Development of a shelf stable synbiotic formulation containing Lactic acid bacteria and Fructooligosaccharides

Transcription

1 Chapter: 3 Development of a shelf stable synbiotic formulation containing Lactic acid bacteria and Fructooligosaccharides 102

2 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 4 o C. 103

3 Section: 3A Shelf stable synbiotic spray-dried formulation of selected Lactic acid bacteria with Fructooligosaccharides 104

4 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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 <2.8% (w/w), respectively) causes bacterial inactivation (Zayed and Roos 2004). Presently, industrial production of dried lactobacilli cultures is achieved mainly by freeze-drying, that applies gentle, low-temperature drying. However, freeze drying is a discontinuous and expensive process with low yields, time and energy demanding (Knorr 1998; Meng et al. 2008). 105

5 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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; O Riordan 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 of Bifidobacterium and Lactobacillus in the gut, thereby increasing the host s 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 30 o 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. 106

6 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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 selected L. plantarum and 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 10 8 cells 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 food supplement 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; O Riordan 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 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. plantarum CFR 2194 spray dried using different carrier media was investigated. 107

7 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation Materials and methods Materials Maltodextrin was procured from Nimesh Corporation (Mumbai, India). Non-fatted skimmed milk was obtained from a local retailer. FOS-90, the prebiotic used for spray drying studies, was prepared as described in the previous section 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 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 (LGG) was used as a reference strain. The LAB strains were stored at 60 o C 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 37 o C for 12 h 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 37 o C (control and to obtain initial count), 55, 58, 59, 60 and 61 o C. 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 108

8 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation plated in MRS agar. Survivors were enumerated after 3 days of incubation at 37 o C. 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) 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 of L. plantarum was 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 90 o C for 30 min, tempered at 37 o C prior to the addition of the cell suspension Spray drying L. plantarum in 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. 109

9 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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 μl each) were spread plated on MRS agar. Colonyforming 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/N 0 100, Where N 0 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 N 0 were 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 30 o C. 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 30 o C Moisture content The moisture content of spray-dried powders was determined by oven drying at 102 o C. 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) 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) 110

10 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation using an accelerating voltage of 20 kv. Micrographs were taken at different magnifications 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 spraydried L. plantarum was plated on MRS agar plates supplemented separately either with 5% (w/v) NaCl, 10 mg/ml lysozyme, 0.75 μg/ml penicillin G or 0.25% (w/v) bile salt. The plates were examined after 2 3 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-dried L. plantarum. One ml of fresh or rehydrated spraydried L. plantarum was transferred to 49 ml of MRS or milk, with yeast extract previously equilibrated at 37 C (final concentration was approx CFU/ml). At regular intervals, samples were withdrawn and ph of the samples was recorded 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 P 0.05 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). 111

11 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation Results Thermal tolerance of probiotic lactobacilli The most important aspect that needs special attention in the preparation of spray-dried powders 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 o C was compared. At 59 o C, a decrease of 1.92 log 10 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 61 o C, L. plantarum CFR 2194 was most thermal tolerant, with an observed decrease of 1.59 log 10 CFU/ml. Whereas, in case of L. fermentum CFR 2192 and L. rhamnosus GG, a decrease of 2.99 log 10 CFU/ml and 3.70 log 10 CFU/ml respectively was observed. The D-values of 6.21 min calculated from the above data confirmed L. plantarum CFR 2194 to be the most thermotolerant among the three strains studied, following exposure to 61 o C. In contrast, D-values for L. fermentum CFR 2192 and L. rhamnosus GG were 2.7 and 1.6 min, respectively. Similarly, there was a considerable difference in D-values among the three strains studied, at 59 ºC (8.8 min for L. planatrum, and 3.9 and 3.3 min for L. fermentum and L. rhamnosus GG). 112

12 Survivors log 10 CFU/ml Survivors log 10 CFU/ml Survivors log 10 CFU/ml Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation (a) Time (min) (b) Time (min) (c) Time (min) Figure 3.1.1: Survival of L. plantarum CFR 2194 (a), L. fermentum CFR 2192 (b) and L. rhamnosus GG ATCC (c) heated in maltodextrin (20% w/v) at 55 o C ( ), 58 o C ( ), 59 o C ( ), 60 o C ( ), 61 o C ( ). The results are based on data from triplicate heat challenge experiments. 113

13 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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 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 log 10 CFU/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. plantarum showed greater survival of over 85%, when MDX and NFSM were used as carriers, yielding powder with viable counts of and log 10 CFU/g. Cell damage was not observed in spraydried powders (Figure 3.1.3). Under dehydration conditions used, L. plantarum showed very little reduction of viability (0.89 and 1.6 log 10 CFU/g) in MDX+FOS and NFSM+FOS powders. Whereas, significant (P 0.05) reduction in viability of 2.08 and 2.6 log 10 CFU/g was found in dehydrated powders of MDX and NFSM. No significant (P 0.05) 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 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% H 2 O/g in order to be categorized as stable (Masters 1985). 114

14 Survival (%) Moisture Content (%) Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation MDX MDX + FOS NFSM NFSM+ FOS Carrier Adjuncts 2.9 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 ± 2 o C and inlet temperature of 140 ± 2 o C. 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. 115

15 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation Cells covered with carriers Figure 3.1.3: Scanning electron micrographs of the spray-dried L. plantarum Acidification rates of spray-dried L. plantarum CFR 2194 The decrease of ph in MRS and milk medium seeded with spray-dried L. plantarum was determined and the results are shown in the Figure Acidification rates in both growth media by the activity of fresh (before SD) and rehydrated (after SD) L. plantarum showed significant difference (P 0.05). 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 (P 0.05) 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. 116

16 ph ph Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation (a) (b) Time (h) Time (h) Figure 3.1.4: Acidification activity by fresh and spray-dried L. plantarum CFR a), L. plantarum spray dried with maltodextrin and b), L. plantarum spray dried with nonfatted skimmed milk growing in MRS and milk with added 1% (w/v) yeast extract. Rehydrated L. planatarum + 20% MDX or NFSM ( ); LP + 10 % MDX + 10 % FOS or NFSM ( ) and fresh ( ) strain growing in milk, and rehydrated L. planatrum + 20% MDX or NFSM ( ); L. plantarum + 10 % MDX + 10 % FOS or NFSM (x) and fresh ( ) strain growing in MRS. Each point represents the mean of triplicates. 117

17 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation Tolerance of cells to NaCl, lysozyme and bile salt Retention of the probiotic functional properties of L. plantarum CFR 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. plantarum after SD appear to be sensitive to NaCl, as revealed by decrease in the cell number. The decrease was insignificant (P 0.05) 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, penicillin but showed significant (P 0.05) sensitivity to bile salt. This suggests that the FOS might form a glassy layer around the cell membrane of L. plantarum CFR 2194 during the SD, thus causing less membrane damage and thus resulting in higher viability. 118

18 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation Table 3.1.1: Number of viable bacteria and sensitivity to NaCl, lysozyme, penicillin G and bile salt of L. plantarum CFR 2194 before and after spray drying. Log 10 CFU/ml Initial Count a NaCl b Lysozyme b Pencillin b Bile Salt b Strains Fresh Dried 1% LP + 20% MDX 8.90 ± ± 0.3 a 6.76 ± 0.13 a 6.80 ± 0.7 a 6.77 ± 1.41 a 6.23 ± 1.31 b 1% LP + 10% MDX + 10% FOS 10.2 ± ± 0.15 a 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 ± ± 0.13 a 6.42 ± 1.31 a 6.45 ± 0.34 a 6.43 ± 2.1 a 6.01 ± 3.61 b 1% LP+ 10% NFSM + 10% FOS ± 1.2 a 7.95 ± 0.24 a 8.01 ± 1.2 a 8.04 ± 1.02 a 8.05 ± 2.34 a a The values in the column represent the viable count of L. plantarum before (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-dried L. plantarum on MRS containing the agent. Different small letter superscripts depict the statistical difference within a row, P 0.05 between means for different agents 119

19 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation Storage Studies Following SD, MDX+FOS and NFSM+FOS powders were placed in sealed polythene bags and stored at 4 and 30 o C, 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 4 o C. 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 (P 0.05) 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-dried L. plantarum corresponded to 3.3 and 3.6 log CFU/g with MDX+FOS and NFSM+FOS powders, respectively. 120

20 Log10CFU/g Log10 CFU/g Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation a) Storage Time (Days) b) Storage Time (Days) 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 4 C ( ) and 30 C ( ). Data are the means of triplicate experiments ± SD. Values were significantly different at P<

21 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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 of L. plantarum CFR 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-dried L. plantarum has been investigated. The thermotolerance of the probiotic lactic cultures in the range of o C 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. plantarum CFR 2194 and L. fermentum CFR 2192 are 6.21 min and 2.7 min at 61 o C, respectively. The reported D values for 2 different strains of L. rhamnosus; E800 and GG are 2.7 and 1.32 min, respectively, at 61 o C. 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 75 o C were used, the survival rate was found to be inversely proportional to outlet temperature. The residual moisture content, from % (w/w) in L. rhamnosus GG and % (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 o C (Masters 1985). Based on these reports, SD trials were carried at an outlet temperature of 75 o C and an inlet temperature of 140 o C to 122

22 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation obtain good quality powders having a moisture content of <4%. Spray-dried powders with this moisture content are known to exhibit prolonged storage stability. Stationary phase L. plantarum CFR 2194 in the presence of MDX or NFSM as adjuncts was used for the SD studies. The suitability of partially substituting MDX and NFSM solids by prebiotic FOS was also evaluated. The total solids content of the SD medium was kept constant at 20% (w/v). This solid content has often been used and regarded as optimal for assuring high residual viability of different strains of LAB (Desmond et al. 2001; Gardiner et al. 2000; Johnson and Etzel 1993; Mauriello et al. 1999). The viability of L. plantarum during SD with different carrier adjuncts was compared. Over 85% survival was observed in MDX+FOS and NFSM+FOS powders compared to that of MDX/NFSM. No significant differences in survivability (P 0.05) between the spray-dried L. plantarum with MDX+FOS and NFSM+FOS were observed (Figure 3.1.2). Significant loss (P 0.05) of viability was observed when either MDX or NFSM was used alone as carrier medium (Figure 3.1.2). These experimental data demonstrated that prebiotic substances could be successfully incorporated in the SD medium without affecting the cell viability in the final product. Although skim milk appears to be more protective, the use of MDX during SD could be cost effective in the preparation of spray dried powders of probiotic microorganisms and incorporation into sugar-based foods. Residual moisture contents of 4% (w/w) were achieved upon SD at an outlet temperature of 75 o C. These results are in agreement with that of Gardiner et al. (2000). Cell survival as a function of storage was inversely related to temperature, in parallel with the previous studies (Gardiner et al. 2000; Desmond et al. 2002). The spray-dried L. plantarum in MDX+FOS and NFSM+FOS demonstrated over 60% survivability after 8 weeks storage at 4 o C. The survivability of spray-dried L. plantarum rapidly decreased in both the powders stored at 30 o C. Over 50% of viability has been recorded in both the spray-dried powders. Previous studies have also shown that the temperature is critical for microbial survival during storage, and higher survival rates have been obtained at lower storage temperatures (Abd El Gawad et al. 1989; Johnson and Etzel 1993; Teixeira et al. 1995b). It is apparent from the results of this study and other studies that refrigerated 123

23 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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-dried L. plantarum CFR 2194 in both the growth media (MRS and Milk) showed a significant (P 0.05) difference. However, rehydrated L. 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 dehydrated L. delbrueckii ssp 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 (P 0.05) between the viable count of L. plantarum in 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 dried L. rhamnosus GG (LGG) and L. rhamnosus E-800 (E800). In contrast, in this study, taking into account that L. plantarum CFR 2194 was more resistant to the drying 124

24 Chapter: 3A Shelf Stable Synbiotic Spray dried Formulation 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 10 9 CFU/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 4 o C, when MDX+FOS were used. However, the viability during storage at 30 o C 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 powders could be tailored for specialized functional food applications. 125

25 Section: 3B Development of synbiotic yogurt containing selected Lactic acid bacteria and Fructooligosaccharides: Comparative study during refrigerated storage 126

26 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt 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 more than 10 6 CFU/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 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. 127

27 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt 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. bulgaricus and 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 10 6 CFU/ml is necessary for deriving the health benefits (Ervin et al. 2002). To meet the National Yogurt Association s criteria for live and active culture yogurt, the finished yogurt must contain live LAB in amounts 10 8 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. animalis ssp. lactis and B. longum ssp. longum (Ramachandran and Shah 2010; Sheu et al. 2010). The conventional yogurt starter bacteria L. delbrueckii ssp. bulgaricus and 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, 128

28 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt 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 B 12 and significant antagonistic activity against the intestinal pathogen like Escherichia coli, Listeria monocytogens and 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 lactobacillus strains, and the probiotic LAB strains used as food supplements may have a substantial 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 Materials and methods 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 All other chemicals and solvents used were of analytical grade 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 o C in De Man-Rogosa Sharpe (MRS) broth, supplemented with 40% (v/v) glycerol as a 129

29 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt cryoprotectant. Prior to use, the cultures [1% (v/v)] were transferred to MRS broth: LB was incubated at 40 o C and ST, LP and LF were incubated at 37 o C 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 37 o C before inoculation into milk. Fructooligosaccharides (FOS) [70 o B, containing 90-93% (w/w) FOS] was used as a prebiotic in the preparation of synbiotic yogurt 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 40 o C 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 40 o C for 6-8 h. After incubation yogurt samples were stored at 4 o C for 28 days. Samples were drawn at weekly intervals up to fourth week. 130

30 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt Table 3.2.1: Preparation of probiotic, prebiotic and synbiotic yogurt samples Groups Probiotic yogurt Synbiotic yogurt Regular control A B C D RC Combination of organism (1% v/v) and Cell concentration, log CFU/ml S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp. bulgaricus CFR2028 (7.38) + L. plantarum CFR2194 (7.51) S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp. bulgaricus CFR2028 (7.38) + L. fermentum CFR 2192 (7.42) S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp. bulgaricus CFR2028 (7.38) + L. plantarum CFR2194 (7.51) + 1% (w/v) Fructooligosacharides S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp. bulgaricus CFR2028 (7.38) + L. fermentum CFR 2192 (7.42) + Fructooligosacharides 1% (w/v) S. thermophilus ATCC19258 (7.92) + L. delbrueckii ssp. bulgaricus CFR2028 (7.38) 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 37 o C for 48 h) and reinforced clostridia agar (RCA) (anaerobic incubation at 42 o C for 48 h), respectively. LP was enumerated on Lactobacillus plantarum selective medium (LPSM), under anaerobic incubation at 37 o C for 48 h (Bujalance et al. 2006). LF was enumerated on Columbia Agar Base (CAB) at ph 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. 131

31 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt 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 Texture analysis The gel strength of yogurt samples was determined at 4-6 o C 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 cm 2 ; penetration speed, 1.0 mm/s; penetration distance, 20 mm into surface. The gel strength was determined in triplicate and expressed as N/cm 2 of probe area 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, b values represent brightness/darkness, green/red and yellow/blue respectively (Krishnamurthy and Kantha 2005). The operating conditions were illuminant D65 and 10 o observer. An average of 5 values was taken per replication. The values represent an average of three readings 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 μl), 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 ± 2 o C). The proteolytic activity of these bacterial cultures was expressed as the 132

32 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt free amino groups measured at 340 nm as a difference in absorbance between probiotic, synbiotic and control batches Antioxidant activity assay 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 µl) was mixed with 0.2 ml of yogurt sample or 95% ethanol (Control), vortexed well and incubated for 30 min at RT (28 ± 2 o C). The samples were centrifuged for 5 min at g at 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] 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 ± 2 o C) in a shaking water bath. To this, FCR reagent (1 ml) (1:2 dilution) and 10% Na 2 Co 3 (2 ml) was added. The mixture was centrifuged at g 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 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 µl of sample was added and incubated at

33 Chapter: 3B Preparation of Probiotic and Synbiotic Yogurt o C 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 ( mm/l)] and results were expressed as mg Fe 2+ /100 ml (FRAP value) 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< Results Effect of FOS on the Antioxidant properties of yogurt samples The DPPH scavenging activity of yogurt samples is shown in Table The probiotic and synbiotic yogurt samples had a higher antioxidant activity when compared to control yogurt. In synbiotic yogurt samples containing LP and LF the DPPH radical inhibition were 85 and 82%, respectively, at day 1 when compared to that of control sample (72%) and the values were higher throughout the storage period. These results indicate that the metabolic end products of LAB, resulting from the selective utilization of FOS might be contributing to the higher antioxidant activity of the synbiotic yogurt in comparison with that of control. 134