J Food Sci Technol (June 2020) 57(6):2038–2050

https://doi.org/10.1007/s13197-020-04238-3

ORIGINAL ARTICLE

Development of probiotic yogurt: effect of strain combination

on nutritional, rheological, organoleptic and probiotic properties
Richa Soni1 • Nayan K. Jain1 • Vidhi Shah1 • Jinal Soni1 • Dipali Suthar1 •

Priyal Gohel1

Revised: 15 November 2019 / Accepted: 3 January 2020 / Published online: 14 January 2020
Ó Association of Food Scientists & Technologists (India) 2020

Abstract Seven combinations of yogurt; C1 [yogurt starter combination found to be even more effective. BB seemed
culture (YSC)], T1, [YSC ? Lactobacillus acidophilus more stable than three other probiotic strains. The present
(LA)], T2 [YSC ? Bifidobacterium bifidum (BB)], T3 study can be helpful to dairy industry in developing new
[YSC ? Lactobacillus plantarum (LP)], T4 [YSC ? Lac- probiotic products and may provide a rational for selecting
tobacillus casei (LC)], T5 [YSC ? LA ? BB] and T6 a combination of probiotic strains.
[YSC ? LP ? LC] were developed. Nutritional [proximate
and minerals], rheological [total soluble solids (TSS), pH, Keywords Probiotics
Nutritional properties
Strain
titratable acidity (TA), water holding capacity, synersis, combination
Rheological properties
Probiotic potential
viscosity] organoleptic and probiotic properties [viability,
acid tolerance, bile salt tolerance] were assessed with
standard methods. Nutritional composition differed sig- Introduction
nificantly among samples except for the iron and zinc
(P \ 0.05). Yogurt containing LP as single or in combi- Probiotics are ‘‘live microorganisms, when administered in
nation with LC resulted in significantly higher ash, protein, adequate amounts confer a health benefit on the host’’
calcium and phosphorous level. Probiotic combination also (FAO/WHO 2001).
significantly affected the rheological properties of yogurts Different probiotics have been shown to exhibit certain
(P \ 0.05). Yogurt with LP and LC as single or in com- health benefits common to most or all probiotic species
bination lead to significantly higher TSS and viscosity known as ‘‘core benefits’’ includes regulation of intestinal
while significantly low syneresis, whereas yogurt with LA transit, normalization of perturbed microbiota, turnover of
as single or in combination resulted in low pH and high TA enterocytes, competitive exclusion of pathogens, colo-
(P \ 0.05). Interestingly, combination of LA and BB nization resistance, and short-chain fatty acid production.
increased TSS, reduced pH and syneresis as compare to Other health benefits are probiotic strain specific this
these bacteria as single probiotic source. Panel experts includes neurological effects, immunological effects,
found yogurt with LP more flavourful. Combination of endocrinological effects, and the production of bioactives
multi-strain and multi-species probiotic resulted in (Scourboutakos et al. 2017).
improved texture but we found no significant difference in The Joint FAO/WHO Working Party Report in 2002
overall acceptability. Combination of probiotic strains also suggested that microbes should have a minimum set of
resulted in better probiotic potential with multi-species characteristics that could predict probiotic potential. These
included the ability to resist passage through the stomach in
the presence of acid and pepsin, and the ability to grow in
& Richa Soni the proximal small intestine in the presence of pancreatin
[email protected] and bile salts (FAO, WHO 2002). Traditional yogurt star-
1 ters have nonhuman origins, and they (especially strepto-
Present Address: Food Science and Nutrition, Department of
Life Science, University School of Science, Gujarat cocci) are known to suffer from exposure to gastric acidic
University, Ahmedabad, India conditions (Elli et al. 2006).

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Substantial amount of scientific research has been con- and cooled to 37 °C). The sterilized skimmed milk bottles
ducted in the past to assess the effectiveness of individual were inoculated with freeze dried cultures and incubated at
probiotic strain on improving various disease conditions, 37 °C for 8 h. All the cultures were then subsequently
assessing knowledge of probiotics among health profes- inoculated in another set of sterilized RSM test tubes and
sionals (Soni et al. 2018) and understanding their mecha- incubated at 37 °C for 8 h, thereafter stored at 4 °C. The
nism of action. Probiotic research and development have propagation of stock cultures (37 °C) was done once in
traditionally focused on single strains for specific health 15 days to maintain their activity, whereas working cul-
application and little information exists on the influence of tures were freshly prepared in RSM as and when needed.
probiotic strain combination on nutritional, rheological Skimmed milk was medium heated before propagation.
properties and sensory characteristics of yogurts and fer- Cultures were stored at 4 °C between the transfers. Starter
mented milks. Some studies have reported multi-strain cultures were maintained and propagated individually and
probiotics as more efficient than single strain for gut and were mixed just before use.
immune function (Chapman et al. 2011; Wu et al. 2013).
Additionally, it has been suggested that multi-strain pro- Development of probiotic yogurt
biotic produces better texture and nutritional properties in
cheese than mono-strain probiotic (Setyawardani et al. Fresh toned milk (with 3% fat and 8.5% SNF) was heat
2016). That being said, it should be noted that not all strain treated at 90 °C for 15 min followed by immediate cooling
mixtures are beneficial, as strains can antagonize one at 37 °C. This milk was inoculated with a culture combi-
another. Therefore, research is needed to verify if mixtures nation comprising yogurt starter culture (ST and LB at the
are synergistic or antagonistic (Scourboutakos et al. 2017). ratio of 1:1 at 2% v/v) and probiotic cultures at the level of
There is a lack of research on multi-strain probiotics 2% v/v of final milk volume, filled in disposable cups and
because such research is more difficult to conduct and thus sealed with cup sealing machine followed by incubation at
more expensive. Yogurt physical and sensory properties 37 °C for 10 h or until pH reached 4.5 then refrigerated at
are important aspects for consumer acceptability and can 4 °C. Total seven types of yogurts were developed T1
be altered by the addition of some ingredients, acid pro- (YSC ? LA), T2 (YSC ? BB), T3 (YSC ? LP), T4
duction and microbial growth during fermentation and (YSC ? LC), T5 (YSC ? LA ? BB), T6 (YSC ? LP ? LC)
storage (Da Silva et al. 2017). and C1 (YSC only) served as control.
The main objectives of this study were to produce pro-
biotic yogurt with combination of probiotic strain cultures Assessment of nutritional composition
and to investigate the effect of these combinations on some
nutritional, rheological, sensory and probiotic properties The yogurt samples were analyzed for proximate including
during refrigerated storage. moisture, ash, protein, fat, carbohydrate and minerals
including calcium, phosphorous iron and zinc as described
in the Association of Official Analytical Chemist proce-
Methods and materials dures (AOAC 2002). Ash was assessed by taking the dried
samples and using a muffle furnace to burn off all non-
Procurement of cultures mineral matter. Protein content was analyzed using the
Kjeldahl digestion method and a nitrogen conversion factor
Freeze dried bacterial cultures namely; Lactobacillus del- of 6.38. Fat was examined using Mojonnier method for fat
brueckiisubsp. bulgaricus (LB) (NCDC-253), Streptococ- analysis of dairy products. Carbohydrate content was then
cus thermophilus (ST) (NCDC-199), Lactobacillus obtained by determining the variance in total solids to the
acidophilus (LA) (NCDC-13), Bifidobacterium bifidum other solid components. Calcium, Phosphorous, iron and
(NCDC-229) (BB), Lactobacillus casei (LC) (NCDC-17) zinc were analysed by using atomic absorption spec-
and Lactobacillus plantarum (LP) (NCDC-20) were pro- trophotometer after wet digestion (Lindsey and Norwal
cured from National Dairy Research Institute, Karnal, 1969). Three trials from each of the formulations were
India. All the reagents and glassware were sterilized either examined for chemical content.
by autoclaving or hot air oven for each set of experiment.
Assessment of rheological properties
Preparation of starter and mother culture
Total soluble solids
All NCDC cultures were maintained in sterilized recon-
stituted skimmed milk (RSM-11 g skimmed milk powder The total soluble solids (TSS) were determined as per
in 100 mL distilled water, pasteurized at 82 °C for 30 min method given by Mazumdar and Majumder (2003) using

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Digital-Bench Refrectometer. Instrument was cleaned with Assessment of probiotic potential

distilled water and adjusted to zero at 20 °C. Sample was
placed on the prism plate of the refractometer with the help Acid tolerance
of the glass rod. The reading appeared on the screen was
directly recorded as total soluble solids. For each sample Acid tolerance of the probiotic bacteria used to prepare
instrument was calibrated using distilled water. yogurt was estimated via method given by Conway et al.
(1987) and Sahadeva et al. (2011). 1 mL of yogurt sample
Assessment of pH was inoculated in MRS broth in tubes with varying pH 1,
1.5, 2, 3, 4 adjusted with 1 M HCl. The tubes were incu-
The pH of different yogurt samples was determined in bated 37 °C for 3 h. At 0 h (immediately after inoculation)
duplicate by using M-tronics digital pH meter. The pH was 1 mL sample from each pH tube was inoculated in 9 mL
determined by inserting a pH probe, directly into a broth and plated on MRS agar plates. After 1.5, 2, 2.5 and
homogenized sample. 3-h 1 mL sample was again inoculated in 9 mL broth form
each tube and plated on MRS agar plates at 37 °C for 48 h.
Titratable acidity Broth with pH 6.2 served as a control for the study. Each
assay was performed in duplicates. Acid tolerance was
5 mL of diluted yogurt samples were mixed with 100 mL estimated by comparing the growth of viable cell counts in
of boiling water and titrated with 0.1 N (NaOH) until all the MRS agar plates after 48 h.
reaching the pale pink end point with phenolphthalein
indicator. Bile salt tolerance

Water holding capacity The effects of bile on the growth of probiotic strains were
examined using methods given by Tsai et al. (2007). Bile
Water-holding capacity (WHC) was determined using a salt tolerance was estimated at the end of the third hour of
procedure given by Guzman-Gonzalez et al. (1999). 20 g acid tolerance test. 5 mL sample from acid tolerance
of yoghurt (Y) was centrifuged for 30 min at 12509 g at sample pH 1, 1.5, 2, 3 and 4 was taken in centrifuge tubes
20 °C. The whey expelled (WE) was removed and and centrifuged at 4000 rpm for 10 min at 25 °C. After that
weighed. The WHC was determined as. the supernatant was discarded and pallets were washed
100  ðY-WEÞ with PBS and centrifugation was repeated again at
WHC ¼ 4000 rpm for another 10 min at 25 °C, supernatant was
Y
again discarded and the remaining sample was re-sus-
pended into three MRS broths with different bile salt
Syneresis concentrations (0.3, 0.5 and 2.0%), incubated aerobically/
anaerobically at 37 °C for 24 h. Subsequently, 0.1 mL was
Syneresis of the yogurt was assessed through the cen- pipetted out from each of the MRS broth and serial dilu-
trifugation procedure given by Motoki and Seguro (1998). tions were performed for plating (duplicates). All the plates
20 g of yogurt was taken into a 50 mL glass tube and was were incubated aerobically/anaerobically at 37 °C for 48 h.
centrifuged at 3500 rpm for 15 min at 20 °C. The syneresis Bile tolerance was determined by comparing the viable cell
was estimated as the percentage of the released whey over counts on MRS agars with and without bile salt. Broth with
the initial gel weight. 0% bile concentration serves as a control for the study.
weight of supernatant
Syneresis% ¼  100 Viability of probiotic bacteria during storage
weight of yogurt

The survival rate of the probiotic bacteria was investigated

Viscosity over 10 days of cold storage at 4 °C at day 0, and 10th.
Yogurt sample was added to phosphate-buffered saline
The viscosity was measured using rotational viscometer (PBS) and the appropriate serial dilutions were prepared.
(Brookfield model DV II, USA). Samples were put in a LA was enumerated selectively using deMan, Rogosa,
stainless measuring cylinder and viscosity readings taken Sharpe (MRS) agar, LB on MRS agar at pH (5.4) 1 M HCl
on the viscometer at 600 rev/min. Viscosity measurements was used to adjust the pH of the medium, STM-17 agar,
were carried out after yogurt production and during LPSM (L. plantarum selective medium) was used to enu-
10 days storage at temperature 4 °C using a Brookfield LV merate LP, MRS-clindamycin-ciprofloxacin (MRS-CC)
spindle no. 4 at 10 rpm. agar for selective enumeration of LC and for BB 0.05% L-

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cysteine hydrochloride was added in the MRS medium. combination had significant effect on nutritional composi-
Enumeration was carried out using the pour plate tech- tion. There was approximately 86-88% moisture, 0.6–0.8 g
nique. The plates were incubated at 37 °C for at least 72 h ash, 3.19–3.93 g protein, 2.0 g fat, 5.0–8.0 g carbohydrate,
under both aerobiosis and anaerobiosis (Bujalance et al. 53–63 calories, 98–125 mg calcium, 75–93 mg phospho-
2006). After incubation, colonies were counted. rous, 0.32–0.36 mg zinc and 0.2–0.23 mg iron per 100 g
probiotic yogurts.
Assessment of storage stability Nutritional composition differed significantly among
samples except for the iron and zinc (P \ 0.05). LA and BB
The packaged yogurt samples were stored at 4 °C for as single probiotic strain or in combination had signifi-
10 days. Samples were monitored for rheological, micro- cantly higher moisture, and low carbohydrate level than LP
bial, organoleptic properties at 0 day, and at 10th day. and LC (P \ 0.05). Yogurt containing LP as single (T3) or
Analysis of microbial count was done by estimating: total in combination with LC (T6) resulted in significantly higher
bacterial count (TBC), total mould count (TMC) and total ash, protein, calcium and phosphorous level in comparison
yeast count (TYC), coliform count, staphylococcus count, with other probiotic yogurt samples (P \ 0.05). Reeta et al.
salmonella and shigella count on nutrient agar, potato (2015) and Setyawardani et al. (2016) had also reported
dextros agar, mannitol agar, MRS agar, MRS broth, higher protein content in goat cheese produced with mixed
MacConckey agar and Eosin-methylene blue agar culture (L. rhamnosus and L. plantarum) and varied
respectively. nutritional composition of yogurt according to the starter
Assessment of sensory properties was done by a panel of culture used and length of the fermentation.
24 members including students and staff of the university.
Yogurt samples were given two-digit codes and were Effect on rheological properties
served chilled. Panellists were provided with a glass of
water and, instructed to rinse their palate with water and Gel strength and viscosity are important quality indicators
drink water between samples. They were given written related to consistency and mouth feel of fermented dairy
instructions and asked to rate the coded samples on color, products and stability of viscosity during the storage is
flavour, texture, aroma, consistency, appearance, mouth- important qualitative characteristic of yoghurt (Stijepić
feel, sourness, sweetness and overall acceptability, using a et al. 2013). ST plays an important role in the production of
nine-point hedonic scale [1 = like extremely to 9 = dislike exocellular texturing agents called exopolysaccharides that
extremely]. might interact with the protein content of milk and increase
the viscosity and rheological quality of products.
Statistical analysis
Total soluble solids
The mean value of three measurements was taken for each
parameter assessed in the study. Data obtained from the Total soluble solids were found to be significantly higher in
nutrient analysis, rheological properties of the samples yogurt with LP and LC and their combination (T3, T4 and
were evaluated statistically using a variance analysis T6) as compare to LA and BB (P \ 0.05). It was also
(ANOVA) and the Duncan’s new multiple range test. observed that combination of LA and BB improved TSS.
Survivability percentage was calculated to assess acid and Purwandari et al. (2007) in his study reported that ST helps
bile salt tolerance and paired‘t’test was used to assess the in the development of yoghurt texture through
effect of refrigerated storage on rheological properties, exopolysaccharide (EPS) production which tends to
microbial quality, organoleptic properties and viability of increase the total solids. Ahluwalia and Kumar (2013) have
probiotic bacteria in different yogurt formulations. All the also reported ST and LB significantly increases total solids
statistical analysis was done using SPSS version 17.0 of the products as compare to LA and Bifidobacterium.
(Chicago, USA). The level of significance was set at These results are similar with the findings of Istikhar et al.
P \ 0.05. (2009) that the natural yogurt contain higher total solids as
compared to probiotic yogurt with LA and BB but less
when compared with LP and LC yogurt.
Results
pH
Effect on nutritional composition
Table 1 shows the pH values of the seven yogurt formu-
The nutritional analysis of the developed probiotic dairy lations which lie within the range 4.38 to 4.49. Yogurt
products revealed (Table 1) that the probiotic strain inoculated with LA and BB produced the lowest yogurt pH,

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Table 1 Nutrient composition and rheological properties of yogurt

S. No. Properties C1 T1 T2 T3 T4 T5 T6

Nutrient composition*
1. Moisture (%) 87.32 ± 4.40b 87.46 ± 2.63b 88.13 ± 2.61b 85.81 ± 3.90a 86.03 ± 2.34a 88.52 ± 3.52b 86.22 ± 2.99a
a a a c b a
2. Ash (g) 0.67 ± 0.07 0.71 ± 0.02 0.70 ± 0.21 0.85 ± 0.06 0.75 ± 0.04 0.70 ± 0.03 0.81 ± 0.01b
3. Protein (g) 3.21 ± 0.98b 3.10 ± 0.91b 2.91 ± 0.87a 3.93 ± 0.21c 3.04 ± 0.73b 3.19 ± 0.51b 3.33 ± 0.63c
b a c b b a
4. Fat (g) 2.15 ± 0.02 2.03 ± 0.14 2.23 ± 0.26 2.07 ± 0.05 2.14 ± 0.31 2.02 ± 0.13 2.09 ± 0.17b
a a a b b a
5. Carbohydrate (g) 6.65 ± 0.08 6.70 ± 0.31 6.03 ± 0.82 7.34 ± 0.95 8.04 ± 1.83 5.57 ± 0.04 7.55 ± 0.16b
b a a b b a
6. Energy (Kcal) 59 ± 5.03 57 ± 4.51 55 ± 6.12 63 ± 6.1 63.58 ± 7.31 53 ± 4.31 62 ± 3.36b
c a a b c a
7. Calcium (mg) 110.07 ± 9.83 100.16 ± 5.31 99.56 ± 2031 125.61 ± 8.13 111.23 ± 7.41 98.77 ± 6.47 119.07 ± 6.14b
8. Phosphorous (mg) 78.55 ± 14.82a 70.61 ± 11.31a 79.37 ± 9.15a 85.31 ± 8.49b 88.41 ± 7.41b 75.44 ± 12.41a 93.15 ± 10.21b
a a a a a a
9 Zinc (mg) 0.31 ± 0.06 0.35 ± 0.03 0.31 ± 0.09 0.33 ± 0.05 0.36 ± 0.06 0.33 ± 0.03 0.36 ± 0.01a
10. Iron (mg) 0.21 ± 0.01a 0.24 ± 0.17a 0.29 ± 0.21a 0.27 ± 0.03a 0.29 ± 0.08a 0.23 ± 0.16a 0.20 ± 0.12a
Rheological properties
1. Total soluble solids % 12.11 ± 1.8b 10.34 ± 0.62a 10.31 ± 0.62a 13.91 ± 0.91c 13.10 ± 1.75c 11.03 ± 1.77b 13.11 ± 0.90c
b b b b b a
2. pH 4.43 ± 0.31 4.39 ± 0.01 4.44 ± 0.03 4.40 ± 0.31 4.49 ± 0.52 4.38 ± 0.28 4.40 ± 0.3b
3. Titratable acidity 0.12 ± 0.06b 0.14 ± 0.05a 0.11 ± 0.03b 0.13 ± 0.01b 0.10 ± 0.02b 0.16 ± 0.03a 0.14 ± 0.01a
a a a a a a
4. Water-holding capacity % 56.5 ± 3.99 54.74 ± 3.44 55.91 ± 2.83 58.85 ± 1.86 56.91 ± 4.13 55.5 ± 5.11 57.0 ± 4.81a
b c c a b b
11.08 ± 2.11b

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5. Syneresis (mL) 12.40 ± 1.36 13.91 ± 2.94 13.99 ± 1.83 10.31 ± 2.66 12.39 ± 3.65 12.58 ± 1.24
b a a c b a
6 Viscosity (mPa s) 816 ± 77 776 ± 71 811 ± 28 866 ± 41 823 ± 72 809 ± 82 831 ± 42c
*Nutrient compositions are per 100 g of the yogurt (wet)
Values with different alphabet within the same row differs significantly according to Analysis of varience (ANOVA) and Duncun’s multiple range test
C1—containing yogurt starter culture (S. thermophilus ? L. bulgaricus), T1—(yogurt starter culture ? L. acidophilus), T2—(yogurt starter culture ? Bifidobacterium bifidum), T3—(yogurt
starter culture ? L. plantarum), T4—(yogurt starter culture ? L. casei), T5—(yogurt starter culture ? L. acidophilus ? Bifidobacterium bifidum), T6—(yogurt starter culture ? L. plan-
tarum ? L. casei)
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followed by yogurt with LA as single probiotic culture. pH Effect on probiotic potential

values of the yogurt with LP, LC, BB, their combinations
and control yogurt (C1) were significantly higher Acid tolerance
(P \ 0.05).
When seven yogurt formulations were subjected to differ-
Titratable acidity ent pH concentrations results are given in Table 2. At pH
1.0 no growth was observed after 1.5 h in any of the for-
Titratable acidity shows opposite trends to pH. For yogurt mulation. At pH 1.5 yogurts with LA and BB as single
T5 which showed lowest pH titratable acidity was highest. probiotic or in consortium showed growth after 2.5 h.
Lowest titratable acidity was noted in case of yogurt T4. Control yogurt failed to show any growth after 1.5 h and
yogurt with LP and LC as single or in consortium also
Water holding capacity and syneresis unsuccessful to grow after 2 h. Only test yogurts were
capable of surviving pH 3.0 and 4.0 after 3 h and only LA
Water holding capacity was found to be highest in case of and BB survived pH 1.5 till 2 h. Yogurt with LA and BB
yogurt T3 while it was lowest for yogurt T1. Highest was found to be more acid tolerance with 66.1% surviv-
syneresis was observed in case of T2 and lowest was in case ability rate after 3 h at pH 3.0 as compare to yogurt with
of T3 (P \ 0.05). Syneresis and water holding capacity LP and LC with only 44.6% survivability rate. Multispecies
were inversely proportional. The yogurt which had highest combination resulted in better acid tolerance.
water holding capacity showed lowest syneresis. Ahluwalia
and Kumar (2013) have also reported signified positive Bile salt tolerance
effect of LB and ST on syneresis while LA and Bifidobac-
terium had a negative effect. Results were similar to the trend as acid tolerance. Bacteria
that could not survive at low pH also failed to grow in
Viscosity subsequent bile test. No growth was observed in case of
sample from pH 1.0 and 1.5 at different bile salt concen-
Table 1 portrays the viscosity of the seven yogurt formu- trations. Highest survivability (99.7) was observed in case
lations which was in the range of 776 to 866 mPa s. of yogurt T5 at bile salt concentration 0.3% from the
According to Lee and Lucey (2010), gelation occurs when sample of pH 3.0 after that there was a gradual decline in
pH is just above the dairy milk isoelectric point (pH 5.2) viable count as bile concentration increased. Higher inhi-
and the observed viscosities are indicative of gelation. The bition of growth seen as bile salt concentration increased
addition of probiotic organisms, especially LP, resulted in a (Table 3).
significantly (P \ 0.05) higher viscosity compared to Upon exposure to the bile acids cellular homeostasis
yoghurt with LA, BB, LC and starter culture only. The disruption causes dissociation of lipid bilayer and integral
initial viscosity of yogurt containing combination of LP protein of their cell membrane that result in leakage of
and LC was substantially higher than that containing LA bacterial content and cell death (Hassanzadazar et al.
and BB or control yogurt (C1). This may have been caused 2012).
by the production of exopolysaccharide (EPS), although All the bacterial strains except LB and ST showed good
this was not measured in this study. Conversely LB seems probiotic potential. The survival at pH 3.0 was good in case
to decrease yogurt viscosity which was also reported by of all four probiotic strains but decline in viable count was
Dahlan and Sani (2017). Similar to our findings, higher observed when samples were subjected to increased acid-
viscosity values were observed by Donkor et al. (2007). ity. Four strains used in this study have met the criteria to
Our study also showed a substantial effect of the probiotic be considered as good source of probiotics.
organisms on viscosity.
Overall it is stated that yogurt with LP and LC and their Effect on viability of probiotic bacteria
combination had significantly higher TSS, viscosity, pH during storage
and low synersis whereas, yogurt with LA and BB in alone
or in combination had low pH. Good viability is a prerequisite for the functionality of
probiotics. It is very important that probiotic strains retain
their viability and functional activity throughout the shelf
life of product. The survival rate of the probiotic bacteria
was assessed as viable counts (log CFU/mL) of all the
bacterial strains used for the development of probiotic
yogurts during storage at 4 °C over 10 days. The percent of

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Table 2 Total plate count for probiotic yogurt on MRS agar at different pH values over 3 h period
pH value Yogurt Total plate count (log CFU/mL)
0h 1.5 h 2h 2.5 h 3h

1.0 C1 3.01 ± 0.71 (37.1) – – – –

T1 3.51 ± 0.19 (47.9) – – – –
T2 3.63 ± 1.81(44.3) – – – –
T3 3.51 ± 1.0 (50.6) – – –
T4 3.59 ± 0.71 (47.5) – – –
T5 4.12 ± 0.01 (45.1) – – –
T6 3.61 ± 0.18 (47.6) – – –
1.5 C1 4.01 ± 2.03 (49.4) – – – –
T1 4.52 ± 0.15 (61.7) 1.14 ± 1.34 (15.2) 0.88 ± 0.61 (11.7) 0.41 ± 0.04 (5.6) –
T2 4.03 ± 0.66 (49.2) 0.93 ± 0.02 (11.6) 0.63 ± 0.93 (7.7) 0.21 ± 0.02 (2.6) –
T3 4.16 ± 0.28 (60.0) 2.13 ± 1.11 (29.8) 1.01 ± 0.63 (14.4) – –
T4 4.44 ± 0.71(58.8) 0.86 ± 0.23 (11.4) 0.73 ± 0.20 (10.0) – –
T5 5.24 ± 0.14 (57.4) 2.68 ± 1.87 (29.6) 2.01 ± 1.47 (26.2) 1.01 ± 0.56 (11.1) –
T6 4.12 ± 0.64 (54.4) 2.01 ± 0.13 (26.7) 1.98 ± 0.67 (21.9) – –
2.0 C1 5.91 ± 1.16 (72.8) 2.35 ± 0.01 (27.1) – –
T1 4.86 ± 1.05 (66.3) 3.16 ± 0.07 (42.3) 2.99 ± 0.82 (39.8) 2.22 ± 0.17 (30.4) 2.03 ± 0.68 (28.0)
T2 4.01 ± 1.01 (48.9) 2.94 ± 0.81 (36.7) 2.16 ± 0.65 (26.6) 1.98 ± 0.12 (24.9) 1.72 ± 0.36 (21.4)
T3 4.42 ± 0.93 (63.7) 3.30 ± 0.51 (46.2) 2.86 ± 0.16 (40.7) 2.84 ± 1.03 (40.5) 2.27 ± 0.49 (34.0)
T4 4.47 ± 0.86 (59.2) 3.56 ± 0.16 (47.4) 2.55 ± 0.53 (35.0) 2.01 ± 0.61 (28.4) 1.96 ± 0.13 (26.6)
T5 5.01 ± 0.51 (54.9) 4.18 ± 0.85 (46.1) 4.06 ± 0.16 (44.9) 4.01 ± 0.09 (44.4) 4.91 ± 0.25 (54.4)
T6 4.49 ± 0.21 (59.3) 3.31 ± 0.07 (44.0) 3.10 ± 0.91 (41.5) 3.17 ± 0.17 (42.6) 3.19 ± 1.12 (44.6)
3.0 C1 5.11 ± 0.07 (63.0) 2.37 ± 0.81 (27.4) – – –
T1 5.28 ± 0.33 (72.1) 4.81 ± 0.92 (65.0) 4.03 ± 1.52 (53.7) 3.63 ± 1.41 (49.7) 3.52 ± 0.82 (48.5)
T2 4.83 ± 0.36 (58.9) 4.01 ± 1.25 (50.1) 4.12 ± 1.83 (50.5) 3.68 ± 1.30 (50.4) 3.43 ± 0.77 (42.8)
T3 4.62 ± 1.27 (66.6) 4.42 ± 1.27 (61.9) 4.16 ± 0.73 (59.3) 3.81 ± 0.62 (54.4) 3.53 ± 0.41 (53.0)
T4 4.95 ± 1.61 (65.5) 4.41 ± 1.31 (58.7) 3.91 ± 1.22 (53.7) 3.01 ± 0.22 (42.0) 3.33 ± 0.63 (45.2)
T5 6.86 ± 0.45 (75.2) 6.17 ± 0.23 (68.1) 6.19 ± 0.72 (68.4) 5.87 ± 1.45 (65.0) 5.96 ± 0.41 (66.1)
T6 5.12 ± 0.02 (67.6) 5.13 ± 0.72 (68.3) 5.26 ± 0.91 (70.5) 4.30 ± 0.81 (57.7) 3.19 ± 0.05 (44.6)
4.0 C1 5.49 ± 1.65 (67.6) 3.35 ± 0.05 (38.7) 2.61 ± 1.40 (34.0) – –
T1 5.91 ± 1.41 (80.7) 5.80 ± 1.61 (77.7) 5.61 ± 1.4 (74.8) 5.71 ± 0.59 (78.3) 5.31 ± 1.82 (73.2)
T2 5.50 ± 1.21 (67.1) 5.42 ± 1.73 (67.7) 5.11 ± 0.86 (62.6) 4.72 ± 1.02 (59.5) 4.16 ± 1.05 (51.9)
T3 5.27 ± 1.04 (76.0) 5.21 ± 0.93 (73.0) 4.93 ± 2.01 (70.3) 4.41 ± 0.83 (63.0) 4.00 ± 1.26 (60.6)
T4 5.00 ± 1.22 (66.2) 4.83 ± 0.85 (64.3) 4.44 ± 1.71 (61.0) 4.12 ± 0.66 (57.4) 4.12 ± 0.74 (55.9)
T5 6.71 ± 0.34 (73.5) 6.49 ± 0.14 (71.7) 6.32 ± 0.61 (69.9) 6.04 ± 0.14 (66.8) 5.81 ± 0.23 (64.4)
T6 5.81 ± 0.81 (76.7) 5.17 ± 0.77 (68.8) 4.73 ± 0.49 (63.4) 4.12 ± 0.51 (55.3) 3.61 ± 1.12 (50.4)
Control C1 8.11 ± 0.26 (100) 8.64 ± 0.41 (100) 7.67 ± 0.96 (100) 8.42 ± 0.99 (100) 7.41 ± 1.30 (100)
(pH 6.2) T1 7.32 ± 0.06 (100) 7.46 ± 0.82 (100) 7.50 ± 0.71 (100) 7.29 ± 0.94 (100) 7.25 ± 0.68 (100)
T2 8.19 ± 0.31 (100) 8.00 ± 0.93 (100) 8.15 ± 0.61 (100) 7.93 ± 0.15 (100) 8.01 ± 0.52 (100)
T3 6.93 ± 0.26 (100) 7.13 ± 0.64 (100) 7.01 ± 0.18 (100) 7.00 ± 0.74 (100) 6.66 ± 0.29 (100)
T4 7.55 ± 0.23 (100) 7.51 ± 0.33 (100) 7.27 ± 0.18 (100) 7.16 ± 0.39 (100) 7.36 ± 0.72 (100)
T5 9.12 ± 0.08 (100) 9.05 ± 0.07 (100) 9.04 ± 0.06 (100) 9.03 ± 0.12 (100) 9.01 ± 0.71 (100)
T6 7.57 ± 0.05 (100) 7.51 ± 0.06 (100) 7.46 ± 0.08 (100) 7.44 ± 0.03 (100) 7.15 ± 0.05 (100)
Values in parenthesis show percent survivability. C1—containing yogurt starter culture (S. thermophilus ? L. bulgaricus), T1—(yogurt starter
culture ? L. acidophilus), T2—(yogurt starter culture ? Bifidobacterium bifidum), T3—(yogurt starter culture ? L. plantarum), T4—(yogurt
starter culture ? L. casei), T5—(yogurt starter culture ? L. acidophilus ? Bifidobacterium bifidum), T6—(yogurt starter culture ? L. plan-
tarum ? L. casei)

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Table 3 Total plate count for probiotic yogurt on MRS agar at different bile salt concentration
S. No. Bile concentration Yogurt Total plate count (log CFU/mL) at different pH value
1.0 1.5 2.0 3.0 4.0

1. 0.0% C1 – – – – –
T1 – – 1.47 ± 0.05 (100) 2.91 ± 0.07 (100) 3.69 ± 0.76 (100)
T2 – – 1.03 ± 0.61 (100) 2.83 ± 0.64 (100) 3.33 ± 0.48 (100)
T3 1.61 ± 0.72 (100) 2.22 ± 0.04 (100) 3.69.0 ± 0.41 (100)
T4 1.11 ± 0.41 (100) 2.17 ± 0.04 (100) 3.91 ± 0.62 (100)
T5 2.19 ± 0.07 (100) 3.61 ± 0.05 (100) 4.29 ± 0.81(100)
T6 1.87 ± 0.43 (100) 2.61 ± 0.01 (100) 3.99 ± 0.06 (100)
2. 0.3% C1 – – – – –
T1 – – 1.23 ± 0.12 (83.6) 2.91 ± 0.65 (100) 3.66 ± 0.19 (99.1)
T2 – – 1.01 ± 0.88 (98.0) 1.78 ± 0.41 (62.8) 2.65 ± 0.48 (79.5)
T3 1.51 ± 0.58 (93.7) 2.11 ± 0.06 (95.0) 3.68 ± 0.83 (92.9)
T4 1.11 ± 0.72 (100) 1.99 ± 0.91 (91.7) 3.83 ± 0.79 (97.9)
T5 2.02 ± 0.14 (92.2) 3.60 ± 0.45 (99.7) 4.11 ± 0.21 (97.8)
T6 1.81 ± 0.08 (96.7) 2.58 ± 0.33 (98.8) 3.91 ± 0.85 (97.9)
3. 0.5% C1 – – – – –
T1 – – 1.10 ± 0.72 (74.8) 1.99 ± 0.60 (68.3) 2.81 ± 0.82 (76.1)
T2 – – 0.98 ± 0.04 (95.1) 1.23 ± 0.32 (43.4) 1.84 ± 0.11 (55.2)
T3 1.00 ± 0.06 (62.1) 2.14 ± 0.58 (96.3) 2.98 ± 0.62 (80.7)
T4 1.01 ± 0.81 (90.9) 1.83 ± 0.84 (84.3) 3.13 ± 0.59 (80.0)
T5 1.61 ± 0.86 (73.5) 2.98 ± 0.49 (82.5) 3.63 ± 0.14 (84.8)
T6 1.12 ± 0.77 (59.8) 2.15 ± 0.46 (82.3) 3.13 ± 0.96 (78.4)
4. 2.0% C1 – – – – –
T1 – – 0.81 ± 0.28 (55.1) 0.95 ± 0.41 (32.6) 1.57 ± 0.41 (42.5)
T2 – – 0.77 ± 0.22 (74.7) 1.10 ± 0.42 (38.8) 1.98 ± 0.37 (59.4)
T3 0.69 ± 0.29 (42.8) 0.99 ± 0.81 (44.5) 1.81 ± 0.27 (49.0)
T4 0.72 ± 0.21 (64.8) 1.10 ± 0.36 (50.6) 1.86 ± 0.50 (47.5)
T5 1.10 ± 0.37 (50.2) 1.45 ± 0.07 (40.1) 2.17 ± 0.21 (50.5)
T6 0.78 ± 0.81 (41.7) 1.11 ± 0.85 (42.5) 1.94 ± 0.36 (48.6)
Values in parenthesis show percent survivability. C1—containing yogurt starter culture (S. thermophilus ? L. bulgaricus), T1—(yogurt starter
culture ? L. acidophilus), T2—(yogurt starter culture ? Bifidobacterium bifidum), T3—(yogurt starter culture ? L. plantarum), T4—(yogurt
starter culture ? L. casei), T5—(yogurt starter culture ? L. acidophilus ? Bifidobacterium bifidum), T6—(yogurt starter culture ? L. plan-
tarum ? L. casei)

viable bacteria at 10th day ranged 87.9 to 98.8. The viable Effect on organoleptic properties
counts of probiotics were decreased by less than 1 log cycle
in all treatments during storage. Significant reduction was The probiotic combination had no significant effect on
observed in the viability of all the probiotic bacteria during color, consistency, appearance, mouthfeel and overall
storage expect LA with highest viability of 98.80% acceptability of yogurt while flavour, texture, aroma,
(P \ 0.05) LP had the poorest viability, recorded highest sourness and sweetness were significantly altered by the
reduction and only 87% its initial viable population at the probiotic strain used (P [ 0.05). Yogurt with LP was found
end of storage followed by LC and BB. This decline varied to be significantly better in flavour and yogurt with LA was
among probiotic species during storage because of differ- sourer (P [ 0.05) (Table 4). Combination of probiotic
ent sensitivity to environmental stresses. strains resulted in better textural qualities. LP has mini-
mum effect on textural qualities of fermented milk.

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Table 4 Organoleptic properties of developed yogurts

S. No. Organoleptic qualities C1 T1 T2 T3 T4 T5 T6

1. Color 8.60 ± 1.47a 8.66 ± 1.21a 8.02 ± 1.0a 8.36 ± 0.81a 8.51 ± 1.4a 8.40 ± 1.2a 8.00 ± 0.34a
a b a c a b
2. Flavour 7.49 ± 0.88 8.51 ± 0.65 7.23 ± 1.40 9.0 ± 0.07 7.63 ± 0.04 8.60 ± 1.63 7.96 ± 1.58a
a a a a a b
3. Texture 7.30 ± 1.36 8.00 ± 1.41 7.98 ± 1.69 7.31 ± 1.31 7.51 ± 1.24 8.96 ± 1.24 8.77 ± 1.2b
4. Aroma 7.67 ± 0.81b 7.51 ± 0.52a 7.20 ± 0.37a 8.85 ± 0.38b 7.60 ± 0.83b 7.21 ± 1.31a 8.16 ± 0.47b
a a a a a a
5. Consistency 8.27 ± 0.84 8.71 ± 0.36 8.54 ± 0.93 8.06 ± 1.41 8.22 ± 1.02 8.88 ± 1.61 8.0 ± 1.35a
a a a a a a
6. Appearance 8.0 ± 1.21 8.41 ± 0.63 8.37 ± 0.57 8.01 ± 0.83 8.13 ± 0.99 8.46 ± 0.81 8.28 ± 1.71a
a a a a a a
7. Mouthfeel 7.95 ± 0.92 8.23 ± 1.31 7.92 ± 1.11 7.58 ± 0.69 7.68 ± 1.03 8.13 ± 1.64 7.92 ± 0.81a
a c b b a b
8. Sourness 6.52 ± 0.34 8.58 ± 0.83 7.27 ± 1.36 7.51 ± 1.53 6.91 ± 1.41 7.91 ± 0.95 7.77 ± 0.89b
9. Sweetness 8.26 ± 0.98b 6.66 ± 1.05a 7.53 ± 1.24b 8.21 ± 1.48b 8.51 ± 1.40b 6.96 ± 1.31a 8.81 ± 1.39b
a a a a a a
10 Overall acceptability 8.14 ± 0.34 7.91 ± 0.22 7.63 ± 0.89 8.51 ± 1.37 8.12 ± 1.56 8.77 ± 1.03 8.30 ± 1.54a
Values with different alphabet within the same row differ significantly according to Analysis of varience (ANOVA) and Duncun’s multiple range
test
Values in parenthesis show percent survivability. C1—containing yogurt starter culture (S. thermophilus ? L. bulgaricus), T1—(yogurt starter
culture ? L. acidophilus), T2—(yogurt starter culture ? Bifidobacterium bifidum), T3—(yogurt starter culture ? L. plantarum), T4—(yogurt
starter culture ? L. casei), T5—(yogurt starter culture ? L. acidophilus ? Bifidobacterium bifidum), T6—(yogurt starter culture ? L. plan-
tarum ? L. casei). 9-point hedonic scale was used to assess the organoleptic qualities of the developed probiotic products
Rating scale (9 = like extremely; 1 = dislike extremely)

Storage stability Different probiotic strain (single or in combination) can

produce yogurts with varied nutritional, rheological and
Table 5 and Fig. 1 shows the effect of refrigerated storage sensory behaviour according to starter and probiotic culture
on rheological, microbial and organoleptic properties of used, therefore interactive behaviour amongst probiotic and
developed yogurts. No significant change in TSS, total yogurt cultures must be evaluated prior to their commercial
viable counts, flavour, aroma, appearance, mouthfeel was application.
observed in all seven formulation during storage Probiotic strains combination had significant effect on
(P \ 0.05). While, in yogurt with LA and BB, water nutritional composition of the probiotic yogurts. Yogurt
holding capacity, sourness, and sweetness changed signif- with LA and BB produced yogurt with higher moisture and
icantly (P \ 0.05). Yogurt T3 and T6 with LP and LC noted low calorie while fermentation of yogurt LP and LC
significant reduction in viscosity and consistency resulted in higher ash, protein, carbohydrate, energy, cal-
(P \ 0.05). No significant reduction in any parameters of cium and phosphorous content.
rheological properties was observed in control yogurt while The rheological and physical characteristics of non-fat
the sensory attributes like color, texture and sourness or low-fat yogurt are key parameters for assessing its
changed significantly (P \ 0.05). quality because reducing the fat content of yogurt results in
Overall it can be concluded that probiotic bacteria have alteration in its physico-chemical and sensory properties.
more significant changes during storage rather than yogurt It was observed in this study that addition of probiotic
starter culture. strain with starter culture exhibited significant alteration in
rheological properties. Addition of LA and BB produced
yogurt with lowest pH and water holding capacity and
Discussion highest syneresis and acidity, On the other hand addition of
LP and LC produced yogurt with more soluble solids,
Yogurt has been a part of human diet in many parts of the highest water holding capacity and viscosity and lowest
world because of acceptance of its taste (along with syneresis.
remarkable beneficial effects). Textural properties of Syneresis could be influenced by solids content and the
yogurt, such as viscosity, smoothness, thickness acquiring type of starter culture used for the preparation of
natural flavours and structural resistance to stress are yogurt. The increased total solid increases yogurt gel
important attributes to determine its consumer acceptance, strength and thereby increases the density and reduces the
and these attributes nowadays are accompanied with cer- pore size, with the result water is bound more firmly which
tain health benefits (Han et al. 2016). increases the firmness of the yogurt. Other researchers
(Ahluwalia and Kumar 2013; Dahlan and Sani 2017;

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Table 5 Rheological and microbial properties of developed yogurt during storage

Sample Storage days pH Titratable acidity Total Solids WHC % Syneresis (mL) Viscosity

Rheological properties
C1 0 day 4.43 ± 0.31b 0.21 ± 0.41a 12.11 ± 1.80a 56.5 ± 3.99a 12.40 ± 1.36a 816 ± 77a
b a a a a
10th day 4.41 ± 0.91 0.19 ± 0.62 11.99 ± 1.35 54.31 ± 4.11 12.41 ± 1.20 811 ± 68a
T1 0 day 4.39 ± 0.01b 0.14 ± 0.05a 10.34 ± 0.62a 54.74 ± 3.44a 13.91 ± 2.94b 776 ± 71a
a b a a c
10th day 4.25 ± 0.05 0.29 ± 0.06 10.38 ± 0.05 52.11 ± 2.82 14.81 ± 1.98 788 ± 69a
b a a a b
T2 0 day 4.44 ± 0.03 0.11 ± 0.03 10.31 ± 0.62 55.91 ± 2.83 13.99 ± 1.83 811 ± 28a
b a a a c
10th day 4.41 ± 0.06 0.12 ± 0.01 10.22 ± 0.31 54.21 ± 3.21 14.21 ± 1.20 801 ± 39a
b a a a a
T3 0 day 4.40 ± 0.31 0.13 ± 0.01 13.91 ± 0.91 58.85 ± 1.86 10.31 ± 2.66 866 ± 41b
10th day 4.35 ± 0.22a 0.16 ± 0.02a 13.71 ± 0.82a 58.21 ± 1.72a 10.79 ± 1.42a 801 ± 72a
b a a a a
T4 0 day 4.49 ± 0.52 0.10 ± 0.02 13.10 ± 1.75 56.91 ± 4.13 12.39 ± 3.65 823 ± 72b
b a a a a
10th day 4.46 ± 0.71 0.11 ± 0.07 13.11 ± 0.84 56.62 ± 3.82 12.60 ± 2.14 800 ± 68a
a a a a a
T5 0 day 4.38 ± 0.28 0.16 ± 0.01 11.03 ± 1.77 55.5 ± 5.11 12.58 ± 1.24 809 ± 82a
a a a b b
10th day 4.31 ± 0.34 0.18 ± 0.03 11.01 ± 0.99 52.1 ± 4.21 13.47 ± 1.11 798 ± 92a
b a a a a
T6 0 day 4.40 ± 0.3 0.17 ± 0.01 13.11 ± 0.90 57.0 ± 4.81 11.08 ± 2.11 831 ± 42b
10th day 4.37 ± 0.19b 0.15 ± 0.34a 12.58 ± 0.85a 54.05 ± 4.27a 12.12 ± 1.81b 811 ± 63a
Sample Storage days Total viable count Mould count Yeast Count Coliform count Salmonella count Shigella count

Microbial quality (log cfu/mL)

C1 0 day 6.41 ± 0.30a Nil Nil Nil Nil Nil
10th day 6.29 ± 0.26a Nil Nil Nil Nil Nil
T1 0 day 5.46 ± 0.41a Nil Nil Nil Nil Nil
10th day 5.11 ± 0.25a Nil Nil Nil Nil Nil
T2 0 day 6.77 ± 0.57a Nil Nil Nil Nil Nil
10th day 5.93 ± 0.31a Nil Nil Nil Nil Nil
T3 0 day 5.33 ± 0.28a Nil Nil Nil Nil Nil
10th day 4.91 ± 0.29a Nil Nil Nil Nil Nil
T4 0 day 5.44 ± 0.31a Nil Nil Nil Nil Nil
10th day 5.41 ± 0.28a Nil Nil Nil Nil Nil
T5 0 day 6.81 ± 0.26b Nil Nil Nil Nil Nil
a
10th day 6.48 ± 0.26 Nil Nil Nil Nil Nil
T6 0 day 5.98 ± 0.91a Nil Nil Nil Nil Nil
10th day 5.61 ± 0.53a Nil Nil Nil Nil Nil

The packaged yoghurt samples were stored at 4 °C for 10 days. Values in parenthesis show percent survivability. C1—containing yogurt starter
culture (S. thermophilus ? L. bulgaricus), T1—(yogurt starter culture ? L. acidophilus), T2—(yogurt starter culture ? Bifidobacterium bifi-
dum), T3—(yogurt starter culture ? L. plantarum), T4—(yogurt starter culture ? L. casei), T5—(yogurt starter culture ? L. aci-
dophilus ? Bifidobacterium bifidum), T6—(yogurt starter culture ? L. plantarum ? L. casei). Values with different alphabet within the same
cell differs significantly according to paired ‘t’ test

Donkor et al. 2007; Istikhar et al. 2009) have also reported the emulsifying effect of bile salts, and reach the site of
the varied rheological properties in yogurt fermented with action in a feasible physiological state. The pH in human
different probiotic strains. stomach ranged from 1 during fasting to 4.5 after a meal
In order to survive passage through the gastrointestinal (Soliman et al. 2015) and food ingestion can take up to 3 h.
tract, resistance to low pH is important. Acid and bile have It was observed in our study that combination of LA and
separate and combine effects on the growth of bacteria. As BB survived at pH 1.5 during an incubation period of 1.5 h
bile stress takes place after pH stress in the stomach. Sub and then the growth was delayed till 2.5 h and after 3 h no
lethally injured microorganisms may have a different and growth was observed in any of the yogurt sample. At pH
unpredictable resistance to new stress factor. The probiotic 2.0, 3.0 and 4.0 the survivability rate for of LA and BB was
strain must be able to overcome the extremely low pH and noted to be 54, 66 and 64% respectively. This is due to the

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Fig. 1 Representation of quantitative organoleptic parameters of acidophilus ? Bifidobacterium bifidum), T6—(yogurt starter cul-
developed yogurts during storage. C1—containing yogurt starter ture ? L. plantarum ? L. casei). 9-point hedonic scale was used to
culture (S. thermophilus ? L. bulgaricus), T1—(yogurt starter cul- assess the organoleptic qualities of the developed probiotic products.
ture ? L. acidophilus), T2—(yogurt starter culture ? Bifidobac- Rating scale (9 = like extremely; 1 = dislike extremely) was used for
terium bifidum), T3—(yogurt starter culture ? L. plantarum), T4— sensory evaluation
(yogurt starter culture ? L. casei), T5—(yogurt starter culture ? L.

fact that Bifidobacterium stimulates the growth of aci- bacteria is also thought to play an important role in the
dophilus due to the production of acetate (Gomes et al. protection of microbial cells against low pH and bile salts
1998). Similarly, survivability rate for LP and LC at pH (Ruiz et al. 2013).
2.0, 3.0 and 4.0 was found to be 44, 44 and 50%. In con- Overall it can be said that the bile salt did not inhibit the
trast exposure to pH 2.0 and 3.0 eliminated more than 73% growth of bacteria completely as when subjected to 2% bile
of LB and ST during an incubation period of 2 h. All four of salt there was still a high number of bacterial count. May
our probiotic strains were able to survive pH 3.0 for 3 h be because of stress adaptation mechanism explains the
and exhibit good probiotic potential. Good acid survivable increased growth with longer incubation hours after pre
abilities of selected lactic acid bacteria (LP, LC, LA) was exposure to acid stress. Other reason could be enhanced
also reported by Srinu et al. (2013) and Soliman et al. survival capabilities appeared to be due to acclimatization
(2015). of bacteria to the low pH environment therefore, mini-
Bile salt tolerance is the most crucial property as it mizing the relative toxicity to glycoconjugates in the
determines the ability of bacteria to survive in the small intestine. The probiotic strains proved to exhibit an
intestine, and consequently their capacity to play their excellent quality of bile salt tolerance.
functional role as probiotics. A concentration of 0.3% of For dairy products, the sensory properties depend lar-
bile salt closely appropriates the bile salt level found in the gely on the relative balance of flavour compounds derived
gastro intestinal tract (Soliman et al. 2015). All strains from carbohydrate, protein or fat in the milk and specific
showed considerable difference with regards to growth in compounds produced from milk fermentation. Starter cul-
different bile salt concentrations. Highest survivability was tures make key contributions to the formation of the flavour
in case of LA and BB combination at 0.3% bile salt con- compounds in yogurt (Chen et al., 2017).
centration while LP, LC also showed good survivability at In case of traditional yogurt starter cultures ST and LB,
0.3% level. As the bile salt concentration increased the these two strains have a symbiotic interaction called
survival rate declined. Survival rate reported in the study at ‘‘proto-cooperation’’ in mixed cultures, which means that
0.3 and 0.5% bile salt concentration are similar to the rates they are mutually beneficial during fermentation. It has
reported by other researchers (Jamaly et al. 2011; soliman been suggested that the level of flavour compounds is much
et al. 2015). These researchers also reported higher bile salt greater in mixed cultures than single culture due to their
tolerance by LA as compare to LP and LC. This can be due associative growth and mutual stimulation (Tamime and
to the fact that acidophilus and bifidobacterium display a Robinson 1999). In our study the probiotic combination
variety of proteins devoted to the efflux of bile salts or had no significant effect on color, consistency, appearance,
protons to modify sugar metabolism or to prevent protein mouthfeel and overall acceptability while flavour, aroma,
misfolding. Exopolysaccharides produced by lactic acid sourness and sweetness were significantly affected.

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These results suggest that in co-fermentation with tra- rheological and sensory properties throughout the storage
ditional starters, the probiotic strains do not influence the period and the changes were less significant.
physical appearance of yogurt, but produce different
amount of metabolic products and key aroma-forming
volatile metabolites that results in varied flavour, aroma Conclusion
and taste.
Other researchers have reported the presence of unique So, after considering all the results it can be concluded that
volatile flavour compounds (2,3 Pentanedione, acetalde- different bacterial combination significantly affects the
hyde, acetate) in yogurt supplemented with LP. These nutritional, rheological, organoleptic and probiotic prop-
compounds are result of LP metabolism and were absent in erties. LA and BB in combination or alone produces yogurt
control yogurt (Cheng 2010, Changkun et al. 2017). with higher moisture low calorie, more acidic and more
As also reported by other researchers that bifidobacteria syneresis, exhibited higher acid tolerance and consumer
often exhibit a characteristic aroma and a slightly acidic acceptability with more sourness and less sweetness.
flavour. Fermentation of dairy products with LC alone Whereas, the consortium of LP and LC produced yogurt
resulted in formation of acetic acid, acetoin, butyric acid, with more protein, carbohydrate, calcium, higher viscosity
caproic acid, 2-pentanone, and 2-butanone, while the and lower syneresis. It was also observed that probiotic
volatile compounds typical of yogurt were absent. Fer- yogurt exhibits more changes in rheological and sensory
mentation with LC and yogurt cultures resulted in greatly characteristics than traditional yogurt during storage. Strain
increased levels of 3-hydroxy-2-butanone and hexanoic combination affects nutritional, rheological and
acid (Zhuang et al. 2010; Prasanna et al. 2014). organoleptic properties more than the viability and probi-
In order to provide the claimed health benefits to otic potential.
humans, the minimum viable count of probiotic bacteria in Combination of multi-strain and multi-species probiotic
the fermented milks should be C 106 colony forming units resulted in improved texture and better probiotic potential
(CFU)/g at the end of the shelf-life of the product. The with multi-species combination found to be even more
international standard FIL/IDF describe that the probiotic effective. Therefore, the selection of mono or multi-strain
products should contained minimum of 106 viable probi- probiotics should be based on the required rheological and
otic bacteria per gram of product at the time of consump- organoleptic properties in final product. The present study
tion (Daneshi et al. 2013). In the present study the count of can be helpful to dairy industry in developing new probi-
viable cells was in the range of 5.61 to 6.64 log CFU/mL otic products and may provide a rational for selecting a
which was more than minimum count required to provide combination of probiotic strains.
probiotic benefit. In addition, the decline in viability was
dependent on the strain of probiotic. All six strains showed Acknowledgements Authors are thankful to the University Grants
Commission for providing financial assistance for this research work
an acceptable viability with less than one log CFU/mL [Grant no.F.15-1/2011-12/PDFWM-2011-12-O B-RAJ-11103(SA-
reduction at refrigerator temperature for 10 days with II)]. We are also grateful to the Department of Foods and Nutrition
highest viability was reported for LA (98.80%) and lowest and Department of Fisheries, Maharana Pratap University of Agri-
for LP (87.98%). culture and Technology, Udaipur for providing technical assistance in
estimation of nutrients.
Yogurt bacteria can suppress probiotics during yogurt
storage via ‘post-acidification’ process which is noticeably
intensified in temperatures of more than 5 °C. Ferdousi References
et al. (2013) and Mani-López et al. (2014) had also
reported strain specific survivability during storage with Ahluwalia S, Kumar P (2013) Effect of yoghurt cultures and probiotic
highest survivability reported in case of LA. cultures on physicochemical and sensory properties of mango
Furthermore, like many other dairy products, yogurt is soy fortified probiotic yoghurt (Msfpy). J Food Process Technol.
https://doi.org/10.4172/2157-7110.1000239
prone to deterioration, especially under improper storage AOAC (2002) Official methods of analysis of the association of
conditions. Generation of volatile by-products leads to off- official analytical chemists. AOAC International, Washington
flavours and makes the product unsatisfactory for con- Bujalance C, Jiménez-Valera M, Moreno E, Ruiz-Bravo A (2006)
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