Effect of Synbiotic Interaction of

Fructooligosaccharide and Probiotics on

the Acidification Profile, Textural and
Rheological Characteristics of Fermented
Soy Milk
Shalini Mishra & H.N.Mishra

Food and Bioprocess Technology

An International Journal

ISSN 1935-5130

Food Bioprocess Technol

DOI 10.1007/s11947-012-1021-4

1 23
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 Author's personal copy
Food Bioprocess Technol
DOI 10.1007/s11947-012-1021-4

ORIGINAL PAPER

Effect of Synbiotic Interaction of Fructooligosaccharide

and Probiotics on the Acidification Profile, Textural
and Rheological Characteristics of Fermented Soy Milk
Shalini Mishra & H. N. Mishra

Received: 29 May 2012 / Accepted: 3 December 2012

# Springer Science+Business Media New York 2012

Abstract Preparation of yoghurt-like product from non-dairy TA Texture profile

raw material, such as soy with probiotic and prebiotic is a EPS Exopolysaccharide
novel development in the field of fermented functional foods.
This research work aimed at finding the new combinations of
probiotics Lactobacillus acidophilus, Lactobacillus plantarum
Introduction
and Lactobacillus rhamnosus, which can give good product
characteristics to fermented soy milk. Fructooligosaccharide
The consumption of functional foods and beverages con-
was added in an attempt to reduce the after-taste of soymilk,
taining probiotic microorganisms is a growing worldwide
improve acidification rates and growth of probiotics.
trend (Verbeke 2005). Probiotics are live microorganisms
Acidification rate was enhanced with L. acidophilusL.
which, when administered in adequate amounts, provide a
plantarum and L. acidophilusL. plantarumL. rhamno-
confer health benefit to the host (Araya et al. 2002).
sus, resulting in a shorter time to reach pH 4.5.
Prebiotics are non-digestible substances, such as fructooli-
Hardness was significantly (P < 0.05) higher for soy
gosaccharide (FOS) and inulin, which provide beneficial
yoghurt fermented by binary co-culture followed by
physiological effect on the host by selectively stimulating
mixed cultures. All the samples showed higher G
the favourable growth or activity of a limited number of
(1,279.701405 Pa) and lower tan (0.2730.346) val-
indigenous bacteria (FAO/WHO 2001). A food product
ues which signifies firmer and solid-like character of the
containing both probiotics and prebiotics is named as syn-
gel formed by probiotic bacteria. Soy yoghurt made
biotic (Homayouni et al. 2007) resulting in an increase in the
with L. acidophilusL. plantarum resulted in improved
probiotic count and the reduction of pathogen microorgan-
product characteristics with shorter tpH 4.5 (4.28 h). Soy
isms in the gut (Vernazza et al. 2006). Incorporation of
yoghurt fermented with L. acidophilusL. plantarum
prebiotics also improves the sensory profile, physicochem-
showed more than 9 log cfu/ml count which is required
ical and rheological characteristics of probiotic-fermented
for probiotic functional food.
products (Mohammadi and Mortazavian 2011). Various
food products have been developed as carriers for probiot-
Keywords Probiotics . Fructooligosaccharide . Synbiotic .
ics, mainly of dairy origins because consumers are com-
Soy yoghurt . Binary co-culture . Mix culture
monly associated with fermented dairy products and
perceive health benefit in the presence of probiotic cultures
Abbreviations
(Sanders 2000). Few drawbacks associated with the fer-
FOS Fructooligosaccharide
mented milk, mainly the increasing prevalence of the lactose
TPA Texture profile analysis
intolerance and the level of cholesterol, make essential to
explore some other non-dairy sources as suitable substrate
for the probiotics (Prado et al. 2008).
Soy is an excellent raw material for the development of
S. Mishra (*) : H. N. Mishra
probiotic non-dairy functional foods to overcome the limita-
Agricultural and Food Engineering Department, Indian Institute of
Technology Kharagpur, Kharagpur 721302 West Bengal, India tions associated with dairy products. The benefits of soy have
e-mail: sharahine@gmail.com drawn much attention recently and numerous soy products
 Author's personal copy
have been evaluated as possible probiotic vehicles. Soy-based
fermented foods may provide additional benefits for the con-
sumer due to their various functional properties such as hypo-
lipidemic, anticholesterolemic and antiatherogenic and
reduced allergenicity (Lopez-Lazaro and Akiyama 2002).
Soy milk has low acidification rate and slow growth of
probiotic bacteria which take longer time to complete the
fermentation and produces undesirable changes in the prod-
uct which is not acceptable to the consumer (Donkor et al.
2007). A rapid acidification of the raw material prevents
growth of undesirable microorganisms and is also essential
for aroma, texture and flavour of the end-product (Vuyst
2000). According to Champagne et al. (2009), the develop-
ment of a fermented soy product containing probiotics
requires strain selection for the ability to grow in the sub-
strate, as well as the ability to compete or even establish a
synergy between strains. Donkor et al. (2005) reported that
the protein in fermented soymilk could encourage the
growth of many probiotic strains such as Lactobacillus
acidophilus, Lactobacillus casei and Streptococcus thermo-
philus. Scientific research has shown that probiotic contain-
ing soy-fermented beverages present a good sensory
acceptance by potential consumers (Shimakama et al. 2003).
FOS is fructose oligomer linked to glucose and/or fruc-
tose molecules that contain up to ten sugar moieties. With
prebiotic properties, FOS is highly soluble in water (Franck
2002) and low-calorie carbohydrates (Roberfroid 1993).
Dosage levels of FOS in the range of 250 % (w/w) are
recommended for various food formulations (Franck 2002).
Hauly et al. (2005) reported that soy yoghurt supplemented
with FOS presented acceptance index above 70 %.
The texture (Cobos et al. 1995) and taste of soy yoghurt
are essential attributes for product acceptability by the con-
sumers (Donkor et al. 2007). There are very few studies on
the textural and rheological properties of soy yoghurt con-
taining probiotics (Donkor et al. 2007). Gel formation of soy
milk proteins is a key process step in the manufacture of
non-dairy-fermented product like yoghurt. The rheological
properties of set gels determine the texture, organoleptic
properties and shelf-life of the product (Cayot et al. 2008;
Lee and Lucey 2006). However, not much information is
available in the literature about the effect of probiotic and
their combinations without yoghurt culture (Streptococcus
salivarius subsp. thermophilus and Lactobacillus del-
brueckii subsp. bulgaricus) on the fermentation kinetics
and textural characteristics of fermented soy milk.
Preliminary experiments were conducted on probiotic soy
yoghurt without any supplementation, but due to poor textural
and rheological characteristics product had poor sensory ac-
ceptance. Therefore, the effect of FOS and their synbiotic
interactions with various probiotic and their combinations
was studied for the production of soy yoghurt with improved
acidification, textural and rheological characteristics. A
Food Bioprocess Technol
minimum concentration of 2 % FOS as recommended in dairy
products (Franck 2002) was fixed to study the effect of pro-
biotic on the various characteristics of soy yoghurt. Also FOS
prebiotic effect on the growth of L. acidophilus, L. rhamnosus
and L. plantarum, either in pure cultures or in binary co-
cultures or in a combination was examined.
Materials and Methods
Microbial Cultures
Probiotic cultures, L. plantarum NCDC414, L. acidophilus
NCDC11, L. rhamnosus NCDC24 and yoghurt culture, S.
salivarius subsp. thermophilus NCDC118 and L. del-
brueckii subsp. bulgaricus NCDC290 procured in freeze
dried from National Collection of Dairy Culture, National
Dairy Research Institute, Karnal, India were used in this
study. The cultures of Lactobacilli spp. were activated by
inoculating in MRS and S. thermophillus in M17 broth
followed by incubation for 24 h at 37 C. The activated
cultures were used after three successive transfers for the
preparation of stock cultures for soy yoghurt making. Stock
cultures were prepared by mixing 24-h grown-activated
cultures with sterilized 80 % glycerol (v/v) in a 1:1 ratio in
Eppendorf tubes and stored at 40 C for further analysis.
For the preparation of the inocula, 100 mL of sterile MRS
and M17 broths were inoculated with 1 mL of a thawed
stock culture and incubated at 37 C until a pH of 4.5 was
reached (Champagne et al. 2009). The incubation time var-
ied between 12 to 14 h as a function of the strain. The
cultures were centrifuged (4,500g, 10 min, 4 C), pallet
washed in peptone water and re-suspended with peptone
water to obtained the final concentration of inoculum
10.74 to 10.98 log cfu/ml. This concentration was selected
on the basis of preliminary experiments to reduce the fer-
mentation time and maintain the desirable viable culture
concentration during storage.
Production of Soy Milk and Soy Yoghurt
Soybeans (100 g) were soaked in one litre water for 16 h at
25 C. Soaked beans were mixed with 350 ml warm water
(6570 C) and macerated in mixer grinder for 3 min, fil-
tered with single-layer muslin cloth and steamed for 15 min
at 10 psi/g pressure. The soy milk preparation was standard-
ized to adjust the protein and fat per cent as recommended
for set type of yoghurt (Tamime et al. 1995; Codex 2008).
Soymilk samples thus prepared contained total solid (10
12 %), total soluble solids (810Brix), protein (4.89
5.15 %) and fat (1.832.23 %) having pH 6.106.22. To
observe the effect of prebiotics 2 % (w/v) FOS (Swetoos,
EnSigns Health Care Pvt. Ltd., India) was mixed in soy milk
 Author's personal copy
Food Bioprocess Technol
before steaming. Batches of yoghurt were prepared in
100 ml glass beaker, containing 50 ml of soymilk by inoc-
ulation of culture in sterilized condition. Probiotic yoghurts
and yoghurt with basic cultures were prepared by inoculat-
ing soy milk with about 10.94, 10.72, 10.98, 10.74 and
10.90 log cfu/ml each of L. acidophilus, L. plantarum, L.
rhamnosus, S. salivarius subsp. thermophilus and L. del-
brueckii subsp. bulgaricus cultures respectively. After inoc-
ulation, beakers were transferred to incubator and batch
fermentations were performed at 40 C up to pH 4.5 (se-
lected as the condition to stop the fermentation).
Acidification Kinetics Parameters
The changes in pH during fermentation were carried out in
triplicates monitored continuously by means of a glass elec-
trode pH meter (Model: 361; Make: Systronics) at an inter-
val of 15 min (Varghese and Mishra 2008). The acidification
rate (Vmax) was calculated as the time variation of pH (dpH/
dt) and expressed as pH units/min. At the end of incubation,
the following kinetic parameters were calculated: (1) tVmax ,
time to reached Vmax (h); (2) pHVmax , pH at Vmax (3) tpH 4. 5,
time to complete the fermentation (in hours).
Post-acidification
On completion of fermentation, post-acidification was de-
termined by pH measurement on 1st, 7th and 28th days of
storage at 4 C.
TPA
Texture profile analysis (TPA) of fermented soymilk was
measured using a Texture Analyzer (Model: TA.XT-2,
Make: Stable Microsystems, UK) equipped with 25 kg load
cell (Kumar and Mishra 2004) at 200.5 C. The data
obtained from the texture profile (forcetime) curve were
used for the calculation of hardness (in grams), cohesive-
ness, adhesiveness (in grams second), springiness and gum-
miness (in grams) (Kumar and Mishra 2004).
Rheological Measurements
Small amplitude oscillatory measurements were used to mon-
itor the gelation process and viscoelastic properties of the set
gels as per method given by Ferragut et al. (2009). The
rheological properties were monitored on a Bohlin rheometer
(Model: Gemini 200 Make: Bohlin Instruments UK,
Cirencester, Gloucestershire, UK) using a parallel plate ar-
rangement with 2.5 mm gap (Model No. PP 20) at a constant
temperature of 200.1 C. The temperature was regulated by
a viscotherm VT 2 circulating bath and controlled with a
Peltier system. The data of all rheological measurements were
analyzed with the supporting software Bohlin Gemini 200.
After gentle stirring with a plastic spoon, a small sample of
soy yoghurt was deposited on the inset plate. First, amplitude
sweep test was performed using a frequency of 1 Hz to
ascertain viscoelastic properties followed by frequency sweep
test (0.1 to 10 Hz at a maximum strain of 0.06). Measurements
were carried out within the linear viscoelastic range, previous-
ly determined by a stress sweep test from 0.01 to 100 Pa.
Dynamic moduli (G, G and G*) and tan were calculated
from frequency sweep test. G is related to the molecular
events of elastic nature; G to the viscous character of mate-
rial; G* (complex modulus), corresponds to (G2 +G2)1/2 and
phase angle (tan ), is calculated as G/G (values closer to 0
have more solid like structure) (Ferragut et al. 2009).
Microbial Analyses
Serial ten-fold dilutions were prepared in a solution of 0.9 %
NaCl(w/v) and 0.1 % (w/v) bactopeptone and suitable dilutions
were plated on appropriate media. Enumeration of L. del-
brueckii subsp. bulgaricus and S. salivarius subsp. thermophi-
lus was done on Lactobacilli MRS media and Streptococcus
Selection HiVeg Agar respectively which were aerobically
incubated at 37 C for 48 h (Dave and Shah 1996). L. acid-
ophilus was enumerated on a modified MRS agar with clinda-
mycin (Oliveira et al. 2011). Selective media for L. rhamnosus
was prepared as per method given by Farnworth et al. (2007).
Enumeration of L. plantarum was done in modified MRS agar
with ciproflaxin antibiotics (Bujalance et al. 2006).
Statistical Analysis
ANOVA was carried out to determine significant differences
between samples at the 95 % level of confidence, using the
SPSS 17 System for windows (SPSS Statistical Software,
Inc., Chicago, IL) software package. Data were obtained
from three independent experiments using Duncan test.
Results and Discussion
Acidification Kinetics
The acidification kinetics of yoghurt were characterized by
Vmax, tVmax , pHVmax and tpH 4.5 (Spinnler and Corrieu 1989).
Acidification profile of FOS-supplemented soy milk fer-
mented by single, co-cultures and mixed culture of L. acid-
ophilus, L. plantarum, L. rhamnosus and yoghurt culture are
given in Table 1. The highest value of Vmax (23.33103 pH
units/min) was obtained with the L. acidophilusL. plantarum
and S. thermophilusL. bulgaricus. The lowest Vmax value
was observed with the L. plantarum (14.0103 pH units/
min) demonstrating its poor acidification ability as compared
 Author's personal copy
Food Bioprocess Technol

Table 1 Acidification kinetic parameters of FOS-supplemented soy milk fermented by single, co-cultures and mixed culture of Lactobacillus
acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus and yoghurt culture (Streptococcus thermophilusLactobacillus bulgaricus)

S. No. Culture combination Vmax (103 pH units/min) tVmax (h) pHVmax tpH 4.5 (h)

1 L. acidophilus 18.00.34 c 3.000.2 b 5.540.13 b 5.750.19 c

2 L. plantarum 14.01.00 a 2.751.00 a, b 5.480.27 b 5.50.15 c
3 L. rhamnosus 15.330.33 a, b 2.500.12 a 5.600.28 b 5.830.37 c
4 L. acidophilusL. plantarum 23.331.33 d 3.530.36 c 5.150.15 a, b 4.280.08 b
5 L. acidophilusL. rhamnosus 17.331.1 c 3.550.45 c 4.900.40 a 4.470.23 b
6 L. plantarumL. rhamnosus 16.330.67 b, c 3.980.05 c 5.270.15 a, b 4.570.55 b
7 L. acidophilusL. plantarumL. rhamnosus 22.01.00 d 3.770.23 c 5.370.13 b 4.680.20 b
8 S. thermophilusL. bulgaricus 23.330.67 d 2.300.30 a 5.560.33 b 3.470.11 a

All values are meansstandard deviations of data from three independent experiments. Different lowercase letters (ad) in the same column
indicate significant difference (P<0.05)

with others. However, L. plantarum showed better acidifica-
tion rate in combination with L. acidophilus, L. rhamnosus
and mixture of all. Binary and mixed cultures of the probiotic
microorganism increased the average acidification rate of
sample significantly (P<0.05) by about 16.1 and 28 % as
compared with single culture. The Vmax value for yoghurt
culture (S. thermophilusL. bulgaricus) was 32.36, 15.8 and
6 % higher to average Vmax of single, binary and mixed
cultures of probiotic, respectively. The largest increase in this
parameter was observed for L. acidophilus as single, binary
and mix culture. The ability of L. acidophilus to ferment FOS
more effectively than other microorganisms (Barrangou et al.
2003) might be the primary reason for the higher acidification
rate which was significantly (P<0.05) higher from L. planta-
rum and L. rhamnosus. Another probable reason for higher
acidification rate of L. acidophilusL. plantarum, L. acid-
ophilusL. rhamnosus and L. acidophilusL. plantarumL.
rhamnosus might be release of compounds like peptides by
L. acidophilus during fermentation which could have stimu-
lated the metabolic process of other lactic acid bacteria
(Oliveira et al. 2011).
Champagne et al. (2000) and Fonseca et al. (2000) stud-
ied the acidification kinetics of different strains of S. ther-
mophilus and Lactobacilli and reported Vmax, tVmax , pHVmax
and tpH 4.5 are strain dependent. These parameters are also
dependent on the buffering capacity of milk (Xanthopoulos
et al. 1994). The tVmax value for probiotic cultures ranged
from 2.50 to 3.98 h. It was minimum (2.50 h) for L. rham-
nosus and maximum (3.98 h) for L. plantarumL. rhamno-
sus. The L. acidophilus and L. plantarum showed no
significant difference in the values of tVmax . The tVmax value
of L. rhamnosus was significantly (P<0.05) lower than that
of L. acidophilus (3.00 h). Average tVmax (3.69 h) for the all
the probiotic binary co-cultures was significantly (P<0.05)
higher than S. thermophilusL. bulgaricus binary co-
cultures (2.30 h). Soy milk containing mixture of all the
probiotics showed similar tVmax values as binary co-cultures.
The tVmax value was higher by 0.07 times for L. acidophilus,
1.13 times for L. plantarum and 1.18 times for L. rhamnosus
compared with the average of all the binary co-cultures.
Oliveira et al. (2009c) studied the effect of culture combi-
nations on the acidification kinetic parameters and reported
similar findings. Apparently, there was no direct relationship
between Vmax and tVmax , in the presence of single probiotic
culture or in the combination.
Highest Vmax and lowest tVmax were seen for the S. thermo-
philusL. bulgaricus which might be due to symbiotic growth
patterns of the yogurt cultures in soy milk. Tamime and
Robinson (1985) reported that the Streptococci initiate the
milk fermentation and that Lactobacilli contribute to acidifi-
cation later in the incubation. S. thermophilus is well able to
grow in soy beverages because of its ability to use sucrose and
other oligosaccharides present in soy milk (Chumchuere and
Robinson 1999). Similar behaviour might be explained for L.
acidophilusL. plantarum which showed highest Vmax and
lower tpH 4.5.
The pHVmax value represented the H+ ion concentration at
the time of Vmax and maximum acid-producing ability of
cultures. All the probiotic culture in single and mixture
showed no significant difference in pHVmax . The pHVmax value
was significantly (P<0.05) lower for L. acidophilusL. rham-
nosus as compared with single, mix and yoghurt cultures.
Time to reach pH 4.5 ranged between 3.47 and 5.83 h,
which was significantly (P<0.05) different for all the pro-
biotic as well as yoghurt culture. Minimum fermentation
time was taken by yoghurt culture (S. thermophilusL. bul-
garicus) followed by L. acidophilusL. plantarum which
was statistically significant (P<0.05). L. acidophilus, L.
plantarum and L. rhamnosus took longer time to complete
the fermentation which was 5.75, 5.5 and 5.83 h, respec-
tively; however this difference was not significant among
each other. All the binary combination and mixture of pro-
biotic showed significant (P<0.05) lower tpH 4.5 as com-
pared with single culture. Cultures in binary combination
 Author's personal copy
Food Bioprocess Technol
and in mixture resulted in increased rate of fermentation and
reduced fermentation time (Marafon et al. 2010; Kristo et al.
2003) might be due to simultaneous symbiotic activity of
two bacteria. It is possible that the simultaneous presence of
two or more than two microorganisms could have led to
synergistic effects and associated to the release of metabolic
products stimulating the metabolism of the others (Radke-
Mitchell and Sandine 1986). Oliveira et al. (2009a) explored
various prebiotic effects on the probiotic-fermented milk
and reported the improved acidification kinetics with FOS.
These results as a whole not only confirm that S. thermo-
philusL. bulgaricus is the best co-culture for FOS-
supplemented soy yoghurt preparation but also that the
alternative probiotics employed in this study (L. acidophi-
lus, L. plantarum and L. rhamnosus) in binary and mixture
combination would be able to ensure satisfactory results in
terms of acidification and fermentation time.
Post-acidification Changes
The results of post-acidification study of probiotic-
fermented soy milk are shown in Fig 1. This analysis was
conducted for 28 days of storage at an interval of 7 days at
4 C after the completion of fermentation. Accordingly to
Souza (1991), post-acidification study (pH after the com-
pletion of fermentation) and lactic acidity is the best criteria
to express the acidity and acceptability of yoghurts during
storage at 4 C. Measurement of pH is often preferable to
titratable acidity methods, because of the convenience and
high repeatability of pH measurement (Zourari et al. 1991).
Results of post-acidification study showed that all the sam-
ples fermented by probiotic culture in single, binary and
mixture combinations varied little in pH during storage. It
1st day
7th day
28thday
4.6
4.4
4.2
pH
4.0
3.8
3.6
3.4
LA LP LR LA-LP LA-LR LP-LRLA-LP-LRST-LB
Culture Combinations
Fig. 1 Post-acidification (pH) of FOS-supplemented soy milk fermented
by single, co-cultures and mixed culture of L. acidophilus (LA), L.
plantarum (LP), L. rhamnosus (LR) and yoghurt culture (ST-LB)
might be due to less activity of microbes at 4 C. On the first
day of storage the pH of soy yoghurts fermented by single,
binary and mix combinations of probiotics and yoghurt culture
decreased from 4.54 to 4.39. Among single cultures L. acid-
ophilus and L. plantarum showed significant (P<0.05) reduc-
tion in pH on the 28th day whereas, in L. rhamnosus
significant (P<0.05) reduction was observed on the 7th day
of storage. L. acidophilusL. rhamnosus showed significant
change in pH throughout the storage. However, binary cultures
(L. acidophilusL. plantarum and L. plantarumL. rhamno-
sus) and mix cultures (L. acidophilusL. plantarumL. rham-
nosus) showed significant changes in pH on the 7th and 28th,
respectively. Not much change was observed in pH for S.
thermophilusL. bulgaricus during storage. The pH of com-
mercial yoghurt is largely variable, ranging from 3.7 to 4.6.
Nevertheless, to avoid insipidness or excess acidity to the taste,
optimal values of pH should be in the range 4.04.4 (Souza
1991). Probiotics used in binary combinations (L. acidophilus
L. plantarum and L. acidophilusL. rhamnosus) and in mixture
showed the production of more acid in soy yoghurts with
respect to single and basic yoghurt cultures. The highest values
of pH obtained with L. rhamnosus and L. rhamnosusL.
plantarum with respect to the other co-cultures are consistent
with the heterolactic nature of L. rhamnosus (Jyoti et al. 2003)
and L. plantarum (Koistinen et al. 2007). Oliveira et al.
(2009b) reported that post-acidification of yoghurt is less af-
fected by prebiotic as compared with culture combinations.
Post-acidification phenomenon can be interpreted on the
basis of the metabolic behaviour of each microorganism.
The highest production of lactic acid either during fermen-
tation or after fermentation by L. acidophilus and its binary
combination with L. plantarum and L. rhamnosus with
respect to the other co-cultures was the likely result of the
homolactic metabolism of L. acidophilus (Zhao et al. 2007).
The lower lactic acidity associated with S. thermophilusL.
bulgaricus could have been due to the shift from homolactic
to heterolactic behaviour of L. bulgaricus consequent to the
change of environmental pH (Rhee and Pack 1980). Post-
acidification study reflected that all the culture in single and
combination showed pH in the range of optimal values.
Textural Characteristics
TPA is an imitative test in which the sample is compressed
twice, mimicking the action of the jaw in the mouth
(Friedman et al. 1963). Results of TPA tests showed that
addition of probiotics had marked effect on the textural
characteristics of soy yoghurt containing FOS (Table 2).
As a critical parameter for evaluation of textural properties
of yoghurt, hardness is used to estimate the maximum force
of the first compression. The hardness (in grams) of the soy
yoghurt ranged from 89.21 to 103.00 depending upon the
varying combinations of probiotic cultures as well as single
 Author's personal copy
Food Bioprocess Technol

Table 2 Texture profile of FOS-supplemented soy milk fermented by single, co-cultures and mixed culture of Lactobacillus acidophilus,
Lactobacillus plantarum, Lactobacillus rhamnosus and yoghurt culture (Streptococcus thermophilusLactobacillus bulgaricus)

S. No. Culture combinations Hardness (g) Cohesiveness Adhesiveness (gs) Springiness Gumminess (g)

1 L. acidophilus 93.403.60 a, b 0.350.010 b 23330 b, c 0.980.014 a 33.950.37 a, b

2 L. plantarum 90.903.10 a 0.360.010 b, c 23817 a, b, c 0.980.046 a 32.552 a, b
3 L. rhamnosus 89.216.21 a 0.370.010 c 228.527.5 c 1.630.084 c 33.071.38 a, b
4 L. acidophilusL. plantarum 103.004.00 c 0.360.007 b, c 201.727.3 c 0.980.020 a 37.540.78 c
5 L. acidophilusL. rhamnosus 93.003.03 a, b 0.350.007 b, c 260.717.6 a, b 1.000.045 a 32.831.09 a, b
6 L. plantarumL. rhamnosus 94.650.48 a, b 0.330.014 a 246.710.4 a, b 1.340.020 b 31.481.23 a
7 L. acidophilusL. plantarumL. rhamnosus 93.772.45 a, b 0.360.005 b, c 267.412.9 a, b 1.450.194 b, c 34.011.19 b
8 S. thermophilusL. bulgaricus 98.601.44 b, c 0.330.008 a 27624 a 1.500.083 c 32.72.36 a, b

All values are meansstandard deviations of data from three independent experiments. Different lowercase letters (ad) in the same column
indicate significant difference (P<0.05)

culture. Production of exopolysaccharide (EPS) by probiotic
bacteria (Welman and Maddox 2003) could increase the vis-
cosity, water retention and interaction with other components
of soy milk, resulting in increased hardness of the protein
matrix in the final product (Duboc and Mollet 2001). L. acid-
ophilusL. plantarum combination might have produced
some additional bioactive components which were responsi-
ble for increased hardness of the samples. L. acidophilus
showed higher hardness as compared with L. plantarum and
L. rhamnosus which was not significant. Yoghurt culture gave
firmer texture to soy yoghurt in the presence of FOS as
compared with single culture, binary culture (L. acidophi-
lusL. rhamnosus and L. plantarumL. rhamnosus) and mix-
ture of all probiotics. Lowest hardness was observed for the
single culture L. rhamnosus (89.21 g) followed by L. planta-
rum (90.90 g).
The main factor responsible for milk gelation is reduction
in high-net negative charge on the casein micelles because
of the liberation of acids from microbial activity. During
fermentation, casein micelles, together with denatured whey
proteins, aggregate into chains and clusters through hydro-
phobic and electrostatic bonds, governing the structure of
yogurt. Aggregation of casein micelles starts at a pH of
5.3, which also causes the solubilization of colloidal cal-
cium phosphate. Further pH reduction to below 5.0 causes a
more complex and extensive interconnection of casein
micelles, and the gel attains its maximum firmness at
pH 4.6, the isoelectric point of casein (Tamime 2006).
Same phenomenon responsible for gel formation in fer-
mented soy milk as, isoelectric point of soy protein is
similar to casein (Yang and Li 2010). It is generally accepted
that soybean glycinin is a hexamer consisting of five kinds
of subunits, each subunit containing acidic and basic chains
linked by a disulphide bond (Adachi et al. 2003).
Consequently, the tight and rigid molecular structure finally
results in a firm protein gel (Yang and Li 2010). FOS could
have also provided additional energy to potentiate EPS
biosynthesis, thus improving firmness and viscosity
(Oliveira et al. 2011). The increased firmness is related to
an improvement of the texture and makes the yoghurt less
susceptible to rearrangements within its network, shrinkage
and whey expulsion (Brennan and Tudorica 2008). The
firmness of yogurt is directly dependent on its total solids
and specifically protein content and the type of proteins.
Higher protein content would cause higher degree of cross-
linkage of the gel network, resulting in a much denser and
more rigid gel structure (Tamime 2006).
Other texture parameters, such as cohesiveness, adhe-
siveness, springiness and gumminess are important for set-
type yoghurts (Domagala et al. 2006), as these products
should be spoonable, firm and free from slimy or deadhead
textures (Tamime and Robinson 1999). Cohesiveness is
defined as the extent to which a material can be deformed
before it rupture and depends upon the strength of internal
bonds. Average cohesiveness value of single culture is 0.36
which was 5 % higher than the average value of binary co-
cultures. L. plantarumL. rhamnosus and S. thermophilus
L. bulgaricus showed significant (P<0.05) lower cohesive-
ness as compared with single, binary and mix cultures.
Single culture (L. acidophilus and L. plantarum) and binary
combination of L. acidophilus with L. rhamnosus and L.
plantarum showed no significant change in the strength of
internal bonds present in soy yoghurt.
Adhesiveness is the force necessary to remove the mate-
rial that adheres to the mouth during eating and is defined as
the negative force area for the first bite, representing the
work (gram second) necessary to pull the plunger away
from the food sample. The adhesiveness of all the samples
ranged from 201.7 to 276 gs. Highest value of adhesive-
ness was observed in sample having S. thermophilusL.
bulgaricus and lowest for the L. acidophilusL. plantarum
and this difference was statistically significant (P<0.05).
Lower adhesive value of yoghurt like sample makes the
product more acceptable among the consumers (Tamime
 Author's personal copy
Food Bioprocess Technol

Dynamic viscosity (Pas)

Table 3 Rheological parameters of FOS-supplemented soy milk fermented by single, co-cultures and mixed culture of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus
and Robinson 1999). Adhesive and cohesive yoghurts may
be pulled into threads or strings and may have a greater
degree of stickiness in the mouth, influencing the consisten-
cy and texture negatively (Kailasapathy 2006).

43.170.94 d

31.372.02 b
39.091.44 c

48.682.34 e
48.542.19 e

26.862.48 a

48.562.21 e

All values are meansstandard deviations of data from three independent experiments. Different lowercase letters (ad) in the same column indicate significant difference (P<0.05)
53.472.15 f
Springiness is the rate at which the sample returns to its
original shape when the deforming force is removed. L.
rhamnosus showed significantly (P<0.05) higher springi-
ness as compared with other single, binary and mixture of

a, b
b, c

b, c

b, c
probiotic bacteria. Among binary culture L. plantarumL.

c
a

a
a
rhamnosus showed significantly (P<0.05) higher springi-

0.2950.005
0.3310.014
0.3460.016
0.2880.008
0.3320.008
0.2730.026
0.2790.034
0.3240.006
ness as compared with L. acidophilusL. plantarum and L.
acidophilusL. rhamnosus. Gumminess also increased with

Tan
rhamnosus and yoghurt culture (Streptococcus thermophilusLactobacillus bulgaricus) under oscillatory testing at frequency 0.1 to 10 Hz, strain 0.06
increased value of hardness and cohesiveness. The values
obtained for the TPA of FOS-supplemented soy yoghurt were

Complex modulus G* (Pa)

consistent with soy fortified buffalo milk yoghurt (Kumar and
Mishra 2004). Textural profile analysis of probiotic soy yo-

1,351.9238.08 a, b
1,301.2253.58 a, b

1,342.3940.16 a, b
1,346.8059.94 a, b
1,408.8141.58 b, c
ghurt indicates that S. thermophilusL. bulgaricus and other

1,462.6424.59 c

1,326.4526.45 a
1,298.6222.2 a
probiotic binary combination gave best texture to the product
with firmer gel, lower adhesiveness and springiness, higher
gumminess and average cohesiveness.
These results suggest that the potential advantages asso-
ciated to the use of probiotics in various combinations
would not be limited to the improvement of growth and Viscous modulus G (Pa)
fermentation kinetics, but include also the textural character-
istics of fermented soy milk. 382.9812.98 a, b, c

360.7648.56 a, b
408.9028.67 b, c

415.0616.72 b, c
424.2827.95 c

349.0638.83 a
405.3327 b, c
443.407.48 c
Rheological Characteristics

The rheological parameters of all the samples studied under

oscillatory testing are presented in Table 3. Frequency
sweep tests were used to determine the viscoelastic charac-
Elastic modulus G (Pa)

1,279.7020.30 a, b, c

teristics of soy yoghurts. Storage (G) and loss modulus (G)

1,296.5445.69 b, c

1,293.0117.22 b, c
1,281.2534.25 b, c
1,235.3635.30 a, b
1,227.3624.64 a

1,337.2153.02 c

characterize the degree of solid- (elastic) and liquid-like

1,405.3627.0 d

(viscous) characteristics of a gel respectively. FOS-

supplemented soy milk fermented by different probiotics
showed a clear gel-like behaviour, in which G was higher
than G (Donkor et al. 2007). Higher G and lower tan
values were observed for all the samples which, signifies
L. acidophilusL. plantarumL. rhamnosus

firmer and solid-like character of the gel. S. thermophilus,

Lactobacillus spp. and Bifidobacterium spp. are well known
to influence the rheological properties of fermented milk,
and the effects of these microorganisms and low tempera-
S. thermophilusL. bulgaricus
L. acidophilusL. rhamnosus

ture storage on viscous modulus have been reported (Kristo

L. acidophilus-L. plantarum

L. plantarumL. rhamnosus

et al. 2003; Tamime 2005). Since milk composition was

kept constant in the present study, the differences observed
Culture combination

were mainly due to change in the cultures type. All the

L. acidophilus

L. rhamnosus

culture showed significant (P<0.05) effect on the rheolog-

L. plantarum

ical parameters of FOS-supplemented soy yoghurt. Among

the pure culture G value was higher for L. acidophilus
(1,296.54 Pa) followed by L. plantarum (1,235.36 Pa) and
L. rhamnosus (1,227.36 Pa). L. rhamnosus-fermented soy
S. No.

milk showed lower value of G in pure culture but higher

value in combination with L. acidophilus and L. plantarum.
1
2
3
4
5
6
7
8
 Author's personal copy
Food Bioprocess Technol

The G value of yoghurt culture-fermented soy milk was
almost similar to sample fermented by mixture of probiotics.
Viscous modulus (G) showed minimum value for L. plan-
tarumL. rhamnosus (349.06 Pa) and maximum for L. acid-
ophilusL. rhamnosus (443.40 Pa). Among the samples
containing single cultures, sample fermented with L. rham-
nosus showed higher G followed by L. plantarum.
Significant (P<0.05) difference was observed in G values
for all the three co-cultures with L. plantarumL. rhamnosus
showing higher viscous behaviour.
The development of rigidity was estimated from G* values
which represents the total resistance of a material against the
applied strain. L. acidophilusL. plantarum showed highest
G* among all the studied culture (Table 3). Probiotic showed
better rigidity in the presence of FOS as they can better utilize
FOS and metabolise it. A culture in binary combination
resulted in more rigid gel as compare to other combinations.
This result correlates well with the results of TPA. The slight
variation in the rheological parameters and texture profile data
can be attributed to the different magnitude of force applied in
the mechanical evaluation. G* is obtained from a test in which
structure of the network is not broken. Thus its sensitivity for
detecting the contribution of different interactions is higher
than in the puncture test, in which macroscopic failure of the
structure is experienced (Ferragut et al. 2009). Lower phase
angle (tan ) value indicates that the entire sample showed
more solid characteristics which provide more firm network to
gel. L. rhamnosus showed significant (P<0.05) lower tan as
compared with L. acidophilus and L. plantarum. No significant
change was observed in the springiness value of binary culture
(L. acidophilusL. plantarum and L. plantarumL. rhamno-
sus) and mix culture-fermented soy yoghurt. However, these
values significantly (P<0.05) lower from yoghurt culture and
L. acidophilusL. rhamnosus-fermented soy yoghurt.
The dynamic viscosity () represented the flow charac-
teristics of soy yoghurt (in Pascal second). Probiotic cultures
both in pure form and in combinations showed statistically
significant (P<0.05) effect on dynamic viscosity. L. planta-
rum- and L. rhamnosus-fermented samples showed signifi-
cantly (P<0.05) higher as compared with L. acidophilus,
binary cultures and mixture of all. The increase in viscosity
due to addition of fibre has also been attributed to interac-
tions between oligo- or poly-saccharides, like inulin and
proteins (Sodini et al. 2002). Steffe (1996) reported values
of 129, 153, 0.140 Pas and 1.19 for G, G, and tan (),
respectively, in concentrated solution whereas values of
5,187, 363, 36.3 Pas and 0.0699 were typical for gels which
indicated prepared fermented soy milk was set-type yoghurt.

Table 4 Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus and yoghurt culture (Streptococcus thermophilusLacto-
bacillus bulgaricus) count in FOS-supplemented soy yoghurt

S. No. Culture combinations Log cfu/ml of the product during storage period

1st day 7th day 28th day

1 L. acidophilus 13.561.83 a, G 12.672.65 a, B, C,D , E, F, G 11.631.07 a, b, A, B, C, D, E, F, G

2 L. plantarum 13.641.75 a, G 12.981.75 a, D, E, F, G 12.081.37 b, A, B, C, D, E, F, G
3 L. rhamnosus 13.81.8 a, G 12.042.17 a, A, B, C, D, E, F, G 9.510.72 a, A
4 L. acidophilusL. plantarum
L. acidophilus 13.211.2 a, E, F, G 12.981.64 a, D, E, F, G 11.531.32 a, b, A, B, C, D, E, F, G
L. plantarum 13.451.53 a, F, G 12.670.07 a, F, G 11.841.27 a, b, A, B, C, D, E, F, G
5 L. acidophilusL. rhamnosus
L. acidophilus 13.410.85 a, F, G 13.011.33 a, D, E, F, G 11.341.87 a, b, A, B, C, D, E, F, G
L. rhamnosus 13.081.06 a, D, E, F, G 12.281.26 a, B, C, D, E, F, G 10.010.69 a, b, A, B
6 L. plantarumL. rhamnosus
L. plantarum 13.341.16 a, F G 12.870.84 a, C, D, E, F, G 11.021.15 a, b, A, B, C, D, E, F, G
L. rhamnosus 12.890.85 a, D, E, F, G 12.631.42 a, B, C, D, E, F, G 10.141.24 a, b, A, B, C
7 L. acidophilusL. plantarumL. rhamnosus
L. acidophilus 13.151.04 a, E, F, G 12.981.85 a, D, E, F, G 11.430.45 a, b, A, B, C, D, E, F, G
L. plantarum 13.311.23 a, E, F, G 12.781.59 a, C, D, E, F, G 11.121.04 a, b, A, B, C, D, E, F, G
L. rhamnosus 12.891.11 a, A B,C D, E F, G 12.681.55 a, A, B, C, D, E, F, G 10.210.98 a, b, E, F, G
8 S. thermophilusL. bulgaricus
S. thermophilus 12.741.26 a, A, B, C, D, E 12.031.42 a, A, B, C, D, E, F, G 10.761.22 a, b, E, F, G
L. bulgaricus 12.690.51 a, A, B, C, D 11.762.24 a, A, B, C, D, E, F, G 11.231.97 a, b, F, G

All values are meansstandard deviations of data from three independent experiments. Different lowercase letters (ac) in the same row indicate
significant difference (P<0.05). Different uppercase letters (AG) in the same column indicate significant difference (P<0.05)
 Author's personal copy
Food Bioprocess Technol
Rheological analysis of probiotic soy yoghurt indicates
that binary combination showed good gel characteristics
with higher complex modulus, lower tan and lower dy-
namic viscosity as compared with single and mix cultures.
Probiotic (L. acidophilus, L. plantarum and L. rhamnosus)
and Yoghurt Culture (S. thermophilusL. bulgaricus) Count
Variation in viable counts of probiotic bacteria and yoghurt
cultures during storage are presented in Table 4. The count of
L. acidophilus, L. plantarum and L. rhamnosus in single,
binary and mixture varied within a very narrow range at the
1st and 7th days of storage. On the 28th day of storage, L.
acidophilus (11.63 log cfu/ml) and L. rhamnosus (9.51 log
cfu/ml) counts were, lower than L. plantarum (12.08 log cfu/
ml) in soy milk fermented by single culture. In binary and mix
combination, L. acidophilus and L. plantarum count did not
varied significantly at the 7th and 28th days of storage. There
was significant (p<0.05) reduction in L. rhamnosus count in
binary and mixture on the 28th day as compared with the 7th
and 1st day of storage. A high number of the probiotic culture
at the end of fermentation is one of the prerequisites to
maintain high levels of the probiotic during storage. These
results indicated stimulating effect of FOS on viability of
probiotics. The prebiotic can stimulate the metabolism of
probiotics, as the likely result of the increased level of fructose
released from its partial hydrolysis, which was metabolized as
an additional carbon and energy source (Tamime 2005).
Similar results were reported by Donkor et al. (2007) in soy
yoghurt supplemented with inulin and raffinose. Oliveira et al.
(2009b) and Kristo et al. (2003) studied the growth of pro-
biotic bacteria in combinations and reported good growth of
bacteria in combination at 4 C storage.
Conclusions
Among all the studied combinations, binary cultures were
most suitable probiotic starter cultures for fermented soy milk
as they showed lower fermentation time with improved prod-
uct characteristics. L. acidophilusL. plantarum among binary
culture was the best combination as it was showed good
acidification profile, textural and rheological characteristics.
Presence of FOS in soy-fermented milk can also improve the
sensory characteristics of products by providing sweetness.
Industrially, short fermentation times are preferable in order to
increase plant output as well as to reduce microbial contami-
nation. Probiotic cell count achieved was more than 9 log cfu/
mL in fermented soy milk after the 28th day of storage at 4 C
which meets the desirability for probiotic functional foods.
This fermented soy milk could be used as an application for
the production of non dairy lactic beverages after conducting
appropriate scale up and pilot plant study.
Acknowledgements The authors acknowledge the financial assis-
tance of the Department of Biotechnology, Government of India,
New Delhi, for funding this research work. Author would also like
to thank Mr. Sabarish Warrier, EnSigns Health Care Pvt. Ltd., India for
providing FOS (Swetoos) as prebiotic.
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