The traditional diversity in the preparation of fermented foods and substrates is crucial for their microbiota and unique compositions [6]. Most of the members of these microbial communities were capable of surviving gastrointestinal transit and helping to strengthen the gut microbiome [1]. Several studies have indicated that fermented foods are a good source of probiotic bacteria [44]. However, isolation of probiotic bacteria from local and traditional fermented foods remained under-reported. In this study, we investigated probiotic attributes, adhesion ability, safety, antimicrobial potential, exopolysaccharide production, biofilm formation, and whole genome sequence analysis of fermented milk (Dahi) isolate L. delbrueckii subsp. indicus DC-3.
The survival under gastro-intestinal conditions is one of the prerequisite properties for probiotic bacteria. In order survive under these conditions lactic acid bacteria stimulate the activity of F0F1 ATP proton pumps to maintain cytoplasmic pH, produce alkaline compounds in the cell cytoplasm, modify the cell membrane integrity and fluidity, and upregulate the genes for amino acid decarboxylation and repair or protection of macromolecules [45]. In this study, strain DC-3 showed 83% viability for up to 3 hours in gastric juice and 71% viability for 6 hours in intestinal juice, which falls between a reasonable survival rate of 70 − 80% for in vitro static experiments [46, 47]. Studies suggest that the bacterial survival rate for gastro-intestinal conditions is largely dependent on exposure time and strain [32, 48]. Moreover, the gastric and intestinal juice viability of strain DC-3 was comparatively higher (> 70%) than the gastric and intestinal juice viability (< 70%) reported for L. delbrueckii subsp. indicus CRL1447 [47].
Adhesion of probiotic candidates to the intestinal epithelial cells for colonization is crucial for inhibition of pathogens, nutrient absorption, and immunity [49, 50]. It has been suggested that probiotic bacteria interact with epithelial cells through electrostatic and hydrophobic interactions mediated through teichoic (TA) and lipoteichoic acid (LTA), environmental DNA (eDNA), polysaccharides, and complex polymers, or by producing bioactive metabolites [51]. In this study, the higher percentage of DC-3 autoaggregation suggested the ability of the strain to interact with or recognize surface proteins, TA, LTA, organelles, eDNA, or exopolysaccharides (EPS) for colonization. Besides this, the DC-3 adhesion to the polar solvent xylene demonstrated hydrophobic cell surfaces. The negatively charged membrane potential indicated the presence of TA and LTA, a phosphate-rich cell surface glycolpolymer essential for the attachment of bacterial cells to epithelial cells [52]. Moreover, adhesion to mucin and CaCo-2 cells further confirmed the adhesion potential of the L. delbrueckii subsp. indicus DC-3, which is well coordinated with previous findings [47].
In order to establish safety, candidate probiotics must have data on genotypic and phenotypic identity, whole genome sequencing (WGS), studies on virulence, toxin production, and antibiotic resistance [10]. Besides this, when a candidate probiotic doesn’t have a history of safe use and is not listed in the qualified presumption of safety (QPS), extensive safety testing is required, including animal and human studies [9]. The strain DC-3 reported in this study was isolated from traditional fermented milk (Dahi), which has a long history of safe use. It doesn’t produce hemolysin to lyse red blood cells, gelatinase to break down extracellular matrix, and is incapable of degrading mucin, a core structural element of the mucosal surfaces of the digestive tract. The cytotoxic effect of DC-3 observed against hepato- and colon-carcinoma cells could be due to their anti-carcinogenic abilities. The MICs of ampicillin, chloramphenicol, clindamycin, and erythromycin observed against DC-3 were as per the cut-off values provided by EFSA [30]. However, higher MICs of gentamycin, kanamycin, tetracycline, and vancomycin could not pose the risk of transmissible antibiotic resistance genes from food and feed, as it is an outcome of the intrinsic resistance of the Lactobacillus [53]. Similarly, resistance to selected antibiotics in the classes aminoglycosides, coumarin glycosides, macrolactams, steroids / derivatives, and ofloxacin, a quinolone, could be intrinsic, i.e., on the chromosome. These findings are well aligned with antibiotic resistance in probiotic microorganisms [54]. Moreover, the antibiotic- and antifungal (triazole)-resistant trait of the strain could be useful for its application during or along with antibiotic or antifungal agents. Overall, based on in vitro safety and antibiotic susceptibility profiles, strain DC-3 might be regarded a safe strain for probiotic use.
The antimicrobial activity of the probiotics is essential to eliminating pathogens, which are the main competitors for nutrients and colonization in the gut [55]. Studies indicated that probiotic bacteria are capable of producing lactic acid, acetic acid, diacetyl, bacteriocins and bacteriocin-like inhibitory substances (BLIS), hydrogen peroxide, and surface active molecules to inhibit the pathogens [56]. In this investigation, strain DC-3 inhibited the growth of both Gram-positive and Gram-negative bacteria as well as yeast. Based on the zones of growth inhibition and the strains' inability to inhibit bacterial growth following neutralization of cell-free supernatant, it was determined that the action was attributable to lactic acid and/or hydrogen peroxide rather than bacteriocins. The production of a higher amount of lactic acid and hydrogen peroxide further confirmed the findings. Karnaouri et al. [57] showed that, as starter cultures in dairy fermentation, L. delbrueckii subsp. are efficient lactic acid producers. Similarly, the hydrogen peroxide production ability of L. delbrueckii subsp. bulgaricus was previously reported by Kot et al. [58].
Besides antimicrobial production, co-aggregation of probiotic bacteria with pathogens is one of the important properties of manipulating the aberrant intestinal microbiota [59]. This property increases the colonization efficiency of probiotic bacteria. In this study, strain DC-3 showed co-aggregations with E. coli, P. mirabilis, and C. albicans, indicating its ability to lower both bacteria and yeast colonization in the gut. The co-aggregation percentages were mostly dependent on strains and the time of the assay, and the results obtained in this study are well coordinated with previous findings [59, 25].
The exopolysaccharide of lactic acid bacteria (LAB) is known to enhance their tolerance to harsh gastro-intestinal conditions and colonization in the gut [60]. It is also reported for numerous other health benefits like immunomodulation, antiviral, anti-cancer, anti-inflammatory, anti-yeast, and putative antimicrobial activities [61]. In this study, strain DC-3 showed its ability to produce EPS, which is important for its survival and colonization in the gut. The quantity of EPS reported in this study was comparatively lower than that of L. delbrueckii ssp. indicus WDS-7 [62]. In addition to EPS production, strain DC-3 exhibited biofilm formation, which is considered an advantageous trait for efficient colonization. Studies have shown that LAB-biofilms enhance their antimicrobial capacity and could act as a biocontrol agent for pathogens and pathogenic biofilms [63]. The strain DC-3 biofilm could be an ideal combination of a good probiotic strain and its antimicrobials (lactic acid and hydrogen peroxide) to inhibit invading pathogens.
To date, regulatory standards for probiotic safety have been well established in Europe and some Asian countries [9]. According to those standards, whole genome sequencing (WGS) is one of the requirements for establishing the safety of probiotic bacteria [9, 10]. In this study, WGS of strain DC-3 isolated from traditional fermented milk (Dahi) showed a single circular chromosome (3,145,837 bp) with a GC content of 56.73% and was identified as L. delbrueckii subsp. indicus. The higher genomic GC content of the DC-3 than previously reported L. delbrueckii subsp. indicus JCM 15610T (49.4%) indicated high thermal stability [64]. Recently, Teng et al. [65] showed that genomic GC has a considerable impact on the average amino acid characteristics of proteomes, including the N/C ratio and hydrophobicity, which may determine bacterial fitness. In comparative genome analysis with the closest neighbours (L. delbrueckii subsp. indicus, L. equicursoris, and L. porci), strain DC-3 showed a higher number of accessory and unique genes, which might be useful to enhance the strains' ability to adapt to their hosts or the surrounding environment [66]. Moreover, the open pan-genome of the DC-3 could indicate a probability of finding novel function genes. These results corroborate well with previous findings that the pan-genomes of most species of the Lactobacillaceae family are moderately open compared to other bacterial species [67]. In coding sequence analysis, each of the 15% genes was found to contribute to amino acid and carbohydrate metabolism, 10% to protein metabolism, 9% to cofactors and vitamins, 5% to membrane transport, 3% to stress response, 2% to regulation of cell signaling, 1% to chemotaxis, etc. No virulence factor genes, mobile and insertion elements, or plasmids were detected or identified in DC-3, which indicated the strain’s safety. The antibiotic resistance gene profiling results were well coordinated with the in vitro findings of the strain and further confirmed the strain's safety. Furthermore, the lanthipeptide class III identified in the WGS analysis was not detected in vitro as, most of the class III lanthipeptides often lack antibacterial activities [68]. Lactobacillus delbrueckii often possessed Lanthipeptide III and their conserved residues, i.e., lyase, kinase, and cyclase enzymes [69].