KR102808726B1 - Fructophilic lactic acid producing bacteria - Google Patents

Abstract

The present invention discloses a novel fructophilic lactic acid producing bacterium, Bacillus coagulans strain FF-7 (MTCC 25235), and methods for isolation and characterization of the bacterium. The present invention also discloses biological applications/therapeutic uses of the fructophilic lactic acid producing bacterium for increasing the utilization of fructose from foods and for managing diseases associated with high fructose intake.

Description

Fructophilic lactic acid producing bacteria

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

Probiotics, and their importance in health and prevention of many diseases, have already been reported. Fructophilic lactic acid bacteria (FLAB) are a special group of lactic acid bacteria that use fructose as a growth substrate. Because of their unique characteristic of not growing well on glucose and preferring oxygen, they are considered "unconventional" lactic acid bacteria (LAB). Their unusual growth characteristics are due to an incomplete gene encoding a bifunctional alcohol/acetaldehyde dehydrogenase (adhE). This results in an imbalance of NAD/NADH, requiring additional electron acceptors for glucose metabolism. Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB have far fewer genes for carbohydrate metabolism than other LAB, particularly because they lack a complete phosphotransferase system (PTS) transporter. FLAB was originally classified as a Leuconostoc species, but was later reclassified as a Fructobacillus species based on its 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. nov. Int J Syst Evol Microbiol 58:2195-2205).

Fructose metabolism is initiated in the liver by the enzyme fructase. Fructose load is converted to lactate in the enterocytes and liver. In addition, excess fructose in the liver is directed to peripheral tissues and is taken up by GLUT 4, an insulin-dependent glucose transporter present in adipose tissue, and converted to fatty acids. GLUT 4 has been reported to play an important role in the development of fructose-induced hepatic steatosis and dyslipidemia. In addition, several fructose-induced metabolic diseases have been reported, such as NASH, NAFLD, dyslipidemia, ectopic lipid deposition in the liver and skeletal muscle, uric acid metabolism, hypertension, and 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 has also been associated with increased cardiovascular risk. Increased fructose intake can also lead to an excessive decrease in blood pH, which can lead to lactic acidosis.

Probiotics play an important role in intestinal fructose metabolism. Orally ingested low doses of fructose reach the intestines and are metabolized in the presence of different enzymes and a unique microbiota. However, when high doses of fructose are present, the fructose burden shifts from the intestines to the liver. To reduce the outflow of fructose from the intestines to the liver, probiotics play an important role in converting fructose into SCFAs (short-chain fatty acids).

FLABs have been isolated from various sources such as fruits and vegetables, human fecal cultures, natural antimicrobials, cheese, kefir grains, dairy and non-dairy products, fermented and raw milk, feces of breast-fed infants, lactating milk, sheep, buffalo and cow milk, yogurt, beverages, poultry sources, rumen contents of animals, cecum of Pengging Duck, chicken intestine and fecal samples, chicken feed, enzymes, fermented rice, curd, meat and yeast extracts, glucose and sucrose, human intestine, human colonial epithelial cells, vaginal and oral extracts of humans and animals, human baby diapers, pineapple waste, industrial sausages, ice cream, small intestine of piglets, corn slurry, crops and intestinal duck. The following prior art documents describe the isolation of different FLABs:

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 (see section 3.1, "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." in 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), there is still a need to find superior fructophilic probiotic bacteria with improved biological functions. The present invention relates to a novel fructophilic lactic acid bacterium, Bacillus The above problem is solved by introducing Coagulans MTCC 25235.

The main object of the present invention is to provide a novel fructophilic lactic acid producing bacteria Bacillus Disclosed is a method for producing coagulans MTCC 25235 and its isolation process.

Another object of the present invention is to provide a fructophilic lactic acid producing bacterium, Bacillus The production of short-chain fatty acids by Coagulans MTCC 25235 is initiated.

Another object of the present invention is to provide a fructophilic lactic acid producing bacterium, Bacillus Initiates the antibacterial effect of Coagulans MTCC 25235.

The present invention solves the above-mentioned objects and provides additional related advantages.

Deposit of biological material

Bacillus biological substance having the accession number MTCC 25235 mentioned in this application Coagulans strain FF7 was deposited on 28 February 2019 at the Microbial Type Culture Collection & Gene Bank (MTCC), Institute of Microbial Technology, Sector, CSIR, Sector 39-A, Chandigarh - 160036, India.

The present invention discloses fructophilic lactic acid producing bacteria. The present invention further discloses the isolation process, characterization and biological applications/therapeutic uses of the fructophilic lactic acid producing bacteria.

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

The patent or application file shall contain 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 required fee.
Figures 1a, 1b, 1c, and 1d show phase contrast microscopy images (× 1000 magnification) of Bacillus coagulans MTCC 25235 ( Figure 1a ), a wet mount of cells with spores ( Figure 1b ), a Gram stain of vegetative cells ( Figure 1c ), a spore stain of sporulated cells ( Figure 1d ), and colonies grown on a GYE agar plate.
Figure 2 is a graphical representation of the comparison of growth patterns of Bacillus coagulans FF7, MTCC 25235 and B. coagulans ATCC 31284 in the presence of fructose and dextrose as carbon sources. Values are means (±SD) of three independent measurements.
Figure 3 is a graphical representation of the utilization of fructose by Bacillus coagulans MTCC FF7, 25235 after 24 h of incubation in the presence of media and fructose-rich foods (honey, fruit juice). Values are means (±SD) of three independent measurements.
Fig. 4 is Bacillus A phylogenetic tree based on 16S rRNA sequences showing the relative positions of Coagulans FF7 is shown.
Figure 5 shows the effect of different pHs on the growth of B. coagulans FF7, MTCC 25235 and B. coagulans ATCC 31284, and the optimum pH for growth was found to be pH 7.5 and pH 6.5, respectively. Values are means (±SD) of three independent measurements.
Figure 6 graphically illustrates the effect of different temperatures on the growth of B. coagulans MTCC FF7, 25235 and B. coagulans ATCC 31284. Values are means (±SD) of three independent measurements.
Figure 7 shows the effect of GIT hostile conditions on the survival of B. coagulans MTCC 25235 after gastric treatment in an in vitro experiment mimicking in vivo conditions. Sterile saline was used as an untreated control.
Fig. 8 is Bacillus Indicates BSH activity of B. coagulans MTCC 25235. ( Figure 8a ) Measurements using soft MRS agar supplemented with ox bile (0.3%, w/v) and CaCO ₃ (0.3%, w/v) and MRS agar without bile salts were used as negative controls ( Figure 8b ). The hollow area suggests BSH activity of B. coagulans MTCC 25235.
Figure 9 is a graphical representation of the survival of Bacillus coagulans MTCC FF7, 25235 at various pH values (1.5-8.0) in gastric acid buffer. Values are expressed as Log ₁₀ spores/g. Data represent the mean and standard deviation (±SD) of two different experiments performed.
Figure 10 is a graphical representation of the in vitro effect of bovine bile salts on the growth of B. coagulans FF7, MTCC 25235, and B. coagulans ATCC 31284. Fresh overnight cultures of B. coagulans MTCC 25235 and B. coagulans ATCC 31284 were inoculated into MRS broth with (0.1, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9, and 1%, w/v) bovine bile salts and without (%, w/v) bovine bile salts. Values are means (±SD) of three independent measurements.
Figure 11 graphically represents the production of L-lactic acid by B. coagulans FF7, MTCC 25235 in the presence of two standardized preparations corresponding to 6Х10 ⁹ (preparation 1) and 15Х10 ⁹ cfu/g (preparation 2). Values are means (±SD) of three independent measurements.
Figures 12a, 12b and 12c show Bacillus fermenting fructose, FOS, cranberry seed fiber and fenugreek seed fiber. The production of acetic acid ( Fig. 12a ), butyric acid ( Fig. 12b ), and propionic acid ( Fig. 12c ) from Coagulans MTCC 25235 is graphically represented. Samples were collected after 4, 8, 12, 18, and 24 h of fermentation. Values are the average of three replicates performed at two different times and are expressed in mg/g.
Figure 13 is a graphical representation of the viability of B. coagulans MTCC 25235 during storage at 40 ± 2°C and RH 60% ± 5%. Two standardized preparations corresponding to 15Х10 ⁹ (preparation 1) and 6Х10 ⁹ cfu/g (preparation 2) were studied. The mean spore counts are expressed as log10 cfu/g. Each time point represents the mean _Log10 standard deviation (±SD) of three different experiments performed in duplicate.

In a most preferred embodiment, the present invention discloses a method for isolating and identifying novel probiotic bacteria using fructose from honey, comprising the following steps:

a) A step of obtaining a suspension by mixing honey and saline solution in a ratio of 1:10 w/v;

b) a step of thoroughly mixing the suspension of step a) and subjecting it to a heat shock at 50-70°C for 30 minutes for selective isolation of spores;

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

d) selecting and incubating morphologically distinct colonies in a suitable medium containing fructose as a carbon source to purify the bacterial isolate;

e) Identifying the bacterial strain as Bacillus coagulans strain FF7 with accession number MTCC 25235 by biochemical analysis and 16S rRNA sequencing.

In a related aspect, the honey is selected from the group including but not limited to raw honey, filtered honey, acacia honey, alfalfa honey, aster honey, avocado honey, basswood honey, beechwood 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, orangeblossom honey, pine honey, sourwood honey, sage honey, and tupelo honey. In another related aspect, the culture medium is selected from the group comprising MRS (De Man, Rogosa and Sharpe agar), GYA (Glucose Yeast Extract Agar), TSB (Tryptone Soya Broth), Sporulation medium and Mueller Hinton Agar.

In another related aspect, the isolated probiotic strains returned positive for biochemical tests such as catalase, oxidase, methyl red, voges proskauer, 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 and for biochemical tests such as sorbose malonate utilization, citrate utilization, xylitol, methyl mannoside, melezitose, erythritol, adonitol, inositol, dulcitol, glycerol, blood hemolysis, citrate and indole. Back to voice.

In another preferred embodiment, the present invention discloses a novel probiotic bacteria of the genus Bacillus isolated from honey for increasing the utilization of fructose from fructose-rich foods. In another related aspect, the fructophilic probiotic bacteria are gram-positive. In another aspect, the optimal pH and temperature recorded for the growth of the fructophilic bacteria are 7.5 and 40°C, respectively. In another aspect, the fructophilic probiotic bacteria are bile-resistant, gastric acid-resistant and produce lactic acid. In a related aspect, the fructophilic probiotic bacteria are Bacillus Coagulans . In another related implementation, Bacillus Coagulans strains are Bacillus Coagulans MTCC 25235. In a related aspect, the fructose-rich food is selected from the group comprising 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, and ice cream. In a related aspect, the fructophilic probiotic bacteria are used in the therapeutic management of a disease associated with high fructose intake. In a related aspect, the disease associated with high fructose intake is selected from the group comprising but 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 fructophilic probiotic bacteria are in the form of an inoculum, a freeze-dried powder, a micropowder, a tablet, a capsule, a suspension, a solution, an emulsion, a gummy, a chewable or an edible food, administered alone or in combination with a fructose-rich food selected from the group comprising 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, and ice cream.

In another preferred embodiment, the present invention provides a method for inhibiting a pathogenic microorganism, wherein the microorganism is a fructophilic probiotic bacterium, Bacillus A method comprising the step of contacting a coagulans MTCC 25235 is disclosed. In a related aspect, the pathogenic microorganism is Salmonella abony , Micrococcus Luteus ( Micrococcus luteus ) , Escherichia coli ( coli ) , Pseudomonas aeruginosa , Bacillus cereus , Propionibacterium acnes ​ ​ acnes ), Streptococcus Streptococcus mutans , Staphylococcus ​ aureus (Staphylococcus aureus ), and Staphylococcus Selected from the group containing Staphylococcus epidermidis .

In another preferred embodiment, the present invention comprises a fructophilic probiotic bacterium, Bacillus subtilis , together with plant fibers selected from the group consisting of fructose, fenugreek seed fiber, cranberry seed fiber, and fructooligosaccharides (FOS). A method for producing short-chain fatty acids by culturing Coagulans MTCC 25235 is disclosed.

The specific examples included in this specification below illustrate the most preferred embodiments of the present invention.

Example

Example 1: Fructophilic Isolation and identification of bacteria

method

Unfiltered raw honey was used in this study to isolate spore-forming fructophilic lactic acid bacteria. For the isolation of fructophilic bacteria, de Man, Rogosa, and Sharpe (MRS) (dextrose replaced by fructose) was used in this study (Table 1).

1 g of unfiltered raw honey was placed in a test tube containing 10 ml of saline solution. It was mixed well and heat shocked at 70°C for 30 min for selective isolation of spores. Isolation of bacteria was performed 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 h. After incubation, morphologically distinct colonies were picked for further testing and purification of the bacterial isolate. Bacillus Coagulans MTCC 25235 was isolated and streaked on different MRS agar plates containing fructose as a carbon source.

Fructophilic Biochemical characterization of bacteria

Bacterial isolates were grown on de Man, Rogosa and Sharpe Agar (MRSA) at 37°C for 24 h. One to three well-isolated colonies were picked to prepare bacterial inoculum and a homogeneous suspension was prepared in sterile saline. The density of the suspension was ≥ 0.5 OD at 620 nm. This inoculum (50 μl) was used and the test was performed according to the kit manufacturer's instructions (HiMedia, Mumbai, India). Bacillus Biochemical characterization of Bacillus coagulans MTCC 25235 and B. coagulans ATCC 31284 was performed 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 tests were performed according to the manufacturer's instructions. Bacillus Bacterial suspensions of B. coagulans MTCC 25235 and B. coagulans ATCC 31284 were prepared as mentioned in the above section. Additionally, IMViC (using indole, methyl red, Boggys Proskauer, and citrate) test, oxidase and Gram staining of B. coagulans ATCC 31280 were performed according to the methods of Majeed et al., (2016); (Cappucino G and Natalie Sherman. Microbiology: A Laboratory manual 5th edition. 1998).

16S rDNA Sequencing

16S rDNA sequencing Genomic DNA of B. coagulans MTCC 5856 was prepared as described previously (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). A fragment of the 16S rDNA gene was sequenced using an ABI 3500 Genetic Analyzer automated DNA sequencer as described previously (Heyrman and Swings 2001). The sequencing primers used were 5-AGHGTBTGHTCMTGNCTCAS-3 (forward primer) and 5-TRCGGYTMCCTTGTWHCGACTH-3 (reverse primer). An amplified DNA fragment of approximately 1.5 kb was separated on a 1% agarose gel and purified using Qiagen spin columns. The purified fragment was used directly for DNA sequencing. The sequence was used for BLAST searches ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ).

Bacillus Coagulans MTCC Growth conditions of 25235

Bacillus The growth conditions for Coagulans MTCC 25235 were optimized at various temperatures and pH. MRSB medium was prepared, and the pH was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 using 2 N HCl and 2 N NaOH. The overnight culture (1%, v/v) was inoculated into the pH-adjusted medium, and incubated for 24 h in a shaking incubator at 120 rpm. Growth was monitored every 6 h by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan). MRSB medium was prepared, the pH was adjusted to 6.5, and the overnight culture (1%, v/v) was inoculated into the medium. Additionally, the flasks were incubated in a shaking incubator at 120 rpm at different temperatures (20, 30, 37, 40, 50, and 60°C) for 24 h. Growth was monitored every 6 h by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

result

Fructophilic Identification of bacteria

Bacillus Coagulans The spores of MTCC 25235 were oval terminal spores ( Fig. 1a ) and the vegetative cells were Gram-positive rod-shaped as shown by Gram staining ( Fig. 1b ). Colonies of B. coagulans MTCC 25235 grew on the medium and formed uniform white to cream colored smooth colonies measuring 1-3 mm in diameter, which contained vegetative rod-shaped cells ( Figs. 1c and 1d ). The growth of the isolated bacterium was better on fructose-rich medium than on dextrose-rich medium ( Fig. 2 ), demonstrating its fructophilic properties and fructose utilization potential compared to other Bacillus coagulans strains. The growth of the bacteria was also tested on fructose-rich food and showed inference from the fructose content suggesting that the bacterium utilizes fructose as a carbon source for growth and development ( Fig. 3 ). Therefore, these bacteria can be used to manage and prevent diseases associated with high fructose consumption, since they utilize the excess fructose in foods to metabolize it. The bacteria can be administered alone or in combination with foods rich in fructose, 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 creamers, jams and jellies, energy drinks, condiments, and ice cream (Braverman J, List of Foods high in fructose, https://www.livestrong.com/article/30454-list-foods-high-fructose/ , accessed May 3, 2019).

Biochemical characterization

Bacillus Biochemical characterization of Bacillus coagulans MTCC 25235 and B. coagulans ATCC 31284 was performed 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 results were compared with the commercial strain B. coagulans ATTCC 3128 and are presented in Table 2.

The results showed that the isolated probiotic strain was positive for biochemical tests such as catalase, oxidase, methyl red, bogis proskauer, lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melobiose, sucrose, melobiose, arabinose, mannose, inulin, sodium gluconate, salicin, sorbitol, mannitol, arabitol, methyl glucoside, rhamnose, cellobiose, ONPG, esculin hydrolysis and negative for biochemical tests such as sorbose malonate utilization, citrate utilization, xylitol, methyl mannoside, melezitose, erythritol, adonitol, inositol, dulcitol, glycerol, blood hemolysis, citrate and indole.

16S rDNA Sequencing

Bacterial 16S rDNA was sequenced and sequence information (SEQ ID) was obtained as follows:

A BLAST (Basic local alignment search tool) search was performed with the above sequences, and the results of the first 10 aligned sequences are presented in Table 3.

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

Fructophilic Optimal growth conditions for bacteria

The optimal pH and temperature recorded for the growth of fructophilic bacteria were 7.5 and 40°C, respectively ( Figures 5 and 6 ).

Example 2: Bacillus Coagulans MTCC In the test tube of 25235 Probiotic evaluation

Resistance to stomach acid

Bacillus The survival of Coagulans MTCC 25235 spores was studied by adding 1 ml of the suspension to 100 ml of sterile electrolyte solution (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl ₂ , and 1.2 g/L NaHCO ₃ ) containing 0.01% lysozyme (Sigma-Aldrich) and 0.3% pepsin (Sigma-Aldrich) and incubating for 5 min. Additionally, the pH was adjusted to 1.5, 3, 4, 5, 6, 7 and 8 (adjusted using 1 N NaOH and 1 N HCl). The incubation temperature was monitored at 37°C for 4 h. 1 ml samples were withdrawn at different time intervals: 0, 1.0, 2.0, 3.0 and 4.0 h. After incubation, serial dilutions were performed with sterile saline (0.89% w/v), plated on glucose yeast extract agar (HiMedia), and viable counts were calculated. The experiment was performed three times at two different times.

Cholelithiasis Bile salt tolerance

Bacillus The bile tolerance of Coagulans MTCC 25235 cells was determined by a previously described method (Gilliland et al. 1984; Hyronimus et al. 2000). Bacillus Approximately 10 ⁶ cfu mL ^{- 1} of an overnight culture of Coagulans MTCC 25235 was inoculated and supplemented with 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) or no bile salts as a control in the experiments. The samples were incubated at 37 °C for 24 h with shaking at 120 rpm. Growth in the control (no bile) and test cultures containing different concentrations of bile was monitored hourly by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Lactic acid production

Bacillus Lactic acid production by Coagulans MTCC 25235 was estimated using the Megazyme kit. Bacillus A loopful of an overnight culture of Coagulans MTCC 25235 was added to glucose yeast extract broth (HiMedia) and incubated at 37°C at 120 rpm for 18 h. After incubation, the broth was filtered through 0.22 micron (Sartorius, India) and assayed for lactic acid content using a Megazyme kit (K-DLATE 10/04) according to the instructions (Megazyme International Ireland, IDA Business Park, Wicklow, Ireland). GYE medium was used as blank in the assay.

Simulated gastric acid tolerance

Bacillus to indicate oral digestive conditions Survival of spores of Coagulans MTCC 25235 was studied. Bacillus Simulated saliva contents consisting 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) were applied to _{1 mL} of Coagulans MTCC 25235 suspension, adjusted to pH 6.8±0.2, and incubated at 37°C for 5 min. 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), and the pH was aseptically adjusted to 3.0±0.2 using 2 N HCl. The samples were additionally incubated at 37°C for 3 h at low rpm. After 3 hours of incubation, the pH was aseptically adjusted to 7.0 using 2N NaOH. Bovine bile (5 g/L) was further incubated at 37°C for 24 hours. After the final stage of the simulated digestion process, samples were collected and incubated with Bacillus coagulans The spore survival of MTCC 25235 was evaluated and counted by the serial dilution method using glucose yeast extract agar medium (HiMedia) ( Figure 7 ).

Antibiotic resistance patterns

MIC was measured according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2012). Bacillus Coagulans MTCC 25235 suspension was prepared by suspending bacterial culture grown for 18 h in sterile saline (0.89% NaCl wt/vol; Himedia, Mumbai India). The turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard (equivalent to 1.5 Х 10 ⁸ colony forming units (CFU)/ml). Antibiotic stock solutions were prepared according to CLSI guidelines and two-fold serial dilutions were diluted in 100 μl volume in 96-well U-bottom microtiter plates (BD Labware, NJ USA) on broth medium ( Bacillus For S. aureus ATCC 6538, glucose yeast extract acetate broth [GYEA, HiMedia, Mumbai India] was prepared and for S. aureus, Mueller Hinton broth [MHB, Difco Laboratories, Detroit, Mich USA] was used. The above mentioned bacterial suspensions were further diluted with MHB and 100 μl volume of this diluted inoculum was added to each well of the plate to yield a final inoculum of 5 x 10 ⁵ CFU/ml in the well, giving a final concentration of antibiotic in the range of 0.0078 to 4 μg/ml. S. aureus ATCC 6538 was used as a reference culture in this study. The plates were incubated at 37°C for 24 h and read visually for absence or presence of turbidity. The lowest concentration of compound that did not show turbidity was reported as MIC.

Ames (AMES) Test

The experimental data suggested that B. coagulans MTCC 25235 spores did not increase the number of revertants in five Salmonella strains (TA98, TA100, TA102, TA1535, and TA1537) compared to the negative control, regardless of the absence or presence of the S9 metabolic activation system. Furthermore, there was no dose-dependent mutagenic effect induced by B. coagulans MTCC 25235 spores (up to 5000 μg/plate). B. coagulans MTC 25235 spores did not exhibit any mutagenic activity under the experimental conditions.

BSH Active

Bacillus on agar plates containing bovine bile and calcium carbonate Growth of Coagulans MTCC 25235 was observed, indicating resistance to bovine bile and the presence of BSH activity. As shown in Figures 8a and 8b , there was a clear zone of 19±2 mm on the soft agar plate, indicating the presence of BSH activity.

Antibacterial activity against human pathogens

Antibacterial activity was performed by a well diffusion assay as described previously with slight 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, 5 mL lawn of soft (0.7% agar ) glucose yeast extract (HiMedia, India) containing 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. aviny NCIM 2257) was poured onto concentrated hard (1.5% agar) tryptic soy agar (HiMedia, India). Overnight grown culture of B. coagulans MTCC 25325 was added to MRS medium and incubated at 37°C for 24 h at 120 rpm. After 24 h, the culture was centrifuged (10,000 × 9 g) to remove cells, the supernatant was collected, concentrated 10-fold by lyophilization, and filter sterilized through a 0.22 micron filter (Sartorius, India). The concentrated supernatant (50 μL) was added to 6 mm wells punched into solidified double layer agar. The plates were kept in the refrigerator (4 ± 2°C) for 5 h to allow diffusion of the sample into the agar and then incubated at 37°C for 18 to 20 h. After incubation, the zone of inhibition was measured and recorded in mm.

Production of short chain fatty acids

Bacillus In vitro fermentation using Coagulans MTCC 25235 was performed according to the method described by McBurney and Thompson (1987) with some modifications. Briefly, 2.0 g of fructose, fenugreek seeds, cranberry seed fiber, and 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, anaerobic conditions were induced by adding oxygen reductase (Oxyrase® for broth, Oxyrase, Inc, OH, USA) to each flask. Bacillus grown overnight Five percent of the culture of Coagulans MTCC 25235 was inoculated into all flasks and incubated at 37°C with gentle shaking for 24 h. The bottles were tightly closed and sealed with parafilm to maintain the anaerobic conditions created by the enzyme supplement. The pH values at 0 h of incubation and after fermentation (24 h) were also recorded. To inhibit further microbial growth, 1 ml of copper sulfate (10 g/L) was added to each sample (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 with 3 M H ₂ SO ₄ . 10 ml of chilled diethyl ether (-20°C) was added to the samples and vortexed for 1 min. Sodium chloride was added and centrifuged at 3000Хg for 10 min. After centrifugation, the organic layer was separated and transferred to a new vial. This was used to quantify SCFAs. SCFA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and processed similarly. SCFA production (acetate, propionate, and butyrate) was measured by gas chromatography (GC) using an Agilent technologies 6890N gas chromatograph (Stevens Creek Blvd Santa Clara, CA, USA) containing a DB-FFAP (terephthalic acid modified polyethylene glycol) column. The column temperature was 200 °C. The injector and detector port temperatures were 250 °C. The carrier gas was N ₂ 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 as milligrams per gram (mg/g) of galactomannan from fenugreek seeds.

result

Resistance to stomach acid

There was no significant difference (2-5%) in spore counts at pH 3 to pH 8.0 compared to the initial spore counts up to 4 h of the study ( Fig. 9 ). However, a 0.44 and 2.036 log ₁₀ reduction was observed at 1 h and 4 h, respectively, at pH 1.5. The results demonstrated the stability of Bacillus coagulans MTCC 25235 spores under acidic and alkaline pH conditions.

Bile tolerance test

Bacillus on agar plates containing bile salts (1% w/v) Growth of Bacillus coagulans MTCC 25235 was observed, indicating its tolerance to bile salts. In addition, a bile tolerance assay was performed by supplementing bovine bile (0.1-2.0%) to MRS broth in another flask. Bacillus Growth of Bacillus coagulans MTCC 25235 was observed in the presence and absence of bovine bile up to 2%, w/v. In contrast, growth of Bacillus coagulans ATCC 31284 was observed at 0.8% w/v ( Fig. 10 ). Similarly, growth of Bacillus coagulans in the presence and absence of bile salts There was no significant difference in the viability of B. coagulans MTCC 25235 and B. coagulans ATCC 31284.

Lactic acid production

Bacillus Lactic acid production by Coagulans MTCC 25235 was estimated using the Megazyme kit. Bacillus The total lactic acid produced by Coagulans MTCC 25235 was 4.487 g/L. L-lactic acid was 4.12 g/L. On the other hand, D-lactic acid Bacillus Coagulans MTCC 25235 is 0.367 g/L. ( Fig. 11 )

Antibiotic resistance

Bacillus The MIC results of clindamycin, kanamycin, ampicillin, streptomycin, vancomycin, erythromycin, gentamicin, tetracycline and chloramphenicol against Bacillus coagulans MTCC 25235 and S. aureus ATCC 6538 are given in Table 4. All antibiotics tested were bactericidal against Bacillus The MIC range for Coagulans MTCC 25235 was 0.0078 to 1.0 μg/ml. All the tested antibiotics showed an MIC range of 0.031 to 2 μg/ml against S. aureus ATCC 6538.

Antibacterial activity

The antibacterial activity of B. coagulans MTCC 25235 was evaluated. The antibacterial activity was measured by the well diffusion assay described by Cintas et al. (1995). The results showed that the probiotic bacteria had a high antibacterial activity against Salmonella aviani , Micrococcus luteus , Escherichia coli , Pseudomonas aeruginosa , Bacillus cereus, Propionibacterium acnes, Streptococcus Mutans , Staphylococcus aureus , and staphylococcus It has been shown to be an effective antibacterial agent against Epidermidis .

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

Short story fatty acid production

The results of the analysis are shown in Figs. 12a, 12b, and 12c . Acetic acid production was high in B. coagulans MTCC 25235 using FOS, followed by fenugreek seed fiber, cranberry fiber, and fructose ( Fig. 12a ). Similarly, butyric acid production was high in B. coagulans MTCC 25235 using fructose, followed by FOS, fenugreek seed fiber, and cranberry fiber ( Fig. 12b ). Propionic acid production was similar in B. coagulans MTCC 25235 cultured with fructose, FOS, and cranberry seed fiber, and was lower in the case of fenugreek seed fiber ( Fig. 12c ).

Storage and Survival Abilities

Two standardized preparations corresponding to 15Х10 ⁹ (preparation 1) and 6Х10 ⁹ cfu/g (preparation 2) were studied. B. coagulans MTCC 25235 showed enhanced viability during storage at 40 ± 2°C and RH 60% ± 5% ( Figure 13 ).

Other modifications and variations of the present invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Accordingly, while only specific embodiments of the present invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be construed only in conjunction with the appended claims.

SEQUENCE LISTING <110> Majeed, Muhammed Nagabhushanam, Kalyanam Majeed, Shaheen Ali, Furqan Arumugam, Sivakumar Beede, Kirankumar <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 ctgagaacacg 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 600 gaaactggga gggctttgag tgcaagaaag aggagagtgg aatttccacg tgtagcggtg 660 aaatgcgtaa agatgtggag gaacaccagt ggcgaaggcg gctctctggt ctgtaactga 720 cgctgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780 aaacgatgag tgctaagtgt tagagggttt ccgcccttta gtgctgcagc taacgcatta 840 agcactccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acggggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960 gacatcctct gacctccctg gagacagggc cttccccttc gggggacaga gtgacaggtg 1020 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080 acccttgacc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1140 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1200 tgctacaatg gatggtacaa agggctgcga gaccgcgagg ttaagccaat cccagaaaac 1260 cattcccagt tcggattgca ggctgcaacc cgcctgcatg aagccggaat cgctagtaat 1320 cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt acggagccag ccgccga 1437

Claims (18)

A fructophilic probiotic bacterium, Bacillus coagulans strain FF7, having the accession number MTCC 25235, isolated from honey to increase the utilization of fructose in foods rich in fructose. In claim 1, the fructophilic probiotic bacteria are gram-positive probiotic bacteria. In the first paragraph, a probiotic bacteria, wherein the optimal pH and temperature for growth of the fructophilic probiotic bacteria are 7.5 and 40°C, respectively. In claim 1, the fructophilic probiotic bacteria are probiotic bacteria that have cholestasis and gastric acid resistance and produce lactic acid. Probiotic bacteria in claim 1, wherein 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 foods, canned foods, cereals, fruit juice, coffee creamer, jams and jellies, energy drinks, seasonings, and ice cream. In claim 1, the fructophilic probiotic bacteria are in the form of an inoculum, a freeze-dried powder, a micropowder, a tablet, a capsule, a suspension, a solution, an emulsion, a gummy, a chewable or an edible food, and are administered alone or together 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 foods, canned foods, cereals, fruit juices, coffee creamers, jams and jellies, energy drinks, seasonings, and ice cream. As a probiotic bacteria, the probiotic bacteria is Bacillus coagulans MTCC 25235, a fructophilic probiotic bacteria having pathogenic microorganism inhibitory activity. A probiotic bacteria in claim 7, wherein 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, and Staphylococcus epidermidis . A method for producing short-chain fatty acids by culturing fructophilic probiotic bacteria, Bacillus coagulans MTCC 25235, together with plant fibers selected from the group consisting of fructose, fenugreek seed fiber, cranberry seed fiber, and fructooligosaccharides (FOS). A method in claim 9, wherein the short-chain fatty acid is selected from the group consisting of acetic acid, butyric acid and propionic acid.
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