JOURNAL OF PURE AND APPLIED MICROBIOLOGY, June 2014. Vol. 8(3), p.

Proof

Impact of Banana Puree on Viability of Probiotic

Soy Yoghurt During Storage
Dilip Kumar*, S.N. Thakur and Dinker Singh
1
Department of Animal Husbandry and Dairying, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi - 221 005, India.

(Received: 18 November 2013; accepted: 09 February 2014)

The present investigation was made with an attempt to manufacture banana

based probiotic soy yoghurt with addition of probiotic culture and banana puree, were
examined. Probiotic soy yoghurt samples were produced from soy milk fermented with
yoghurt and probiotic culture i.e. Streptococcus thermophilus NCDC-074, Lactobacillus
delbrueckii ssp. bulgaricus NCDC-009, Lactobacillus acidophilus NCDC-15 and
Bifidobacterium bifidum NCDC-235. The viability (SPC) of these four strains were analyzed
during storage at refrigerated temperature with the selective media of MRS, ST, MRS-
sorbitol and MRS-maltose agar. These counts generally decreased during the storage
period. Supplementation of banana fortified soy yoghurts with 5, 10 and 15% of banana
puree, improved the viability of probiotic bacteria during storage period. However, the
yoghurt supplemented with banana puree, at 15% level, showed highest viability (106 cfu/
g) among all treatment combinations up to four weeks of refrigerated storage.

Key words: Soy yogurt, Banana, Storage.

This research aims to expand soybean supplementation of Lactobacillus and

and banana utilization by developing new product Bifidobacterium sp., resulting in a product termed
banana based probiotic soy yogurt fermented by AB yoghurt (Shah, 2000). L. acidophilus, and
typical yoghurt bacteria and probiotic bacteria Bifidobacterium sp. are considered probiotic
(Lactobacillus acidophilus and Bifidobacterium organisms since they are believed to exert
bifidum). These products are particularly popular beneficial health effects in the host by modulating
among children, aged people and health conscious the intestinal microflora (Schrezenmeir and De
consumers (Garcia et al., 1998; Corte, 2008). The Vrese, 2001). A number of health benefits have been
therapeutic dietetic properties of fermented dairy proposed including antimicrobial, antimutagenic,
foods have been attracting the human population anticarcinogenic and antihypertensive properties,
since ages. and reduction in serum cholesterol, alleviation of
Fermented dairy foods have long been lactose intolerance, and reduction of allergic
considered safe and nutritious. The beneficiary symptoms (Schrezenmeir and De Vrese, 2001;
role of yoghurt may be further enhanced by the Lourens-Hattingh and Viljoen, 2001).
Probiotic organisms are ‘live micro-
organisms which, when consumed in adequate
amounts, confer a health benefit on the host’ (FAO/
* To whom all correspondence should be addressed. WHO, 2002). To observe a positive health effect of
E-mail: dilip.bhu@live.in their consumption, a minimum level of live
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microorganisms is required; this level, depending particularly lactobacilli and bifidobacteria (Davis
on the strains used and the required health effect, and Milner, 2009). FOS are dietary fibers that help
is usually between 108 and 1011 cfu/d (Vanderhoof keep the stomach and bowels healthy. They do
and Young, 1998). Therefore, assuming a daily this by nourishing and promoting the naturally
consumption of fermented dairy products of 100 present, “friendly” bacteria (Bifidobacteria and
g, they should contain between 106 cfu/g to 109 Lactobacilli in particular) capable of warding off
cfu/g of these live bacteria at the time of infection in the digestive system. Because of these
consumption. While this is the case for a limited properties FOS is considered a “Prebiotic”. The
number of strains, the precise mechanism by which dietary intake of fibers and probiotics exert a
probiotic organisms exert a health effect in vivo is positive impact on the development of the intestinal
not clearly understood. One aspect that is clear, micro-biota and are reported to relieve constipation
however, is that some strains produce certain and reduce the incidence of colon cancer
health-promoting metabolites (including proteins (Farnworth, 2008).
and fatty acids) which are desirable from a Some foods naturally contain prebiotics.
nutritional and/or physiological perspective. Examples are wheat, chicory, onions, bananas,
However, it should be emphasized that the garlic, asparagus, Jerusalem artichokes and leeks,
ingestion of probiotic fermented foods opens up although the amount of prebiotics in these foods
the possibility that these health-promoting varies casement P. A. (1999) and Roberfroid M. B.
metabolites may also be produced in vivo (Tamime, (2000).
2005). The combination of soy and prebiotics
Soybeans are less expensive and more may lower cholesterol and boost heart health.
plentiful than bovine milk. Moreover, soymilk has Banana (Musa sp., Musaceae) is a good sources
no cholesterol. Soy based products have already of probiotics, having 1% prebiotic fiber, 22% sugar,
been developed which compete favorably with no harm in eating a banana and the soybean is an
dairy products. Soy yogurt can command a market always typical source of prebiotics. Banana by
on the strength of being cholesterol free. Soy product contains around 43–49 g of total dietary
products contain the isoflavone phytoestrogens fiber, 1 g of inulin, 6 g of fructo-oligosaccharide
with potential anticarcinogenic activity (Wolfe et and 10–20 g of pectin per 100 g of dry matter, in
al., 2003). addition to significant amounts of ±-linolenic acid
On the other hand, soymilk, which serves (ALA), essential amino acids and micronutrients
as a base for a variety of beverages, contains such as Mg, K, P and Ca (Emaga et al., 2007, 2008;
indigestible oligosaccharides such as raffinose and Mohapatra et al., 2010).
stachyose, which are usually associated with Throughout the world, probiotics and
stomach discomfort and flatulence (Rackis et al., prebiotics are leaders in sales in the functional
1970). Desai et al, (2002) reported high foods category. Prebiotics are nonviable food
concentrations of flatulent sugars (raffinose, components that exert a benefit on the health of
stachyose) in soy yogurt because the yogurt the host, associated with modulation of the
bacteria cannot utilize these sugars. Some strains intestinal flora (FAO/WHO, 2007). On the other
of Bifidobacterium are able to reduce the hand, probiotics are live microorganisms,
concentration of raffinose and stachyose, administered in quantities adequate to confer
eliminating the potential cause of flatulence, and health benefits (FAO/WHO, 2001). Synbiotic may
also decrease the levels of pentanal and n-hexanal be defined as the combination of probiotics (the
responsible for the beany flavour (Desai et al., 2002; live bacteria) and the prebiotics (the food
Tsangalis and Shah, 2004). components they live on), being mainly used
Prebiotics nourish Probiotic bacteria and because a true probiotic, without its prebiotic food
yeasts so that they can grow faster. Banana source does not survive well in the digestive
content of insoluble and soluble dietary fibers (DF), system (Panesar et al., 2009). Functional foods are
pectin and fructooligosaccharides. These products that have been enriched with added
prebiotics are in fact able to selectively stimulate nutrients or other substances that are considered
the growth and activity of the gut microbiota, to provide health benefits over and above their

J PURE APPL MICROBIO, 8(3), JUNE 2014.

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nutritional value. This term covers a broad range % fat, 12% serum solids, 6% sugar and total solids
of products: typical examples are probiotic yogurts, adjusted to 22.06 %. The soy yoghurt mix was
cholesterol-lowering spreads, and oligosaccharide- prepared by dissolving cream and skim milk powder
added foods (Williamson, 2009). (SMP) in soy milk at 60°C for adjusting of fat and
This study aims to formulate high protein, solid not fat (SNF) respectively. The mix was
synbiotic action and acid production in probiotic homogenized and then pasteurized at 85°C for 30
soy yoghurt which add health benefits and value min followed by cooling (37°C) and aseptically
addition by incorporation of banana puree and inoculating with 0.5% (v/v) of each strains L.
probiotic bacteria. In the addition of probiotic bulgaricus NCDC 009, S. thermophilus NCDC 074,
bacteria in to the products provides an extra L. acidophilus NCDC15, and B. bifidum NCDC235,
beneficial aspect to the product and therapeutic after that incubated at 37°C until the pH of 4.9 is
value. reached. The fermentation time ranged between 8
to 12 hrs for probiotic soy yoghurt. The pH of soy
MATERIALSAND METHODS yoghurt samples were recorded using (Eutech and
Oakton instruments, Malaysia) digital pH meter.
This study was carried out at “Warner The banana puree stirred at 0, 5, 10 and 15% with
School of Food and Dairy Technology, Sam plain yoghurt T0, T1, T2 and T3 respectively and
Higginbottom Institute of Agriculture, Technology stored at 4°C for during 28 days.
and Sciences, Allahabad, U.P., India”, with The microbiological analysis i.e. Std.
appropriate methodology. Plate Count (SPC) of Banana Based Soy Yoghurt
Fresh mixed milk and fresh cream (50%) were estimated by using standardized Procedure
was obtained from the “Student’s Training Dairy” of laid down in I.S(1847) part 2 and manual of dairy
WSFDT, SHIATS, Allahabad. Skimmed milk powder bacteriology, ICAR publication (1972 ).
(brand name ‘Anikspray’), sugar, soybean and Microbiological analyses
banana were procured from local market in The count of S. thermophiles, L.
Allahabad, U.P., India. Freeze dried Yoghurt culture bulgaricus, L. acidophilus and B. bifidum in to
(S. thermophilus NCDC 074 and L. bulgaricus the formulated soy yoghurt was evaluated on 1st,
NCDC 009) and Probiotic culture (L. acidophilus 7th, 14th, 21st and 28th day. One ml sample was taken
NCDC 15 and B. bifidum NCDC 235) which has from each yogurt sample for serial dilution. Serial
proven therapeutic benefits (Reddy et al., 2006), tenfold dilutions were prepared in a solution of
were purchased from the National Dairy Research 0.9% NaCl (w/v) and 0.1% (w/v) bactopeptone and
Institute (NDRI), Karnal, Haryana, India. suitable dilutions were plated on appropriate media.
Soy milk preparation L. bulgaricus were enumerated on Lactobacilli
100 g of hand washed soybeans were MRS agar, when the incubation is carried out at
soaked for overnight in 1,000 ml of tap water. After 45°C for 72 h. S. thermophiles were enumerated on
that 0.5% sodium bicarbonate added and stand for ST agar under aerobic incubation at 37°C for 24 h.
2 hours at 50°C, then blanched with the same For determining B. bifidum by differential counts
carbonated water for 4 minutes. The soybeans were between L. acidophilus enumerated on MRS-
immediately separated and circulated over by tap sorbitol agar and the total counts of L. acidophilus
water. The beans were blended in a high speed and B. bifidum obtained from MRS-maltose agar
blender with 1,000 ml hot water (90°C) for 3 minutes. were done by Dev and Shah (1960); Shah (2000).
The mix was filtered with 3 sheets of cheese cloth Statistical analysis
then boiled for 10-15 minutes. The filtered mixed A data of the samples were investigated
with 11% nonfat dry milk at the ratio of 80:20 (final for microbiological on 1st, 7th, 14th, 21st and 28th day.
concentration of 2.2%) for better fermentation. For All analyses were conducted twice. Data obtained
soy yoghurt manufacturing, heat treatment of from analysis of the samples were evaluated by
soymilk is usually carried out at 95-100°C for 30 variance analysis, and the differences among means
min for soymilk (Liu, 1997). were detected by Duncan’s multiple range tests
Preparation of Soy yoghurt samples (SPSS 1999).
Soy yoghurt mix were standardized to 4.0

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RESULT AND DISCUSSION soy yoghurt during storage period (4ºC). Even the
high level of growth of all those bacteria when a
Growth of bacterial culture high percentage (15%) of banana was used.
Fig. 1 shows the growth pattern that Insufficient growth occurred with 5 and 10%
occurred when the yogurt producing bacteria S. banana. The total colony counts after fermentation
thermophilus and L. bulgaricus were together with of the soy yoghurt to pH 4.9 were 4 x 108 cfu/g for
probiotic bacteria L. acidophilus and B. bifidum all types of bacteria. Initial freezing of the soy
inoculated into the soy yoghurt. Twelve hrs were yoghurt in the freezer at 4ºC for 4 weeks caused a
required to reach a pH of 4.3. These four strains reduction of less than three log cycle in total colony
used in this study grew well in the banana based counts.
soy yoghurt and their population stabilized 10 hrs The L. bulgaricus, S. thermophiles, B.
incubation period. bifidum and L. acidophilus count (log10 cfu/g) of
Microbial counts formulated soy yoghurt during storage period
The differentiation of S. thermophiles, L. (Table. 1) showed a significant difference. The
bulgaricus, L. acidophilus and B. bifidum has growth of all four bacteria was better in soy yoghurt
been a problem in cultured dairy foods. The supplemented with 15% banana. This might be due
difficulty of cultivating bifidobacteria in milk, to the growth promoting effect of prebiotics. There
because of lack of acid tolerance or oxygen was a significant difference in the count of L.
sensitivity, was not encountered in these bulgaricus, S. thermophiles, B. bifidum and L.
experiments. In this study, yoghurt and probiotic acidophilus between different treatments during
bacteria grew to high numbers in banana based storage period at 4ºC.

Table 1. Average score of the viability (10-7cfu/g) of

bacterial culture in banana based soy yoghurt

Days T0 T1 T2 T3

S. thermophilus (10-7cfu/g)
1st 8.38±0.08a 8.44±0.05ab 8.52±0.04bc 8.58±0.08c
th
7 8.02±0.08a 8.06±0.05a 8.12±0.08ab 8.22±0.08c
14th 7.20±0.07a 7.400.07b 7.46±0.05b 7.64±0.11c
21th 6.52±0.21a 6.94±0.11b 7.12±0.13bc 7.30±0.12c
th
28 6.26±0.28a 6.64±0.11b 6.80±0.12bc 6.94±0.16c
L. bulgaricus (10-7cfu/g)
1st 8.18±0.13a 8.34±0.08b 8.42±0.08bc 8.48±0.08c
th
7 7.48±0.08a 7.56±0.11a 7.84±0.05b 7.90±0.07b
th
14 6.98±0.08a 7.28±0.08b 7.42±0.14b 7.64±0.16c
21th 6.32±0.14a 6.72±0.21b 6.86±0.18bc 7.04 ±0.21c
28th 6.02±0.17a 6.24±0.15b 6.44±0.15bc 6.60±0.12c
B. bifidum (10-7cfu/g)
1st 8.18±0.13a 8.44±0.05b 8.50±0.07bc 8.60±0.07c
7th 7.10±0.15a 7.78±0.08b 8.04±0.16c 8.10±0.19c
th
14 6.38±0.24a 6.92±0.21b 7.18±0.21b 7.24±0.20b
th
21 6.08±0.08a 6.60±0.07b 6.90±0.25c 6.90±0.12c
28th 5.34±0.33a 6.08±0.08b 6.42±0.17c 6.26±0.16bc
L. acidophilus (10-7cfu/g)
1st 8.40±0.10a 8.56±0.08a 8.60±0.07a 8.68±0.26a
7th 7.80±0.12a 7.92±0.08a 8.06±0.11b 8.28±0.08c
14th 6.94±0.11a 7.24±0.08b 7.40±0.07c 7.58 ±0.08d
th
21 6.0±0.50a 7.06±0.11ab 7.32±0.08bc 7.50±0.07c
28th 6.02±0.08a 6.34±0.05b 6.56±0.08c 6.74±0.11d

Means bearing different superscripts with in the row differ significantly (P< 0.05)

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On 7 th day of storage at 4ºC, S. x l07 cfu/g and L. acidophilus was at 8 x l07 cfu/g.
thermophilus was at a level of 1 x 108 cfu/g, L. Whereas, on 28th day of storage at 40C, the S.
bulgaricus was at 5 x l07 cfu/g, B. bifidum was at 1 thermophilus decreased by two log cycles to 3 x

Fig. 1. Average score of the viability of bacterial count on 1st, 7th, 14th, 21st and 28th day

Fig. 2. Average growth rate of the viability of Streptococcus thermophiles on storage period

Fig. 3. Average growth rate of the viability of Lactobacillus delbrueckii subsp. bulgaricus on storage period
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Fig. 4. Average growth rate of the viability of Bifidobacterium bifidum on storage period

Fig. 5. Average growth rate of the viability of Lactobacillus acidophillus on storage period

106 cfu/g, L. bulgaricus decreased by two log cycle others T1, T0 and T3. Whenever, on 7th day T0 and
to 1 x l06 cfu ml, B. bifidum decreased by three log T1 have not significance difference but T2 and T3
cycles to 3 x 105 cfu/g, L. acidophilus decreased (higher) have significance difference. On 14th, 21st
by two log cycle to 1 x l06 cfu/g viability in the soy and especially 28 th day, T3 sample showed
yoghurt presented in Table 1and Figure 1. significantly higher than compared to the others
With regard to the S. thermophiles counts, T2, T1 and T0 and that day T3 sample are more
on 1st day and 7th day T3 sample was showed effectively compare to 1st and on 7th day (Fig.3).
significantly (P< 0.05) higher than compared to the The counts of B. bifidum, on 1st day T3
others T 2, T1 and T0 sample. On 14th, 21 st and sample was showed significantly higher than
especially 28th day, T3 sample showed significantly compared to the other formulation. Whenever on
(P< 0.05) higher than followed by T2, T1 and T0, 7 th day T 0 between T 1 T 2 and T 3 have higher
respectively and that day T3 sample are more significance difference. On 14th, 21st and especially
effectively compare to 1st and on7th day (Fig.2). 28th day, T3 sample showed significantly
With regard to the L. bulgaricus counts, (P< 0.05) higher than compared to the others T2, T1
on 1st day T2 banana based soy yoghurts showed and T 0 and in which shows banana are more
significantly (P< 0.05) higher than compared to the effectively compare to control (Fig.4).

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The L. acidophillus counts, on 1st, 7th, 14th thereby increasing its effectiveness.” (Macfarlane
th
and 28 day of T3 banana based soy yoghurts et al., 2006).
showed significantly higher than compared to the
others samles. On 21st day showed T0 between T1, CONCLUSION
T2, T3 sample significantly higher difference and
that day T3 sample in which shows banana are This study find that banana based soy
more effectively compare to control (Fig.5). yoghurt may be a suitable medium for delivery of
The average scores of the L. bulgaricus, probiotic bacteria and their numbers stability
S. thermophiles, B. bifidum and L. acidophilus steadily with added banana puree in soy yoghurt
count on 1st, 7th, 14th, 21st and 28th day of during during stored at refrigerator temperature. Thus,
storage also showed (Fig. 2, 3, 4 and 5) a significant prebiotic approach through diet increases
difference between T1, T2 and T3 respectively and resident bacteria which are beneficial to human
more effectivaly that control. Donkor et al. (2006) health. In general, all selected probiotic culture
observed that a banana fibers increased the counts showed appreciable viability and above therapeutic
of all probiotics by no less than 1 log cfu/g value during prolonged storage at 4°C which is an
compared to controls yoghurts, especially at 28 additional of beneficial effect of banana soy
days (Table No. 1). yoghurt.
The number of probiotic organisms in a
probiotic product should meet the suggested ACKNOWLEDGEMENTS
minimum value of >6 log cfu/g to achieve optimal
potential therapeutic effects (Rasic et al., 1978). The author thank to supervisor and
Vinderola et al. (2003) also stated that probiotic departments for providing part of the material
microflora counts decrease during storage. The resources employed in this study. The author
rate of this loss in cell viability depended on the gratefully acknowledge Er. S. N. Thakur for his
yogurt type and the use of lactic starter. assistance with some analyses performed in the
Finally, in this study was found the present study.
banana was a good medium for the enhancing the
growth of yoghurt and probiotic bacteria. Banana References
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