Note: Descriptions are shown in the official language in which they were submitted.
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Fructophilic lactic acid producing bacteria
BACKGROUND OF THE INVENTION
Field of the invention
[Para0011 The invention in general relates to fructophilic lactic acid
producing bacteria. More
specifically, the present invention relates to isolation, characterization and
biological applications
of fructophilic probiotic bacteria Bacillus coagulans.
Description of prior art
[Para002] Probiotics and its importance in health and prevention of many
disorders have been
already reported. Fructophilic lactic acid bacteria (FLAB) are a special group
of lactic acid
bacteria which utilizes fructose as a growth substrate. Due to their unique
characteristics of poor
growth on glucose and preference of oxygen, they are regarded as
"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 the
imbalance of
NAD/NADH and the requirement of additional electron acceptors to metabolize
glucose.
Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB have
significantly fewer
genes for carbohydrate metabolism than other LAB, especially due to the lack
of complete
phosphotransferase system (PTS) transporters. FLABs were originally classified
as Leuconostoc
species and later reclassified as Fructobacillus species based on their
phylogenetic positions 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 .1 Syst Evol Microbiol 58:2195-2205).
[Para003] The metabolism of fructose starts with the enzyme fructase in the
liver. The fructose
load is converted into lactate in enterocytes and in the liver. Further,
excess fructose in the liver
is directed toward peripheral tissues, and is taken up by the insulin-
dependent glucose
transporter, GLUT 4, present on adipose tissue is converted into fatty acids.
GLUT 4 is reported
to play an important role in the development of fructose-induced hepatic
steatosis and
dyslipidemia. Further, several fructose induced metabolic disorders are
reported like NASH,
NAFLD, Dyslipidemia, ectopic lipid deposition in the liver and skeletal
muscle, Uric Acid
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Metabolism, High Blood Pressure, 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 Jim; 9(6):
549). Excessive
fructose intake is also associated with increased cardiovascular risk.
Increase in fructose intake
may also lead to lactic acidosis by excessively decreasing the pH in blood.
[Para0041 Probiotics play an important role in metabolizing intestinal
fructose. The low dose of
fructose that is orally consumed, reach the intestine and is metabolized in
the presence of
different enzymes and inherent microbiota. However, in the presence of high
dose of fructose,
the load of fructose spills over from intestine to liver. In order to reduce
the spillover of fructose
from intestine to liver, probiotics play an important role to convert fructose
to SCFAs (Short
chain fatty acids).
[Para005] FLABs are isolated from various sources such as fruits and
vegetables, human stool
culture, natural antimicrobial agent, 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, animal rumen contents, Pengging Duck's caecum,
chicken intestine
and fecal samples, chicken feed, enzymes, fermented rice, curd, meat and yeast
extracts, glucose
and sucrose, human gut, human colonial epithelial cells, human and animal
vagina and mouth
extraction, diapers of human babies, pineapples wastes, industrial sausages,
ice-cream, small
intestines of piglets, corn slurry, crop and intestinal ducks. The following
prior art documents
describes 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, Fnictophilic 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
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[Para0061 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, See section 3.1
indicating that "The
current state of evidence suggests that probiotic effects are strain specific.
Strain identity is
important to link a strain to a specific health effect as well as to enable
accurate surveillance and
epidemiological studies."), there still exists a need to find a superior
fructophilic probiotic
bacteria with improved biological functions. The present invention solves the
above problem by
disclosing a novel fructophilic lactic acid bacteria Bacillus coagulans MTCC
25235.
[Para007] It is the principal objective of the invention to disclose a novel
fructophilic lactic acid
producing bacteria Bacillus coaguhins MTCC 25235 and its process of isolation.
[Para008] It is another objective of the invention to disclose the production
of short chain fatty
acid by fructophilic lactic acid producing bacteria Bacillus coagulans MTCC
25235.
[Para009] It is yet another objective of the invention to disclose the anti-
microbial effects of
fructophilic lactic acid producing bacteria Bacillus coagulans MTCC 25235.
[Para00101 The present invention solves the above mentioned objectives and
provides further
related advantages.
Deposit of Biological Material
[Paraffin] The deposit of biological material Bacillus coagulans strain FF7
bearing accession
number MTCC 25235, mentioned in the instant application has been made on 28th
February 2019
at Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of
Microbial
Technology, Sector 39-A, Chandigarh ¨ 160036, India.
Summary of the Invention
[Para00121 The present invention discloses the fructophilic lactic acid
producing bacteria. The
invention further discloses the process of isolation, characterization and
biological
applications/therapeutic use of fructophilic lactic acid producing bacteria.
[Para0013] Other features and advantages of the present invention will become
apparent from
the following more detailed description, which illustrate, by way of example,
the principle of the
invention.
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BRIEF DESCRIPTION OF DRAWINGS
[Para00141 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.
[Para0015] FIG. 1A, 1B, 1C and 1D shows the phase contrast microscopic image
(x 1000
magnification) of Bacillus coagulans MTCC 25235 (FIG. 1A), wet mount of cells
with the
spores (FIG. 1B), Gram staining of vegetative cells (FIG. 1C), Spore staining
of sporulated
cells and (FIG. 1D), colony grown on GYE agar plate.
[Para00161 FIG. 2 shows graphical representation of comparison of growth
pattern of Bacillus
coagulans FF7, MTCC 25235 and B. coagulans ATCC 31284 in presence of fructose
and
dextrose as carbon source. Values are mean ( SD) from three independent
determinations.
[Para0017] FIG. 3 shows graphical representation of utilization of fructose by
Bacillus
coagulans MTCC FF7, 25235 after 24 h of incubation in presence of media and
fructose rich
foods (Honey, fruit juice). Values are mean ( SD) from three independent
determinations.
[Para00181 FIG. 4 shows phylogenetic tree based on the sequence of 16S rRNA
showing the
relative positions of Bacillus coagulans FF7.
[Para0019] FIG. 5 shows effect of different pH on the growth of B. coagulans
FF7, MTCC
25235 and B. coagulans ATCC 31284, optimum pH for the growth was found to be
pH 7.5 and
pH 6.5 respectively. Values are mean ( SD) from three independent
determinations.
[Para00201 FIG. 6 shows graphical representation of effects of different
temperatures on the
growth of B. coagulans MTCC FF7, 25235 and B. coagulans ATCC 31284. Values are
mean
( SD) from three independent determinations.
[Para0021] FIG. 7 shows Effect of GIT hostile conditions on the viability of
B. coagulans
MTCC 25235 after gastric treatment in an in-vitro experiment mimicking invivo
conditions.
Sterile saline was taken as untreated control.
[Para0022] FIG.8 shows BSH activity of Bacillus coagulans MTCC 25235. (FIG.
8A)
Determination using soft MRS agar supplemented with ox bile (0.3%, w/v) and
CaCO3 (0.3%,
w/v) and MRS agar without bile salts served as the negative control (FIG. 8B).
Hollow zones
suggest the BSH activity of B. coagulans MTCC 25235.
[Para0023] FIG. 9 shows graphical representation of the survival of Bacillus
coagulans MTCC
FF7, 25235 at various pH values (1.5-8.0) in gastric juice buffer. The values
are expressed in
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Lot..)-10 spores/g. Data represent the mean and standard deviations ( SD) of
two different
experiments performed.
[Para0024] FIG. 10 shows graphical representation of in-vitro effect of ox
bile salt on the
growth of B. coagulans FF7, MTCC 25235 and B. coagulans ATCC 31284. The
overnight grown
fresh culture of B. coagulans MTCC 25235 and B. coagulans ATCC 31284 were
inoculated in
MRS broth with (0.1, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9 and 1%, w/v) and without ox
bile salt (%, w/v).
Values are mean ( SD) from three independent determinations.
[Para00251 FIG. 11 shows graphical presentation of production of L-Lactic acid
by B.
coagulans FF7, MTCC 25235 in presence of two standardized preparations
equivalent to 6x 109
(preparation 1) and 15x109 cfu/g (preparation 2) were studied. Values are mean
( SD) from
three independent determinations.
[Para0026] FIG. 12A, 12B and 12 C shows graphical presentation of Acetic FIG.
12A, butyric
FIG. 12 B and propionic acid FIG. 12C production from the Bacillus coagulans
MTCC 25235
while fermenting fructose, FOS, cranberry seed fibre, fenugreek seeds fibre.
Samples were
collected after 4, 8, 12, 18 and 24 h of fermentation. Values are average mean
of triplicate
performed at two different occasions and represented in mg/g.
[Para0027] FIG. 13 shows graphical presentation of Viability of B. coagulans
MTCC 25235
during the storage at temperature 40 2 C with RH 60 % 5 `)//0. Two
standardized preparations
equivalent to 15x 109 (preparation 1) and 6x109 cfu/g (preparation 2) were
studied. Average
means of spore viable counts are expressed in log10 cfu/g. Each time point
represents the mean
Logio standard deviations ( SD) of three different experiments performed in
duplicate.
DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
[Para0028] In the most preferred embodiment, the present invention discloses a
method for
isolating and identifying novel fructose utilizing probiotic bacteria from
honey, comprising step
of:
a) Mixing honey with saline in the ratio of 1:10 w/v to obtain a
suspension;
b) Thoroughly mixing the suspension of step a) and providing heat shock at
50-70 C for
30 minutes for selective isolation of spores;
c) Isolating the bacterial colonies by incubating 1-2 ml of the suspension
from step b) in
suitable culture media containing fructose for 48 hrs at 35-37 C;
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d) Purifying bacterial isolates by selecting and incubating morphologically
distinct
colonies in a suitable media containing fructose as carbon source;
e) Identifying the bacterial strain by biochemical analysis and 16S rRNA
sequencing as
Bacillus coagulans strain FF7 bearing accession no. MTCC 25235.
[Para00291 In a related aspect, the honey is selected from the group
comprising, 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, pinetree honey, sourwood honey, sage honey, and tupelo
honey. In
another related aspect, the culture media is selected from the group
comprising MRS (De
Man, Rogosa and Sharpe agar), GYA (Glucose Yeast Extract Agar), TSB (Tryptone
Soya
Broth), Sporulation media and Mueller Hinton Agar.
[Para00301 In another related aspect, the isolated probiotic strain returns
positive for
biochemical tests catalase, oxidase, methyl red, voges proskauers, 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 negative for biochemical tests
sorbose malonate
utilization, citrate utilization, xylitol, methyl mannoside, melezitose,
erythritol, adonitol, inositol,
dulcitol, glycerol, blood hemolysis, citrate and indole.
[Para0031] In another preferred embodiment, the invention discloses a novel
probiotic bacteria
of the genus Bacillus, isolated from honey for increased utilization of
fructose from foods rich in
fructose. In another related aspect, the fructophilic probiotic bacterium is
Gram positive. In yet
another aspect, optimum pH and temperature recorded for the growth of
fructophilic bacteria is
7.5 and 40 C respectively. In another aspect, the fructophilic probiotic
bacteria is bile tolerant,
gastric acid resistance and produces lactic acid. In a related aspect, the
fructophilic probiotic
bacteria is Bacillus coagulans. In yet another related embodiment, the
Bacillus coagulans strain
is Bacillus coagulans MTCC 25235. In a related aspect, the foods rich in
fructose are selected
from the group comprising 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 creamer, jams and jellies, energy drinks, condiments, ice
cream. In a related
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aspect, the fructophilic probiotic bacteria is used for the therapeutic
management of disorders
related to high fructose intake. In a related aspect, diseases related to high
fructose intake are
selected from the group comprising, but not limited, obesity, non-alcoholic
steatohepatitis
(NASH), insulin resistance, metabolic syndrome, cardio-vascular complications,
diabetes,
hyperlipidemia, hypertension, inflammation and hyperuricemia. In another
related aspect, the
fructophilic probiotic bacteria is present in the form of an inoculum, freeze-
dried powder, fine
powder, tablet, capsule, suspension, solution, emulsion, gummy, chewable or
edible foods and
administered as a stand-alone or in combination with foods rich in fructose
selected from the
group comprising 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 creamer, jams and jellies, energy drinks, condiments, ice
cream.
[Para0032] In yet another preferred embodiment, the invention discloses a
method of inhibiting
pathogenic microbes said method comprising step of bringing to contact said
microbes with the
fructophilic probiotic bacteria Bacillus coagulans MTCC 25235.
In a related aspect, the
pathogenic microbes are selected from the group comprising Salmonella abony,
Micrococcus
luteus, Es'cherichict coli, Pseudomonas aeruginosa, Bacillus cereus,
Propionibacterium acnes,
Streptococcus inutans, Staphylococcus aureusõS'taphylococcus epidermidis.
[Para0033] In another preferred embodiment, the invention discloses a method
of producing
short chain fatty acids by culturing the fructophilic probiotic bacteria
Bacillus coagulans MTCC
25235 along with plant fibers selected from the group consisting of fructose,
fenugreek seed
fibers, cranberry seed fibers, fructooligosaccharides (FOS).
[Para00341 The specific examples included herein below illustrate the
aforesaid most preferred
embodiments of the present invention.
[Para00351 EXAMPLES
[Para0036] Example 1: Isolation and identification of fructophilic bacteria
[Para00371 Methods
[Para00381 Raw unfiltered honey was used in this study to isolate the spore
forming
fructophilic lactic acid bacteria. For the isolation of fructophilic bacteria,
de Man, Roaosa and
Sharpe (MRS) (dextrose replaced by fructose) was used in this study (table 1).
[Para0039] Table 1: Media composition
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Sr. No. Ingredients Gms / Liter
1 Proteose peptone 10.000
2 Beef extract 10.000
Yeast extract 5.000
4 Fructose 20.000
Polysorbate 80 1.000
6 Ammonium citrate 2.000
7 Sodium acetate 5.000
8 Magnesium sulphate 0.100
9 Manganese sulphate 0.050
Dipotassium phosphate 2.000
11 Agar 12.000
12 Final pH ( at 25 C) 6.5 0.2
[Para0040] One gm of unfiltered raw honey was taken into the test tubes
containing 10 ml of
saline. This was mixed well and heat shock was given at 70 C for 30 minutes
for selective
isolation of spores. Isolation of bacteria was carried out by adding 1 ml of
above sample from
each dilution onto the MRS agar plates containing fructose. Plates were
further incubated at
37 C for 48 h. After incubation, morphologically distinct colonies were picked
up for further
testing and purification of bacterial isolates. Bacillus coagulans MTCC 25235
was isolated and
streaked on other MRS agar plate containing fructose as carbon source.
[Para00411 Biochemical characterization of fructophilic bacteria
[Para00421 Bacterial isolates were grown in de Man, Rogosa and Sharpe Agar
(MRSA) for 24
h at 37 C. Bacterial inoc-ulums were prepared by picking 1 to 3 well isolated
colonies and a
homogenous suspension in sterile saline was prepared. The density of
suspension was > 0.5 OD
at 620 nm. This inoculum (50 Ml) of was used and test was performed as per the
kit
manufacturer's instructions (HiMedia, Mumbai, India). Biochemical
characterization of Bacillus
coagulans MTCC 25235 and B. 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
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and phenotypic consistency of Bacillus coagulans MTCC 5856: A commercial
probiotic strain.
World Journal of Microbiology & Biotechnology, 32,60). HiCarbohydratem4 Kit
(code-KB009)
was procured from HiMedia, Mumbai, India and tests were performed as per the
manufacturer's
instructions. Bacterial suspension of Bacillus coagulans MTCC 25235 and B.
coagulans ATCC
31284 was prepared as mentioned in above section. Further, INIViC (indole,
methyl red, Voges
Proskauer, Citrate utilization) test, oxidase and Gram staining of and B.
coagulans ATCC 31280
were performed as per the method Majeed et al., (2016); (Cappucino G and
Natalie Sherman.
Microbiology: A Laboratory manual 5th edition. 1998).
[Para00431 16S rDNA sequencing
[Para004.11 16S rDNA sequencing Genomic DNA of the B. coagulans MTCC 5856 was
prepared as previously described by (William J. Bruno, Nicholas D. Socci, and
Aaron L. Halpern
(2000). Weighted Neighbor Joining: A Likelihood-Based Approach to Distance-
Based
Phyloaeny Reconstruction, Mol. Biol. Evol. 17 (1): 189-197). A fragment of the
16S rDNA gene
was sequenced using an ABI 3500 genetic analyser automated DNA sequencer as
described
earlier (Heyrman and Swings 2001). The sequencing primers used were 5¨
AGHGTBTGHTCMTGNCTCAS 3 (Forward Primer) and 5¨
TRCGGYTMCCTTGTWHCGACTH ¨ 3 (Reverse Primer). The amplified DNA fragment of
approximately 1.5 kb separated on a 1 % agarose gel and purified by using
Qiagen spin columns.
The purified fragment was used directly for DNA sequencing. This sequence was
used in a
BLAST search (http://bla s t.ncbi.nlm.nih. gov/Blast. c gi).
[Para0045] Growth conditions of Bacillus coagulans MTCC 25235
[Para00461 The optimization of growth conditions for Bacillus coagulans MTCC
25235 was
analyzed for different temperature and pH. MRSB media was prepared and
adjusted pH to 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 with 2 N HC1 and 2N NaOH. Overnight grown
culture (1%,
v/v) inoculated to pH adjusted media and incubated for 24h in a shaking
incubator at 120 rpm.
Growth was monitored at every six hour interval of time by measuring
absorbance at 600 nm
using spectrophotometer (Shimadzu Corporation, Kyoto, Japan). MRSB media was
prepared
adjusted pH to 6.5 and inoculated with overnight grown culture (1%, v/v).
Further, flasks were
incubated for 24h in shaking incubator at 120 rpm at different temperatures
(20, 30, 37, 40, 50
and 60 C). Growth was monitored at every six hour interval of time by
measuring absorbance at
600 nm using spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
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[Para00471 Results
[Para00481 Identification of fructophilic bacteria
[Para0049] The spores of Bacillus coagulans MTCC 25235 were ellipsoidal
terminal spores
(FIG 1A) and vegetative cells were Gram positive rod shaped as indicated in
Gram's stain (FIG
1B). Colonies from B. coagulans MTCC 25235 were grown on media, yielding
uniform, 1-3 mm
in diameter, white to cream, smooth colonies that contain vegetative rod
shaped cells (FIG. 1C
and D). The growth of the isolated bacteria was better in fructose rich media
rather than dextrose
rich media (FIG. 2), indicating its fructophilic nature and fructose
utilization potential when
compared to other Bacillus coagulans strains. The growth of the bacterial was
also tested in
fructose rich food stuff and showed a deduction in the fructose content (FIG.
3) implying that
the bacterium utilizes fructose as a carbon source for its growth and
development. Thus, the
bacterium can be used in managing and preventing disorders related to high
fructose intake by
utilizing the excess fructose in the food stuff for its metabolism. The
bacteria can be
administered as a stand alone or in combination with foods rich in fructose
which include 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 creamer,
jams and jellies, energy drinks, condiments, ice cream (Braverman J, List of
Foods high in
fructose, https://www.livestrong.com/article/30454-list-foods-high-fructosel,
accessed 31( May
2019)
[Para00501 Biochemical characterization
[Para0051] Biochemical characterization of Bacillus coagulans MTCC 25235 and
B. 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 compared with commercial strain B.
coagulans ATTCC
3128 and tabulated in Table 2.
[Para00521 Table 2: Biochemical characterization of B. coagulans ATTCC 3128
and Bacillus
coagulans MTCC 25235
S.No. Tests B.
coagulans ATTCC 3128 B. coagulans MTCC 25235
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1. Catalase Positive
Positive
2. Oxidase Positive
Positive
3. Indole Negative
Negative
4. Methyl Red
Positive Positive
5. Voges Proskauers
Positive Positive
6. Citrate Negative
Negative
7. Blood Hemolysis
Negative Negative
8. Gram staining Gram
Positive Gram Positive
9. Lactose Positive
Positive
10. Xylose Positive Positive
11. Maltose Positive Positive
12. Fructose Positive Positive
13. Dextrose Positive Positive
14. Galactose Positive Positive
15. Raffinose Positive Positive
16. Trehalose Positive Positive
17. Melobiose Positive Positive
18. Sucrose Positive Positive
19. L-Arabinose Positive Positive
20. Mannose Positive Positive
21. Inulin Positive Positive
22. Sodium gluconate
Positive Positive
23. Glycerol Negative Negative
24. Salicin Positive Positive
25. Dulcitol Negative Negative
26. Inositol Negative Negative
27. Sorbitol Positive
Positive
28. Mannitol Positive
Positive
29. Adonitol Positive Negative
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30. Arabitol Positive
Positive
31. Erythritol Negative
Negative
32. a-Methyl-D-glucoside Positive
Positive
33. Rhanmose Positive
Positive
34. Cellobiose Positive
Positive
35. Melezitose Negative
Negative
36. a-Methyl-D- Negative
Negative
mannoside
37. Xylitol Negative
Negative
38. ONPG Positive
Positive
39. Esculin hydrolysis
Positive Positive
40. D-Arabinose Positive
Positive
41. Citrate utilization
Negative Negative
42. Malonate
utilization Negative Negative
43. Sorbose Negative
Negative
[Para0053] The results indicated that the isolated probiotic strain is
positive for biochemical
tests catalase, oxidase, methyl red, voges proskauers, lactose, xylose,
maltose, fructose, dextrose,
galactose, raffinose, trehalose, melobiose, sucrose, melobiose, arabinose,
mannose,
sodium gluconate, salicin, sorbitol, mannitol, arabitol, methyl glucoside,
rhamnose, cellobiose,
ONPG, esculin hydrolysis and negative for biochemical tests sorbose malonate
utilization, citrate
utilization, xylitol, methyl mannoside, melezitose, erythritol, adonitol,
inositol, dulcitol, glycerol,
blood hemolysis, citrate and indole.
[Para00541 16S rDNA sequencing
[Para0055] The bacterial 16S rDNA was sequenced and the sequence information
(SEQ ID)
was obtained as below:
LOCUS seq FF7 1437 hp
ORIGIN
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 OTACGGGAGG CAGCAGTAGG GAATCTTCCG CAATGGAOGA AAGTOTGACG GAGCAACGCC
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361 GCGTGAGTGA AGAAGGCCTT CGGGTCGTAA AACTCTGTTG CCGGGGAAGA ACAAGTGCCG
421 TTCGAACAGG GCGGCGCCTT GACGGTACCC GGCCAGAAAG CCACGGCTAA CTACGTGCCA
491 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 CGTCAAATCA 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
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A BLAST (Basic local alignment search tool) search was performed with the
above sequence
and results of the first 10 alignment sequences are tabulated in table 3.
Table 3: Alignment view using combination of NCBI GenBank - Distribution of 10
Blast
Hits on the Query Sequence
S.No. Description Max Total Query E value Ident
Accession
score score cover
1 Bacillus coagulans 2562 2562 100% 0.0 98.96%
MF077122.1
strain 55-LR4 16S
ribosomal RNA gene,
partial sequence
2 Bacillus coatmlans 2556 2556 100% 0.0 98.89%
MF992239.1
strain KCCM203098
16S ribosomal RNA
gene, partial sequence
3 Bacillus sp. MC-02 2556 2556 99% 0.0 98.95%
AB849115.1
gene for 16S ribosomal
RNA, partial sequence
4 Bacillus coagulans gene 2556 2556 100% 0.0 98.89%
AB696800.1
for 16S ribosomal RNA,
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partial sequence, strain:
NTUIOB YUN2
Bacillus coagulans gene 2556 2556 100% 0.0 98.89% AB240205.1
for 16S rRNA, strain:
T5
6 Bacillus coagulans 2553 2553 100% 0.0
98.82% MH392659.1
strain ICMP 22322 16S
ribosomal RNA gene,
partial sequence
7 Bacillus sp. IMM05 2553 2553 100% 0.0 98.82%
FR727705.1
partial 16S rRNA gene,
strain IMM05
8 Uncultured Bacillus sp. 2551 2551 100% 0.0 98.82%
MG557779.1
clone Bco 16S
ribosomal RNA gene,
partial sequence
9 Bacillus coagulans 2529 2529 100% 0.0
98.54% CP009709.1
DSM 1 = ATCC 7050,
complete genome
Bacillus coagulans 2505 2505 100% 0.0 98.19% NR 115727.1
strain ATCC 7050 16S
ribosomal RNA gene,
partial sequence
[Para0056] The results indicated that the isolated organism is a new strain of
Bacillus coagulans
having 98.96% identity with Bacillus coagulans strain 55-LR4. The results of
the phylogenetic
analysis also indicated that the organism is a new strain of Bacillus
coagulans (FIG. 4)
[Para0057] Optimum of the growth conditions for fructophilic bacteria
[Para00581 The optimum pH and temperature recorded for the growth of
fructophilic bacteria
was 7.5 and 40 C respectively (FIG. 5 and FIG. 6)
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[Para00591 Example 2: In vitro probiotic evaluation of Bacillus coagulans MTCC
25235
[Para00601 Resistance to gastric acid
[Para0061] The survival of Bacillus coagulans MTCC 25235 spores was studied by
addition of
1 ml of suspension into 100 ml of a sterile electrolyte solution (6.2 g/L
NaCl, 2.2 g/L KC1, 0.22
g/L CaCl2. and 1.2 g/L NaHCO3) containing 0.01% lysozyme (Sigma-Aldrich) and
0.3% pepsin
(Sigma-Aldrich) and incubated for 5 mm. Further, pH was adjusted to 1.5, 3, 4,
5, 6, 7 and 8
(adjusted using 1 N NaOH and 1 N HCl). Incubation temperature was monitored to
37 C for 4h.
One ml of sample was withdrawn at different time intervals at 0, 1.0, 2.0, 3.0
and 4.0 h. After
incubation, serial dilution was done in sterile saline (0.89 % wily) and the
viable count was
enumerated by plating on glucose yeast extract agar (HiMedia). Experiments
were performed in
triplicate at two different occasions.
[Para0062] Bile salt tolerance
[Para00631 Bile tolerance of Bacillus coagulans MTCC 25235 cells was
determined by the
method described earlier (Gilliland et al. 1984; Hyronimus et al. 2000). MRS
broth (HiMedia)
was inoculated with approximately 106 cfu mL-1 of Bacillus coagulans MTCC
25235 overnight
grown culture and then supplemented with (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 /0, w/v) bile salt and without bile salt as control in the experiment.
Samples were incubated
for 24 h at 37 C with shaking at 120 tpm. Growth in control (no bile) and
test cultures at
different concentrations of bile was monitored hourly by measuring absorbance
at 600 nm using
spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
[Para0064] Production of lactic acid
[Para00651 Lactic acid production by Bacillus coagulans MTCC 25235 was
estimated by using
a Megazyme kit. A loopfill of an overnight grown culture of Bacillus coagulans
MTCC 25235
was added to glucose yeast extract broth (HiMedia) and incubated at 37 C for
18 h with 120
rpm. After incubation, the broth was filtered through 0.22 micron (Sartori-us,
India) and analyzed
for lactic acid content by using Megazyme kit (K-DLATE 10/04) as per
instructions (Megazyme
International Ireland, IDA Business Park, Wicklow, Ireland). GYE media was
taken as blank in
the assay.
[Para00661 Simulated gastric juice tolerance
[Para0067] The survival of Bacillus coagulans MTCC 25235 spores was studied to
represent
buccal digestion conditions. One mL of the suspension of Bacillus coagulans
MTCC 25235 was
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subjected to stimulated salivary juice contents consist of KC1 (0.8946 g/l),
CO (Nf2)2 (0.1981
g/L), Na2SO4 (0.5681 g/L), NaHCO3 (1.680 g/L), Nall24304(0.8878 g/L), pH
adjusted to 6.8 0.2
and incubated at 37 C for 5 min. Further, simulated gastric juice was prepared
by adding 9 g/L
of sodium chloride and 3 g/L of pepsin (Sigma¨Aldrich, St. Louis, MO, USA) and
then the pH
was adjusted to aseptically 3.0 0.2 by using 2 N HCI. Samples were further
incubated at 37 C
for 3 h with low r.p.m. After 3 h of incubation, pH was adjusted aseptically
to 7.0 using 2 N
NaOH. Ox bile (5 g/L) this was further incubated at 37 C for 24 h. After
final step of the
simulated digestion process, samples were collected and evaluated for the
spore survival of
Bacillus coagulans MTCC 25235 was enumerated by serial dilution method using
glucose yeast
extract agar media (HiMedia) (FIG. 7).
[Para0068] Antibiotic resistance pattern
[Para00691 MIC was determined as per the guidelines of Clinical and Laboratory
Standards
Institute (CLSI 2012). Bacillus coagulans MTCC 25235 suspensions were prepared
by
suspending 18 h grown bacterial culture in sterile normal saline (0.89% NaCl
wt/vol; Himedia,
Mumbai India). The turbidity of the bacterial suspension was adjusted to 0.5
McFarland
standards (equivalent to 1.5 x 108 colony forming units (CFU)/m1). The
antibiotics stock
solutions were prepared as per CLSI guidelines and 2-fold serial dilutions
were prepared in broth
medium (glucose yeast extract acetate broth [GYEA, HiMedia, Mumbai India for
Bacillus
coagulans MTCC 25235 and Mueller Hinton Broth [MHB, Difco Laboratories,
Detroit, Mich
USA] for S`. aureus) in 100 p.1 volume in 96-well U bottom microtiter plates
(BD Labware, NJ
USA). The above-mentioned bacterial suspension was further diluted in the MHB
and 100 pl
volume of this diluted inoculum was added to each well of the plate resulting
in the final
inoculum of 5 x 105 CFUlml in the well and the final concentration of
antibiotics ranged from
0.0078 to 4 pg/ml. S. aureus ATCC 6538 was used as reference culture in this
study. The plates
were incubated at 37 C for 24 h and were visually read for the absence or
presence of turbidity.
The minimum concentration of the compound concentration showing no turbidity
was recorded
as MIC.
[Para0070] AMES TEST
[Para0071] The data of the experiment suggested that the B. coagulans MTCC
25235 spores
did not increase the number of revertants in five Salmonella strains (TA98,
TA100, TA102,
TA1535 and TA1537), compared with their negative controls, either absence or
presence of the
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S9 metabolic activation system. Further, no dose- dependent mutagenic effects
were caused by
the B. coaguhins MTCC 25235 spores (up to 5000 us/plate). B. coagulans MTC
25235 spores
did not show any mutagenic activity under the experimental conditions.
[Para0072] BSH activity
[Para00731 Bacillus coagulans MTCC 25235 growth was observed on an agar plate
containing
ox bile and calcium carbonate, which indicates its tolerance against ox bile
and the presence of
BSH activity. There was 19 2 mm clear zone in the soft agar plate as shown in
(FIG. 8A and
8B) which indicated the presence of BSH activity.
[Para00741 Antimicrobial activity against human pathogens
[Para0075] The antimicrobial activity was performed by a well diffusion assay
as previously
described 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.
App! Environ
Microbiol 61:2643-2648). Briefly, a 5 mL lawn of soft (0.7 % agar) glucose
yeast extract
(HiMedia, India), containing 106 cfil mL-I) of the indicator strains (S.
epidennidis ATCC 14990,
S. inuons MTCC 1943õS`. wireus ATCC 29213, B. cereus ATCC 14579, P. acnes ATCC
11827,
E. coli ATCC 25922, P. aeruginosa ATCC 9027, M luteus NCIM 216 and S. abony
NCIM
2257) was poured on top of an enriched hard (1.5 % agar) tryptic soya agar
(HiMedia, India). A
loopful of overnight grown culture B. coagulans MTCC 25325 was added to MRS
media and
incubated for 24 h at 37 C with 120 rpm. After 24 h, the culture was
centrifuged (10,000x9g) to
remove the cells and the supernatant was collected, concentrated tenfold by
lyophilization and
filter-sterilized through a 0.22 micron filter (Sartorius, India).
Concentrated supernatant (50 IlL)
was added to 6-mm wells punched in the solidified hi-layer agar. Plates were
kept in the
refrigerator (4 2 C) for 5 h to allow the sample to diffuse into the agar
and subsequently
incubated at 37 C for 18-20 h. After incubation, the zone of inhibition was
measured and
recorded in mm.
[Para00761 Production of short chain fatty acids
[Para00771 The in vitro fermentation with the Bacillus coagulans MTCC 25235
was carried out
by following method described by McBumey and Thompson (1987) with some
modifications.
Briefly, 2.0 g of fructose, fenugreek seeds, cranberry seed fiber, FOS, were
added to 100 ml of
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demineralised water. The pH was adjusted to 7.0 0.2 and autoclaved at 121 C
for 20 mm. After
sterilization, oxygen reducing enzyme Oxyrase (Oxyrase for Broth, Oxyrase,
Inc, OH, USA)
was added to each flask, to induce anaerobic conditions. Five percent of
overnight grown
Bacillus coagulans MTCC 25235 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. The 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-Aldrich,
St. Louis, MO,
USA). Further, 5.0 ml of samples were added to 5 ml of distilled water and pH
was adjusted to
1.5 using 3 M H2504. 10 ml of chilled diethyl ether (-20 C) was added to
samples and then
vortex for 1 minute. Sodium chloride was added and then centrifuged at 3000x g
for 10 minutes.
After centrifugation, organic layer was separated and transferred to the fresh
vial. This was used
to quantify SCFAs. The SCFA standards were purchased from Sigma-Aldrich (St.
Louis, MO,
USA) and similarly processed. SCFA production (acetate, propionate and
butyrate) was
measured by gas chromatography (GC) with the use of a Agilent technologies
6890N gas
chromatograph (Stevens Creek Blvd Santa Clara, CA, USA) containing a DB-FFAP
(Terephthalic acid modified poly ethylene Glycol) column. 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/gram of
galactomannan
from fenugreek seeds.
[Para00781 Result
[Para00791 Resistance to gastric acid
[Para0080] There was no significant difference (2-5%) in spore count at pH 3
to pH 8.0 in
comparison to the initial spore count up to 4 h of the study (FIG. 9).
However, 0.44 and 2.036
logo reduction was observed at pH 1.5 in 1 and 4 h respectively. Results of
the study confirmed
the stability of Bacillus coagzilans MTCC 25235 spores in acidic as well as
alkaline pH
conditions.
[Para0081] Bile tolerance test
[Para0082] Bacillus coag-ulansIVITCC 25235 growth was observed on the agar
plate containing
bile salt (1 % w/v) which indicated its tolerance against bile salt. Further,
bile tolerance assay
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was performed by supplementing (0.1-2.0%) ox bile to the MRS broth in
different flask. Bacillus
coagulans MTCC 25235 growth observed in presence and absence of ox bile up to
2%, w/v.
Whereas, growth of B. coagulans ATCC 31284 was found in 0.8% w/v (FIG. 10).
Similarly,
there was no significant difference in the viability of Bacillus coagulans
MTCC 25235 and B.
coagulans ATCC 31284 in the presence and absence of bile salt.
[Para00831 Production of lactic acid
[Para0084] Lactic acid production by Bacillus coagulans MTCC 25235 was
estimated by using
a Megazyme kit. The total lactic acid produced by Bacillus coagulans MTCC
25235 was 4.487
g/L. L-form of lactic acid was 4.12 g/L. Whereas, D-form of lactic acid
Bacillus coagulans
MTCC 25235 is 0.367 g/L. (FIG. 11)
[Para00851 Antibiotic resistance
[Para0086] MIC results of clindamycin, kanamycin, ampicillin, streptomycin,
vancomycin,
erythromycin, gentamicin, tetracycline and chloramphenicol against the
Bacillus coagulans
MTCC 25235 and S. aureus ATCC 6538 is given in table 4. All the antibiotics
tested showed a
MIC range of 0.0078 to 1.0 1,ig/m1 against Bacillus coagulans MTCC 25235. All
tested
antibiotics exhibited a MIC range of 0.031 to 2 tg/m1 against S. aureus ATCC
6538.
[Para00871 Table 4: Minimum inhibitory concentrations of antibiotics against
Bacillus
coagulans MTCC 25235 cultures and S. aureus ATCC 6538
S.No. Antibiotics MIC (fig/m1)*
Bacillus coagulans MTCC 25235 S. aureus ATCC 6538
1 Clindamycin hydrochloride 0.25 0.062
2 Kanamycin sulphate 0.062 2.0
3 Ampicillin sodium salt 0.125 0.031
4 Streptomycin sulphate 0.062 4.0
Vancomycin hydrochloride 0.25 0.5
6 Erythromycin 0.125 0.125
7 Gentamicin sulphate 0.06 0.25
8 Tetracycline hydrochloride 0.03 0.062
9 Chloramphenicol 0.031 2.0
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[Para00881 Antimicrobial activity
The antimicrobial activity of B. coagulans MTCC 25235 was evaluated. Anti-
microbial activity
was determined by well diffusion assay as described by Cintas et al. (1995).
The results
indicated that the probiotic bacteria is an effective antimicrobial agent
against Salmonella
abony, Micrococcus luteus, Escherichia coil, Psettdomonas cieruginosci,
Bacillus cereus,
Propionibacterium acnes, Streptococcus mutansõS'taphylococcus aureus,
Staphylococcus
epidermidis.
[Para00891 Table 4: Antimicrobial activity of B. coagulans MTCC 25235 against
tested
bacteria
S. no. Tested organism Zone of inhibition
1 Salmonella abony NCIM 2257 14.00 1.0
2 Micrococcus luteus NCIM 2169 18.50 1.6
3 Escherichia coil ATCC 25922 19.00 2.0
4 Pseudomonas aeruginosa ATCC 9027 17.42 1.8
Bacillus cereus ATCC 14579 17.00 2.1
6 Propionibacteriurn acnes ATCC 11827 16.50 1.2
7 Streptococcus mutans MTCC 1943 17.50 1.75
8 Staphylococcus aureus ATCC 29213 15.85 1.25
9 Staphylococcus epidermidis ATCC 14990 14.85 1.40
Data represent the mean SD of three independent experiments performed in
triplicate
[Para00901 Short chain fatty acid production
[Para00911 The results of the analysis are presented in FIG. 12A, 12B and 12C.
The
production of acetic acid was high for B. coagulans MTCC 25235 with FOS,
followed by
fenugreek seed fibers, cranberry fibers and fructose (FIG. 12 A). Similarly,
the production of
butyric acid was high for B. coagulans MTCC 25235 with fructose, followed by
FOS, fenugreek
seed fibers and cranberry fibers (FIG. 12 B). The production of propionic acid
was similar for B.
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coagulans MTCC 25235 cultured with fructose, FOS and cranberry seed fibers and
lower for
fenugreek seed fibers (FIG. 12 C).
[Para00921 Storage and viability
[Para00931 Two standardized preparations equivalent to 15x 109 (preparation 1)
and 6x109
cfii/g (preparation 2) were studied. B. coagulans MTCC 25235 showed enhanced
viability during
the storage at temperature 40 2 DC with RH 60 c',70 5 Ã)./O (FIG. 13)
[Para00941 Other modifications and variations to the invention will be
apparent to those skilled
in the art from the foregoing disclosure and teachings. Thus, while only
certain embodiments of
the 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 is to be interpreted only in conjunction with the
appended claims.
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