TY - JOUR
AU - Beede, Kirankumar
AB - ABSTRACT Thermal stability (D-value and pasteurization) and gastric acid resistance of spore forming and nonspore forming probiotic strains were evaluated in this study. Bacillus coagulans MTCC 5856 spores showed highest thermal resistance (D-value 35.71 at 90 °C) when compared with other Bacillus strains and Lactobacillus species. B. coagulans strains exhibited significantly higher resistance to simulated gastric juice (pH 1.3, 1.5, and 2.0) compared to Lactobacillus strains. It also showed high resistance to cooking conditions of chapati (whole wheat flour-based flatbread) (88.94% viability) and wheat noodles (and 94.56% viability), suggesting remarkable thermal resistance during food processing. Furthermore, B. coagulans MTCC 5856 retained 73% viability after microwave cooking conditions (300 s, at 260 °C) and 98.52% in milk and juice at pasteurization temperature (420 min, at 72 °C). Thus, B. coagulans MTCC 5856 clearly demonstrated excellent resistance to gastric acid and high temperature (90 °C), thereby suggesting its extended application in functional foods (milk, fruit juices, chapati, and wheat noodles) wherein high temperature processing is involved. Graphical Abstract Open in new tabDownload slide Study of thermostability and gastrointestinal survival of probiotic Bacillus coagulans MTCC 5856. Graphical Abstract Open in new tabDownload slide Study of thermostability and gastrointestinal survival of probiotic Bacillus coagulans MTCC 5856. probiotics, Bacillus coagulans, D-value, functional food, LactoSpore® Globally, there has been exponential increase in manufacturing, marketing, and consumption of functional foods containing probiotics lately (Khan and Arshad 2011). The challenge for the usage of probiotics in food is stability during manufacturing, processing, storage, and also survivability in the gastrointestinal (GI) tract, all of which need to be met for the market demand (Bezkorovainy 2001). World Health Organization (WHO) and Food and Agriculture Organization of the United Nations (FAO) define probiotics as “live microorganisms while ingested in adequate amounts could offer a health benefit on the host” (FAO/WHO 2002). There is no specific recommended minimum level of probiotics proposed to meet the functionality of ingested probiotics (Champagne, Gardner and Roy 2005). Nevertheless, the administration of probiotics in human studies has been reported to inhibit the carcinogens produced during metabolism, lowering the cholesterol, improving mucosal immunity, anti-inflammatory, irritable bowel syndrome, antibiotic associated diarrhea, stimulate the immune responses, and thereby providing clinical effectiveness (Markowiak and Śliżewska 2018). Therefore, probiotic strains, belonging to Bifidobacterium, Enterococcus, Lactobacillus, Bacillus, and some fungal strains are the most generally used groups of probiotics (Majeed et al.2016b). Nonspore forming probiotics such as Bifidobacterium and Lactobacillus show poor survival during thermal processing and are unstable at ambient temperature during storage (Succi et al.2017). Eventually, this became a drawback for the usage of nonspore forming probiotics to formulate in food to retain its viability and shelf life. Several methods are available to increase the stability of probiotics such as microencapsulation and refrigeration, but this results in a cost burden to consumers. The applications of spore-forming probiotics, including the Bacillus species, are gaining importance over nonspore formers as they possess better tolerance to production, storage, and protection against gastrointestinal conditions, apart from antimicrobial activities and competitive exclusion (Elshaghabee, Rokana and Gulhane 2017). The most commonly used spore-forming probiotics for humans and animals are Bacillus coagulans, Bacillus licheniformis, Bacillus polyfermentans, Bacillus subtilis, and Bacillus clausii (Elshaghabee, Rokana and Gulhane 2017). Among many Bacillus species, only a few strains are within reach for the usage of human probiotics, one of them is B. coagulans (Nithya and Halami 2013). B. coagulans is the most widely clinically studied probiotic for the control of hypercholesterolemia, lactose intolerance, gastrointestinal disorders, and diarrhea (Majeed et al.2016b). Specifically, genetic and physical composition of spore-forming bacterium B. coagulans MTCC 5856 (LactoSpore®) was consistent over 2 decades of commercial production (Majeed et al.2016b). B. coagulans MTCC 5856 was studied for irritable bowel syndrome (IBS) and symptoms of major depressive disorder (MDD) with IBS and found to be effective in both the clinical trials at a dose of 2 billion spores per day (Majeed et al.2016c, 2018a). In another human clinical trial, B. coagulans MTCC 5856 was found to be safe at a dose of 2 billion spores in a day (Majeed et al.2016d). Further, in vitro studies are evident for the cholesterol-lowering effect, survivability in gastric and colonization in the intestine, and also for excellent immunomodulatory efficacy (Majeed et al.2018b). Furthermore, in another 2 different studies probiotic strain B. coagulans MTCC 5856 in combination with fenugreek and cranberry seed fiber reported a potential for an ideal synbiotic product (Majeed et al.2018c; 2018d). During the preparation and storage of functional foods such as tea, coffee, and baked foods in the presence of B. coagulans MTCC 5856 was found to be stable (Majeed et al.2016a). Given the consumer demand across the globe toward functional foods, it is crucial to assess a probiotic strain for its stability during application. Hence, this study was designed to evaluate the stability of B. coagulans MTCC 5856 during gastrointestinal hostile conditions at high temperatures (for the D-value). B. coagulans MTCC 5856 stability was also evaluated during the manufacturing process of functional food such as chapati and noodles, which are common foods in day-to-day life in the Asian continent. Material and methods Bacterial growth Commercial probiotic spore forming bacteria strains of B. coagulans MTCC 5856 used in this study is a proprietary strain of Sabinsa Corporation/Sami Labs Limited manufactured in a good manufacturing practice (GMP) facility in Bangalore, India (Majeed et al.2016b). B. coagulans MTCC 5856 spores were standardized to contain minimum 1.5 × 1010 CFU/g using food-grade maltodextrin (Sanwa Starch Co. Ltd. Nara, Japan) as a diluent. The viable spore count of B. coagulans MTCC 5856 was enumerated as per the method described previously (Majeed et al.2016b). In this study, we have also used B. coagulans ATCC 31284 and L. casei ATCC 393 procured from American Type Culture Collection (ATCC, Manassas, VA, USA). B. subtilis MTCC 441 was procured from Microbial Type Culture Collection and Gene Bank (MTCC, Chandigarh, India). Spores of B. subtilis MTCC 441 were produced using tryptone soya broth (Himedia) supplementing with minerals as described by Donnellan, Nags and Levins (1964). Lactobacillus rhamnosus NRRL B-442 and L. helveticus NRRL B-734 were procured from USDA Agriculture Research Service (NRRL) Culture Collection (Peoria, IL, USA). Cultures were grown in MRS broth at 37 °C for 24 h and centrifuged. Cell biomass was freeze-dried (VirTis 2 K Freeze Dryer, SP Industries, Inc., Warminster, PA USA). Freeze-dried powder was diluted using maltodextrin to obtain viable count equal to 1.5 × 1010 CFU/g. Enumeration of L. casei ATCC 393, L. rhamnosus NRRL B-442, and L. helveticus NRRL B-734 was performed as per the method described previously (Karu and Sumeri 2016). Probiotic strains viability in simulated gastric Juice Simulated gastric juice was prepared as previously described (Majeed et al.2019), with minor modifications. To represent the buccal digestion condition, Na2SO4 (0.56 g/L), KCl (0.89 g/L), NaH2PO4 (0.88 g/L), NaHCO3 (1.68 g/L), NaCl (9 g/L), and 3 g/L of pepsin (Sigma–Aldrich, St. Louis, MO, USA) were used in the study. The pH was aseptically adjusted to 1.3, 1.5, and 2.0 ± 0.1 by using 2 N HCl. Each probiotic strain (1 g) containing 1.5 × 1010 CFU/g (B. coagulans MTCC 5856, L. rhamnosus NRRL B-442, L. helveticus NRRL B-734, and L. casei ATCC 393, respectively) was added separately to 100 mL of gastric juice and incubated at 37 °C for 2 h with 50 rpm. After 2 h of incubation, pH was adjusted to 7.0, and the viable count of each probiotic strain was enumerated by plate count agar method. The analysis was performed in duplicate for 2 independent experiments. The average mean of viable counts is expressed in Log10 CFU/g. Thermostability of the probiotic strains Thermostability of the probiotic strain was performed according to the modified method described earlier (Janstova and Lukasova 2001). In 3 different sets of experiments, Butterfield's phosphate buffer (pH 7.0 ± 0.05), milk (pH 6.7 ± 0.05), and juice (commercial orange juice, pH 4.05 ± 0.05) (100 mL) were taken in 3 different flasks, and 1 g of B. coagulans MTCC 5856 (1.5 × 1010 CFU/g) was added to make a suspension. Crimp cap vial (20 mL capacity) was used in the study. 10 mL of the suspension of B. coagulans MTCC 5856 was added to 6 different vials and sealed with the crimp cap. A thermometer was inserted to check the temperature inside in one of the crimp cap vials. All the vials with the sample were kept in the oil bath (Vision Lab Equipment, India), and the time was recorded. Each crimp cap vial was taken out at different time intervals (0, 60, 120, 180, 240, 300, 360, and 420 min) and immediately cooled in ice-cold water. B. coagulans MTCC 5856 and B. coagulans ATCC 31284 strains were analyzed at different temperatures (63, 72, 90, 95, 100, 105, and 110 °C). L. rhamnosus NRRL B-442, L. helveticus NRRL B-734, and L. casei ATCC 393 were analyzed at 63 and 72 °C. The holding time at every temperature varied and was dependent on the survival at each temperature. The holding time countdown started when the desired temperature was reached inside the control vial. Enumeration of spore count was performed using glucose yeast extract agar (HiMedia) for B. coagulans MTCC 5856, B. coagulans ATCC 31284, and B. subtilis MTCC 441. deMan, Rogosa, and Sharpe Agar (MRSA) were used to analyze L. rhamnosus NRRL B-442, L. helveticus NRRL B-734, and L. casei ATCC 393. The average mean of viable counts is presented in Log10 CFU/mL. D-value The D-value (decimal reduction time) is defined as time required, at a given condition (eg temperature) or set of conditions, to achieve a Log reduction. D-values were estimated graphically from the slope of the regression line obtained by plotting Log of survival counts versus their corresponding heating times. Only survival curves with a coefficient of correlation (r) ≥0.98 and with more than 4 values in the straight portion of the line were used. Survival curves with a straight portion, including less than 1 Log cycle, were rejected. Thermostability of B. coagulans MTCC 5856 in foods The thermostability of the B. coagulans MTCC 5856 was analyzed during the preparation of chapati (composed of whole wheat flour, vegetable oil, salt and water) and noodles (composed of wheat flour, tapioca starch, water, vegetable oil, mineral salts, salt, vegetable gum, and color [riboflavin]). Chapati was prepared by using 100 g of whole wheat flour mixed with 400 mg of B. coagulans MTCC 5856 spores (1.5 × 1010 CFU/g) powder and 50 mL of water to make a soft and supple dough. The dough was divided into small balls (approximate 3 inches in diameter), each ball lightly flattened using the rolling pin and drenched with wheat flour on both sides and continued rolling the dough to make evenly flat (approximately 8-9 inches in diameter). Using a frying pan (tawa) over high temperature, the chapati was cooked on both sides however temperature inside will be less than 100 °C. The dough was analyzed for B. coagulans MTCC 5856 before and after the preparation of the chapati using mass balance by accounting for the moisture content in the respective samples. Enumeration of B. coagulans MTCC 5856 was described previously (Majeed et al.2016b). The experiment was repeated twice, and values are expressed in mean Log10 CFU spores of B. coagulans MTCC 5856 per gram of chapati. Noodles were purchased from the local market, and B. coagulans MTCC 5856 spores (1.5 × 1010 CFU/g) powder (200 mg) was mixed with the tastemaker (masala). Initial CFU count was 3 billion spores in the preparation. Water (350 mL) was boiled in a pan, and then Noodle (noodle block) (70 g) was added and cooked (temperature 90-95 °C) for 1 min. After 1 min, “tastemaker” containing B. coagulans MTCC 5856 was added and allowed simmering for 1 min till the noodles were fully cooked. Whole cooked Noodles were added to 1000 mL of diluent phosphate buffer (pH 7.0 with 0.1 m). The viable spore count of B. coagulans MTCC 5856 was enumerated as per the method described previously (Majeed et al.2016b). The experiment was repeated twice, and values are expressed in mean in Log10 CFU spores of B. coagulans MTCC 5856 per gram of noodles before and after cooking, considering the mass balance. As B. coagulans MTCC 5856, which at 2 billion/g is clinically approved for safety and efficacy, we note that the same clinical data is not available for B. coagulans ATCC 31284. Dry heat thermostability of B. coagulans MTCC 5856 in a microwave oven Dry heat thermostability of B. coagulans MTCC 5856 was analyzed in a microwave oven (Make: LG All In One Microwave Oven; Model: MC2886SFU) at high temperature (260 °C). B. coagulans MTCC 5856 spores were standardized to contain the concentration of 1.5 × 1010 CFU/g which was taken in the 6 different sterile beakers containing 5 g each. Initially, all the beakers were kept in the microwave oven, and the temperature was adjusted to (260 °C). After 10 min, B. coagulans MTCC 5856 spores were added in the beaker. At different time intervals (60, 120, 180, 240, and 300 s) each beaker containing 5 g of B. coagulans MTCC 5856 was taken out of the microwave oven. Viable spore enumeration of B. coagulans MTCC 5856 was described previously (Majeed et al.2016b). Statistical analysis Enumeration of B. coagulans MTCC 5856 before and after cooking chapati and noodles were expressed in Log10 CFU. Student's t-test was calculated differences between the 2 mean values. The data presented are the average of 3 determinations. Statistical significance was set at P < .05. Results and discussion Thermostability of B. coagulans MTCC 5856 The viability of probiotics is the major concern during manufacturing and food processing, which undergoes harsh conditions when exposed to high temperatures (Bezkorovainy 2001). According to Food and Agriculture Organization (FAO) and the World Health Organization (WHO), one of the qualifications of a probiotic formulation is the number of live cells present in the formulation (FAO/WHO 2002). In view of this, the selection of the stable probiotics strain is a very crucial factor to gain maximum health benefits. Thus, the current study demonstrated B. coagulans MTCC 5856 to be stable at high temperatures, which benefits the application of probiotics in food during processing and storage. The thermostability (wet heat) of B. coagulans MTCC 5856 was analyzed at various temperatures (63, 72, and 90 °C) in different media such as buffer, milk, and juice, to correlate with milk pasteurization which is normally practiced at 2 different temperatures (63 and 72 °C) for different intervals of time. Based on the pasteurization temperature, we analyzed the stability of B. coagulans MTCC 5856 at 63 and 72 °C at different time intervals. Figure 1 shows at 63 °C, no significant change was observed in the viability of B. coagulans MTCC 5856 up to 420 min in all the 3 media (milk, buffer and juice). At 72 °C, the medium containing milk showed no significant difference in the viability of B. coagulans MTCC 5856. However, B. coagulans MTCC 5856 count was reduced to 9.93 Log10 CFU/mL from the initial 10.17 Log10 CFU/mL in buffer and 9.92 Log10 CFU/mL from the initial 10.17 Log10 CFU/mL in juice after 420 min of incubation. Further, there was no reduction in viable count of B. coagulans MTCC 5856 up to 100 min in milk at 90 °C, while in buffer and juice, the spore count was reduced significantly (P < .05) after 50 min of incubation compared to untreated control. Thus, the current study demonstrated the ability of B. coagulans MTCC 5856 to be stable at high temperatures, which benefits the application of probiotics in food during processing and storage. Although viability of B. coagulans MTCC 5856 spores was reduced in acidic juice (pH 4), it exhibited high thermotolerance at pasteurization temperatures 63, 72, and 90 °C in buffer and milk (pH 7) suggesting its wider application in beverages. The heat resistance of B. coagulans was affected by the combination of heat and acidic environment especially at high temperatures. Most authors have revealed that heating medium in acidic conditions causes a decrease in the microbial heat resistance (Palop, Raso and Pagán 1999). The thermostability of the nonspore forming bacterial strains (L. rhamnosus NRRL B-442, L. helveticus NRRL B-734, and L. casei ATCC 393) was evaluated at 63 and 72 °C (Figure 2). All 3 strains were stable only up to 10 min, with no significant decrease in the viable count at temperature 63 °C and up to 5 min at 72 °C. Thus, this study suggested that spores had higher stability at temperature 63, 72 and 90 °C compared to nonspores forming bacterial strains evaluated in the study. Previous studies also confirmed that the spore-forming probiotic exhibited acceptable performance during cooking or heat treatments in processed foods in comparison to traditional vegetative probiotic strains such as Lactobacillus and Bifidobacterium (Jafari et al.2017). B. coagulans MTCC 5856 has shown significantly higher tolerance (P < .05) compared to the B. coagulans ATCC 31 284 in neutral and acidic pH (Figure 3) indicating strain specific difference of same species. However, there was a decrease in the microbial heat resistance of B. coagulans MTCC 5856 at pH 4.0 in juice compared to the pH 7.0 with buffer in the B. coagulans MTCC 5856 suggesting that the effects of pH during heating on microbes are cumulative. In a similar study reported by Leguerinel and Mafart (2001) (Leguerinel and Mafart 2001), B. subtilis showed specific effects on the heat resistance at pH 4 in the presence of organic acid. However, due to the ability to form spores at the dormant stage of growth, B. coagulans MTCC 5856 exhibited a remarkable heat resistance property. Sporulation is a process that helps the strain to survive at reduced levels of nutrients in the environment. Spore coat consists of mainly hydrophobic spore core, mineral ions, and spore proteins (Hayes and Setlow 2001). Therefore, the resistance may be due to saturation of spore DNA with a/b-type small acid-soluble spore proteins which protect DNA against wet heat damage (Nicholson et al.2000). Figure 1. Open in new tabDownload slide Thermostability of the B. coagulans MTCC 5856 at (a) 63 °C, (b) 72 °C, and (c) 90 °C in Butterfield's phosphate buffer, milk, and juice were analyzed at different time intervals. Figure 1. Open in new tabDownload slide Thermostability of the B. coagulans MTCC 5856 at (a) 63 °C, (b) 72 °C, and (c) 90 °C in Butterfield's phosphate buffer, milk, and juice were analyzed at different time intervals. Figure 2. Open in new tabDownload slide Effect of temperature on the viability of L. rhamnosus NRRL B-442, L. casei ATCC 393, and L. helveticus NRRL B-734 at (a) 63 °C and (b) 72 °C. Figure 2. Open in new tabDownload slide Effect of temperature on the viability of L. rhamnosus NRRL B-442, L. casei ATCC 393, and L. helveticus NRRL B-734 at (a) 63 °C and (b) 72 °C. Figure 3. Open in new tabDownload slide D-values for B. coagulans MTCC 5856, B. coagulans ATCC 31284, and B. subtilis MTCC 441 at temperatures ranging from 90 to 110 °C in the presence of (a) Butterfield's phosphate buffer (pH 7), (b) milk (pH 6.7), and (c) orange juice (pH 4.05). Figure 3. Open in new tabDownload slide D-values for B. coagulans MTCC 5856, B. coagulans ATCC 31284, and B. subtilis MTCC 441 at temperatures ranging from 90 to 110 °C in the presence of (a) Butterfield's phosphate buffer (pH 7), (b) milk (pH 6.7), and (c) orange juice (pH 4.05). The D-value for the B. coagulans MTCC 5856 was calculated in comparison to the B. coagulans ATCC 31284 and B. subtilis MTCC 441 in different media (Butterfield's phosphate buffer, milk, and juice) at various temperatures (90, 95, 100, 105, and 110 °C). In the presence of milk as the medium, there was a significant increase in the D-value of B. coagulans MTCC 5856, B. coagulans ATCC 31284 and B. subtilis MTCC 441 in comparison to the Butterfields phosphate buffer and juice. The D-value of B. coagulans MTCC 5856 was higher in the Butterfields phosphate buffer, milk, and juice in comparison to the B. coagulans ATCC 31284 (P < .05). However, the D-value of B. subtilis MTCC 441 was higher in juice at 95, 100, 105, and 110 °C compared to B. coagulans MTCC 5856 and the B. coagulans ATCC 31284. Therefore, B. coagulans MTCC 5856 being a spore-forming bacterium exhibited the highest D-value (48.85 min) when added to milk at 90 °C. Milk being a natural vehicle for bioactives with the proteins present in it binds to ions and small molecules. Its gelation properties, excellent surface, and self-assembly properties help probiotics to safeguard textural properties thereby providing microbiological stability (Livney 2010). B. coagulans MTCC 5856 showed much higher (P < .05) stability than the strain B. coagulans ATCC 8038 reported previously by Peng et al. (2012). The rise in temperatures from 90 to 110 °C resulted in a continual decrease of the D-value of spores among inter and intra genus of Bacillus. The results reported earlier by Janstova and Lukasova, 2001 (Janstova and Lukasova 2001) suggested that the spores of B. coagulans and B. subtilis did not only survived at 100 °C, but were also able to germinate. Similar observations were made in another study wherein, bacillus spores, such as B. cereus, B. subtilis, B. licheniformis, B. megaterium, B. alvei, B. amylolyticus, were evaluated for the D-value and found to be stable at higher temperature (Janstova and Lukasova 2001, Rodriguez, Cousin and Nelson 1993). This difference in the thermostability could be due to the strain specificity, method of manufacturing which has also been indicated in the previous studies (Peng et al.2012). Comparative viability in simulated gastric juice of Probiotic strains Another important aspect of the selection of proper probiotic strain is to ensure the survivability at gastric pH. This study was carried out to evaluate the survivability of B. coagulans and Lactobacillus strains in 3 different simulated gastric fluids characterized by different pH 1.3, 1.5, and 2.0. In this study, we selected gastric pH range from 1.3 to 2.0 based on the clinical and in vitro studies reported earlier wherein gastric pH in postprandial for 2 h, was reported to be 1.3 and 1.5 (Vo et al.2005). Figure 4 depicts that B. coagulans MTCC 5856 and B. coagulans ATCC 31284 had the highest survival rate over the 120 min exposure to simulated gastric juice at pH 1.3, 1.5, and 2.0. After 120 min of incubation, cfu Log count was 9.3, 9.3, and 9.8 Log10 CFU/mL at pH 1.3, 1.5 and 2.0 respectively, which was less than the 1 Log cfu reduction from the initial count of 10.20 Log CFU/mL. However, in case of L. casei ATCC 393, the count was reduced to 5.01, 5.21, and 5.55 Log10 CFU/mL at gastric pH 1.3, 1.5, and 2.0 respectively after 120 min of incubation which was more than 4 Log cfu reduction from the initial count of 10.1 Log CFU/mL. Similarly, it was evident that L. rhamnosus NRRL B-442, and L. helveticus NRRL B-734 at pH 1.3 showed poor resistance, wherein the count was reduced to 6.2 and 5.1 Log10 CFU/mL respectively from the initial count of 10.1 Log CFU/mL after 120 min of incubation. Overall, the data showed that the survival rates of B. coagulans differ from other Lactobacillus species and those differences are also evident at the strain level. The resistance of B. coagulans MTCC 5856 was not only in the gastric pH but also in digestive enzyme (pepsin) suggesting its high resistance to GIT environmental stresses. Our results are in agreement with the earlier results reported by Majeed et al. (2016b) for gastric resistant and also bile tolerant which provided additional evidences that the B. coagulans MTCC 5856 is highly gastric resistant. Therefore, these studies further prove that spores confer superior resistance to hostile gastric, intestinal conditions (pH and digestive enzymes) and the stresses encountered during the industrial production and storage (Gu et al.2015). Resistance to the digestion process is crucial to ensure that the ingested dose of probiotics reaches the gastrointestinal tract to provide probiotic attributes (Hong et al.2008). Although, there are studies suggesting Bacillus spores such as B. subtilis that only few percentage of orally administered spores germinate in the small intestine and few vegetative cells detected in the large intestine (Nguyen and Nguyen 2006) but administrations of such spores has been effective against opportunistic pathogens such as Staphyocuccus aureus (Piewngam and Zheng 2018) suggesting effectiveness of spores upon admiration. Figure 4. Open in new tabDownload slide Effect of gastric acid stress on the viability of B. coagulans MTCC 5856, B. coagulans ATCC 31284, L. rhamnosus NRRL B-442, L. helveticus NRRL B-734, and L. casei ATCC 393 at (a) pH 1.3, (b)1.5, and (c) 2.0. Figure 4. Open in new tabDownload slide Effect of gastric acid stress on the viability of B. coagulans MTCC 5856, B. coagulans ATCC 31284, L. rhamnosus NRRL B-442, L. helveticus NRRL B-734, and L. casei ATCC 393 at (a) pH 1.3, (b)1.5, and (c) 2.0. Effect of cooking condition in Indian bread (chapati) and noodles on viability of B. coagulans MTCC 5856 Foods act as a carrier for the delivery of probiotics which provides buffering through the gastrointestinal tract and regulate the probiotics to colonize the human gut (Ranadheera, Baines and Adams 2010). Among foods, flour obtained from the grains may also help to protect probiotics through the gastrointestinal tract and to increase colonization (Martins et al.2013). The survivability of probiotics is not only in the gastrointestinal tract, but it is important to survive during food manufacturing and storage as well (Ranadheera, Baines and Adams 2010). Concerning this, the current experiment was conducted to evaluate the survivability of the spores the B. coagulans MTCC 5856 in chapati and noodles preparation. B. coagulans MTCC 5856 spores were found to be stable during the preparation of chapati, which retained 6.92 Log10 CFU/g from the initial count of 7.78 Log10 CFU/g. In the noodles, the spores count reduced to 6.78 Log10 CFU/g from an initial count of 7.17 Log10 CFU/g (Figure 5). Upon addition of B. coagulans MTCC 5856 spores to the chapati and Noodles during cooking, no significant difference (P > .05) in the viability of B. coagulans MTCC 5856 spores was observed in this study. It was determined that B. coagulans MTCC 5856 survived 88.94 and 94.56% after cooking in chapati and Noodles respectively. The results are in agreement with an experiment conducted earlier with Bacillus strain included in bread (Soares et al.2019). Though, in the present study, direct heat was involved wherein chapati was cooked in the frying pan (tawa) at high temperatures (∼205-230 °C) compared to typical bread making procedures, spore survival against high processing temperature was very high suggesting its wider application in food processing wherein high temperature is employed. Thus, spores of B. coagulans MTCC 5856 are highly resistant to food processing temperatures and gastrointestinal hostile environment, which does not require encapsulation for their stability (Majeed et al. 2016a). Figure 5. Open in new tabDownload slide The viability of B. coagulans MTCC 5856 before and after cooking of chapati (Indian bread) and noodles was analyzed. The average of viable counts is expressed in Log10 CFU/g. Error bars represent the ±SD. Figure 5. Open in new tabDownload slide The viability of B. coagulans MTCC 5856 before and after cooking of chapati (Indian bread) and noodles was analyzed. The average of viable counts is expressed in Log10 CFU/g. Error bars represent the ±SD. Thermostability of B. coagulans MTCC 5856 in a microwave oven at high temperature Thermostability of B. coagulans MTCC 5856 in the microwave oven was analyzed at a high temperature of 260 °C for different time intervals (0, 60, 120, 180, 240, and 300 s). The survivability of B. coagulans MTCC 5856 was 96% after 60 s, and even after 300 s of treatment, the survivability was 73% in comparison to the untreated control (Figure 6). Additionally, technological processing of functional foods which frequently are based on the application of drastic procedures, such as pasteurization, sterilization, blanching, cooking, frying, stewing, and microwave heating that may affect the bioactive constituents or the probiotics in the functional food (Grajek, Olejnik and Sip 2005). B. coagulans MTCC 5856 stability is likely to give added benefits during the preparation of functional foods wherein microwave oven heating is involved at such higher temperatures. Moreover, the stability of B. coagulans MTCC 5856 has been reported along with various preparations and storage of foods such as tea, coffee, cranberry seed fiber, muffins, chocolates, cake, and apple juice (Majeed et al.2016a), but our results in this study may further extend the probiotic application in foods such as noodles, Indian bread, juices and milk. Figure 6. Open in new tabDownload slide The thermostability of B. coagulans MTCC 5856 in a microwave oven at 260 °C for different time intervals (0, 60, 120, 180, 240, and 300 s). Figure 6. Open in new tabDownload slide The thermostability of B. coagulans MTCC 5856 in a microwave oven at 260 °C for different time intervals (0, 60, 120, 180, 240, and 300 s). Conclusion In conclusion, B. coagulans MTCC 5856 spores have greater thermal resistance (D-values) than B. coagulans ATCC 31284 and nonspore forming Lactobacillus bacteria. Therefore, it is considered to be the ideal probiotic strain for developing and validating thermal processes for making high temperature/high processing foods that require both short- and long-term shelf life. These results also collectively indicate that the ingestion of B. coagulans MTCC 5856 spores can reach the colon unaffected by gastric acids, and enzymes without encapsulation. Author contribution M.M., S.M., and S.A. sorted and analyzed data and revised the manuscript critically for important intellectual content. F.A. and K.B. contributed to the design of experiments, drafted the manuscript, and revised it. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work. Funding None declared. Data Availability The data that support the findings of this study are available from the corresponding author, upon reasonable request. Disclosure statement No potential conflict of interest was reported by the authors. 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Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Comparative evaluation for thermostability and gastrointestinal survival of probiotic Bacillus coagulans MTCC 5856
JF - Bioscience Biotechnology and Biochemistry
DO - 10.1093/bbb/zbaa116
DA - 2020-12-24
UR - https://www.deepdyve.com/lp/oxford-university-press/comparative-evaluation-for-thermostability-and-gastrointestinal-gqRKWslTg4
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -