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ORIGINAL ARTICLE
Development of probiotic yogurt: effect of strain combination
on nutritional, rheological, organoleptic and probiotic properties
Richa Soni
1
•Nayan K. Jain
1
•Vidhi Shah
1
•Jinal Soni
1
•Dipali Suthar
1
•
Priyal Gohel
1
Revised: 15 November 2019 / Accepted: 3 January 2020
ÓAssociation of Food Scientists & Technologists (India) 2020
Abstract Seven combinations of yogurt; C
1
[yogurt starter
culture (YSC)], T
1
,[YSC ?Lactobacillus acidophilus
(LA)], T
2
[YSC ?Bifidobacterium bifidum (BB)], T
3
[YSC ?Lactobacillus plantarum (LP)], T
4
[YSC ?Lac-
tobacillus casei (LC)], T
5
[YSC ?LA ?BB] and T
6
[YSC ?LP ?LC] were developed. Nutritional [proximate
and minerals], rheological [total soluble solids (TSS), pH,
titratable acidity (TA), water holding capacity, synersis,
viscosity] organoleptic and probiotic properties [viability,
acid tolerance, bile salt tolerance] were assessed with
standard methods. Nutritional composition differed sig-
nificantly among samples except for the iron and zinc
(P\0.05). Yogurt containing LP as single or in combi-
nation with LC resulted in significantly higher ash, protein,
calcium and phosphorous level. Probiotic combination also
significantly affected the rheological properties of yogurts
(P\0.05). Yogurt with LP and LC as single or in com-
bination lead to significantly higher TSS and viscosity
while significantly low syneresis, whereas yogurt with LA
as single or in combination resulted in low pH and high TA
(P\0.05). Interestingly, combination of LA and BB
increased TSS, reduced pH and syneresis as compare to
these bacteria as single probiotic source. Panel experts
found yogurt with LP more flavourful. Combination of
multi-strain and multi-species probiotic resulted in
improved texture but we found no significant difference in
overall acceptability. Combination of probiotic strains also
resulted in better probiotic potential with multi-species
combination found to be even more effective. BB seemed
more stable than three other probiotic strains. The present
study can be helpful to dairy industry in developing new
probiotic products and may provide a rational for selecting
a combination of probiotic strains.
Keywords Probiotics Nutritional properties Strain
combination Rheological properties Probiotic potential
Introduction
Probiotics are ‘‘live microorganisms, when administered in
adequate amounts confer a health benefit on the host’’
(FAO/WHO 2001).
Different probiotics have been shown to exhibit certain
health benefits common to most or all probiotic species
known as ‘‘core benefits’’ includes regulation of intestinal
transit, normalization of perturbed microbiota, turnover of
enterocytes, competitive exclusion of pathogens, colo-
nization resistance, and short-chain fatty acid production.
Other health benefits are probiotic strain specific this
includes neurological effects, immunological effects,
endocrinological effects, and the production of bioactives
(Scourboutakos et al. 2017).
The Joint FAO/WHO Working Party Report in 2002
suggested that microbes should have a minimum set of
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
the proximal small intestine in the presence of pancreatin
and bile salts (FAO, WHO 2002). Traditional yogurt star-
ters have nonhuman origins, and they (especially strepto-
cocci) are known to suffer from exposure to gastric acidic
conditions (Elli et al. 2006).
&Richa Soni
richasoniricha@gmail.com
1
Present Address: Food Science and Nutrition, Department of
Life Science, University School of Science, Gujarat
University, Ahmedabad, India
123
J Food Sci Technol
https://doi.org/10.1007/s13197-020-04238-3
Substantial amount of scientific research has been con-
ducted in the past to assess the effectiveness of individual
probiotic strain on improving various disease conditions,
assessing knowledge of probiotics among health profes-
sionals (Soni et al. 2018) and understanding their mecha-
nism of action. Probiotic research and development have
traditionally focused on single strains for specific health
application and little information exists on the influence of
probiotic strain combination on nutritional, rheological
properties and sensory characteristics of yogurts and fer-
mented milks. Some studies have reported multi-strain
probiotics as more efficient than single strain for gut and
immune function (Chapman et al. 2011; Wu et al. 2013).
Additionally, it has been suggested that multi-strain pro-
biotic produces better texture and nutritional properties in
cheese than mono-strain probiotic (Setyawardani et al.
2016). That being said, it should be noted that not all strain
mixtures are beneficial, as strains can antagonize one
another. Therefore, research is needed to verify if mixtures
are synergistic or antagonistic (Scourboutakos et al. 2017).
There is a lack of research on multi-strain probiotics
because such research is more difficult to conduct and thus
more expensive. Yogurt physical and sensory properties
are important aspects for consumer acceptability and can
be altered by the addition of some ingredients, acid pro-
duction and microbial growth during fermentation and
storage (Da Silva et al. 2017).
The main objectives of this study were to produce pro-
biotic yogurt with combination of probiotic strain cultures
and to investigate the effect of these combinations on some
nutritional, rheological, sensory and probiotic properties
during refrigerated storage.
Methods and materials
Procurement of cultures
Freeze dried bacterial cultures namely; Lactobacillus del-
brueckiisubsp. bulgaricus (LB) (NCDC-253), Streptococ-
cus thermophilus (ST) (NCDC-199), Lactobacillus
acidophilus (LA) (NCDC-13), Bifidobacterium bifidum
(NCDC-229) (BB), Lactobacillus casei (LC) (NCDC-17)
and Lactobacillus plantarum (LP) (NCDC-20) were pro-
cured from National Dairy Research Institute, Karnal,
India. All the reagents and glassware were sterilized either
by autoclaving or hot air oven for each set of experiment.
Preparation of starter and mother culture
All NCDC cultures were maintained in sterilized recon-
stituted skimmed milk (RSM-11 g skimmed milk powder
in 100 mL distilled water, pasteurized at 82 °C for 30 min
and cooled to 37 °C). The sterilized skimmed milk bottles
were inoculated with freeze dried cultures and incubated at
37 °C for 8 h. All the cultures were then subsequently
inoculated in another set of sterilized RSM test tubes and
incubated at 37 °C for 8 h, thereafter stored at 4 °C. The
propagation of stock cultures (37 °C) was done once in
15 days to maintain their activity, whereas working cul-
tures were freshly prepared in RSM as and when needed.
Skimmed milk was medium heated before propagation.
Cultures were stored at 4 °C between the transfers. Starter
cultures were maintained and propagated individually and
were mixed just before use.
Development of probiotic yogurt
Fresh toned milk (with 3% fat and 8.5% SNF) was heat
treated at 90 °C for 15 min followed by immediate cooling
at 37 °C. This milk was inoculated with a culture combi-
nation comprising yogurt starter culture (ST and LB at the
ratio of 1:1 at 2% v/v) and probiotic cultures at the level of
2% v/v of final milk volume, filled in disposable cups and
sealed with cup sealing machine followed by incubation at
37 °C for 10 h or until pH reached 4.5 then refrigerated at
4°C. Total seven types of yogurts were developed T
1
(YSC ?LA), T
2
(YSC ?BB), T
3
(YSC ?LP), T
4
(YSC ?LC), T
5
(YSC ?LA ?BB), T
6
(YSC ?LP ?LC)
and C
1
(YSC only) served as control.
Assessment of nutritional composition
The yogurt samples were analyzed for proximate including
moisture, ash, protein, fat, carbohydrate and minerals
including calcium, phosphorous iron and zinc as described
in the Association of Official Analytical Chemist proce-
dures (AOAC 2002). Ash was assessed by taking the dried
samples and using a muffle furnace to burn off all non-
mineral matter. Protein content was analyzed using the
Kjeldahl digestion method and a nitrogen conversion factor
of 6.38. Fat was examined using Mojonnier method for fat
analysis of dairy products. Carbohydrate content was then
obtained by determining the variance in total solids to the
other solid components. Calcium, Phosphorous, iron and
zinc were analysed by using atomic absorption spec-
trophotometer after wet digestion (Lindsey and Norwal
1969). Three trials from each of the formulations were
examined for chemical content.
Assessment of rheological properties
Total soluble solids
The total soluble solids (TSS) were determined as per
method given by Mazumdar and Majumder (2003) using
J Food Sci Technol
123
Digital-Bench Refrectometer. Instrument was cleaned with
distilled water and adjusted to zero at 20 °C. Sample was
placed on the prism plate of the refractometer with the help
of the glass rod. The reading appeared on the screen was
directly recorded as total soluble solids. For each sample
instrument was calibrated using distilled water.
Assessment of pH
The pH of different yogurt samples was determined in
duplicate by using M-tronics digital pH meter. The pH was
determined by inserting a pH probe, directly into a
homogenized sample.
Titratable acidity
5 mL of diluted yogurt samples were mixed with 100 mL
of boiling water and titrated with 0.1 N (NaOH) until
reaching the pale pink end point with phenolphthalein
indicator.
Water holding capacity
Water-holding capacity (WHC) was determined using a
procedure given by Guzman-Gonzalez et al. (1999). 20 g
of yoghurt (Y) was centrifuged for 30 min at 12509gat
20 °C. The whey expelled (WE) was removed and
weighed. The WHC was determined as.
WHC ¼100 Y-WEðÞ
Y
Syneresis
Syneresis of the yogurt was assessed through the cen-
trifugation procedure given by Motoki and Seguro (1998).
20 g of yogurt was taken into a 50 mL glass tube and was
centrifuged at 3500 rpm for 15 min at 20 °C. The syneresis
was estimated as the percentage of the released whey over
the initial gel weight.
Syneresis%¼weight of supernatant
weight of yogurt 100
Viscosity
The viscosity was measured using rotational viscometer
(Brookfield model DV II, USA). Samples were put in a
stainless measuring cylinder and viscosity readings taken
on the viscometer at 600 rev/min. Viscosity measurements
were carried out after yogurt production and during
10 days storage at temperature 4 °C using a Brookfield LV
spindle no. 4 at 10 rpm.
Assessment of probiotic potential
Acid tolerance
Acid tolerance of the probiotic bacteria used to prepare
yogurt was estimated via method given by Conway et al.
(1987) and Sahadeva et al. (2011). 1 mL of yogurt sample
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-
bated 37 °C for 3 h. At 0 h (immediately after inoculation)
1 mL sample from each pH tube was inoculated in 9 mL
broth and plated on MRS agar plates. After 1.5, 2, 2.5 and
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.
Broth with pH 6.2 served as a control for the study. Each
assay was performed in duplicates. Acid tolerance was
estimated by comparing the growth of viable cell counts in
all the MRS agar plates after 48 h.
Bile salt tolerance
The effects of bile on the growth of probiotic strains were
examined using methods given by Tsai et al. (2007). Bile
salt tolerance was estimated at the end of the third hour of
acid tolerance test. 5 mL sample from acid tolerance
sample pH 1, 1.5, 2, 3 and 4 was taken in centrifuge tubes
and centrifuged at 4000 rpm for 10 min at 25 °C. After that
the supernatant was discarded and pallets were washed
with PBS and centrifugation was repeated again at
4000 rpm for another 10 min at 25 °C, supernatant was
again discarded and the remaining sample was re-sus-
pended into three MRS broths with different bile salt
concentrations (0.3, 0.5 and 2.0%), incubated aerobically/
anaerobically at 37 °C for 24 h. Subsequently, 0.1 mL was
pipetted out from each of the MRS broth and serial dilu-
tions were performed for plating (duplicates). All the plates
were incubated aerobically/anaerobically at 37 °C for 48 h.
Bile tolerance was determined by comparing the viable cell
counts on MRS agars with and without bile salt. Broth with
0% bile concentration serves as a control for the study.
Viability of probiotic bacteria during storage
The survival rate of the probiotic bacteria was investigated
over 10 days of cold storage at 4 °C at day 0, and 10th.
Yogurt sample was added to phosphate-buffered saline
(PBS) and the appropriate serial dilutions were prepared.
LA was enumerated selectively using deMan, Rogosa,
Sharpe (MRS) agar, LB on MRS agar at pH (5.4) 1 M HCl
was used to adjust the pH of the medium, STM-17 agar,
LPSM (L. plantarum selective medium) was used to enu-
merate LP, MRS-clindamycin-ciprofloxacin (MRS-CC)
agar for selective enumeration of LC and for BB 0.05% L-
J Food Sci Technol
123
cysteine hydrochloride was added in the MRS medium.
Enumeration was carried out using the pour plate tech-
nique. The plates were incubated at 37 °C for at least 72 h
under both aerobiosis and anaerobiosis (Bujalance et al.
2006). After incubation, colonies were counted.
Assessment of storage stability
The packaged yogurt samples were stored at 4 °C for
10 days. Samples were monitored for rheological, micro-
bial, organoleptic properties at 0 day, and at 10th day.
Analysis of microbial count was done by estimating: total
bacterial count (TBC), total mould count (TMC) and total
yeast count (TYC), coliform count, staphylococcus count,
salmonella and shigella count on nutrient agar, potato
dextros agar, mannitol agar, MRS agar, MRS broth,
MacConckey agar and Eosin-methylene blue agar
respectively.
Assessment of sensory properties was done by a panel of
24 members including students and staff of the university.
Yogurt samples were given two-digit codes and were
served chilled. Panellists were provided with a glass of
water and, instructed to rinse their palate with water and
drink water between samples. They were given written
instructions and asked to rate the coded samples on color,
flavour, texture, aroma, consistency, appearance, mouth-
feel, sourness, sweetness and overall acceptability, using a
nine-point hedonic scale [1 = like extremely to 9 = dislike
extremely].
Statistical analysis
The mean value of three measurements was taken for each
parameter assessed in the study. Data obtained from the
nutrient analysis, rheological properties of the samples
were evaluated statistically using a variance analysis
(ANOVA) and the Duncan’s new multiple range test.
Survivability percentage was calculated to assess acid and
bile salt tolerance and paired‘t’test was used to assess the
effect of refrigerated storage on rheological properties,
microbial quality, organoleptic properties and viability of
probiotic bacteria in different yogurt formulations. All the
statistical analysis was done using SPSS version 17.0
(Chicago, USA). The level of significance was set at
P\0.05.
Results
Effect on nutritional composition
The nutritional analysis of the developed probiotic dairy
products revealed (Table 1) that the probiotic strain
combination had significant effect on nutritional composi-
tion. There was approximately 86-88% moisture, 0.6–0.8 g
ash, 3.19–3.93 g protein, 2.0 g fat, 5.0–8.0 g carbohydrate,
53–63 calories, 98–125 mg calcium, 75–93 mg phospho-
rous, 0.32–0.36 mg zinc and 0.2–0.23 mg iron per 100 g
probiotic yogurts.
Nutritional composition differed significantly among
samples except for the iron and zinc (P\0.05). LA and BB
as single probiotic strain or in combination had signifi-
cantly higher moisture, and low carbohydrate level than LP
and LC (P\0.05). Yogurt containing LP as single (T
3
)or
in combination with LC (T
6
) resulted in significantly higher
ash, protein, calcium and phosphorous level in comparison
with other probiotic yogurt samples (P\0.05). Reeta et al.
(2015) and Setyawardani et al. (2016) had also reported
higher protein content in goat cheese produced with mixed
culture (L. rhamnosus and L. plantarum) and varied
nutritional composition of yogurt according to the starter
culture used and length of the fermentation.
Effect on rheological properties
Gel strength and viscosity are important quality indicators
related to consistency and mouth feel of fermented dairy
products and stability of viscosity during the storage is
important qualitative characteristic of yoghurt (Stijepic
´
et al. 2013). ST plays an important role in the production of
exocellular texturing agents called exopolysaccharides that
might interact with the protein content of milk and increase
the viscosity and rheological quality of products.
Total soluble solids
Total soluble solids were found to be significantly higher in
yogurt with LP and LC and their combination (T
3
,T
4
and
T
6
) as compare to LA and BB (P\0.05). It was also
observed that combination of LA and BB improved TSS.
Purwandari et al. (2007) in his study reported that ST helps
in the development of yoghurt texture through
exopolysaccharide (EPS) production which tends to
increase the total solids. Ahluwalia and Kumar (2013) have
also reported ST and LB significantly increases total solids
of the products as compare to LA and Bifidobacterium.
These results are similar with the findings of Istikhar et al.
(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.
pH
Table 1shows the pH values of the seven yogurt formu-
lations which lie within the range 4.38 to 4.49. Yogurt
inoculated with LA and BB produced the lowest yogurt pH,
J Food Sci Technol
123
Table 1 Nutrient composition and rheological properties of yogurt
S. No. Properties C
1
T
1
T
2
T
3
T
4
T
5
T
6
Nutrient composition*
1. Moisture (%) 87.32 ±4.40
b
87.46 ±2.63
b
88.13 ±2.61
b
85.81 ±3.90
a
86.03 ±2.34
a
88.52 ±3.52
b
86.22 ±2.99
a
2. Ash (g) 0.67 ±0.07
a
0.71 ±0.02
a
0.70 ±0.21
a
0.85 ±0.06
c
0.75 ±0.04
b
0.70 ±0.03
a
0.81 ±0.01
b
3. Protein (g) 3.21 ±0.98
b
3.10 ±0.91
b
2.91 ±0.87
a
3.93 ±0.21
c
3.04 ±0.73
b
3.19 ±0.51
b
3.33 ±0.63
c
4. Fat (g) 2.15 ±0.02
b
2.03 ±0.14
a
2.23 ±0.26
c
2.07 ±0.05
b
2.14 ±0.31
b
2.02 ±0.13
a
2.09 ±0.17
b
5. Carbohydrate (g) 6.65 ±0.08
a
6.70 ±0.31
a
6.03 ±0.82
a
7.34 ±0.95
b
8.04 ±1.83
b
5.57 ±0.04
a
7.55 ±0.16
b
6. Energy (Kcal) 59 ±5.03
b
57 ±4.51
a
55 ±6.12
a
63 ±6.1
b
63.58 ±7.31
b
53 ±4.31
a
62 ±3.36
b
7. Calcium (mg) 110.07 ±9.83
c
100.16 ±5.31
a
99.56 ±2031
a
125.61 ±8.13
b
111.23 ±7.41
c
98.77 ±6.47
a
119.07 ±6.14
b
8. Phosphorous (mg) 78.55 ±14.82
a
70.61 ±11.31
a
79.37 ±9.15
a
85.31 ±8.49
b
88.41 ±7.41
b
75.44 ±12.41
a
93.15 ±10.21
b
9 Zinc (mg) 0.31 ±0.06
a
0.35 ±0.03
a
0.31 ±0.09
a
0.33 ±0.05
a
0.36 ±0.06
a
0.33 ±0.03
a
0.36 ±0.01
a
10. Iron (mg) 0.21 ±0.01
a
0.24 ±0.17
a
0.29 ±0.21
a
0.27 ±0.03
a
0.29 ±0.08
a
0.23 ±0.16
a
0.20 ±0.12
a
Rheological properties
1. Total soluble solids % 12.11 ±1.8
b
10.34 ±0.62
a
10.31 ±0.62
a
13.91 ±0.91
c
13.10 ±1.75
c
11.03 ±1.77
b
13.11 ±0.90
c
2. pH 4.43 ±0.31
b
4.39 ±0.01
b
4.44 ±0.03
b
4.40 ±0.31
b
4.49 ±0.52
b
4.38 ±0.28
a
4.40 ±0.3
b
3. Titratable acidity 0.12 ±0.06
b
0.14 ±0.05
a
0.11 ±0.03
b
0.13 ±0.01
b
0.10 ±0.02
b
0.16 ±0.03
a
0.14 ±0.01
a
4. Water-holding capacity % 56.5 ±3.99
a
54.74 ±3.44
a
55.91 ±2.83
a
58.85 ±1.86
a
56.91 ±4.13
a
55.5 ±5.11
a
57.0 ±4.81
a
5. Syneresis (mL) 12.40 ±1.36
b
13.91 ±2.94
c
13.99 ±1.83
c
10.31 ±2.66
a
12.39 ±3.65
b
12.58 ±1.24
b
11.08 ±2.11
b
6 Viscosity (mPa s) 816 ±77
b
776 ±71
a
811 ±28
a
866 ±41
c
823 ±72
b
809 ±82
a
831 ±42
c
*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
C
1
—containing yogurt starter culture (S. thermophilus ?L. bulgaricus), T
1
—(yogurt starter culture ?L. acidophilus), T
2
—(yogurt starter culture ?Bifidobacterium bifidum), T
3
—(yogurt
starter culture ?L. plantarum), T
4
—(yogurt starter culture ?L. casei), T
5
—(yogurt starter culture ?L. acidophilus ?Bifidobacterium bifidum), T
6
—(yogurt starter culture ?L. plan-
tarum ?L. casei)
J Food Sci Technol
123
followed by yogurt with LA as single probiotic culture. pH
values of the yogurt with LP,LC,BB, their combinations
and control yogurt (C
1
) were significantly higher
(P\0.05).
Titratable acidity
Titratable acidity shows opposite trends to pH. For yogurt
T
5
which showed lowest pH titratable acidity was highest.
Lowest titratable acidity was noted in case of yogurt T
4
.
Water holding capacity and syneresis
Water holding capacity was found to be highest in case of
yogurt T
3
while it was lowest for yogurt T
1
. Highest
syneresis was observed in case of T
2
and lowest was in case
of T
3
(P\0.05). Syneresis and water holding capacity
were inversely proportional. The yogurt which had highest
water holding capacity showed lowest syneresis. Ahluwalia
and Kumar (2013) have also reported signified positive
effect of LB and ST on syneresis while LA and Bifidobac-
terium had a negative effect.
Viscosity
Table 1portrays the viscosity of the seven yogurt formu-
lations which was in the range of 776 to 866 mPa s.
According to Lee and Lucey (2010), gelation occurs when
pH is just above the dairy milk isoelectric point (pH 5.2)
and the observed viscosities are indicative of gelation. The
addition of probiotic organisms, especially LP, resulted in a
significantly (P\0.05) higher viscosity compared to
yoghurt with LA,BB,LC and starter culture only. The
initial viscosity of yogurt containing combination of LP
and LC was substantially higher than that containing LA
and BB or control yogurt (C
1
). This may have been caused
by the production of exopolysaccharide (EPS), although
this was not measured in this study. Conversely LB seems
to decrease yogurt viscosity which was also reported by
Dahlan and Sani (2017). Similar to our findings, higher
viscosity values were observed by Donkor et al. (2007).
Our study also showed a substantial effect of the probiotic
organisms on viscosity.
Overall it is stated that yogurt with LP and LC and their
combination had significantly higher TSS, viscosity, pH
and low synersis whereas, yogurt with LA and BB in alone
or in combination had low pH.
Effect on probiotic potential
Acid tolerance
When seven yogurt formulations were subjected to differ-
ent pH concentrations results are given in Table 2.AtpH
1.0 no growth was observed after 1.5 h in any of the for-
mulation. At pH 1.5 yogurts with LA and BB as single
probiotic or in consortium showed growth after 2.5 h.
Control yogurt failed to show any growth after 1.5 h and
yogurt with LP and LC as single or in consortium also
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
and BB survived pH 1.5 till 2 h. Yogurt with LA and BB
was found to be more acid tolerance with 66.1% surviv-
ability rate after 3 h at pH 3.0 as compare to yogurt with
LP and LC with only 44.6% survivability rate. Multispecies
combination resulted in better acid tolerance.
Bile salt tolerance
Results were similar to the trend as acid tolerance. Bacteria
that could not survive at low pH also failed to grow in
subsequent bile test. No growth was observed in case of
sample from pH 1.0 and 1.5 at different bile salt concen-
trations. Highest survivability (99.7) was observed in case
of yogurt T
5
at bile salt concentration 0.3% from the
sample of pH 3.0 after that there was a gradual decline in
viable count as bile concentration increased. Higher inhi-
bition of growth seen as bile salt concentration increased
(Table 3).
Upon exposure to the bile acids cellular homeostasis
disruption causes dissociation of lipid bilayer and integral
protein of their cell membrane that result in leakage of
bacterial content and cell death (Hassanzadazar et al.
2012).
All the bacterial strains except LB and ST showed good
probiotic potential. The survival at pH 3.0 was good in case
of all four probiotic strains but decline in viable count was
observed when samples were subjected to increased acid-
ity. Four strains used in this study have met the criteria to
be considered as good source of probiotics.
Effect on viability of probiotic bacteria
during storage
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
J Food Sci Technol
123
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)
0 h 1.5 h 2 h 2.5 h 3 h
1.0 C
1
3.01 ±0.71 (37.1) – – – –
T
1
3.51 ±0.19 (47.9) – – – –
T
2
3.63 ±1.81(44.3) – – – –
T
3
3.51 ±1.0 (50.6) – – –
T
4
3.59 ±0.71 (47.5) – – –
T
5
4.12 ±0.01 (45.1) – – –
T
6
3.61 ±0.18 (47.6) – – –
1.5 C
1
4.01 ±2.03 (49.4) – – – –
T
1
4.52 ±0.15 (61.7) 1.14 ±1.34 (15.2) 0.88 ±0.61 (11.7) 0.41 ±0.04 (5.6) –
T
2
4.03 ±0.66 (49.2) 0.93 ±0.02 (11.6) 0.63 ±0.93 (7.7) 0.21 ±0.02 (2.6) –
T
3
4.16 ±0.28 (60.0) 2.13 ±1.11 (29.8) 1.01 ±0.63 (14.4) – –
T
4
4.44 ±0.71(58.8) 0.86 ±0.23 (11.4) 0.73 ±0.20 (10.0) – –
T
5
5.24 ±0.14 (57.4) 2.68 ±1.87 (29.6) 2.01 ±1.47 (26.2) 1.01 ±0.56 (11.1) –
T
6
4.12 ±0.64 (54.4) 2.01 ±0.13 (26.7) 1.98 ±0.67 (21.9) – –
2.0 C
1
5.91 ±1.16 (72.8) 2.35 ±0.01 (27.1) – –
T
1
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)
T
2
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)
T
3
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)
T
4
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)
T
5
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)
T
6
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 C
1
5.11 ±0.07 (63.0) 2.37 ±0.81 (27.4) – – –
T
1
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)
T
2
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)
T
3
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)
T
4
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)
T
5
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)
T
6
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 C
1
5.49 ±1.65 (67.6) 3.35 ±0.05 (38.7) 2.61 ±1.40 (34.0) – –
T
1
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)
T
2
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)
T
3
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)
T
4
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)
T
5
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)
T
6
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
(pH 6.2)
C
1
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)
T
1
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)
T
2
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)
T
3
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)
T
4
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)
T
5
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)
T
6
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. C
1
—containing yogurt starter culture (S. thermophilus ?L. bulgaricus), T
1
—(yogurt starter
culture ?L. acidophilus), T
2
—(yogurt starter culture ?Bifidobacterium bifidum), T
3
—(yogurt starter culture ?L. plantarum), T
4
—(yogurt
starter culture ?L. casei), T
5
—(yogurt starter culture ?L. acidophilus ?Bifidobacterium bifidum), T
6
—(yogurt starter culture ?L. plan-
tarum ?L. casei)
J Food Sci Technol
123
viable bacteria at 10th day ranged 87.9 to 98.8. The viable
counts of probiotics were decreased by less than 1 log cycle
in all treatments during storage. Significant reduction was
observed in the viability of all the probiotic bacteria during
storage expect LA with highest viability of 98.80%
(P\0.05) LP had the poorest viability, recorded highest
reduction and only 87% its initial viable population at the
end of storage followed by LC and BB. This decline varied
among probiotic species during storage because of differ-
ent sensitivity to environmental stresses.
Effect on organoleptic properties
The probiotic combination had no significant effect on
color, consistency, appearance, mouthfeel and overall
acceptability of yogurt while flavour, texture, aroma,
sourness and sweetness were significantly altered by the
probiotic strain used (P[0.05). Yogurt with LP was found
to be significantly better in flavour and yogurt with LA was
sourer (P[0.05) (Table 4). Combination of probiotic
strains resulted in better textural qualities. LP has mini-
mum effect on textural qualities of fermented milk.
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% C
1
––– – –
T
1
– – 1.47 ±0.05 (100) 2.91 ±0.07 (100) 3.69 ±0.76 (100)
T
2
– – 1.03 ±0.61 (100) 2.83 ±0.64 (100) 3.33 ±0.48 (100)
T
3
1.61 ±0.72 (100) 2.22 ±0.04 (100) 3.69.0 ±0.41 (100)
T
4
1.11 ±0.41 (100) 2.17 ±0.04 (100) 3.91 ±0.62 (100)
T
5
2.19 ±0.07 (100) 3.61 ±0.05 (100) 4.29 ±0.81(100)
T
6
1.87 ±0.43 (100) 2.61 ±0.01 (100) 3.99 ±0.06 (100)
2. 0.3% C
1
––– – –
T
1
– – 1.23 ±0.12 (83.6) 2.91 ±0.65 (100) 3.66 ±0.19 (99.1)
T
2
– – 1.01 ±0.88 (98.0) 1.78 ±0.41 (62.8) 2.65 ±0.48 (79.5)
T
3
1.51 ±0.58 (93.7) 2.11 ±0.06 (95.0) 3.68 ±0.83 (92.9)
T
4
1.11 ±0.72 (100) 1.99 ±0.91 (91.7) 3.83 ±0.79 (97.9)
T
5
2.02 ±0.14 (92.2) 3.60 ±0.45 (99.7) 4.11 ±0.21 (97.8)
T
6
1.81 ±0.08 (96.7) 2.58 ±0.33 (98.8) 3.91 ±0.85 (97.9)
3. 0.5% C
1
––– – –
T
1
– – 1.10 ±0.72 (74.8) 1.99 ±0.60 (68.3) 2.81 ±0.82 (76.1)
T
2
– – 0.98 ±0.04 (95.1) 1.23 ±0.32 (43.4) 1.84 ±0.11 (55.2)
T
3
1.00 ±0.06 (62.1) 2.14 ±0.58 (96.3) 2.98 ±0.62 (80.7)
T
4
1.01 ±0.81 (90.9) 1.83 ±0.84 (84.3) 3.13 ±0.59 (80.0)
T
5
1.61 ±0.86 (73.5) 2.98 ±0.49 (82.5) 3.63 ±0.14 (84.8)
T
6
1.12 ±0.77 (59.8) 2.15 ±0.46 (82.3) 3.13 ±0.96 (78.4)
4. 2.0% C
1
––– – –
T
1
– – 0.81 ±0.28 (55.1) 0.95 ±0.41 (32.6) 1.57 ±0.41 (42.5)
T
2
– – 0.77 ±0.22 (74.7) 1.10 ±0.42 (38.8) 1.98 ±0.37 (59.4)
T
3
0.69 ±0.29 (42.8) 0.99 ±0.81 (44.5) 1.81 ±0.27 (49.0)
T
4
0.72 ±0.21 (64.8) 1.10 ±0.36 (50.6) 1.86 ±0.50 (47.5)
T
5
1.10 ±0.37 (50.2) 1.45 ±0.07 (40.1) 2.17 ±0.21 (50.5)
T
6
0.78 ±0.81 (41.7) 1.11 ±0.85 (42.5) 1.94 ±0.36 (48.6)
Values in parenthesis show percent survivability. C
1
—containing yogurt starter culture (S. thermophilus ?L. bulgaricus), T
1
—(yogurt starter
culture ?L. acidophilus), T
2
—(yogurt starter culture ?Bifidobacterium bifidum), T
3
—(yogurt starter culture ?L. plantarum), T
4
—(yogurt
starter culture ?L. casei), T
5
—(yogurt starter culture ?L. acidophilus ?Bifidobacterium bifidum), T
6
—(yogurt starter culture ?L. plan-
tarum ?L. casei)
J Food Sci Technol
123
Storage stability
Table 5and Fig. 1shows the effect of refrigerated storage
on rheological, microbial and organoleptic properties of
developed yogurts. No significant change in TSS, total
viable counts, flavour, aroma, appearance, mouthfeel was
observed in all seven formulation during storage
(P\0.05). While, in yogurt with LA and BB, water
holding capacity, sourness, and sweetness changed signif-
icantly (P\0.05). Yogurt T
3
and T
6
with LP and LC noted
significant reduction in viscosity and consistency
(P\0.05). No significant reduction in any parameters of
rheological properties was observed in control yogurt while
the sensory attributes like color, texture and sourness
changed significantly (P\0.05).
Overall it can be concluded that probiotic bacteria have
more significant changes during storage rather than yogurt
starter culture.
Discussion
Yogurt has been a part of human diet in many parts of the
world because of acceptance of its taste (along with
remarkable beneficial effects). Textural properties of
yogurt, such as viscosity, smoothness, thickness acquiring
natural flavours and structural resistance to stress are
important attributes to determine its consumer acceptance,
and these attributes nowadays are accompanied with cer-
tain health benefits (Han et al. 2016).
Different probiotic strain (single or in combination) can
produce yogurts with varied nutritional, rheological and
sensory behaviour according to starter and probiotic culture
used, therefore interactive behaviour amongst probiotic and
yogurt cultures must be evaluated prior to their commercial
application.
Probiotic strains combination had significant effect on
nutritional composition of the probiotic yogurts. Yogurt
with LA and BB produced yogurt with higher moisture and
low calorie while fermentation of yogurt LP and LC
resulted in higher ash, protein, carbohydrate, energy, cal-
cium and phosphorous content.
The rheological and physical characteristics of non-fat
or low-fat yogurt are key parameters for assessing its
quality because reducing the fat content of yogurt results in
alteration in its physico-chemical and sensory properties.
It was observed in this study that addition of probiotic
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
highest syneresis and acidity, On the other hand addition of
LP and LC produced yogurt with more soluble solids,
highest water holding capacity and viscosity and lowest
syneresis.
Syneresis could be influenced by solids content and the
type of starter culture used for the preparation of
yogurt. The increased total solid increases yogurt gel
strength and thereby increases the density and reduces the
pore size, with the result water is bound more firmly which
increases the firmness of the yogurt. Other researchers
(Ahluwalia and Kumar 2013; Dahlan and Sani 2017;
Table 4 Organoleptic properties of developed yogurts
S. No. Organoleptic qualities C
1
T
1
T
2
T
3
T
4
T
5
T
6
1. Color 8.60 ±1.47
a
8.66 ±1.21
a
8.02 ±1.0
a
8.36 ±0.81
a
8.51 ±1.4
a
8.40 ±1.2
a
8.00 ±0.34
a
2. Flavour 7.49 ±0.88
a
8.51 ±0.65
b
7.23 ±1.40
a
9.0 ±0.07
c
7.63 ±0.04
a
8.60 ±1.63
b
7.96 ±1.58
a
3. Texture 7.30 ±1.36
a
8.00 ±1.41
a
7.98 ±1.69
a
7.31 ±1.31
a
7.51 ±1.24
a
8.96 ±1.24
b
8.77 ±1.2
b
4. Aroma 7.67 ±0.81
b
7.51 ±0.52
a
7.20 ±0.37
a
8.85 ±0.38
b
7.60 ±0.83
b
7.21 ±1.31
a
8.16 ±0.47
b
5. Consistency 8.27 ±0.84
a
8.71 ±0.36
a
8.54 ±0.93
a
8.06 ±1.41
a
8.22 ±1.02
a
8.88 ±1.61
a
8.0 ±1.35
a
6. Appearance 8.0 ±1.21
a
8.41 ±0.63
a
8.37 ±0.57
a
8.01 ±0.83
a
8.13 ±0.99
a
8.46 ±0.81
a
8.28 ±1.71
a
7. Mouthfeel 7.95 ±0.92
a
8.23 ±1.31
a
7.92 ±1.11
a
7.58 ±0.69
a
7.68 ±1.03
a
8.13 ±1.64
a
7.92 ±0.81
a
8. Sourness 6.52 ±0.34
a
8.58 ±0.83
c
7.27 ±1.36
b
7.51 ±1.53
b
6.91 ±1.41
a
7.91 ±0.95
b
7.77 ±0.89
b
9. Sweetness 8.26 ±0.98
b
6.66 ±1.05
a
7.53 ±1.24
b
8.21 ±1.48
b
8.51 ±1.40
b
6.96 ±1.31
a
8.81 ±1.39
b
10 Overall acceptability 8.14 ±0.34
a
7.91 ±0.22
a
7.63 ±0.89
a
8.51 ±1.37
a
8.12 ±1.56
a
8.77 ±1.03
a
8.30 ±1.54
a
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. C
1
—containing yogurt starter culture (S. thermophilus ?L. bulgaricus), T
1
—(yogurt starter
culture ?L. acidophilus), T
2
—(yogurt starter culture ?Bifidobacterium bifidum), T
3
—(yogurt starter culture ?L. plantarum), T
4
—(yogurt
starter culture ?L. casei), T
5
—(yogurt starter culture ?L. acidophilus ?Bifidobacterium bifidum), T
6
—(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)
J Food Sci Technol
123
Donkor et al. 2007; Istikhar et al. 2009) have also reported
the varied rheological properties in yogurt fermented with
different probiotic strains.
In order to survive passage through the gastrointestinal
tract, resistance to low pH is important. Acid and bile have
separate and combine effects on the growth of bacteria. As
bile stress takes place after pH stress in the stomach. Sub
lethally injured microorganisms may have a different and
unpredictable resistance to new stress factor. The probiotic
strain must be able to overcome the extremely low pH and
the emulsifying effect of bile salts, and reach the site of
action in a feasible physiological state. The pH in human
stomach ranged from 1 during fasting to 4.5 after a meal
(Soliman et al. 2015) and food ingestion can take up to 3 h.
It was observed in our study that combination of LA and
BB survived at pH 1.5 during an incubation period of 1.5 h
and then the growth was delayed till 2.5 h and after 3 h no
growth was observed in any of the yogurt sample. At pH
2.0, 3.0 and 4.0 the survivability rate for of LA and BB was
noted to be 54, 66 and 64% respectively. This is due to the
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
C
1
0 day 4.43 ±0.31
b
0.21 ±0.41
a
12.11 ±1.80
a
56.5 ±3.99
a
12.40 ±1.36
a
816 ±77
a
10th day 4.41 ±0.91
b
0.19 ±0.62
a
11.99 ±1.35
a
54.31 ±4.11
a
12.41 ±1.20
a
811 ±68
a
T
1
0 day 4.39 ±0.01
b
0.14 ±0.05
a
10.34 ±0.62
a
54.74 ±3.44
a
13.91 ±2.94
b
776 ±71
a
10th day 4.25 ±0.05
a
0.29 ±0.06
b
10.38 ±0.05
a
52.11 ±2.82
a
14.81 ±1.98
c
788 ±69
a
T
2
0 day 4.44 ±0.03
b
0.11 ±0.03
a
10.31 ±0.62
a
55.91 ±2.83
a
13.99 ±1.83
b
811 ±28
a
10th day 4.41 ±0.06
b
0.12 ±0.01
a
10.22 ±0.31
a
54.21 ±3.21
a
14.21 ±1.20
c
801 ±39
a
T
3
0 day 4.40 ±0.31
b
0.13 ±0.01
a
13.91 ±0.91
a
58.85 ±1.86
a
10.31 ±2.66
a
866 ±41
b
10th day 4.35 ±0.22
a
0.16 ±0.02
a
13.71 ±0.82
a
58.21 ±1.72
a
10.79 ±1.42
a
801 ±72
a
T
4
0 day 4.49 ±0.52
b
0.10 ±0.02
a
13.10 ±1.75
a
56.91 ±4.13
a
12.39 ±3.65
a
823 ±72
b
10th day 4.46 ±0.71
b
0.11 ±0.07
a
13.11 ±0.84
a
56.62 ±3.82
a
12.60 ±2.14
a
800 ±68
a
T
5
0 day 4.38 ±0.28
a
0.16 ±0.01
a
11.03 ±1.77
a
55.5 ±5.11
a
12.58 ±1.24
a
809 ±82
a
10th day 4.31 ±0.34
a
0.18 ±0.03
a
11.01 ±0.99
a
52.1 ±4.21
b
13.47 ±1.11
b
798 ±92
a
T
6
0 day 4.40 ±0.3
b
0.17 ±0.01
a
13.11 ±0.90
a
57.0 ±4.81
a
11.08 ±2.11
a
831 ±42
b
10th day 4.37 ±0.19
b
0.15 ±0.34
a
12.58 ±0.85
a
54.05 ±4.27
a
12.12 ±1.81
b
811 ±63
a
Sample Storage days Total viable count Mould count Yeast Count Coliform count Salmonella count Shigella count
Microbial quality (log cfu/mL)
C
1
0 day 6.41 ±0.30
a
Nil Nil Nil Nil Nil
10th day 6.29 ±0.26
a
Nil Nil Nil Nil Nil
T
1
0 day 5.46 ±0.41
a
Nil Nil Nil Nil Nil
10th day 5.11 ±0.25
a
Nil Nil Nil Nil Nil
T
2
0 day 6.77 ±0.57
a
Nil Nil Nil Nil Nil
10th day 5.93 ±0.31
a
Nil Nil Nil Nil Nil
T
3
0 day 5.33 ±0.28
a
Nil Nil Nil Nil Nil
10th day 4.91 ±0.29
a
Nil Nil Nil Nil Nil
T
4
0 day 5.44 ±0.31
a
Nil Nil Nil Nil Nil
10th day 5.41 ±0.28
a
Nil Nil Nil Nil Nil
T
5
0 day 6.81 ±0.26
b
Nil Nil Nil Nil Nil
10th day 6.48 ±0.26
a
Nil Nil Nil Nil Nil
T
6
0 day 5.98 ±0.91
a
Nil Nil Nil Nil Nil
10th day 5.61 ±0.53
a
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
J Food Sci Technol
123
fact that Bifidobacterium stimulates the growth of aci-
dophilus due to the production of acetate (Gomes et al.
1998). Similarly, survivability rate for LP and LC at pH
2.0, 3.0 and 4.0 was found to be 44, 44 and 50%. In con-
trast exposure to pH 2.0 and 3.0 eliminated more than 73%
of LB and ST during an incubation period of 2 h. All four of
our probiotic strains were able to survive pH 3.0 for 3 h
and exhibit good probiotic potential. Good acid survivable
abilities of selected lactic acid bacteria (LP,LC,LA) was
also reported by Srinu et al. (2013) and Soliman et al.
(2015).
Bile salt tolerance is the most crucial property as it
determines the ability of bacteria to survive in the small
intestine, and consequently their capacity to play their
functional role as probiotics. A concentration of 0.3% of
bile salt closely appropriates the bile salt level found in the
gastro intestinal tract (Soliman et al. 2015). All strains
showed considerable difference with regards to growth in
different bile salt concentrations. Highest survivability was
in case of LA and BB combination at 0.3% bile salt con-
centration while LP,LC also showed good survivability at
0.3% level. As the bile salt concentration increased the
survival rate declined. Survival rate reported in the study at
0.3 and 0.5% bile salt concentration are similar to the rates
reported by other researchers (Jamaly et al. 2011; soliman
et al. 2015). These researchers also reported higher bile salt
tolerance by LA as compare to LP and LC. This can be due
to the fact that acidophilus and bifidobacterium display a
variety of proteins devoted to the efflux of bile salts or
protons to modify sugar metabolism or to prevent protein
misfolding. Exopolysaccharides produced by lactic acid
bacteria is also thought to play an important role in the
protection of microbial cells against low pH and bile salts
(Ruiz et al. 2013).
Overall it can be said that the bile salt did not inhibit the
growth of bacteria completely as when subjected to 2% bile
salt there was still a high number of bacterial count. May
be because of stress adaptation mechanism explains the
increased growth with longer incubation hours after pre
exposure to acid stress. Other reason could be enhanced
survival capabilities appeared to be due to acclimatization
of bacteria to the low pH environment therefore, mini-
mizing the relative toxicity to glycoconjugates in the
intestine. The probiotic strains proved to exhibit an
excellent quality of bile salt tolerance.
For dairy products, the sensory properties depend lar-
gely on the relative balance of flavour compounds derived
from carbohydrate, protein or fat in the milk and specific
compounds produced from milk fermentation. Starter cul-
tures make key contributions to the formation of the flavour
compounds in yogurt (Chen et al., 2017).
In case of traditional yogurt starter cultures ST and LB,
these two strains have a symbiotic interaction called
‘‘proto-cooperation’’ in mixed cultures, which means that
they are mutually beneficial during fermentation. It has
been suggested that the level of flavour compounds is much
greater in mixed cultures than single culture due to their
associative growth and mutual stimulation (Tamime and
Robinson 1999). In our study the probiotic combination
had no significant effect on color, consistency, appearance,
mouthfeel and overall acceptability while flavour, aroma,
sourness and sweetness were significantly affected.
Fig. 1 Representation of quantitative organoleptic parameters of
developed yogurts during storage. C
1
—containing yogurt starter
culture (S. thermophilus ?L. bulgaricus), T
1
—(yogurt starter cul-
ture ?L. acidophilus), T
2
—(yogurt starter culture ?Bifidobac-
terium bifidum), T
3
—(yogurt starter culture ?L. plantarum), T
4
—
(yogurt starter culture ?L. casei), T
5
—(yogurt starter culture ?L.
acidophilus ?Bifidobacterium bifidum), T
6
—(yogurt starter cul-
ture ?L. plantarum ?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) was used for
sensory evaluation
J Food Sci Technol
123
These results suggest that in co-fermentation with tra-
ditional starters, the probiotic strains do not influence the
physical appearance of yogurt, but produce different
amount of metabolic products and key aroma-forming
volatile metabolites that results in varied flavour, aroma
and taste.
Other researchers have reported the presence of unique
volatile flavour compounds (2,3 Pentanedione, acetalde-
hyde, acetate) in yogurt supplemented with LP. These
compounds are result of LP metabolism and were absent in
control yogurt (Cheng 2010, Changkun et al. 2017).
As also reported by other researchers that bifidobacteria
often exhibit a characteristic aroma and a slightly acidic
flavour. Fermentation of dairy products with LC alone
resulted in formation of acetic acid, acetoin, butyric acid,
caproic acid, 2-pentanone, and 2-butanone, while the
volatile compounds typical of yogurt were absent. Fer-
mentation with LC and yogurt cultures resulted in greatly
increased levels of 3-hydroxy-2-butanone and hexanoic
acid (Zhuang et al. 2010; Prasanna et al. 2014).
In order to provide the claimed health benefits to
humans, the minimum viable count of probiotic bacteria in
the fermented milks should be C106 colony forming units
(CFU)/g at the end of the shelf-life of the product. The
international standard FIL/IDF describe that the probiotic
products should contained minimum of 106 viable probi-
otic bacteria per gram of product at the time of consump-
tion (Daneshi et al. 2013). In the present study the count of
viable cells was in the range of 5.61 to 6.64 log CFU/mL
which was more than minimum count required to provide
probiotic benefit. In addition, the decline in viability was
dependent on the strain of probiotic. All six strains showed
an acceptable viability with less than one log CFU/mL
reduction at refrigerator temperature for 10 days with
highest viability was reported for LA (98.80%) and lowest
for LP (87.98%).
Yogurt bacteria can suppress probiotics during yogurt
storage via ‘post-acidification’ process which is noticeably
intensified in temperatures of more than 5 °C. Ferdousi
et al. (2013) and Mani-Lo
´pez et al. (2014) had also
reported strain specific survivability during storage with
highest survivability reported in case of LA.
Furthermore, like many other dairy products, yogurt is
prone to deterioration, especially under improper storage
conditions. Generation of volatile by-products leads to off-
flavours and makes the product unsatisfactory for con-
sumers (Chen et al. 2017). It was observed that most of the
sensory and rheological properties were maintained during
refrigerated storage of 10 days among all seven yogurt
formulations. While in LA and BB combination sourness
increased but sweetness and WHC decreased. In contrast
the combination of LP and LC resulted in decreased vis-
cosity and consistency. Control yogurt retained its
rheological and sensory properties throughout the storage
period and the changes were less significant.
Conclusion
So, after considering all the results it can be concluded that
different bacterial combination significantly affects the
nutritional, rheological, organoleptic and probiotic prop-
erties. LA and BB in combination or alone produces yogurt
with higher moisture low calorie, more acidic and more
syneresis, exhibited higher acid tolerance and consumer
acceptability with more sourness and less sweetness.
Whereas, the consortium of LP and LC produced yogurt
with more protein, carbohydrate, calcium, higher viscosity
and lower syneresis. It was also observed that probiotic
yogurt exhibits more changes in rheological and sensory
characteristics than traditional yogurt during storage. Strain
combination affects nutritional, rheological and
organoleptic properties more than the viability and probi-
otic potential.
Combination of multi-strain and multi-species probiotic
resulted in improved texture and better probiotic potential
with multi-species combination found to be even more
effective. Therefore, the selection of mono or multi-strain
probiotics should be based on the required rheological and
organoleptic properties in final product. The present study
can be helpful to dairy industry in developing new probi-
otic products and may provide a rational for selecting a
combination of probiotic strains.
Acknowledgements Authors are thankful to the University Grants
Commission for providing financial assistance for this research work
[Grant no.F.15-1/2011-12/PDFWM-2011-12-O B-RAJ-11103(SA-
II)]. We are also grateful to the Department of Foods and Nutrition
and Department of Fisheries, Maharana Pratap University of Agri-
culture and Technology, Udaipur for providing technical assistance in
estimation of nutrients.
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