The present invention relates to the development of a novel multi-strain synergistic anti-inflammatory
synbiotic formulation comprising of two anti-inflammatory putative probiotic bacteria along with its
preferable non-digestible carbohydrates such as isomaltooligosaccharides and anti-inflammatory, nondigestible millet dietary fiber, the arabinoxylan. The synergistic anti-inflammatory synbiotic formulation
confers better protection against IBD and more specifically against ulcerative colitis rather than bacteria or
non-digestible carbohydrates alone.
4.2. BACKGROUND OF THE INVENTION
Dysbiosis in the gastrointestinal microbiota causes obesity, diabetes, cancer and many other inflammatory
conditions such as inflammatory bowel disease (IBD) including Crohn’s disease and Ulcerative colitis (UC).
IBD involves inflammation of the digestive tract. Crohn’s disease can occur anywhere between mouth and
the anus in the form of patches while UC is limited to the colon and causes long-lasting inflammation and
ulcers in the colon. The multifactorial causal agents of IBD include genetic factors like polygenic disease,
susceptible loci and HLA gene association; environmental factors like nutritional and social factors; and
disrupted microflora-immune response (Ananthakrishnan, 2015). According to National Center for Chronic
Disease Prevention and Health Promotion (NCCDPHP) report, internationally, the prevalence for IBD is 396
cases per 100,000 persons annually and the numbers are escalating as a result of the shift in life style
including decreased physical activity, eating habits and irrational intake of antibiotics. An increasing
prevalence of the inflammatory diseases is observed in India as well which is due to rapid urbanization and
westernization of diets and poor physical activity (Kedia & Ahuja, 2017; Ng et al., 2013). Increased levels of
Th1 and decreased levels of Th2 cells in patients suffering from IBD have been suggested (Zang & Li, 2014).
However, understanding the exact etiology of IBD is still challenging and gut microbial involvement in the
development of IBD is strongly believed (Ni et al., 2017).
Therapeutic regimens for UC such as the use anti-inflammatory drugs, immune suppressors, biological
therapy and the last substitute, colectomy i.e. surgical removal of the inflamed colonic part, is being in
usage. However, side-effects of the therapeutics such as inflammation of pancreas, kidney disease, and
increase risk of infections, insomnia, weight gain, hypertension, osteoporosis and cataract have been
reported (Ardizzone et al., 2006; Ordas et al., 2012). The major side effect of biological therapy like
monoclonal antibodies, against inflammatory cytokines is remission of the symptoms (Neurath et al.,
2014). Hence, there is a need for newer therapeutic and preventive approaches with minimum side
effects. Dietary manipulations like intake of prebiotics and probiotics; and fecal transplant are suggested
as alternative and safe approach for mitigating IBD and associated comorbidities (Sunkara et al., 2018).
Balanced diets that are rich in dietary fiber, prebiotics, proteins, omega fatty acids and fermented foods
have been shown beneficial under inflammatory due to their beneficial impact on the resident gut
microbial population. Intake of dairy products and fermented foods could deliver beneficial bacteria such as
Lactobacillus, Bifidobacterium and several others that impart beneficial effects against many inflammatory
and metabolic complications like diabetes and obesity (Porras et al., 2018; Zhang et al., 2018). Prebiotic
substances and dietary fibers not only help in the development of healthy gut bacteria by selectively
stimulating the growth and/or activating the metabolism of one or a limited number of health promoting
bacteria in the GIT but also demonstrate antimicrobial activity against many pathogenic bacteria such as
Clostridium difficile (Kondepudi et. al, 2012). A synbiotic is a mixture of probiotics and prebiotics that can
selectively stimulates the growth and activity of one or limited number of health- promoting bacteria at the
same time implants the exogenous bacteria in the gut. Fermentation of the prebiotics and dietary fibers by
the exogenously administered and gut resident bacteria helps in the production of secondary metabolites
such as acetate, propionate and butyrate and these help in the improvement of the host’s immunity and
mucosal health. A study suggested the cross-feeding association between Bifidobacterium infantis and
butyrate producing Anaerostipes caccae. Acetate produced by Bifidobacterium infantis was utilized by
A.caccae to produce butyrate, a key SCFA that helps in improving gut-barrier integrity (Chia et al., 2018). In
another study, Belenguer et. al, (2006) has showed functional potential of co-cultured Bifidobacterium
adolescentis and butyrate-producing anaerobes along with fructoligosacharides as prebiotic source against
gut dysbiosis. However, there are no multi-strain synergistic synbiotic formulations that are made based on
Indian origin indigenous strains, prebiotics and especially in a blend with millet dietary fiber. Therefore, the
present innovation is aimed at developing a novel multi-strain synergistic anti-inflammatory synergistic
synbiotic formulation comprising probiotics with its preferable prebiotic and millet dietary fibre that can
prevent DSS induced IBD more specifically ulcerative colitis.
4.3. STATEMENT OF INVENTION
For achieving the beneficial modulation of the gut microbiota through dietary approach, a novel multistrain synergistic anti-inflammatory synergistic synbiotic formulation comprising two anti-inflammatory
putative probiotic Bifidobacterium strains that are compatible with each other blended with a prebiotic,
that is isomaltooligosaccharides, and an anti-inflammatory dietary fiber from millets especially from finger
millet or popularly known as ragi. Both the bacterial strains could reduce the lipopolysaccharide induced
inflammation in the murine macrophage and human intestinal epithelial cell lines either as single cultures
or as in combination. Isomaltooligosaccharides is being metabolized by both the bacterial strains making it
a synergistic synbiotic while the millet dietary fiber isolated from the bran exerts anti-inflammatory activity
besides promoting healthy gut bacteria. The formulation is more potent in preventing dextran sodium
sulphate (DSS) induced colitis symptoms in Balb/c mice than the individual ingredients of the formulation
in.
4.4. DETAILED DISCLOSURE OF THE INVENTION
In detail, the process of synbiotic preparation and its preventive effect against colitis in rodent model
comprises of the following steps.
4.4.1 Identification and prioritization of Anti-inflammatory Bifidobacterium strains
i. Bacterial strains were isolated from the human infant feces after taking consent from their parents.
ii. Strains having an ability to reduce nitric oxide production and reduction in proinflammatory
cytokines by the LPS treated murine macrophage cells (RAW264.7) and intestinal epithelial cell
(Caco2) respectively were selected for animal studies.
iii. Probiotic attributes of the selected strains were evaluated as per ICMR-DBT guidelines using
established protocols.
4.4.2 Selection of preferable prebiotic sugar for the formulation
Preference of the selected strains for a prebiotic and a dietary fiber was establish using agar plate
assay and growth profiles were determined using batch fermentation assays. The most preferable
prebiotic i.e. isomaltooligosaccharides and the anti-inflammatory millet dietary fiber i.e. arabinoxylan
were included along with the bacterial mix.
4.4.3 Protective efficiency of Synbiotic formulation against IBD Synbiotic preparation:
Preventive ability of the synbiotic formulation was evaluated in DSS induced ulcerative colitis in Balb/c
mice for 25 days. Post one-week acclimatization on normal pellet diet (NPD), the mice were
randomized and allocated to different groups according to their weights. In the intervention groups
probiotics (Bif10+Bif11), prebiotics (AX/IMOS) and synbiotic (Bif10+Bif11+AX+IMOS) were gavaged
orally on every day right from the zeroth day till the end of experiment. DSS (2.5% w/v) was mixed in
drinking water and provided ad-libitum on day 11th to 18
th day as per the schedule. At the end of the
experiment, blood was collected from the mice and plasma was used to determine the levels of
proinflammatory markers. Fresh feces, cecal content and colonic tissues were collected in aseptic vials
and snap frozen and stored at -80o
c until further use.
Example 1: Identification and prioritization of Anti-inflammatory Bifidobacterium strains:
(i) Identification of the bacterial strains
Infant faecal samples were collected from Post Graduate Institute of Medical Education and Research
(PGIMER), Chandigarh following the laws and guidelines properly. Consent from the infant’s parents and
ethical approval from the institute’s ethical committee was taken using serial dilution on MRS agar plates
supplemented with 0.05% L-Cystine and mupirocin. Plates were incubated under anaerobic conditions
(80% N2, 10% CO2 and 10% H2) in an anoxomat jar at 37°C for 48 h. To get the pure culture, a single colony
of the strains was streaked on to fresh MRSC agar plates. A total of 25 strains were isolated, which were
sub-culture and stored as glycerol stock cultures at −80 °C. The presence of Fructose 6-phosphate
phosphoketolase gene confirmed the genus of the strains. Species level of identification of the strains was
carried out using 16sRNA gene sequencing followed by nucleotide Blast analysis at the NCBI database.
Sequences obtained were submitted GenBank.
(ii) Protection against LPS induced inflammation in RAW264.7
RAW264.7 cells obtained from National Centre for Cell Science (NCCS), Pune, India were cultured in DMEM
medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO2 in a
humidified incubator. Media was changed every second day until full confluence was attained. Antiinflammatory activity of all the isolated strains was investigated in LPS-induced pro-inflammatory stress in
RAW 264.7 cells. Briefly, confluent macrophages in 96-well plate were treated with bacteria, either in
presence or absence of LPS. Nitric Oxide (NO) production was checked via adding 100μl of Griess reagent
in 150μl of the supernatants. The level of NO was determined by measuring OD at 540 nm using ELISA
reader. RAW 264.7 macrophages showed 100% viability when treated with Bifidobacterial strains at 1 ×
1010 CFU/ml.
Cell culture supernatants treated with LPS and the bacterial strains showed a decreased production of
TNF-α, IL-6 and IL-1β while per-se treatment with these strains did not stimulate the production of these
cytokines except with Bif 4 where its per-se treatment resulted in the increased production of TNF-α.
Based on the maximum reduction in NO and TNF-α, IL-1β and IL-6 production upon LPS and Bifidobacteria
co-treatment and lack of higher production relative to macrophages upon per-se treatment with
Bifidobacteria, twelve strains were shortlisted and their biochemical properties and probiotic attributes
were determined. Bif4, 6, 10, 11, 12, 14, 16, 17, 20, 29, 30 and 40 strains showed highest reduction of NO
production upon co-treatment with LPS on the macrophages. Per-se treatment of macrophages with Bif
strains did not stimulate the NO production by all the strains except Bif 4 and 29.
Figure 1. Nitric Oxide production in RAW 264.7 cells after 16 hours of with and without LPS treatment in
top twelve strains of Bifidobacterium. (* P < 0.05 versus control; # P < 0.05 versus LPS)
Effect on intestinal epithelial cell (Caco2) inflammation:
To confirm the anti-inflammatory effect of the selected four bacterial strains, epithelial cell line (Caco2)
procured from National Centre for Cell Science (NCCS), Pune, India were cultured in DMEM medium
containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO2 in a humidified
Cnrl LPS Bif 1 Bif 1 + LPS Bif 2 Bif 2 + LPS Bif 3 Bif 3 + LPS Bif 4 Bif 4 + LPS Bif 6 Bif 6 + LPS Bif 10 Bif 10 + LPS Bif 11 Bif 11 + LPS Bif 12 Bif 12 + LPS Bif 14 Bif 14 + LPS Bif 15 Bif 15 + LPS Bif 16 Bif 16 + LPS Bif 17 Bif 17 + LPS Bif 18 Bif 18 + LPS Bif 19 Bif 19 + LPS Bif 20 Bif 20 + LPS Bif 21 Bif 21 + LPS Bif 22 Bif 22 + LPS Bif 23 Bif 23 + LPS Bif 24 Bif 24 + LPS Bif 25 Bif 25 + LPS Bif 26 Bif 26 + LPS Bif 29 Bif 29 + LPS Bif 30 Bif 30 + LPS Bif 34 Bif 34 + LPS Bif 37 Bif 37 + LPS Bif 38 Bif 38 + LPS Bif 40 Bif 40 + LPS
0
15
30
45
60
75
90
105
120
#
#
#
# #
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
*
*
*
*
*
*
*
*
*
*
*
* *
*
* %
* Nitric Oxide Production
incubator. Media was changed every second day until confluence was attained (15days). The cells were
then treated with TNF-α for 24 h to induce inflammation. On the next day cells were treated with fixed O.D
of bacterial suspension in DMEM with or without LPS for 24 h. Next day supernatants were collected from
the respective treatment groups and the levels of IL-8 was determined using commercially available ELSIA
kits as per the manufacturer instructions. Results obtained suggested all the strains showed reduction in
LPS induced inflammation.
Figure 2. Assessment of IL-8 levels in LPS inflamed Caco2 cells after treatment with top four strains
Bif10, Bif11, Bif12 and Bif16 strains (TNF-α stimulated) (* P < 0.05 versus control; # P < 0.05 versus LPS)
(iii) In-vitro probiotic characterization of the selected strains:
Probiotic attributes of the twelve selected strains were checked as per ICMR guidelines. For stress
tolerance test (Table 1.A.) percentage viability was checked in presence of gastric (pH 2.5-3.0) and bile
juices (0.2%-0.4%). Also antimicrobial activity, cholesterol lowering and adhesion to mucin (Table 1.C.),
and cell surface hydrophobicity (Table 1.B.) were determined as described in Singh et al. (2018). On the
basis of the above experiments, four potent strains Bif10, Bif11, Bif12 and Bif16 were shortlisted for
further studies.
Table 1. Comparison of major probiotic attributes like; Stress tolerance (%viability) (A), Cell surface
properties (B), and Antimicrobial activity, Mucin binding (%) and Cholesterol lowering assay (%) (C)of the
top twelve strains: Bif4, 6, 10, 11, 12, 14, 16, 17, 20, 29, 30 and 40.
1.A.
Stress tolerance (% viability)
Strains Identity Source Acid (pH) Bile
2.5 3.0 3.5 0.2 % 0.4%
Bif 4 B. pseudocatenulatum Infant 2 82 80 85 87 87
Bif 6 B. longum Infant 3 - 74 84 79 60
Bif 10 B. longum Infant 4 56 80 84 82 73
CntrlLPSBif 10 Bif 10 + LPSBif 1 Bif 1 + LPSBif 12 Bif 12 + LPSBif 16 Bif 16 + LPS
0
5
10
15
20
25
30
* # #
# C
# onc.(pg/ml)
Bif 11 B. breve infant 4 60 86 91 96 73
Bif 12 B. longum Infant 5 63 57 73 80 60
Bif 14 B. longum Infant 5 70 59 82 79 75
Bif 16 B. longum Adult 1 69 80 90 80 69
Bif 17 B. longum Adult 1 - 61 64 83 83
Bif 20 B. longum Adult 1 - 68 87 84 77
Bif 29 B. longum Adult 2 - 69 72 78 84
Bif 30 B. longum Adult 2 - 78 77 72 74
Bif 40 B. breve VSL-3 - 76 81 89 88
1.B.
Cell surface properties
Salt aggregation CRB (%)
% Autoaggregation
CSH
Strains
Agar
grown
Broth
grown
Agar
grown
Broth
grown Xylene Hexane
Chloroform
Hexadecane
Bif 4 ≥ 3.2 M ≥ 3.2 M 23 26 25 30 31 27
Bif 6 ≥ 3.2 M ≥ 3.2 M 23 26 11 16 30 25 27
Bif 10 ≥ 0.02 M ≥ 0.02 M 22 25 13 61 55 58 40
Bif 11 ≥ 3.2 M ≥ 3.2 M 29 22 41 16 17 14 13
Bif 12 ≥ 3.2 M ≥ 3.2 M 19 31 7 40 28 56 39
Bif 14 ≥ 4 M ≥ 4 M 13 16 22 27 31 31 16
Bif 16 ≥ 3.2 M ≥ 3.2 M 17 52 13 10 8 12 17
Bif 17 ≥ 3.2 M ≥ 3.2 M 28 19 5 17 13 46 23
Bif 20 ≥ 3.2 M ≥ 3.2 M 16 18 8 9 8 7 10
Bif 29 ≥ 3.2 M ≥ 3.2 M 32 32 5 1.2 9.2 14.3 11.3
Bif 30 ≥ 3.2 M ≥ 3.2 M 16 54 11 4 3 44 43
Bif 40 ≥ 3.2 M ≥ 3.2 M 13 15 26 15 6 17 13
1.C.
Anti-microbial activity (AMA)
S. typhi E. coli S. aureus Cholesterol Mucin
Strains
Inhibitio
n
zone
(mm) Inhibition
zone
(mm) Inhibition
zone
(mm)
Binding lowering assay
Bif 4 ++ 17 ++ 14 + 16 71 65
Bif 6 ++ 18.5 ++ 17 ++ 17.5 14 42
Bif 10 ++ 17 ++ 15.5 ++ 14 11 34.4
Bif 11 ++ 16 ++ 15 ++ 16 56 28.35
Bif 12 ++ 13 ++ 15 ++ 14 25 38.05
Bif 14 ++ 16.5 ++ 17 + 15.5 10 22.75
Bif 16 ++ 20 ++ 17.5 + 18.5 13 44.4, 8.4
Bif 17 ++ 20 ++ 19 + 16 25.3 21.6
Bif 20 ++ 20 ++ 19 + 20 24.6 17.9
Bif 29 - - - 43 61.7
Bif 30 ++ 14.5 ++ 15.5 ++ 13.5 37 18.2
Bif 40 ++ 19 ++ 14.5 + 16 55.4 32.4
Note: 1+ weak (1 mm); 2+, moderate (2 mm); 3+, strong degradation (3 mm).
Example 2: Selection of preferable prebiotic sugar for the formulation:
Prebiotic metabolism efficiency of the strains was determined using spot assay on modified MRSC agar
plates of pH 6.8 and having 0.5% (w/v) prebiotic, either isomaltooligosaccharides (IMOS) or
fructooligosaccharides (FOS) or inulin or starch or resistant starch (RS) or arabinoxylan and 0.05% phenol
red as an indicator dye. A single colony of each strain was spotted on the plates and incubated at 37oC for
48 h. Color change from red to yellow indicates the metabolism of the prebiotic or dietary fiber. An
arbitrary score from 1 to 3 was given depending upon the width (mm) of the yellow zone around the
bacterial colony. The selected strains of were able to utilize IMOS efficiently which was further confirmed
by level of carbohydrate utilization using phenol sulphuric assay. Growth profiles of the four
Bifidobacterium strains were also generated in IMOS and AX supplemented Basal MRSC broth. For the
synbiotic formulation development the most preferable prebiotic i.e. isomaltooligosaccharides (IMOS) is
selected and the dietary fiber i.e. arabinoxylan (AX) is selected owing to its anti-inflammatory activity in
vitro on the murine macrophages when co-treated with LPS.
Table 2. Prebiotic profiling of top four strains Bif10, Bif11, Bif12 and Bif16 strains using
Isomaltooligosaccharides (IMOS); Fructooligoasaccharides (FOS); Inulin; Soluble starch (SS); and
Resistant Starch (RS)
Strain Bacteria (Patent
Deposited)
Isolation
Source
MTCC No.
Assigned
IMOS FOS INULIN SS RS
Bif10 Bifidobacterium
longum
Infant
(faeces)
MTCC
25245
2+ 2+ - - -
Bif11 Bifidobacterium
breve
Infant
(faeces)
MTCC
25246
3+ - - 2+ 2+
Bif12 Bifidobacterium
longum
Infant
(faeces)
- 3+ - - - -
Bif16 Bifidobacterium
longum
Infant
(faeces)
MTCC
25247
3+ 1+ - - -
Note: 1+ weak (1 mm); 2+, moderate (2 mm); 3+, strong degradation (3 mm).
Figure 3. Growth rates of Bif10 (a), Bif11 (b), Bif12 (c), and Bif16 (d) in MRSC-BB supplemented with 1% (v/v)
of Glucose and prebiotic-IMOS. Significance, P*< 0.05, calculated in comparison with MRSC-BB with glucose.
Relative bacterial growth rate
Biomass (g/L) of the strains in MRSC broth supplemented with either 1% glucose or 1% IMOS was also
calculated by measuring the dry weight of washed cells in their log phase of growth i.e. after 36 h of
incubation. Relative growth rates of all the strains in IMOS were prepared by taking biomass in glucose as
100%. Overall the relative bacterial growth rate of bacterial culture at 48 hours of incubation in MRSC-BB
supplemented with IMOS was comparable to the growth observed Glucose supplemented MRSC-BB.
Figure 4. Relative bacterial growth rate of Bif10 (a), Bif11 (b), Bif12 (c), and Bif16 (d) in MRSC-BB
supplemented with 1% (v/v) of Glucose and prebiotic-IMOS. Significance, P*< 0.05, calculated in
comparison with MRSC-BB with glucose.
Based on the results from the probiotic profiling in Table 1, prebiotic profiling in Table 2, growth rates in
Figure 1 and relative bacterial growth rates figure 4, probiotic strains presented in Table 3 were selected
for further experiments.
Table 3. Selected list of probiotic candidates and Culture Collection accession number
Strain Criteria of Selection Culture Collection
accession number
B. longum
Bif10
Bif10 was selected based on efficient probiotic
attributes such as
1. Anti-inflammatory activity
2. AMA against S. typhi, E. coli and S. aureus
3. Utilization of prebiotics such as IMOS and FOS
MTCC 25245*
B. breve
Bif11
Bif10 was selected based on efficient probiotic
attributes such as
1. Anti-inflammatory activity
2. Mucin binding and
3. Utilization of prebiotics like IMOS, and dietary fiber
like resistant starch (RS) and soluble starch (SS)
MTCC 25246*
Note: MTCC: Microbial Type Culture Collection, CSIR-IMTECH, Chandigarh
*Patent Deposited under Budapest Treaty
Example 3: In-vivo Preventive effect of synbiotic formulation in DSS induced colitis
Preventive ability of the synbiotic formulation was evaluated in DSS induced ulcerative colitis in Balb/c
mice for 25 days. The steps involved are as follows
Step 1: Preparation of synbiotic formulation:
Overnight grown cultures of Bif10 and Bif11 were centrifuged at 8000 rpm for 10 min at 4°C, washed twice
with PBSC and re-suspended in the fresh PBSC buffer. Each strain was maintained at 1 × 1010 CFU in
trypticase soy broth (TSB) with 15% glycerol and stored in -80°C. At the time of dosing, stored bacterial
stocks of each strain was centrifuged and mixed together in PBSC to achieve 2 × 1010 CFU and IMOS and AX
are added to at 1g/kg body weight to the bacterial blend to get the synbiotic formulation.
This suspension was gavaged orally on every day from the beginning till the end of experiment in the
probiotic group (i.e. Bif10+Bif11 group) while DSS is provided ad-libitum on day 11th to 18th day as per
schedule.
Step 2: Evaluation of the Protective Efficacy of the formulation against DSS induced colon inflammation
Male C57 Black mice were acclimatized for a week to the experimental conditions and normal pellet diet
(NPD) was provided ad libitum. Mice were randomized and allocated to different groups according to their
weights. All animals were provided free access to food and water. Details on the various experimental
groups are given in Fig…. In the intervention groups probiotics (Bif10+Bif11), prebiotics (AX/IMOS) and
synbiotic (Bif10+Bif11+AX+IMOS) were gavaged orally for the animals in the corresponding groups on
every day from the beginning (day 0) till the end of experiment (day 25) while 2.5% DSS is provided adlibitum on day 11th to 18th day as per schedule. In the per se groups, the mice were fed with probiotic
(Bif10+Bif11), prebiotics (AX/IMOS) and synbiotic (Bif10+Bif11+AX+IMOS) respectively for the entire
experimental period. Change in body weights, rectal bleeding, feces consistency and the survival rate of
the studied animals were monitored throughout the study. Using the following formula change in body
weights was analyzed.
% body weight change = [(weight Ds – weight D0 / weight D0) × 100]; where, Ds is body weight at specific
day and D0 is body weight at day 0.
The level of lipocalin-2 was quantified in feces collected at the 7th day of after DSS treatment using ELISA
kit.
All samples including Blood, ileum, proximal colon, distal colon, cecum, cecum content, feces and spleen
were harvested from each animal at the end of the experiment. Colon length, weight and spleen weights
were determined.
A disease activity index (DAI) was calculated taking three major parameters in account including the data
on body weight loss (0 for <1%; 1 for 1-5%; 2 for 5-10%; 3 for 10-20%; 4 for >20%), stool consistency (0 for
normal; 2 for loose; 4 for diarrhea) and gross bleeding (0 for normal color; 2 for reddish color; 4 for bloody
stool) divided by 3 for each mouse (Toumi et al.,2014).
Figure 5. Schematic illustration of the experimental protocol used in this study. A total of 70 male
BALB/c mice were randomly assigned into 10 groups. DSS colitis (n = 8), normal Control (n = 6), AX (n =
8), IMOS (n = 8), Bif10+Bif11 (n = 8), Synbiotic (Bif10+Biff11+AX+IMOS) (n = 8) and four perse groups of
the respective four treatments (n = 6).
Figure 6. Evaluation of protective efficacy of synbiotic formulation in DSS induced colitis in mice. (a)
Percentage survival of animals (b) Body weight change as the percentage baseline weight of each mice
(c) Disease Activity Index (DAI) was determined in each group of mice and (d) Colon length measured in
cm.
Figure 7. Images of colons in each group of mice to determine the observed the changes occurring due to
DSS induced colitis and its prevention via different treatments.
Mice in DSS group showed decreased body weight and colonic lengths. It also caused 38% mortality with
increased disease activity index as compared to normal, whereas, mice groups fed with different
treatments of probiotics, prebiotics and synbiotic showed protection against inflammation caused by DSS.
DSS treatment decreased the SOD activity and GSH levels; whereas catalase levels were increased as
compared to normal mice. Mice pre-treated with probiotics, prebiotics and synbiotic resulted in increased
SOD and GSH levels; levels of catalase were observed to decrease.
Figure 8. Oxidative stress parameters including SOD, GSH and catalase from DSS induced colitis study
on Balb/c mice. (*P < 0.05 versus DSS)
Approx. 100 mg of proximal colon was taken to prepare 10% tissue homogenate in ice cold PBS containing
protease inhibitor cocktail with the help of handheld homogenizer. The resulting homogenate was
centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected and stored at -20°C for further
use. Protein content in the homogenate was determined by Bradford method. DSS treatment enhanced
the inflammatory markers like TNF-α, IL-1β and IL-6, whereas levels of anti-inflammatory marker like IL-22
declined. Mice in synbiotic group showed synergistic effect of both probiotic and prebiotic with maximum
decline in TNF-α, IL-1β, IL-6 levels and amplified IL-22 levels.
Figure 9. Levels of inflammatory marker like; IL-6, IL-1β, TNF-α and IL-22 levels in colon tissue samples of
different groups of animal study. (* P < 0.05 versus DSS)
Serum levels of IL-10, Lipocalin, IL-6, IL-1β, LPS and CRP levels were determined using commercially
available ELISA kits as per the manufacturer’s instructions. DSS treatment enhanced the systemic levels of
inflammatory markers like lipocalin, IL-1β, IL-6, LPS and CRP compared to the normal mice while the
treatment groups showed lower levels of the same. Mice in synbiotic group showed synergistic effect of
both probiotic and prebiotic with maximum decline in lipocalin levels and increased anti-inflammatory IL10.

Figure 10. Levels of inflammatory marker like; IL-10, Lipocalin, IL-6, IL-1β, LPS and CRP levels in serum
samples of different groups of animal study. (* P < 0.05 versus control; # P < 0.05 versus DSS)
Further, short chain fatty acids in the cecum contents were determined using High performance liquid
chromatography as described by Singh et al., (2016). Mice in DSS group had lower levels of lactate,
acetate, propionate and butyrate while mice in the intervention groups showed higher levels of SCFA
relative to the DSS group. Synbiotic group showed the additive effect of both the probiotics and prebiotics
with maximum levels of SCFA.

Figure 11. Lactic acid, Acetic acid, Propionic acid and Butyric acid levels in mM/mg of cecum samples of
different groups of animal study. (* P < 0.05 versus control; # P < 0.05 versus DSS)
Thin sections (5 μm) of the paraffin embedded proximal colon tissues was processed for histological
analysis using hematoxylin and eosin staining and microscopic evaluation while alcian blue and eosin
staining of the sections was carried out to assess the number of mucus producing goblet cells. On the basis
of architecture of colonic sections scoring was done for each slide as per Toumi et al., (2014). DSS
treatment significantly affected the colonic integrity and architecture along with disruption of crypt
structure. Further, epithelial layer showed derangements with increased submucosal thickness and
decreased number of goblet cells upon DSS treatment. Supplementation with probiotics, prebiotics and
synbiotic prevented these harmful changes in the colon and conferred protection against colitis.
Preventive effect in the synbiotic supplemented group was profound in the mice that were fed with
synbiotic formulation. The two selected strains Bif10 and Bif11 were patent deposited under Budapest
treaty at Microbial Type Culture Collection, CSIR-IMTECH with the following accession number MTCC 25245
and MTCC 25246 respectively.

Figure 12. Histopathological analysis of proximal colon was performed using ImageScope X64 software.
(a) Submucosal thickness of the mice colon samples of different groups (µm); (b) Number of goblet cells
in colon crypts; and (c) Alcian Blue straining of colons of mice of different groups (5X Magnification).
4.4 References
1. Ananthakrishnan A.N. (2015). Epidemiology and Risk Factors for IBD. Nat Rev Gastroenterol
Hepatol. 12(4):205-17.
2. Arboleya S., Watkins C., Stanton C. and Ross R.P. (2016). Gut Bifidobacteria Populations in Human
Health and Aging. Front Microbiol, Vol. 7, Article 1204.
3. Belenguer A., Duncan S.H., Calder A.G., Holtrop G., Louis P., Lobley G.E. and Flint H.J. (2006). Two
Routes of Metabolic Cross-Feeding between Bifidobacterium adolescentis and Butyrate-Producing
Anaerobes from the Human Gut. Appl Environ Microbiol. 72: 3593-3599.
4. Besten G.D., Eunen K.V., Groen A.K., Venema K., Reijngoud D.J., and Bakker B.M. (2013). The role
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We claim

1. A novel multi-strain synergistic anti-inflammatory synbiotic formulation comprising of
Bifidobacterium strains and two or more non-digestive carbohydrates for protection against colon
inflammation
2. According to claim 1, the synergistic synbiotic composition has at least two Bifidobacterium strains
wherein the strains are capable of metabolizing isomaltooligosaccharides (IMOS) and one of the
strain, Bif11 (MTCC 25246) should metabolize starch and resistant starch (RS).
3. According to claim 1, the synergistic synbiotic formulation consists of oligosaccharides ranging from
isomaltooligosaccharides, fructooligosaccharides and millet derived dietary fiber like arabinoxylan
and others like resistant starch.
4. According to claim 1, the multi-strain synergistic anti-inflammatory synbiotic formulation for the use
in the prevention and amelioration of symptoms of dextran sodium sulphate induced ulcerative
colitis, mucin depletion and intestinal inflammation on the mammalian subjects.