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Viability and activity of bifidobacteria in yogurt containing

fructooligosaccharide during refrigerated storage

Article  in  International Journal of Food Science & Technology · May 2004

DOI: 10.1111/j.1365-2621.2004.00829.x

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International Journal of Food Science and Technology 2004, 39, 613–621 613

Viability and activity of bifidobacteria in yoghurt

containing fructooligosaccharide during refrigerated
storage

Ayşe Sibel Akalın,* Serap Fenderya & Necati Akbulut

Dairy Technology Department, Agricultural Faculty, Ege University, 35100, Bornova, Izmir, Turkey
(Received 16 April 2003; Accepted in revised form 9 February 2004)

Summary The viability of yoghurt bacteria and two commercial strains of bifidobacteria was assessed
in either yoghurt containing chicory fructooligosaccharide (FOS) or without any prebiotic,
during 28 days storage at 4
C. All the products showed a decrease in the viable count of
yoghurt bacteria and bifidobacteria during storage. Numbers of Lactobacillus delbrueckii
ssp. bulgaricus decreased faster than those for Streptococcus thermophilus. The viability of
bifidobacteria in yoghurt was affected by the strain type and the presence of FOS.
Bifidobacterium animalis exhibited better stability in the yoghurt than B. longum. The
recommended level of 1 million cells was exceeded for B. animalis throughout storage. The
highest viable number of bifidobacteria (3.59–2.25 · 107 CFU g)1) was obtained in the
product containing B. animalis and FOS. Viability of B. longum in yoghurt containing FOS
remained above 106 CFU g)1 for up to 21 days, whereas this level was maintained for only
7 days for that organism in yoghurt without any prebiotic.
Keywords Bifidobacteria, fructooligosaccharide, viability, yoghurt.

viable and ingested in numbers ‡106 cells per gram

Introduction
of yoghurt (Kurman & Rasic, 1991). Despite the
Bifidobacteria constitute a major part of the importance of viability of these beneficial bifido-
human intestinal microflora and play an import- bacteria, surveys have shown poor viability of
ant role in maintaining good health. The potential bifidobacteria in yoghurt preparations (Iwana
health benefits ascribed to bifidobacteria include et al., 1993; Medina & Jordano, 1994; Shah et al.,
inhibition of bacterial pathogens, reduction of 1995; Rybka & Fleet, 1997). Several factors have
serum cholesterol levels and colon cancer risks, been claimed to affect the viability of probiotic
stimulation of the immune response, improvement bacteria in yoghurt, including acidity, pH, hydro-
of lactose tolerance, calcium absorbtion and gen peroxide, oxygen content, concentrations of
vitamin synthesis (Mitsuoka, 1990; Gibson & lactic and acetic acids, temperature of storage etc.
Roberfroid, 1995; Kailasapathy & Rybka, 1997; during manufacture and storage of yoghurt
Tannock, 1999). At present, bifidobacteria are (Samona & Robinson, 1994; Lankaputhra &
increasingly incorporated into fermented dairy Shah, 1995; Lankaputhra et al., 1996a). Thus,
products. Bifidobacteria grow slowly in milk and maintaining viability of bifidobacteria until the
the usual practice is to add yoghurt starter products are consumed in order to ensure the
bacteria to enhance the fermentation process for delivery of live organisms has been of much
making probiotic yoghurt (Samona & Robinson, interest.
1994). For bifidobacteria to provide therapeutic Presently, five species of Bifidobacterium have
benefits, it has been recommended that they be attracted attention in the dairy industry for
manufacturing probiotic milk products – Bifido-
*Correspondent: Fax: +90 232 3881864; bacterium adolescentis, B. bifidum, B. breve,
e-mail: sakalin21@[Link] B. infantis and B. longum (Shah, 1997). Medina
doi:10.1111/j.1365-2621.2004.00829.x
 2004 Blackwell Publishing Ltd
614 Viability of bifidobacteria in yoghurt A. S. Akalın et al.

& Jordano (1994) proposed that using an acid- Certain properties that come from the use of these
resistant Bifidobacterium strain was important for oligosaccharides by the resident saccharolytic
the manufacture of bifidus-fermented milks. microflora, especially bifidobacteria, include pro-
According to Shah (1997), use of B. longum for duction of short chain fatty acids and vitamins,
manufacturing fermented products should be antimicrobial activity, and stimulation of the
encouraged because of the better tolerance to acid immune system (Perrin et al., 2002). They are also
and hydrogen peroxide than other strains of used to enhance the survivability and colonization
bifidobacteria. On the other hand, Meile et al. of probiotic bacteria, that are added in food
(1997) described a new species, B. lactis, and there products, in the gut (Ziemmer & Gibson, 1998).
is currently some confusion regarding B. animalis Fructooligosaccharides (FOS) are one of the most
and B. lactis. According to Klein et al. (1998) the studied prebiotics as they resist digestion by gastric
taxonomic position of B. lactis must be reconsid- acid and pancreatic enzymes in vivo and increase
ered and it should be ruled out that B. lactis the population of bifidobacteria in the colon
strains are in fact B. animalis strains, which are (Tomamatsu, 1994; Cummings et al., 2001). In
adapted to milk. Ventura & Zink (2002) proposed Japan, FOS are considered to be food, not food
that B. lactis should be reclassified as a subspecies ingredients, and are found in more than 500 food
of B. animalis. Iwana et al. (1993) isolated bifido- products (Spiegel et al., 1994).
bacteria from commercial yoghurt sold in Europe Although the effects of oligosaccharides on
and found that most of the yoghurts contained colonic bifidobacteria have been investigated, there
B. animalis. Of sixteen strains of Bifidobacterium are no published reports on the effects of oligosac-
isolated from fifteen dairy products eleven strains charides on bifidobacteria in yoghurt during stor-
were identified as B. animalis and three strains age. Therefore, the objective of this study was to
were B. longum on the basis of DNA similarities evaluate the effects of FOS on the viability and
(Yaeshima et al., 1996). In addition, according to activity of two different commercial and widely
the phenotypic features and confirmed by DNA- used Bifidobacterium species in yoghurt during
DNA hybridization most of the bifidobacteria refrigerated storage. For this purpose, changes in
strains from dairy origin belonged to B. animalis, pH, concentration of lactic and acetic acids and the
although they were often declared as B. longum viable counts of S. thermophilus, L. delbrueckii ssp.
by the manufacturer (Klein et al., 1998). Thus, bulgaricus, and bifidobacteria were monitored
B. longum and B. animalis have been frequently during storage of yoghurts for 28 days at 4
C.
seen in commercial dairy products.
Studies have shown that yoghurt bacteria
Materials and methods
(Streptococcus salivarius ssp. thermophilus and
Lactobacillus delbrueckii ssp. bulgaricus) survive
FOS, starter and probiotic cultures
well in yoghurt until the use-by-date (Hamann &
Marth, 1984; Rohm et al., 1990). However, Bifi- The commercial yoghurt starter culture containing
dobacterium spp. showed a rapid decline after S. thermophilus and L. delbrueckii ssp. bulgaricus
manufacture of yoghurt made by commercial was selected because its mild acidity protects
starter cultures (Dave & Shah, 1997). After bifidobacteria. Both yoghurt starter culture and
ingestion of the fermented milk product is con- two commercial strains of bifidobacteria [B. ani-
cluded, the exogenous bacteria are rapidly excreted malis (Bb-12) and B. longum (Bb-46)] were
(Bouhnik et al., 1992). An alternative approach is obtained from Chr. Hansen A/S, Hørsholm,
the adddition to foods of substrates which have a Denmark. The cultures were in freeze-dried direct
specific stimulatory effect on the growth of bifido- vat set (DVS) form and stored after procurement
bacteria already resident in the colon. It was according to the recommendation of the manu-
determined that certain oligosaccharides had bene- facturer. Commercially available FOS (Fibru-
ficial effects on human health and the potential to lose F97, Cosucra S.A., Fontenoy, Belgium),
increase bifidobacteria in the colon without being which is extracted from chicory roots and has a
utilized by other intestinal bacteria (Gibson et al., degree of polymerization from 2 to 30, with an
1995; Ballongue et al., 1997; Bouhnik et al., 1997). average of 5.

International Journal of Food Science and Technology 2004, 39, 613–621  2004 Blackwell Publishing Ltd
 Viability of bifidobacteria in yoghurt A. S. Akalın et al. 615

yoghurt samples were transferred to a refrigerator

Preparation of yoghurt
at 4
C and stored at this temperature over 28 days
Twenty litres of regular homogenized and pasteur- for the analyses of pH, cell counts and concentra-
ized milk was tempered to 45
C and divided into tions of lactic and acetic acids.
two lots. To adjust milk solids to 15%, one lot was All the yoghurt samples were uniformly mixed
fortified with 3.5% (w/v) non-fat dried milk and in a sterile glass beaker, a sample was aseptically
the other with 1.5% (w/v) non-fat dried milk and taken for microbiological and other analyses. The
2.0% (w/v) FOS as recommended by the manu- 0-day period represents analyses after overnight
facturer. The mixtures were heated to 85
C for storage of yoghurt samples, and periods 7–
30 min and cooled to 40–43
C, and then divided 28 days represent analyses of yoghurt samples
into two lots again. Thus, four lots of mixture were after 7, 14, 21 and 28 days of storage respect-
obtained. Yoghurt starter culture and B. animalis ively.
were added to each of two lots (containing FOS or Total solids content was determined by drying
no FOS) and yoghurt starter culture and B. longum duplicate samples at 110
C for 2 h and was in the
were added to each of the remaining two lots range of 15.00–15.29% in yoghurt samples.
(containing FOS or no FOS) as outlined in Fig. 1.
All the cultures were used according to the
pH value
manufacturer’s instructions. The mixtures were put
into 100-mL plastic cups. The cups were incubated The pH values of the yoghurt samples were
at 40
C as recommended by the culture supplier measured at 17–20
C using a pH meter (model
and terminated at pH 4.55. After fermentation, SS-3, Beckman, Fullerton, CA, USA).

Homogenized and pasteurized milk

Addition of skimmed milk powder Addition of skimmed milk powder (1.5%) and
(3.5%) Fructooligosaccharide (2.0%)

Pasteurization Pasteurization
(85 ºC 30 min) (85 ºC 30 min)

Cooling Cooling
(43 ºC) (43 ºC)

Inoculation Inoculation

Yoghurt culture Yoghurt culture Yoghurt culture Yoghurt culture

+B. animalis +B. longum +B. animalis +B. longum

Filling Filling Filling Filling

Incubation Incubation Incubation Incubation

(40 ºC until pH 4.55) (40 ºC until pH 4.55) (40 ºC until pH 4.55) (40 ºC until pH 4.55)

Cooling and storage Cooling and storage Cooling and storage Cooling and storage
(+4 ºC) (+4 ºC) (+4 ºC) (+4 ºC)
A B C D

Figure 1 Production line of yoghurt samples.

 2004 Blackwell Publishing Ltd International Journal of Food Science and Technology 2004, 39, 613–621
616 Viability of bifidobacteria in yoghurt A. S. Akalın et al.

0.5 mL min)1. The wavelength of detection was

Enumeration of yoghurt and probiotic bacteria
optimized at 210 nm for the quantification of
One gram of each yoghurt sample was diluted organic acids. Duplicate (10 lL each) injections
with 9 mL of sterile 0.1% (w/v) peptone water were performed for all samples. The standard
(Oxoid, Hampshire, UK) and mixed uniformly solutions of lactic and acetic acids (Sigma,
with a vortex mixer. Subsequent serial dilutions St Louis, MO, USA) were prepared in distilled
were made and viable yoghurt and bifidobacteria water to establish elution times and calibration
numbers enumerated using the pour plate tech- curves. The retention time for lactic acid was
nique. The counts of S. thermophilus were enu- 6 min and acetic acid 7.15 min. The standard
merated on ST agar after incubating the plates curve coefficients were 0.999 and 0.995 for lactic
aerobically at 37
C for 24 h (Dave & Shah, and acetic acids, respectively.
1996). MRS agar (Merck, Darmstadt, Germany)
adjusted to pH 5.2, and anaerobic incubation at
Statistical analysis
43
C for 72 h were used for the differential
enumeration of L. delbrueckii ssp. bulgaricus Each experiment was independently replicated
(Dave & Shah, 1996). Bifidobacteria were en- three times in a completely randomized design.
umerated according to the method of Lankapu- All analyses and enumerations were done in
thra et al. (1996b) using MRS-NNLP (nalidixic duplicate.
acid, neomycin sulphate, lithium chloride and All data were analysed by anova using the
paramomycin sulphate) agar. The inoculated general models procedure of SAS (1989). Differ-
plates were incubated anaerobically at 37
C for ences among means were tested for significance
72 h using an oxygen-free milieu and a CO2 (P > 0.05) by Duncan’s multiple range test.
atmosphere in anaerobic jars (Merck). Plates
containing twenty-five to 250 colonies were enu-
Results and discussion
merated and recorded as colony forming units
(CFU) per gram of sample.
Changes in pH in yoghurt
The pH values of yoghurt samples during refri-
Lactic and acetic acid concentrations
gerated storage are shown in Fig. 2.
The quantification of lactic and acetic acids was
done by high-performance liquid chromatography
(HPCL; LC-900 Series, Jasco International Co.,
4.52
Hachioji, Tokyo, Japan) according to the modifi-
cation of the methods described by Fernandez- 4.5
Garcia & McGregor (1994) and Akalin et al.
(2002). For extraction of acids, 4 g of yoghurt 4.48
sample was diluted to 25 mL with 0.1 n H2SO4,
pH

homogenized and centrifuged at 5000 · g for 4.46

10 min. The supernatant was filtered through
Whatman # 1 filter paper and through a 0.20 lm 4.44
membrane filter (Millipore Corp., Bedford, MS,
4.42
USA), and 2-mL aliquots were stored in HPLC
vials at )20
C until HPLC analysis. The HPLC 4.4
system consisted of a (model IH-980–01) holder 0 7 14 21 28
that accepts Rheodyne valves, 7124 injector fitted Storage days
with a 20-lL sample loop, a Jasco PU-980 solvent
Figure 2 Changes in pH values in yoghurt A ()) con-
delivery system, and a Jasco UV-980 detector. A
taining B. animalis as control, yoghurt B (h) containing
Nucleosil 120-5C18 column (Macherey Nagel, B. longum as control, yoghurt C (n) containing FOS
Düren, Germany) was used. The degassed mobile and B. animalis and yoghurt D (s) containing FOS and
phase of 0.009 n H2SO4 was used at a flow rate of B. longum.

International Journal of Food Science and Technology 2004, 39, 613–621  2004 Blackwell Publishing Ltd
 Viability of bifidobacteria in yoghurt A. S. Akalın et al. 617

The initial pH values for the different yoghurt probiotic yoghurts during refrigerated storage
types ranged from 4.51 to 4.48. The pH of all are presented in Table 1. There were minimal
samples decreased slightly during storage and did differences in the viability of S. thermophilus
not drop under 4.4 at the end of storage, possibly among yoghurt types and the viable counts in
due to the low acidifying activity of the yoghurt general decreased slightly during the storage. After
and probiotic cultures. The drop in the pH was 28 days of storage, the counts of S. thermophilus
similar for all the yoghurt samples, and between decreased by only 10–15% in all the samples and
0.06 and 0.09 pH units throughout storage. As were not statistically significant (P > 0.05) from
there was no major difference in pH values initial counts. Similar viable counts of S. thermo-
(P > 0.05) or relative drop in pH values at 4
C philus were also found in yoghurts containing
for up to 28 days, it is unlikely that these changes bifidobacteria by Medina & Jordano (1994), Dave
would affect viability. & Shah (1997), Rybka & Fleet (1997) and
Shah et al. (1995, 2000) also found similar Vinderola et al. (2000).
decreases in pH values during storage of commer- Lactobacillus delbrueckii ssp. bulgaricus showed
cial yoghurts containing L. acidophilus and B. bifi- a more marked decrease than S. thermophilus
dum. Similarly, the initial pH values in yoghurts during refrigerated storage (Table 1). The initial
containing L. acidophilus and bifidobacteria numbers (between 0.9 and 1.1 · 108 CFU g)1)
decreased from 4.33–4.41 at day 0 to 4.16–4.22 at were found to be almost 50% of the viable counts
the end of 35 days of storage (Dave & Shah, 1997). of S. thermophilus. After 7 days of storage, the
counts of L. delbrueckii ssp. bulgaricus showed a
sharp decline (6–10-fold) which was statistically
Changes in the counts of yoghurt bacteria and
significantly different (P < 0.05) from the initial
bifidobacteria
counts. The counts of L. delbrueckii ssp. bulgaricus
The changes in the viable counts of S. thermo- had decreased by 97–99% in all samples at the end
philus and L. delbrueckii ssp. bulgaricus in of storage. Similarly, S. thermophilus counts were
higher by at least 1 log order than those for
Table 1 Changes in viable counts of S. thermophilus,
L. delbrueckii ssp. bulgaricus in yoghurts con-
L. delbrueckii ssp. bulgaricus B. animalis or B. longum during taining probiotic bacteria (Vinderola et al., 2000).
refrigerated storage of yoghurtsa On average, the survival rate of S. thermophilus
was better than that of both L. delbrueckii ssp.
Storage days
bulgaricus and bifidobacteria. These observations
Product b
0 7 14 21 28 are in line with those of Kim et al. (1993), Medina
& Jordano (1994), Lim et al. (1995) and Dave &
S. thermophilus (·106)
A 231 229 225 210 197
Shah (1997).
B 205 203 205 192 175 The bifidobacteria were initially present
C 196 190 182 180 177 between the levels of 2.89 · 107 and 4.06 ·
D 197 191 195 183 170 107 CFU g)1 for yoghurts A and D respectively.
L. delbrueckii ssp. bulgaricus (·105)
As the growth of bifidobacteria was affected by the
A 1015 167 67 50 27.7
B 1148 114 38 25 14
addition of FOS, the initial viable cell counts
C 949 112 40 38 20 varied prior to refrigerated storage. The initial
D 1023 95 13 12 8.5 counts of both strains of bifidobacteria were
B. animalis or B. longum (·105) higher in yoghurts C (3.6 · 107 CFU g)1) and D
A 289 235 185 152 107
(4.1 · 107 CFU g)1) containing FOS than those in
B 291 23 4.8 2.5 2
C 359 353 323 252 225
the control samples. All the yoghurt samples
D 406 32 24 15 2 showed a steady decline in the numbers of
bifidobacteria. The decline was more rapid for
a
Means of three trials, and each trial was examined in
the yoghurts B and D containing B. longum.
duplicate.
b
Yoghurts A, B, C and D contains B. animalis as control,
Therefore, the stability of B. animalis in yoghurt
B. longum as control, FOS and B. animalis, and FOS and was found to be higher than B. longum during the
B. longum, respectively. storage.

 2004 Blackwell Publishing Ltd International Journal of Food Science and Technology 2004, 39, 613–621
618 Viability of bifidobacteria in yoghurt A. S. Akalın et al.

There were no significant differences (P > 0.05) These results are consistent with previous
in the viable counts obtained for B. animalis at the reports on the ability of FOS to stimulate the
end of storage. The viable counts of B. animalis viability of bifidobacteria in reconstituted non-fat
remained well above the recommended limit of dried milk during 4 weeks of refrigerated storage
1 million cells per gram of yoghurt throughout the at 4
C. FOS was the most effective prebiotic
storage period. However, a significant decrease among the carbohydrate sources tested and the
(P < 0.05) in the B. longum population in both effect of FOS increased with increasing carbohy-
yoghurts (B and D) was observed at 7 days of drate concentration (maximal at 5%) (Shin et al.,
refrigerated storage. The counts of B. longum 2000b). Similarly, the viability of bifidobacteria
decreased by 99% in those yoghurts at the end of strains in reconstituted skimmed milk during
storage. Martin & Chou (1992) observed that 4 weeks of storage was significantly higher
viability of Bifidobacterium spp. was species and (P < 0.05), when they were inoculated in the
strain dependent and that viability varied greatly. presence of prebiotics as compared with controls
Similarly, it was reported that the bifidobacterial without any prebiotic. The highest viability of
strain used in yoghurt manufacture affected the 75.3% was found using strains of B. longum (Bb-3)
viability (Dave & Shah, 1997). The final pH of (Morinaga Milk Industry Co. Ltd, Tokyo, Japan)
yoghurt can affect the viability of bifidobacteria and B. animalis (Bb-5) (Chr. Hansen Pty. Ltd,
(Shah et al., 1995). Vinderola et al. (2000) repor- Boyswater, Australia) using hi-maize (Bruno
ted that pH values of 4.5 or lower jeopardized the et al., 2002). Stimulation of bifidobacteria in the
viability of probiotic organisms in yoghurt stored human colon by FOS has also been demonstrated
at 5
C. Similarly, Sakai et al. (1987) concluded in human-feeding trials (Gibson et al., 1995;
that the most important factor in bifidobacterial Roberfroid et al., 1998). On the other hand, the
mortality was the low pH of the yoghurt and any influence of fructan-type oligosaccharides (as pre-
drop in pH below 4.3 greatly affected the viability biotics) on growth and acidifying activity of
of bifidobacteria (Lankaputhra et al., 1996a). Shin Bifidobacterium strains has been studied in vitro,
et al. (2000a) observed that viability of bifidobac- using minimal nutrition media. The results showed
teria in commercial yoghurts remained above that the majority of Bifidobacterium species util-
106 CFU g)1 until the expiration date. At low ized FOS, but only eighteen of thirty strains tested
pH values fermentation acids like lactic and acetic (mostly of B. longum and B. animalis) were
are powerful antimicrobial agents and may have a stimulated (Bielecka et al., 2002). The degradation
role in modulating survival of bifidobacteria. of FOS by fructofuronasidases of bifidobacteria
Hence the higher levels of these acids in yoghurts can increase growth and short-chain FOS like
A and B resulted in lower counts of bifidobacteria. Fibrulose F97 are fermented more quickly by
The viability of bifidobacteria in yoghurt during bifidobacteria (Perrin et al., 2002).
the storage was higher when they were grown in
the presence of FOS as compared with the control
Changes in lactic and acetic acid contents
samples containing no prebiotic (Table 1).
Although a significant decrease (P < 0.05) was These changes, as determined by HPLC, are
observed after 7 days in yoghurt D, viable B. shown in Fig. 3. The lactic acid contents of
longum remained above 106 CFU g)1 until yoghurt samples at 0 day varied between 13.10
21 days of storage. However, in yoghurt B con- and 13.86 mg g)1. A noticeable increase was
taining no FOS, the recommended level of 1 observed, especially for yoghurts A and B during
million cells was maintained only for 7 days. The the first 7 days of storage (P < 0.05), followed by
highest viability (P < 0.05) and lowest decrease of a gradual increase throughout the storage period.
37.3% throughout the storage was retained by The lactic acid contents of yoghurts C and D
strain B. animalis in yoghurt C containing FOS. gradually increased during storage. In general, the
On the other hand, despite the initial number of B. contents of lactic acid were higher in yoghurts A
animalis decreasing by 63%, the organism was still and B than those found in C and D (P < 0.05)
1 · 107 CFU g)1 after 28 days of storage in yog- possibly due to the higher lactose content in
hurt A. yoghurts enriched only by addition of dried milk.

International Journal of Food Science and Technology 2004, 39, 613–621  2004 Blackwell Publishing Ltd
 Viability of bifidobacteria in yoghurt A. S. Akalın et al. 619

16.5 0.6 probiotic bacteria. The results reported show a

great variability in the survival ability of B. longum
16 0.55 and B. animalis and that the addition of FOS
improved the viability of both bifidobacteria
15.5 0.5
strains.

Acetic acid (mg/g)

Lactic acid (mg/g)

15 0.45
Acknowledgments
14.5 0.4
The authors thank the Ege University, Agricul-
14 0.35 tural Faculty, Research Fund Council and Cosu-
cra S.A. for funding this project.
13.5 0.3

13 0.25 References
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Figure 3 Changes in lactic acid (closed symbols) and acetic cheese. Journal of Dairy Science, 85, 1670–1676.
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