development of a shelf stable synbiotic formulation containing lactic acid bacteria and...

7/24/2019 Development of a shelf stable synbiotic formulation containing Lactic acid bacteria and Fructooligosaccharides

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Chapter: 3

Development of a shelf stable synbiotic formulation containing Lactic

acid bacteria and Fructooligosaccharides


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Overview

Food formulations of live probiotic cultures with prebiotics are most convenient for

functional food and nutraceutical applications. The present chapter details the

development of synbiotic formulation containing probiotic LAB and prebiotic FOS. The

first section is focused on the preparation of shelf stable synbiotic formulation of selected

LAB and FOS, which are able to survive during spray drying and to study the effects of a

preliminary mild heat treatment and different food matrices on post-drying survival.

Effect of several factors, identified to be critical for microbial cell survival during drying

and storage period, including initial cell mass, growth conditions, drying media and

rehydration conditions were studied in detail. The second section details on the viability,

proteolysis and antioxidant properties of probiotic and synbiotic yogurt in comparison

with that of control yogurt during 28 days of storage at 4oC.


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Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation

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3.1.1. Introduction

Probiotics are associated with beneficial health effects, and thus may find potential

application in the prevention and treatment of selected diseases (Alvarez-Olmos and

Oberhelman 2001; Shanahan 2002; Guarner and Malagelada 2003). Research in this

direction has stimulated interest in dairy products containing beneficial bacteria for the

general population, children and high risk groups (FAO/WHO 2001). Lactic acid

bacteria, specifically lactobacilli and bifidobacteria are the principal representatives of

probiotics in the functional food industry (Holzafpel and Schillinger 2002). Suitable

strain selection necessitates consideration of three essential premises, encompassing

general aspects (origin, identity, safety and acid/bile resistance), technical aspects

(growth characteristics, in vitro properties and survival during processing and storage)and functional/beneficial features (Collins et al. 1998; Holzafpel and Schillinger 2002;

Stanton et al. 2003). The human derived L. rhamnosus GG is a commercial probiotic

strain with recognized health benefits (Marteau et al. 2001) has been exploited for use in

the development of functional foods (Erkkila et al. 2001; Ahola et al. 2002).

The market diversification of probiotic foods relies on the availability of new strains or

new formats of probiotic cultures. Until now, fermented dairy products, mainly fermented

milks, have been used as the most successful commercial food products for the delivery

of probiotic bacteria (Saxelin 2008; Figueroa-Gonzalez et al. 2011). The commercially

available formats of starter and probiotic bacteria are generally frozen/freeze-dried. In

particular, the production of dried cell cultures is interesting because, unlike frozen

cultures, dehydrated cultures demand less storage space and lower cost of transport and

refrigeration. However, the maintenance of cell viability during drying and storage is a

major challenge. Insufficient or too extensive dehydration (moisture >5.0% (w/w) or


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Researchers have been investigating the use of spray drying (SD) as a convenient method

for producing large quantities of certain bacterial probiotic cultures (Gardiner et al. 2000;

Desmond et al. 2001; Silva et al. 2002; Corcoran et al. 2004; Golowczyc et al. 2011). It

has been estimated that the cost of SD is six times lower per kilogram of water removed

than the cost of freeze-drying (Knorr 1998). The principal advantages of SD are that it is

less expensive and faster for producing large quantities of dried cells, than other

techniques used to preserve microorganisms (Teixeira et al. 1995a, 1995b; Gardiner et al.

2000; Silva et al. 2005). However, the SD of probiotic bacteria presents a number of

challenges, in particular, the requirement to maintain culture viability, given the high

processing temperatures encountered (Daemen and van der Stege 1982; Stanton et al.

2003). Cell membrane damage is often evident following SD, and this has been attributed

primarily to the effects of heat and dehydration (Lievense et al. 1994; Teixeira et al.

1995b; To and Etzel 1997a, 1997b). Parameters affecting the survival of LAB during SD

include process airflow configuration (cocurrent or countercurrent), outlet temperature of

spray dryer, strain, carrier medium and its solids content and pre-adaptation of culture

(Johnson and Etzel 1993; Bielecka and Majkowska 2000; Gardiner et al. 2000; Desmond

et al. 2001; ORiordan et al. 2001; Lian et al. 2002).

Prebiotics may potentially be exploited as carrier media for the purposes of SD and may

be useful for enhancing probiotic survival during processing. The defining effect of

prebiotics concerns selective stimulation ofBifidobacteriumandLactobacillusin the gut,

thereby increasing the hosts natural resistance to invading pathogens (Cummings and

Macfarlane 2002). Carbohydrates such as gum acacia and soluble starch have been used

as SD carriers (Desmond et al. 2002; Lian et al. 2002). SD of probiotic L. paracasei

NFBC 338 in conjunction with the soluble fibre, gum acacia, increased its viability

compared with reconstituted skimmed milk (RSM) control during powder storage both at

15 and 30o

C (Desmond et al. 2002). However, it should be mentioned that cell

dehydration may inevitably cause membrane damage and inactivation depending on the

technological conditions applied. During SD, bacterial cultures are exposed to different

stresses (osmotic, heat, oxidative) due to the quite harsh conditions of temperature

required for product dehydration, which can cause a partial thermal inactivation of cells.


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Since some probiotic properties are closely related to the structure of the bacterial

surface, it is very important to evaluate the cellular damage after SD to determine

whether the spray-dried microorganisms maintain their functional properties. Therefore,

the objective of the present study was to investigate the injuries caused by the SD process

on selectedL. plantarumand their effect on functional properties.

Probiotic bacteria represent a unique group of LAB which is claimed to benefit human

health upon consumption, provided that a sufficiently high number of viable and

functional cells are consumed regularly (Fooks and Gibson 2002; Mattila-Sandholm et al.

2002). A daily therapeutic minimum of 108cells was proposed to ensure probiotic effects

on consumer health (Lourens-Hattingh et al. 2001). These aspects must therefore be

considered in the production of dried probiotic preparations either for use as a foodsupplement or as a constituent of starter cultures, i.e. high level of viability and

maintenance of the health-related functionality during their storage. Studies on the

evaluation of SD processes to produce probiotic culture preparations focused on the

optimization of processing parameters, selection of an appropriate drying medium, and

monitoring the loss of viability during storage under various conditions (Desmond et al.

2002; Gardiner et al. 2000; Lian et al. 2002). Furthermore, feasible approaches, such as

microencapsulation and preconditioning treatment have been assessed on their potential

to improve tolerance against adverse drying conditions (Desmond et al. 2001; Lian et al.

2003; ORiordan et al. 2001).

The aim of the present study was to investigate the SD as a method for laboratory scale

production of dry powders of L. plantarum CFR 2194. In addition, the suitability of

prebiotic substances as a part of the drying medium was assessed so as to demonstrate the

possibility of producing pro and synbiotic preparations. The storage stability of L.

plantarumCFR 2194 spray dried using different carrier media was investigated.


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3.1.2. Materials and methods

3.1.2.1. Materials

Maltodextrin was procured from Nimesh Corporation (Mumbai, India). Non-fattedskimmed milk was obtained from a local retailer. FOS-90, the prebiotic used for spray

drying studies, was prepared as described in the previous section 2.1.2.3. Lysozyme,

penicillin G and bile salt were procured from Sigma, St. Louis, MO, USA.

Microbiological culture media and media ingredients were obtained from Hi-media

Laboratories Pvt. Ltd., Mumbai, India. All other chemicals and solvents used were of

analytical grade.

3.1.2.2. Bacterial and culture conditions

L. plantarum (LP) CFR 2194 and L. fermentum (LF) CFR 2192, isolated during the

preparation of kanjika, an ayruvedic lactic acid fermented preparation, was used in this

study (Reddy et al. 2007). L. rhamnosus GG ATCC 53103 (LGG) was used as a

reference strain. The LAB strains were stored at 60oC in De Man-Rogosa Sharpe

(MRS) broth, supplemented with 40% (v/v) glycerol as a cryoprotectant. Prior to use, the

cultures [1% (v/v)] were transferred twice to MRS broth and incubated at 37oC for 12 h.

3.1.2.3. Heat challenge experiments

The thermal tolerance of L. plantarum CFR 2194, L. fermentum CFR 2192 and L.

rhamnosus GG were compared. The heating menstrum used consisted of maltodextrin

(MDX, 20% w/v), supplemented with yeast extract (0.5% w/v) and was heat-treated (90

C, 30 min); in 50 ml each in two 100 ml bottles agitated by magnetic stirrer bars, were

placed in a water bath at the appropriate test temperatures of 37oC (control and to obtain

initial count), 55, 58, 59, 60 and 61oC. One bottle was used to monitor the temperature,

while to the second bottle, an inoculum (1% (v/v)) of overnight cultures of either L.

plantarum CFR 2194, L. fermentum CFR 2192 or L. rhamnosus GG was added after

temperature equilibrium. At appropriate intervals (between 30 s and 4 min), 1 ml aliquots

were removed from the test bottle, serially diluted in saline (0.9% NaCl) and spread


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plated in MRS agar. Survivors were enumerated after 3 days of incubation at 37oC.

Experiments were conducted in triplicates and mean log survivor counts were plotted as a

function of heating time for each test temperature. At each temperature, a best fit straight

line was obtained by regression analysis, and D-values, which represent the time (min)

required to kill 90% of cells, were determined by taking the absolute value of the inverse

of the slope of this line (Stumbo 1965).

3.1.2.4. Preparation of feed solutions for spray-drying application

For SD studies, two types of feed solutions were prepared. In the first type, overnight

grown culture ofL. plantarumwas inoculated into MRS broth (1% v/v) and incubated at

37 C until the stationary phase was reached. After centrifugation at 9000 g for 15 min at

4 C, the cells [1% (w/v) on a wet weight basis] were re-suspended in 20% non-fatted

skimmed milk (NFSM) or 10% NFSM + 10% FOS (NFSM+FOS). In the second type,

the cells [1% (w/v) on a wet basis] were re-suspended in 20% maltodextrin (MDX) or

10% MDX + 10% FOS (MDX+FOS). The pH of the feed solution varied from 4.0 to 4.5.

These feed solutions were directly spray-dried. Each trial was conducted in triplicate. The

final feed solution used for SD consisted of an equal ratio of NFSM/MDX and FOS

(20%, w/v, total solids). The feed solutions were heat treated in a water bath at 90oC for

30 min, tempered at 37 oC prior to the addition of the cell suspension.

3.1.2.5. Spray drying

L. plantarumin different carrier media was spray-dried using bowen spray drier (Model

No. BE 1216, Bowen Engineering, INC. Somerville, New Jersey, USA). The inlet air,

heated to 140 2 C by an electrical heater, flowed concurrently with the spray into a

12.5-L drying chamber with an outlet temperature of 75 2 C. Feed solution was

delivered by a peristaltic pump into two fluid stainless steel atomizers. The spray-dried

powder was collected at the bottom of a cyclone. Spray drying was carried out in

triplicate, and the properties of samples from each trial of SD were studied.


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3.1.2.6. Cell survival and storage

The residual viability of L. plantarum after SD was determined by the standard plate

count method. The spray-dried powder (1 g) was rehydrated with 10 ml of sterile distilled

water to about the same solid content as that of the feed solution. The rehydrated samples

were kept on a shaker for 30 min to get a homogeneous suspension. Suitably diluted feed

solution and rehydrated samples (100 l each) were spread plated on MRS agar. Colony-

forming units were determined after an incubation of 48 h at 37 C. The percentage

survival of the spray-dried sample was calculated as follows:

% survivors = N/N0 100,

Where N0 represented the number of bacteria in the feed solution before drying and N

was the number of bacteria in the spray-dried powder. Both N and N0were expressed as

per gram of dry matter. The probiotic and synbiotic spray-dried powders in sealed

polythene bags were stored in aluminum coated bags, and stored at 4 and 30oC. Viability

of the probiotic strains was determined on the first day of preparation of spray-dried

powder and during storage for 8 weeks, at 4 and 30oC.

3.1.2.7. Moisture content

The moisture content of spray-dried powders was determined by oven drying at 102oC.

This involved determination of the difference in weight before and after drying,

expressed as a percentage of the initial powder weight, according to the International

Dairy Federation Bulletin (IDF 1993).

3.1.2.8. Scanning electron microscopy

Spray-dried powder was attached to brass stubs and coated with gold using a scanning

electron microscopy coating system (Polaron Sputter coat system, Model 5001, Polaron

Equipment Ltd., Watfort, UK). Samples were examined with a Leo electron microscope

(Model Leo-435 VP, Leo Electron Microscopy Ltd., Carlzeiss SMT, Cambridge, UK)


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using an accelerating voltage of 20 kV. Micrographs were taken at different

magnifications.

3.1.2.9. Sensitivity test

To study the potential cellular damage arising due to SD, the sensitivity of L. plantarum

CFR 2194 to NaCl, lysozyme, bile salt and penicillin G, before and after drying processes

was determined (Gardiner et al. 2000; Teixeira et al. 1995a, b). All additives except NaCl

were added to MRS molten agar, after filter sterilization. Fresh and rehydrated spray-

dried L. plantarumwas plated on MRS agar plates supplemented separately either with

5% (w/v) NaCl, 10 mg/ml lysozyme, 0.75 g/ml penicillin Gor 0.25% (w/v) bile salt.

The plates were examined after 23 days of incubation and viable cell counts were

compared with that obtained on unsupplemented MRS plates (without these selective

agents).

Acidification rate of MRS and milk with added yeast extract (1% w/v) was performed

with fresh and rehydrated spray-driedL. plantarum. One ml of fresh or rehydrated spray-

dried L. plantarum was transferred to 49 ml of MRS or milk, with yeast extract

previously equilibrated at 37 C (final concentration was approx. 2106 CFU/ml). At

regular intervals, samples were withdrawn and pH of the samples was recorded.

3.1.2.10. Statistical Analysis

Experiments were carried out in triplicates. The mean and standard deviation were

calculated for n=3. One-way analysis of variance (ANOVA) at P0.05 was used to

express the statistical differences between the spray dried and the fresh cells. ANOVA

was carried out using the statistical software (Origin 6, USA).


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3.1.3. Results

3.1.3.1. Thermal tolerance of probiotic lactobacilli

The most important aspect that needs special attention in the preparation of spray-driedpowders of probiotic lactobacilli is the tolerance of the microorganisms to very high

temperatures encountered during SD. Tolerance to high temperature has been shown to

vary among strains of probiotic lactobacilli (Gardiner et al. 2000). Initially, the thermal

tolerance of three probiotic lactic cultures in MDX (20% w/v), in the range of 5561oC

was compared. At 59oC, a decrease of 1.92 log10 CFU/ml of L. rhamnosus GG was

observed (Figure 3.1.1c), while the two other strains exhibited comparatively higher heat

resistance at this temperature (Figure 3.1.1a, b). At 61oC, L. plantarumCFR 2194 was

most thermal tolerant, with an observed decrease of 1.59 log10CFU/ml. Whereas, in case

ofL. fermentumCFR 2192 andL. rhamnosus GG,a decrease of 2.99 log10CFU/ml and

3.70 log10CFU/ml respectively was observed.

The D-values of 6.21 min calculated from the above data confirmed L. plantarumCFR

2194 to be the most thermotolerant among the three strains studied, following exposure to

61oC. In contrast, D-values forL. fermentumCFR 2192 andL. rhamnosusGG were 2.7

and 1.6 min, respectively. Similarly, there was a considerable difference in D-valuesamong the three strains studied, at 59 C (8.8 min for L. planatrum, and 3.9 and 3.3 min

forL. fermentumandL. rhamnosusGG).


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Figure 3.1.1: Survival ofL. plantarumCFR 2194 (a),L. fermentumCFR 2192 (b) andL.

rhamnosus GG ATCC 53103 (c) heated in maltodextrin (20% w/v) at 55oC (), 58

oC

(), 59oC (), 60

oC (), 61

oC (). The results are based on data from triplicate heat

challenge experiments.

(a)

(b)

(c)

4

5

6

7

8

9

10

0 1 2 3 4

Survivorslo

g10CFU/ml

Time (min)

3

4

5

6

7

8

9

10

0 1 2 3 4

Survivorslog10CFU/ml

Time (min)

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4

Survivorslog10CFU/ml

Time (min)


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3.1.3.2. Effect of prebiotic FOS on the survival of L. plantarum CFR 2194 during

spray drying

The survival of L. plantarum at stationary phase, spray-dried with different carrier

medium is shown in Figure 3.1.2. Percent survival was more than 75, when MDX (20%

w/v) and NFSM (20% w/v) were used as a carrier medium. The viable counts of resultant

spray-dried powders were 8.90 and 9.11 log10CFU/g respectively. In order to evaluate

the efficacy of the prebiotic, FOS in enhancing the survival of the test organism during

SD and its protective effect against cell damage, subsequent SD trials were carried out

with FOS as an additional ingredient of the feed solution [MDX+FOS(20% w/v) and

NSFM+FOS (20% w/v)]. In case of the presence of FOS, L. plantarumshowed greater

survival of over 85%, when MDX and NFSM were used as carriers, yielding powder withviable counts of 10.50 and 9.631 log10CFU/g. Cell damage was not observed in spray-

dried powders (Figure 3.1.3). Under dehydration conditions used, L. plantarum showed

very little reduction of viability (0.89 and 1.6 log10 CFU/g) in MDX+FOS and

NFSM+FOS powders. Whereas, significant (P0.05) reduction in viability of 2.08 and

2.6 log10CFU/g was found in dehydrated powders of MDX and NFSM. No significant

(P0.05) difference in viability was observed between the two samples of MDX+FOS

and NFSM+FOS powders. From the results obtained it can be concluded that, inclusion

of FOS as one of the carrier adjuncts in the SD medium offered an increased survival or

protection from cell damage during SD.

3.1.3.3. Moisture content of spray-dried L. plantarum

The moisture content of all the spray-dried powders under this study was found to be less

than 4% (Figure 3.1.2). The results obtained are in line with the general requirement that

spray-dried powder should contain less than 4% H2O/g in order to be categorized as

stable (Masters 1985).


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Figure 3.1.2: Effect of different carrier adjuncts on the survival of L. plantarum CFR

2194 (black bars) and moisture content of powder (line plot) prepared by spray drying

various carrier feed solution at an outlet temperature of 75 2oC and inlet temperature of

140 2oC . MDX= Maltodextrin powder (20%, w/v, total solids); MDX/FOS (10%

Maltodextrin + 10% Fructooligosaccharides); NFSM= Non-fatted skimmed milk powder

(20%, w/v, total solids); NFSM/FOS (10% Non-fatted skimmed milk powder + 10%

Fructooligosaccharides). Data are the means of triplicate spray drying trials SD.

2.9

2.95

3

3.05

3.1

3.15

3.2

3.25

65

70

75

80

85

90

95

MDX MDX + FOS NFSM NFSM+ FOS

Mois

tureContent(%)

Survival(%)

Carrier Adjuncts


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Figure 3.1.3: Scanning electron micrographs of the spray-driedL. plantarum

3.1.3.4. Acidification rates of spray-dried L. plantarumCFR 2194

The decrease of pH in MRS and milk medium seeded with spray-dried L. plantarumwas

determined and the results are shown in the Figure 3.1.4. Acidification rates in both

growth media by the activity of fresh (before SD) and rehydrated (after SD) L. plantarum

showed significant difference (P0.05). However, rehydrated L. plantarum spray-dried

either with MDX or NFSM showed a delay in acidifying activity, both in MRS and milk

medium. However, presence of prebiotic FOS showed the better acidification activity in

both the growth medium. MDX+FOS and NFSM+FOS samples showed a significant

(P0.05) and faster reduction in pH as compared with that of MDX and NFSM samples.

Comparatively, as expected, fresh culture of L. plantarum showed better acidification

activity in comparison with all the above rehydrated samples.

Cells covered

with carriers


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Figure 3.1.4: Acidification activity by fresh and spray-driedL. plantarumCFR 2194. a),

L. plantarumspray dried with maltodextrin and b), L. plantarum spray dried with non-

fatted skimmed milk growing in MRS and milk with added 1% (w/v) yeast extract.RehydratedL. planatarum+ 20% MDX or NFSM (); LP + 10 % MDX + 10 % FOS or

NFSM () and fresh () strain growing in milk, and rehydrated L. planatrum + 20%

MDX orNFSM ();L. plantarum+ 10 % MDX + 10 % FOS orNFSM (x) and fresh ()

strain growing in MRS. Each point represents the mean of triplicates.

(b)

(a)

4

4.5

5

5.5

6

6.5

7

0 4 8 12 16 20 24 30 34 38 42 46

pH

Time (h)

4

4.5

5

5.5

6

6.5

7

0 4 8 12 16 20 24 30 34 38 42 46

pH

Time (h)


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3.1.3.5. Tolerance of cells to NaCl, lysozyme and bile salt

Retention of the probiotic functional properties of L. plantarumCFR 2194 after SD was

investigated. Functional properties such as tolerance to NaCl, lysozyme, bile salt and

penicillin G are associated with membrane and cell wall integrity. SD could affect these

characteristics, and thus results in cell wall damage. Sensitivity to different agents was

studied as a measure of membrane damage (Table 3.1.1). As can be seen from the results,

L. plantarumafter SD appear to be sensitive to NaCl, as revealed by decrease in the cell

number. The decrease was insignificant (P0.05) in MDX+FOS and NFSM+ FOS

samples. Exposure of the MDX+FOS and NFSM+FOS samples to lysozyme, bile salt

and penicillin G did not result in any reduction in viability. However on rehydration, both

the samples of MDX and NFSM showed insignificant sensitivity to lysozyme, penicillinbut showed significant (P0.05) sensitivity to bile salt. This suggests that the FOS might

form a glassy layer around the cell membrane of L. plantarumCFR 2194 during the SD,

thus causing less membrane damage and thus resulting in higher viability.


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Table 3.1.1:Number of viable bacteria and sensitivity to NaCl, lysozyme, penicillin G and bile salt ofL. plantarumCFR 2194 before

and after spray drying.

a The values in the column represent the viable count ofL. plantarumbefore (fresh) and after (dried) spray drying on unsupplemented

MRS (survival of strains).

b These values are the mean of three independent assays SD and represent the viable counts of spray-driedL. plantarumon MRS

containing the agent.

Different small letter superscripts depict the statistical difference within a row, P0.05 between means for different agents

Strains

Log10CFU/ml

Initial Counta NaCl

b Lysozyme

b Pencillin

b Bile Salt

b

Fresh Dried

1% LP + 20% MDX 8.90 0.12 6.82 0.3a 6.76 0.13

a 6.80 0.7

a 6.77 1.41

a 6.23 1.31

1% LP + 10% MDX + 10% FOS 10.2 0.32 9.31 0.15a 9.24 0.21

a 9.29 0.42

a 9.32 1.23

a 9.34 0.9

a

1% LP + 20% NFSM 9.11 1.4 6.51 0.13a 6.42 1.31

a 6.45 0.34

a 6.43 2.1

a 6.01 3.61

1% LP+ 10% NFSM + 10% FOS 9.63 + 0.12 8.03 1.2a 7.95 0.24a 8.01 1.2a 8.04 1.02a 8.05 2.34a


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3.1.3.6. Storage Studies

Following SD, MDX+FOS and NFSM+FOS powders were placed in sealed polythene

bags and stored at 4 and 30oC, during which viability was assessed to identify the

optimal temperature for storage over 60 days, and to investigate the probable beneficial

effect of FOS on viability during storage. The optimal survival of L. plantarum was

found to be 60% and 52% in both the powders (MDX+FOS and NFSM+FOS) during

storage at 4oC. During storage at 4 C, a reduction in the viability of 0.5 and 1.2 log

CFU/g was observed in case of MDX+FOS and NFSM+FOS powders (Figure 3.1.5).

However, during storage at 30 C for 60 days, a significant (P0.05) reduction in the

viability was observed. The survivability in case of MDX+FOS and NFSM+FOS

powders was observed to be only 50% and 45%, respectively. In terms of log survivors

the reduction in the viability of spray-driedL. plantarumcorresponded to 3.3 and 3.6 log

CFU/g with MDX+FOS and NFSM+FOS powders, respectively.


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Figure 3.1.5: Survival of spray-dried L. plantarum CFR 2194 and

Fructooligosaccharides with maltodextrin (MDX) (a); and nonfat skimmed milk (NFSM)

(b) as a carrier during storage at 4C () and 30C (). Data are the means of triplicateexperiments SD. Values were significantly different at P


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3.1.4. Discussion

Successful application of probiotic cultures as functional food ingredient essentially

depends on the availability of viable, stable cultures in large scale. SD is an economical

process for preparing industrial scale food adjuncts. The main focus of the present

chapter is on the effect of additives such as MDX, NFSM, most commonly used carriers

on the SD ofL. plantarumCFR 2194. Studies on the effect of FOS, a prebiotic prepared

in our laboratory has been addressed as a case study. Viability, functional properties and

shelf stability of spray-driedL. plantarumhas been investigated.

The thermotolerance of the probiotic lactic cultures in the range of 55-61oC was

compared in the MDX (20%) menstrum (Gardiner et al. 2000). From the data presented

in the current study, it can be inferred that L. plantarum CFR 2194 is thermotolerant

strain, followed by L. fermentum CFR 2192, L. rhamnosus GG. The D-values for L.

plantarumCFR 2194 and L. fermentumCFR 2192 are 6.21 min and 2.7 min at 61oC,

respectively. The reported D values for 2 different strains of L. rhamnosus; E800 and

GG are 2.7 and 1.32 min, respectively, at 61oC. Thus it can be said that the L. plantarum

strain used in the current study exhibits comparatively a higher thermotolerance.

Additionally, stationary phase cultures were used for SD, which might have contributed

to increased heat resistance, as it has been demonstrated previously that stationary-phase

cultures are more resistant to heat stress than cells in the exponential phase of growth

(Gardiner et al. 2000; Teixeira et al. 1994).

Sunny-Roberts and Knorr (2009) conducted preliminary SD experiments to determine the

optimum outlet temperature for maximum probiotic viability and to get powders with

moisture contents not exceeding 4% (Masters 1985). When outlet temperatures between

60 and 75oC were used, the survival rate was found to be inversely proportional to outlet

temperature. The residual moisture content, from 3.57-4.2% (w/w) in L. rhamnosusGG

and 3.57-4.43% (w/w) in L. rhamnosus E800, was seen to decrease as the outlet air

temperature increased. A residual moisture content of 3.79% and 4.1% has been achieved

upon SD at an outlet temperature of 65-70oC (Masters 1985). Based on these reports, SD

trials were carried at an outlet temperature of 75oC and an inlet temperature of 140

oC to


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obtain good quality powders having a moisture content of


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storage is necessary for optimal culture viability in spray-dried powders over time,

although impractical from a commercial point of view.

Acidification activity of fresh and spray-driedL. plantarum CFR 2194 in both the growth

media (MRS and Milk) showed a significant (P0.05) difference. However, rehydratedL.

plantarum strain showed a delay in acidifying activity both in MRS and milk medium

when MDX and NFSM samples were used. Similar results were obtained by Teixeira et

al. (1995a) with dehydratedL. delbrueckiissp bulgaricus. In the present study rehydrated

L. plantarum(MDX+FOS and NFSM+FOS) showed faster rate of acidifying activity by

lowering the pH of the both media.

It is known that during and after SD and also during subsequent storage in the dried

state, cells can suffer from a variety of stresses including heat, osmotic and oxidative

stress that result in the loss of cellular viability and activity (Teixeira et al. 1995a, 1995b;

Gardiner et al. 2000; Silva et al. 2002, 2005; Golowczyc et al. 2011). After SD it is

necessary to investigate whether viable bacteria maintain their properties and

functionality, and these characteristics will be associated with membrane and cell wall

integrity. Increased sensitivity of sub-lethally injured bacteria to NaCl, lysozyme,

penicillin G and bile salt has been associated with cell membrane damage (Brennan et al.

1986; Teixeira et al. 1995b, 1997; Sunny-Roberts and Knorr 2009). Sensitivity to

different agents was studied as a measure of membrane damage (Table 3.1.1). No

significant difference (P0.05) between the viable count of L. plantarumin the presence

of FOS on MRS containing each agent before and after SD was observed. Using these

indirect approaches, we can infer that no cell damage was observed in spray-dried

stationary phase cells and in the presence of prebiotic FOS. These results are in

agreement to Corcoran et al. (2004). Gardiner et al. (2000) demonstrated that L.

paracasei was more resistant to drying than L. salivarius and concluded that this is

directly related with greater membrane damage in L. salivarius(evaluated by sensitivity

to NaCl). These results are similar to the results of Sunny-Roberts and Knorr (2009) on

spray driedL. rhamnosusGG (LGG) andL. rhamnosusE-800 (E800). In contrast, in this

study, taking into account that L. plantarumCFR 2194 was more resistant to the drying


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process (showing > 80% viability), the viable microorganisms that survive drying process

have not demonstrated membrane damage under the conditions tested.

In conclusion, we found that the inclusion of the prebiotic FOS in the feed solution

resulted in increased probiotic survival during SD. It has been successfully shown that

spray-dried powder with a viable count of 109CFU/g could be prepared using stationary

phase cultures and MDX/NFSM, along with the prebiotic FOS in the feed media.

Furthermore, probiotic cultures retained good viability during storage at 4oC, when

MDX+FOS were used. However, the viability during storage at 30oC declined rapidly.

Powders consisting of MDX+FOS afforded better protection to probiotic lactobacilli

during storage. Given the broad applicability of maltodextrin powders and the health

benefits associated with both prebiotics and probiotics, it is possible that these powderscould be tailored for specialized functional food applications.


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Section: 3B

Development of synbiotic yogurt containing selected Lactic acid bacteria

and Fructooligosaccharides: Comparative study during refrigerated storage


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Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt

127

3.2.1. Introduction

The role of food in the maintenance of health and well-being and in the prevention of

disease continues to receive increased scientific and commercial interest which has

strengthened the concept of functional foods. In general it is accepted, that a functional

food provides health benefits beyond general nutritional benefits (Shortt et al. 2004).

Probiotics, prebiotics, and synbiotics are considered as the main dietary products

marketed under the category of functional foods. Continued efforts are being made to

improve the human health by modulating the intestinal microbiota using live microbial

adjuncts, probiotics. Probiotic organisms require a vehicle to reach the site of action, GIT

of the human body. The vehicle is generally a food product, which contains these live

bacteria. The products should have a good shelf-life and should have a cell count morethan 10

6CFU/ml till the end of recommended period of storage. The product should also

sustain the harsh conditions of gastric acid and bile salts before it reaches the GIT.

Scientific evidence suggests that the probiotic bacteria consumed at a level of 10910

11

CFU/day can decrease the incidence and severity of some intestinal disorders (Zubillaga

et al. 2001). In the current market scenario, dairy products such as yogurt, fermented milk

and cheese dominate the probiotic food sector.

The yogurt and fermented milks represents 55 billion and 20 million tons produced per

year with North America, Europe and Asia accounting for 50% of the market. Sales of

yogurt and fermented milks also continues to expand worldwide, most noticeably in

emerging markets such as China, India and Russia as well as in countries of the Middle

east, North Africa and Latin America, in the span of 5 years, the global sales value

increased about 25%. The functional fermented milks grew from 2, 13 billion in 2005

to 3, 3 billion in 2010, which means an increase of 55%. In particular, probiotic drinks

have contributed to fermented milks market growth, leading to some of the most

innovative new products in the dairy sector today (FAO/WHO 2012). The popularity of

fermented milks is due, at least in part to various health claims and therapeutic benefits

that have been associated with some of these products.


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A variety of fermented milk products are produced throughout the world, among which

yogurt (or yoghurt) is most popular. Yogurt is one of the well-known foods that contain

probiotics. Yogurt is defined by the Codex Alimentarius, (1992) as a coagulated milk

product that results from the fermentation of lactic acid in milk by L. bulgaricusand S.

thermophilus(Bourlioux and Pochart 1988). Of late, other LAB species are often used for

imparting the desired characteristics to the final product. It is generally assumed that,

consumption of probiotic yogurt should be more than 100 g/day containing more than 106

CFU/ml is necessary for deriving the health benefits (Ervin et al. 2002). To meet the

National Yogurt Associations criteria for live and active culture yogurt, the finished

yogurt must contain live LAB in amounts 108

CFU/g at the time of manufacture

(Chandan et al. 1993), and the cultures must remain active at the end of the stated shelf

life, as ascertained using specific activity test.

In the past two decades, there has been a significant increase in the popularity of yogurt,

emphasizing the incorporation of L. acidophilus, L. casei, B. animalisssp. lactisand B.

longumssp. longum(Ramachandran and Shah 2010; Sheu et al. 2010). The conventional

yogurt starter bacteriaL. delbrueckiissp. bulgaricusand S. thermophilus,lack the ability

of surviving the passage through the GIT and consequently do not play a role in the

human gut (Gibson and Roberfroid 1995). Many studies suggest that the consumption of

synbiotic products has a greater beneficial effect on the human health than probiotic or

prebiotic products (Gibson and Roberfroid 1995; Gmeiner et al. 2000; Roberfroid 1998;

Schaafsma et al. 1998). Synbiotics, suggesting synergism, refers to the combined use of a

prebiotic compound that selectively favors a probiotic organism. The principle aim of

adding prebiotic/probiotics or synbiotics to the diet is to beneficially affect the consumer

by improving the intestinal microflora balance that might lead to improved nutrition and

health (Kolida et al. 2002; Rastall 2004). Moreover, the synbiotic product may allow an

efficient implantation of probiotic bacteria in the colon, because prebiotic has a

stimulating effect on the growth and/or activities of the exogenous and the endogenous

colonic bacteria (Roberfroid 1998). In synbiotic fermented milks, the strains of L.

acidophilus, L. casei and Bifidobacterium ssp. (B. animalis, B. bifidum, B. breve, B.

infantis and B. longum) are widely used as probiotics, whereas, FOS, GOS, lactulose,


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inulin-derived products etc. are widely used as prebiotics (Klaenhammer and Kullen

1999; Ziemer and Gibson 1998).

L. plantarum CFR 2194 and L. fermentum CFR 2192 used in the present study are

isolates from kanjika, a rice-based ayurvedic fermented food preparation (Reddy et al.

2007). The organisms under study has shown important probiotic properties like acid and

bile tolerance, ability for the production of vitamin B12 and significant antagonistic

activity against the intestinal pathogen like Escherichia coli, Listeria monocytogensand

Salmonella(Madhu et al. 2010; Madhu et al. 2011). Besides the probiotic properties, the

antioxidative ability of LAB, including yogurt starters, has been reported (Kullisaar et al.

2002; Kullisaar et al. 2003; Lin and Chang 2000). The antioxidative activity of some

lactobacillusstrains, and the probiotic LAB strains used as food supplements may have asubstantial impact on human health (Lin and Chang 2000; Oxman et al. 2000). The

present chapter focuses on the characterization and antioxidative functionality of

probiotic and synbiotic yogurt samples during refrigerated storage for 28 days.

3.2.2. Materials and methods

3.2.2.1. Materials

Reconstituted skimmed milk and pasteurized milk were obtained from a local retailer.

Microbiological culture media and media ingredients were obtained from Hi-media

Laboratories Pvt. Ltd., Mumbai, India. FOS-90, the prebiotic used was prepared as

described in the previous section 2.1.2.3. All other chemicals and solvents used were of

analytical grade.

3.2.2.2. Microorganisms and culture conditions

The regular starters namely S. thermophilus (ST) ATCC19258 and L. delbrueckii ssp.

bulgaricus (LB) CFR 2028 along with probiotic L. plantarum (LP) CFR 2194 and L.

fermentum(LF) CFR 2192, isolated from kanjika(Reddy et al. 2007; Madhu et al. 2010;

Madhu et al. 2011), were used for the yogurt preparation. The cultures were stored at 60

oC in De Man-Rogosa Sharpe (MRS) broth, supplemented with 40% (v/v) glycerol as a


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cryoprotectant. Prior to use, the cultures [1% (v/v)] were transferred to MRS broth: LB

was incubated at 40oC and ST, LP and LF were incubated at 37

oC for 12 h. The active

cultures after two successive transfers were further inoculated [1% (v/v)] to 10 ml

aliquots of reconstituted skimmed milk medium (RSM) supplemented with glucose (2%),

yeast extract (1%) and incubated for 4-6 h at 37oC before inoculation into milk.

Fructooligosaccharides (FOS) [70oB, containing 90-93% (w/w) FOS] was used as a

prebiotic in the preparation of synbiotic yogurt.

3.2.2.3. Yogurt preparation and storage

Fresh, pasteurized milk containing 3% fat, collected from local market was used for the

preparation of yogurt. The milk was preheated to 63 C for 30 min, at which stage the

FOS (1g/100 ml) was added, followed by cooling to 40oC before inoculation. The milk

was divided into 3 groups and 5 different portions (Table 3.2.1)and 100 ml of same was

poured into each of the polystyrene cups under aseptic conditions. This was followed by

inoculation with ST (7.92 log CFU/ml), LB (7.38 log CFU/ml), LP (7.51 log CFU/ml)

and LF (7.42 log CFU/ml), each at 1 % (v/v). The preparation was mixed thoroughly and

kept for incubation at 40oC for 6- 8 h. After incubation yogurt samples were stored at 4

oC for 28 days. Samples were drawn at weekly intervals up to fourth week.


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Table 3.2.1: Preparation of probiotic, prebiotic and synbiotic yogurt samples

3.2.2.4. Determination of viability

The colony counts of LB, ST were determined as described elsewhere (Dave and Shah

1996; Tharmaraj and Shah 2003). The viability of ST and LB was determined using M17

agar medium (aerobic incubation at 37oC for 48 h) and reinforced clostridia agar (RCA)

(anaerobic incubation at 42oC for 48 h), respectively. LP was enumerated on

Lactobacillus plantarumselective medium (LPSM), under anaerobic incubation at 37oC

for 48 h (Bujalance et al. 2006). LF was enumerated on Columbia Agar Base (CAB) atpH 5.1, supplemented with 0.5 g cysteine, 5 g raffinose, 2 g LiCl and 3 g sodium

propionate per litre (Champagne et al. 1997). Plates were incubated at 37 C for 48 h

under anaerobic conditions.

Groups Combination of organism (1% v/v) and Cell

concentration, log CFU/ml

Probiotic

yogurt

A S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp.

bulgaricus CFR2028 (7.38) + L. plantarum CFR2194 (7.51)

B S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp.

bulgaricus CFR2028 (7.38) + L. fermentum CFR 2192 (7.42)

Synbiotic

yogurt

C S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp.

bulgaricus CFR2028 (7.38) + L. plantarum CFR2194 (7.51)

+ 1% (w/v) Fructooligosacharides

D S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp.

bulgaricus CFR2028 (7.38) + L. fermentum CFR 2192 (7.42)

+ Fructooligosacharides 1% (w/v)

Regular

control

RC S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp.

bulgaricus CFR2028 (7.38)


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3.2.2.5. Chemical Analyses

The pH values of yogurt samples were measured using a pH meter (Fisher Scientific,

model 955, India). Titratable acidity (TA) was determined by titration with 0.1N NaOH

solution and expressed as percent lactic acid (AOAC 1984). TA and pH were measured

on a weekly basis during storage of yogurt samples.

3.2.2.6. Texture analysis

The gel strength of yogurt samples was determined at 4-6oC by penetration

measurements (Stevens-L.F.R.A. Texture Analyser, CNS Farnell, Borehamwood, UK).

The instrument was adjusted to the following conditions: cylindrical probe, probe area

5.07 cm2; penetration speed, 1.0 mm/s; penetration distance, 20 mm into surface. The gel

strength was determined in triplicate and expressed as N/cm2of probe area.

3.2.2.7. Color analysis

The color values of yogurt samples were measured using a Hunter Lab color measuring

system (Lab scan XE, Hunter Ass. Lab, Virginia, USA), using the L, a, b color

scheme. The L, a, bvalues represent brightness/darkness, green/red and yellow/blue

respectively (Krishnamurthy and Kantha 2005). The operating conditions were illuminant

D65 and 10o observer. An average of 5 values was taken per replication. The values

represent an average of three readings.

3.2.2.8. Determination of proteolytic activity

The extent of proteolysis was determined by measuring the liberated amino acids and

peptides using the o-phthaldialdehyde (OPA) method of Leclerc et al. (2002) with some

modifications. Yogurt samples (2.5 ml) were mixed with trichloroacetic acid (0.75%; 5

ml) and the mixture was filtered using a filter paper (Whatman No.1). To the permeate

(150 l), OPA reagent (3 ml) was added and the absorbance of the solution was measured

spectrophotometrically (UV-1601, Shimadzu Corporation, Japan) at 340 nm after 2 min

at RT (28 2oC). The proteolytic activity of these bacterial cultures was expressed as the


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free amino groups measured at 340 nm as a difference in absorbance between probiotic,

synbiotic and control batches.

3.2.2.9. Antioxidant activity assay

3.2.2.9.1. Measurement of DPPH free radical scavenging activity

The antioxidant activity of each yogurt sample was determined as the ability of the

extract to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. A 0.1 mM DPPH

radical solution in 95% ethanol was prepared. Ethanolic DPPH solution (800 l) was

mixed with 0.2 ml of yogurt sample or 95% ethanol (Control), vortexed well and

incubated for 30 min at RT (28 2oC). The samples were centrifuged for 5 min at 10000

gat RT and the absorbance of samples was measured at 517 nm. The antioxidant activity

was expressed as percentage (%) DPPH scavenging = [(control absorbance - sample

absorbance)/ (control absorbance) 100].

3.2.2.9.2. Determination of total phenolics

The method of Zheng and Wang (2001) was used for the determination of total phenolic

compounds in yogurt samples using Folin-Ciocalteu reagent (FCR) and gallic acid as

standard. The sample (0.1 ml) was mixed with 0.9 ml of distilled water and was

incubated for 2 h at RT (28 2oC) in a shaking water bath. To this, FCR reagent (1 ml)

(1:2 dilution) and 10% Na2Co3(2 ml) was added. The mixture was centrifuged at 10000g

for 20 min and the supernatant was decanted and filtered through filter paper (Whatman

No. 1). The absorbance of the clear supernatant solution was measured at 765 nm.

Experiments were carried in triplicates. Results were expressed as milligrams gallic acid

equivalent; (GAE) mg/100 ml extract.

3.2.2.9.3. Measurement of ferric reducing antioxidant power (FRAP)

The total antioxidant potential of the sample was determined by the ferric reducing ability

(FRAP assay) as a measure of the antioxidant power (Benzie and Strain 1996). To the

freshly prepared FRAP solution (3 ml), 100 l of sample was added and incubated at 37


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oC for 10 min. The absorbance of reaction mixture was measured at 593 nm. FRAP

values were calculated with reference to a standard curve [ferrous sulphate

(FeSO4.7H2O) solutions (0.1-3.0 mM/l)] and results were expressed as mg Fe2+

/100 ml

(FRAP value).

3.2.2.10. Statistical analysis

The experiments were organized as a randomized blocked split-plot in time design,

exploring the influence of prebiotics and time as the main effects. All experiments were

carried out in triplicates. Results were analyzed using the general linear model (GLM)

procedure of the SAS system (SAS 1996). The level of significance is presented at

P


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Table 3.2.2: Scavenging effects of yogurt samples on the 1,1-diphenyl-2-pyrylhydrazyl

radical

Samples* Inhibition Percentage (%)

Day 1 Day 7 Day 14 Day 21 Day 28

A 82.4

83.1a

84.3a

84.4a

84.4a

B 81.4a

82.0

82.1

82.1

82.1

C 84.7c

85.4c

86.2c

86.2c

86.2c

D 82.1 82.8 83.0 83.3 83.0

RC 71.2a

73.5a

74.2a

75.5a

75.2a

Results presented as a mean (n = 3) pooled standard error of the mean (0.014).

Different small letter superscripts depict the statistical difference within a row, P


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Figure 3.2.1: Effect of storage time on phenolic compound content in yogurt samples.

Error bars represent a pooled standard error of the mean, SEM=0.02 mg/100ml. The

significant difference in different samples when compared to that of control at respective

time intervals was analysed and indicated as P


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Figure 3.2.2: Effect of storage time on antioxidant power (FRAP value) of yogurt

samples. Error bars represent a pooled standard error of the mean, SEM=0.03 mg/100ml.

The significant difference in different samples when compared to that of control at

respective time intervals was analysed and indicated as P0.05). The viable cell counts

of LB declined from 7.89 to 6.73 log CFU/g in control yogurt; however, it was

maintained ~7 log CFU/g in probiotic and synbiotic yogurt samples throughout the

storage. There were no observable changes in the viability of probiotic cultures in

probiotic batches. However, supplementation of FOS resulted in a significant (P


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increase in the total count of LP and LF from 9.16 and 9.17 log CFU/g to 9.52 and 9.45

log CFU/g, respectively. The viable cell counts of all probiotics by the end of 28 days of

storage were 9 log CFU/g and thus the yogurt developed could be considered as a

probiotic product.


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Table 3.2.3: Effect of FOS on viability of yogurt starter cultures (L. delbruekii ssp.

bulgaricus LB 2028 and S. thermophilus ST 19258) and probiotic organisms (L.

plantarum CFR2194,L. fermentumCFR 2192)

Cultures Samples Period of Storage, day (Log CFU/g)1

1 7 14 21 28

L. delbruekii ssp. bulgaricusLB 2028

Control Batches RC 7.89a

7.46a

6.51 6.67a

6.73c

Probiotic Batches A 7.91a

7.93a

8.06a

8.14 8.11a

B 7.89a

7.95a

8.12 8.17a

8.15c

Synbiotic Batches C 7.93a

8.01a

8.09a

8.02a

7.96a

D 7.89

a

7.91

a

7.95

a

7.92

a

7.93

a

S. thermophilusST 19258

Control Batches RC 8.79a

8.81a

8.85a

8.86a

8.78a


8.48a

8.52a

8.55a

8.49a

B 8.39a

8.44a

8.59a

8.45a

8.36a


8.63a

8.62a

8.59a

8.57a

D 8.50a

8.55a

8.53a

8.49a

8.45a

L. plantarumCFR2194


8.13a

7.67 7.93a

8.32a


9.45a

9.55a

9.56 9.52

L. fermentumCFR 2192

Probiotic Batches B 7.44a

7.67a

7.48a

7.33a

7.03a

Synbiotic Batches

SEM

D 9.17a

9.23a

9.30

0.24

9.31 9.45a

1Results presented as a mean (n = 3) pooled standard error of the mean (0.243).

Different small letter superscripts depict the statistical difference within a row, P


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3.2.3.3. Changes in pH & TA

Changes in pH and TA during refrigerated storage of yogurt samples are shown in Table

3.2.4. The pH of the samples ranged from 4.62 to 4.42 units. Both synbiotic samples

with LP and LF showed a significant (P


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Table 3.2.4:Changes in pH and titratable acidity (TA; percentage lactic acid) of yogurt samples during storage.

Results presented as a mean (n = 3) pooled standard error of the mean (0.036). Different small letter superscripts depict the

statistical difference within a row, P


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3.2.3.4. Changes in extent of proteolysis

During fermentation, milk proteins are hydrolysed by extracellular proteinases produced

by LAB resulting in an increase in the amount of free amino groups. Proteolytic activity

of mixed culture supplemented with prebiotic FOS during prolonged cold storage was

estimated by determining the free amino groups using the OPA method (Figure 3.2.3).

The extent of proteolysis was similar in probiotic and synbiotic samples until day 14.

Thereafter, it was higher (P


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Figure 3.2.3: Changes in extent of proteolysis (A340) in control, probiotic and synbiotic

yogurt samples stored at 4oC for 28 days. Error bars represent a pooled standard error of

the mean, SEM=0.02 Abs340nm. Values were significantly different when compared to

control sample at P


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Figure 3.2.4:Gel strength of control, probiotic and synbiotic yogurt samples. Error bars

represent a pooled standard error of the mean, SEM=0.04 N/cm2. Abbrevations are as per

Table 3.2.1.

In case of all yogurt samples, the Lvalues were observed to be very high throughout the

storage period indicating that the product retained the appealing whiteness. There was a

slight increase in the b value of synbiotic yogurt samples resulting in a slight yellowness

of the product, which may be due to the presence of FOS. However, the change in color

was negligible. All samples had a negative a (greenness) values (Table 3.2.5). The

addition of FOS did not affect the avalues of the product.

0

0. 1

0. 2

0. 3

0. 4

0. 5

0. 6

0. 7

0. 8

1 7 14 21 28

GelStrength(N/cm2)

Storage Time (Days)

A B C D RC


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Table 3.2.5: Color properties (measured as Hunter L, a and b) of yogurt samples at

different storage time.

Storage

(Days)

Samples**

Visual color of Yogurt Samples*

L a b

7

A 82.11 3.10 7.09

B 82.31 2.98 10.12

C 80.41 3.05 10.58

D 79.36 3.01 10.63

RC 79.08 3.10 11.01

14

A 82.91 3.38 8.15

B 81.66 3.09 10.56

C 85.48 3.10 10.92

D 82.78 3.03 10.93

RC 81.16 3.17 11.49

21

A 83.31 3.61 8.52

B 82.09 3.98 10.91

C 86.06 3.81 10.98

D 83.55 3.89 11.01

RC 82.25 3.63 11.72

28

A 83.81 3.82 8.92

B 82.79 4.01 11.01

C 86.76 3.95 11.18

D 83.75 3.89 11.06

RC 82.75 3.89 11.79

L: (lightness); a: (+a; redness and a; greenness); b: (+b; yellowness and b; blueness)

*Each value is the mean of triplicate experiments.

**Abbrevations are as per Table 3.2.1.


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3.2.4. Discussion

The physiological state of the probiotic organisms is of special importance when

selecting a strain for a specific application. Several investigations have shown that

bacteria in the logarithmic phase are much more susceptible to environmental stresses as

compared to those in stationary phase (Heller 2001). A significant (P0.05). The


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addition of FOS had no adverse effect on the growth of yogurt cultures. These

observations are in line with a report with similar findings but different cultures (Gee et

al. 2007; Vasiljevic et al. 2007). As expected there were no observable changes in the

viability of probiotic cultures in probiotic samples. However, supplementation of FOS

resulted in a significant (P


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duration. Similar to previous findings (Donkor et al. 2007), FOS enhanced the liberation

of peptides and amino acids substantially (Figure 3.2.3), suggesting that the inherent

proteolytic activity of probiotic strains used for the study can be enhanced using FOS.

This is further corroborated strongly with the increased amount of free NH3groups in the

yogurt during storage irrespective of final pH. Ramachandran and Shah (2010) observed

significantly higher proteolysis in synbiotic lowfat yogurt containing inulin as a

prebiotic.

The probiotic and synbiotic yogurt samples showed the significant (P


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Fe2+

/100 ml at 12 days storage (Gad et al. 2010). Considering the earlier reports and

present observations it can be concluded that the synbiotic supplementation, significantly

influences and maintains the antioxidant status in yogurt samples.

development of a shelf stable synbiotic formulation containing lactic acid bacteria and...

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