fat % in the milk sample.
(IS 10083-1982)
3.3 Preparation of Paneer Whey:
The Paneer Whey was obtained by coagulating standardized milk (fat-4.5%, SNF-8.5%)
as described by Singh et al., 2014 with some modifications. The milk was heated up to 90 oC and
then coagulated by 2% citric acid (Hi-Media, Mumbai) after cooling the milk till 70 ⁰C with
continuous stirring for complete coagulation. The citric acid added was heated up to 70 ⁰C before
mixing it into milk. Then the milk with added coagulant was left undisturbed for 10 minutes in
order to get complete separation of Paneer Whey. Subsequently, the clear whey was separated
through muslin cloth (procured from a textile dealer at local market) and stored for further use.
Standardized Milk ( fat- 4.5%, SNF- 8.5%) Whey
Heating (90 ̊C – No hold ) Settling for 10 min
(90 ̊C – No hold )
Cooling (70 ̊C/ pH- 5.3 to 5.35) Continuous stirring till whey separates out
Addition of Citric Acid (2%)
3.4 Analysis of Whey:
The whey obtained was analyzed for various physical attributes including pH, Acidity
and protein. The procedure involved for the same are delineated below:
3.4.1 Physical Attributes Analysis
[Link] pH
The pH of whey was assessed using a digital pH meter (EUTECH Instruments). First, a
standard buffer solution with pH values of 4, 7, and 9 was used to standardize the pH meter. The
pH meter's electrode was then placed directly into the whey and the pH of the whey was shown
on the pH meter's display and recorded. The readings were in accordance with the temperature of
the sample of Whey as measured by the temperature electrode of the pH meter.
[Link] Titratable Acidity (%LA)
It was measured by titration with 0.1N Standard Sodium Hydroxide Solution (Hi- Media,
Mumbai). 10ml of whey was taken in a suitable conical flask to which equal amount of distilled
water was added. Then 1 ml of phenolphthalein indicator was added and shaked well. It was
then titrated against standard NaOH solution within 20 seconds. Titratable acidity was calculated
as per the formula (% lactic acid):-
= 9 AN
W
Where, A = Volume of standard NaOH required for titration
N = Normality of Standard NaOH solution
W = weight of the sample taken for test
[Link]. Protein
The protein content of the beverage was estimated as per the AOAC method, 2005 with some
modifications by using automatic digestion and distillation unit (Kel Plus- KES12L, Pelican
Industries,Chennai). Kjeldhal digestion tubes were first filled with 2ml of sample. To which, 10
ml of pure, nitrogen-free sulfuric acid was added with 3 to 4 grams of digestion mixture
(potassium sulphate and copper sulphate in ratio 5:1) and run in the digestion unit at two
programs 250ºC for 7 minutes and 420ºC for 75minutes. After digestion, the sample was cooled
and diluted with 10 to 20ml distilled water. The content of digestion flask were then transferred
to distillation apparatus and made alkaline by adding excess of 40 per cent NaOH
solution(40ml).The ammonia liberated during the process was collected in 4% boric acid
solution(10ml) placed at the receiver end of the distillation unit. The obtained distillate was
titrated against standard 0.1 N Hydrochloric acid added with 2 drops of methyl red as an
indicator till a light pink color appeared. The percent protein was calculated using formula:-
Protein(%) = 14× Normality of acid used ×Volume of acid used × 6.38
Weight of sample in grams × 10
3.5 Development of Paneer Whey Based Kefir Drink
The Paneer Whey collected after coagulation was neutralized using 1N NaOH till pH 6.5
was achieved followed by filtration to get clear whey. The prebiotic fructo-oligosaccharides
(FOS) at three different concentrations (0.5%, 1% and 1.5%) was added to whey at 25˚C
followed by pasteurization of mix at 65˚C for 30 min. Subsequently, Kefir starter cultures (Zoh
Probiotics, Amazon) were inoculated and the fermentation was carried at 25˚C for 18 hours.
Following fermentation, various amounts of refined sugar (6%, 7%, and 8%) were added, and the
resulting fermented product (kefir) was chilled and stored at 4˚ C until additional examination.
The flowchart for the manufacturing of Paneer Whey based Kefir Drink is as follows:-
Addition of FOS (0.5%, 1%, 1.5%) at 25oC
Paneer Whey
Neutralization by 1N NaOH till pH 6.5 Pasteurization of Paneer Whey at 65oC for 30 min
Filtration Addition of Kefir Starter Cultures into the Whey
Clear Whey
Incubation at 25oC for 18 hours
To fermented Paneer whey-Sugar Addition (6 %,7% and 8%)
Cooling and Storage at 4oC
Fig: 3.5.1: Flow chart of Manufacturing of Paneer Whey based Kefir Drink
3.6 Sensory Analysis of the Product:
The final kefir beverages were evaluated by 25 semi-trained tasters. Randomized,
refrigerated samples of 10ml was served and labeled with random numbers. Distilled water was
provided for rinsing of the palate during the testing. Tasters was asked to indicate how much
they liked or disliked each product on a 9-point hedonic scale according to following
characteristics:-
Sensory Properties Sample-1 Sample-2 Sample-3 Sample-4
1. Flavor/Taste
2. Body and Texture
3. Color and Appearance
4. Container and Closer
5. Overall Acceptability
(9 = ‘like extremely’, 8=‘like very much’, 7=‘like moderately’, 6=‘like slightly’, 5= ‘neither
like nor dislike’ 4= ‘dislike slightly’. 3=‘dislike moderately’,2= ‘dislike very much’, 1 =
‘dislike extremely’)
3.7 Parameters studied for the Paneer Whey Based Kefir Drink
3.7.1 Fat:
A.O.A.C 17th edn, 2000 Official method 932.06 Fat in Dried Milk was used for
determination of fat content of the newly developed Kefir Drink with slight modifications. 1 ml
of a well-mixed sample was measured and transferred to a small beaker. Next, 9 ml water was
added and mixed well. Following this, 1 – 1.25 ml NH 4 OH were added, and the beaker was
warmed on a steam bath. The mixture was then transferred to a fat extraction flask or tube. Once
it cooled, fat extraction was carried out using the Roese-Gottleib method (A.O.A.C, 17th
edn,2000 Official method 905.02 Fat in milk) after adding 10 ml of alcohol and calculated using
the formula:
Weight of fat percent w/w = Weight of Fat x 100
Weight of Whey based kefir Drink
3.7.2. Total Solids:
As per method suggested under IS:1479(1961),5 ml of the kefir drink sample was poured
in a dry and clean previously weighed flat bottomed aluminium dish and the weight of sample
was recorded. The dish was then placed in boiling water bath for at least 30 minutes followed by
drying of the dish at 100˚C for 3 hours in the microwave oven. After the drying phase, the dish
was removed from the oven and placed in dessicator for 30 minutes for cooling. At last, the dish
was weighed again and reading was recorded. The total solids were calculated in the beverage by
the following formula:-
Total solids, percent by weight = 100× w
W
Where,
w = weight in g of the residue after drying, and
W= weight in g of the Kefir drink sample taken for the test.
3.7.3 pH
At a temperature of 25oC, the pH of Paneer whey based kefir drink was assessed using a
digital pH meter (EUTECH Instruments). First, a standard buffer solution with pH values of 4, 7,
and 9 was used to standardize the pH meter. The pH meter's electrode was placed directly into
the beverage and the pH of the beverage was shown on the pH meter's display and recorded.
3.7.4 Acidity:
As per IS :1479 (Part I) 1960 method ,10ml of completely mixed kefir beverage sample
were introduced together with the same amount of distilled water. The sample was then added
with 1.0 ml of phenolphthalein indicator. Titration of the dish's contents against 0.1 N NaOH
continued until the solution developed a bright pink tint that lasted for 30 seconds. Titratable
acidity was calculated as per the formula(% lactic acid):-
= 9 AN
W
Where, A = Volume of standard NaOH required for titration
N = Normality of Standard NaOH solution
W = weight of the sample taken for test
3.7.5. Alcohol
The total alcohol of the Alcoholic beverage samples was determined by using following
formula (AOAC, 2000)
ABV (alcohol by volume) % = [1.05 × (OG – FG ) ÷FG] × 100
0.79
Where,
OG = Initial specific gravity measurement of whey
FG = Final specific gravity measurement of Paneer whey based kefir beverage
Also, the alcoholic content was also determined using Gas Chromatography.
3.7.6. CO2
A sample of the carbonated beverage was prepared in a clean and dry container, and the
container and sample were weighed and recorded. The volume of the sample was measured
using a calibrated pipette or burette. The CO 2 was then released from the sample by shaking the
container vigorously for at least one minute. Next, the container was placed in a water bath at a
temperature of 20°C. After 30 minutes, the container was removed from the water bath and
weighed again. The difference between the initial weight and the final weight of the container
and the sample was calculated. The CO 2 content in grams per liter was then calculated using the
following formula (IS12544:1988):
CO2 = (weight difference x 1000) / volume of the sample in milliliters.
3.7.7 Reducing Sugar
The Lane-Eynon method was used for estimating lactose which was based on lactose's
reduced ability to be found in milk. The capacity of these sugars to reduce metal ions,
particularly copper, iron, or silver in alkaline solution, serves as a typical indicator of their
reducing characteristics.
Reagents
Fehling's Solution: A (Copper sulphate) and B (Sodium and potassium Tartrate)
Acetic acid solution: 10% (w/v; aqueous) solution.
Methylene blue indicator: 1% (w/v; aqueous) solution.
Standard lactose solution: Weigh accurately 5 g of this dried lactose and dissolve in freshly
boiled and cooled distilled water. Transfer the solution quantitatively to a 1 L volumetric flask
and dilute up to mark with distilled water. Mix well and store it in a cool place. One ml of this
solution contains 5 mg lactose.
Preparation of filtrate of sample:
10 ml of the whey sample was pipette-measured into a 100 ml volumetric flask. After the
contents of the flask have been warmed to 40 to 45°C by adding 30 to 40 ml of distilled water,
1.5 ml of 10% acetic acid was added right away. The final volume was made up to the mark with
distilled water. The flask was left undisturbed for 30 minutes followed by filteration of flask's
contents using Whatman Grade 42 filter paper. Filtrate was then collected in a clean and dry
flask with a stopper after the first few milliliters were discarded.
Determination of lactose:
5 ml of Fehling’s solution A and 5 ml of Fehling's solution B were added using two
separate pipettes in a 250 ml Erlenmeyer flask. Subsequently, the content of the flask were
boiled over burner or heater for 2 min followed by addition of 3 to 4 drops of methylene blue
indicator without removing from the flame. The standard lactose solution was filled in the
burette for titration of the content of the flask against standard lactose solution from the burette
until the blue colour disappears and the bright brick-red colour of precipitated Cu 2O appears.
Volume of lactose solution required for the standardization of Fehlings solution was being noted
as [Link] the same procedure for the sample filtrate and noted the volume of the sample
filtrate required for titration as V2. The formula for calculation of % reducing sugar used was as
follows:-
% lactose = 5 x V1
V2
Where,
V1 = volume in ml, of standard lactose solution taken to reduce Fehling's solution
V2 = volume in ml, of prepared Kefir Whey based filtrate taken to reduce Fehling's solution.
3.7.8 Protein
Protien estimation of the Paneer Whey based kefir drink was being carried out by the
same procedure as followed in the case of analysis of physical attributes of the Paneer Whey in
the section [Link].
3.8 Study of Storage Stability
The shelf life study was conducted for 12 days (0 th day, 3rd day, 6th day, 9th day and 12th
day) in which coliform, lactobacilli, and lactococci was enumerated by pour plating method.
Also, the change in physiochemical parameters(pH, acidity, lactose, color and viscosity) were
investigated.
3.8.1 Microbiological Analysis for Storage Stability
The media were used for microbiological analysis were Violet Red Bile Agar (VRBA)
for enumeration of coliforms, M-17 for lactococcus and MRS for lactobacillus from Hi-media
Laboratories private limited, Mumbai and distilled water as a diluent were used for enumeration.
[Link] Preparation of Dilutions
9 test tubes were taken with 9ml saline (0.85grams of NaCl in 100ml distilled water) in
each of them. Then 1ml of sample was being added to the ist test tube and mixed followed by
addition of 1ml sample from the ist test tube to the 2 nd test tube with the help of micro pipette.
Similarly, serial dilution till 10 -9 were made through the same procedure.
[Link] Enumeration of Coliforms:
It subsumed the preparation of VRBA media, inoculation and incubation procedure of plates at
suitable temperature for the enumeration of colliforms as specified below:
Preparation of Media
All the components were completely mixed in the water and allowed to stand for
sometime. The media was then autoclaved at 121˚C for 15min.
Inoculation and Incubation
Using a fresh sterile pipette, 1 ml of the first decimal dilution (10 –1 ) of the test
sample was transferred to each of the two sterile Petri dishes.15 ml of the VRBA media
at 45˚C ± 0.˚5C was poured into each Petri dish. The inoculum was mixed thoroughly
with media by rotating the petri plates first clockwise and then anti- clockwise and
allowed to solidify. A control plate was also prepared by pouring 15ml of media for
checking the sterility of the media. The solidified dishes were inverted and incubated at
37˚C for 24 h. After incubation, purplish red colonies surrounded by a reddish zone of
precipitated bile of coliform were counted and calculated as cfu/ml (Colony forming
unit)by multiplying the dilution factor of each dish with number of colony.
[Link] Enumeration of lactobacillus:
It subsumed the preparation of VRBA media, inoculation and incubation
procedure of plates at suitable temperature for the enumeration of lactobacillus as
specified below:
Preparation of Media
Suspended 5.51grams of MRS(deMan, Rogosa and Sharpe) broth media in 100ml
distilled water and autoclaved at 121 ˚C for 15mins
Inoculation and Incubation
The dilutions were made as per the procedure stated above. After dilution, 1ml of
sample from each of three dilution of 10 -9 , 10 -8 , 10 -7 were taken in duplicates of
sterile petri dishes followed by pouring the sterile molten and cooled MRS media to the
petri dishes. After mixing and solidification of plates, they were placed upside down in
the incubator at 37˚C for 24 to 48 hours. The colonies were counted after incubation
period.
[Link] Enumeration of lactococcus:
It subsumed the preparation of VRBA media, inoculation and incubation
procedure of plates at suitable temperature for the enumeration of colliforms as
specified below:
Preparation of Media
Suspended 4.22 grams of M17 broth media in 100ml distilled water and
autoclaved at 121 ˚C for 15mins.
Inoculation and Incubation
The dilution was made as per the procedure stated above. After dilution, 1ml of
sample from each of three dilution of 10 -9 ,10 -8 ,10 -7 were taken in duplicates of sterile
petri dishes followed by pouring the sterile molten M17 broth media to the petri dishes.
After mixing and solidification of plates, they were placed upside down in the incubator
at 37˚C for 24 to 48 [Link] colonies were counted after incubation period.
3.8.2 Physio-chemical Parameters:
[Link] pH
pH estimation of the Paneer Whey based kefir drink was being carried out by the same
procedure as followed in the case of analysis of pH of Paneer Whey Based Kefir Drink in the
section 3.7.3.
[Link] Acidity
It was being carried out by the same procedure as followed in the case of analysis of
acidity of Paneer Whey Based Kefir Drink in the section 3.7.4.
[Link] Lactose
The lactose content was being evaluated using the same method as the one used
for the analysis of lactose content of Kefir Drink in the previous section 3.7.7.
[Link] Color
Chroma Meter CR-400 was used for color assessment of the product throughout
the storage study. The software in the meter collected data on the L* (lightness), a*
(redness), and b* (yellowness) values of a freshly prepared Whey Based Kefir Drink.
The L* values range from 0 (black) to 100 (white), while the a* values range from +60
(red) to -60 (green) and the b* values range from +60 (yellow) to -60 (blue). The
sample was tempered at 25°C for 30 minutes, then placed in a sample cup and measured
using an analysis glass. The readings were taken in triplicate to ensure accuracy.
[Link] Viscosity
It was assessed using a Brookfield Viscometer Model RVT at an RPM of 60 for 1min
with spindle size no. 2.
3.9 Free Fatty acid Profiling
Free fatty acid profiling was done to estimate the health promoting SCFAs in the
developed product. It was identified and quantified by gas Chromatography- Mass
Spectrophotometry (GCMS). For sample preparation for the GCMS, 1g of sample was
weighed in 50 ml Falcon tubes to which 50 μl of internal standard (heptadecanoic acid
and sorbic acid- 6 mg/ml), 4 ml methanol, 2 ml dichloromethane, and a trace amount of
BHT were added. It was being assessed in the Sigma Test and Research Center, New
Delhi.
3.10 Evaluation of functional attributes/properties
The functional attributes included the evaluation of anti-microbial and anti-oxidant
properties. The antimicrobial and antioxidant properties were evaluated using the protocols
described by (Wang et al., 2017) and (Leon-Lopez et al., 2020 and Sreeja Mudgil,2020) ,
respectively
The samples from four different preparations such as
i) whey
ii) fermented product A (Whey+Sugar+Kefir grains) and
iii) fermented product B (Whey+Sugar+Prebiotic+Kefir grains)
The samples were taken in triplicates to abstain the errors.
3.10.1 Anti-Oxidant Activity:
With some modifications, DPPH Assay was used to assess antioxidant activity. It is based on
total radical scavenging capacity, a compound's capacity to scavenge the stable DPPH radical in
10 minutes.
DPPH method
Preparation of sample
Two grams of the kefir samples were mixed with 20 ml of extracting solvent
(methanol/water, 70:30 v/v) and blended thoroughly on a magnetic stirrer stored at 20±1 °C for 4
h in a dark place. Then it was centrifuged at 10,000 rpm for 10 min and filtered through
Whatman™ 12.5 cm Grade 2 cellulose filter paper (Diameter: 12.5 cm, Pore Size: 8 µm). The
obtained supernatants were used to determine antioxidant activity by DPPH. The following three
combinations of samples were used for estimation:
Extract prepared Control samples Blank samples Sample
Whey Individual DPPH(1ml) Individual
extracts(1ml) +Methanol(3ml) extracts(1ml)
Whey Based Kefir +Methanol (3ml) +Methanol (2ml)
Drink +DPPH(1ml)
FOS+ Whey Based
Kefir Drink
Preparation of DPPH Stock Solution
10 −3 M of stock solution- 0.39432g/lt of DPPH
Procedure
1 ml of kefir Drink extract prepared was added to 1 ml of DPPH reagent (10 −3 M of stock
solution) in a tube and mixed. Then, methanol was added to obtain the final volume of 4 ml. The
tube was kept in the dark for 30 min and the absorbance was read using a spectrophotometer at
517 nm against a blank.
% DPPH Scavenging Activity(%) = 1 - A1-A0 × 100
A2
Where,
A0= Absorbance at 517nm for control or Initial absorbance
A1= Absorbance at 517 nm for the sample or Final absorbance
A2 = Absorbance at 517 nm for the Blank Values.
3.10.2 Anti- Microbial Activity
The anti-microbial activity of the Paneer whey based kefir drink was tested by agar well
diffusion method as followed by Chaudhary & Mudgal, (2020) with slight modifications. It was
tested against [Link] and Salmonella.
[Link] Media
BHI Hard Agar- 2%Agar Powder
BHI Soft Agar- 0.8% Agar Powder
[Link] Procedure
The petri dish was filled with 30 to 40ml of BHI (Brain Heart Infusion) hard agar and left
to solidify for half an hour. The dish was then overlayed with 100μl of indicator strain of [Link]
and Salmonella previously mixed with 50ml of soft BHI agar separately. Prior to using a sterile
borer to create wells in the agar, the plates were chilled at 5 °C for 10 to 15 minutes. The wells
were then filled with 100 μl of sample of whey based kefir beverage and whey to test the
inhibitory effects on each of the two bacteria. The plates were once more chilled for 10 to 15
minutes at 5 °C to enable the spread of samples of whey based kefir beverage, and incubated for
24 hours at 37°C. The anti-microbial activity of the beverage samples on the indicator strains
were identified by the clear zone surrounding the agar wells and this region of Inhibition in the
vicinity of the wells was measured in mm.
3.11 Bio- active Peptides Extraction
The bioactive peptides fractions was derived from the developed paneer whey
based kefir after neutralization of product with 1N NaOH. Afterwards, three fractions
(>5KDa, 5-3KDa, 3-1 KDa,) were obtained through fractionation by using filters of the
three different sizes respectively (>5KDa, 5-3KDa, 3-1 KDa,) through refrigerated
centrifugation at 12,000rpm for 15minutes. The fractions derived were analyzed for
their protein concentration using Lowry method (Waterborg, 2009) followed by
evaluation for anti-microbial and anti-oxidant activities using same protocols as
mentioned above.
3.11 Anti-microbial and Anti-oxidative activities of fractions:
The fractions derived were analyzed for both anti-microbial and anti-oxidant
activities by evaluation of their protein content through lowry method followed by
equalization of concentration of protein in each fraction.
3.11.1 The lowry Method
This method is based on the principle that when peptide bonds of proteins react with
copper under alkaline conditions to produce Cu+, which subsequently reduced the Folin
reagent( Folin- Ciocalteay phosphomolybdic phosphotungstic acid) to heteropolymolybdenum
blue by the copper-catalyzed oxidation of aromatic amino acids results in a strong blue color. It
is best suited for concentration of protein between 0.01–1.0 mg/ml.
Reagents
1. Complex forming Reagent:
Mixing the following stock solutions in the proportion [Link] (by vol):
Solution A: 2% (w/v) Na2CO3 in distilled water.
Solution B: 1% (w/v) CuSO4·5H2O in distilled water.
Solution C: 2% (w/v) sodium potassium tartrate in distilled water.
2. 2 N NaOH
3. Folin reagent (1 N concentration)
4. Standard - BSA( Bovine Serum Albumin )- 2 mg/ml protein in distilled water frozen at –20°C.
Procedure
1. Initially, 0.1 ml of sample or standard was taken to which 0.1 ml of 2 N NaOH was added
followed by Hydrolysis at 100°C for 10 min in a heating block or boiling water bath.
2. Then the hydrolysate was cooled to room temperature and 1ml of freshly mixed complex-
forming reagent was added to it.
3. The solution was then left to stand at room temperature for 10 min.
4. 0.1 ml of Folin reagent was added using a vortex mixer and the mixture was again left to
stand at room temperature for 30–60 min .
5. The absorbance was recorded at 750 nm
6. Standard curve of absorbance as a function of initial protein concentration was used to
determine the unknown protein concentrations.
3.11.2 Effect of proteinase k enzyme on fraction activity
Proteinase treatment is a common method used in various fields of research to cleave
proteins at specific sites. This technique is used to break down proteins into smaller fragments,
which can be analyzed further. Therefore, each fraction was treated with proteinase k for
breakdown of proteins for the confirmation of the entity responsible for functional activities in
the product.
3.11.3 Treatment with Proteinase k
Each fraction(equalized in concentration through dilution) was treated with protienase k
at a concentration of 1 ul/100 ul of sample. The mixture was then left undisturbed for 1 hour at
37˚C. After incubation, they were checked for anti- oxidant and anti-microbial activities.
3.11.4 Anti-oxidative activities of proteinase treated fractions
The estimation of anti-oxidative activities of proteinase treated fractions was being
carried out by the same procedure as followed in the section 3.10.1.
3.11.5 Anti-microbial activities of proteinase treated fractions
The estimation of anti-microbial activities of proteinase treated fractions was being
carried out by the same procedure as followed in the section 3.10.2.
