Introduction

Probiotics are “live microorganisms” which when administered in adequate amounts confer a health benefits on the host [1]. Milk and milk-based products are the main ingredients of food in India. In Indian subcontinent, during 7500 BC, the Aryans used to eat “ghee” (cow’s milk-based product) prior and later butter-milk (cow’s milk-based product) during Mahabharata epoch [2]. Furthermore, peoples started to consume milk and milk products of other animals such as goat, sheep, and mare, which are the possible sources of probiotic bacteria. The viable microorganisms with probiotic profile play an important role in maintaining the integrity for immunological, digestive, and respiratory functions. Exploration of effective probiotic bacteria bears high score of immunomodulation and safety appraisal being carried out to develop safer food products [19].

Probiotic microorganisms must possess the desired properties, such as survival in the gastrointestinal (GI) tract, persistence in host, and safety assurance for consumer [3]. They favorably maintain the balance of intestinal microflora, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria, and increase resistance against infections [4]. The survival ability contributed by adherence in GI tract influenced with gastric and bile ambience further competitive advantage and become beneficial to maintain microbial homeostasis. These traits have been proven beneficial in competitive exclusion of pathogenic bacteria due to niche occupation by lactic acid bacteria (LAB) [5, 6]. Milk and milk-based products bear huge diversity of probiotic microorganisms, in which these can easily utilize lactose as an energy sources for their growth and proliferation. The mode of action of probiotics is cherished with milk protein, providing protection during passage, binding ability, and elimination of microbial pathogens of GI tract [7, 8].

The lactic acid bacteria (LABs) as producer of myriad antimicrobial substances exhibit antimicrobial activity against wide range of pathogenic bacteria. Kumis, a fermented dairy product, is traditionally made from mare’s milk in Central Asia. Globally, the traditional milk-derived products are gaining importance as a rich source of probiotics containing functional lactobacilli [9, 10]. Now-a-days, drug administration imbalance normal microflora of intestine; therefore, it needs to be balanced soon with probiotic supplementation. In the light of above facts, the present study was undertaken to investigate the probiotic potential and safety assessment necessary to characterize the lactic acid bacteria from milk and milk-based products from different animals and to evaluate in vitro and in vivo probiotic potential based on intestinal colonization and survival profile.

Materials and Methods

Isolation and Purification of Lactobacilli

A total of 23 milk samples from different pet animals such as cow, buffalo, goat, sheep, and mare were collected from neighboring regions of Haridwar hills. An aliquot of 1 ml milk sample was enriched with De Man Rogosa (MRS) medium (1% peptone, 1% beef extract, 0.5% yeast extract, 2% dextrose, 0.1% Tween 80, 2% tri-ammonium citrate, 5% sodium acetate, 0.05% magnesium sulfate, 0.02% manganese sulfate, 2% di-potassium phosphate, pH 6.8) at 37 °C for 24 h. Decimal dilutions were made up to 10−8, and respective dilutions were poured on MRS agar medium and incubated at 37 °C for 36 h. The colonies identical to the Lactobacillus were further streaked on MRS agar plates and purified. Routine cultures maintained on MRS agar slants were kept at 4 °C for further studies. Stock cultures were maintained in 50% glycerol stock and preserved at − 20 °C [11].

Identification of Lactobacilli

Selected isolates were examined for Gram’s staining, colony morphology, and potential growth in presence of salt. These isolates were further evaluated for catalase, oxidase, and endospore tests [11].

Secondary Screening for Probiotic Attributes

Acid Tolerance

Selected isolates were grown in MRS broth (HiMedia, Mumbai, India) at 37 °C. Growth of overnight grown culture was measured at 550 nm using a spectrophotometer (Shimadzu, Japan). The actively growing cells were harvested by centrifugation at 5488×g at 4 °C for 10 min and suspended in MRS broth. The pH was adjusted to 2.0, 3.0, and 6.5 by using 1 N HCl [12]. MRS broth having pH 6.5 was used as control. Viable cells were counted at an interval of every 1 h up to 3 h. Cell viability was assessed by plate count method and expressed as log cfu/ml.

Bile Tolerance

Overnight grown cultures of isolates were harvested and suspended in 3 ml of MRS broth supplemented with 0.3, 0.5, and 1.0% oxgall. MRS medium without oxgall acted as control [13]. Samples were withdrawn at 0 min, 3 h, and 24 h of incubation, and cell viability was measured by plate count method.

Inhibitory Effect Against Enteric Pathogens

Inhibitory effect of bacterial isolates against the selected enteric pathogens was carried out by agar well diffusion method [14]. Four standard human enteric pathogenic bacteria, viz., Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 27853, Klebsiella pneumoniae MTCC 432, and Salmonella typhi MTCC 733 (procured from IMTECH, Chandigarh, India), were used in this study. Each bacterial suspension was swabbed on Müller-Hinton agar (MHA) medium. Wells were made by using a cork borer (6 mm) on plates. Overnight grown cultures of isolates were individually centrifuged at 5488×g for 10 min. Cell-free supernatant (CFS) was poured into wells on plates by using micropipette and incubated at 37 °C. Zone of inhibition against pathogens was observed after 24 h of incubation.

Determination of Antibiotic Resistance

Antibiotic susceptibility profile of isolates was determined by agar disk diffusion assay on MHA medium [15]. The MHA plates were prepared; freshly grown culture of isolates was spread on surface of medium and allowed to dry. Antibiotic disks were placed on agar surface and incubated at 37 °C. Zone of inhibition against each disk was measured after 48 h of incubation.

Resistance to Phenol

Phenol resistance is an essential characteristic for survival of probiotic bacteria in GI tract [16]. Overnight grown culture of lactobacilli was inoculated in MRS broth containing 0.4% phenol. Samples were withdrawn at an interval of 0 min, 3 h, and 24 h, and cell viability was tested by cell plate count method.

Bacterial Adhesion to Hydrocarbons

Hydrophobicity was measured following the method of Reniero et al. [17] with some modification. Overnight grown culture of isolates was harvested by centrifugation at 5488×g at 4 °C for 5 min. Cell pellets were washed twice, suspended in phosphate urea magnesium (PUM) buffer (pH 6.5), and absorbance of bacterial suspension was adjusted to 0.7–1.0 at 560 nm. Bacterial suspension (3 ml) was transferred into culture tubes containing 1 ml of each hydrocarbon separately and incubated at 37 °C for 10 min and further vortexed for 1–2 min. Suspension was kept undisturbed for 1 h for phase separation. Thereafter, aqueous phase was separated with pipette and absorbance was measured at 560 nm using a spectrophotometer. Hydrophobicity was calculated by using the following formula given by Lee et al. [18].

$$ \mathrm{Hydrophobicity}\left(\%\right)=\frac{\mathrm{Initial}\kern0.17em \mathrm{absorbance}-\mathrm{final}\kern0.17em \mathrm{absorbance}}{\mathrm{Initial}\kern0.17em \mathrm{absorbance}}\times 100 $$

Safety Assessment of Isolates

All the selected five isolates were observed for DNase activity, hemolytic activity, and ability to liquefy the gelatin following the method of Gupta and Malik [19].

Molecular Identification

Among the five isolates, MMP4 was the isolate with the highest probiotic potential; it was used for molecular identification and in vivo studies. Pure culture of the isolate MMP4 was grown in MRS media until log phase was achieved. Genomic DNA was extracted following the method of Pospiech and Neumann [20]. Genomic DNA was amplified through a PCR machine (Applied Biosystems, India) by using genus-specific universal primers. PCR product was sent to Agile Life Science Technologies India Pvt. Ltd. (Maharashtra) for DNA sequencing. DNA sequence was matched in EMBL/GeneBank/DDBJ libraries by using BLASTn. Sequence of bacterial strains nearest to the isolate MMP4 was retrieved and aligned using ClustalW. Phylogenetic analysis was performed by using MEGA version 7. 16S rRNA bacterial gene sequence was submitted to the GenBank of NCBI to get the accession number.

Molecular Study of Bacterial Adhesion to Substratum

Colony-PCR analysis was performed to identify the Mub, Map, EF-Tu, and Bsh genes of the most potential isolate Lactobacillus pentosus MMP4. Overnight grown bacterial colonies of the isolate MMP4 were transferred in lysis buffer (TE and 0.1% Triton-X 100) and incubated at 90–100 °C for 10 min. The mixture was centrifuged at 11,200×g for 5 min and supernatant was used as template for colony PCR. PCR mix containing DNA template, short-strand RNA F/R primers of Map gene (Map A F-TGGATTCTGCTTGAGGTAAG, R-GACTAGTAATAACGCGACCG), Mub gene (MubF-GTAGTTACTCAGTGACGATCAATG R-TAATTGTAAAGGTATAATCGGAGG), EF-Tu(EF-TuF-TTCTGGTCGTATCGATCGATCGTG, R-CCACGTAATAACGCACCAAC), Bsh gene (BshF-ATCACCGCTACATTGGTTGG, R-AGTCCGCCCATTCCTCTACT), dNTPs, Taq polymerase, Taq buffer, and nuclease-free water was pooled and allowed for PCR, followed by 35 cycles of denaturation at 95 °C for 5 min, annealing at Tm as required for 45 s, extension at 72 °C for 5 min, and final extension at 72 °C for 7 min. Gel electrophoresis was performed using 0.9% agarose gel, and bands of expected size were visualized under UV Gel-doc system.

In Vivo Surface Attachment Study in Intestine of Albino Mice

The experiment on albino mice was carried out in the Department of Pharmaceutical Sciences, Gurukula Kangri Vishwavidyalaya, Haridwar (India), after approval granted from the Institutional Animal Ethical Committee (Animal House Reg. No. 1324/a/10/CPCSEA, Government of India). Albino mice (4–5 weeks old with 15–20-g body weight) were kept under standard conditions of light and dark cycle at suitable temperature. A standard pellet diet (Hindustan Lever Products, Limited, Kolkata, India) was provided and water ad libitum. Before supplementing L. pentosus MMP4 water, feed and animals were observed to be free from any contamination and pathogenic infection. Five subsequent doses of L. pentosus MMP4 were prepared in saline having 106–107 cfu/ml and given to mice orally after initiating the artificial infection by consortium of different pathogens.

Scanning Electron Microscopy of Excised Mice Intestine

Albino mice were sacrificed by cervical dislocation after 7 days of administrating the doses, and their small intestines were gently removed. The segmented intestine was opened and washed with PBS and fixed in 4% glutaraldehyde for 60–70 min. The fixed samples were dehydrated gradually by different concentration of alcohol. Finally, the fixed and dehydrated samples were mounted on aluminum stubs further coated with gold palladium. Attachment of L. pentosus MMP4 on intestinal surface was observed by scanning electron microscope at 30 kV in a LEO 485 VPSEM (ZEISS, USA), and photomicrographs were taken with the same microscope.

Statistical Analysis

All the experiments were carried out in triplicates. Data were analyzed statistically by using Microsoft Excel 2010, Microsoft Corporation (USA).

Results

Isolation of Bacteria

A total 53 bacterial isolates were screened from 23 milk samples collected from different animals. Initially, these isolates revealed acid and bile tolerance but only 21 were able to survive in gastrointestinal ambience. Out of 21 isolates, only five isolates were screened on the basis of physiological and biochemical characters along with the degrees of salt and temperature tolerance. These were Gram’s positive and found negative for catalase, oxidase, and endospore formation.

Acid Tolerance

Survival of potential probiotic bacteria in gastrointestinal environment is an important selection criterion of potential probiotic strains. All the isolates showed good viability at pH 2.0 and 3.0, but a marginal cease in growth was recorded at 3 and 24 h. The isolate MMP4 survived well with the maximum cfu on broad range of pH as compared to the other isolates (Table 1).

Table 1 Effect of different pH on survival of bacterial isolates
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Bile Tolerance

All selected isolates were further evaluated for total viable counts at different concentration of bile salt (0.3, 0.5, and 1.0%). All the five isolates survived and proliferated on wide concentrations of bile salt. The isolate MMP4 exhibited the maximum cell viability (11 cfu/ ml at 1% concentration) for 24 h, followed by the isolates LP2 and CP5 (Table 2).

Table 2 Bile tolerance by bacterial isolates
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Inhibitory Effect Against Food-Borne Pathogens

All the isolates assessed for antimicrobial activity against the four human pathogens showed different degree of sensitivity. The isolates PP1 and LP2 caused the maximum zone of inhibition against E. coli, but S. aureus was more sensitive against GMP3, MMP4, and CP5. The isolate MMP4 splashed the maximum zone of inhibition of 12, 10, 8, and 12 mm against E. coli, K. pneumonia, S. aureus, and S. typhi, respectively (Table 3).

Table 3 Inhibitory effect of bacterial isolates against some food-borne pathogens
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Assessment of Antibiotic Susceptibility

The isolate PP1 was sensitive to some antibiotic, namely, chloramphenicol, novobiocin, and methicillin, and resistant to tetracyclin, streptomycin, erythromycin, penicillin, and fusidic acid. A similar result was observed with the isolate LP2, which showed sensitivity to erythromycin in addition to PP1. The isolate CP5 was found sensitive to most of the antibiotics used in this study, while the GMP3 showed resistance against all the antibiotics used, except novobiocin and fusidic acid. But, the isolate MMP4 exhibited the resistance to all the antibiotics such as chloramphenicol, novobiocin, methicillin, tetracyclin, streptomycin, erythromycin, penicillin, and fusidic acid. Hence, isolate MMP4 showed paramount resistance to all antibiotics.

Resistance to Phenol

Survivability of all isolates was evaluated at constant (0.4%) concentration of phenol supplemented in MRS broth during variable time intervals such as 0 min, 3 h, and 24 h. The isolates PP1 moderately survived in presence of phenol, while growth rate of LP2 was slow in presence of phenol as compared to others. Isolate PP1 showed 9 cfu/ml at 0 min, but after 24 h, the growth of PP1 was reduced to 6 cfu/ml. The growth of CP5 and GMP3 up to 3 h remains unaffected in presence of phenol but ceased rapidly thereafter as observed on 24 h. The isolate MMP4 showed the maximum tolerance to phenol and maintained 19.6, 19.8, and 19.7 cfu/ml at 0 min, 3 h, and 24 h, respectively, with standard deviation (SD ± 0.1). Hence, MMP4 exhibited growth stability in the presence of phenol than that of other isolates.

Cell Surface Hydrophobicity

The isolates showed different level of adhesion against each hydrocarbon. The isolate LP2 exhibited moderate hydrophobicity (39.25%) to n-octane but GMP3 showed more affinity to n-hexane (55.72%), while MMP4 exhibited the highest affinity to each hydrocarbon as compared to the other isolates (Table 4).

Table 4 Hydrophobicity (%) of bacterial isolates
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Safety Assessment

All the selected isolates were non-endospore forming Gram-positive rods having catalase and oxidase negative properties. None of the isolates were positive for hemolytic, DNase, and gelatinase activities.

Molecular Identification

The most potential isolate MMP4 was identified as L. pentosus on the basis of 16S rRNA gene sequencing (accession number MF000890). The phylogenetic analysis exhibited a close evolutionary similarity with L. pentosus (Fig. 1). Therefore, the isolate MMP4 has been designated in the text as L. pentosus MMP4.

Fig. 1
figure 1

Phylogenetic and evolutionary relationships with reference type strains as optimal tree with the sum of branch length = 0.24898856 clustered together in the bootstrap test (500 replicates) next to the branches. The tree was computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The analysis involved 17 nucleotide sequences. All positions containing gaps and missing data were eliminated (1392 positions in the final dataset)

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Molecular Study of Bsh and Other Genes Involved in Attachment to Intestine

The Bsh gene is considered as an important probiotic marker that helps the organisms to resist against toxicity of bile salt in intestinal conditions. The Bsh, Mub, MapA, and EF-Tu genes were detected in the presence of bile at low pH by using colony PCR. The DNA bands of expected size were observed on agarose gel electrophoresis. Bsh gene was observed between 300- and 200-bp band of marker with average size ~231 bp. Whereas, Mub gene appeared slightly lower than 200 and significantly above 100-bp band of marker, hence showing average size ~150 bp. Similarly, Map gene was detected with the size ~156 bp. The EF-Tu gene appeared with size ~161 bp, which was slightly above then both previous genes.

Scanning Electron Microscopy of Excised Mice Intestine

Scanning electron micrograph of small intestine of mice (control) showed the presence of large number of coccus-shaped bacteria on the upper surface, while the treated mice displayed huge number of rods of L. pentosus MMP4. Well-organized and firm microvilli can be observed in L. pentosus MMP4-treated mice (Fig. 2).

Fig. 2
figure 2

Scanning electron microscopy (SEM) of excised mice GI showing presence of large number of coccus-shaped bacteria on the upper surface (a). The mice administrated with L. pentosus MMP4 displayed huge number of rods on the microvilli of GI tract (b). Enlarged images of L. pentosus MMP4 showing single bacterial cell (inset) (c)

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Discussion

The complex behavior of Lactobacillus as probiotic was studied by isolating a wide variety of isolates from different animal’s milk. However, few bacteria during primary screening were not found to possess characteristics of probiotics. Only L. pentosus MMP4 from mere’s lactation proved it’s excellence on different portrayals. In this study, L. pentosus MMP4 showed acid tolerance and sounded proactive stability against acid gradient. Hence, the survival of probiotic strains was considered crucial to its functionality and supposed to be able to survive against the inevitable biological barriers, such as acidic environment of the stomach and bile acids in the duodenum, after ingestion [21]. The basic environment of stomach with pH value 2.0–3.0 due to gastric juice causes mortality of ingested microorganism at very initial stage [8]. Hence, preliminary screening of isolates was based on the growth behavior in the presence of acid and bile [22]. Human gastric juice at very low pH is a robust cidal agent for lactobacilli, against which very few strains could survive [23]. Therefore, constancy during permeation in GI tract is an important selection criterion for probiotic microorganisms because probiotic strains must survive in such harsh acidic environment to express their health-promoting functions [24]. In this study, L. pentosus MMP4 showed pragmatic candidature in such environmental stresses. Similarly, in vitro evaluation has also been carried out by Chou and Weimer [5].

Bile acids are synthesized in liver from cholesterol and secreted from gall bladder into duodenum in conjugated form. Probiotic strain must be able to tolerate and grow in the presence of bile acids and thus able to pass through stomach passage to reach small intestine, which is the real niche. Previously, Gupta and Tewari [11] examined the effect of bile concentration in the range of 0–0.4% on survival of various species of Lactobacillus. Our result is better than that of Gupta and Tiwari as L. pentosus MMP4 showed a wide range of bile tolerance (0.3–1.5%) in contrast to 0–0.4%. In the present study, L. pentosus MMP4 survived on bile concentration, which is almost equivalent to the physiological concentration of bile in duodenum (0.2–0.7%). The isolate L. pentosus MMP4 was more resistance to bile salts. The growth of L. pentosus MMP4 increased even in strong bile concentrations.

According to FAO/WHO, every probiotic strain must be assessed for safety before to use as food supplement [24]. Extracellular DNase production correlates the pathogenicity of bacteria. [25]. In this study L. pentosus MMP4 did not exhibit hemolysis, gelatinase, and DNase activity, and thus, it was considered as non-pathogenic followed by resistant to various antibiotics. Hence, such bacterial strains may be considered as safe for probiotic consumption. Saarela et al. [21] have evaluated the mentioned traits of Lactobacillus before recommending as probiotics. The deamination of aromatic amino acids by intestinal microbiota either autogenously or through diets leads to phenol production. The phenol compounds enact bacteriostatic effect against some probiotic strains. In the present investigation, L. pentosus MMP4 resisted 0.4% phenol and showed peerless growth. Vizoso-Pinto et al. [26] reported phenol tolerance in four out of ten isolates of Lactobacillus.

Production of antimicrobial compound by probiotic bacteria is an excellent feature for modulation of intestinal microbiota [27], reduction of Helicobacter pylori load, and some other infectious diseases [28]. Antagonistic activity of LABs increases their possible potential to be utilized as preservative for certain foods [29]. During this study, L. pentosus MMP4 caused the highest zone of inhibition against both Gram-positive and Gram-negative pathogens displaying inhibitory properties.

Essentially, epigenomes found in bacteria are capable of acquiring resistance (r) genes and promoting their transmission to those that differ with the genus of the pathogen. There are similarities but clear differences between the Gram-positive and Gram-negative bacteria also exist. Surprisingly, bacteriophages carrying antibiotic r genes have rarely been identified in the environment or probiotic strains. Sharma et al. [30] also discussed that antibiotic resistance is prevalent in different species of probiotic strains, which may pose a food safety concern. Besides, it is still unknown how long it will take and if ever resistance (r) gene determinants will be lost from the populations of trillions of bacterial cells that administrated or residing in human gastrointestinal tracts worldwide [31].

The anticipated problem of r gene transfer can be solved if naturally occurring r genes are not present on mobile genetic elements. Thus, r genes cannot be transferred to pathogenic organisms in the gut. Vancomycin resistance has been reported as intrinsic in Lactobacillus species and is thought to derive from point mutations rather than gene transfer [32]. In this paper, antibiotic resistance in probiotic strains was observed against antibiotics as innate ability, which is found among all species of the same genus. The confined intrinsic antibiotic resistance in probiotic as earlier has also been reported by several workers [33, 34]. The lactobacilli isolated from commercial products in Europe comprised of certain strains resistant to tetracycline (29.5%), chloramphenicol (8.5%), and erythromycin (12%), and overall, more than 68% of the isolates exhibited resistance to two or more antibiotics [35]. Resistance to such antibiotics was considered natural and intrinsic in lactobacilli as chromosomally encoded [36, 37].

LAB strains showed intrinsic resistance with various antibiotics, but this is not a concern of transfer to the pathogens [38]. The occurrence of antibiotic resistance in LAB strains has earlier been reported [39,40,41]. De Souza et al. also accepted the antibiotic resistance as a positive character of probiotic bacteria [38]. It is clear that simultaneous application of susceptible probiotics with oral antibiotics is generally unreasonable, in the case of microbial infection. Possibly, such intrinsic antibiotic-resistant probiotics can be used during or post-antibiotic treatment [42]. Hence, antibiotic resistance is considered as an important aspect of safety assessment for the evaluation of probiotics [43]. A recommendation by EFSA panel on biological hazards (BIOHAZ) has included L. pentosus in the updated Qualified Presumption of Safety (QPS) list of the EFSA, and in this regard is a species regarded as safe [44].

Physical and chemical properties of the surface of bacterial cells depend mainly on its hydrophobicity [45]. Surface adhesion of bacteria is one of the important in vitro characteristics to proof them as potential probiotic strains. In fact, colonization in the GI tract has been considered as the prerequisite of the probiotic bacteria. Hydrophobicity refers to competitive ability for adhesion which contributes to the adhesive reaction between microorganisms and a suitable substrate [46]. In this study, L. pentosus MMP4 exhibited maximum hydrophobicity (72, 67, 43%) in xylene, n-octane, and n-hexane, respectively, which establishes L. pentosus MMP4 as a strong colonizer with adhesion properties to several organic hydrocarbons. Several studies report on cell line assay to establish adhesion properties but useful indication of adhesion rely on reflection of the ability to adhere intestinal cell covered with mucus [47], adhesion to mucosa, and further immune system development.

Furthermore, an adhesion-like protein classified as elongation factor Tu (EF-Tu) has been isolated and reported as adhesion protein. This protein binds to mucin and mediates the colonization of human intestinal cells [48]. In lactobacilli, EF-Tu may function as an “envelop-associated protein” released during the osmotic shock [49]. The concentration gradient of EF-Tu on the intestinal surface suggests possible gradient expression [48]. The presence of EF-Tu molecule in L. pentosus MMP4 correlates the capability to adapt the normal healthy intestine and has the genetic characteristics required for an effective probiotic. Some other genes like Mub, Map, Fbp, and CnBP have been reported as factors responsible for adhesion to probiotic bacteria. Roos and Jonsson [50] reported that Mub gene secretes mucin-binding protein which supports the attachment of bacteria to intestinal cell wall.

PCR amplification revealed the presence of Bsh gene in the chromosome L. pentosus MMP4 that conferred hydrolysis of bile salts helping in colonization of bacterial cells in GI tract. This finding suggests that MMP4 could inhabit and survive the passage through GI tract and serves as a potential indigenous probiotic bacterium. Due to the presence of putative adhesion protein encoding gene like EF-Tu, Map, and Mub, L. pentosus MMP4 may potentially affect the binding of pathogenic microorganisms to the GI tract lining by competitive exclusion. L. pentosus had long life span in both ethenic and commercial probiotic preparation. It may be concluded that L. pentosus MMP4 accomplished all the attributes necessary for a good probiotic bacterial strain including the safety aspect of colonization and health-promoting effects. Therefore, it can serve as potential probiotic culture in the development of neutraceutical products, prophylactic, and therapeutic purposes combined with immunomodulatory studies.