Dr Vishwas Kaveeshwar, Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, Karnataka 580009, India
Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain lactic acid bacteria (LAB) which are considered as GRAS and probiotic bacteria. The present study aimed to investigate indigenous LAB from homemade fermented milk samples collected in remote areas of Karnataka (India), in order to isolate the most potent and well-adapted to local environmental conditions bacteria, which were then evaluated using a step-by-step approach focused on the evaluation of probiotic traits and β-galactosidase-producing ability. LAB were screened using 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) and
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 (
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 (
Probiotics are live microorganisms, which when administered in an adequate amount, confer health benefits to the host (FAO/WHO) (
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 (
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.
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 (
β-galactosidase assay of eight isolates was performed (
Where, A1560 denotes the absorbance before the test and A2560 denotes the absorbance of the reaction mixture.
β-galactosidase-producing isolates were identified by colony characteristics
From the selected bacterial isolates, DNA was isolated using the CTAB protocol (
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) (
Pancreatic enzyme tolerance was calculated according to a study by Rashmi and Gayathri (
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 (
For the hydrophobicity assay, two different solvents
Where, A0 and A are the absorbance before incubation and after incubation, respectively.
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
Antibiotic disc diffusion method was performed according to Kumara
The ability of bacteria to auto-aggregate and co-aggregate was assessed according to Armas
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 [
Where, Aprobiotic bacteria: Absorbance of the
Apathogen: Absorbance of the pathogen as a control
Amix: Absorbance of both probiotic bacteria and the pathogen in a single tube.
Bacterial adhesion with human colon cancer cells was performed (
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) (
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.
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 (
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
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
The results of assessment of hydrophobicity indicated that the eight isolates were hydrophobic, as revealed in
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
Antibiotic discs were used to assess antibiotic sensitivity/resistance of the eight isolates, and the assessed antibiotics suppressed the growth of
The percentage of auto-aggregation was measured after every hour of incubation, and
Adhesion assay of eight potential isolates to human colorectal adenocarcinoma intestinal epithelial HT-29 cells was determined and graphically represented in
All eight isolates were investigated for the suppression of
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 (
API50 CHL and 16S rRNA sequence analysis may also be used to identify LAB (
The eight isolates in the present study with the highest hydrophobicity demonstrated maximum adherence to xylene and chloroform solvents, with
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 (
The antimicrobial activity against human pathogens is considered as a main characteristic of probiotic strains that maintain gut health (
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
Probiotic bacteria exhibit cell line attachment and can colonize with intestinal epithelial cells in order to establish themselves in the gut (
Vinderola and Reinheimer (
Not applicable.
Sequence data that support the findings of the present study have been deposited in GenBank with the primary accession no. MN686265: MN696220-MN696226 (
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.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
(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
Carbohydrate fermentation profile of
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.
Gastric, bile, and pancreatic juice tolerance tests of
(A) Hydrophobicity of
(Aa) Control HT-29 cell line monolayer and (Ab) arrow showing adhered Gram-positive
Antimicrobial activity against human pathogenic bacteria.
Human pathogenic bacteria with zone of inhibition in diameter (mm) | |||||
---|---|---|---|---|---|
Isolates | |||||
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 |
Zone of inhibition in mm. ND, not detected.
Assessment of antibiotics for
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 |
Zone of inhibition in mm. AMP, ampicillin; AK, amikacin; OF, ofloxacin; P, penicillin; CFM, cefixime; CIP, ciprofloxacin; E, erythromycin; AZM, azithromycin; ND, not detected.