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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

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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
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Food Bioprocess Technol

have been evaluated as possible probiotic vehicles. Soy-based minimum concentration of 2 % FOS as recommended in dairy
fermented foods may provide additional benefits for the con- products (Franck 2002) was fixed to study the effect of pro-
sumer due to their various functional properties such as hypo- biotic on the various characteristics of soy yoghurt. Also FOS
lipidemic, anticholesterolemic and antiatherogenic and prebiotic effect on the growth of L. acidophilus, L. rhamnosus
reduced allergenicity (Lopez-Lazaro and Akiyama 2002). and L. plantarum, either in pure cultures or in binary co-
Soy milk has low acidification rate and slow growth of cultures or in a combination was examined.
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. Materials and Methods
2007). A rapid acidification of the raw material prevents
growth of undesirable microorganisms and is also essential Microbial Cultures
for aroma, texture and flavour of the end-product (Vuyst
2000). According to Champagne et al. (2009), the develop- Probiotic cultures, L. plantarum NCDC414, L. acidophilus
ment of a fermented soy product containing probiotics NCDC11, L. rhamnosus NCDC24 and yoghurt culture, S.
requires strain selection for the ability to grow in the sub- salivarius subsp. thermophilus NCDC118 and L. del-
strate, as well as the ability to compete or even establish a brueckii subsp. bulgaricus NCDC290 procured in freeze
synergy between strains. Donkor et al. (2005) reported that dried from National Collection of Dairy Culture, National
the protein in fermented soymilk could encourage the Dairy Research Institute, Karnal, India were used in this
growth of many probiotic strains such as Lactobacillus study. The cultures of Lactobacilli spp. were activated by
acidophilus, Lactobacillus casei and Streptococcus thermo- inoculating in MRS and S. thermophillus in M17 broth
philus. Scientific research has shown that probiotic contain- followed by incubation for 24 h at 37 C. The activated
ing soy-fermented beverages present a good sensory cultures were used after three successive transfers for the
acceptance by potential consumers (Shimakama et al. 2003). preparation of stock cultures for soy yoghurt making. Stock
FOS is fructose oligomer linked to glucose and/or fruc- cultures were prepared by mixing 24-h grown-activated
tose molecules that contain up to ten sugar moieties. With cultures with sterilized 80 % glycerol (v/v) in a 1:1 ratio in
prebiotic properties, FOS is highly soluble in water (Franck Eppendorf tubes and stored at 40 C for further analysis.
2002) and low-calorie carbohydrates (Roberfroid 1993). For the preparation of the inocula, 100 mL of sterile MRS
Dosage levels of FOS in the range of 250 % (w/w) are and M17 broths were inoculated with 1 mL of a thawed
recommended for various food formulations (Franck 2002). stock culture and incubated at 37 C until a pH of 4.5 was
Hauly et al. (2005) reported that soy yoghurt supplemented reached (Champagne et al. 2009). The incubation time var-
with FOS presented acceptance index above 70 %. ied between 12 to 14 h as a function of the strain. The
The texture (Cobos et al. 1995) and taste of soy yoghurt cultures were centrifuged (4,500g, 10 min, 4 C), pallet
are essential attributes for product acceptability by the con- washed in peptone water and re-suspended with peptone
sumers (Donkor et al. 2007). There are very few studies on water to obtained the final concentration of inoculum
the textural and rheological properties of soy yoghurt con- 10.74 to 10.98 log cfu/ml. This concentration was selected
taining probiotics (Donkor et al. 2007). Gel formation of soy on the basis of preliminary experiments to reduce the fer-
milk proteins is a key process step in the manufacture of mentation time and maintain the desirable viable culture
non-dairy-fermented product like yoghurt. The rheological concentration during storage.
properties of set gels determine the texture, organoleptic
properties and shelf-life of the product (Cayot et al. 2008; Production of Soy Milk and Soy Yoghurt
Lee and Lucey 2006). However, not much information is
available in the literature about the effect of probiotic and Soybeans (100 g) were soaked in one litre water for 16 h at
their combinations without yoghurt culture (Streptococcus 25 C. Soaked beans were mixed with 350 ml warm water
salivarius subsp. thermophilus and Lactobacillus del- (6570 C) and macerated in mixer grinder for 3 min, fil-
brueckii subsp. bulgaricus) on the fermentation kinetics tered with single-layer muslin cloth and steamed for 15 min
and textural characteristics of fermented soy milk. at 10 psi/g pressure. The soy milk preparation was standard-
Preliminary experiments were conducted on probiotic soy ized to adjust the protein and fat per cent as recommended
yoghurt without any supplementation, but due to poor textural for set type of yoghurt (Tamime et al. 1995; Codex 2008).
and rheological characteristics product had poor sensory ac- Soymilk samples thus prepared contained total solid (10
ceptance. Therefore, the effect of FOS and their synbiotic 12 %), total soluble solids (810Brix), protein (4.89
interactions with various probiotic and their combinations 5.15 %) and fat (1.832.23 %) having pH 6.106.22. To
was studied for the production of soy yoghurt with improved observe the effect of prebiotics 2 % (w/v) FOS (Swetoos,
acidification, textural and rheological characteristics. A EnSigns Health Care Pvt. Ltd., India) was mixed in soy milk
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before steaming. Batches of yoghurt were prepared in analyzed with the supporting software Bohlin Gemini 200.
100 ml glass beaker, containing 50 ml of soymilk by inoc- After gentle stirring with a plastic spoon, a small sample of
ulation of culture in sterilized condition. Probiotic yoghurts soy yoghurt was deposited on the inset plate. First, amplitude
and yoghurt with basic cultures were prepared by inoculat- sweep test was performed using a frequency of 1 Hz to
ing soy milk with about 10.94, 10.72, 10.98, 10.74 and ascertain viscoelastic properties followed by frequency sweep
10.90 log cfu/ml each of L. acidophilus, L. plantarum, L. test (0.1 to 10 Hz at a maximum strain of 0.06). Measurements
rhamnosus, S. salivarius subsp. thermophilus and L. del- were carried out within the linear viscoelastic range, previous-
brueckii subsp. bulgaricus cultures respectively. After inoc- ly determined by a stress sweep test from 0.01 to 100 Pa.
ulation, beakers were transferred to incubator and batch Dynamic moduli (G, G and G*) and tan were calculated
fermentations were performed at 40 C up to pH 4.5 (se- from frequency sweep test. G is related to the molecular
lected as the condition to stop the fermentation). events of elastic nature; G to the viscous character of mate-
rial; G* (complex modulus), corresponds to (G2 +G2)1/2 and
Acidification Kinetics Parameters phase angle (tan ), is calculated as G/G (values closer to 0
have more solid like structure) (Ferragut et al. 2009).
The changes in pH during fermentation were carried out in
triplicates monitored continuously by means of a glass elec- Microbial Analyses
trode pH meter (Model: 361; Make: Systronics) at an inter-
val of 15 min (Varghese and Mishra 2008). The acidification Serial ten-fold dilutions were prepared in a solution of 0.9 %
rate (Vmax) was calculated as the time variation of pH (dpH/ NaCl(w/v) and 0.1 % (w/v) bactopeptone and suitable dilutions
dt) and expressed as pH units/min. At the end of incubation, were plated on appropriate media. Enumeration of L. del-
the following kinetic parameters were calculated: (1) tVmax , brueckii subsp. bulgaricus and S. salivarius subsp. thermophi-
time to reached Vmax (h); (2) pHVmax , pH at Vmax (3) tpH 4. 5, lus was done on Lactobacilli MRS media and Streptococcus
time to complete the fermentation (in hours). Selection HiVeg Agar respectively which were aerobically
incubated at 37 C for 48 h (Dave and Shah 1996). L. acid-
Post-acidification ophilus was enumerated on a modified MRS agar with clinda-
mycin (Oliveira et al. 2011). Selective media for L. rhamnosus
On completion of fermentation, post-acidification was de- was prepared as per method given by Farnworth et al. (2007).
termined by pH measurement on 1st, 7th and 28th days of Enumeration of L. plantarum was done in modified MRS agar
storage at 4 C. with ciproflaxin antibiotics (Bujalance et al. 2006).

TPA Statistical Analysis

Texture profile analysis (TPA) of fermented soymilk was ANOVA was carried out to determine significant differences
measured using a Texture Analyzer (Model: TA.XT-2, between samples at the 95 % level of confidence, using the
Make: Stable Microsystems, UK) equipped with 25 kg load SPSS 17 System for windows (SPSS Statistical Software,
cell (Kumar and Mishra 2004) at 200.5 C. The data Inc., Chicago, IL) software package. Data were obtained
obtained from the texture profile (forcetime) curve were from three independent experiments using Duncan test.
used for the calculation of hardness (in grams), cohesive-
ness, adhesiveness (in grams second), springiness and gum-
miness (in grams) (Kumar and Mishra 2004). Results and Discussion

Rheological Measurements Acidification Kinetics

Small amplitude oscillatory measurements were used to mon- The acidification kinetics of yoghurt were characterized by
itor the gelation process and viscoelastic properties of the set Vmax, tVmax , pHVmax and tpH 4.5 (Spinnler and Corrieu 1989).
gels as per method given by Ferragut et al. (2009). The Acidification profile of FOS-supplemented soy milk fer-
rheological properties were monitored on a Bohlin rheometer mented by single, co-cultures and mixed culture of L. acid-
(Model: Gemini 200 Make: Bohlin Instruments UK, ophilus, L. plantarum, L. rhamnosus and yoghurt culture are
Cirencester, Gloucestershire, UK) using a parallel plate ar- given in Table 1. The highest value of Vmax (23.33103 pH
rangement with 2.5 mm gap (Model No. PP 20) at a constant units/min) was obtained with the L. acidophilusL. plantarum
temperature of 200.1 C. The temperature was regulated by and S. thermophilusL. bulgaricus. The lowest Vmax value
a viscotherm VT 2 circulating bath and controlled with a was observed with the L. plantarum (14.0103 pH units/
Peltier system. The data of all rheological measurements were min) demonstrating its poor acidification ability as compared
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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- The tVmax value was higher by 0.07 times for L. acidophilus,
tion rate in combination with L. acidophilus, L. rhamnosus 1.13 times for L. plantarum and 1.18 times for L. rhamnosus
and mixture of all. Binary and mixed cultures of the probiotic compared with the average of all the binary co-cultures.
microorganism increased the average acidification rate of Oliveira et al. (2009c) studied the effect of culture combi-
sample significantly (P<0.05) by about 16.1 and 28 % as nations on the acidification kinetic parameters and reported
compared with single culture. The Vmax value for yoghurt similar findings. Apparently, there was no direct relationship
culture (S. thermophilusL. bulgaricus) was 32.36, 15.8 and between Vmax and tVmax , in the presence of single probiotic
6 % higher to average Vmax of single, binary and mixed culture or in the combination.
cultures of probiotic, respectively. The largest increase in this Highest Vmax and lowest tVmax were seen for the S. thermo-
parameter was observed for L. acidophilus as single, binary philusL. bulgaricus which might be due to symbiotic growth
and mix culture. The ability of L. acidophilus to ferment FOS patterns of the yogurt cultures in soy milk. Tamime and
more effectively than other microorganisms (Barrangou et al. Robinson (1985) reported that the Streptococci initiate the
2003) might be the primary reason for the higher acidification milk fermentation and that Lactobacilli contribute to acidifi-
rate which was significantly (P<0.05) higher from L. planta- cation later in the incubation. S. thermophilus is well able to
rum and L. rhamnosus. Another probable reason for higher grow in soy beverages because of its ability to use sucrose and
acidification rate of L. acidophilusL. plantarum, L. acid- other oligosaccharides present in soy milk (Chumchuere and
ophilusL. rhamnosus and L. acidophilusL. plantarumL. Robinson 1999). Similar behaviour might be explained for L.
rhamnosus might be release of compounds like peptides by acidophilusL. plantarum which showed highest Vmax and
L. acidophilus during fermentation which could have stimu- lower tpH 4.5.
lated the metabolic process of other lactic acid bacteria The pHVmax value represented the H+ ion concentration at
(Oliveira et al. 2011). the time of Vmax and maximum acid-producing ability of
Champagne et al. (2000) and Fonseca et al. (2000) stud- cultures. All the probiotic culture in single and mixture
ied the acidification kinetics of different strains of S. ther- showed no significant difference in pHVmax . The pHVmax value
mophilus and Lactobacilli and reported Vmax, tVmax , pHVmax was significantly (P<0.05) lower for L. acidophilusL. rham-
and tpH 4.5 are strain dependent. These parameters are also nosus as compared with single, mix and yoghurt cultures.
dependent on the buffering capacity of milk (Xanthopoulos Time to reach pH 4.5 ranged between 3.47 and 5.83 h,
et al. 1994). The tVmax value for probiotic cultures ranged which was significantly (P<0.05) different for all the pro-
from 2.50 to 3.98 h. It was minimum (2.50 h) for L. rham- biotic as well as yoghurt culture. Minimum fermentation
nosus and maximum (3.98 h) for L. plantarumL. rhamno- time was taken by yoghurt culture (S. thermophilusL. bul-
sus. The L. acidophilus and L. plantarum showed no garicus) followed by L. acidophilusL. plantarum which
significant difference in the values of tVmax . The tVmax value was statistically significant (P<0.05). L. acidophilus, L.
of L. rhamnosus was significantly (P<0.05) lower than that plantarum and L. rhamnosus took longer time to complete
of L. acidophilus (3.00 h). Average tVmax (3.69 h) for the all the fermentation which was 5.75, 5.5 and 5.83 h, respec-
the probiotic binary co-cultures was significantly (P<0.05) tively; however this difference was not significant among
higher than S. thermophilusL. bulgaricus binary co- each other. All the binary combination and mixture of pro-
cultures (2.30 h). Soy milk containing mixture of all the biotic showed significant (P<0.05) lower tpH 4.5 as com-
probiotics showed similar tVmax values as binary co-cultures. pared with single culture. Cultures in binary combination
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and in mixture resulted in increased rate of fermentation and might be due to less activity of microbes at 4 C. On the first
reduced fermentation time (Marafon et al. 2010; Kristo et al. day of storage the pH of soy yoghurts fermented by single,
2003) might be due to simultaneous symbiotic activity of binary and mix combinations of probiotics and yoghurt culture
two bacteria. It is possible that the simultaneous presence of decreased from 4.54 to 4.39. Among single cultures L. acid-
two or more than two microorganisms could have led to ophilus and L. plantarum showed significant (P<0.05) reduc-
synergistic effects and associated to the release of metabolic tion in pH on the 28th day whereas, in L. rhamnosus
products stimulating the metabolism of the others (Radke- significant (P<0.05) reduction was observed on the 7th day
Mitchell and Sandine 1986). Oliveira et al. (2009a) explored of storage. L. acidophilusL. rhamnosus showed significant
various prebiotic effects on the probiotic-fermented milk change in pH throughout the storage. However, binary cultures
and reported the improved acidification kinetics with FOS. (L. acidophilusL. plantarum and L. plantarumL. rhamno-
These results as a whole not only confirm that S. thermo- sus) and mix cultures (L. acidophilusL. plantarumL. rham-
philusL. bulgaricus is the best co-culture for FOS- nosus) showed significant changes in pH on the 7th and 28th,
supplemented soy yoghurt preparation but also that the respectively. Not much change was observed in pH for S.
alternative probiotics employed in this study (L. acidophi- thermophilusL. bulgaricus during storage. The pH of com-
lus, L. plantarum and L. rhamnosus) in binary and mixture mercial yoghurt is largely variable, ranging from 3.7 to 4.6.
combination would be able to ensure satisfactory results in Nevertheless, to avoid insipidness or excess acidity to the taste,
terms of acidification and fermentation time. optimal values of pH should be in the range 4.04.4 (Souza
1991). Probiotics used in binary combinations (L. acidophilus
Post-acidification Changes L. plantarum and L. acidophilusL. rhamnosus) and in mixture
showed the production of more acid in soy yoghurts with
The results of post-acidification study of probiotic- respect to single and basic yoghurt cultures. The highest values
fermented soy milk are shown in Fig 1. This analysis was of pH obtained with L. rhamnosus and L. rhamnosusL.
conducted for 28 days of storage at an interval of 7 days at plantarum with respect to the other co-cultures are consistent
4 C after the completion of fermentation. Accordingly to with the heterolactic nature of L. rhamnosus (Jyoti et al. 2003)
Souza (1991), post-acidification study (pH after the com- and L. plantarum (Koistinen et al. 2007). Oliveira et al.
pletion of fermentation) and lactic acidity is the best criteria (2009b) reported that post-acidification of yoghurt is less af-
to express the acidity and acceptability of yoghurts during fected by prebiotic as compared with culture combinations.
storage at 4 C. Measurement of pH is often preferable to Post-acidification phenomenon can be interpreted on the
titratable acidity methods, because of the convenience and basis of the metabolic behaviour of each microorganism.
high repeatability of pH measurement (Zourari et al. 1991). The highest production of lactic acid either during fermen-
Results of post-acidification study showed that all the sam- tation or after fermentation by L. acidophilus and its binary
ples fermented by probiotic culture in single, binary and combination with L. plantarum and L. rhamnosus with
mixture combinations varied little in pH during storage. It respect to the other co-cultures was the likely result of the
homolactic metabolism of L. acidophilus (Zhao et al. 2007).
1st day The lower lactic acidity associated with S. thermophilusL.
7th day bulgaricus could have been due to the shift from homolactic
28thday
to heterolactic behaviour of L. bulgaricus consequent to the
4.6
change of environmental pH (Rhee and Pack 1980). Post-
4.4
acidification study reflected that all the culture in single and
combination showed pH in the range of optimal values.
4.2

Textural Characteristics
pH

4.0

3.8 TPA is an imitative test in which the sample is compressed


twice, mimicking the action of the jaw in the mouth
3.6
(Friedman et al. 1963). Results of TPA tests showed that
3.4
addition of probiotics had marked effect on the textural
characteristics of soy yoghurt containing FOS (Table 2).
LA LP LR LA-LP LA-LR LP-LRLA-LP-LRST-LB As a critical parameter for evaluation of textural properties
Culture Combinations of yoghurt, hardness is used to estimate the maximum force
Fig. 1 Post-acidification (pH) of FOS-supplemented soy milk fermented
of the first compression. The hardness (in grams) of the soy
by single, co-cultures and mixed culture of L. acidophilus (LA), L. yoghurt ranged from 89.21 to 103.00 depending upon the
plantarum (LP), L. rhamnosus (LR) and yoghurt culture (ST-LB) varying combinations of probiotic cultures as well as single
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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 biosynthesis, thus improving firmness and viscosity
bacteria (Welman and Maddox 2003) could increase the vis- (Oliveira et al. 2011). The increased firmness is related to
cosity, water retention and interaction with other components an improvement of the texture and makes the yoghurt less
of soy milk, resulting in increased hardness of the protein susceptible to rearrangements within its network, shrinkage
matrix in the final product (Duboc and Mollet 2001). L. acid- and whey expulsion (Brennan and Tudorica 2008). The
ophilusL. plantarum combination might have produced firmness of yogurt is directly dependent on its total solids
some additional bioactive components which were responsi- and specifically protein content and the type of proteins.
ble for increased hardness of the samples. L. acidophilus Higher protein content would cause higher degree of cross-
showed higher hardness as compared with L. plantarum and linkage of the gel network, resulting in a much denser and
L. rhamnosus which was not significant. Yoghurt culture gave more rigid gel structure (Tamime 2006).
firmer texture to soy yoghurt in the presence of FOS as Other texture parameters, such as cohesiveness, adhe-
compared with single culture, binary culture (L. acidophi- siveness, springiness and gumminess are important for set-
lusL. rhamnosus and L. plantarumL. rhamnosus) and mix- type yoghurts (Domagala et al. 2006), as these products
ture of all probiotics. Lowest hardness was observed for the should be spoonable, firm and free from slimy or deadhead
single culture L. rhamnosus (89.21 g) followed by L. planta- textures (Tamime and Robinson 1999). Cohesiveness is
rum (90.90 g). defined as the extent to which a material can be deformed
The main factor responsible for milk gelation is reduction before it rupture and depends upon the strength of internal
in high-net negative charge on the casein micelles because bonds. Average cohesiveness value of single culture is 0.36
of the liberation of acids from microbial activity. During which was 5 % higher than the average value of binary co-
fermentation, casein micelles, together with denatured whey cultures. L. plantarumL. rhamnosus and S. thermophilus
proteins, aggregate into chains and clusters through hydro- L. bulgaricus showed significant (P<0.05) lower cohesive-
phobic and electrostatic bonds, governing the structure of ness as compared with single, binary and mix cultures.
yogurt. Aggregation of casein micelles starts at a pH of Single culture (L. acidophilus and L. plantarum) and binary
5.3, which also causes the solubilization of colloidal cal- combination of L. acidophilus with L. rhamnosus and L.
cium phosphate. Further pH reduction to below 5.0 causes a plantarum showed no significant change in the strength of
more complex and extensive interconnection of casein internal bonds present in soy yoghurt.
micelles, and the gel attains its maximum firmness at Adhesiveness is the force necessary to remove the mate-
pH 4.6, the isoelectric point of casein (Tamime 2006). rial that adheres to the mouth during eating and is defined as
Same phenomenon responsible for gel formation in fer- the negative force area for the first bite, representing the
mented soy milk as, isoelectric point of soy protein is work (gram second) necessary to pull the plunger away
similar to casein (Yang and Li 2010). It is generally accepted from the food sample. The adhesiveness of all the samples
that soybean glycinin is a hexamer consisting of five kinds ranged from 201.7 to 276 gs. Highest value of adhesive-
of subunits, each subunit containing acidic and basic chains ness was observed in sample having S. thermophilusL.
linked by a disulphide bond (Adachi et al. 2003). bulgaricus and lowest for the L. acidophilusL. plantarum
Consequently, the tight and rigid molecular structure finally and this difference was statistically significant (P<0.05).
results in a firm protein gel (Yang and Li 2010). FOS could Lower adhesive value of yoghurt like sample makes the
have also provided additional energy to potentiate EPS 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 structure is experienced (Ferragut et al. 2009). Lower phase
almost similar to sample fermented by mixture of probiotics. angle (tan ) value indicates that the entire sample showed
Viscous modulus (G) showed minimum value for L. plan- more solid characteristics which provide more firm network to
tarumL. rhamnosus (349.06 Pa) and maximum for L. acid- gel. L. rhamnosus showed significant (P<0.05) lower tan as
ophilusL. rhamnosus (443.40 Pa). Among the samples compared with L. acidophilus and L. plantarum. No significant
containing single cultures, sample fermented with L. rham- change was observed in the springiness value of binary culture
nosus showed higher G followed by L. plantarum. (L. acidophilusL. plantarum and L. plantarumL. rhamno-
Significant (P<0.05) difference was observed in G values sus) and mix culture-fermented soy yoghurt. However, these
for all the three co-cultures with L. plantarumL. rhamnosus values significantly (P<0.05) lower from yoghurt culture and
showing higher viscous behaviour. L. acidophilusL. rhamnosus-fermented soy yoghurt.
The development of rigidity was estimated from G* values The dynamic viscosity () represented the flow charac-
which represents the total resistance of a material against the teristics of soy yoghurt (in Pascal second). Probiotic cultures
applied strain. L. acidophilusL. plantarum showed highest both in pure form and in combinations showed statistically
G* among all the studied culture (Table 3). Probiotic showed significant (P<0.05) effect on dynamic viscosity. L. planta-
better rigidity in the presence of FOS as they can better utilize rum- and L. rhamnosus-fermented samples showed signifi-
FOS and metabolise it. A culture in binary combination cantly (P<0.05) higher as compared with L. acidophilus,
resulted in more rigid gel as compare to other combinations. binary cultures and mixture of all. The increase in viscosity
This result correlates well with the results of TPA. The slight due to addition of fibre has also been attributed to interac-
variation in the rheological parameters and texture profile data tions between oligo- or poly-saccharides, like inulin and
can be attributed to the different magnitude of force applied in proteins (Sodini et al. 2002). Steffe (1996) reported values
the mechanical evaluation. G* is obtained from a test in which of 129, 153, 0.140 Pas and 1.19 for G, G, and tan (),
structure of the network is not broken. Thus its sensitivity for respectively, in concentrated solution whereas values of
detecting the contribution of different interactions is higher 5,187, 363, 36.3 Pas and 0.0699 were typical for gels which
than in the puncture test, in which macroscopic failure of the 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 Acknowledgements The authors acknowledge the financial assis-
tance of the Department of Biotechnology, Government of India,
that binary combination showed good gel characteristics
New Delhi, for funding this research work. Author would also like
with higher complex modulus, lower tan and lower dy- to thank Mr. Sabarish Warrier, EnSigns Health Care Pvt. Ltd., India for
namic viscosity as compared with single and mix cultures. providing FOS (Swetoos) as prebiotic.

Probiotic (L. acidophilus, L. plantarum and L. rhamnosus)


and Yoghurt Culture (S. thermophilusL. bulgaricus) Count
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