JP2022536252A - Fructophilic lactic acid-producing bacteria - Google Patents

Abstract

The present invention discloses a novel fructophylic lactic acid-producing bacterium Bacillus coagulans strain FF-7 (MTCC 25235) and methods for isolation and characterization of said bacterium. The present invention also discloses the biological/therapeutic use of fructophylic lactic acid-producing bacteria in increasing the utilization of fructose from food and in managing disorders associated with high fructose intake.

Description

The present invention generally relates to fructophilic lactic acid-producing bacteria. More specifically, the invention relates to the isolation, characterization, and biological applications of the fructophilic probiotic bacterium Bacillus coagulans.

Probiotics and their importance in health and prevention of many disorders have already been reported. Fructophilic lactic acid bacteria (FLAB) are a special group of lactic acid bacteria that utilize fructose as a growth substrate. Due to their unique properties of poor growth on glucose and preference for oxygen, they are considered "unconventional" lactic acid bacteria (LAB). Their abnormal growth properties are due to a defective gene encoding a bifunctional alcohol/acetaldehyde dehydrogenase (adhE). As a result, the NAD/NADH balance is disrupted and additional electron acceptors are required to metabolize glucose. Oxygen, fructose and pyruvate are used as electron acceptors. FLABs have significantly fewer genes for carbohydrate metabolism than other LABs, especially due to the lack of complete phosphotransferase system (PTS) transporters. FLAB was originally classified as Leuconostoc species and subsequently reclassified as Fructobacillus species based on FLAB's phylogenetic position and 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. Evol Microbiol 58:2195-2205).

Fructose metabolism begins with the liver enzyme fructase. The fructose load is converted to lactate in enterocytes and liver. In addition, excess fructose in the liver is directed to peripheral tissues and taken up and converted to fatty acids by the insulin-dependent glucose transporter, GLUT4, present in adipose tissue. GLUT4 has been reported to play an important role in the development of fructose-induced hepatic steatosis and dyslipidemia. In addition, several fructose-induced metabolic disorders have been reported, such as NASH, NAFLD, dyslipidemia, ectopic lipid deposition in 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 increased cardiovascular risk. Increased fructose intake can also cause lactic acidosis by excessively lowering blood pH.

Probiotics play an important role in intestinal fructose metabolism. Low doses of orally ingested fructose reach the intestine and are metabolized in the presence of various enzymes and indigenous microflora. However, in the presence of high doses of fructose, the fructose load spills from the intestine to the liver. Probiotics play an important role in converting fructose to SCFAs (short chain fatty acids) to reduce fructose spillover from the gut to the liver.
FLAB can be found in fruits and vegetables, human faecal cultures, natural antimicrobials, cheese, kefir grains, dairy and non-dairy products, fermented and raw milk, faeces of nursing infants, lactating milk, sheep, Buffalo milk and milk, yoghurt, beverages, poultry sources, animal rumen contents, Pengging Duck cecum, chicken intestine and fecal samples, chicken feed, enzymes, fermented rice, curds, meat extracts and yeast extracts, glucose and sucrose, human gastrointestinal tract, human colony epithelial cells, human and animal vaginal and oral extracts, human infant diapers, pineapple waste, industrial sausages, ice cream, piglet small intestine , corn slurries, crops and intensinal ducks. The isolation of different FLABs is described in the following prior art documents.

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

Given that 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, "Current evidence suggests that probiotic effects are strain-specific. Strain identification is important to associate strains with specific health effects, as well as to enable accurate surveillance and epidemiological studies.”), biological There remains a need to find superior fructophilic probiotic bacteria with improved function. The present invention solves the above problems by disclosing a novel fructophylic lactic acid bacterium, Bacillus coagulans MTCC 25235.

The main object of the present invention is to disclose a novel fructophylic lactic acid-producing bacterium Bacillus coagulans MTCC 25235 and its isolation method.
Another object of the present invention is to disclose the production of short chain fatty acids by the fructophilic lactic acid producing bacterium Bacillus coagulans MTCC 25235.
Yet another object of the present invention is to disclose the antibacterial effect of the fructophylic lactic acid-producing bacterium Bacillus coagulans MTCC 25235.
The present invention solves the above objectives and provides further related advantages.
Deposit of Biological Material The deposit of the biological material Bacillus coagulans strain FF7 having accession number MTCC 25235, referred to in this application, was made on February 28, 2019 at CSIR-Institute of Microbiological Technology, Sector 39-A. , Chandigarh-160036, India, Microbial Type Culture Collection & Gene Bank (MTCC).

The present invention discloses fructophylic lactic acid-producing bacteria. The present invention further discloses methods of isolation, characterization, and biological/therapeutic use of fructophylic lactic acid-producing bacteria.

Other features and advantages of the invention will become apparent from the following more detailed description, which illustrates, by way of example, the principles of the invention.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Phase-contrast microscopy images of Bacillus coagulans MTCC 25235 (1000x magnification) (Fig. 1A), wet mounts of spore-bearing cells (Fig. 1B), Gram staining of vegetative cells (Fig. 1C), spore staining of sporulating cells, and (Fig. 1D) Colonies grown on GYE agar plates. Bacillus coagulans FF7, MTCC 25235, and B. coagulans in the presence of fructose and dextrose as carbon sources. FIG. 2 is a graphical representation of a comparison of growth patterns of coagulans ATCC 31284. FIG. Values are means (±SD) from three independent measurements. FIG. 3 is a graphical representation of fructose utilization by Bacillus coagulans MTCC FF7, 25235 after 24 hours of incubation in the presence of medium and fructose-rich foodstuffs (honey, fruit juices). Values are means (±SD) from three independent measurements. FIG. 1 shows a phylogenetic tree based on the sequence of 16S rRNA showing the relative positions of Bacillus coagulans FF7. B. coagulans FF7, MTCC 25235, and B. FIG. 2 is a graphical representation showing the effect of different pH on the growth of coagulans ATCC 31284. FIG. The optimum pH for growth was found to be pH 7.5 and pH 6.5 respectively. Values are means (±SD) from three independent measurements. B. Coagulans MTCC FF7, 25235 and B.I. FIG. 2 is a graphical representation of the effect of different temperatures on the growth of coagulans ATCC 31284. FIG. Values are means (±SD) from three independent measurements. B. after gastric treatment in an in vitro experiment mimicking in vivo conditions. FIG. 4 is a graphical representation showing the effect of GIT harsh conditions on coagulans MTCC 25235 viability. Sterile saline was taken as an untreated control. FIG. 2 shows the BSH activity of Bacillus coagulans MTCC 25235; (FIG. 8A) MRS soft agar supplemented with calf bile (0.3%, w/v) and CaCO3 (0.3%, w/v) was measured, MRS agar without bile salts was negative. Served as a control (Fig. 8B). The cavity area is B.I. suggesting BSH activity of coagulans MTCC 25235. FIG. 2 is a graphical representation of survival of Bacillus coagulans MTCC FF7, 25235 at various pH values (1.5-8.0) in gastric buffer. Values are expressed in Log10 spores/g. Data represent the mean and standard deviation (±SD) of two different experiments performed. B. coagulans FF7, MTCC 25235, and B. FIG. 2 is a graphical representation of the in vitro effect of calf bile salts on the growth of coagulans ATCC 31284. FIG. B. coagulans MTCC 25235 and B.I. An overnight grown fresh culture of coagulans ATCC 31284 was added to calf bile salts (0.1, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9 and 1%, w/v). MRS broth was inoculated with and without (%, w/v). Values are means (±SD) from three independent measurements. B. in the presence of two standardized preparations corresponding to 6×10 ⁹ (preparation 1) and 15×10 ⁹ cfu/g (preparation 2) studied. FIG. 4 is a graphical representation of the production of L-lactic acid by coagulans FF7, MTCC 25235. FIG. Values are means (±SD) from three independent measurements. FIG. 12A is a graphical representation of acetate (FIG. 12A), butyrate (FIG. 12B) and propionate (FIG. 12C) production from Bacillus coagulans MTCC 25235 during fermentation of fructose, FOS, cranberry seed fiber, fenugreek seed fiber. Samples were collected after 4, 8, 12, 18 and 24 hours of fermentation. Values are the mean of 3 replicates performed on 2 different occasions and are expressed in mg/g. B. during storage at 40±2° C. and 60%±5% RH. FIG. 4 is a graphical representation of coagulans MTCC 25235 survival. Two standardized preparations corresponding to 15×10 ⁹ (preparation 1) and 6×10 ⁹ cfu/g (preparation 2) were studied. Mean spore viability is expressed as log10 cfu/g. Each time point represents the mean Log ₁₀ standard deviation (±SD) of three different experiments performed in duplicate.

In a most preferred embodiment, the present invention is a method for isolating and identifying novel fructose-utilizing probiotic bacteria from honey comprising the steps of:
a) mixing honey with saline in a ratio of 1:10 w/v to obtain a suspension;
b) thoroughly mixing the suspension of step a) and subjecting it to heat shock at 50-70° C. for 30 minutes to selectively detach the spores;
c) isolating the bacterial colonies by incubating 1-2 ml of the suspension from step b) for 48 hours at 35-37° C. in a suitable culture medium containing fructose;
d) purifying the bacterial isolate by selecting and incubating morphologically distinct colonies in a suitable medium containing fructose as carbon source;
e) identifying the bacterial strain as Bacillus coagulans strain FF7 having accession number MTCC 25235 by biochemical analysis and 16S rRNA sequencing.

In a related aspect, the honey is raw honey, filtered honey, acacia honey, alfalfa honey, aster honey, avocado honey, basswood honey, beech honey, blueberry honey, bluegum honey, buckwheat honey, clover honey, dandelion honey, eucalyptus honey , fireweed honey, heather honey, ironbark honey, jarrah honey, leatherwood honey, linden honey, macadamia honey, manuka honey, orange blossom honey, pine tree honey, sourwood honey, sage honey, and tupelo honey. is selected from the group that is not limited to In another related aspect, the culture medium is selected from the group comprising MRS (Agar by De Man, Rogosa and Sharpe), GYA (Glucose Yeast Extract Agar), TSB (Tryptone Soy Broth), Sporulation Medium and Mueller Hinton Agar. be done.

In another related embodiment, the isolated probiotic strain is biochemically tested catalase, oxidase, methyl red, Voges proscauel, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melobiose , sucrose, arabinose, mannose, inulin, sodium gluconate, salicin, sorbitol, mannitol, arabitol, methylglucoside, rhamnose, cellobiose, ONPG, aesculin hydrolysis, biochemical test sorbose malonate utilization, citric Negative for acid utilization, xylitol, methylmannoside, melezitose, erythritol, adonitol, inositol, dulcitol, glycerol, hemolysis, citrate and indole.

In another preferred embodiment, the present invention discloses a novel probiotic bacterium of the genus Bacillus isolated from honey to increase fructose utilization from fructose-rich foods. In another related aspect, the fructophilic probiotic bacterium is Gram positive. In yet another aspect, the optimum pH and temperature recorded for growth of fructophilic bacteria are 7.5 and 40° C., respectively. In another aspect, the fructophilic probiotic bacterium is bile-tolerant, acid-resistant, and produces lactic acid. In a related aspect, the fructophilic probiotic bacterium is Bacillus coagulans. In yet another related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 25235. In related embodiments, the fructose-rich food product is high fructose corn syrup, honey, agave, maple syrup, coconut sugar, palm sugar, molasses, soda, candy, sweetened yogurt, frozen food, canned food, cereals, fruit juices, coffee. Selected from the group comprising creamers, jams and jellies, energy drinks, condiments, ice creams. In a related aspect, fructophilic probiotic bacteria are used in the therapeutic management of disorders associated with high fructose intake. In a related aspect, diseases associated with high fructose intake include obesity, nonalcoholic steatohepatitis (NASH), insulin resistance, metabolic syndrome, cardiovascular complications, diabetes, hyperlipidemia, hypertension, inflammation and hypertension. Selected from the group including but not limited to uricemia. In another related aspect, the fructophilic probiotic bacterium is in the form of an inoculum, lyophilized powder, micronized powder, tablet, capsule, suspension, solution, emulsion, gummy, chewable or edible, Alone or High Fructose Corn Syrup, Honey, Agave, Maple Syrup, Coconut Sugar, Palm Sugar, Molasses, Soda, Candies, Sweetened Yogurt, Frozen Foods, Canned Foods, Cereals, Fruit Juices, Coffee Creamers, Jams and Jellies, Energy Administered in combination with a fructose-rich food selected from the group comprising drinks, condiments and ice creams.

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

In another preferred embodiment, the present invention provides a method for producing short chain fatty acids, comprising fructose, fenugreek seed fiber, cranberry seed fiber, fructo-oligosaccharides (FOS) together with vegetable fiber selected from the group consisting of A method is disclosed by culturing the philic probiotic bacterium Bacillus coagulans MTCC 25235.

The specific examples included herein below demonstrate the foregoing most preferred embodiments of the invention.

Example 1 Isolation and Identification of Fructophilic Bacteria Methods In this study, unfiltered raw honey was used to isolate spore-forming fructophilic lactic acid bacteria. In this study, MRS (de Man, Rogosa and Sharpe) (dextrose replaced by fructose) was used for the isolation of fructophilic bacteria (Table 1).

１ｇｍの未ろ過の生蜂蜜を１０ｍｌの生理食塩水を含む試験管に入れた。これをよく混合して、胞子の選択的単離のため、７０℃で３０分間熱ショックを与えた。細菌の単離は、上記の試料１ｍｌを各希釈液からフルクトースを含むＭＲＳ寒天プレートへ加えることによって行った。プレートをさらに３７℃で４８時間インキュベートした。インキュベーション後、細菌単離株のさらなる試験と精製のために、形態学的に異なるコロニーをピックアップした。バチルス・コアグランスＭＴＣＣ ２５２３５を単離し、フルクトースを炭素源として含む他のＭＲＳ寒天培地プレートにストリークした。

1 gm of unfiltered raw honey was placed in a test tube containing 10 ml of saline. This was mixed well and heat shocked at 70°C for 30 minutes for selective isolation of spores. Bacterial isolation was performed by adding 1 ml of the above samples from each dilution to MRS agar plates containing fructose. Plates were further incubated at 37°C for 48 hours. After incubation, morphologically distinct colonies were picked for further testing and purification of bacterial isolates. Bacillus coagulans MTCC 25235 was isolated and streaked onto other MRS agar plates containing fructose as carbon source.

Biochemical Characterization of Fructophilic Bacteria Bacterial isolates were grown on MRSA (agar by de Man, Rogosa and Sharpe) at 37° C. for 24 hours. Bacterial inoculum was prepared by picking 1-3 well-isolated colonies and preparing a uniform suspension in sterile saline. The concentration of the suspension was ≧0.5 OD at 620 nm. This inoculum (50 μl) was used to perform the test according to the kit manufacturer's instructions (HiMedia, Mumbai, India). Bacillus coagulans MTCC 25235 and B. Biochemical characterization of coagulans ATCC 31284 was carried out as described (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 HiCarbohydrate™ Kit (Code-KB009) was procured from HiMedia, Mumbai, India and the test was performed according to the manufacturer's instructions. Bacillus coagulans MTCC 25235 and B. A bacterial suspension of coagulans ATCC 31284 was prepared as described in the section above. Furthermore, B. The IMViC (Indole, Methyl Red, Voges-Proscawell, Citrate Utilization) test, oxidase and Gram staining of Coaglance ATCC 31280, Majeed et al. (2016); (Cappucino G and Natalie Sherman. Microbiology: A Laboratory manual 5th edition 1998).

16S rDNA sequencingB. Genomic DNA for 16S rDNA sequencing of coagulans MTCC 5856 was obtained from (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). Fragments of the 16S rDNA gene were sequenced using the ABI 3500 Gene Analyzer Automated DNA Sequencer as previously described (Heyrman and Swings 2001). The sequencing primers used were 5-AGHGTBTGHTCMTGNCTCAS-3 (forward primer) and 5-TRCGGYTMCCTTGTWHCGACTH-3 (reverse primer). Amplified DNA fragments of approximately 1.5 kb were separated on a 1% agarose gel and purified by using Qiagen spin columns. Purified fragments were used directly for DNA sequencing. This sequence was used in a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Growth Conditions for Bacillus coagulans MTCC 25235 Optimization of the growth conditions for Bacillus coagulans MTCC 25235 was analyzed for different temperatures and pHs. MRSB medium was prepared and adjusted to pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 with 2N HCl and 2N NaOH. Overnight growth broth (1%, v/v) was inoculated into pH adjusted medium and incubated for 24 hours at 120 rpm in a shaking incubator. Growth was monitored at 6 hour intervals by measuring absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan). MRSB medium was prepared with pH adjusted to 6.5 and inoculated with overnight growth culture (1%, v/v). In addition, flasks were incubated at different temperatures (20, 30, 37, 40, 50 and 60°C) for 24 hours at 120 rpm in a shaking incubator. Growth was monitored at 6 hour intervals by measuring absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Results Identification of Fructophilic Bacteria Bacillus coagulans MTCC 25235 spores were elliptical, concentric spores (Fig. 1A), and vegetative cells were Gram-positive, rod-shaped as shown by Gram staining (Fig. 1B). . B. Colonies of coagulans MTCC 25235 were grown on the medium to give uniform, 1-3 mm diameter, white to cream smooth colonies containing vegetative rod cells (FIGS. 1C and D). Growth of the isolated bacteria was better on fructose-rich media than on dextrose-rich media (Fig. 2), which is due to their fructophilic nature when compared to other Bacillus coagulans strains. and the possibility of fructose utilization. Growth of this bacterium was also tested on fructose-rich foods, which showed a decrease in fructose content (Fig. 3), indicating that the bacterium utilizes fructose as a carbon source for growth and growth. implying that. Thus, this bacterium can be used in the management and prevention of disorders associated with high fructose intake by utilizing excess fructose in food for its metabolism. This bacterium can be found alone or in 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 creamers, and jams. and fructose-rich foods, including jellies, energy drinks, condiments, ice cream (Braverman J, List of Foods high in fructose, https://www.livestrong.com/article/30454- list-foods-high-fructose/, viewed May 3, 2019)

Biochemical characterization Bacillus coagulans MTCC 25235 and B. Biochemical characterization of coagulans ATCC 31284 was performed by the method described (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 analyzed using commercially available B. Table 2 shows the comparison with the coagulans ATTCC 3128 strain.

The results show that the isolated probiotic strains are biochemically tested catalase, oxidase, methyl red, Voges-Proscauel, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, merobiose, sucrose, merobiose, Positive for arabinose, mannose, inulin, sodium gluconate, salicin, sorbitol, mannitol, arabitol, methylglucoside, rhamnose, cellobiose, ONPG, aesculin hydrolysis, biochemical tests sorbose malonate utilization, citrate utilization, xylitol , methylmannoside, melezitose, erythritol, adonitol, inositol, dulcitol, glycerol, hemolysis, citrate and indole.

16S rDNA Sequencing Bacterial 16S rDNA was sequenced to obtain sequence information (SEQ ID NO) as follows:

locus seq_FF7 1437 bp
starting point
1 ACTTGCAAGT CGTGCGGCCC TTTTTTAAAA GCTTGCTTTT TAAAAGGTTA GCGGCGGACG
61 GGTGAGTAAC ACGTGGGCAC CCTGCCTGTA AGATCGGGAT AACGCCGGGA AACCGGGGCT
121 AATACCGGAT AGTTTTTTCC TCCGCATGGA GGAAAAAGGA AAGACGGCTT CTGCTGTCAC
181 TTACAGATGG GCCCGCGGCG CATTAGCTAG TTGGTGGGGT AACGGCTCAC CAAGGCAACG
241 ATGCGTAGCC GACCTGAGAG GGTGATCGGC CACATTGGGA CTGAGACACG GCCCAAACTC
301 CTACGGGAGG CAGCAGTAGG GAATCTTCCG CAATGGACGA AAGTCTGACGGAGCAACGCC
361 GCGTGAGTGA AGAAGGCCTT CGGGTCGTAA AACTCTGTTG CCGGGGAAGA ACAAGTGCCG
421 TTCGAACAGG GCGGCGCCTT GACGGTACCC GGCCAGAAAG CCACGGCTAA CTACGTGCCA
481 GCAGCCGCGG TAATACGTAG GTGGCAAGCG TTGTCCGGAA TTATTGGGCG TAAAGCGCGC
541 GCAGGCGGCT TCTTAAGTCT GATGTGAAAT CTTTGCGGGC TCACCCGCAA GCGGTCATTG
601 GAAACTGGGA GGGCTTTGAG TGCAAGAAAG AGGAGAGTGG AATTTCCACG TGTAGCGGTG
661 AAATGCGTAA AGATGTGGAG GAACACCAGT GGCGAAGGCG GCTCTCTGGT CTGTAACTGA
721 CGCTGAGGCG CGAAAGCGTG GGGAGCAAAC AGGATTAGAT ACCCTGGTAG TCCACGCCGT
781 AAACGATGAG TGCTAAGTGT TAGAGGGTTT CCGCCCTTTA GTGCTGCAGC TAACGCATTA
841 AGCACTCCGC CTGGGGAGTA CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC
901 GCACAAGCGG TGGAGCATGT GGTTTAATTC GAAGCAACGC GAAGAACCTT ACCAGGTCTT
961 GACATCCTCT GACCTCCCTG GAGACAGGGC CTTCCCCTTC GGGGGACAGA GTGACAGGTG
1021 GTGCATGGTT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT TAAGTCCCGC AACGAGCGCA
1081 ACCCTTGACC TTAGTTGCCA GCATTCAGTT GGGCACTCTA AGGTGACTGC CGGTGACAAA
1141 CCGGAGGAAG GTGGGGATGA CGTAAATCA TCATGCCCCT TATGACCTGG GCTACACACG
1201 TGCTACAATG GATGGTACAA AGGGCTGCGA GACCGCGAGG TTAAGCCAAT CCCAGAAAAC
1261 CATTCCCAGT TCGGATTGCA GGCTGCAACC CGCCTGCATG AAGCCGGAAT CGCTAGTAAT
1321 CGCGGATCAG CATGCCGCGG TGAATACGTT CCCGGGCCTT GTACACACCG CCCGTCACAC
1381 CACGAGAGTT TGTAACACCC GAAGTCGGTG AGGTAACCTT ACGGAGCCAG CCGCCGA
//
A BLAST (Basic local alignment search tool) search was performed with the above sequences and the results of the first ten aligned sequences are shown in Table 3.

この結果は、単離された生物がバチルス・コアグランス菌株５５－ＬＲ４と９８．９６％の同一性を有するバチルス・コアグランスの新しい菌株であることを示した。系統発生分析の結果も、生物がバチルス・コアグランスの新しい菌株であることを示した（図４）。

The results indicated that the isolated organism was a new strain of Bacillus coagulans with 98.96% identity to Bacillus coagulans strain 55-LR4. Phylogenetic analysis results also indicated that the organism was a new strain of Bacillus coagulans (Fig. 4).

Optimal Growth Conditions for Fructophilic Bacteria The optimal pH and temperature recorded for the growth of fructophilic bacteria were 7.5 and 40° C., respectively (FIGS. 5 and 6).

Example 2: In vitro probiotic evaluation of Bacillus coagulans MTCC 25235 Resistance to gastric acid For viability of Bacillus coagulans MTCC 25235 spores, 1 ml of the suspension was mixed with 0.01% lysozyme (Sigma-Aldrich) and was added to 100 ml of sterile electrolyte solution (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2, and 1.2 g/L NaHCO3) containing 0.3% pepsin (Sigma-Aldrich). was studied by incubating for 5 min. Additionally, the pH was adjusted to 1.5, 3, 4, 5, 6, 7 and 8 (adjusted using 1N NaOH and 1N HCl). Incubation temperature was monitored at 37° C. for 4 hours. 1 ml samples were taken at different time intervals of 0, 1.0, 2.0, 3.0 and 4.0 hours. After incubation, serial dilutions were made in sterile saline (0.89% w/v) and viable counts were counted by plating on glucose yeast extract agar (HiMedia). Experiments were performed in triplicate on two different occasions.

Bile Salt Tolerance Bile salt tolerance of Bacillus coagulans MTCC 25235 cells was determined by previously reported methods (Gilliland et al. 1984; Hyronimus et al. 2000). MRS broth (HiMedia) was inoculated with approximately 10 ⁶ cfu mL ⁻¹ of Bacillus coagulans MTCC 25235 overnight growth medium followed by (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) supplemented with bile salts and experimental controls without supplemented bile salts. and Samples were incubated for 24 hours at 37°C with shaking at 120 rpm. The growth of control (no bile) and test cultures with different concentrations of bile was monitored hourly by measuring absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Lactic Acid Production Lactic acid production by Bacillus coagulans MTCC 25235 was estimated using the Megazyme kit. One loopful of overnight growing culture of Bacillus coagulans MTCC 25235 was added to glucose yeast extract broth (HiMedia) and incubated at 37° C. for 18 hours at 120 rpm. After incubation, the broth was filtered through 0.22μ (Sartorius, India) and analyzed for lactate content using the Megazyme kit (K-DLATE 10/04) according to the manufacturer's instructions (Megazyme International Ireland, IDA Business Park, Wicklow, Ireland). GYE medium was used as a blank in the assay.

Artificial Gastric Juice Resistance To demonstrate oral digestion conditions, survival of Bacillus coagulans MTCC 25235 spores was studied. 1 mL of Bacillus coagulans MTCC 25235 suspension, adjusted to pH 6.8±0.2, KCl (0.8946 g/l), CO(NH ₂ ) ₂ (0.1981 g/L), Na ₂ SO An artificial saliva content consisting of ₄ (0.5681 g/L), NaHCO ₃ (1.680 g/L), NaH ₂ PO ₄ (0.8878 g/L) was applied and incubated at 37° C. for 5 minutes. In addition, simulated gastric fluid was prepared by adding 9 g/L sodium chloride and 3 g/L pepsin (Sigma-Aldrich, St. Louis, Mo., USA), after which the pH was aseptically adjusted using 2N HCl. Adjusted to 3.0±0.2. Samples were incubated at 37° C. for 3 hours at low r.m. p. m. was further incubated with After 3 hours of incubation, the pH was aseptically adjusted to 7.0 using 2N NaOH. Ox bile (5 g/L) was further incubated at 37° C. for 24 hours. After the final step of the artificial digestion process, samples were collected and evaluated for spore survival of Bacillus coagulans MTCC 25235. Enumerations were performed by serial dilution using glucose yeast extract agar (HiMedia) (Fig. 7).

Antibiotic Resistance Patterns MICs were determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2012). Bacillus coagulans MTCC 25235 suspension was prepared by suspending an 18 hour growing bacterial culture in sterile normal saline (0.89% NaCl wt/vol; Himedia, Mumbai India). The turbidity of the bacterial suspension was adjusted to a McFarland standard solution of 0.5 (equivalent to 1.5×10 ⁸ colony forming units (CFU)/ml). Antibiotic stock solutions were prepared according to CLSI guidelines and two-fold serial dilutions were made in 96-well U-bottom microtiter plates (BD Labware, NJ USA) in a volume of 100 μl in broth medium (glucose yeast extract acetate broth [Bacillus Coagulans MTCC 25235 was prepared in GYEA, HiMedia, Mumbai India, Staphylococcus aureus in Mueller Hinton Broth [MHB, Difco Laboratories, Detroit, Mich USA]). The bacterial suspension described above was further diluted in MHB and a 100 μl volume of this diluted inoculum was added to each well of the plate for a final inoculum of 5×10 ⁵ CFU/ml in the well and Final concentrations of substances ranged from 0.0078 to 4 μg/ml. In this study, S. Aureus ATCC 6538 was used as a reference culture. Plates were incubated at 37° C. for 24 hours and visually inspected for turbidity. The lowest compound concentration that did not show turbidity was recorded as the MIC.

AMES Testing Experimental data were obtained from B.E. Coagulans MTCC 25235 spores did not increase the number of revertants of five Salmonella strains (TA98, TA100, TA102, TA1535 and TA1537) compared to negative controls with or without S9 metabolic activation. suggested that Furthermore, B. Coagulans MTCC 25235 spores (up to 5000 μg/plate) produced no dose-dependent mutagenic effects. B. Coagulans MTC 25235 spores showed no mutagenic activity under the experimental conditions.

BSH Activity Growth of Bacillus coagulans MTCC 25235 was observed on agar plates containing calf bile and calcium carbonate, indicating resistance to calf bile and the presence of BSH activity. As shown in (FIGS. 8A and 8B), the soft agar plates had clear areas of 19±2 mm, indicating the presence of BSH activity.

Antimicrobial activity against human pathogens Antimicrobial activity was performed by a previously described well diffusion assay 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 ⁶ cfu mL ⁻¹ of indicator strains (S. epidermidis ATCC 14990, S. mutans MTCC 1943, S. aureus ATCC 29213, B. cereus ATCC 14579, P. acnes ATCC 11827, E. coli ATCC 25922, P. aeruginosa ATCC 9027, M. luteus NCIM 216 and S. abonii NCIM 2257) were mixed with 5 mL of a lawn of soft (0.7% agar) glucose yeast extract (HiMedia, India) with enhanced hardness (1.5 % agar) was poured on top of tryptic soy agar (HiMedia, India). 1 platinum loop of overnight growth mediumB. Coaglans MTCC 25325 was added to the MRS medium and incubated at 37° C. for 24 hours at 120 rpm. After 24 h, the culture was centrifuged (10,000 x 9 g) to remove cells, the supernatant collected, concentrated 10-fold by lyophilization and passed through a 0.22μ filter (Sartorius, India). Filter sterilized. Concentrated supernatant (50 μL) was added to 6 mm wells punched in solidified double layer agar. Plates were stored in a refrigerator (4±2° C.) for 5 hours and samples were allowed to spread in agar before incubation at 37° C. for 18-20 hours. After incubation, the zone of inhibition was measured and recorded in mm.

Production of Short Chain Fatty Acids In vitro fermentation with Bacillus coagulans MTCC 25235 was performed by the following method with some modifications to the method described by McBurney and Thompson (1987). Briefly, 2.0 g fructose, fenugreek seeds, cranberry seed fiber, FOS were added to 100 ml deionized water. The pH was adjusted to 7.0±0.2 and autoclaved at 121° C. for 20 minutes. After sterilization, oxygen reductase oxylase (Oxyrase® for Broth, Oxyrase, Inc, OH, USA) was added to each flask to induce anaerobic conditions. All flasks were inoculated with 5% overnight growing Bacillus coagulans MTCC 25235 culture and incubated at 37° C. with gentle shaking rpm for 24 hours. The bottles were tightly closed and sealed with parafilm to maintain the anaerobic conditions created by enzyme supplementation. The pH values at 0 hours of incubation and after fermentation (24 hours) were also recorded. 1 ml of copper sulfate (10 g/L) was added to each sample 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 the pH was adjusted to 1.5 using 3M _H2SO4 . 10 ml of cold diethyl ether (-20°C) was added to the sample and vortexed for 1 minute. Sodium chloride was added and centrifuged at 3000 xg for 10 minutes. After centrifugation, the organic layer was separated and transferred to a new vial. This was used to quantify SCFA. SCFA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and treated similarly. SCFA production (acetate, propionate and butyrate) was gas chromatographed using an Agilent technologies 6890N gas chromatograph (Stevens Creek Blvd Santa Clara, CA, USA) containing a DB-FFAP (terephthalic acid modified polyethylene glycol) column. Measured by lithography (GC). The column temperature was 200°C. The injector and detector port temperatures were 250°C. The carrier gas was _N2 at a flow rate of 1.0 ml/min. SCFA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). SCFA (acetate, propionate and butyrate) concentrations were expressed in mg/g galactomannan from fenugreek seeds.

Results Resistance to Gastric Acid There was no significant difference (2-5%) in spore counts from pH 3 to pH 8.0 compared to initial spore counts in studies up to 4 hours (FIG. 9). However, at pH 1.5, 0.44 and 2.036 log ₁₀ reductions were observed at 1 and 4 hours, respectively. The results of the study confirmed the stability of Bacillus coagulans MTCC 25235 spores in acidic as well as alkaline pH conditions.

Bile Tolerance Test Growth of Bacillus coagulans MTCC 25235 was observed on agar plates containing bile salts (1% w/v), demonstrating tolerance to bile salts. In addition, a bile tolerance assay was performed by supplementing the MRS broth with bovine bile (0.1-2.0%) in different flasks. Growth of Bacillus coagulans MTCC 25235 was observed in the presence and absence of up to 2% w/v calf bile. On the other hand, B. Growth of coagulans ATCC 31284 was seen at 0.8% w/v (Figure 10). Similarly, in the presence and absence of bile salts, Bacillus coagulans MTCC 25235 and B. There was no significant difference in coagulans ATCC 31284 survival.
Lactic Acid Production Lactic acid production by Bacillus coagulans MTCC 25235 was estimated using the Megazyme kit. The total amount of lactic acid produced by Bacillus coagulans MTCC 25235 was 4.487 g/L. L-lactic acid was 4.12 g/L. On the other hand, D-lactic acid by Bacillus coagulans MTCC 25235 is 0.367 g/L (Fig. 11).

Antibiotic resistance Clindamycin, kanamycin, ampicillin, streptomycin, vancomycin, erythromycin, gentamicin, tetracycline and chloramphenicol, Bacillus coagulans MTCC 25235 and S. The MIC results for Aureus ATCC 6538 are shown in Table 4. All antibiotics tested showed an MIC range of 0.0078-1.0 μg/ml against Bacillus coagulans MTCC 25235. All tested antibiotics are S. It exhibited an MIC range of 0.031-2 μg/ml for Aureus ATCC 6538.

抗菌活性
Ｂ．コアグランスＭＴＣＣ ２５２３５の抗菌活性を評価した。抗菌活性は、Ｃｉｎｔａｓら（１９９５）に記載されるように、ウェル拡散アッセイによって決定した。結果は、プロバイオティクス細菌がサルモネラ・アボニー、ミクロコッカス・ルテウス、大腸菌、シュードモナス・エルギノーサ、バチルス・セレウス、プロピオニバクテリウム・アクネス、ストレプトコッカス・ミュータンス、スタフィロコッカス・アウレウス、スタフィロコッカス・エピダーミディスに対して効果的な抗菌剤であることを示した。

Antibacterial activity B. The antibacterial activity of coagulans MTCC 25235 was evaluated. Antimicrobial activity was determined by a well diffusion assay as described by Cintas et al. (1995). The results showed that the probiotic bacteria were Salmonella avonii, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, and Staphylococcus epidermidis. was shown to be an effective antibacterial agent against

データは、3反復で実行された3回の独立した実験の平均±SDを表す

Data represent the mean ± SD of three independent experiments performed in triplicate

Production of Short Chain Fatty Acids The results of the analysis are shown in Figures 12A, 12B and 12C. The production of acetic acid has been reported in B. FOS was high for coagulans MTCC 25235, followed by fenugreek seed fiber, cranberry fiber and fructose (Figure 12A). Similarly, the production of butyrate is associated with B. It was high in fructose for coagulans MTCC 25235, followed by FOS, fenugreek seed fiber and cranberry fiber (Figure 12B). Propionate production was confirmed by B. culturing with fructose, FOS and cranberry seed fiber. Similar for coagulans MTCC 25235, but lower for fenugreek seed fiber (Fig. 12C).
Storage and Viability Two standardized preparations corresponding to 15×10 ⁹ (preparation 1) and 6×10 ⁹ cfu/g (preparation 2) were studied. B. Coaglans MTCC 25235 showed improved viability during storage at 40±2° C. and 60%±5% RH (FIG. 13).
Other modifications and variations to the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Therefore, while only certain embodiments of the invention have been specifically described herein, it will be apparent that many changes can be made thereto without departing from the spirit and scope of the invention. deaf. The scope of the invention should be interpreted only in conjunction with the appended claims.

Production of Short Chain Fatty Acids The results of the analysis are shown in Figures 12A, 12B and 12C. The production of acetic acid has been reported in B. FOS was high for coagulans MTCC 25235, followed by fenugreek seed fiber, cranberry fiber and fructose (Figure 12A). Similarly, the production of butyrate is associated with B. It was high in fructose for coagulans MTCC 25235, followed by FOS, fenugreek seed fiber and cranberry fiber (Figure 12B). Propionate production was confirmed by B. culturing with fructose, FOS and cranberry seed fiber. Similar for coagulans MTCC 25235, but lower for fenugreek seed fiber (Fig. 12C).
Storage and Viability Two standardized preparations corresponding to 15×10 ⁹ (preparation 1) and 6×10 ⁹ cfu/g (preparation 2) were studied. B. Coaglans MTCC 25235 showed improved viability during storage at 40±2° C. and 60%±5% RH (FIG. 13).
Other modifications and variations to the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Therefore, while only certain embodiments of the invention have been specifically described herein, it will be apparent that many changes can be made thereto without departing from the spirit and scope of the invention. deaf. The scope of the invention should be interpreted only in conjunction with the appended claims.
Another aspect of the present invention may be as follows.
[1] A method for isolating and identifying novel fructose-utilizing probiotic bacteria from honey, comprising:
f) mixing honey with saline in a ratio of 1:10 w/v to obtain a suspension;
g) thoroughly mixing the suspension of step a) and subjecting it to heat shock at 50-70° C. for 30 minutes to selectively detach the spores;
h) isolating the bacterial colonies by incubating 1-2 ml of the suspension from step b) for 48 hours at 35-37° C. in a suitable culture medium containing fructose;
i) purifying the bacterial isolate by selecting and incubating morphologically distinct colonies in a suitable medium containing fructose as carbon source;
j) identifying the bacterial strain as Bacillus coagulans strain FF7 with accession number MTCC 25235 by biochemical analysis and 16S rRNA sequencing;
A method, including
[2] Honey includes raw honey, filtered honey, acacia honey, alfalfa honey, aster honey, avocado honey, basswood honey, beech honey, blueberry honey, bluegum honey, buckwheat honey, clover honey, dandelion honey, eucalyptus honey, fire selected from the group comprising weed honey, heather honey, ironbark honey, jarrah honey, leatherwood honey, linden honey, macadamia honey, manuka honey, orange blossom honey, pine tree honey, sourwood honey, sage honey, and tupelo honey; The method according to [1] above.
[3] the culture medium is selected from the group comprising MRS (agar by De Man, Rogosa and Sharpe), GYA (glucose yeast extract agar), TSB (tryptone soy broth), sporulation medium and Mueller Hinton agar; The method according to [1].
[4] the isolated probiotic strain is biochemically tested catalase, oxidase, methyl red, Voges proscauel, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, merobiose, sucrose, arabinose , mannose, inulin, sodium gluconate, salicin, sorbitol, mannitol, arabitol, methylglucoside, rhamnose, cellobiose, ONPG, positive for aesculin hydrolysis, biochemical tests sorbose malonate utilization, citrate utilization, The method of [1] above, wherein xylitol, methylmannoside, melezitose, erythritol, adonitol, inositol, dulcitol, glycerol, hemolysis, citrate and indole are negative.
[5] A fructophilic probiotic bacterium of the genus Bacillus isolated from honey to increase fructose utilization from fructose-rich foods.
[6] The probiotic bacterium of [5] above, which is Gram-positive.
[7] The probiotic bacterium according to [5] above, wherein the optimum pH and temperature for growth of the fructophilic bacterium are 7.5 and 40°C, respectively.
[8] The probiotic bacterium of [5] above, which is resistant to bile and gastric acid and produces lactic acid.
[9] The probiotic bacterium of [5] above, which is Bacillus coagulans.
[10] The probiotic bacterium of [5] above, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 25235.
[11] The fructose-rich food is high fructose corn syrup, honey, agave, maple syrup, coconut sugar, palm sugar, molasses, soda, candy, sweet yogurt, frozen food, canned food, cereal, fruit juice, coffee creamer, The probiotic bacterium according to [5] above, which is selected from the group including jams and jellies, energy drinks, seasonings and ice creams.
[12] The probiotic bacterium of [5] above, which is used for therapeutic management of a disorder associated with high fructose intake.
[13] selected from the group comprising obesity, non-alcoholic steatohepatitis (NASH), insulin resistance, metabolic syndrome, cardiovascular complications, diabetes, hyperlipidemia, hypertension, inflammation and hyperuricemia; Disorders associated with high fructose intake according to [12] above.
[14] the fructophilic probiotic bacterium is present in the form of an inoculum, freeze-dried powder, fine powder, tablet, capsule, suspension, solution, emulsion, gummy, chewable or edible food, and or 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 creamers, jams and jellies, energy drinks, condiments The probiotic bacterium according to [5] above, which is administered in combination with a fructose-rich food selected from the group including food, ice cream.
[15] A method of inhibiting a pathogenic microorganism comprising the step of contacting said microorganism with the fructophilic probiotic bacterium Bacillus coagulans MTCC 25235.
[16] The pathogenic microorganism is Salmonella abony, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacteriaceae Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, selected from the group including Staphylococcus epidermidis [15] above [15] the method of.
[17] A method for producing short-chain fatty acids, comprising fructophilic probiotic bacterium Bacillus coagulans with a plant fiber selected from the group consisting of fructose, fenugreek seed fiber, cranberry seed fiber, fructo-oligosaccharides (FOS) A method by culturing MTCC 25235.
[18] The method of [17] above, wherein the short-chain fatty acid is selected from the group containing acetic acid, butyric acid and propionic acid.

Claims (18)

A method for isolating and identifying novel fructose-utilizing probiotic bacteria from honey, comprising:
f) mixing honey with saline in a ratio of 1:10 w/v to obtain a suspension;
g) thoroughly mixing the suspension of step a) and subjecting it to heat shock at 50-70° C. for 30 minutes to selectively detach the spores;
h) isolating the bacterial colonies by incubating 1-2 ml of the suspension from step b) for 48 hours at 35-37° C. in a suitable culture medium containing fructose;
i) purifying the bacterial isolate by selecting and incubating morphologically distinct colonies in a suitable medium containing fructose as carbon source;
j) identifying the bacterial strain as Bacillus coagulans strain FF7 having accession number MTCC 25235 by biochemical analysis and 16S rRNA sequencing. Raw honey, filtered honey, acacia honey, alfalfa honey, aster honey, avocado honey, basswood honey, beech honey, blueberry honey, bluegum honey, buckwheat honey, clover honey, dandelion honey, eucalyptus honey, fireweed honey, Claims selected from the group comprising heather honey, iron bark honey, jarrah honey, leatherwood honey, linden honey, macadamia honey, manuka honey, orange blossom honey, pine tree honey, sourwood honey, sage honey, and tupelo honey Item 1. The method according to item 1. 2. The method of claim 1, wherein the culture medium is selected from the group comprising MRS (Agar by De Man, Rogosa and Sharpe), GYA (Glucose Yeast Extract Agar), TSB (Tryptone Soy Broth), Sporulation Medium and Mueller Hinton Agar. described method. The isolated probiotic strain is characterized by biochemical tests catalase, oxidase, methyl red, Voges proscauel, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, merobiose, sucrose, arabinose, mannose, Inulin, sodium gluconate, salicin, sorbitol, mannitol, arabitol, methylglucoside, rhamnose, cellobiose, ONPG, positive for aesculin hydrolysis, biochemical tests sorbose malonate utilization, citrate utilization, xylitol, methylmannoside , melezitose, erythritol, adonitol, inositol, dulcitol, glycerol, hemolysis, citrate and indole. A fructophilic probiotic bacterium of the genus Bacillus isolated from honey to increase fructose utilization from fructose-rich foods. 6. A probiotic bacterium according to claim 5, which is Gram-positive. 6. Probiotic bacteria according to claim 5, wherein the optimum pH and temperature for growth of fructophilic bacteria are 7.5 and 40<0>C respectively. 6. The probiotic bacterium of claim 5, which is bile-tolerant, gastric-acid-resistant and produces lactic acid. 6. A probiotic bacterium according to claim 5, which is Bacillus coagulans. 6. The probiotic bacterium of claim 5, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 25235. Fructose rich foods include 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 creamers, jams and jellies. , energy drinks, condiments, ice cream. 6. A probiotic bacterium according to claim 5 for use in the therapeutic management of disorders associated with high fructose intake. 12. Selected from the group comprising obesity, non-alcoholic steatohepatitis (NASH), insulin resistance, metabolic syndrome, cardiovascular complications, diabetes, hyperlipidemia, hypertension, inflammation and hyperuricemia. Disorders associated with high fructose intake as described in . Said fructophilic probiotic bacteria are present in the form of an inoculum, lyophilized powder, micronized powder, tablet, capsule, suspension, solution, emulsion, gummy, chewable or edible food, either alone or high fructose Corn Syrup, Honey, Agave, Maple Syrup, Coconut Sugar, Palm Sugar, Molasses, Sodas, Candies, Sweetened Yogurt, Frozen Foods, Canned Foods, Cereals, Fruit Juices, Coffee Creamers, Jams and Jellies, Energy Drinks, Condiments, Ice 6. A probiotic bacterium according to claim 5, administered in combination with a fructose-rich food product selected from the group comprising creams. A method of inhibiting pathogenic microorganisms comprising contacting said microorganisms with the fructophilic probiotic bacterium Bacillus coagulans MTCC 25235. The pathogenic microorganism is Salmonella abony, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes 16. The method of claim 15 selected from the group comprising Propionibacterium acnes, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis. A method of producing short chain fatty acids, comprising the use of the fructophilic probiotic bacterium Bacillus coagulans MTCC 25235 with a plant fiber selected from the group consisting of fructose, fenugreek seed fiber, cranberry seed fiber, fructo-oligosaccharides (FOS). A method by culturing. 18. The method of claim 17, wherein the short chain fatty acid is selected from the group comprising acetic acid, butyric acid and propionic acid.

Images