Bacillus Coagulans SKB LAB-19: A Potential Probiotic in Humans and Animal Healthcare

Probiotics are live microorganism which when administered in adequate amounts confer health benets to the host. Different probiotic strains are being used in animal feeds. The main goal of current study was to screen the isolate for potential probiotic characteristics viz. different enzyme production, antimicrobial properties, pH/bile salt tolerance, temperature stability, antidiarrheal activity against E. coli and castor oil induced diarrhoea in non-ruminant’s Swiss albino mice and Wistar rats; and acute oral toxicity in mice using OECD guidelines. The results showed that the isolate SKB LAB-19 produces 8 potential enzymes, effective against E. coli and C. perfringensis, tolerant to bile salt and gastric pH, stable at 40-90 o C and shows no cytotoxicity. In in vivo studies, SKB LAB19 was found to be safe and displayed promising results to reverse E. coli and castor oil induced diarrhoea in Albino mice and Wistar rats, respectively. An attempt was made to explain mechanism of action and immunomodulatory effects of Bacillus coagulans SKB LAB-19 on reversal of diarrhoeal symptoms, haematological parameters, concentration of immunoglobulins in blood and increases the weight of spleen and thymus. Histopathological pictures showed repair to damaged mucosal epithelium cells and also improves integrity of the goblet cells of colon. Thus, Bacillus coagulans SKB LAB-19 could be an effective probiotic alternative to standard anti-diarrhoeal drugs in humans and animals.


Introduction
As per FAO (Food and Agriculture Organisation of the United Nation) and WHO (World Health Organisation), probiotics are de ned as healthy living microorganisms, which when administered in adequate amounts confer a health bene t on the host [1]. There has been growing interest in probiotics over last two decades results in more than 6000 publications in biomedical literatures, with more than 60% research papers published in last 5 years [2,3]. Lactic acid bacteria and Bi dobacteria are most commonly used as probiotics, although yeast strains and other bacteria are also used [4]. The bene cial effects of the probiotics are speci c to strain and success/failure of one probiotic strain cannot be correlated with another strain. Therefore, identi cation and characterization of the probiotic strain using novel techniques are necessary [5].
As per guidelines suggested by ICMR DBT for evaluation of probiotic in food [6]; A probiotic strain to qualify as safe; needs to pass following criteria's viz. A) Genus, species and strain identi cation, B) in Methods Isolation and identi cation of the lactic acid producing bacteria Soil samples were collected from of the industrial area of the S K Biobiz Pvt Ltd. Nashik, India. Soil samples were collected from the top layer of the soil and transferred aseptically into sterile bag. Soil sample (1 g) was inoculated aseptically into glucose yeast extract (GYE) broth and incubated in an incubator shaker (160 rpm, 37°C for 48 h). The 48-h grown culture was streaked on glucose yeast extract agar (GYEA) and incubated in an incubator (37°C for 24 h). Based on morphology and growth characteristics, ve different types of colonies were isolated. These colonies were picked up individually and inoculated into GYE broth under aseptic condition and further incubated in an incubator shaker (160 rpm, 37 °C and 24 h). The isolated colonies were further puri ed on GYE agar plates.
Morphological, physiological and biochemical characteristics of the soil isolate SKB LAB-19 isolate was assessed for its shape, motility, and purity using a light microscope (Olympus CX31). The morphology and growth characteristic of the isolated colonies were assessed on GYE agar plate. The biochemical tests and Gram staining characteristics were carried out by using KB020 HiLacto Identi cation Kit (HiMedia, India).
Partial gene sequencing by 16S rRNA and phylogenetic analysis SKB LAB-19 isolate was identi ed by 16S rRNA partial gene sequencing. The extraction and isolation of DNA from the SKB LAB-19 isolate was carried out as per the reported protocol [12]. The extracted DNA was analysed by agarose gel (1.2%) electrophoresis, results showed the presence of single band of high molecular weight DNA. The resultant 1525 nucleotide bases were compared with GenBank database using the BLAST server at NCBI. Neighbour-joining method was adopted for construction of phylogenetic tree, while Molecular Evolutionary Genetic Analysis (MEGA) phylogenetic software was used for genetic analysis. The nucleotide sequences were submitted to GenBank databases under Accession No. MW750514.
In vitro determination of enzyme activity SKB LAB-19 isolate was screened for production of different enzymes viz. catalase, α-amylase, lipase, protease, phytase, uricase, phosphatase, cellulase and nitrate reductase. SKB LAB-19 was grown in GYE broth (HiMedia M963) at 37°C, 160 RPM for 24 h in a shaking incubator (Remi CIS-24 PLUS, India). The 24 h old culture broth was subjected for centrifugation (2000 × g for 15 min), the cells so obtained were washed thrice with a sterile saline solution (0.9%w/v) to remove the residual media components. Then, different dilutions (10 -1 to 10 -10 ) of the inoculums was placed on GYEA (HiMedia 963) and incubated in an incubator (37°C for 48 h) to determine the concentration of viable cells (cfu/mL). For evaluation of enzyme activity, 20 μl (10 8 cfu/mL) suspension of SKB LAB-19 was placed at the centre of well dig in each selective media plate. After incubation, zones of clearance were measured in millimetres (mm). Relative enzyme activity (REA) was evaluated by using the following formula, All the in-vitro enzyme activity determination experiments were evaluated in triplicate and values of relative enzyme activities were represented in Table 2.
Production of catalase SKB LAB-19 culture was grown on GYEA plates for 48 h at 37 o C and then ooded with 3 mL of 10% v/v hydrogen peroxide solution. Production of gas bubbles on the colonies indicates production catalase enzyme by the culture [13].

Production of amylase
Amylase activity was determined using starch agar media composed of (g/L) of tryptone 10, starch soluble 3, KH 2 PO 4 5, yeast extract 10, and agar 15. For evaluation of amylase activity, 20 µL of 48 h old culture of SKB LB-19 (grown in GYE broth) was inoculated in 8 mm well in starch agar plates and were incubated in an incubator (37°C for 48 h). For observation of zone of clearance, the plates were ooded with Gram's iodine solution (5 mL) [14].

Production of lipase
The production of lipase was evaluated on tributyrin agar composed (g/L) of tributyrin-10 and nutrient agar-13 with pH-7. Twenty µL of 48 h old culture of SKB LB-19 (grown in GYE broth) was inoculated in 8 mm well on tributyrin agar plates. These plates were subjected for incubation in an incubator (37°C for 24-48 h). The zone of clearance around the halo indicates the lipase production [14,15,16].

Production of protease
Protease activity was evaluated using skim milk agar (SMA) containing (g/L) of skim milk-25, bacteriological agar-20. Twenty µL of 48 h old culture of SKB LAB-19 (grown in GYE broth) was inoculated in 8 mm well in SMA agar plates. These plates were subjected for incubation in an incubator (37°C for 24 h). The zone of clearance was observed around the halo [17].

Production of phytase
For determination of phytase activity SKB LAB-19 was inoculated in phytate agar medium containing (g/L) of dextrose-10, ammonium sulphate-0.3, magnesium sulfate-0.5, calcium chloride-0.1, manganese sulfate-0.01, ferrous sulphate-0.01, sodium phytate-5, and bacteriological agar-15. Twenty µL of 48 h old culture of SKB LAB-19 (grown in GYE broth) was inoculated in 8 mm well in phytate agar plates. These plates were subjected for incubation in an incubator (37°C for 120 h) and zone of clearance was observed after incubation [18].

Production of uricase
Uricase activity was determined using uric acid agar composed of (g/L) of nutrient agar 10, uric acid 10, bacteriological agar 10 with pH 6.8. Twenty μL of 48 h old culture of SKB LAB-19 in GYE broth was inoculated in 8mm diameter wells cut using a well cutter and the plates were subjected for incubation in an incubator (37°C for 24-48 h), and the diameter of the zone of clearance was determined [19].
Phosphate solubilization assay Phosphate solubilization assay was performed using Pikovskaya agar. Twenty μL of SKB LAB-19 (grown in GYE broth) was inoculated in 8 mm well in agar plates and these plates were subjected for incubation in an incubator (37°C for 7 days). The zone of clearance around the colonies was observed after 5 days of incubation [20].

Screening for cellulase producers
Cellulase activity was evaluated using carboxymethyl cellulose agar containing (g/L) of yeast extract 0.5, potassium dihydrogen orthophosphate 1, sodium carboxymethyl cellulose 1.2, magnesium sulphate 0.5, sodium nitrate 0.3, potassium chloride 0.5, bacteriological agar 15, and supplemented with marine salts 15 [21]. Twenty μL of 48 h old culture (grown in GYE broth) was inoculated in 8 mm well in cellulose agar plates and then plates were incubated for 5-10 days (37°C) in an incubator. For determination of cellulase activity, the plates were ooded with 0.01% w/v Congo red dye for 15 minutes, and then plates were washed with 1M NaCl solution to observe cellulase activity. The cellulase activity was indicated by presence of an orange zone surrounding the colony against a red media background [22].

Acid and bile salt tolerance
For acid tolerance assay, 1 mL of SKB LAB-19 culture (10 Billion cfu/mL) was transferred into 9 mL of phosphate buffer saline (PBS) with pH 2.5 (adjusted with 5 M HCl) and then subjected for incubation in an incubator (30°C). The number of viable cells in the PBS was determined after 0, 1, 2 and 3 hrs of incubation periods on a GYE agar plates in triplicates.
For the bile salt tolerance assay, 1 mL of SKB LAB-19 culture (10 Billion cfu/mL) was transferred into 9 mL of GYE broth containing bile salt (0.05 -0.3% w/v), and then subjected for incubation in an incubator (37°C). The number of viable cells was enumerated after 0 and 4 hrs of incubation periods [23].

Hemolysis
The safety assessment of SKB LAB-19 culture towards its use as potential probiotics was con rmed by its non-hemolytic activity on sheep blood agar plate. SKB LAB-19 culture was streaked on sheep blood agar plates, and then subjected for incubation in an incubator (30°C for 48 h) [24]. The hemolysis assay was performed in duplicate.

Evaluation of antimicrobial activity of probiotic bacteria
The antimicrobial activity of the SKB LAB-19 isolate was performed using Mueller-Hinton Agar (MHA). The inhibitory and antagonistic effects of probiotic bacteria on the E. coli and Clostridium perfringens were evaluated using well diffusion agar methods [25].
Well diffusion agar method for antimicrobial e cacy against E. coli Suspension of E. coli cultured in tryptic soya broth (TSB) were spread on Mueller-Hinton Agar, then 10 μL of 48 h old SKB LAB-19 culture (grown in GYE broth) was inoculated in 8 mm well in the MHA plates and incubated in an incubator (37°C for 24 h). The growth inhibition zone was observed after incubation [25].
Well diffusion agar method for antimicrobial e cacy against Clostridium perfringens Clostridium perfringens ATCC 13124 were inoculated into uid thioglycolate broth and subjected for incubation under anaerobic conditions (37°C). A 12-hr old culture of C. perfringens was streaked on the surface of tryptone soya yeast extract agar using a sterile cotton swap. SKB LAB19 culture was grown in GYE broth and subjected for incubation in an incubator (37°C under 5% CO 2 ). Twenty μL of 48 h old SKB LAB-19 culture was inoculated into the 8 mm wells of tryptone soya yeast extract agar plates previously streaked with C. perfringens and incubated in an incubator (37°C under anaerobic conditions) for 18 h. The diameter of growth inhibition zone was determined after incubation.
Effect of temperature on viability SKB LAB-19 spore count A 100 mL spore suspension of SKB LAB-19 (10 Billion cfu/mL) was prepared in previously sterilized saline solution (0.9% w/v). This spore solution was divided into 10 sterile test tubes each containing 10 mL of suspension and then subjected to heat treatment at 45 o C for 60 minutes, 60 o C for 45 minutes, 70 o C for 30 minutes, 80 o C for 20 minutes and 90 o C for 10 minutes. The viability of the spores in the treated sample was assessed by serial dilution method. The serial dilution method was performed until the appropriate dilution was attained (approximately 30-100 cfu/mL). The enumeration of spore was carried out by pour plate technique. The plates were subjected for incubation in an incubator (37°C for 48 h). At least six plates were counted and the average count per plate was calculated. The number of cfu per unit (mL or gram) of sample was calculated by employing the following equation [11].
In-vitro antioxidant activity DPPH radical scavenging activity Diphenylpicryl hydrazine (DPPH) radical scavenging activity is an indicator of hydrogen-donating capability. A 0.8 mL of 0.1mM DPPH solution (prepared in 95% methanol) was added to 0.2 mL of different concentrations (5, 10, 20, 50 mg/mL) of SKB LAB-19 suspension and subjected for incubation (30 ± 2 °C for 30 min). After incubation the samples were centrifuged (8385 × g for 5 min) and supernatant so obtained was used for measuring the absorbance at 517 nm. The antioxidant activities of the different samples of the SKB LAB-19 were compared with ascorbic acid (50 mg/mL) [26, 27,28]. The antioxidant activity was determined by following equation, DPPH scavenging activity (%) = [Absorbance sample / Absorbance control] -1) × 100 Reducing power assay Different concentrations (5, 10, 20, 50 mg/mL) of SKB LAB-19 (0.2 mL) were added to 0.2 mL of phosphate buffer (0.2 M, pH 6.6) and 1% w/v potassium ferricyanide. The reaction mixture was allowed to incubate at 50 °C for 20 min, after incubation 0.5 mL of trichloroacetic acid (10% w/v) was added. This reaction mixture was subjected for centrifugation (755 × g, 10 min). The supernatant was separated and added to deionized water and mixed with ferric chloride (0.1% w/v) at ratio of 1:1: In vivo studies Acute oral toxicity assay Nulliparous and non-pregnant female albino mice (8-10 weeks age and 25 ± 2 g weight) were randomly selected according to OECD test guidelines 425. The mice were kept under standard laboratory conditions for seven days. The limit test was performed using as single dose (2000 mg/kg p.o.) and mice were fasted for 3-4 h before dosing but allowed to take water ad libitum. The mice were monitored for rst 30 min, then for 4 h and then food was given 1-2 h of postdosing. The treated mouse was checked for its survival and four additional mice were administered with same dose under same conditions. Vehicle control group (5 mice) were administered with same volume of saline solution as like treated group. Both the groups were observed for any toxic effects initially for 4 h and then for 14 days at regular interval. Body weight and behavioural pattern was observed [29].
Castor oil induced diarrhoea Swiss albino mice were screened for induction of diarrhoea by administering castor oil (0.5 mL). The mice suffered from diarrhoea were subjected for further studies. Mice were randomly assigned as 5 animals in each group (n = 5). Mice were administered with three different doses of SKB LAB-19 (5, 20 and 50 Billion cfu/kg) for 8 days. On eighth day, Group I were administered with 10 mL/kg, p.o. of sterile water for injection, Group II were administered with 0.5 mL castor oil p.o. for induction of diarrhoea, Group III were administered with 3 mg/kg, p.o. of loperamide, Group IV were administered with SKB LAB-19 (5 Billion cfu/kg, p.o.), Group V were administered with SKB LAB-19 (20 Billion cfu/kg, p.o.), and Group VI were administered with SKB LAB-19 (50 Billion cfu/kg p.o.). After 1h, all mice were administered with castor oil p.o (0.5 mL) and all mice were evalauted for diarrhoeal index. The parameters of evaluation included, onset of diarrhoea (latency), frequency of defecation, total faecal weight and percentage inhibition of defecation [30. 31, 32].

E. coli induced diarrhoea
Wistar rats were screened and standardized for induction of diarrhoea by administering E. coli suspension. Rats were administered with three different doses of SKB LAB-19 for 15 days. E. coli suspension was administered for 3 subsequent days for induction of diarrhoea on eleventh day. Group I (vehicle control) were administered with sterile water for injection (10 mL/kg p.o.), Group II (diarrhoeal control) were administered with E. coli suspension (1 mL, 1 Billion cfu/mL), Group III were administered with SKB LAB-19 (5 Billion cfu/kg, p.o.), Group IV were administered with SKB LAB-19 (20 Billion cfu/kg, p.o.) and Group V were administered with SKB LAB-19 (50 Billion cfu/kg, p.o.) by oral gavage. The parameters monitored were total faecal output, faecal consistency, faecal water content on 12 th , 13 th and 14 th day and all rats were sacri ced on 15 th day of its treatment. Blood was collected and analysed for immunoglobulin level and WBC count. Colon tissues were isolated for histopathological investigation whereas spleen and thymus were isolated for evaluation of immune organ index [12,33,34].

Haematoxylin and Eosin staining
Colons isolated from the rats were deep in neutral buffered formalin solution (10% v/v) for xing the tissues. The colon tissues were xed in para n and divided into 3 µm slices on glass slides. The slides were depara nized and stained with haematoxylin followed by eosin. The slides were dehydrated in alcohol, cleared in xylene, and covered for microscopic examination. The slides were observed blindly by a pathologist and the colonic damage was evaluated [34].

Statistical analysis
Page 10/28 The experimental data were analysed by PRIMER statistical software and expressed as Mean ±SEM.
Statistical analysis was performed using one-way ANOVA, followed by Dunnett's test. # P value < 0.05 and ## P value < 0.01was considered statistically signi cant.

Results And Discussions
Isolation and identi cation of the lactic acid producing bacteria The lactic acid producing bacteria was isolated from the soil. Based on the single colony characteristics, ve different cultures were isolated.
Morphological, physiological and biochemical characteristics of lactic acid producing bacteria  Fig S1). Therefore, it was identi ed as Bacillus coagulans SKB LAB-19 and considered as a new strain of Bacillus coagulans.
In vitro determination of enzyme activity Animal feed are complex and contain material rich in nutrient though hard to digest. The main constituents of the commercial animal feed are grains such as soybeans, corn, sorghum, oats and barley etc. Supplementation of animal diet with a probiotic enhanced digestibility of dry matter (DM), weight gain, feed intake, average daily gain, and feed conversion ratio (FCR). Moreover, probiotics enhanced apparent ileal digestibility (AID) of the essential amino acids, and increased bioavailability of essential ions like calcium [33].
Spore forming bacteria such as B. subtilis and B. amyloliquefaciens secrets extracellular enzymes such as α-amylase, protease, metalloproteases and cellulase etc. [35,36] enhances nutrient digestion. Supplementation of probiotics improved enzyme activity in the gastrointestinal tract of animals. It is evident from Fig. 1 and Table 1 that Bacillus coagulans SKB LAB-19 is capable of producing eight extracellular enzymes at good concentrations when a suitable substrate is available. The enzyme produced by SKB LAB-19 can increase the digestibility of complex carbohydrates, non-starch polysaccharides, proteins, fats and also provide protections against pathogens, thereby increasing overall health of the animal. Probiotic survival at lower pH is very crucial for resisting initial stress in the stomach [37]. Table 2 illustrates the survivability of Bacillus coagulans SKB LAB-19 strain under simulated conditions of low pH. The results indicate that the studied strain resist a simulated phosphate buffer saline solution (pH 2.5) showed an over 87% survival rate after 3h. Additionally SKB LAB-19 was also found to be stable in presence of 0.05-0.3% bile salt with 95% and 70% retention of activity in presence of 0.05 and 0.3% bile salt, respectively (Table 3). It is evident from the results that spores of SKB LAB-19 quali es as potential probiotic  Table 4, SKB LAB-19 when exposed to variable time and temperature incubation conditions was found to be stable and retained more than 80 % activity at 90 o C and 10 minutes and more than 90% activity when exposed to 70 o C for 30 minutes. These properties of SKB LAB-19 make it a probiotic of choice to be incorporated in animal feed during pelletization. Evaluation of antimicrobial activity of SKB LAB-19 Inhibition of growth of food borne pathogen is one of the desirable characteristics of probiotic bacteria.
Recently there is lot of development on Lactobacillus probiotics in food and feed industry, however there is still an urgent need for commercial Direct Feed Microbials (DFM) that are cost-effective and shelfstable. In this context, different Bacillus spp. has been isolated as an active member from poultry and pigs [40]. Furthermore, endospores of some Bacillus spp. have been extensively studied as DFM; results showed that these spores are safe and reliable prophylactic agents to reduce GI diseases in livestock and humans [41,42].
In current study, SKB LAB-19 showed antimicrobial activity against E. coli and C. perfringens (Fig. 3). This might be due to the capability of SKB LAB-19 to synthesize antimicrobial metabolites. Clostridium is an anaerobic spore forming bacteria and is a major etiological agent of nosocomial diarrhoea in patients under antibiotic therapy. Therefore, Bacillus coagulans SKB LAB-19 could be an appropriate alternative as DFM to diminish the occurrence of bacterial GI diseases in humans and animals, including cases of E. coli and Clostridium infections.
In-vivo studies  New practice for management of diarrhoea by using probiotics is very common. Probiotics containing lactic acid bacteria reported to have various health bene ts such as counteract with harmful microorganisms in gut to restore the gut epithelial and immune homeostasis [27,47,48]. SKB LAB-19 administered for 8 days with three different doses, signi cantly (P < 0.01) controls the onset of diarrhoea compared with diarrhoeal control group (Fig. 5a). There was signi cant reduction in frequency of defecation (P < 0.05) (Fig. 5b) and weight of stool (P < 0.01) (Fig. 5c) in medium and high dose (SKB LAB-19) treated group compared to diarrhoeal control group. The percent inhibition of defecation with three different doses of SKB LAB-19 were found to be 54.55%, 71.29%, 72.25% and was more or less similar to Loperamide treatment group (68.42%) (Fig. 5d). SKB LAB-19 signi cantly reduces the diarrhoeal index and strongly inhibits diarrhoea in low, medium and high doses.
In case of castor oil induced diarrhoea, castor oil induces the release of prostaglandins which stimulates vasodilation, contraction of smooth muscle and secretion of mucus which results in diarrhoea. SKB LAB-19 prevents release of prostaglandin which in turn delayed onset of diarrhoea, reduced frequency of defecation and faecal output. It is also assumed that antidiarrheal activity of SKB LAB-19 probably involves the nitric oxide (NO) pathway. Bacillus coagulans LAB-19 may inhibit the production of NO which leads to inhibition of diarrhoea. From the observed results, it is clearly evident that Bacillus coagulans LAB-19 proves to be strong anti-diarrhoeal through the anti-secretory properties.
E. coli (5×10 9 cfu/mL) induced diarrhoea: 15 days treatment of SKB LAB-19 E. coli induced diarrhoea is a well-known model for assessing anti-diarrhoeal potential of probiotic strains against the infectious diarrhoea. Researchers have discovered that pathogenesis of E coli occurs due to different virulence factors which leads to different pathological processes. E. coli adheres to intestinal mucosa results in destruction of microvilli and formation of 'pedestals' of epithelial cell membrane at site of EPEC adhesion [49]. EPEC confers rstly to enterocytes microvilli, causing their localized effacement.
Colonized E. coli attaches to intestinal cell surface results in swelling of plasma membrane around attached bacteria. Changes in cell organelles indicates intracellular damage as a result of heavily colonized enterocytes, nally leads to death and loss of cells from villus surface. These observations also evident in histopathology studies of colon in diarrhoeal control rats (Fig. 6a-6e).
E. coli induced diarrhoea, results in disruption of absorptive and secretary mechanism of intestinal uid and electrolytes which further destructs mucosa and increases intestinal permeability. SKBLAB-19 acts by secreting various antimicrobial peptides like as bacteriocins, reuterin, and hydrogen peroxide. Lactic acid produced by SKB LAB-19 prevents the growth of pathogens by producing the acidic environment and probably by its bactericidal effect. It was also evident in our in vitro study that SKB LAB-19 showed good antimicrobial activity against E. coli (Fig. 3a). SKB LAB-19 reduced faecal output (Fig. 7a), improved faecal consistency (Fig. 7b) and faecal water content (Fig. 7c) in a dose dependent manner. It is also evident from histopathology study of SKB LAB-19 treated group, which exhibited no damage to the mucosal epithelium with retention of complete crypt cell architecture and integrity of goblet cells and no erosion nor cell in ltration observed (Fig. 6, Table 6 and Fig. 7a-7d).  [27,50,51]. Thymus is related with differentiation, development and maturation of T lymphocytes.
Spleen produces lymphocytes, puri es the blood and store white blood cells [52]. SKB LAB-19 treated group showed signi cant increase in relative organ weight index of thymus and spleen compared with diarrhoeal and vehicle control group. Different probiotic strains reported to increase the spleen and thymus index which further improves the immune system of the animals. Present results showed that Bacillus coagulans SKB LAB 19 promotes anti-infective capability by enhancing growth of spleen and thymus and also promotes spleen recovery in rats infected with E. coli. (Fig. 7d).
Leukocyte count is measure of infectious process in the body. A reduced WBC count results in leukopenia which indicates altered immunity or existence of infection that limits the supply of certain WBC's. A reduced WBC count in diarrhoeal control compared to vehicle control explains resolution of infection. In the present study, there was no signi cant difference in WBC count in treated group as compared to vehicle control. However, WBC count was signi cantly higher in all SKB LAB-19 treated (5 Billion cfu/kg, 20 Billion cfu/kg and 50 Billion cfu/kg) groups as compared to diarrhoea control group (Fig. 8a).
The results can be better explained considering the differential WBC count. Neutrophils are primary defence system able to respond in different ways through chemotaxis, phagocytosis, exocytosis, intracellular and extracellular killing of microorganisms [53]. In present study, neutrophils percentage was signi cantly higher (P = 0.001), suggesting that supplementation of probiotic results in modulation of non-speci c immune response. The present results corroborate with the earlier study by Babar et al who reported signi cant increase in neutrophils in Wistar rats after administration of probiotic [54].
The results also showed there is a decrease in lymphocytes in infected group (Fig. 8a) which may be due to stress of infection, which produces a moderate to marked absolute decrease in lymphocytes.
Lymphocytes play an important role in immune system function and helps body to ght against infection. SKB LAB-19 stimulated signi cantly increased levels of lymphocytes (P = 0.001) in all treated rats as compared to the diarrhoeal control. This is also in correlation to our observation with immune organ development which shows increased thymus weight/development which is major site for production of lymphocytes. Thus, Bacillus coagulans SKB LAB-19 shows a probiotic effect by relieving the stress form infection by increasing lymphocyte count.
The production of antibodies in the serum via soluble antigens could be promoted by probiotics and that plays crucial role to enhance the innate immunity [55]. It can be hypothesised from the results that, SKB LAB-19 improved host defence in the gut by production in speci c antibodies (Ab) against pathogens, and the overall increase in total IgA and IgM in a dose dependent manner. There was no signi cant effect observed in secretion of IgG antibodies in the blood (Fig. 8b)

Conclusions
A new strain of B. coagulans SKB LAB-19 was isolated and characterized. Owing to its properties viz. enzyme production, gastric pH and bile salt stability, temperature stability, non-cytotoxic nature, production of antimicrobial metabolites, its proven in vivo e cacy towards treatment and reversal of castor oil and E. coli induced diarrhoea in mice and rats and its safe nature based on acute toxicity studies and its immunomodulatory effects. Bacillus coagulans SKB LAB-19 can serve as an ideal candidate to qualify as probiotic in human and animal feed industry. Further studies to show e cacy in humans and animals is warranted.

Declarations Ethical Approval
The protocols for experimentation and handling of animals were approved by Institutional Animal Ethics   Reducing power activity (C) Ascorbate auto-oxidation inhibition assay (The dissimilar alphabets (a to e) above the bar indicate that the corresponding mean value belongs to different subsets at 95% con dence interval of the mean).

Figure 7
Page 28/28 Effect of B. coagulans SKB LAB-19 on (A) faecal output in 24 hrs in E. coli induced diarrhoea in rat (B) faecal consistency in E. coli induced diarrhoea in rats (C) faecal water content (%) in E. coli induced diarrhoea in rats (D) relative weight index of spleen and thymus in E. coli induced diarrhoea in rats. (All values are expressed as mean ±SEM (n=5), #p 0.05 and ##p 0.01 as compared with the diarrhoeal control group and * p 0.05 and **p 0.01 as compared with the vehicle control group. All data are analysed by one-way ANOVA followed by Dunnett's test).

Figure 8
Effect of Bacillus coagulans SKB LAB-19 on (A) differential leukocytes count in E. coli induced diarrhoea in rats (B) immunoglobulin level on E. coli induced diarrhoea in rats (All values are expressed as mean ±SEM (n=6), *p 0.05 and **p 0.01 as compared with the diarrhoeal control group. All data are analysed by one-way ANOVA followed by Dunnett's test).

Supplementary Files
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