Functional and molecular characterization of millet associated probiotic bacteria

Abstract

The lactic acid bacteria are one of the sustainable ways of food production. As the native lactic acid bacteria (LAB) easily manipulate the substrate, helps in production of health essential probiotics with enhancing the bioavailability of the substrate. Here also, in present study, the native LAB isolates isolated from the millets and characterize them for the functional analysis for the human health association. In the present study, fermented millet-associated lactic acid bacteria were screened and characterized for their probiotic potential, safety evaluation and antimicrobial activity. A total of 33 isolates were purified as lactic acid bacteria based on colony shape and biochemical assays. However, only 13 isolates were found to be catalase-negative. Among the 13 isolates, 5 isolates exhibited optimum growth at 6.5% and 9.5% of salt concentrations, pH of 4.5 to 8.5 and 17 °C to 40 °C of the temperature. The probiotic properties of the five isolates exhibited that the survival rates in acid and bile salt concentration ranged from 56.2 to 73.7% and 55.3 to 70.3%, respectively. Similarly, the surface hydrophobicity of the isolates was 41–75%. Antibiotic assay revealed that all five isolates were resistant to Amoxicillin, Cloxacillin, and Penicillin-V. Interestingly, all the isolates except ME26 displayed susceptibility towards Penicillin (2 units) and Tetracycline (10 µg). Further, the four isolates (ME25, ME26, ME9, and ME2) had more antifungal activity against Aspergillus flavus. However, only three, except ME1 and ME2, showed maximum antibacterial activity and produced more antimicrobial compounds compared to reference strain L. plantarum Pb3. The potential probiotic isolates were identified as Weisella cibaria ME9, Weisella cibaria ME26, and Weisella confusa ME25.

Introduction

The recent transition towards the consumption of nutritional and healthy fermented foods among the global population has prompted researchers to search for newer food products with multiple benefits [1]. Owing to the disadvantages, such as high glycemic index, lactose intolerance, and dyslipidemia, etc., of dairy-based fermented foods, non-dairy like legume, millet, and cereal-based fermented products have been considered globally [2]. However, natural fermentation using spontaneous organisms would have both positive and negative health benefits. More recently, functional fermented foods incorporate probiotic and prebiotic substances for improving gut health and reducing the threat of non-communicable diseases are preferred by consumers globally [3]. Probiotics are live microorganisms that offer health advantages to the host beyond the consumption of the inherent nutrition by enhancing the microbial balance of the intestine [4]. Lactic acid bacteria (LAB) are a group of microorganisms that serve as potential probiotics in humans. LAB consists of genus Lactobacillus, Enterococcus, Weissella, Oenococcus, Pediococcus, Streptococcus, Lactococcus, Leuconostoc, Tetragenococcus and Carnobacterium. These LAB colonize gastrointestinal tract of humans (GIT) and confer health benefits [5].

Besides promoting health benefits to humans, these LAB are also used for fermentation of food products. Their inherent functional property in fermented foods including antibacterial, antifungal, anti-inflammatory, antidiabetic and anticancer, which enhances their consumption [6]. One of the promising approaches in food fermentation is to use of native strains, as they are more abundant, adopted to the food conditions and are capable of converting the sugars in the food materials efficiently. Many studies have screened LAB for effective fermentation and functional property supplementation. For instance, Szutowska [7] evaluated the functional role of LABs in the fruits and vegetable juices. They found that LABs enhance the functional property of juices along with the improvement in nutrient content of the juices. LABs are screened for probiotic foods in poultry foods by Reuben et al. [8]. In this study the authors emphasised the isolation of native LAB from poultry intestine enhanced the disease resistance of poultry besides improving yield.

Therefore, it is imperative to isolate screen LAB from native flora of food material to harness the full benefit. In this study LAB from millet native flora has been isolated and characterized. Further this study not only aimed at improving functional and nutritional property of fermented food but also control of aflatoxin contamination in the millet’s grains,

Materials and methods

Sources of strains and millets

The bacterial positive culture, Lactobacillus plantarum Pb3 was obtained from the Department of Agricultural Microbiology TNAU, Coimbatore. Aflatoxin (AFB1, AFB2) producing fungal positive culture Aspergillus flavus (NRRL3357) was received from ARS Culture Collection, National Center for Agricultural Utilization Research 1815 N. University St. Peoria, IL6160. Different types of millet samples such as finger millet (Eleusine coracana), Kodo millet (Paspalum scrobiculatum), little millet (Panicum sumatrense) was obtained from Department of Millets, TNAU (Coimbatore) which served as a basis for the isolation of Lactic Acid Bacteria (LAB). In sterile covers the samples were collected aseptically and ensured that the millet samples are fresh and free from any visible signs of contamination.

Enrichment isolation of LAB from fermented millets

The collected millet samples from different sources were devoid of visible signs of spoilage or contamination. LAB were isolated from millets by enrichment isolation [9]. Ten grams of each millet were surface sterilized using 0.1% sodium hypochlorite and 70% ethanol. Then, the millets were ground with sterile pestle and mortar, mixed in 100 ml of MRS (de Man Rogosa and Sharpe) broth incubated for four to six days at room temperature. After seven days the samples were serially diluted upto 10⁸ and then spread on MRS agar media plates and incubated for 48 h (h) at 37 °C. After incubation, observed the microbial growth on MRS agar plates for distinct colonies.

Morphological and biochemical characterization of LAB isolates

Isolated LAB were observed for their phenotypic characteristics like Gram’s staining and biochemical characteristics viz., catalase test, citrate, MR-VP and fermentation of carbohydrate (includes glucose, fructose, arabinose, sucrose, sorbitol, and xylose) were performed as per the method described by Ismail et al. [10]. The salt resistance ability of the LAB isolates was studied as described by Nath et al. [11] with some modification of different NaCl concentrations 3.5%, 6.5%, 9.5%. The 1% overnight grown cultures were inoculated into 5 ml of MRS broth and incubated for seven days. The sedimentation and biomass were estimated using UV-Vis spectrophotometer at wavelength of 600 nm in respective days. Different temperatures and pH levels were optimized for the growth of bacterial isolates by optical density at 600 nm using UV-Vis spectrophotometer [10]. The selected cultures after showing the probiotic activities for further parameter optimization, taken when the cultures were at there exponential stage (12–14 h of the incubation).

Optimization of LAB growth at different temperatures and pH

The interactions between the two factors such as pH and temperature that influence the bacterial growth were assessed through central composite design (Response Surface Methodology) using Design-Expert^® Version 13 software. The range limit fixed for two variables, pH (4.5–8.5) and temperature (17 –40 °C) and initial bacterial suspension of 1 × (10⁶ CFU/ml) was used to perform the present experiment. The 24 h grown cultures were inoculated into MRS broth and then incubated at various temperatures, including 17 °C, 27 °C, and 40 °C. The pH of the broth was adjusted to 4.5, 6.5, and 8.5 using 1 M HCl and 1 M NaOH for acidic and alkaline pH respectively. The growth was monitored at 600 nm using UV-Vis spectrophotometer. The measurements were taken from 0 to 6 h after inoculation, by every 1 h interval.

Acid and bile salts tolerance test

According to Guo et al. [12] the probiotic properties of the screened LAB isolates evaluated at both low pH value and 0.3% bile salts concentration. In brief, the cultures were inoculated into MRS broth with 0.3% ogall and pH of the broth adjusted to 2.0. The broth without oxgall considered as a control, the optical density at 600 nm was estimated suing UV-Vis spectrophotometer. The following formula was used to calculate the survival rate (%) as per the method described by Feng et al. [13].

Hydrophobicity of the cell surface

The hydrophobicity of bacterial cell surface assessed with the procedure described by Rokana et al. [14], it was tested in vitro by assessing adhesion of cell surface to hydrocarbons (xylene and hexane). Cultures were maintained in MRS broth, centrifuged at 6000 rpm for 15 min to remove pellets, washed them in phosphate buffer solution (pH 7.2), and then resuspended them in the same buffer. Initial absorbance (A₀) was measured at 600 nm. The equal volumes of hydrocarbons and cell suspension (v/v) were added to separate aqueous and organic phases and allowed to settle for 1 h at 37 °C. The aqueous phase (2 ml) was separated, and absorbance (A₁) was measured at 600 nm. The reduction in absorbance was used to determine the % hydrophobicity and was computed with the given formula.

Safety evaluation of bacterial isolates

Haemolytic activity

The 5% (w/v) sheep blood media was used to determine the haemolytic activity of the isolates. The classification of haemolytic activity of isolates was based on the extent of the red blood cells lysis in the medium surrounding the colonies. On sheep blood agar plates, the green-coloured zones around colonies indicate α-haemolysis, clear zones indicate β-haemolysis, and around colonies absence of zones indicates γ-haemolysis. According to Yadav et al. [15], the strains exhibiting γ-haemolysis will be considered as safe.

Antibiotic susceptibility

The disc diffusion method was used to assess the antibiotic susceptibility of the isolates as described by Bauer et al. [16] and Hudzicki [17] on MRS agar plates. The isolates were tested against eight antibiotics (Hi-media): Amoxycillin (10 µg), Cloxacillin (5 µg), Erythromycin (15 µg), Tetracycline (10 µg), Penicillin (2units), Co-Trimoxazole (25 µg), Penicillin-V(3 µg), Cefalexin(30 µg). The overnight cultures were swabbed on prepared MRS agar media plates, allowed to dry for 10–20 min then placed the discs with sterilized forceps and incubated for 24 h at 37 °C, observed for inhibition zone around the discs. The results were shown as susceptible or resistant.

Screening for antibacterial and antifungal activity

Antibacterial activity

Agar well method with slight modifications was used for antibacterial activity estimation as described by Schillinger & L cke [18]. Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus were used as test pathogens and were swabbed on Luria-Bertani (LB) agar plates. The overnight-grown LAB isolates were centrifuged for 15 min at 4 °C at 8,000 rpm. 80 µL of the filtered cell-free supernatant was added to the wells made on the LB agar plates after the supernatant had been filtered with a 0.2-micron pore size syringe filter. LB broth alone served as a control.

Antifungal activity

Detection of aflatoxin from Aspergillus flavus (NRRL3357) was performed by HPLC. The fungal culture was grown on potato dextrose agar (PDA) media plates for 7 days. The fungal cultures were harvested using a 0.1% solution of tween 80 and collected in 50 ml tubes. Next, the equal volumes (v/v) of 2 ml spore suspension were mixed with 2 ml of chloroform, vigorously shaken for 20 min, and then centrifuged at 7000 rpm for 2.5 min. The lower organic layer was carefully transferred into clean new glass vials. The same process was repeated by re-exacting with 1.5 ml of chloroform, and the two extracts were combined and dried. The resulted material was then reconstituted using mobile phase water: methanol: acetonitrile in the ratio of 50:40:10 for HPLC analysis, as per the method described by Ahmed & Asghar [19]. The sample was analysed using Agilent 12,006 HPLC (USA), reverse phase HPLC silica packed C18 column for separation and FLD (fluorescence detector) with an excitation/emission wavelength of 365/425 nm respectively. The samples were eluted with the mobile phase of water: methanol: acetonitrile at a flow rate of 1 mL/min. The injection volume was 50 µL, Peak areas of AFB₁ were recorded. The formula 100x (Peak area of AFB1 in the supernatant/Peak area of AFB1 in the positive (control)) was used to determine the residue percentage as given by Pierides et al. [20].

The LAB isolates were screened against A. flavus (NRRL3357) using agar overlay method as described by Tagg et al. [21]. Initially LAB isolates were inoculated on MRS agar plates, incubated for 24 h at 37 °C. Following that, 0.2% soft maltose extract agar solution containing spore suspension (10⁶ spores/ml) of A. flavus, and poured onto MRS agar containing plates. After four days of incubation at 28 °C, the inhibition zones around the isolated colonies in the MRS agar plates were observed.

Quantitative estimation of antimicrobial compounds

Lactic acid and acetic acid production were estimated according to Kim et al. [22]. MRS broth (Annexure I) of 15 ml was prepared in 100 ml flasks. Overnight grown cultures were inoculated into the conical flask and incubated for 48 h. After 48 h the samples were titrated against 0.1 N NaOH with 1 ml of 0.5% phenolphthalein indicator. Appearance of the pink color indicates the endpoint. Titrated acidity, representing lactic and acetic acid (% w/v), were calculated. Notably, 1 ml of 1 N NaOH equals 90.08 mg lactic acid and 60.05 mg acetic acid. Titratable acidity was then determined as per AOAC 1998.

ME = 1 ml of NaOH is equal to 90.08 mg of lactic acid production and 60.08 mg of acetic acid production.

The estimation of hydrogen peroxide production was performed according to Wakil & Osamwonyi [23]. Overnight grown cultures were inoculated into the MRS broth and incubated for 48 h. After 48 h, the samples were mixed with 15 ml of 0.1M H₂SO₄ and then titrated against 0.1 N potassium permanganate (KMnO₄). The endpoint was observed. The H₂O₂ production was calculated by using following formula.

ME = 1 ml of KMnO₄ is equal to 1.701 mg of H₂O₂.

Molecular identification of LAB strains by PCR

The genomic DNA was isolated from the bacterial cultures using a method given by Shahriar et al. [24]. The overnight grown cultures were centrifuged at 6000 rpm for 5 min at 4 °C, and treated with 1mL of TE buffer and 0.5 mL of n-butanol, repeated the centrifuge to remove n-butanol. Added the lysozyme with TE buffer and incubated for 10 min, and then added proteinase K, 10% of SDS; incubated for 1 h at 37 °C. 200 µl of 5 M NaCl, 150 µl of CTAB added and again incubated for 10 min at 65 °C. The obtained deproteinized lysate after centrifugation at 6000 rpm for 10 min at 4 °C; mixed in 1 mL of phenol: chloroform. DNA precipitated with ice cold isopropanol, preserved at -20 °C with TE buffer. DNA (A260/A280: 2.0) used for PCR.

The PCR amplification was done using a reaction containing Smart Prime 2X PCR master mix-red, the primers for 16s rRNA sequencing as 27F5’-AGAGTTTGATCATGGCTCAG-3’;1492R5’-GTTACCTTGTTACGACTT-3’, DNA template [25]. Bio-Rad gradient PCR, Thermal cycler (USA) was used with the following conditions: first denaturation in PCR amplification at 95 °C for 5 min, 30 denaturation cycles were maintained, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min, respectively. The PCR amplification was then programmed to continue elongating at 72 °C for up to 7–10 min. The amplified products were confirmed with gel documentation, loaded with 250 to 10,000 bp ladder for all isolates.

Statistical analysis

The experimental dataset subjected to two-way analysis of variance ( ANOVA), and Response surface methodology, (Design-Expert^® Version 13 software) and the means were distinguished using Tukey test at a 0.05 probability, employing the statistical software Origin Pro2.0 version, graphical analysis performed in XLSTAT 2021.3.1, as described by Bressani et al. in 2021 [26]. All the experiments were performed in three replications.

Results and discussion

Characterization of lactic acid bacterial isolates

The current study assessed enrichment isolation, characterization of functional properties and evaluation of putative probiotic LAB from fermented millets as well as their ability to inhibit the growth of pathogenic bacteria and A. flavus, a fungus that produces aflatoxin. This research holds significant importance to expand the pool of potential probiotics, providing new options for developing functional foods and dietary supplements, promoting food security, embracing indigenous and sustainable solutions, understanding microbial diversity, and harnessing health benefits.

In this experiment, a total of 33 isolates were purified from fermented millets based on colony morphology. The colony morphology of the isolates appeared as small to medium size, convex and round shaped colonies and gram-positive rods and cocci. Among the 33 isolates, 13 were catalase negative, which are further subjected to biochemical tests. Among the 13 isolates ,12 was negative for the VP test and citrate reduction test and all 13 isolates fermented the glucose, 12 isolates fermented fructose and xylose, 7 isolates fermented arabinose and sucrose, 7 isolates fermented sorbitol as mentioned in Table 1. Gunkova et al. [24] reported that the disaccharides carbohydrate metabolism of LAB differs in their species composition. On maltose media, the cultures S. thermophilus and L. delbruckii ssp. bulgaricus showed the slowest growth rates. The mixed cultures like L. lactis subspecies cremoris, L. lactis subspecies lactis, and L. lactis subspecies lactis biovar diacetylactis showed rapid fermentation of maltose, sucrose, and lactose. In the present study, findings provide valuable information about the metabolic capabilities of the LAB isolates. Similar results were reported by Nath et al. [11], indicating that all isolates were able to ferment the glucose, specific isolates exhibited the ability to ferment fructose, sucrose, xylose, and sorbitol, and arabinose. These differences indicate diverse metabolic capabilities among the isolates and their too variations in enzymes or regulatory mechanisms.

Table 1.

Morphological and biochemical characteristics of lactic acid bacteria

Isolates	Gram staining	Shape	Catalase	Citrate utilization	MR-VP test	Carbohydrates fermentation						Osmotic resistance
						Glucose	Fructose	Arabinose	Sucrose	Sorbitol	Xylose	3.5%	6.5%	9.5%
ME1	+	Rods and cocci	-	-	-	+	+	-	-	+	+	+++	++	+
ME2	+	Rods and cocci	-	-	-	+	+	-	-	+	+	+++	++	+
ME4	+	Rods and cocci	-	-	-	+	+	-	-	+	+	+++	++	-
ME5	+	Rods and cocci	-	-	-	+	+	-	-	+	+	+++	++	-
ME6	+	Rods and cocci	-	-	-	+	+	-	-	+		+++	++	-
ME9	+	Rods	-	-	-	+	+	+	+	+	+	+++	++	+
ME11	+	Rods	-	-	-	+	+	+	+	+		+++	++	-
ME22	+	Rods	-	+	+	+	-	-	-	-	+	+++	++	-
ME24	+	Rods and cocci	-	-	-	+	+	+	+	-	+	+++	++	-
ME25	+	Rods and cocci	-	-	-	+	+	+	+	-	+	+++	++	+
ME26	+	Rods and cocci	-	-	-	+	+	+	+	-	+	+++	++	+
ME28	+	Rods and cocci	-	-	-	+	+	+	+	-	+	+++	++	-
ME29	+	Rods and cocci	-	-	-	+	+	+	+	-	+	+++	++	-

Based on the result of above-mentioned biochemical tests, 13 isolates were selected and subjected to salt resistance at 4.5, 6.5, and 8.5% concentration. All isolates displayed tolerance to 4.5% and 6.5% salt concentrations. Among them, five isolates (ME1, ME2, ME9, ME25, and ME26) were able to tolerate salt concentration at 8.5% after 7 days of incubation (Fig. 1). The results were similarly matched with Lee et al. [24] reported on the growth kinetics of W. confusa at different salt concentrations. According to their study, the majority of the strains exhibited the tolerance ability to salt concentration at 6.5% but few isolates were at thrive whenever exposed to 8% salt concentration. The findings are also corresponded with Khedid et al. [27], it was observed only 0.5% of the strains exhibited viability in broth with 8% NaCl concentration. The three LAB species named as L. fermentum, Enterococcus faecium, and Enterococcus hirae shown the capability to survive at high-salinity fermentation conditions which reported by Bazireh et al. [28]. These results suggested that the selected LAB isolates are well adapted to survive in varying osmotic conditions.

Fig. 1.

Salt resistance ability of the lactic acid bacteria at different concentrations

Further, the optimization of pH and temperature conditions were conducted and analyzed through response surface methodology (RSM) statistical tool. The author Gorlach-Lira et al. [29], applied this statistical tool, to analyse the data and optimization, by creating 3D response surface plots and they visually examined the pH and temperature influence on bacterial growth. De Lima et al. [30] studied the lactic acid production using this analytical tool, which had positive influence on supplemented media, temperature, and pH control 6.5 and reported that as the temperature increases the production of lactic acid was also increased. At pH 6.0, the production of lactic acid was maximum, for optimum production of lactic acid the range of temperature and pH was vary from 34 °C to 39.6 °C and pH ranges from 5.9 to 6.5, respectively. In the present study, the influence of pH and temperature on bacterial growth were optimized by RSM, revealed that all isolates displayed optimum growth at pH 6.5, except for ME26, which exhibited optimum growth at pH 4.5; while ME9, which exhibited optimum growth at pH 4.5 and 8.5 (Fig. 2). Furthermore, all isolates exhibited optimal growth at 27 °C temperature. The isolate ME25 shown optimum growth at 17 °C and 40 °C temperatures. ME2 and ME26 showed optimum growth at 40^OC temperature (Fig. 3). The RSM analysis showed a high determination coefficient for pH (R² = 0.889), and temperature displayed a strong determination coefficient (R² = 0.9393) indicating the best fit of model to the experimental data. Both pH and temperature had significant influence on the growth of the bacterial isolates, as indicated by the ANOVA results.

Fig. 2.

Optimization of lactic acid bacterial growth at different pH. (A):ME1; (B):ME2; (C): ME9; (D): ME25; (E): ME26

Fig. 3.

Optimization of lactic acid bacterial growth at different temperatures. (A):ME1; (B): ME2; (C): ME9; (D): ME25; (E): ME26

Evaluation of probiotic properties

Probiotic bacteria should have the ability to sustain in the gastric conditions in the stomach, which typically maintains an acidic pH < 2.0 and bile salt concentration. Additionally, they need to remain viable for at least 4 h before reaching the gastrointestinal tract. Consequently, the primary factors within the host that can impact commercial probiotics are the higher levels of acidity in both the proventriculus and ventriculus. Therefore, exhibiting tolerance to acidic conditions becomes a crucial criterion when selecting potential probiotic strains to ensure their viability and effectiveness [28]. Three LAB isolates exhibiting a tolerance to acidic conditions, with a survival rate of 50% or higher at gastric conditions, were categorized as acid-tolerant [31].

In the present study all the six isolates including reference strain (L. plantarum Pb3) showed survival rate above 50% at both acidic pH ( pH 2 and 4). However, isolates ME9, ME25, and ME26 exhibited higher survival rate of 66.7%, 66.5% and 66%, respectively than L. plantarum Pb3 (62%) at acidic pH 2 upto 4 h (Fig. 4). At pH 4, ME25 exhibited the highest survival rate of 73.3%, followed by ME2 and L. plantarum Pb3 (71.1%), ME26 (69.6%), ME9 (68.9%) and ME1 (67.5%) (Fig. 5). Contrary results were obtained [32] who observed the strain Lacticaseibacillus paracasei L2 demonstrated growth and acid tolerance at pH 2 and 2.5, no viable strains were observed after both 2 and 3 h of incubation and they have observed the survival rate at pH 4.

Fig. 4.

The survival rates of isolates in an acidic pH 2 (A) ME1, (B) ME2, (C) ME9, (D) ME25, (E) ME26 and (F) L.plantarum Pb3 from 0 to 4 h at 37 ºC. The data shows mean±SD of triplicate values of independent experiments

Fig. 5.

The survival rates of isolates in an acidic pH 4 (A) ME1, (B) ME2, (C) ME9 ,(D) ME25, (E) ME26 and (F) L.plantarum Pb3 from 0 to 4 h at 37 ºC. The data shows mean±SE of triplicate values of independent experiments

In the bile salt concentration, ME26 showed the highest survival rate at (66.2%), followed by ME9 (61.5%) and ME25 (60.7%) when compared to L. plantarum Pb3 which exhibited 59.1% survival rate. In contrast, ME1, ME2 demonstrated lower survival rates, recording 57.5%, 56.8%, respectively (Fig. 6). Similar observations reported by Li et al. [33] Lactobacillus mucosae exhibited 62.7% survival rate at 0.3% bile salt concentration after 4 h. The viability of the isolates decreased more over time in acidic pH 2 compared to pH 4 and bile salt concentration. Some of the researchers reported as the basis for the assessment of probiotic properties focused on acid and bile salt tolerance. Chou & Weimer [34] reported as their strains were able to cultivate at pH 3.5. Prasad et al. [35] reported that there is a considerable decrease in the sustainability of the strains at pH 2.0 and below. Dave & Shah [36] standardized the pH at 3.0 to screen the LAB strains for acid tolerance. The bile salt concentration of isolates may be due to the presence of proteins on bacterial cell walls [33].

Fig. 6.

The survival rate of isolates at 0.3% bile salt concentration (A) ME1, (B) ME2, (C) ME9, (D) ME25, (E) ME26 and (F) L. plantarum Pb3. Data shown are mean±SE of triplicate values of independent experiments

According to Dey et al. [37], the interaction between host cell surface and the cell membrane of LAB strains is necessary for the attachment to the membrane linings of the gastrointestinal tract. As a result, the physical and chemical characteristics of cell, surface hydrophobicity were important for the colonisation of microbes on the host cells. In the present study, the hydrophobicity of their cell surfaces measured using organic solvents n-hexane and xylene revealed that the isolate, ME2 having the highest cell surface hydrophobicity at 75% followed by ME25 and ME26 which showed 68.5% and 64.3%, respectively with n-Hexane. In case of xylene, ME26 had the highest at 70% followed by ME2 (67.9%) nd ME1 (65.6%). L. plantarum Pb3 and ME25 showed the least affinity to xylene (41%) (Fig. 7). Similar results were obtained by [9] who demonstrated the highest cell surface hydrophobicity of Lactobacillus plantarum strain COORG-6 of (62.6%) with n-hexane Lactobacillus plantarum strain COORG- of (63.6%) with xylene. Henning et al. and Son et al. [38, 39] reported that the important factor for choosing probiotic organisms as starter cultures in fermentation is their ability to inhibit pathogens from binding to gastrointestinal linings through competitive exclusion. This kind of process was typically linked to the existence of antibacterial and fungal compounds.

Fig. 7.

Surface hydrophobicity of the LAB isolates. (A) ME1, (B) ME2, (C) ME9, (D) ME25, (E) ME26 and (F) L. plantarum Pb3. Data shown are mean±SE of triplicate values of independent experiments

Screening for safety evaluation of lactic acid bacteria

The haemolytic activity and antibiotic susceptibility of the isolates were used to evaluate their safety, demonstrating the non-pathogenic character of the probiotic isolates [39]. Anas and coworkers [40] reported that four strains of Lactobacillus pentosus exhibited α haemolysis. According to Argyri et al. [41], non-haemolytic strains showed no signs of virulence and may be recommended for consumption as probiotics, although more extensive research is needed to confirm their usage as probiotic strains in food. The “no zone” in the blood agar test plates inoculated with all five isolates confirmed the results that none of the five isolates had any haemolytic activity. The haemolytic activity of selected isolates was given in Fig.S1. These findings align with prior studies conducted by Asadi et al. [42] and Wei et al. (2022) [43], all of whom isolated various non-haemolytic Lactobacillus species from different dietary sources and millet-based alcoholic beverages. The researchers [44] have reported analogous results, reinforcing the idea that probiotics typically lack haemolytic activity, thus enhancing their safety for probiotic applications.

The antibiotic susceptibility of the five isolates was evaluated against eight antibiotics. Notably, all isolates demonstrated resistance to Amoxicillin (10 µg), Cloxacillin (5 µg), and Penicillin-V (3 µg). The susceptibility was observed towards Erythromycin, Co-Trimoxazole, and Cephalexin. Interestingly, for Penicillin (2 units) and Tetracycline (10 µg), all isolates except ME26 displayed susceptibility. This comprehensive assessment indicates isolate’s varying response to antibiotics and their potential differences in resistance profiles (Table 2). Those LAB strains resistant to antibiotics were being classified into potential probiotics and usually, do not cause the safety concern. In these case, Rojo-Bezares et al. [45] reported that the antibiotic resistance is not a transmissible type and possess health benefits to consumers, despite the strains isolated from fermented millets may induce some health benefits to consumers.

Table 2.

Antibiotic sensitivity of lactic acid bacterial isolates

S. No	Antibiotics	Name of the LAB
		ME1	ME2	ME9	ME25	ME26	L. plantarum Pb3
1	Amoxycillin(10 µg)	R	R	R	R	S	R
2	Cloxacillin(5 µg)	R	R	R	R	S	S
3	Erythromycin(15 µg)	S	S	S	S	S	S
4	Tetracycline(10 µg)	R	S	S	S	S	S
5	Penicillin (2units)	R	S	R	S	S	S
6	Co-Trimoxazole(25 µg)	S	S	S	S	S	S
7	Penicllin-V(3 µg)	R	R	R	S	S	R
8	Cefalexin(30 µg)	S	S	S	S	S	S

(R) = Resistance, (S) = Susceptible. Data noted as per EFSA guidelines

Screening for antibacterial activity of lactic acid bacteria

The pathogenic indicator bacteria Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Escherichia coli MC1400, and Listeria sp. were used to determine the antibacterial activity of LAB and Bifidobacterium strains [46]. In the present study, the antibacterial activity of LAB isolates towards bacterial pathogens was evaluated and results showed that the investigated pathogens viz., E. coli, P. aeruginosa, B. subtilis and S. Aureus were inhibited by three isolates ME9, ME25, and M26 among five isolates tested (Table 3). The cell-free supernatant of ME25 demonstrated the highest antibacterial activity, with a zone of inhibition of (15.1 ± 1.8 mm) against E.coli, followed by ME26 (14.1 ± 1.0 mm). Similarly, against P. aeruginosa, ME25 displayed inhibition (10.1 ± 1.2 mm). However, the isolate ME26 exhibited notable inhibition against B. subtilis (12.6 ± 2.0 mm), followed by ME25 (11.1 ± 1.1 mm) and ME9 (10.9 ± 1.0 mm). Regarding inhibition against S. aureus culture, ME26 and L. plantarum Pb3 showed considerable inhibition activity (10 ± 1.7 mm) and (10.1 ± 1.1 mm) respectively, followed by ME9 (8.4 ± 1.3 mm). On the other hand, ME1 and ME2 did not demonstrate any antibacterial activity.

Table 3.

Antibacterial activity of lactic acid bacterial isolates

Isolates	Zone of inhibition (cm)
	Escherichia coli	Pseudomonas aeruginosa	Bacillus subtilis	Staphylococcus aureus
ME1	ND	ND	ND	ND
ME2	ND	ND	ND	ND
ME9	8.7 ± 1.0	7.3 ± 1.0	10.9 ± 1.0	8.4 ± 1.3
ME25	15.1 ± 1.8	10.1 ± 1.2	11.1 ± 1.1	7.1 ± 1.1
ME26	14.1 ± 1.0	10.5 ± 1.1	12.6 ± 2.0	10 ± 1.7
L. plantarum Pb3	14.1 ± 1.8	9.1 ± 1.2	10.1 ± 1.1	10.1 ± 1.1

Values are mean (± standard error) (n = 3) of inhibition zone values of cell free supernatant. ND- Not deducted

Henning et al. and Son et al. [38, 39] reported that the important factor for choosing probiotic organisms as starter cultures in fermentation is their ability to inhibit pathogens from binding to gastrointestinal linings through competitive exclusion. This kind of process is typically linked to the existence of antibacterial and fungal compounds.

Analysis of antifungal activity of lactic acid bacteria

The Aspergillus flavus (NRRL3357) strain has been confirmed to produce toxins AFB₁, using Agilent 12,006 reverse phase HPLC (USA). The peak area percentage of 81.16% at 7.9 to 8.0 indicates AFB₁ (Fig. S2). The results on the antifungal activity of new isolates revealed that among the five isolates, only two, ME25 and ME29 showed maximum inhibition zone against fungal pathogen at all incubation temperature (17 °C, 27 °C and 40 °C), compared with reference strain. Whereas at 27 °C, all exhibited a zone of inhibition. The isolate ME25 exhibited a significant inhibition zone (11.0 ± 1.0 mm) followed by ME26 (7.3 ± 1.2 mm) under 17 °C. The inhibition zone at 27 °C, ME25 and ME26 showed a considerable inhibition zone (9.5 ± 1.8 mm) and (9.3 ± 2.1 mm), respectively, which was comparable with L. plantarum Pb3 (Table 4). The result was in accordance with Somashekaraiah et al. [47] and they assessed the antifungal activity of LAB isolates over a period of seven days, MYSN 106 exhibited the significant antifungal activity. It demonstrated a zone of inhibition measured as 10.45 ± 0.70 mm against A. flavus. This antagonism ability of strains may be due to the production of organic acid, H₂O_2, etc., The author’s [48] stated the antagonistic effect of hydrogen peroxide may be resulted from sulfhydryl groups oxidation causing denaturation of many of enzymes, and from membrane lipids peroxidation thus increases the permeability of membranes. Peptides, reuterin, organic acids, hydrogen peroxide, phenolic antioxidants, diacetyl, and other substances were listed as several antifungal agents by Sadiq et al. [46]. This demonstrates the potential of these strains to counter A. flavus growth, suggesting their applicability in antifungal strategies.

Table 4.

Screening of isolates for inhibition of aflatoxin producing Aspergillus flavus

Isolates	Pathogen	Zone of inhibition at Incubation temperature (cm)
		17 °C	27 °C	40 °C
ME1	Aspergillus flavus (NRRL3357)	ND	8.1 ± 2.3	ND
ME2		ND	9.1 ± 1.4	ND
ME9		ND	7.3 ± 1.5	ND
ME25		11 ± 1.0	9.5 ± 1.8	7.3 ± 1.8
M26		7.3 ± 1.2	9.8 ± 2.1	7.9 ± 1.9
L. plantarum Pb3		6.7 ± 1.0	8.8 ± 0.9	7.1 ± 1.2

Values are mean (± standard error) (n = 3) of inhibition zone values of cell free supernatant. ND- Not deducted

Quantification of antimicrobial compounds

The antimicrobial compounds produced by the isolates were presented in Table 4. The isolates, ME1 and ME9 generated lactic acid exclusively, while ME2 produced acetic acid alone. The isolates ME25 and ME26 produced both lactic acid and acetic acid. ME25 produced a substantial amount of lactic acid at 5.07 g/l, followed by ME9 at 4.8 g/l, compared to L. plantarum Pb3 (4.5 g/l) at 48 h (Table 5). The same trend was observed in acetic acid production. Whereas, none of the isolates produced H₂O₂ at a concentration of 0.6 g/l. Gharib et al. [49] reported that antagonist properties of LAB due to production of organic acids and bacteriocins. Similar results were obtained in the study performed for organic acids production [50] and the strain W. confusa WM36 produced lactic acid and acetic acid up to (2.6% w/v) and (1.6% w/v), respectively.

Table 5.

Quantification of antimicrobial compounds of the lactic acid bacteria isolates

Isolates	Time (h)	Antimicrobial compounds (g/l)
		Lactic acid	Acetic acid	H2O2
ME1	24 h	2.3 ± 0.9	ND	ND
	48 h	4.9 ± 1.5	ND	ND
	72 h	4.1 ± 1.9	ND	ND
ME2	24 h	ND	1.3 ± 2.3	ND
	48 h	ND	4.8 ± 0.7	ND
	72 h	ND	4.4 ± 1.3	ND
ME9	24 h	1.9 ± 1.3	ND	ND
	48 h	3.4 ± 0.9	ND	ND
	72 h	4.8 ± 0.9	ND	ND
ME25	24 h	2.6 ± 0.9	3.4 ± 1.2	ND
	48 h	5.07 ± 1.8	5.9 ± 1.6	ND
	72 h	4.9 ± 2.8	4.9 ± 1.9	ND
ME26	24 h	3.4 ± 1.2	1.3 ± 0.9	ND
	48 h	4.7 ± 2.3	3.8 ± 1.0	ND
	72 h	4. 0 ± 1.7	3.0 ± 2.3	ND
L. plantarum Pb3	24 h	4.2 ± 0.0	3.2 ± 0.3	ND
	48 h	4.5 ± 0.7	3.9 ± 1.8	ND
	72 h	4.0 ± 2.6	3.2 ± 1.3	ND

Values are mean ± standard error (n = 3) of antimicrobial compounds; ND- Not deducted

Molecular identification LAB isolates

Limited studies were performed for isolation and characterization of LAB from fermented millets. Lactococcus lactis subspecies lactis, Streptococcus lutetiensis, Lactobacillus plantarum, Lactobacillus fermentum, Paenibacillus species, Enterococcus faecium, Enterococcus lactis and Pediococcus acidilactici were isolated from fermented finger millet [51]. Manovina and Coworkers (2022) [52] isolated LAB from fermented millet porridge and identified as Enterococcus lactis and Weisella cibaria. Huligere et al. (2023) [53] isolated LAB from fermented batters and characterized as potential probiotics. Further they have identified as Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Lacticaseibacillus casei and Lacticaseibacillus paracasei based on molecular characterization. In the present study, the bacterial isolates from fermented millets were recognized by matching the 16 S rRNA partial sequences with the sequences from NCBI GeneBank and the sequence similarity showed from 91.3 to 94.3%. The isolates were confirmed as Weisella cibaria strain ME9, Weisella cibaria strain ME26, and Weisella confusa strain ME25 with accession numbers OR346143; OR220340; OR335813 respectively by NCBI GenBank. The evolutionary relation of the strains was analysed by Neighbour-Joining method and results were given in Fig. 8.

Fig. 8.

The phylogenetic analysis of lactic acid bacterial isolates (ME9, ME25, and ME26) from fermented millets in comparison to reference strains. (The percentage of replicate trees, in which related taxa grouped together in bootstrap test with 1000 replicates, is indicated next to the branches)

Conclusion

The LAB, Weisella cibaria strain ME26, and Weisella confusa strain ME25 obtained from fermented millets displayed considerable probiotic properties as well as had antibacterial and antifungal activity when compared to reference strain, L. plantarum, Pb4. Among them, ME26 had best probiotic properties since, it exhibited osmotic tolerance, optimal growth at specific pH and temperature conditions, antagonistic activity against pathogens, acid and bile salt tolerance, non-haemolytic activity, and different antibiotic sensitivity profiles. These findings contributed to understand the probiotic potential of LAB from fermented millets and emphasized their potential may be used for application in functional foods. Further research is warranted to explore their specific mechanisms of action and their potential to promote human health benefits and food safety.

Author contributions

BG : methodology, investigation, conceptualization, writing-original draft. AR: conceptualization, methodology, investigation, supervision, formal analysis, writing-review & editing. MB: supervision, resources, writing-review & editing. SK: conceptualization, Methodology. IM: formal analysis, writing-review & editing. JSP: methodology, writing-review.

Funding

This work was supported by DST-SHRI under cluster, New Delhi and ICAR-AICRP on PHET, Ludhiana.

Data availability

The date will be provided from the corresponding author upon reasonable request. The sequence data generated in the present study has been submitted to NCBI GenBank with accession numbers OR346143; OR220340; OR335813.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.