Drink formulations with Bacillus coagulans MTCC 5856

Description

Field of the invention

[0001] The present invention in general relates to probiotics. More specifically, the present invention relates to a beverage comprising probiotic Bacillus coagulans strain MTCC 5856.Description of prior art

[0002] Probiotics are gaining importance as a dietary supplement owing to their ability to modify the gut microflora yielding increased health benefits. These probiotics can survive the acidic environments of the stomach and small intestine, but are not observed to colonize the gastro-intestinal tract. Thus, there exists a need for constantly supplementing probiotics for the therapeutic benefits to persist.

[0003] Beverages are widely consumed food constituents and can be looked upon as a source for co-administering probiotics. It must be noted that beverage compositions are subjected to treatments under extreme stress, temperature and pressure conditions like brewing or aeration wherein the viability of the probiotic spores should be maintained post treatment to result in a successful administration. Majeed, M. et al, (2016), Evaluation of the stability of Bacillus coagulans MTCC 5856 during processing and storage of functional foods. Int J Food Sci Technol, 51: 894-901 concerns viability of Bacillus coagulans MTCC 5856 during processing and storage of functional foods. The document discloses a beverage obtained from brewing a composition comprising Bacillus coagulans MTCC 5856. As shown on page 896, subsection "Brewed coffee", coffee powder was mixed with Bacillus coagulans MTCC 5856 spores at room temperature and subsequently added to water preheated at 90 °C for 2 min followed by incubation at 77 °C for four hours. As shown on page 897, subsection "Brewed coffee" and page 898, Fig. 1, the viability of Bacillus coagulans MTCC 5856 showed a 13 % reduction in viability already 2 min after brewing. Furthermore, the subsequent incubation at 77 °C resulted in a 30 % and 34 % viability reduction after three and four hours, respectively. US 2016 / 0058805 A1 describes (i) the growth promotional activity of natural plant based fibres on Bacillus coagulans MTCC 5856; (ii) the combination of natural plant based fibres and Bacillus coagulans MTCC 5856 to inhibit Gram Negative pathogenic bacteria and (iii) the production of short chain fatty acids (SCFA) by Bacillus coagulans MTCC 5856 using plant based natural fibres. US 2012 / 0251512 A1 relates to a sports nutritional composition comprising a protein source and an isolated Bacillus coagulans spore. US 2015 / 0313951 A1 relates to a composition comprising a non-dairy milk-like product and an isolated Bacillus coagulans spore.

[0004] United States Patent Application 20090232941 discloses a beverage composition comprising Bacillus coagulans strains GBI-30, GBI-20 and GBI-40. United States Patent Application 20130295226 discloses a fruit beverage composition comprising Lactobacillus paracasei. Both these applications do not disclose the method of brewing and the survival of the probiotic bacteria in harsh conditions. Also, it is well known in the scientific art that that biological effects of probiotics or products thereof are strain specific and cannot be generalised among genera, species and strains (Probiotics: In Depth / NCCIH, U.S. Department of Health and Human Services, National Institutes of Health). Hence, there still exists a need to find a probiotic strain that is more efficient and viable when subjected to extreme stress, temperature and pressure conditions.

[0005] The present invention solves the above technical problem by disclosing a probiotic strain that is viable and efficient when co-administered with beverages.

[0006] The principle objective of the inventions is to disclose beverage compositions comprising Bacillus coagulans MTCC 5856 wherein the spore viability is maintained post treatments.

[0007] The present invention fulfils aforesaid objectives and provides further related advantages.SUMMARY OF THE INVENTION

[0008] The present invention discloses beverage compositions that have been subjected to treatments under extreme stress, temperature and pressure conditions like brewing or aeration and comprise Bacillus coagulans MTCC 5856 spores and water soluble prebiotic fibers wherein the spore viability is maintained post said treatments.DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

[0009] In a principle embodiment, the present invention discloses a beverage as specified in claim 1 which beverage is obtained by brewing of a composition comprising Bacillus coagulans MTCC 5856 in the form of spores. The beverage is tea. It comprises Bacillus coagulans in the form of spores and viable or heat killed or dead or lysed vegetative cells of Bacillus coagulans MTCC 5856.

[0010] An embodiment not covered by the invention is a coffee composition comprises Bacillus coagulans MTCC 5856 in the form of spore and bacterium wherein said bacterial spore is heat resistant having more than 95% viability after brewing coffee. In a related embodiment not covered by the invention, the coffee is selected from the group consisting of decaffeinated coffee, unroasted green coffee and roasted coffee. In another related embodiment not covered by the invention, the spores include viable or heat killed or dead spores of Bacillus coagulans MTCC 5856. In yet another related embodiment not covered by the invention, the bacterium includes viable or heat killed or dead or lysed vegetative cells of Bacillus coagulans MTCC 5856.

[0011] The tea includes green tea, black tea, oolong tea, yellow tea, white tea or decaffeinated tea, herbal tea selected from the group consisting of rosehip tea, chamomile tea, jiaogulan tea, pep permint tea, rooibos tea, ginger tea, ginseng tea, or lemon grass tea.

[0012] In another embodiment not covered by the invention, the beverage is fruit juice. The fruit component of the fruit juice is selected from the group consisting of orange, apple, pears, strawberry, raspberry, cranberry, blue berry, apricot, pineapple, peach, banana, mango, lime, grape, tomato, pomegranate, papaya, tender coconut water, kiwi and a fruit mixture.

[0013] Another preferred embodiment not covered by the invention is an aerated soda composition comprising Bacillus coagulans MTCC 5856 in the form of spore and bacterium wherein the said bacterial spore has the ability to survive aeration. In another related embodiment not covered by the invention, the spores include viable or heat killed or dead spores of Bacillus coagulans MTCC 5856. In yet another related embodiment not covered by the invention, the bacterium includes viable or heat killed or dead or lysed vegetative cells of Bacillus coagulans MTCC 5856.

[0014] In another preferred embodiment, the composition comprises a water soluble prebiotic fiber.

[0015] Another embodiment not covered by the invention is a synergistic coffee composition comprising a prebiotic water soluble fiber and Bacillus coagulans in the form of spore and bacterium wherein the said bacterial spore is heat resistant having more than 95% viability after brewing coffee. In a related embodiment not covered by the invention, coffee includes decaffeinated coffee, unroasted green coffee and roasted coffee. In another related embodiment not covered by the invention the water soluble prebiotic fiber is selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharide (GOS), inulin and polydextrose. In another related embodiment not covered by the invention, the spores include viable or heat killed or dead spores of Bacillus coagulans. In yet another related embodiment not covered by the invention, the bacterium includes viable or heat killed or dead or lysed vegetative cells of Bacillus coagulans. In a preferable related embodiment not covered by the invention, the Bacillus coagulans strain is preferably Bacillus coagulans MTCC 5856 and also the strains derived from Bacillus coagulans Hammer strain accession number ATCC 31284 and ATCC 7050

[0016] In another related embodiment of the invention the water soluble probiotic fiber is selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharide (GOS), inulin and polydextrose.

[0017] The aforesaid most preferred embodiments incorporating the technical features and technical effects of instant invention, are explained through illustrative examples herein under.EXAMPLE 1: Methods employed for brewing coffee (not according to the invention) and tea with Bacillus coagulans MTCC 5856 Bacillus coagulans MTCC 5856 stability during Coffee brewing (not according to the invention)

[0018] Commercial preparation of Bacillus coagulans MTCC 5856 spores was standardized to 15 × 10 9< cfu (spores) per gram and added to grounded unroasted green coffee or roasted coffee and blended at room temperature with low r.p.m. for 60 min. Further, 1.0 g of water soluble prebiotic fibres (fructooligosaccharide, galacto-oligosacharides, inulin and polydextrose) were added to 4.0 gm of unroasted green coffee or roasted coffee and blended at room temperature with low r.p.m. for 60 min (Table 1). Approximately 5.0 g of the compositions were brewed using electric brewer and tested for viable spore count as per method described earlier (Majeed et al., World J Microbiol Biotechnol (2016) 32:60). Average mean of spore viable counts are expressed in log 10 CFU. Table 1. Formulation of Coffee beverages FormulationBacillus coagulans MTCC 5856 (15 × 10 9< spores / gm)#Coffee and beverage excipient + Prebiotic Fibre* (grams)Total weight per serving010.144 g3.96 + 0.04 + 0.05.144020.072 g3.96 + 0.04 +1.05.072030.144 g3.96 + 0.04 + 1.05.144040.288 g3.96 + 0.04 + 1.05.288# where coffee is unroasted green coffee beans and Roasted coffee beans. *Where the prebiotic fibres are FOS, GOS, Inulin and polydextrose Bacillus coagulans MTCC 5856 stability during Tea (Black, Green and Oolong tea) brewing

[0019] Commercial preparation of Bacillus coagulans MTCC 5856 was standardized to 15 × 10 9< cfu (spores) per gram and added to black, green or oolong tea and blended at room temperature with low r.p.m. for 60 min. Further, 1.0 g of water soluble prebiotic fibres (fructooligosaccharide, galacto-oligosacharides, inulin and polydextrose) were added to 4.0 gm of black, green or oolong tea and blended at room temperature with low r.p.m. for 60 min (Table 2). Approximately 5.0 g of these tea compositions were brewed using electric brewer and tested for viable spore count as per method described earlier (Majeed et al., World J Microbiol Biotechnol (2016) 32:60). Average mean of spore viable counts are expressed in log10 CFU. Table 2. Formulation of tea beverages FormulationBacillus coagulans MTCC 5856 (15 × 10 9< spores / gm)#Tea and beverage excipient+ Prebiotic Fibre* (grams)Total weight per serving010.15 g3.96 + 0.04 + 0.04.15020.075 g3.96 + 0.04 + 1.05.075030.15 g3.96 + 0.04 + 1.05.15040.30 g3.96 + 0.04 + 1.05.30#Where the tea are black, green and oolong. *Where the prebiotic fibres are FOS, GOS, Inulin and polydextrose Effect of Gastric stress and brewing conditions on the germination of Bacillus coagulans MTCC 5856 spores

[0020] The effect of gastric stress on the Bacillus coagulans MTCC 5856 spores was studied by treating brewed coffee (not according to the invention) or tea (as described in above paragraph) in acidic conditions. The pH of brewed coffee or tea was adjusted aseptically to 1.5 using 2 N HCl and incubated at 37 °C for 3 h with low r.p.m. After 3 h of incubation, again pH was adjusted aseptically back to 7.0 using 2 N NaOH and 0.5% (w / v) ox bile was added to tea or coffee flask. Oxygen reducing enzyme Oxyrase (Oxyrase ®< for Broth, Oxyrase, Inc, Mansfield, OH, USA) was also added to each flask. Further flasks were incubated at 37 °C for 24 h with low r.p.m. pH values at 0 h of incubation and after 24 h of incubation were also recorded. Optical density (OD) at 600 nm was recorded at 0 h and after 24 of incubation by diluting the sample into sterilized demineralized water to 1:5.EXAMPLE 2: Results for brewing coffee (not according to the invention) and tea with Bacillus coagulans MTCC 5856 Brewed Coffee with Bacillus coagulans MTCC 5856 (not according to the invention)

[0021] The thermo-stability and compatibility of Bacillus coagulans MTCC 5856 spores in coffee was evaluated during brewing using electric brewer. Stainless steel and paper filter were used in brewer to understand the feasibility of probiotic brewing. There was 0.5 Log 10 cfu (spores) reduction after brewing which remained almost same even after 10 min of incubation at room temperature (Table 3). The data suggested that Bacillus coagulans MTCC 5856 spores remained 94.83% viable after brewing using Stainless steel filter. There was no significant difference observed when coffee was brewed using stainless steel or paper filters. Table 3. Viability of B. coagulans MTCC 5856 during Coffee brewing using electric brewerTime Brewed and filtered using SS filter Brewed and filtered using paper filter Log 10 CFU / 10 gViability (%)Log 10 CFU / 10gViability (%)Post Brew9.8388 ± 0.0121009.8388 ± 0.012100After Brew9.3304 ± 0.01194.839.2600 ± 0.01494.11After 10 min9.3096 ± 0.01394.639.2504 ± 0.01294.01 Brewed Tea with Bacillus coagulans MTCC 5856

[0022] Black tea, green tea and oolong tea were supplemented with Bacillus coagulans MTCC 5856 spores and brewed to determine the spore survival during brewing. Bacillus coagulans MTCC 5856 spores were found to be highly stable and the viability remained above 99% after brewing (Table 4 & 5). Table 4. Viability of B. coagulans MTCC 5856 during black tea brewing using electric brewerTime Formulation -01 Formulation -02 Formulation -03 Log 10 CFU / servingViability (%)Log 10 CFU / servingViability (%)Log 10 CFU / servingViability (%)Initial count8.9309 ± 0.011100.009.1182 ± 0.012100.009.4667 ± 0.010100.00Post Brew9.1760 ± 0.014102.749.1846 ± 0.011100.739.6570 ± 0.014102.01After 10 min9.1583 ± 0.011102.559.1760 ± 0.010100.639.6683 ± 0.011102.13After 72 h chilling8.6570 ± 0.01296.938.6739 ± 0.01295.139.2878 ± 0.01198.11

[0023] Same brewed tea samples were chilled for 72 h and then spore viability was determined. The viability of spores in black, green and oolong tea after chilling for 72 h was95.13%, 93.58% and 94.51 % respectively (Table 4 & 5). Table 5. Viability of B. coagulans MTCC 5856 during green and oolong tea brewing using electric brewerTime Green Tea Oolong Tea Log 10 CFU / servingViability (%)Log 10 CFU / servingViability (%)Post Brew9.4471 ± 0.0111009.4313 ± 0.012100After Brew9.3830 ± 0.01299.329.3921 ± 0.01199.58After 10 min9.371 ± 0.01399.199.3842 ± 0.01499.50After 72 h chilling8.841 ± 0.01493.588.914 ± 0.01294.51 Germination of spores

[0024] To deliver the right amount of viable Bacillus coagulans MTCC 5856 spores in the human gut, the effect of brewing conditions and gastric stress on the germination of Bacillus coagulans MTCC 5856 spores was conducted. Tea and coffee along with the Bacillus coagulans MTCC 5856 spores were brewed and then subjected to gastric stress (acid and bile) followed by anaerobic incubation at 37°C with bile salts to mimic human conditions. This set of experiment was conducted to understand the spore germination when Bacillus coagulans MTCC 5856 is brewed and digested by human. Additionally, along with tea and coffee B. coagulans MTCC 5856 was brewed with water soluble prebiotic fibres (fructooligosaccharide, galacto-oligosacharides, inulin and polydextrose). Results of the experiment revealed that B. coagulans MTCC 5856 spores could germinate after brewing and surviving in harsh condition of gastric stress by using tea or coffee as sole nutritional source (Table 6). Additionally, there was further increase in the germination by the addition of water soluble prebiotic fibres (Table 6). This suggested that soluble prebiotic fibres have synergistic effect along with tea and coffee on the germination of B. coagulans MTCC 5856 spores. Table 6. Germination of Bacillus coagulans MTCC 5856 in gastric stress using tea and coffee or in combination with water soluble prebiotic fibres as nutritional sourceS.No. Probiotic Composition OD at 600 nm(1: 5 d ilution) % Increase in growth 0 h 24 h 1Black Tea + B. coagulans MTCC 58560.083 ± 0.0010.156 ± 0.0146.792Black Tea + FOS + B. coagulans MTCC 58560.091± 0.0020.185 ± 0.0150.813Black Tea + GOS + B. coagulans MTCC 58560.085 ±0.0010.187 ± 0.0254.544Black Tea + Inulin + B. coagulans MTCC 58560.088 ±0.0010.191 ± 0.0153.925Black Tea + Polydextrose + B. coagulans MTCC 58560.092 ±0.0030.188 ± 0.0251.066Green Tea + B. coagulans MTCC 58560.075 ± 0.0010.112 ± 0.0233.037Green Tea + FOS + B. coagulans MTCC 58560.078 ± 0.0020.125 ± 0.0137.608Green Tea + GOS + B. coagulans MTCC 58560.075 ± 0.0010.145 ± 0.0348.279Green Tea + Inulin + B. coagulans MTCC 58560.079 ± 0.0030.141 ± 0.0143.9710Green Tea + Polydextrose + B. coagulans MTCC 58560.074 ± 0.0020.142 ± 0.0247.8811Oolong Tea + B. coagulans MTCC 58560.084 ± 0.0010.123 ± 0.0131.7012Oolong Tea + FOS + B. coagulans MTCC 58560.082 ± 0.0010.162 ± 0.0249.3813Oolong Tea + GOS + B. coagulans MTCC 58560.085 ± 0.0030.159 ± 0.0146.5414Oolong Tea + Inulin + B. coagulans MTCC 58560.086 ± 0.0010.159 ± 0.0145.9115Oolong Tea + Polydextrose + B. coagulans MTCC 58560.085 ± 0.0020.157 ± 0.0145.8516Coffee + B. coagulans MTCC 58560.142 ± 0.0010.361 ± 0.0160.6617Coffee + FOS + B. coagulans MTCC 58560.139 ± 0.0010.521 ± 0.0273.3218Coffee + GOS + B. coagulans MTCC 58560.154 ± 0.0020.531 ± 0.0370.9919Coffee + Inulin + B. coagulans MTCC 58560.145 ± 0.0010.516 ± 0.0171.8920Coffee + Polydextrose + B. coagulans MTCC 58560.144 ± 0.0030.532 ± 0.0272.93 EXAMPLE 3 (not according to the invention): Beverage compositions with Bacillus coagulans MTCC 5856

[0025] The viability of Bacillus coagulans MTCC 5856 was also tested in other beverages like aerated soda / cola, fruit juices, mineral water, chocolate, cocoa, fruit drinks, smoothies, milk shakes, cheese spread, honey, tomato ketchup, jam, mayonnaise, protein drink and ice cream. The results are tabulated in tables 7 & 8. Table 7. Stability and recovery of Bacillus coagulans Spores in beveragesTimeSoda drink with B. coagulans MTCC 5856Thums Up with B. coagulans MTCC 5856Orange Juice with B. coagulans MTCC 5856Sprite with B. coagulans MTCC 5856Mineral Water with B. coagulans MTCC 5856Log 10 CFU / servin g Viabi lity (%) Log 10 CFU / servi ng Viability (%) Log 10 CFU / servi ng Viability (%) Log 10 CFU / servin g Viability (%) Log 10 CFU / servin g Viability (%) Expected Recovery9.3011009.3011009.3011009.3011009.301100After 24 h8.61292.598.45492.599.21799.18.20488.219.2699.56 Table 8. Stability of B. coagulans MTCC 5856 spores during the process of preparation of various foods and beverage mix and its recovery S.No.Sample Log 10 CFU / serving % Recovery 1Dry Fruit (Cashew) with B. coagulans MTCC 5856 (20 g serving)8.78194.412Chocolate with B. coagulans MTCC 5856 (50 g serving)9.2599.453Cheese Spread with B. coagulans MTCC 5856 (50g serving)9.2699.564Honey with B. coagulans MTCC 5856 (50 g serving)9.1398.165Mixed Fruit Jam with B. coagulans MTCC 5856 (50 g serving)8.98996.656Tomato Ketchup with B. coagulans MTCC 5856 (50 g serving)9.06597.467Mayonnaise with B. coagulans MTCC 5856 (50 g serving)8.95896.318Protein Drink Mix with B. coagulans MTCC 5856 (50 g serving)9.27499.719Soy Milk with B. coagulans MTCC 5856 (50 g serving)8.23888.5710Shrikhand with B. coagulans MTCC 5856 (50 g serving)9.06497.4511Ice-cream with B. coagulans MTCC 5856 (30 g serving)7.9985.90

[0026] The results indicated that B. coagulans MTCC 5856 is stable in all beverage compositions with increased viability and recovery.

Claims

1. A beverage comprising Bacillus coagulans in the form of spores having more than 95 % viability, wherein the beverage is obtained by brewing of a composition comprising Bacillus coagulans MTCC 5856 in the form of spores, and viable or heat killed or dead or lysed vegetative cells of Bacillus coagulans MTCC 5856, wherein said bacterial spores have the ability to survive stress, temperature and aeration treatments of said beverage composition, wherein the composition comprises tea and the viability is viability 10 minutes after brewing, wherein the tea is selected from the group consisting of green tea, black tea, oolong tea, yellow tea, white tea or decaffeinated tea, herbal tea selected from the group consisting of rosehip tea, chamomile tea, jiaogulan tea, peppermint tea, rooibos tea, ginger tea, ginseng tea, or lemon grass tea.

2. The beverage according to claim 1, wherein the composition further comprises a water soluble prebiotic fiber resulting in a synergistic effect along with the tea on the germination of the spores.

3. The beverage as in claim 2, wherein the water soluble fiber is selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharide (GOS), inulin and polydextrose.