Introduction

Bifidobacteria are one of the early stage colonizer and predominant bacteria of gut known to promote health of the host [30]. Restoration of bifidobacteria counts in patients with lower counts has shown to improve/prevent health disorders [2]. Consequently, supplementation of bifidobacteria as a probiotic has gained significance. Probiotics are defined as live microorganisms when administered in adequate amounts confer health benefits upon the host [11]. However, probiotic properties of the bacteria are strain specific. Hence, there is a necessity to identify new and efficient probiotic strains, which can be studied to address different health disorders.

Bifidobacteria belongs to the phyla Actinobacteria, and currently, 48 species have been identified. A multigene approach can ensure a better identification and classification than the conventional 16SrRNA sequencing [17]. Additionally, molecular tools RAPD, RFLP, Rep-PCR, DGGE, PFGE are also used to validate the identity and investigate the diversity among the species [5]. In case of bifidobacteria, type IVb tight adherence (tad) pili and sortase-dependent pili (SDP), gene clusters have been identified to have a role in adhesion. Genomic studies have revealed the presence and variation in number of SDP in Bifidobacterium sp. [5, 7, 12, 29]. Such genes can serve as an excellent marker for identification of probiotic organisms with adhesion property [19]. Comparative genomic and functional analysis of these genes among the different species and within the species of bifidobacteria will reveal the ability of these organisms to adapt to different ecological niche. Studying the abundance and variation among these genes can help in the targeted hunt for probiotic candidates and for gene editing to improve the existing strains [7]. Serpins are protease inhibitors described to have immunomodulatory function and aid in protection of these bacteria [30]. Using serpins as molecular markers will enable to detect bifidobacteria with such properties rapidly.

Despite the fact that bifidobacteria is one among the widely studied probiotic bacteria, there are very few research works which describe bifidobacteria from India. Traditionally, Indians were known to have a healthy lifestyle which has remained uninfluenced by the modern lifestyle for a longer period of time [25]. Consequently, developing countries like India can be a unique source of beneficial microbes that have been remained impervious to the result of modernisation and use of antibiotics [25, 26].

The present work has focused on isolation and comparison of probiotic as well as functional properties of native isolates of bifidobacteria for their potential use as a probiotic. The work also investigates the presence and diversity of genes related to adhesion and immunomodulation as markers for selection of putative probiotic bifidobacteria.

Materials and Methods

Media, Chemicals, Reagents Bacterial Cultures and Conditions

Microbiological media and chemicals, sugar discs, antibiotic octa disc (Combi 77, Combi 69, Combi IV, G-X-plus) and cell culture media, were supplied by HiMedia Pvt. Ltd., Mumbai, India. All fine chemicals were purchased from Sigma-Aldrich Inc. Other chemicals used were of AR grade unless otherwise specified. Bifidobacteria strains were cultured in deMan Rogosa and Sharpe medium supplemented with 0.05% cysteine hydrochloride (MRS-C). Anaerobic conditions for bifidobacteria cultures during incubation were maintained using Anaerogas pack (HiMedia). Pathogenic strains Kocuria rhizophila ATCC 9341, Yersinia enterocolitica MTCC859, Staphylococcus aureus FRI1722, Escherichia coli MTCC118, Bacillus cereus F4433 and Listeria moncytogenes Scott A maintained in the culture collection of host lab was used. Brain heart infusion (BHI) medium was used for growth and maintenance of pathogens. Bifidobacterium animalis subsp. lactis BB12 used as a standard reference culture was kindly provided by Dr. D.J. O’Sullivan, USA.

Isolation and Identification of Bifidobacteria

Bifidobacterial strains were isolated from newborn infant fecal samples collected from Mysore Medical College, Mysuru, India. Fructose 6 phosphoketolase (F6PPK) assay was performed for isolates showing the typical V or Y shape following the protocol described by Zinedine and Faid [31]. DNA was extracted from the pure cultures as per the protocol described by Baffoni et al. [4]. Molecular characterisation of the isolates were performed by 16S rRNA, xfp and hsp60 gene sequencing and microbial typing by RAPD (Initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 1 min; annealing at 36 °C for 1.45 min followed by 72 °C at 2 min; extension at 72 °C for 7 min). and Rep-PCR(Initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 1 min; annealing at 50 °C for 1 min followed by 72 °C at 2 min; extension at 72 °C for 9 min). PCR-amplified products of isolates were sequenced at Amnion Biosciences Pvt. Ltd., Bengaluru, India. The primers used in the present study and reaction conditions are enlisted in Table 1. All the primers were synthesized and procured from Sigma-Aldrich, Bengaluru, India.

Table 1 Oligonucleotide primers used in the study
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Probiotic Attributes

Acid and Bile Tolerance

Survival efficiency of the isolates under simulated acidic conditions and in the presence of bile salts was determined as described by Archer and Halami [3]. Briefly, 10% of overnight grown bifidobacteria cultures were inoculated in MRS-C broth adjusted to different pH 2, 3, 4 and 5 or bile salt concentrations of 0.3%. At predetermined intervals of 0 h, 1.5 h, 3 h an aliquot of the sample was withdrawn, serially diluted and plated on MRS-C agar. Colonies were enumerated after incubation under anaerobic conditions at 37 °C for 48 h.

Pathogenicity and Safety Evaluation

The isolates were tested for biogenic amine production as per method described by Bover-Cid and Holzapfel [6]. Haemolytic activity of the isolates was evaluated using 7% defibrinated sheep blood agar [8]. Sensitivity of the isolates for commonly used antibiotics Ciprofloxacin (5 mcg), Ofloxacin (5 mcg), Sparfloxacin (5 mcg), Gatifloxacin (5 mcg), Aztreonam (30 mcg), Vancomycin (30 mcg), Doxycycline (30 mcg), Cefotaxime (30 mcg), Amoxycillin (30 mcg), Cefepime (30 mcg), Chloramphenicol (30 mcg), Isepamicin (30 mcg), Ampicillin (10 mcg), Cephalothin (30 mcg), Clindamycin (2 mcg), Gentamycin (10 mcg), Oxacillin (1 mcg), Fusidic acid (10 mcg), Methicillin (10 mcg), Novobiocin (5mcg), Penicillin (1U), Streptomycin (10 mcg) and Tetracycline (25 mcg) was examined using HiMedia Octa discs [8].

In Vitro Adhesion Assay

Adhesion ability of the isolates was analyzed using Caco-2 cell lines purchased from National Centre for Cell Science (NCCS), Pune India. Experimental design was based on the procedure previously described by Serafini et al. [23]. Briefly, the cell line was maintained in Dulbecco’s modified Eagles minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotic and antimycotic solution. The cell line was incubated at 37 °C in a CO2 incubator. The cell culture medium was changed after 2 days, and trypsinization was carried out using 0.25% trypsin–EDTA. For the assay, 105 Caco-2 cells were seeded in six-well tissue culture plates and incubated till confluence. The assay was performed 20 days post confluency. 108–109 bifidobacterial cells were suspended in DMEM without serum and antibiotics. Caco-2 monolayers were washed twice with PBS to remove antibiotics before addition of bacterial cells. The bacterial cells were incubated with Caco-2 cells at 37 °C for 2 h. After washing the monolayer with sterile PBS to remove unattached bacteria, it was trypsinized, and bacterial counts were carried out in MRS-C agar to determine the number of bacteria adhered to the cells. Adhesion percentage was calculated as the number of bacteria adhered with respect to the number of bacteria added. The assays were performed in three independent experiments.

Molecular Characterization of Probiotic Marker Genes

The presence of genes credited for adhesion, i.e. SDP and serpin, proposed to have immunomodulatory function was detected by employing species-specific primers (Table 1). The primers were designed using primer3 software targeting subunits of SDP having highest domains associated with adhesion. Accordingly, primers were designed for fimA, fimP, Major subunit (pili 1 and pili 2 of Bifidobacterium breve). Each 10 µl of PCR reaction mix consists of 1× concentration of PCR buffer, 2 mM of MgCl2, 200 µM of dNTP mix, 0.5 pm of each primer 0.2 U of Taq DNA polymerase.

Evaluation of Antibacterial Activity

Antibacterial activities of the isolates were evaluated against a set of pathogenic bacteria by modification of well diffusion assay described by Archer and Halami [3]. The isolates were grown overnight centrifuged at 8000 rpm for 10 min, supernatant was discarded, and 10 µl of the cell pellet was added to the wells bored on MRS agar. The wells were then sealed with MRS soft agar. After incubation at 37 °C for 24 h under anaerobic conditions, the plates were overlayed with BHI agar seeded with pathogens. Further, incubation was carried out at 37 °C for 24 h under aerobic conditions and was observed for zone of inhibition. To evaluate the proteinaceous nature of the antimicrobial compound, supernatants of 24-h-old MRS-C-grown isolates were vacuum concentrated. An aliquot of the supernatant was neutralized to pH 6.5. The neutralized and non-neutralized supernatants were then added to separate wells bored in BHI soft agar plates seeded with K. rhizophila ATCC 9341. Zone of inhibition was observed after 24 h of incubation at 37 °C.

Functional Properties

Amylase, phytase and bile salt hydrolase (BSH) activity of the isolates was tested as described by Shobharani and Halami [24], Raghavendra and Halami [20] and Archer and Halami [3], respectively. Antioxidant activity of the isolates was determined by radical scavenging assay using 2,2-diphenyl-1-picrylhydrazyl(DPPH) as described by Archer and Halami [3]. The ability of the isolates to utilize different sugars and polyols were tested using HiMedia sugar discs as per the manufacturer’s instructions. Exopolysaccharides (EPS) producing ability of the isolates were evaluated by streaking on ruthenium red agar plate as described by Hongpattarakere et al. [14]. To study the milk coagulation ability of the isolates, 1% of overnight grown culture was inoculated in reconstituted skim milk, incubated under anaerobic conditions at 37 °C and the fermentation was monitored for 24–48 h.

Accession Numbers

The nucleotide sequences of 16S rRNA, xfp, hsp60 and marker genes have been deposited at GenBank database under following accession numbers: 16S rRNA (KU297198, KU297199, KY448275-KY448280); xfp (KU297200, KU297201, KY629958); hsp60 (KX922700, KX922701, KY629959); major subunit pili 1 (KY629951); major subunit pili 2 (629948-50), fimA (KY629952- KY629954); fimP (KY629957, KY629960); serpin (KY629955, KY629956).

Results

Identification and Taxonomic Confirmation of the Bifidobacterial Isolates

Taxonomy of eight isolates with distinct bifid (V or Y) shape and positive for the F6PPK assay were identified by partial 16S rRNA, xfp and hsp60 gene sequencing. These sequences were subjected to BLAST analysis, and their identity was ascertained. The cultures were identified as B. breve 142, Bifidobacterium longum 20, B. longum 24, B. longum 50, B. longum 56, B. longum 815j, B. longum 815k and Bifidobacterium bifidum 96b and have been deposited at National Collection of Industrial Microorganisms, CSIR-NCL, Pune, India under the deposition numbers: B. breve NCIM5671, B. longum NCIM5700, B. longum NCIM5684, B. longum NCIM5685, B. longum NCIM5686, B. longum NCIM5672, B. longum NCIM5687 and B. bifidum NCIM5697, respectively.

RAPD and Rep-PCR were used to differentiate the isolates. Distinct banding pattern was evident in the case of RAPD and Rep-PCR for the three different species and standard culture. Among the B. longum species, similar banding pattern was observed in case of RAPD, for B. longum NCIM5672 and B. longum NCIM5687 as well as for B. longum NCIM5700 and B. longum NCIM5685. However, Rep-PCR was able to differentiate all B. longum isolates (Fig. 1).

Fig. 1
figure 1

Dendrogram constructed employing Mega 5 software using UPGMA model for binary scores obtained from a Rep (GTG)5 profile, b RAPD M13 profile

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Probiotic Attributes

The identified eight bifidobacteria isolates were characterized for the classical probiotic traits (tolerance to GIT conditions, safety, and adhesion). The isolates were found to be sensitive to high acidic conditions (pH 2). Among the isolates, B. breve NCIM5671 showed higher resistance with a decline of 0.8 log cfu at pH 3 after 3 h of exposure. At pH 4, all the strains displayed appreciable survivability with a decrease of 0.7–1.1 log in viability at the end of 3 h. There was only a marginal decline in viability at pH 5 after 3 h of incubation. The isolates were found to be less sensitive to bile salts compared to acidic conditions with a minor decrease in viability after 3 h of exposure. B. longum NCIM5672 exhibited better tolerance with 0.02 log reduction in viability (Fig. S1).

All the isolates were found negative for hemolytic activity and biogenic amine production from lysine, histidine, and ornithine as evidenced by the plate assay. Upon studying the antibiotic resistance of the isolates to 23 different antibiotics, all the isolates were found to be susceptible to the antibiotics tested except for isepamicin to which well-known probiotic B. animalis subsp. lactis BB12 also exhibited resistance.

All the isolates were found to adhere the Caco-2 cell lines and the adhesion percentage varied among the isolates with a maximum of 77% for B. bifidum NCIM5697 followed by B. longum NCIM5672 (Fig. 2; Table S1).

Fig. 2
figure 2

Representative image of bifidobacterial adhesion to Caco-2 cell line: aB. longum NCIM5672, bB. bifidum NCIM5697

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Molecular Characterization of Probiotic Marker Genes

The presence of marker genes associated with adhesion was verified among the isolates. Accordingly, in the case of fimA all B. longum isolates were found to have the desired amplicon size of 1405 bp except for B. longum NCIM5686. Similarly, B. breve NCIM5671 and B. bifidum NCIM5697 also confirmed the presence of fimA gene with amplicon size of 1546 bp. PCR amplification of fimP gene in case of B. bifidum NCIM5697 resulted in product size of 1470 bp revealing the presence of fimP in the isolate. An amplicon size of 1500 bp and 1540 bp validated the presence of major subunit for pili 1 and pili 2 in B. breve NCIM5671. However, presence of major subunit of pili 2 was also detected in case of B. longum NCIM5684 and B. longum NCIM5686 (Fig. S2). These isolates were also verified for the presence of serpin gene. All the isolates were tested positive as evidenced by the PCR amplicon of 1349 bp except for B. bifidum NCIM5697 (Fig. S3).

Evaluation of Antibacterial Activity

All the isolates showed activity against K. rhizophila ATCC 9341, L. moncytogenes ScottA, S. aureus FRI1722, Y. enterocolitica MTCC 859 and B. cereus F4433. Activity against E. coli MTCC118 was variable with B. breve NCIM5671 and B. longum NCIM5700 showing no activity (Table S1). The supernatants were devoid of activity upon neutralization indicating that the activity might be due to the production of organic acids.

Functional Probiotic Properties

All the isolates were found to have phytate-degrading ability while only B. breve NCIM5671 was positive for amylase activity. All the isolates tested for BSH activity, exhibited precipitation zones except for B. bifidum NCIM5697 and B. longum NCIM5700. Antioxidant activity of the isolates was found to be similar, ranging from 76 to 78%. B. longum NCIM5684 and B. longum NCIM5687 demonstrated strong antioxidant activity of 78%. Upon evaluation of the ability of the isolates to utilize different polyols and sugars B. breve NCIM5671 was found to utilize sugar better than the other isolates and B. bifidum NCIM5697 was found to be poor in sugar utilization. B. breve NCIM5671 and B. bifidum NCIM5697 were found to produce white colonies signifying EPS production as analysed by ruthenium red agar. Upon evaluation of milk fermenting ability, B. breve NCIM5671, B. longum NCIM5672 and B. longum NCIM5685 showed faster curdling within 18 h, while all other isolates were found to ferment skim milk within 24 h (Tables S1, S2).

Discussions

The health benefits associated with probiotic bacteria has led to an increased demand for these products, and hence novel probiotic strains are being explored. The present work aimed at studying indigenous bifidobacterial strains for their probiotic properties. The identity of the isolates was confirmed by molecular characterisation using 16S rRNA, xfp and hsp60 genes. Molecular typing tools were successful in differentiating the isolates wherein (GTG)5 Rep-PCR was found to have better discriminatory power than the RAPD corroborating the findings of Jarocki et al. [15] and Křížová et al. [16].

Surviving the gastrointestinal conditions is considered as one of the major criteria for probiotic selection. Our isolates were found to survive acidic conditions above pH 3 and bile conditions of 0.3%. Bifidobacteria are generally considered to be less acid tolerant [18, 21, 27]. Although these species are included in the recommended list of qualified presumption of safety (QPS) and regarded as safe to be included as food additives [9, 10], in vitro tests were carried out to ensure that the native isolates do not raise any safety concerns. The isolates were found free of harmful traits and sensitive to antibiotics, hence can be considered safe for consumption.

Adhesion of probiotic bacteria to gut epithelial cells is of significance as it helps in colonization, host–microbe interactions, immunomodulation, inhibition of adhesion of pathogens by site competition and maintenance of intestinal homeostasis [19, 23]. In our studies, all the isolates were capable of adhering Caco-2 cells and had shown better adherence than the reference strain B. animalis subsp. lactis BB12. Strains with better adhesion than B. animalis subsp. lactis BB12 has also been documented by Arboleya et al. [1] and Serafini et al. [23].

The contribution of SDP in bifidobacterial adherence and colonization of gut has been established. These SDP may be present in varying numbers in different Bifidobacterium species as well as strain, and each cluster is described to possess two subunits and a sortase gene [5, 12, 29]. In the current study, major subunits were used as marker genes to determine the presence of SDP. Considerable variation was noted with respect to presence of these genes in B. longum species. B. longum NCIM5686 was found to be negative for fimA, gene present in all the other B. longum isolates indicating possible absence of fim A subunit or the SDP cluster. Although two-thirds of B. longum strains have been described to possess gene cluster-encoding SDP its absence has been recorded in case of B. longum subsp. infantis ATCC15697 [7, 12]. A comparative genomic study by Bottacini et al. [5] has revealed variation in number of SDP in B. breve. In our study, B. breve NCIM5671 was found positive for major subunits of pili 1, pili 2 and pili 3 indicating the presence of three different SDP. Interestingly, it was also noted that B. longum NCIM5686 and B. longum NCIM5684 were positive for major subunit of pili 2 reported to be found in B. breve. BLAST search of partial sequences of major subunit of pili 2 from B. longum NCIM5686 and B. longum NCIM5684 revealed the presence of similar genes in B. longum BXY01 as well as B. longum JDM301. Adhesion being governed by many factors the role of this variation in genes and its correlation to adhesion in case of B. longum species could not be established in the current study. However, variation in these genes and their profusion may determine the composition of intestinal microbiota [7]. Specific primers designed targeting serpin gene confirmed its presence in all the isolates except B. bifidum NCIM5697. Our findings are in accordance with reports of Turroni et al. [28].

Presence of various functional attributes such as antimicrobial activity, enzyme activity, antioxidant activity, EPS production, etc. among the isolates, adds value to the probiotic microbes and may find potential industrial application.

In conclusion, the native bifidobacterial isolates NCIM5671, NCIM5684, NCIM5685, NCIM5686, NCIM5672, NCIM5687 and NCIM5697 demonstrated desirable probiotic and safety attributes. Though the isolates are less tolerant to high acidic conditions, the safe delivery of these to the intestine can be ensured through food matrix. The isolates also displayed additional functional attributes such as phytase activity, antioxidant activity, antimicrobial activity and BSH activity. However, strain-specific variations were observed in these properties. Molecular tools such as 16S rRNA gene sequencing, gene-specific PCR for xfp and hsp60 genes, along with RAPD and (GTG)5 established the identity and diversity of these isolates. Screening for marker genes has revealed diversity among the isolates for their presence as well as the existence of different types of SDP. Presence of sdp pili in B. longum NCIM5686 and NCIM5684 previously reported to be present in B. breve species indicates possibility of gene transfer or loss of genes during evolution in case of other B. longum sp. Further investigations can be helpful for better understanding of their role in adhesion, interaction with the host and adaptation to a particular environmental niche. Among the isolates, B. breve NCIM5671, B. longum NCIM5672, and B. bifidum NCIM5697 has shown promising attributes and hence can be considered for probiotic functional food formulation.