Patent Grant
10293008
The invention in general relates to Bacillus coagulans (Lactic acid bacteria). More specifically, the invention relates to (i) the growth promotional activity of natural plant based fibers on Bacillus coagulans MTCC 5856; (ii) The production of short chain fatty acids (SCS A) by Bacillus coagulans MTCC 5856 using plant based natural fibers; and (iii) the combination of natural plant based fibers and Bacillus coagulans MTCC 5856 to inhibit Gram Negative pathogenic bacteria.
Combining multistrain probiotics (probiotic bacteria) and probiotics to achieve enhanced immunosupportive effects is well known in the art. Specifically combining probiotics with natural plant based fibers to formulate synbiotics is reported as a promising therapeutic approach (Stig Bengmark and Robert Martindale, “Prebiotics and Synbiotics in Clinical Medicine”. Nutr Clin Pract vol 20 244-261, April 2005). The success of such an approach depends on carefully choosing specific probiotic microorganisms whose viable count is effectively enhanced by natural plant based fibers that are resistant to both enzymatic and acid hydrolysis in the gut. These studies are critically important to accommodate the performance of host animals exposed to symbiotic diet regimes given the teaching that there are limitations to fiber digestion and utilisation by microbes in terms of microbial accessibility to substrates, physical and chemical nature of fibers (forage) and also kinetics of the digestive process (Gabriella A. Varga and Eric S. Kohler, “Microbial and Animal Limitations to Fiber Digestion and Utilization”, J. Nutr. May 1, 1997 vol. 127 no. 5 819S-8235).
It is the principle objective of the present invention to evaluate the performance of selected natural fibers (enzyme and acid hydrolysis resistant) to enhance the viable counts of Bacillus coagulans MTCC 5856.
It is also another objective of the present invention to evaluate the ability of the synbiotic composition (natural fibers and Bacillus coagulans MTCC 5856) to inhibit pathogenic Gram negative bacteria.
It is yet another objective of the present invention to evaluate the ability of the synbiotic composition (natural fibers and Bacillus coagulans MTCC 5856) to produce desired short chain fatty acids, said property having profound therapeutic applications.
The present invention fulfils the aforesaid objectives and provides further related advantages.
The deposit of biological material Bacillus coagoulans SBC37-01 bearing accession number MTCC 5856, mentioned in the instant application has been made on 19 Sep. 2013 at Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Sector 39-A. Chandigarh-160036, India.
Disclosed is (i) the growth promotional activity of natural plant based fibers on Bacillus coagulans MTCC 5856; (ii) the combination of natural plant based fibers and Bacillus coagulans MTCC 5856 to inhibit Gram negative pathogenic bacteria and (iii) the production of short chain fatty acids (SCFA) by Bacillus coagulans MTCC 5856 using plant based natural fibers.
Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principle of the invention.
In the most preferred embodiment, the present invention relates to a method of increasing the viable colony count of Bacillus coagulans MTCC 5856 said method comprising step of growing Bacillus coagulans MTCC 5856 in the presence of natural plant fibers selected from the group consisting of Trigonella foenum-graecum (fenugreek) seed fibers, Lycium barbarum seed fibers, Linum usitatissimum (Flax) seed fibers, Cocos nucifora (Coconut) fibers, Zingiber officinale (Ginger) rhizome fibers, Emblica officinalis (Anila) fruit fibers, Plantago ovata (Psyllium) fibers and Vaccinium oxycoccos (Cranberry) seed fibers.
In another most preferred embodiment, the present invention relates to a method of inhibiting pathogenic Gram negative bacteria said method comprising step of bringing to contact said Gram negative bacteria with Bacillus coagulans MTCC 5856 co-cultured with natural plant fibers selected from the group consisting of Trigonella foenum-graecum (fenugreek) seed fibers, Lycium barbarwn seed fibers, Linum usitatissimum (Flax) seed fibers, Cocos nucifera (Coconut) fibers, Zingiber officinale (Ginger) rhizome fibers, Emblica officinalis (Amla) fruit fibers, Plantago ovata (Psyllium) fibers and Vaccinium oxycoccos (Cranberry) seed fibers.
In yet another most preferred embodiment, the present invention also relates to a method of producing short chain fatty acids by co-culturing Bacillus coagulans MTCC 5856 with natural plant fibers selected from the group consisting of Trigonella foenum-graecum (fenugreek) seed fibers, Lycium barbarum seed fibers, Linum usitatissimum (Flax) seed fibers, Cocos nucifera (Coconut) fibers, Zingiber officinale (Ginger) rhizome fibers, Emblica officinalis (Anila) fruit fibers, Plantago ovata (Psyllium) fibers and Vaccinium oxycoccos (Cranberry) seed fibers. In alternate embodiments, the present invention also relates to a method of protecting against diet-induced obesity and insulin resistance in the mammalian gut by administering composition comprising Bacillus coagulans MTCC 5856 with natural plant fibers selected from the group consisting of Trigonella foenum-graecum (fenugreek) seed fibers, Lycium barbarum seed fibers, Linum usitatissimum (Flax) seed fibers, Cocos nucifera (Coconut) fibers, Zingiber officinale (Ginger) rhizome fibers, Emblica officinalis (Amla) fruit fibers, Plantago ovata (Psyllium) fibers and Vaccinium oxycoccos (Cranberry) seed fibers to bring about the effect of protection against diet induced obesity and insulin resistance.
The specific examples included herein below illustrate the aforesaid most preferred embodiments of the present nvention.
Method of Preparing Fibers
Trigonella foenum-Graecum (Fenugreek) Seed Fibers
Trigonella foenum-graecum (also known as Fenugreek) seeds were collected from local market and milled to course powder (10 mesh pass through). Further, four volumes of n-hexane was added to 100 gm of Trigonella foenum-graecum seeds course powder and extracted at reflux temperature. n-Hexane fraction was filtered through Whatman filter no 1 and again three times same extraction was carried out. After extraction, retenta e was dried at 80° C. for 5 h and then this was milled to obtain 40 mesh pass through powder. In an alternative method, fat or oil from Fenugreek seeds were removed by Super Critical fluid extraction method using liquid CO2 as solvent. To increase the dietary fiber (Galactomarmans) content, the enzymatic hydrolysis using Cellulase was carried out. The galactomannans content was determined by Megazyme kit (K-GALM 03/11) as per the manufacturer's instructions (Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, IRELAND).
Lycium Barbarum Seed Fibers
Goji, Goji berry or Wonerry is the fruit of Lycium barbarum. The fruit of Lycium barbarum was dried and seeds were separated and milled to course powder. Further, four volumes of n-hexane was added to 100 gm of Lycium barbarum course powder and extracted at reflux temperature. n-Hexane fraction was filtered through Whatman filter no 1 and again three times same extraction was carried out. After extraction, retentate was dried at 80° C. for 5 h and then this was milled to obtain 60 mesh pass through powder, Total dietary fiber was determined by Enzyrnatic-Gravimetric Method (AOAC 985.29).
Linum usitatissimum (Flax Seed) Fibers
Linum usitatissimum (also known as common flax or linseed or Flax) seeds were collected and milled to course powder (10 mesh pass through). Further, four volumes of n-hexane was added to 100 gm of Linum usitatissimum course powder and extracted at reflux temperature. n-Hexane fraction was filtered through Whatman filter no 1 and again three times same extraction was carried out. After extraction, retentate was dried at 80° C. for 5 h and then this was milled to obtain 40 mesh pass through powder. Total dietary fiber was determined by Enzymatic-Gravimetric Method (AOAC 985.29).
Cocos nucifera Fibers
A matured coconut was procured from local market and dried. Further, the endosperm (coconut meat) was chopped to course and uniform size material. Further, four volumes of n-hexane was added to 100 gm of Cocos nucifera course material and extracted at reflux temperature, n-Hexane fraction was filtered through Whatman filter no 1 and again three times same extraction was carried out. After extraction, retentate was dried at 80° C. for 5 h and then this was mined to obtain 60 mesh pass through powder. Total dietary fiber was determined by Enzymatic-Gravimetric Method (AOAC 985.29).
Zingiber officinale (Ginger) Rhizome Fibers
Zingiber officinale rhizome, ginger root or simply ginger was dried and milled to course powder (10 mesh pass through). Further, four volumes of n-hexane was added to 100 gm of Zingiber officinale rhizome course powder and extracted at reflux temperature. n-Hexane fraction was filtered through Whitman filter no 1 and again three times same extraction was carried out. In an alternative method, fat or oil from Fenugreek seeds were removed by Super Critical fluid extraction method using liquid CO2 as solvent. After extraction, retentate was dried at 80° C. for 5 h and then this was milled to obtain 40 mesh pass through powder. Total dietary fiber was determined by Enzymatic-Gravimetric Method (AOAC 985.29).
Emblica officinalis (Amla) Fruit Fibers
Emblica officinalis (Phyllanthus emblica), also known as Emblic, Emblic myrobalan, Myrobalan, Indian gooseberry, Malacca tree, or Amla from Sanskrit Amalika. The fruit of Emblica officinalis was procured from local market and dried, pulverized and passed through 60 mesh. This powder was used for the extraction of fibers. Total dietary fiber was determined by Enzymatic-Gravimetric Method (AOAC 985.29).
Two grams of plant based natural fibers listed in Table 1 were dissolved in 100 nil of HCl (0.10 M) and incubated at 37° C. with 100 rpm for 180 min. Samples were taken at 0, 30, 60, 90, 120 and 180 min. Fructooligosaccharide (FOS; Tata Chemicals, India) was also taken in the study as reference to compare with natural fibers and starch (Potato soluble starch; HiMedia, Mumbai, India) was also taken as control. The increase in reducing carbohydrates was measured by Dinitrosalicylate reagent (Nilsson and Bjorck 1988, Journal of Nutrition 118, 1482-1486).
Trigonella
foenum-graecum
Lycium barbarum
Linum usitatissimum
Cocos nucifera
Zingiber officinale
Emblica officinalis
Plantago ovata
Vaccinium
oxycoccos
Table 2 shows the effect of acid hydrolysis on (0.1 M HCl; 37° C., 100 rpm) on Plant Based Natural Fibers. Total reducing sugar was determined by Dinitrosalicylie acid (DNSA) method.
Lycium barbarum seed fibers
100 mg of Pancreatin from Porcine pancreas 4×USP (Sigma-Aldrich Corporation St. Louis Mo., USA) was dissolved in 100 ml of phosphate buffer (50 mM; pH 7.0). Further, plant based natural fibers (2 gm) were dissolved in above Pancreatin solution and incubated at 37° C. with 100 rpm for 180 min, Samples were taken at 0, 30, 60, 90, 120 and 180 min, FOS was also taken in the study as reference to compare with plant based natural fibers and starch was also taken as control. The increase in reducing carbohydrates was measured with a Dinitrosalicylate reagent (Oku et al, 1984, Journal of Nutrition 114, 1574-1581). The effect of enzymatic hydrolysis (0.1% Pancreatin in 20 mM PBS pH 7.0; 37° C., 100 rpm) on plant based natural fibers is represented in Table 3. Total reducing sugar was determined by Dinitrosalicylic acid (DNSA) method.
Lycium barbarum seed fibers
Single isolated colony of Bacillus coagulans MTCC 5856 was inoculated into MRS broth (pH 7.0±20; Himeclia, Mumbai, India) and incubated at 37° C. with 120 rpm for overnight. Plant based natural fibers alone (0.5, 1.0, 2.0%, w/v), and in MRS media (devoid of dextrose) (0.5, 1.0, 2.0%, w/v) were prepared. MRS broth and MRS (devoid of dextrose) were also prepared separately. Similarly, Fructooligosaccharide (FOS) was also taken in the study as reference control to compare with plant based natural fibers. The final pH of all the media was adjusted to 7.0. Five percent of overnight grown Bacillus coagulans MTCC 5856 culture was inoculated to all the flasks and incubated at 37° C. with 100 rpm for 24 h. pH values at 0 h of incubation and after fermentation (24 h) were also recorded. Samples were serially diluted in sterile saline and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia, Mumbai, India) at 0 h and after fermentation (24 h). The plates were incubated at 37° C. for 48 to 72 h. Each analysis was performed in triplicate at two different occasions. Average mean of viable counts are expressed in log 10 cfu/ml (
The in-vitro experiment was designed to evaluate the effect of Plant based natural fibers with probiotic bacteria Bacillus coagulans MTCC 5856 for the inhibition of Gram negative pathogenic bacteria E. coli. Briefly, 2.0 g of plant based natural fibers were added to 100 ml of demineralized water. Psyllium husk Fiber and Flax seed fibers were added 0.5 gm to 100 mL of demineralized water due to high gelling property. The pH was adjusted to 7.0±0.2 and autoclaved at 121° C. for 20 min. After sterilization, oxygen reducing enzyme Oxyrase (Oxyrase® for Broth, Oxyrase, Inc, Mansfield, Ohio, USA) was added to each flask. Bacillus coagulans MTCC 5856 was grown on glucose yeast extract agar (Himedia, Mumbai, India) and E. coli ATCC 25922 was grown on trypticase soya agar (Himedia, Mumbai, India). Single isolated colony of both the cultures was used and the turbidity of the bacterial suspension was adjusted to 0.5 McFarland standards (equivalent to 1.5×108 colony forming units (CFU)/ml). One milliliter of E. coli ATCC 25922 was added to flask containing plant based natural fiber. Similarly, in other group 1 ml of E. coli ATCC 25922 and 1 ml of B. coagulans MTCC 5856 were added to flask containing plant based natural fiber. The flasks were incubated at 37° C. with 100 rpm for 24 h. Samples were serially diluted in sterile saline and the viable count of E. coli ATCC 2592.4. was enumerated by plating on Eosin Methylene Blue Agar (EMB Agar; HiMedia, Mumbai, India) at 0 h and after fermentation (24 h). The plates were incubated at 37° C. for 48 h. Each analysis was performed in triplicate at two different occasions. Average mean of viable counts are expressed in login cfulml (
The in vitro fermentation with the Bacillus coagulans MTCC 5856 was carried out by following method described by McBurney and Thompson (McBurney M I and Thompson L U. (1987) Effect of human faecal inoculum on in vitro fermentation variables. Brit J Nutr 58: 233-243) with some modifications. Briefly, 2.0 g of glucose or Plant Based Natural Fibers were added to 100 mL of demineralised water. Psyllium husk Fiber and Flax seed fibers were added 0.5 gm to 100 ml of demineralised water due to high gelling property. The pH was adjusted to 7.0±0.2 and autoclaved at 121° C. for 20 min, After sterilization, oxygen reducing enzyme Oxyrase (Oxyrase® for Broth, Oxyrase, Inc, Mansfield, Ohio, USA) was added to each flask, to induce anaerobic conditions. Five percent of overnight grown Bacillus coagulans MTCC 5856 culture was inoculated to all the flasks and incubated at 37° C. with gentle shaken rpm for 24 h. The bottles were tightly closed and sealed with parafilm. to maintain anaerobic conditions generated by the enzyme supplement. pH values at 0 h of incubation and after fermentation (24 h) were also recorded. One ml of copper sulphate (10 g/L) was added to each sample to inhibit further microbial growth (Sigma, St. Louis, Mo., USA). The analysis of short chain fatty acids in the aforesaid fermentation samples was done adopting the following parameters.
Reagents
1. Diethyl Ether (AR Grade)
2. H2SO4
3. RO Water
4. Sodium Chloride (AR Grade)
Chromatographic Conditions
Oven:
Inlet
Column
1. DB-FFAP (Terephthalic acid modified poly ethylene Glycol)
2. Dimensions: 30.00 m×250.00 mm×0.25 μm.
3. Carrier gas: Nitrogen
4. Flow: 1.0 ml/min.
Detector
Standard Solution Preparation
100.0 mg of each of Fatty acid standard (Acetic acid, Propionic acid and Butyric acid) was weighed accurately in a 100 ml volumetric flask & dissolved in 50.0 mL of water and made up to the mark with water and mixed well (Stock solution). Further, 10.0 mL of the stock solution was diluted to 100.0 mL with water and mixed well to get standard solution. 5 mL of standard solution was subjected to extraction as described herein below.
1. Taken 5 ml of Standard solution/sample.
2. To Standard solution added 5 ml of water with vortexing for 0.5 min.
3. Adjusted pH to 1-1.5 with 3M H2SO4 with vortexing for 0.5 min.
4. Kept diethyl ether in −20° C. up to 1 hr before adding to the sample/Working Standard.
5. Added 10 ml of diethyl ether with vortexing for 1 min.
6. Added 4 g of Sodium Chloride with vortexing for 1 min.
7. Centrifuged to separate Water Layer & Diethyl Layer
8. Transferred 1.0 mL of Diethyl Ether layer in GC Vial & Injected.
Procedure:
1 μl each of extracted standard solution was injected into the chromatograph in triplicate and recorded the responses of major peaks due to Acetic acid, Propionic acid and Butyric acid. The % Relative Standard Deviation for area of peaks due to Acetic acid, Propionic acid and Butyric acid in triplet injections should not be more than 2.0%. Injected 1.0 μl each of extracted sample solution into the chromatograph. The content of Acetic acid, Propionic acid and Butyric acid was calculated as follows.
The results of the chromatographic analysis are presented in
In Table 4 it may be noted that the production (mg/gram of fiber) of short chain fatty acid (Acetate, Propionate and Butyrate) was from plant based natural fibers as a sole nutritional source in vitro batch—culture fermentation with B. coagulans MTCC 5856. FOS was used as reference control in the study.
In Table 5 it may be noted that the production (mg/gram if fiber) of short chain fatty acid (acetate, propionate and butyrate) was from plant based natural fibers along with other nutrients in vitro batch culture fermentation with B. coagulans MTCC 5856. In MRS media dextrose was replaced by plant based natural fibers for the production of SCFA, FOS was used as reference control in the study. MRS media and Media devoid of Dextrose (MRSD) were also taken to compare for the production of the study.
Lycium barbarum seed fibers
Lycium barbarum seed fibers
While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conj unction with the appended claims.
This application is a continuation of U.S. patent application Ser. No. 14/839,923, filed on Aug. 29, 2015, which in turn claims the benefit of priority to U.S. Provisional Application Nos. 62/043,599, filed on Aug. 29, 2014, and 62/063,453, filed on Oct. 14, 2014. The disclosure of the prior applications is hereby incorporated by reference herein in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20030147857 | Monte | Aug 2003 | A1 |
| 20040197277 | Gonzales | Oct 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| 2014184639 | Nov 2014 | WO |
| Entry |
|---|
| Sudha et al., Biology and Medicine, 2009, vol. 1 (4): Rev4, p. 1-13. |
| Nutra, “Probiotics for Health and Well Being,” 2007, by Majeed and Prakash, 14 pgs. |
| Stig Bengmark et al.; “Prebiotics and Synbiotics in Clinical Medicine”; Nutr Clin Pract; vol. 20; pp. 244-261; Apr. 2005. |
| Gabriella A. Varga et al.; “Microbial and Animal Limitations to Fiber Digestion and Utilization”; J. Nutr; May 1, 1997; vol. 127; No. 5; pp. 819S-823S. |
| Ganguly et al.; Guidelines for Evaluation of Probiotics in Food; Indian Council of Medical Research, Department of Biotechnology; 2011; New Delhi, India. |
| Araya et al.; Guidelines for the Evaluation of Probiotics in Food; Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food; 2002; Food and Agriculture Organization of the United Nations and World Health Organization; London Ontario, Canada. |
| Probiotics: In Depth; National Center for Complementary and Integrative Health; 2016; National Institute of Health, U.S. Department of Health and Human Services; https://nccih.nih.gov/health/probiotics/introduction.htm. |
| National Biodiversity Authority, The Biological Diversity Act, 2002. |
| Number | Date | Country | |
|---|---|---|---|
| 20170274023 A1 | Sep 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62043599 | Aug 2014 | US | |
| 62063453 | Oct 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14839923 | Aug 2015 | US |
| Child | 15622616 | US |
