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Introduction

Lactose intolerance (LI) is a typical condition of dairy food intolerance, that occurs generally when lactase activity is decreased in the brush border of human small intestinal mucosa. LI prevalence shows diversity among regions, human populations, continents and across the globe (1,2). It has been observed that 70% of the global human population exhibit transient lactase activity without LI symptoms, which is influenced by nutritional and genetic factors (1). Prevalence of lactase non-persistence condition in Asian and African countries ranges between 80-100%, however, among Northern European countries the prevalence of LI is observed to be very low (2,3). Furthermore, hypolactasia in the Asian continent has rarely been reported, while in the Western world it is relatively prevalent (1). In the Indian subcontinent, particularly the northern region, the frequency of maldigesters was reported to be 48% per 200 subjects during a breath test, while in the southern region it was observed to be higher (66%) (4). It has been documented that, Indo-Aryan migration brought the lactase persistence (LP) trait to northern India, which was later spread by intermixing of the native population (2). Thus, it is of interest to study whether the distribution of the genetic marker responsible for the LP trait varies between northern and southern Indian populations. In addition, in Northwest Russia lactase non-persistence ranges between 16-23% (1-4). Thus, LI management is a worldwide issue in terms of public health management.

The most essential enzyme in the dairy sector for developing low-lactose food stuffs to overcome LI is β-galactosidase (EC 3.2.1.23) and it is commercially manufactured from microorganisms such as bacteria, yeast, and fungus. Although chemically-synthesized enzymes are gaining significance, bacterial enzymes are preferred as they exhibit high activity and stability (5). β-galactosidase enzyme is produced in the small intestine and its deficiency can cause LI. Diarrhea, stomach discomfort, distention, flatus, and borborgygmi are typical symptoms of LI, affecting both infants and adults, and manifest after 30-120 min of lactose absorption (6). Primary deficiency, also known as hypolactasia, is characterized by partial or complete lack of β-galactosidase in the small intestine of children of various ages, while 70% of the population of the world exhibit evidence of usual symptoms in late adolescence and adulthood. Damage to the small intestine induced by an overgrowth of enterobacteria in secondary deficiency results in the destruction of small intestinal cells. Despite the fact that β-galactosidase is a non-inducible enzyme, it is found in the jejunum and produced in the microvillus membrane of the small intestine, where lactose digestion leads to the production of glucose and galactose monosaccharide, and is absorbed by enterocytes (7).

Probiotics are live microorganisms, which when administered in an adequate amount, confer health benefits to the host (FAO/WHO) (8). The mechanism of action of beneficial organisms include, competition with pathogens for adhesion and nutrients, and in addition to the production of antimicrobial metabolites, enhance host immunity against pathogens in the gut. The association between human health and probiotic gut microbiota has been thoroughly studied, with a particular emphasis on homeostatic and barrier function (9). A wide range of metabolites are produced from probiotic bacteria, including nonspecific fatty acids, and highly specific bacteriocins with antimicrobial properties. Previous studies have shown that probiotic bacteria are progressively renowned as a means for alleviating intestinal disorders and treatments have been successful in mouse models for certain clinical intestinal disorders (10,11). The most prominent probiotics are Lactiplantibacillus and Bifidobacteria genera (10,11) which can be used for prevention and management of numerous disorders including diarrhea, rotaviral diarrhea, Helicobacter pylori infection, hyperlipidemia, colitis, acute and chronic gastroenteritis, irritable bowel syndrome, inflammatory bowel disease, cirrhosis, pouchitis, vaginosis and maldigestion-related conditions such as LI, milk protein allergy and soy protein allergy (6).

In the present study, some of the potential lactic acid bacterial isolates were used to alleviate LI and analysed for probiotic potentiality. β-galactosidase-producing lactic acid bacteria (LAB) were isolated and assessed for acid and bile tolerance, antibiotic susceptibility, antimicrobial activity, auto-aggregation and co-aggregation abilities, cell-surface hydrophobicity, and HT-29 cell adhesion and invasion assays. The 16S rRNA gene consists of highly conserved nucleotide sequences that can be used to distinguish closely related bacterial species and to determine the taxonomy and phylogeny of unknown bacteria by comparing the obtained sequence to known sequences of other bacteria in the GenBank database (12,13). Thus, molecular characterization of selected LAB isolates using 16S rRNA sequence analysis was performed.

Materials and methods

Isolation of LAB from homemade curd samples

A total of 30 homemade curd samples were collected from different rural regions of Karnataka state, India and the samples were stored at 4˚C until further use. With all aseptic precautions, the samples were homogenized, serially diluted (tenfold), 0.1 ml of the sample was plated on de Man Rogosa Sharpe (MRS) agar (Himedia Laboratories Pvt, Ltd.) and incubated for 24 to 48 h at 37˚C. Bacterial colonies developed on MRS media were serially subcultured by following microdilution technique and pure cultures were preserved at 4˚C/MRS agar slants.

Screening of β-galactosidase-producing LAB

A total of 450 LAB isolates were inoculated with MRS agar medium supplemented with 60 µl X-Gal (20 mg/ml in DMSO; Himedia Laboratories Pvt, Ltd.) as a chromogenic substrate and 10 µl of iso-propyl-thio-β-D-galactopyranoside (IPTG) (Himedia Laboratories Pvt, Ltd.) as an inducer for the β-galactosidase. Following incubation for 48 h at 37˚C, development of blue colonies indicated β-galactosidase enzyme activity (10,14).

Quantitative assay for β-galactosidase

β-galactosidase assay of eight isolates was performed (15,16). Briefly, selected isolates were centrifuged at 12,000 x g for 5 min at 4˚C (Eppendorf AG 22331; Eppendorf SE) and washed twice in phosphate-buffered saline (PBS) and cells were adjusted to 1.0 (560 nm). Furthermore, cells were permeabilized with 50 µl of toluene/acetone (1:9 v/v) (HiMedia Laboratories Pvt, Ltd.), vortexed for 7 min and then 100 µl of cell suspension was added to a tube consisting of 900 µl of phosphate buffer and 200 µl of O-nitrophenyl-β-D-galactopyranoside (ONPG; 4 mg/ml) solution (Himedia Laboratories Pvt, Ltd.). Additionally, after a 15-min incubation period at 37˚C, 0.5 ml of 1 M Na2CO3 was added to terminate the reaction, and absorbance values at 420 and 560 nm (NanoDrop 2000C UV-Spectrophotometer; Thermo Fisher Scientific, Inc.) were recorded, with β-galactosidase activity represented in Miller units:

Where, A1560 denotes the absorbance before the test and A2560 denotes the absorbance of the reaction mixture.

Phenotypic and molecular characterization

β-galactosidase-producing isolates were identified by colony characteristics viz., size, shape, color and texture. Furthermore, the isolates were subjected to biochemical tests and characterized based on Bergey's manual of systematic bacteriology (17). Furthermore, carbohydrate fermentation was performed for species level identification using glucose, fructose, sucrose, galactose, lactose, maltose, cellobiose, xylose, arabinose, rhamnose, mannitol, and sorbitol sugars as previously described (18,19).

Identification by 16S rRNA gene sequence

From the selected bacterial isolates, DNA was isolated using the CTAB protocol (20) and the extraction was confirmed by electrophoresis using 0.8% agarose gel. The PCR reaction mixture contained 400 ng of forward primer and 400 ng reverse primer, 4 µl 10X dNTPs (2.5 mM each), 10 µl of DNA polymerase assay buffer and 3U of 1 µl Taq DNA polymerase enzyme (Sigma-Aldrich; Merck KGaA). For 16S rRNA gene amplification, prokaryotic universal primers [Pair 1: ~1,500 bp amplification, forward primer (395), 5'-GGATGAGCCCGCGGCCTA-3' and reverse primer (396), 5'-CGGTGTGTACAAGGCCCGG-3'; Pair 2: ~1,300 bp amplification, forward primer (63F), 5'-CAGGCCTAACACATGCAAGTC-3' and reverse primer (1387R), 5'-GGCGGATGTGTACAAGGC-3'] were used and this experiment was performed at CellKraft Biotech Pvt, Ltd., and designed, using an ABI thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the program: Denaturation at 94˚C for 5 min, followed by 35 cycles at 94˚C for 1 min, annealing at 55˚C for 1 min, and extension at 72˚C for 2 min, and a final extension for 7 min at 72˚C. Amplification was confirmed by electrophoresis of PCR products using 1% agarose gel and then sequenced by Sanger sequencing method with an ABI 3130 genetic analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). 16S rRNA sequences were compared using the Basic Local Alignment Search Tool (BLASTn) program of the National Center for Biotechnology Information (NCBI) database. Furthermore, the multiple sequence alignment was aligned using the MUSCLE program (https://www.drive5.com/muscle/) and the phylogenetic tree was constructed by the neighbor-joining method using MEGA-X software (https://www.megasoftware.net/). Partial nucleotide sequences of 16S rRNA of the identified Lactiplantibacillus isolates were deposited in the NCBI/GenBank (12,13).

In vitro evaluation for potential probiotic bacteria

In vitro studies were undertaken to evaluate the probiotic potentiality of β-galactosidase-producing isolates as per FAO/WHO (8). All eight isolates were subjected to simulated gastric juice and bile tolerance tests. The simulated gastric juice contained 0.3% w/v pepsin and 0.5% w/v NaCl (Himedia Laboratories Pvt, Ltd.), at pH 2 or 4. A total of 1 ml of cell suspension was centrifuged (12,000 x g, 5 min at 5˚C), inoculated into 10 ml of gastric juice with pH 2 or 4, incubated at 37˚C for 3 h and cell viability was measured as colony-forming units (CFU) by plating technique, and the percentage of survival was calculated as follows:

Bile tolerance was determined by inoculating each strain (1% v/v) into MRS broth with 0.3% (w/v) of bile salt (Oxgall; Himedia Laboratories Pvt, Ltd.) and incubated for 3 h at 37˚C. Viability was measured as CFU by plating technique and compared with the control (without bile salt) (13,21).

Pancreatic enzyme tolerance was calculated according to a study by Rashmi and Gayathri (13), with slight modifications. Overnight cultures were centrifuged at 6,000 x g (20 min at 5˚C), inoculated into simulated pancreatic juice (SPJ; bile 3 g/l and pancreatin 0.1 g/l, sodium phosphate dibasic heptahydrate 50.81 g/l, and NaCl 8.5 g/l in a KH2PO4 buffer at pH 8.0; Himedia Laboratories Pvt, Ltd.), incubated at 37˚C for 3 h and the percentage of survival was calculated as follows:

Hemolytic activity

Sheep blood agar (Himedia Laboratories Pvt, Ltd.) was used for inoculation of selected LAB isolates and incubated for 48 h at 37˚C, and then plates were observed for α, β, or γ hemolysis (22,23).

Hydrophobicity

For the hydrophobicity assay, two different solvents viz., non-polar solvent-xylene and polar solvent-chloroform was used. Centrifuged cells (12,000 x g for 5 min at 5˚C) were suspended in 50 mM K2HPO4 (pH 6.5) buffer and adjusted to OD 1.0 at A600 nm and 3 ml of the bacterial suspensions were mixed with 1 ml of solvent and allowed to stand at room temperature for 20 min (13,15). The percentage of bacterial adhesion to the solvent was measured using the aqueous phase at 600 nm.

Where, A0 and A are the absorbance before incubation and after incubation, respectively.

Antimicrobial activity against human pathogenic bacteria

Antimicrobial activity using crude secondary metabolites of LAB against selected pathogenic bacteria was performed using the agar well diffusion technique. Cell-free supernatants (CFS) of each bacterial isolate were prepared and adjusted to pH 6.5. Certain selected strains of pathogenic bacteria viz., Escherichia coli (MTCC no. 433), Staphylococcus aureus (ATCC no. 6538), Pseudomonas aeruginosa (ATCC no. 9027), Salmonella abony (ATCC no. BAA2162) and Listeria monocytogenes (L. monocytogenes; MTCC no. 1143), were purchased from the American Type Culture Collection (ATCC) and Microbial Type Culture Collection (MTCC; IMTech), and 0.1 ml of pathogens were inoculated onto Mueller Hinton agar media (Himedia Laboratories Pvt, Ltd.). Furthermore, 7-mm diameter wells were made in the agar plates and 100 µl of CFS was placed into these wells, and finally the inhibition zone was measured (mm) after 48 h of incubation at 37˚C.

Antibiotic sensitivity test

Antibiotic disc diffusion method was performed according to Kumara et al (23) with modifications. A total of 0.1 ml of each selected isolate was inoculated onto MRS agar media and antibiotic disc containing penicillin (P) 10 mcg, erythromycin (E) 15 mcg, ampicillin (AMP) 10 mcg, amikacin (AK) 30 mcg, ofloxacin (OF) 5 mcg, cefixime (CFM) 5 mcg, ciprofloxacin (CIP) 5 mcg, and azithromycin (AZM) 15 mcg (Himedia Laboratories Pvt, Ltd.) were placed and incubated for 24 h at 37˚C. Subsequently, the diameter of the inhibition zone was measured in mm.

Auto-aggregation and co-aggregation assays

The ability of bacteria to auto-aggregate and co-aggregate was assessed according to Armas et al (22) with slight modifications. Stationary phase cells were centrifuged (5,000 x g for 15 min at 5˚C), pellets were washed thrice and suspended in PBS and the OD was adjusted to 1 (equivalent to 109 CFU m/l) at 600 nm. The bacterial suspension (4 ml) was incubated at 37˚C and monitored at a different time intervals (0 to 5 h) and 0.1 ml of upper suspension was removed and mixed with 3.9 ml of PBS. The percentage of absorbance was then measured at 600 nm using the following formula:

Where, At: Absorbance of the upper layer mix at a particular time (1 to 5 h).

A0: Absorbance at time zero.

To determine co-aggregation, equal volumes (5 ml; 1:1) of each selected isolate and pathogens [E. coli (MTCC no. 433) and L. monocytogenes (MTCC no. 1143] were incubated together at 37˚C for 5 h without disturbance. The absorbance was then calculated at 600 nm and the percentages of co-aggregation were determined as follows:

Where, Aprobiotic bacteria: Absorbance of the Lactiplantibacillus isolates as control,

Apathogen: Absorbance of the pathogen as a control

Amix: Absorbance of both probiotic bacteria and the pathogen in a single tube.

Adhesion assay

Bacterial adhesion with human colon cancer cells was performed (18,24) with some modifications. The human HT-29 cell line (ATCC no. HTB-38; ATCC) was used, and the cell culture work was carried out at the Central Research Laboratory, SDM College of Medical Sciences and Hospital (Dharwad, India). Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (both from Himedia Laboratories Pvt, Ltd.) were used to grow HT-29 cells in 12-well flat-bottom cell culture plates until they reached 80% confluence. Prior to the experiment, HT-29 cells were washed gently with PBS twice. Subsequently, the selected eight isolates were centrifuged (5,000 x g for 15 min at 4˚C) and suspended in DMEM without antibiotics to provide approximately 109 CFU ml of the bacterial suspension. Additionally, 200 µl of each strain was added to separate wells and incubated for 2 h at 37˚C in 5% CO2 atmosphere. The HT-29 cells were then washed twice in sterile PBS to remove non-adherent bacteria before being lysed in 2 ml of 0.1% Triton X-100 in PBS. Cell lysates were tenfold serially diluted and plated with MRS agar and incubated for 24 h at 37˚C. The percentage of adherence was expressed using the formula:

Gram's staining for cell adhesion assay

The adhesion of all eight isolates to HT-29 cells in cell culture plates was assessed using the methanol fix technique for microscopic analysis. Each well received 3 ml of methanol, which was allowed to stand for 10 min. Furthermore, fixed cells were stained with Gram's solution (at 28˚C for 5 min) and examined under oil immersion objective (Olympus Corporation) (24,25).

Invasion assay

In vitro methods evaluated the ability of the eight selected isolates to inhibit the colonization of human pathogens with intestinal cells (13,26). HT-29 cells and the eight isolates were inoculated at a concentration of 109 CFU per well in antibiotic-free DMEM and incubated for 2 h. L. monocytogenes (MTCC no. 1143 and E. coli (MTCC no. 433) (109 CFU/well) were inoculated into each well with the antibiotic-free medium and incubated for 1 h at 37˚C with 5% CO2. Furthermore, extracellular bacteria in the well were eliminated by transferring DMEM to 10% FBS with streptomycin 100 U/ml and incubating for 1 h. Subsequently, 2 ml of 0.1% Triton X-100 in PBS was used to lyse the treated HT-29 cells. Additionally, to count invading bacteria, 0.1 ml of 10-4, 10-5 and 10-6 dilutions of cell lysates were inoculated with brain heart infusion agar (Himedia Laboratories Pvt, Ltd.). Invasion assays were performed on cell lines that had only been exposed to pathogens. The invasion percentage was determined using the following formula:

Statistical analysis

All experiments were conducted in triplicate and the results were reported as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, Inc.). Differences between multiple groups were compared using one-way ANOVA with post hoc Tukey's multiple comparison tests, Brown-Forsythe test and Bartlett's test and two-way ANOVA of grouped multiple t-tests. P<0.05 was considered to indicate a statistically significant difference.

Results

Isolation and screening of β-galactosidase-producing bacteria

A total of 450 LAB isolates were isolated from homemade curd samples collected from various regions of Karnataka (12.97 N 77.50 E), India and all isolates were screened for the β-galactosidase enzyme by qualitative assay using X-gal plates (Fig. 1A). Development of blue-green colored bacterial colonies were selected and the presumptive tests confirmed that selected isolates were Gram-positive, non-motile, non-spore producing Bacillus and the carbohydrate fermentation pattern varied among the isolates (Fig. 2). Based on the quantitative assay of β-galactosidase activity level ranging from 728.25 to 1,203.32 (U/ml) Miller units (P<0.05), it was revealed that Lactiplantibacillus sp. GV66 had the highest value of 1,203.32 (U/ml) and L. fermentum GV254 had the lowest value of 728.25 (U/ml) Miller units.

Figure 1 (A) Bacterial colonies (deep grey colour) were identified on a de Man, Rogosa and Sharpe agar plate containing 5-bromo-4-chloro-3-indole-D-galactopyranoside, for β-galactosidase production. (B) β-Galactosidase production and bar graph represents β-galactosidase activity of Lactiplantibacillus isolates in Miller units. Bar graph was plotted incorporating the sample mean (n=3) and error bar (standard deviation of individual isolate).

Figure 2 Carbohydrate fermentation profile of Lactiplantibacillus isolates.

Identification of β-galactosidase probiotic isolates by 16S rRNA gene sequence

Molecular characterization employing 16S rRNA gene sequence analysis was performed for eight most potential β-galactosidase-producing isolates. To estimate an approximate phylogenetic association, the acquired nucleotide sequences were compared with existing nucleotide gene sequences from GenBank using the BLAST tool. Furthermore, nucleotide sequences were aligned using MUSCLE, and a phylogenetic tree was constructed using the neighbor-joining method in the MEGA-X software. All eight isolates belonged to phylum Firmicutes, showing the highest similarity with the genera Lactiplantibacillus. Fig. 3 illustrates the phylogenetic tree, in which GV54, GV64, GV69, GV418 showed 99% and GV419 showed 98% similarity with L. plantarum. However, GV66 showed 98% similarity with Lactiplantibacillus sp., and GV65 showed 99% similarity with L. pentosus, whereas GV254 showed 100% similarity with L. fermentum. Furthermore, nucleotide sequences were deposited in the GenBank database.

Figure 3 Phylogenetic neighbor-joining tree based on the 16S rDNA sequences (~1,300-1,500 bp) of the eight selected lactic acid bacteria strains of curd samples based on the results of alignment. The scale bar represents 0.10 nucleotide substitutes per position.

In vitro evaluation of potential probiotic bacteria

All eight identified β-galactosidase-producing isolates were subjected to probiotic characterization, in which the isolates exhibited considerably less tolerance to gastric juice at pH 2, while at pH 4 the survival rate was increased, and among them L. plantarum GV64 exhibited the highest tolerance (82.6%) and L. plantarum GV54 exhibited the lowest tolerance (17.77%). For bile tolerance L. plantarum GV418 showed the highest tolerance (99.93%) and L. plantarum GV54 showed the lowest tolerance (44.68%) to bile juice after 4 h of incubation. The percentage of tolerance of simulated gastric juice and bile juice is illustrated in Fig. 4. In the pancreatic enzyme tolerance test, the survival rate ranged from 54 to 77.33%, and among them L. plantarum GV54 exhibited the highest tolerance (77.33%) and L. plantarum GV418 exhibited the lowest tolerance (54%) after 4 h of incubation (Fig. 4). The survival percentage of the selected eight isolates in gastric, bile and pancreatic juices confirmed the resistance to upper gastrointestinal conditions. All eight isolates exhibited γ hemolytic activity and were demonstrated as non-pathogenic.

Figure 4 Gastric, bile, and pancreatic juice tolerance tests of Lactiplantibacillus isolates, expressed as percentages [the bar graph was plotted incorporating the sample mean (n=3) and error bar of individual isolates].

Hydrophobicity

The results of assessment of hydrophobicity indicated that the eight isolates were hydrophobic, as revealed in Fig. 5A. L. plantarum GV419 (63%) and L. plantarum GV69 (56%) with maximum affinity, whereas Lactiplantibacillus sp. GV66 (9%) and L. plantarum GV64 (15%) with minimal affinity to xylene and chloroform, respectively.

Figure 5 (A) Hydrophobicity of Lactiplantibacillus isolates with xylene and chloroform. (B) Auto-aggregation expressed as a percentage after each hour of incubation. (C) Co-aggregation expressed as a percentage after 5 h of incubation with E. coli and L. monocytogenes. (D) HT-29 cell adhesion of Lactiplantibacillus isolates expressed as a percentage after 3 h of incubation. E. coli, Escherichia coli; L. monocytogenes, Listeria monocytogenes.

Antimicrobial activity

The findings of the agar well diffusion method indicated that the eight isolates have an antagonistic impact on human pathogens. All isolates exhibited a zone of inhibition ranging from 4.66±0.57 to 27.00±0.00 mm, whereas L. plantarum GV64 was resistant to L. monocytogenes (MTCC no. 1143) and Lactiplantibacillus sp. GV66 and L. plantarum GV69 were resistant to Pseudomonas aeruginosa (ATCC no. 9027) (Table I).

Table I Antimicrobial activity against human pathogenic bacteria.

Table I

Antimicrobial activity against human pathogenic bacteria.

	Human pathogenic bacteria with zone of inhibition in diameter (mm)
Isolates	Escherichia coli (MTCC no. 433)	Staphylococcus aureus (ATCC no. 6538)	Pseudomonas aeruginosa (ATCC no. 9027)	Salmonella abony (ATCC no. BAA2162)	Listeria monocytogenes (MTCC no. 1143)
GV54	12.33±0.57	12.66±0.57	9.33±0.57	16.00±1.0	10.33±0.57
GV64	4.66±0.57	11.00±1.00	7.33±0.57	10.33±0.57	ND
GV65	12.66±0.57	11.33±0.57	12.33±0.57	12.33±0.57	13.33±0.57
GV66	17.66±0.57	9.33±0.57	ND	14.66±0.57	9.33±0.57
GV69	27.00±0.00	14.66±0.57	ND	12.33±0.57	18.66±0.57
GV254	23.33±0.57	11.33±0.57	13.66±0.57	12.33±0.57	9.33±0.57
GV418	23.66±0.57	7.66±0.57	15.00±0.00	13.66±0.57	10.66±0.57
GV419	23.00±0.00	9.66±0.57	12.33±0.57	15.6667	9.00±1.00

[i] Zone of inhibition in mm. ND, not detected.

Antibiotic sensitivity

Antibiotic discs were used to assess antibiotic sensitivity/resistance of the eight isolates, and the assessed antibiotics suppressed the growth of L. fermentum GV254 and L. plantarum GV418. On the other hand, L. plantarum GV54 was cefixime-resistant; L. plantarum GV64 was resistant to ofloxacin, cefixime, and ciprofloxacin; L. pentosus GV65 was amikacin-resistant; Lactiplantibacillus sp. GV66 was ofloxacin-resistant; L. plantarum GV69 was resistant to ampicillin, ofloxacin, and cefixime; and L. plantarum GV419 was resistant to ofloxacin and cefixime [measured in terms of diameter (mm); Table II].

Table II Assessment of antibiotics for Lactiplantibacillus isolates with probiotic potentiality.

Table II

Assessment of antibiotics for Lactiplantibacillus isolates with probiotic potentiality.

	Zone of inhibition in diameter (mm)
Isolates	AMP	AK	OF	P	CFM	CIP	E	AZM
GV54	10.33±0.57	11.66±1.52	9.66±0.57	10.66±0.57	ND	12.66±0.57	29.33±0.15	24.66±0.57
GV64	11.00±0.00	12.33±0.57	ND	13.66±0.57	ND	ND	30.00±00	26.33±1.15
GV65	9.33±0.57	ND	10.33±0.57	12.66±0.57	9.66±0.57	12.33±0.57	30.00±00	24.33±0.57
GV66	6.00±0.00	15.66±0.57	ND	12.00±0.00	10.33±0.57	10.33±0.57	25.66±0.57	20.00±00
GV69	ND	14.66±0.57	ND	10.66±0.57	ND	10.33±0.57	29.66±0.57	24.66±0.57
GV254	12.33±0.57	14.66±0.57	13.00±0.00	10.00±0.00	10.00±0.00	14.66±0.57	27.66±0.57	23.33±0.57
GV418	12.33±0.57	14.00±0.00	8.66±0.57	7.66±0.57	13.33±0.57	9.00±0.00	30.00±00	23.00±00
GV419	8.00±0.00	14.66±0.57	ND	7.33±0.57	ND	8.33±0.57	24.66±0.57	17.66±0.57

[i] Zone of inhibition in mm. AMP, ampicillin; AK, amikacin; OF, ofloxacin; P, penicillin; CFM, cefixime; CIP, ciprofloxacin; E, erythromycin; AZM, azithromycin; ND, not detected.

Auto-aggregation and co-aggregation assays

The percentage of auto-aggregation was measured after every hour of incubation, and L. plantarum GV54 exhibited the highest rate of auto-aggregation (81%), while L. plantarum GV69 and L. plantarum GV419 showed moderate auto-aggregation (61 and 71%, respectively). L. pentosus GV65 exhibited minimal auto-aggregation 50% (Fig. 5B). Furthermore, the eight isolates exhibited co-aggregative properties with both the pathogens, E. coli (MTCC no. 433) and L. monocytogenes (MTCC no. 1143), after 5 h of incubation at 37˚C. The rate of co-aggregation of E. coli and L. monocytogenes ranged between 10 and 28%, and 14 to 42%, respectively. L. monocytogenes exhibited higher co-aggregation compared to E. coli. Notably, L. plantarum GV419 exhibited higher co-aggregation (28.67%) of E. coli compared to L. monocytogenes, while L. plantarum GV64 (42.71%) exhibited higher co-aggregation of L. monocytogenes compared to E. coli (Fig. 5C).

Adhesion assay

Adhesion assay of eight potential isolates to human colorectal adenocarcinoma intestinal epithelial HT-29 cells was determined and graphically represented in Fig. 5D. Bacterial adherence ability with Lactiplantibacillus sp. GV66 was 99.43% and L. fermentum sp. GV254 exhibited considerably less adhesion at 81.14%. The adhesion of all eight isolates was further verified by direct microscopic observation (Fig. 6A).

Figure 6 (Aa) Control HT-29 cell line monolayer and (Ab) arrow showing adhered Gram-positive Lactiplantibacillus isolate to HT-29 cells (magnification, x1,000). (B) HT-29 cell invasion of human pathogens E. coli and L. monocytogenes expressed as a percentage after 3 h of incubation with Lactiplantibacillus spp. E. coli, Escherichia coli; L. monocytogenes, Listeria monocytogenes.

HT-29 cell invasion assay

All eight isolates were investigated for the suppression of E. coli and L. monocytogenes intracellular invasion, as revealed in Fig. 6B. The inhibition percentage ranged from 3.26 to 37.41% and 2.56 to 31.31%, respectively. L. plantarum GV54 had a low percentage of invasion for both pathogens (3.26 and 2.56%). Lactiplantibacillus sp. GV66 and L. plantarum GV64 exhibited the highest percentage of invasion of E. coli (37.41%) and L. monocytogenes (31.31%), respectively.

Discussion

Fermented foods containing LAB are traditionally used in daily food intake. Curd, prepared by fermentation of milk with an inoculum of previously made curd, is used in most households in India, where it constitutes a significant part of the daily diet. The LAB that ferment the milk are likely to differ slightly in each household as there is no standardized starter culture used to prepare the curd. Although curd is considered to contain probiotics, there is little documentation in this line. The present study was undertaken to evaluate the LAB from homemade curd in southern India for probiotic properties. Probiotic diversity is very vast, therefore, in the present study, potential LAB isolates were selected, which are capable of eliminating or reducing LI.

The key findings in the present study successfully revealed the most promising isolates (GV54, GV64, GV65, GV66, GV69, GV254, GV418, and GV419) with probiotic characteristics and β-galactosidase production. LAB that are found in fermented foods aid lactose digestion by increasing the activity of the β-galactosidase enzyme with distinct health advantages (19). It has been reported that lactose from milk digested by β-galactosidase to hydrolyse glucose and galactose, is absorbed by enterocytes and used as an energy source (4). LI symptoms can be managed with dairy products supplemented with β-galactosidase-producing probiotics, which also inhibit human pathogen adhesion (2). Hence, selected isolates were preferred for β-galactosidase enzyme activity with X-Gal and ONPG as substrates (14). While, Lactococcus casei A13 exhibited negative or zero enzyme activity, L. delbrueckii subsp. bulgaricus Db1 exhibited 2,053±25 Miller units (15), which was the highest enzyme activity reported, and served as a positive control. In the present study, β-galactosidase production was highest (1,203.32 Miller units) in Lactiplantibacillus sp GV66 with 58% of enzyme activity compared to the positive control. Therefore, this isolate is a potential β-galactosidase product and hence the strain can be further improved for industrial production of β-galactosidase.

API50 CHL and 16S rRNA sequence analysis may also be used to identify LAB (27,28). Kumara et al (23) used 16S rRNA gene sequence analysis to identify four LAB isolates, all of which were demonstrated to be L. fermentum. The present study identified eight potential isolates, by molecular characterization using 16S rRNA gene sequencing, including L. plantarum GV54, GV64, GV69, GV418 and GV419, Lactiplantibacillus sp. GV66, L. pentosus GV65, L. fermentum GV254, and deposited them in the GenBank database.

Lactiplantibacillus sp. have been termed probiotic bacteria due to their ability to survive in gut conditions such as gastric juice and bile, and exhibit pancreatic enzyme tolerance, as well as intestinal epithelial cell adhesion (29). However, the aforementioned attributes are not completely the same in in vitro testing as found in the in vivo gut system, but it is still a valuable tool for rapid screening of possible probiotic strains. In vivo investigations are more expensive and time-consuming than in vitro studies for evaluating the probiotic characteristics of unknown bacteria; hence, in vitro testing is selected as an alternative (21). Although, an in vivo validation for the above results is a limitation in this study, further animal studies are in progress. Bacteria must be able to survive in intestinal conditions for considerably long periods to be classified as probiotics (30).

L. fermentum HM3 isolate exhibited better acid tolerance at pH 3 for 3 h than the reference strain L. casai Shirota (31). In addition, Vinderola and Reinheimer (15) reported that S. thermophilus exhibited tolerance while L. lactis and L. delbrueckii subsp. Bulgaricus exhibited tolerance at pH 2 and 3. Hsieh et al (32) reported that Lactobacillus strains TSP05, TSF331 and TSR332 were resistant to gastric acid at pH 3.5 in MRS medium for 3 h, and with regard to the cell viability measured as CFU/ml, Lactobacillus TSF331 exhibited the highest viability (4.45x109) in gastric conditions. In the present study, Lactiplantibacillus strains exhibited better acid tolerance at pH 4 than pH 2 for 4 h. Among them, L. plantarum GV64 exhibited the highest tolerance (82.6%) and L. plantarum GV54 showed the lowest tolerance (17.77%). Several studies have reported that 0.3% (w/v) bile salt concentration in the human gut also varies according to diet and the level of pancreatic enzyme secretion (33). Succi et al (34) also reported that most of the LAB showed resistance to a 0.5% bile concentration, and Byakika et al (35) examined whether the probiotic bacteria were tolerant to 1% bile salt. Hsieh et al (32) also reported that 0.3% bile was used for a bile tolerance test and Lactobacillus TSR332 exhibited the highest viability (4.74x108 CFU/ml) indicating high tolerance to bile salt. In the present study, a 0.3% bile concentration was used and eight isolates that exhibited favourable bile tolerance (99.93%) were selected. Among them, L. plantarum GV418 showed the highest tolerance (99.93%) and L. plantarum GV54 exhibited the lowest tolerance (44.68%). Pancreatic enzymes digest various carbohydrates, proteins, and lipids in the human diet (33). According to Shokryazdan et al (31), LAB strains were incubated for 3 h in growth media containing pancreatic enzymes, and the viability of the L. brevis strain differed. All eight isolates of the present study, exhibited high pancreatic tolerance in this investigation, and the degree of tolerance differed among strains. As aforementioned, the assessed LAB isolates were able to survive at gastric pH. Notably, to increase 100% viability of lactic acid bacterial cells, either encapsulation or coating with inert biomolecules can be performed to improve the likelihood of survival.

The eight isolates in the present study with the highest hydrophobicity demonstrated maximum adherence to xylene and chloroform solvents, with L. plantarum GV419 showing 63% adhesion to xylene and L. plantarum GV69 exhibiting 56% adhesion to chloroform. The adherence of fecal isolates to the affinity for n-hexadecane and toluene was examined in another study (25). The greatest hydrophobicity of L. brevis CCMA 1284, L. plantarum CCMA 0743, L. plantarum CCMA 0359 was reported by Fonseca et al (36), and Behbahania et al (37) analyzed L. plantarum strain L15, which showed 54% adherence to solvents. This suggests that the hydrophobic and hydrophilic appendages, as well as other macromolecule components, contribute to the cell surface mosaic's complexity, resulting in hydrophobicity differences toward hydrocarbons.

The ability of auto-aggregation and co-aggregation increased with time, reaching a maximum level at 24 h of incubation rather than at 5 h (38). The auto-aggregation ability of the L. plantarum strain was shown to be the highest in a similar study reported by Fonseca et al (36), which revealed that L. paracasei CCMA 0504 and L. paracasei CCMA 0505 exhibited the highest percentage of auto-aggregation. All eight isolates in the present study exhibited variable values of auto-aggregation. Notably, L. plantarum GV54 showed the highest rate of auto-aggregation with 81%, and L. plantarum GV69 and L. plantarum GV419 exhibited moderate auto-aggregation with 61 and 71%, respectively. Furthermore, all isolates exhibited co-aggregative properties, and among them, L. plantarum GV419 exhibited a high co-aggregation (28.67%) with E. coli while L. planatrum GV64 (42.71%) with L. monocytogenes.

The antimicrobial activity against human pathogens is considered as a main characteristic of probiotic strains that maintain gut health (21). All eight isolates were antagonistic against human pathogens in the present study, with the zone of inhibition ranging from 4.66±0.57 to 27.66±0.57 mm. Asha and Gayathri (18) assessed Lactiplantibacillus strains from curd samples for their antibacterial activity against E. coli, V. cholerae, Klebsiella strains, Proteus strains, and S. dysenteriae. An additional role of the Lactiplantibacillus strain is to inhibit the colonization of pathogenic bacteria to human and animal intestines by secreting several biochemical compounds and enzymes to prevent various infections. Kumara et al (23) reported that L. fermentum inhibited colonization of S. ebony, S. aureus, E. coli, P. aeruginosa, and these bacteria were susceptible to gentamycin, chloramphenicol, cefoperazone, ampicillin, and resistant to ciprofloxacin and vancomycin. The susceptibility of Lactiplantibacillus strains to antibiotics, using the disc diffusion method, was investigated in the present study and it was revealed that these strains were susceptible to penicillin, cefixime, ofloxacin, ciprofloxacin, amikacin, wherein Lactiplantibacillus sp. GV66 was ofloxacin-resistant, and L. plantarum GV69 was resistant to ampicillin, ofloxacin and cefixime.

The assessed strains were susceptible to at least one of the antibiotics that would prevent the formation of cell wall and proteins. Two strains of L. paracasei were mildly sensitive or susceptible to lincomycin, azithromycin, and penicillin, according to Fonseca et al (36), and L. brevis CCMA1284 strain was resistant to these three antibiotics. Of note, if a gene transfer process was involved, then antibiotic resistance would become a dangerous scenario. Alhough, this process may not be communicable and would not be a unique criteria of the microbial genus or species, it would however be a sort of alerting condition. Furthermore, minimum inhibitory concentration (MIC) values for antibiotic susceptibility of the Lactobacillus strains were tested against antibiotics, including ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol (31) The MIC values of the reference strain and the Lactobacillus strains were less than the MIC breakpoint values. Antibiotic resistance genes have accumulated in probiotics, due to the widespread use of probiotics in combination with antibiotics to restore gut flora. There are significant clinical risks if these resistance genes are transferred from probiotics to pathogens in the colon. The antibiotic sensitivity of a few antibiotics used in the present study were found to be within the European Food Safety Authority (EFSA) breakpoint values for all the examined gluten-hydrolysing bacteria and were thus deemed safe (13). In concurrence with the aforementioned evidence, gentamycin, chloramphenicol, cefoperazone, ampicillin, ciprofloxacin and vancomycin antibiotics were selected to ensure the safety and efficacy of the assessed probiotic bacterial isolates.

Probiotic bacteria exhibit cell line attachment and can colonize with intestinal epithelial cells in order to establish themselves in the gut (31,36). The HT-29 cell line and the selected eight isolates were used in the present study for cell adhesion assay, with Lactiplantibacillus sp. GV66 exhibiting strong adhesion at 99.43% and L. fermentum GV254 exhibiting comparatively less adhesion at 81.14%. In other studies, Lactiplantibacillus strains exhibited strong adherence abilities to the Caco-2 and HT-29 cell lines (39). Rashmi and Gayathri (13) investigated whether gluten-hydrolyzing bacteria adhered to the Caco-2 cell line and inhibited cell invasion by E. coli and L. monocytogenes. Byakika et al (35) used the goat ileum to assess cell adhesion, and Behbahani et al (37) used scanning electron microscopy to identify that the adhesion level of the L. plantarum strain L15 to Caco-2 cells was 12%. Aissi et al (24) employed HT-29, Caco-2 and INT-407 cells, as well as Bifidobacterial strains, and microscopically studied them. HT-29 cell invasion assay by E. coli and L. monocytogenes was performed in the present study using an in vitro approach. L. plantarum GV54 had a low percentage of invasion by the pathogens E. coli and L. monocytogenes (3.26 and 2.56%). By contrast, Lactiplantibacillus sp. GV66 and L. plantarum GV64 exhibited the highest percentage of invasion of E. coli (37.41%) and L. monocytogenes (31.31%), respectively, thus revealing their significant potential as probiotic bacteria.

Vinderola and Reinheimer (15) assessed the β-galactosidase activity in L. delbrueckii substrains bulgaricus, L. acidophilus, and other Lactiplantibacillus stains ranging from 1,301 to 2,053 Miller units. Gheytanchi et al (14) also reported the β-galactosidase enzyme activity in L. delbrueckii substrains bulgaricus and L. casei (ranging from 867 to 1,966 U/ml) isolated from cheese. Lactiplantibacillus strains with substantial β-galactosidase activity were identified in the present study; among them Lactiplantibacillus sp. GV66 had the highest value at 1,203.32 (U/ml) Miller units. All of these positive traits of Lactiplantibacillus sp. render this strain ideal for use in probiotic formulations, either alone or in combination with other advantageous probiotic-bacterial isolates. Lactiplantibacillus sp. that produces β-galactosidase could be used as a probiotic supplement to help individuals with LI. Thus, the use of probiotics may lead to a promising method in prevention or management of LI. In addition, it is possible to improve and optimize the enzyme activity and development of milk products with potential probiotics/enzymes for the management of LI.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

Sequence data that support the findings of the present study have been deposited in GenBank with the primary accession no. MN686265: MN696220-MN696226 (https://www.ncbi.nlm.nih.gov/search/all/?term=MN686265:MN696220-MN696226 [accn]). All data used or analyzed during the present study are included within this article. All other data are available from the corresponding authors upon reasonable request.

Authors' contributions

MV executed the planned experimental work, wrote the manuscript and performed the data analysis. DG conceived and designed the study, as well as acquired and analysed the data. VK, CSP and MB drafted the work, and revised it critically for important intellectual content. DG and VK confirm the authenticity of all the raw data. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References