fructophilic lactic acid producing bacteria

Abstract

The present invention discloses a novel fructose utilizing lactic acid producing bacteria Bacillus coagulans FF-7 (MTCC 25235) and its isolation and characterization method. The present invention also discloses the biological applications / therapeutic uses of fructose utilizing lactic acid producing bacteria in increasing utilization of fructose from food and in managing diseases associated with high fructose intake.

Description

BACKGROUND TECHNICAL FIELD

[0002] The present invention relates generally to fructose-philic lactic acid bacteria. More specifically, the present invention relates to the isolation, characterization and biological applications of the fructose-philic probiotic bacterium, Bacillus coagulans. BACKGROUND

[0004] Probiotics and their importance in health and prevention of a variety of diseases have been widely reported. Fructose-philic lactic acid bacteria (FLAB) are a special class of lactic acid bacteria that utilize fructose as a growth substrate. Due to their unique characteristics of poor growth on glucose substrates and preference for oxygen, they are considered as “unconventional” lactic acid bacteria (LAB). Their unusual growth characteristics are due to an incomplete gene encoding a bifunctional ethanol / acetaldehyde dehydrogenase (adhE). This results in an imbalance of NAD / NADH and a requirement for additional electron acceptors for metabolism of glucose. Oxygen, fructose and pyruvate are used as electron acceptors. The carbohydrate metabolism genes of FLAB are significantly less than other LAB, particularly due to the lack of a complete phosphotransferase system (PTS) transporter protein. FLAB were originally classified as Leuconostoc, and later reclassified as Fructobacillus based on their phylogenetic position as well as biochemical and morphological characteristics (Endo A, Okada S. 2008. Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov. Int J Syst Evol Microbiol 58:2195-2205).

[0005] Metabolism of fructose begins with fructokinase in the liver. Fructose load is converted to lactate in the intestinal cells and liver. In addition, excess fructose in the liver is directed to peripheral tissues and is taken up by insulin-dependent glucose transporter GLUT4 present on adipose tissue, converted to fatty acids. GLUT4 has been reported to play an important role in the development of fructose-induced hepatic steatosis and dyslipidemia. In addition, several metabolic disorders caused by fructose have been reported, such as NASH, NAFLD, dyslipidemia, ectopic lipid deposition in the liver and skeletal muscle, uric acid metabolism, hypertension, mineral metabolism (Prasanthi Jegatheesan and Jean-Pascal De Bandt, Fructose and NAFLD: The Multifaceted Aspects of Fructose Metabolism, Nutrients. 2017 Mar; 9(3): 230; Bidwell AJ, Chronic Fructose Ingestion as a Major Health Concern: Is a Sedentary Lifestyle Making It Worse? A Review, Nutrients. 2017 Jun; 9(6): 549). Excessive fructose intake is also associated with an increased risk of cardiovascular disease. Increased fructose intake can also lead to lactic acidosis by excessively lowering the pH in the blood.

[0006] Probiotics play an important role in intestinal fructose metabolism. Low-dose fructose consumed orally reaches the intestine and is metabolized in the presence of different enzymes and indigenous microbiota. However, in the presence of high-dose fructose, fructose load overflows from the intestine to the liver. To reduce the overflow of fructose from the intestine to the liver, probiotics play an important role in converting fructose to SCFA (short-chain fatty acids).

[0007] FLAB is isolated from various sources such as fruits and vegetables, human fecal culture, natural antibacterial agents, cheese, kefir grain, dairy and non-dairy, fermented and raw milk, feces of breastfed infants, lactation milk, goat milk, buffalo milk and cow milk, yogurt, beverages, poultry sources, animal rumen content, Pengging Duck caecum, chicken intestinal and fecal samples, chicken feed, enzymes, fermented rice, curd, meat and yeast extract, glucose and sucrose, human intestine, human colonocyte epithelial cells, human and animal vaginal and oral extracts, diapers of human infants, pineapple residue, industrial sausage, ice cream, small piglet small intestine, corn syrup, crop and intestinal duck. The following prior art documents describe the isolation of different FLABs.

[0008] 1. Akihito Endo, Fructophilic lactic acid bacteria inhabit fructose-rich niches in nature, Microb Ecol Health Dis. 2012; 23

[0009] 2. Akihito Endo, Shintaro Maeno, Yasuhiro Tanizawa, Wolfgang Kneifel, Masanori Arita, Leon Dicks, Seppo Salminen, Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose Fermenting Microbes, Minireview, Applied and Environmental Microbiology, October 2018 Volume 84 Issue 19 e01290-18

[0010] 3. Arshad F, Mehmood R, Hussain S, Khan MA, Khan MS (2018) Lactobacilli as Probiotics and their Isolation from Different Sources. Br J Res 5(3):43

[0011] Since the biological effects of probiotics are strain specific and cannot be generalized to all strains and species (Guidelines for the evaluation of probiotics in food, Joint FAO / WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food, London, Ontario, Canada, April 30 and May 1, 2002, see section 3.1 which states "The current state of evidence suggests that probiotic effects are strain specific. Strain identity is important to link a strain to a specific health effect as well as to enable accurate surveillance and epidemiological studies."), there is still a need to find superior fructophilic probiotics with improved biological functions. The present invention addresses the above mentioned problem by disclosing a novel fructophilic lactic acid producing bacterium Bacillus coagulans MTCC 25235.

[0012] The main object of the present invention is to disclose a novel fructophilic lactic acid producing bacterium Bacillus coagulans MTCC 25235 and process for its isolation.

[0013] Another object of the present invention is to disclose production of short chain fatty acids by the fructophilic lactic acid producing bacterium Bacillus coagulans MTCC 25235.

[0014] Yet another object of the present invention is to disclose antimicrobial effects of the fructophilic lactic acid producing bacterium Bacillus coagulans MTCC 25235.

[0015] The present invention achieves the above mentioned objects and provides further related advantages.

[0016] Preservation of biological material

[0017] The biological material Bacillus coagulans strain FF7 with accession number MTCC 25235 mentioned in the present application has been deposited at the Microbial Type Culture Collection and Gene Bank (MTCC), Sector 39-A, CSIR-Centre for Biosciences and Biotechnology, Chandigarh-160036, India on 24th December 2018. Invention Overview

[0019] This invention discloses a fructophilic lactic acid-producing bacterium. It also discloses methods for isolating, characterizing, and biological / therapeutic applications of this bacterium.

[0020] Other features and advantages of the invention will become clearer in the following more detailed description, which illustrates the principles of the invention by way of example. Attached Figure Description

[0021] The patent or application document contains at least one color drawing. A copy of this patent or patent application publication with the color drawing will be provided by the patent office upon request and payment of the necessary fees.

[0022] Figure 1A , 1B 1C and 1D show Bacillus coagulans MTCC 25235 ( Figure 1A Phase contrast micrographs (×1000x), wet cell patches with spores ( Figure 1B Gram staining of vegetative cells ( Figure 1C ), spore staining of sporulated cells and colonies growing on GYE agar plates ( Figure 1D ).

[0023] Figure 2 A graph comparing the growth patterns of Bacillus coagulans FF7, MTCC25235, and ATCC 31284 in the presence of fructose and dextrose as carbon sources is shown. Values ​​are the mean (±SD) of three independent determinations.

[0024] Figure 3 A graph shows the fructose utilization of Bacillus coagulans MTCC FF7,25235 after 24 hours of incubation in a culture medium and in the presence of fructose-rich foods (honey, fruit juice). Values ​​are the mean (±SD) of three independent determinations.

[0025] Figure 4 A phylogenetic tree based on the 16S rRNA sequence is shown, which displays the relative position of Bacillus coagulans FF7.

[0026] Figure 5 The effects of different pH values ​​on the growth of Bacillus coagulans FF7, MTCC 25235, and ATCC 31284 were shown, and the optimal pH values ​​for their growth were found to be pH 7.5 and pH 6.5, respectively. Values ​​are the average (±SD) of three independent determinations.

[0027] Figure 6Graphical representation showing the effect of different temperatures on the growth of Bacillus coagulans MTCC FF7, 25235 and Bacillus coagulans ATCC 31284. Values are mean (± SD) from three independent determinations.

[0028] Figure 7 Graphical representation showing the effect of GIT adverse conditions after gastric treatment on the viability of Bacillus coagulans MTCC 25235 in in vitro experiments mimicking in vivo conditions. Sterile saline was used as untreated control.

[0029] Figure 8 shows the BSH activity of Bacillus coagulans MTCC 25235 Figure 8A ). Assay was performed using soft MRS agar supplemented with bovine bile (0.3%, w / v) and CaC03(0.3%, w / v). MRS agar without bile salts was used as negative control Figure 8B ). The hatched area indicates the BSH activity of Bacillus coagulans MTCC 25235.

[0030] Figure 9 Graphical representation showing the survival of Bacillus coagulans FF7, MTCC 25235 in gastric juice buffer at different pH values (1.5-8.0). Values are expressed as Log10spores / g. Data represent the mean and standard deviation (± SD) of two different experiments performed.

[0031] Figure 10 Graphical representation showing the in vitro effect of bovine bile salts on the growth of Bacillus coagulans FF7, MTCC 25235 and Bacillus coagulans ATCC 31284. Fresh cultures of Bacillus coagulans MTCC 25235 and Bacillus coagulans ATCC 31284 grown overnight were inoculated in MRS broth containing bovine bile salts (0.1%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 0.9% and 1%, w / v) and without bovine bile salts (% w / v). Values are mean (± SD) from three independent determinations.

[0032] Figure 11 Graphical representation showing the study of L-lactate production by Bacillus coagulans FF7, MTCC 25235 in the presence of two standardized formulations corresponding to 6 x 10 9 cfu / g (formulation 1) and 15 x 10 9 cfu / g (formulation 2). Values are mean (± SD) from three independent determinations.

[0033] Figure 12A , 12Band 12C shows the graphical representation of production of acetic acid (A), butyric acid (B) and propionic acid (C) by Bacillus coagulans MTCC 25235 in the presence of fermented fructose, FOS, cranberry seed fiber, and psyllium seed fiber. Figure 12A Figure 12B Figure 12C Values are in mg / g and are the average of three replicates from two different occasions.

[0034] Figure 13 Graphical representation showing the viability of Bacillus coagulans MTCC 25235 during storage at temperature 40±2°C and RH 60%±5%. Two standardized formulations equivalent to 15x10 9 cfu / g (formulation 1) and 6x10 9 cfu / g (formulation 2) were studied. The average of spore viable counts is expressed in log10 cfu / g. Each time point represents the average Log 10 standard deviation (±SD) of three different experiments performed in duplicate.

[0035] Description of the most preferred embodiment

[0036] In the most preferred embodiment, the present disclosure discloses a method for isolation and identification of a novel fructose utilizing probiotic bacteria from honey, comprising the steps of:

[0037] a) mixing honey with physiological saline in the ratio of 1:10 w / v to get a suspension;

[0038] b) mixing the suspension of step a) thoroughly and heat shocking at 50-70°C for 30 minutes to selectively isolate spores;

[0039] c) isolating bacterial colonies by plating 1-2 ml of the suspension from step b) in a suitable medium containing fructose at 35-37°C for 48 hours;

[0040] d) purifying the bacterial isolates by selecting and culturing morphologically distinct colonies in a suitable medium containing fructose as a carbon source;

[0041] e) identifying the bacterial strain as Bacillus coagulans FF7 strain, strain number MTCC 25235 by biochemical analysis and 16S rRNA sequencing.

[0042] ​​In a related aspect, the honey includes, but is not limited to, raw honey, filtered honey, acacia honey, alfalfa honey, aster honey, avocado honey, basswood honey, beech honey, blueberry honey, blue gum honey, buckwheat honey, clover honey, dandelion honey, eucalyptus honey, willow herb honey, heather honey, ironbark honey, red ironbark honey, leatherjacket honey, lotus tree honey, macadamia tree honey, orange blossom honey, pine honey, sourwood honey, sage honey, and sloe honey. In another related aspect, the culture medium is selected from the group consisting of MRS (De Man, Rogosa and Sharpe agar), GYA (glucose yeast extract agar), TSB (tryptone soya broth), sporulation medium, and Mueller Hinton agar.

[0043] In another related aspect, the isolated probiotic bacterial strain is positive for hydrogen peroxidase, oxidase, methyl red, VP (vogesproskauers), lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melobiose, sucrose, arabinose, mannose, inulin, sodium gluconate, salicin, sorbitol, mannitol, arabitol, methyl glucoside, rhamnose, cellobiose, ONPG, esculin hydrolysis biochemical tests, and negative for sorbose, malonate utilization, citrate utilization, xylitol, methyl mannose, pine tree trilose, erythritol, adonitol, myo-inositol, galactitol, glycerol, hemolysis, citrate, and indole biochemical tests.

[0044] In another preferred embodiment, the present invention discloses a novel Bacillus probiotic that is isolated from honey, for increasing fructose utilization from fructose rich food. In another related aspect, the fructose loving probiotic is a gram positive bacteria. In another aspect, the optimal pH and temperature for the recorded growth of the fructose loving bacteria are 7.5 and 40°C respectively. In another aspect, the fructose loving probiotic is bile resistant, gastric acid resistant and produces lactic acid. In a related aspect, the fructose loving probiotic is Bacillus coagulans. In yet another related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 25235. In a related aspect, the fructose rich food is selected from the group consisting of high fructose corn syrup, honey, agave, maple syrup, coconut sugar, palm sugar, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereal, juice, coffee creamer, jam and jelly, energy drink, condiment, ice cream. In a related aspect, the fructose loving probiotic is used for therapeutic management of diseases associated with high fructose intake. In a related aspect, the diseases associated with high fructose intake include, but are not limited to, obesity, non-alcoholic steatohepatitis (NASH), insulin resistance, metabolic syndrome, cardiovascular complications, diabetes, hyperlipidemia, hypertension, inflammation and hyperuricemia. In another related aspect, the fructose loving probiotic is present in the form of an inoculum, lyophilized powder, fine powder, tablet, capsule, suspension, solution, emulsion, gel, chewable or edible food and is administered independently or in combination with a fructose rich food selected from the group consisting of high fructose corn syrup, honey, agave, maple syrup, coconut sugar, palm sugar, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereal, juice, coffee creamer, jam and jelly, energy drink, condiment, ice cream.

[0045] In yet another preferred embodiment, the present invention discloses a method of inhibiting a pathogenic microorganism, the method comprising the step of contacting the microorganism with the fructose loving probiotic Bacillus coagulans MTCC 25235. In a related aspect, the pathogenic microorganism is selected from the group consisting of Salmonella abony, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis.

[0046] In another preferred embodiment, the present application discloses a method for production of short chain fatty acids by co-culturing fructose utilizing probiotic Bacillus coagulans MTCC 25235 with plant fibers selected from the group consisting of fructose, fenugreek seed fiber, cranberry seed fiber, fructo-oligosaccharides (FOS).

[0047] The following specific examples, included herein, illustrate the above most preferred embodiment of the present application. Example

[0048] Example 1 : Isolation and identification of fructose utilizing bacteria

[0049] Method

[0050] The present study used unfiltered raw honey for isolation of spore forming fructose utilizing lactic acid bacteria. The present study used de Man, Rogosa and Sharpe (MRS) agar with fructose instead of dextrose for isolation of fructose utilizing bacteria (Table 1).

[0051] Table 1 : Composition of culture medium

[0052] SEQ ID NO Ingredient Grams / Liter 1 Peptone 10.000 2 Beef Extract 10.000 3 Yeast Extract 5.000 4 Fructose 20.000 5 Polysorbate 80 1.000 6 Ammonium Citrate 2.000 7 Sodium Acetate 5.000 8 Magnesium Sulfate 0.100 9 Manganese Sulfate 0.050 10 Dipotassium Phosphate 2.000 11 Agar 12.000 12 Final pH (25°C) 6.5±0.2

[0053] One gram of unfiltered raw honey was added to test tubes containing 10 ml of physiological saline. It was mixed well and heat shocked at 70 °C for 30 minutes for selective isolation of spores. Isolation of bacteria was carried out by adding 1 ml of the above sample from each dilution to MRS agar plates containing fructose. The plates were further incubated at 37 °C for 48 hours. After incubation, morphologically different colonies were picked for further testing and purification of bacterial isolates. Bacillus coagulans MTCC 25235 was isolated and streaked on other MRS agar plates containing fructose as a carbon source.

[0054] Biochemical characteristics of fructose utilizing bacteria

[0055] Bacterial isolates were incubated at 37°C for 24 hours in de Man, Rogosa and Sharpe agar (MRSA). Bacterial inoculum was prepared by picking 1 to 3 well isolated colonies and making a homogenous suspension in sterile saline. The density of the suspension was > 0.5 OD at 620 nm. The test was performed using this inoculum (50 μΐ) and following the kit manufacturer's instructions (HiMedia, Mumbai, India). Biochemical characterization of Bacillus coagulans MTCC 25235 and Bacillus coagulans ATCC 31284 was performed by the methods described in Majeed, M., Nagabhushanam, K., Natarajan, S., Sivakumar, A., Eshuis-de Ruiter, T., Booij-Veurink, J., Janine Booij-Veurink, Ynte P. de Vries, Ali, F. (2016); Evaluation of genetic and phenotypic consistency of Bacillus coagulans MTCC 5856: A commercial probiotic strain. World Journal of Microbiology & Biotechnology, 32, 60. HiCarbohydrat TM The kit (Code - KB009) was purchased from HiMedia, Mumbai, India and tested following the manufacturer's instructions. Bacterial suspensions of Bacillus coagulans MTCC 25235 and Bacillus coagulans ATCC 31284 were prepared as described above. In addition, Bacillus coagulans ATCC 31280 was subjected to IMViC (Indole, Methyl Red, VP, Citrate utilization) test, oxidase and Gram stain according to the methods of Majeed et al. (2016).

[0056] 16S rDNA sequencing

[0057] The 16S rDNA sequencing of the genomic DNA of Bacillus coagulans MTCC 5856 was performed as described earlier (William J. Bruno, Nicholas D. Socci, and Aaron L. Halpern (2000). Weighted Neighbor Joining: A Likelihood-Based Approach to Distance-Based Phylogeny Reconstruction, Mol. Biol. Evol. 17(1): 189-197). The 16S rDNA gene fragments were sequenced using an ABI 3500 Genetic Analyzer automated DNA sequencer as described earlier (Heyrman and Swings 2001). The sequencing primers used were 5- AGHGTBTGHTCMTGNCTCAS-3 (forward primer) and 5-TRCGGYTMCCTTGTWHCGACTH-3 (reverse primer). The amplified DNA fragments of about 1.5 kb were separated on 1% agarose gel and purified using Qiagen spin columns. The purified fragments were directly used for DNA sequencing. The sequence was used for BLAST search http: / / blast.ncbi.nlm.nih.gov / Blast.cgi

[0058] Growth conditions of Bacillus coagulans MTCC 25235

[0059] Optimization of growth conditions of Bacillus coagulans MTCC 25235 was analyzed at different temperatures and pH values. MRSB medium was prepared and pH was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 using 2N HC1 and 2N NaOH. The overnight grown culture (1%, v / v) was inoculated in the medium after pH adjustment and incubated in a shaking incubator at 120 rpm for 24 hours. The growth was monitored at every six hours interval by measuring absorbance at 600 nm using spectrophotometer (Shimadzu Corporation, Kyoto, Japan). MRSB medium was prepared, pH was adjusted to 6.5 and inoculated with the overnight grown culture (1%, v / v). Further, the flasks were incubated in a shaking incubator at 120 rpm for 24 hours at different temperatures (20°C, 30°C, 37°C, 40°C, 50°C and 60°C). The growth was monitored at every six hours interval by measuring absorbance at 600 nm using spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

[0060] Results

[0061] Identification of fructose-utilizing bacteria ​

[0062] Spores of Bacillus coagulans MTCC 25235 are oval endospores Figure 1A ), vegetative cells are gram-positive bacilli as seen by gram staining Figure 1B ). Colonies from Bacillus coagulans MTCC 25235 grow on media producing uniform, 1-3 mm in diameter, white to cream colored, smooth colonies comprising vegetative bacilli Figure 1C and Figure 1D ). The isolated bacteria grow better in fructose-rich media than in dextrose-rich media Figure 2 compared to other Bacillus coagulans strains, indicating its fructophilic nature and potential for fructose utilization. Bacterial growth tests were also performed in fructose-rich foods, and the results showed a decrease in fructose content Figure 3 , which means that the bacteria utilize fructose as a carbon source for their growth and development. Thus, by metabolizing the excess fructose in foods, the bacteria can be used to manage and prevent diseases associated with high fructose intake. The bacteria can be administered alone or in combination with fructose-rich foods, including high fructose corn syrup, honey, agave, maple syrup, coconut sugar, palm sugar, molasses, soda, candy, sweetened yogurt, frozen foods, canned foods, cereals, fruit juices, coffee creamer, jams and jellies, energy drinks, condiments, ice cream (Braverman J, List of Foods high in fructose, https: / / www.livestrong.com / article / 30454-list-foods-high-fructose / , accessed on May 3, 2019).

[0063] Biochemical characterization

[0064] Biochemical characterization of Bacillus coagulans MTCC 25235 and Bacillus coagulans ATCC 31284 was performed by the methods described in Majeed, M., Nagabhushanam, K., Natarajan, S., Sivakumar, A., Eshuis-de Ruiter, T., Booij-Veurink, J., Janine Booij-Veurink, Ynte P. de Vries, Ali, F. (2016); Evaluation of genetic and phenotypic consistency of Bacillus coagulans MTCC 5856: A commercial probiotic strain. World Journal of Microbiology & Biotechnology, 32, 60). The results were compared with commercial strain Bacillus coagulans ATTC 3128 and tabulated in Table 2.

[0065] Table 2: Biochemical characteristics of Bacillus coagulans ATTC 3128 and Bacillus coagulans MTCC 25235

[0066]

[0067]

[0068] The results indicated that the isolated probiotic bacterial strain was positive for catalase, oxidase, methyl red, VP, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melibiose, sucrose, melibiose, arabinose, mannose, inulin, sodium gluconate, salicin, sorbitol, mannitol, arbutin, methyl glucoside, rhamnose, cellobiose, ONPG, esculetin hydrolysis biochemical tests and negative for sorbose, malonate utilization, citrate utilization, xylitol, methyl mannose, melezitose, erythritol, adonitol, inositol, galactitol, glycerol, hemolysis, citrate and indole biochemical tests.

[0069] 16S rDNA sequencing

[0070] The bacterial 16S rDNA was sequenced to obtain the following sequence information (SEQ ID):

[0071]

[0072] A BLAST (Basic Local Alignment Search Tool) search was performed using the above sequence and the results of the top 10 aligned sequences are tabulated in Table 3.

[0073] Table 3: Alignment view using NCBI GenBank portfolio - distribution of 10 BLAST hits against query sequence

[0074]

[0075]

[0076] The results indicated that the isolated organism is a new Bacillus coagulans strain with 98.96% identity to Bacillus coagulans strain 55-LR4. The results of phylogenetic analysis also indicated that the organism is a new Bacillus coagulans strain. Figure 4

[0077] Optimum growth conditions for fructose-utilizing bacteria

[0078] The optimum pH and temperature for growth of fructose-utilizing bacteria were recorded as 7.5 and 40°C, respectively Figure 5 and Figure 6 ).

[0079] Example 2: In vitro probiotic evaluation of Bacillus coagulans MTCC 25235

[0080] Gastric acid resistance

[0081] This was done by adding 1 ml of the suspension into 100 ml of sterile electrolyte solution (6.2 g / L NaCl, 2.2 g / L KC1, 0.22 g / L CaCl2and 1.2 g / L NaHC03) containing 0.01% lysozyme (Sigma-Aldrich) and 0.3% pepsin (Sigma-Aldrich) and incubating for 5 min. Further, the pH was adjusted to 1.5, 3, 4, 5, 6, 7 and 8 (adjusted using 1 N NaOH and 1 N HC1). The incubation temperature was controlled at 37°C for 4 h. At different time intervals of 0, 1.0, 2.0, 3.0 and 4.0 h, 1 ml samples were withdrawn. After incubation, serial dilutions were made in sterile saline (0.89% w / v) and viable counts were enumerated by plating on glucose yeast extract agar (HiMedia). The experiments were performed in triplicates on two different occasions.

[0082] Bile salt tolerance

[0083] Bile tolerance of Bacillus coagulans MTCC 25235 cells was determined by the method described earlier (Gilliland et al., 1984; Hyronimus et al., 2000). About 10 6 cfu mL -1 ​Bacillus coagulans MTCC 25235 was inoculated in MRS broth (HiMedia) and incubated overnight, after which bile salts (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5% and 2.0%, w / v) were added and no bile salts were added as experimental control. The samples were incubated at 37 °C with shaking at 120 rpm for 24 hours. The growth of control (no bile) and test cultures with different concentrations of bile were monitored every hour by measuring absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

[0084] Lactic acid production

[0085] Lactic acid production by Bacillus coagulans MTCC 25235 was evaluated using Megazyme kit. One loop of overnight grown Bacillus coagulans MTCC 25235 culture was added to glucose yeast extract broth (HiMedia) and incubated at 37 °C with shaking at 120 rpm for 18 hours. After incubation, the broth was passed through a 0.22 micron filter (Sartorius, India) and analyzed for lactic acid content using Megazyme kit (K-DLATE 10 / 04) as per the instructions (Megazyme International Ireland, Wicklow IDA Business Park, Ireland). GYE medium was used as blank control in the assay.

[0086] Simulated gastric fluid tolerance

[0087] The survival of *Bacillus coagulans* MTCC 25235 spores was investigated to represent oral digestive conditions. One mL suspension of *Bacillus coagulans* MTCC 25235 was placed in simulated saliva contents composed of KCl (0.8946 g / L), CO(NH2)2 (0.1981 g / L), Na2SO4 (0.5681 g / L), NaHCO3 (1.680 g / L), and NaH2PO4 (0.8878 g / L), and the pH was adjusted to 6.8 ± 0.2 and incubated at 37°C for 5 minutes. Simulated gastric juice was prepared by adding 9 g / L sodium chloride and 3 g / L pepsin (Sigma-Aldrich, St. Louis, MO, USA), followed by aseptic pH adjustment to 3.0 ± 0.2 using 2N HCl. The samples were further incubated at 37°C at low rpm for 3 hours. After 3 hours of incubation, the pH was aseptically adjusted to 7.0 using 2N NaOH. Bovine bile (5 g / L) was then incubated for an additional 24 hours at 37°C. Following this final step of the simulated digestion process, samples were collected and counted using glucose yeast extract agar (HiMedia) via serial dilution to assess the spore viability of Bacillus coagulans MTCC25235. Figure 7 ).

[0088] Antibiotic resistance patterns

[0089] MIC was determined according to the Clinical and Laboratory Standards Institute (CLSI 2012) guidelines. A suspension of Bacillus coagulans MTCC 25235 was prepared by incubating bacterial cultures in sterile physiological saline (0.89% NaCl wt / vol; Himedia, Mumbai, India) for 18 hours. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard (equivalent to 1.5 × 10⁻⁶). 8 Colony forming units (CFU) / ml). Antibiotic stock solutions were prepared according to CLSI guidelines, and serially diluted 100 μl in broth media (glucose yeast extract acetate broth [GYEA, HiMedia, Bombay, India] for Bacillus coagulans MTCC 25235, Mueller Hinton broth [MHB, Difco Laboratories, Detroit, Mich USA] for Staphylococcus aureus) in 96-well U-bottom microtiter plates (BD Labware, NJUSA) to prepare two-fold serial dilutions. The bacterial suspensions were further diluted in MHB, and 100 μl of the diluted inoculum was added to each well of the plate to bring the final inoculum in each well to a concentration of 5 × 10⁻⁶. 5CFU / ml and the final concentration of antibiotic ranged from 0.0078 to 4 pg / ml. Staphylococcus aureus ATCC 6538 was used as reference culture in the present study. The plates were incubated at 37°C for 24 hours and the presence of turbidity was judged by naked eye. The minimum concentration of the compound which showed no turbidity was recorded as MIC.

[0090] AMES test

[0091] The experimental data showed that Bacillus coagulans MTCC 25235 spores did not increase the number of revertants in five Salmonella strains (TA98, TA100, TA102, TA1535 and TA1537) as compared to the negative control, whether S9 metabolic activation system was present or not. Further, Bacillus coagulans MTCC 25235 spores (up to 5000 pg / plate) did not cause a dose dependent mutagenic effect. Bacillus coagulans MTC 25235 spores did not show any mutagenic activity under the experimental conditions.

[0092] BSH activity

[0093] Growth of Bacillus coagulans MTCC 25235 was observed on agar plates containing bovine bile and calcium carbonate, which indicated its tolerance to bovine bile and the presence of BSH activity. As shown in Figure 8A and Figure 8B A transparent zone of 19 ± 2 mm was present in the soft agar plates, indicating the presence of BSH activity.

[0094] Antibacterial activity against human pathogens

[0095] The antibacterial activity was determined by the well diffusion test as described previously with minor modifications (Cintas LM, Rodriguez JM, Fernandez MF, Sletten K, Nes IF, Hernandez PE, Holo H (1995) Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Appl Environ Microbiol 61 :2643-2648). Briefly, 10 6Cfu mL"1indicated the strains (Staphylococcus epidermidis ATCC 14990, Streptococcus mutans MTCC 1943, Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 14579, Propionibacterium acnes ATCC 11827, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Pseudomonas luteola NCIM 216 and Salmonella NCIM 2257) were poured 5 mL of soft (0.7% agar) glucose yeast extract (HiMedia, India) on top of concentrated hard (1.5% agar) tryptone soya agar (HiMedia, India). One loop of overnight grown culture of Bacillus coagulans MTCC 25325 was added to MRS medium and incubated at 37°C at 120 rpm for 24 h. After 24 h, the culture was centrifuged (10,000 x g) to remove the cells and the supernatant was collected, concentrated ten-fold by lyophilization and filter sterilized through 0.22 micron filter (Sartorius, India). Concentrated supernatant (50 μί) was added to 6 mm wells punched in the solidified double layer agar. The plates were kept in the refrigerator (4 ± 2°C) for 5 h to allow the samples to diffuse into the agar and then incubated at 37°C for 18-20 h. After incubation, the zones of inhibition were measured and recorded in millimeters.

[0096] Production of short chain fatty acids

[0097] In-vitro fermentation of Bacillus coagulans MTCC 25235 was carried out by the following method described by McBurney and Thompson (1987) with some modifications. Briefly, 2.0 g of fructose, fenugreek seeds, cranberry seed fiber, FOS were added to 100 ml of demineralized water. The pH was adjusted to 7.0 ± 0.2 and autoclaved at 121°C for 20 min. After sterilization, oxygen reductase dehydrogenase Broth, Oxyrase, Inc, OH, USA) to induce anaerobic conditions. Five percent of overnight grown B. coagulans MTCC 25235 culture was inoculated into all flasks and incubated at 37 °C with gentle shaking rpm for 24 hours. The flasks were closed and sealed with parafilm to maintain the anaerobic conditions created by the enzyme supplement. The pH values at 0 hours of incubation and after fermentation (24 hours) were recorded. To each sample, 1 ml of copper sulfate (10 g / L) was added to inhibit further microbial growth (Sigma-Aldrich, St. Louis, MO, USA). Additionally, 5.0 ml of sample was added to 5 ml of distilled water and pH was adjusted to 1.5 using 3 M H2SO4. To the sample, 10 ml of chilled diethyl ether (-20 °C) was added followed by vortexing for 1 minute. Sodium chloride was added followed by centrifugation at 3000 x g for 10 minutes. After centrifugation, the organic layer was separated and transferred to a new flask. This step was used to quantify SCFA. SCFA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were similarly treated. The yield of SCFA (acetic, propionic, and butyric acids) was measured by gas chromatography (GC) using an Agilent Technologies 6890N gas chromatograph (Stevens Creek Blvd Santa Clara, CA, USA) containing a DB-FFAP (polyethylene glycol modified with terephthalic acid) column. The column temperature was 200 °C. The injector and detector port temperature was 250 °C. N2was used as the carrier gas at a flow rate of 1.0 ml / min. SCFA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). The SCFA (acetic, propionic, and butyric acids) concentration was expressed as milligrams of SCFA produced per gram of galactomannan from fenugreek seeds.

[0098] Results

[0099] Gastric acid resistance

[0100] In the longest 4-hour study, there was no significant difference in spore count at pH 3 to pH 8.0 compared to the initial spore count (2-5%) ( Figure 9 ). However, at pH 1.5, a log 10 reduction of 0.44 and 2.036 in spore count was observed at 1 hour and 4 hours, respectively. The results of the study confirmed the stability of B. coagulans MTCC 25235 spores at acidic and alkaline pH conditions.

[0101] Bile tolerance test

[0102] Growth of B. coagulans MTCC 25235 was observed on agar plates containing bile salts (1% w / v), which indicates its tolerance to bile salts. Further, bile tolerance assay was performed by adding (0.1-2.0%) bovine bile to MRS broth in different flasks. Growth of B. coagulans MTCC 25235 was observed up to 2% w / v in the presence and absence of bovine bile. However, growth of B. coagulans ATCC 31284 was found to be 0.8% w / v Figure 10 ) Similarly, there was no significant difference in the viability of B. coagulans MTCC 25235 and B. coagulans ATCC 31284 in the presence and absence of bile salts.

[0103] Lactic acid production

[0104] Lactic acid production by B. coagulans MTCC 25235 was assessed by using Megazyme kit. Total lactic acid produced by B. coagulans MTCC 25235 was 4.487 g / L. L-type lactic acid was 4.12 g / L. Whereas, D-type lactic acid produced by B. coagulans MTCC 25235 was 0.367 g / L Figure 11 ).

[0105] Antibiotic resistance

[0106] The MIC results of clindamycin, kanamycin, ampicillin, streptomycin, vancomycin, erythromycin, gentamicin, tetracycline and chloramphenicol against B. coagulans MTCC 25235 and S. aureus ATCC 6538 are given in Table 4. The MIC of all the tested antibiotics against B. coagulans MTCC 25235 ranged from 0.0078 to 1.0 μg / ml. The MIC of all the tested antibiotics against S. aureus ATCC 6538 ranged from 0.031 to 2 μg / ml.

[0107] Table 4: Minimum inhibitory concentration of antibiotics against B. coagulans MTCC 25235 culture and S. aureus ATCC 6538

[0108]

[0109] Antibacterial activity

[0110] The Bacillus coagulans MTCC 25235 was evaluated for its antibacterial activity. The antibacterial activity was determined by well diffusion assay as described by Cintas et al. (1995). The results indicated that the probiotic is an effective antibacterial agent against Salmonella abony, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, and Staphylococcus epidermidis.

[0111] Table 4: Antimicrobial activity of Bacillus coagulans MTCC 25235 against test bacteria

[0112]

[0113]

[0114] Data represent mean ± SD of three independent experiments performed in triplicate

[0115] Short chain fatty acid production

[0116] The results of the analysis are shown in Figure 12A , 12B and 12C. The acetic acid production by Bacillus coagulans MTCC 25235 was high with FOS, followed by fenugreek seed fibre, cranberry fibre and fructose Figure 12A . Similarly, the butyric acid production by Bacillus coagulans MTCC 25235 was high with fructose, followed by FOS, fenugreek seed fibre and cranberry fibre Figure 12B . The propionic acid production by Bacillus coagulans MTCC 25235 was similar with fructose, FOS and cranberry seed fibre, respectively, while it was lower with fenugreek seed fibre Figure 12C

[0117] Storage and viability

[0118] Two standardized formulations equivalent to 15 x 10 9 cfu / g (formulation 1) and 6 x 10 9 cfu / g (formulation 2) were investigated. Bacillus coagulans MTCC 25235 showed enhanced viability during storage at a temperature of 40 ± 2 °C and RH 60% ± 5% Figure 13

[0119] In light of the foregoing disclosure and teachings, other modifications and changes will be obvious to those of ordinary skill in the art. Therefore, it is intended that the application be construed as including all such modifications and changes as fall within the scope of the appended claims. The scope of the application should be construed in accordance with the appended claims, rather than the foregoing description, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein. SEQUENCE LISTING ​​<110> Sami-Sabinsa Group, Inc. <120> Fructophilic lactic acid producing bacteria <130> Fructospore <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1437 <212> DNA <213> Bacillus coagulans <400> 1 acttgcaagt cgtgcggccc ttttttaaaa gcttgctttt taaaaggtta gcggcggacg 60 ggtgagtaac acgtgggcac cctgcctgta agatcgggat aacgccggga aaccggggct 120 aataccggat agttttttcc tccgcatgga ggaaaaagga aagacggctt ctgctgtcac 180 ttacagatgg gcccgcggcg cattagctag ttggtggggt aacggctcac caaggcaacg 240 atgcgtagcc gacctgagag ggtgatcggc cacattggga ctgagacacg gcccaaactc 300 ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc 360 gcgtgagtga agaaggcctt cgggtcgtaa aactctgttg ccggggaaga acaagtgccg 420 ttcgaacagg gcggcgcctt gacggtaccc ggccagaaag ccacggctaa ctacgtgcca 480 gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagcgcgc 540 gcaggcggct tcttaagtct gatgtgaaat ctttgcgggc tcacccgcaa gcggtcattg gaaactggga gggctttgag tgcaagaaag aggagagtgg aatttccacg tgtagcggtg aaatgcgtaa agatgtggag gaacaccagt ggcgaaggcg gctctctggt ctgtaactga cgctgaggcg cgaaagcgtg gggagcaaac aggregate accctggtag tccacgccgt 780 aaacgatgag tgctaagtgt tagggtttt ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagacctt accaggtctt gacatcctct gacctccctg gagacagggc cttccccttc gggggacaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt tagtcccgc aacgagcgca 1080 acccttgacc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa agggctgcga gaccgcgagg ttaagccaat cccagaaaac cattcccagt tcggattgca ggctgcaacc cgcctgcatg aagccggaat cgctagtaat 1320 cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt acggagccag ccgccga 1437

Claims

1. A fructoseophlic probiotic Bacillus coagulans isolated from honey for enhancing utilization of fructose from fructose rich food, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 25235.

2. The probiotic of claim 1, wherein the fructoseophlic probiotic is gram positive.

3. The probiotic of claim 1, wherein the optimum pH and temperature for growth of the fructoseophlic bacteria are 7.5 and 40°C, respectively.

4. The probiotic of claim 1, wherein the fructoseophlic probiotic is bile resistant, gastric acid resistant and produces lactic acid.

5. The probiotic of claim 1, wherein the fructose rich food is selected from the group comprising high fructose corn syrup, honey, agave, maple syrup, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereal, fruit juice, coffee creamer, jam and jelly, energy drink, condiment.

6. The probiotic of claim 1, wherein the fructose rich food is selected from the group comprising coconut sugar, palm sugar, ice cream.

7. The probiotic of claim 1, wherein the fructoseophlic probiotic is used for therapeutic management of diseases associated with high fructose intake.

8. The probiotic of claim 7, wherein the diseases associated with high fructose intake are selected from the group comprising obesity, non-alcoholic steatohepatitis (NASH), insulin resistance, metabolic syndrome, cardiovascular complications, diabetes, hyperlipidemia, hypertension, inflammation and hyperuricemia.

9. The probiotic of claim 1, wherein the fructoseophlic probiotic is present in the form of inoculum, lyophilized powder, fine powder, tablet, capsule, suspension, solution, emulsion, gel, chewable or edible food and is administered alone or in combination with fructose rich food selected from the group comprising high fructose corn syrup, honey, agave, maple syrup, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereal, fruit juice, coffee creamer, jam and jelly, energy drink, condiment.

10. The probiotic of claim 1, wherein the fructoseophlic probiotic is present in the form of inoculum, lyophilized powder, fine powder, tablet, capsule, suspension, solution, emulsion, gel, chewable or edible food and is administered alone or in combination with fructose rich food selected from the group comprising coconut sugar, palm sugar, ice cream.

11. Use of fructoseophlic probiotic Bacillus coagulans MTCC 25235 in the manufacture of an antibacterial agent for inhibiting pathogenic microorganisms, wherein the pathogenic microorganisms are selected from the group comprising Salmonella abony, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis.

12. A method of producing short chain fatty acids by culturing the fructoseophlic probiotic Bacillus coagulans MTCC 25235 with plant fibers selected from fructose, fenugreek seed fiber, cranberry seed fiber, fructo-oligosaccharides (FOS).

13. The method of claim 12, wherein the short chain fatty acid is selected from the group comprising acetic acid, butyric acid and propionic acid.

14. Use of fructose-philic probiotic Bacillus coagulans isolated from honey for enhancing utilization of fructose from fructose-rich food, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 25235.

15. The use of claim 14, wherein the fructose-philic probiotic is gram-positive.

16. The use of claim 14, wherein the optimum pH and temperature for fructose-philic bacterial growth are 7.5 and 40°C, respectively.

17. The use of claim 14, wherein the fructose-philic probiotic is bile resistant, gastric acid resistant and lactic acid producing.

18. The use of claim 14, wherein the fructose-rich food is selected from the group comprising high fructose corn syrup, honey, agave, maple syrup, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereal, juice, coffee creamer, jam and jelly, energy drink, condiment.

19. The use of claim 14, wherein the fructose-rich food is selected from the group comprising coconut sugar, palm sugar, ice cream.

20. The use of claim 14, wherein the fructose-philic probiotic is present in the form of inoculum, lyophilized powder, fine powder, tablet, capsule, suspension, solution, emulsion, gel, chewable or edible food and is administered alone or in combination with fructose-rich food selected from the group comprising high fructose corn syrup, honey, agave, maple syrup, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereal, juice, coffee creamer, jam and jelly, energy drink, condiment.

21. The use of claim 14, wherein the fructose-philic probiotic is present in the form of inoculum, lyophilized powder, fine powder, tablet, capsule, suspension, solution, emulsion, gel, chewable or edible food and is administered alone or in combination with fructose-rich food selected from the group comprising coconut sugar, palm sugar, ice cream.

22. Use of fructose-philic probiotic Bacillus coagulans MTCC 25235 for inhibiting pathogenic microorganisms in vitro or ex vivo, wherein the pathogenic microorganism is selected from the group comprising Salmonella abony, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis.

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