Exploring the probiotic potentiality and antibacterial activity of idli batter isolates of lactic acid bacteria from West Bengal, India

Abstract

Background

The lactic acid bacteria (LAB), from diverse sources, are of great importance as probiotics, and several authors from around the globe have reported LAB, isolated from various fermented foods, as potential antimicrobial agents. The current study explored the antibacterial activity and probiotic property of idli batter isolates of LAB, for the first time from Malda (West Bengal, India).

Results

The LAB procured from fresh and fermented idli batter samples had antibacterial activity against pathogenic as well as food-borne bacteria with zone diameter of inhibition of 16, 18 and 23 mm with concentrations 25, 50 and 75 μl/well, respectively, as determined by agar-well diffusion method. The identification of isolated LAB was executed through biochemical tests, 16S rRNA gene sequencing and phylogenetic analysis. The LAB isolates from fresh idli batter: LMEM1001 and LMEM1002, showed maximum (96.81% and 95.20%, respectively) similarities with Lactiplantibacillus pentosus and Lactiplantibacillus plantarum, respectively, whereas the fermented idli batter isolates, LMEM1006 and LMEM1008, showed maximum (96.11% and 98.40%, respectively) similarities with Lactiplantibacillus plantarum and Limosilactobacillus fermentum, respectively. Safety profiling of isolated LAB was executed using antibiogram, DNase and gelatinase tests.

Conclusions

The idli batter-derived lactobacilli have been demonstrated as good probiotics, which displayed excellent antibacterial activity against clinical and food-borne bacteria. Overall, the idli batter isolates of LAB might be useful as probiotics for human consumption and as biotherapeutics in combating bacterial antibiotic resistance.

Background

Multiple antibiotic-resistant (MAR) bacteria are life threatening including the global food-borne infections [1, 2], and most of them are related to fresh-cut fruits, which is caused by Salmonella, Listeria monocytogenes, Staphylococcus aureus, Yersinia sp., Shigella sonnei and Escherichia coli [3, 4]. Antimicrobial resistance (AMR) was one of the top ten worldwide public health issues for human in 2019, and total 143 countries (approximately 90% of the global population) together with 11 member States of the WHO South East Asia region have settled a national action strategy 2019–2023, which identifies containment of AMR as precedence [5]. In the current scenario, nosocomial infections are involved various pathogens, including Enterococcus species, coagulase-negative Staphylococci, S. aureus, Proteus species, Enterobacter sp., Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella oxytoca, Klebsiella pneumoniae, E. coli, whereas multidrug-resistant (MDR) isolates include extended-spectrum cephalosporin-resistant Enterobacter species, vancomycin-resistant E. faecium, and methicillin-resistant S. aureus (MRSA), carbapenem-resistant A. baumannii, Enterobacter sp., E. coli, P. aeruginosa, K. oxytoca, K. pneumoniae [6]. Due to lack of new active compounds in the existing worldwide, treatment regimen has directed to a major upsurge in antibiotic resistance [7]. To counter the global AMR situation, selection of alternative treatment including probiotic therapy is warranted.

Currently the bio-preservation technique is an integrated biotechnology using lactic acid fermentation for humanizing properties of food and vegetables. Two genera such as lactobacilli and lactococcus can show probiotic potentials with large range of bioactivity [8]; by producing antimicrobial peptides (AMP) like bacteriocins, acidic pH, hydrogen peroxide, carbon dioxide or diacetyl [9,10,11], and has strong antagonistic activity on the development and toxin fabrication of other bacteria. Lactic acid bacteria (LAB) are useful beneficial microorganisms, with different well-known probiotic strains, promote good health and generally accepted as safe for human intake [12]. Several researches indicate that fermentation products of probiotics can enhance the synthesis of some bioactive component with beneficial effects that advances the functional value and acceptability of food products [13, 14].

Probiotics isolated from dairy and non-dairy sources can exhibit broad spectrum of antibacterial activity [15,16,17]. As per previous studies, potent LAB can be isolated from cereal dough fermentation [18], traditionally fermented legume products [19], mulkimchi fermentation [20] as well as idli batter [21, 22]. So, probiotic characterization in terms of stress (NaCl, bile salt, low pH, wide range of temperature, etc.) tolerances and antibacterial capacity testing are necessary task to know their beneficial effects on human.

The current investigation was set in order to search the alternative therapeutics against bacterial infection and in combating their antibiotic resistances. In this part of the globe, there is scanty report in terms of isolation, characterization and bioactivity of non-dairy-based probiotics. Thus, isolation and characterization of lactobacilli from non-dairy product, such as idli batter samples (both fermented and non-fermented), were chosen to be explored in order to assess their (lactobacilli) potentiality and inhibition efficacy against infectious bacteria.

Methods

Idli batter sample and lactic acid bacteria

Two idli batter samples (one freshly prepared, and the other was fermented for 24 h) collected from local vendor from Malda district, West Bengal, India, were utilized in the current study. The making of idli batter involved four steps including soaking the rice, urad dal/black gram and a few numbers of fenugreek (methi) seeds, mixing them, and lastly fermenting the batter.

In order to isolate the lactic acid bacteria (LAB), de Man, Rogosa and Sharpe (MRS) broth (Hi-Media, Mumbai, India) was inoculated with freshly collected as well as 24-h fermented idli batter samples separately, and after incubation for 24–48 h at 37 °C, single isolated colonies were procured on MRS agar (Hi-Media, India) plate, from each of the idli batter samples, by streak dilution of the broth culture as described earlier [23].

Phenotypic identity

The size, shape, margin, opacity and colour of the colonies of isolated LAB were documented. Phenotypic and biochemical characterization of the isolates was done following the standard protocol [24, 25]. To study the bio-chemical properties, catalase, citrate utilization, nitrate reduction, indole production, methyl-red (MR), Voges–Proskauer (VP), urease, oxidase tests including different sugar (n = 20, Hi-Media, India): Adonitol (Ad), Arabinose (Ar), Dextrose (De), Dulcitol (Du), Fructose (Fc), Galactose (Ga), Inositol (Is), Inulin (In), Lactose (La), Maltose (Ma), Mannitol (Mn), Mannose (Mo), Melibiose (Mb), Raffinose (Rf), Rhamnose (Rh), Salicin (Sa), Sorbitol (Sb), Sucrose (Su), Trehalose (Tr), and Xylose (Xy), fermentation was performed following Bergey’s manual [25], as described earlier [23].

Molecular identity

The identity validation of the selected idli LAB isolates was done by 16S rRNA gene sequencing and phylogenetic analyses.

The identification of isolated LAB (LMEM1001, LMEM1002, LMEM1006 and LMEM1008) was confirmed based on 16S rRNA gene sequence analysis. The ~ 1.3 kb/1.5 kb, 16 s-rDNA fragment was amplified using high-fidelity PCR polymerase and sequenced bi-directionally using 16S rRNA specific primers (forward primer: 5′-GGATGAGCCCGCGGCCTA–3′ and reverse primer: 5′-CGGTGTGTACAAGGCCCGG–3′) from Biokart Pvt Ltd, India.

The sequence data were aligned using the software “MEGA X” and analysed by ClustalW [26]. The nearest-known relatives of tests sequences obtained using nucleotides homology search through NCBI website with BLAST (Basic Local Alignment Search Tool) technique. Evolutionary distances have been calculated using the method of Nei and Kumar [27], and the phylogenetic trees with the sequences were prepared following the neighbour-joining method using bootstrap with 1000 replicates [28].

Probiotic property

The sodium chloride, bile salt, low-pH and temperature (for 17, 45 and 60 °C) tolerance were determined as probiotic properties of the isolated lactobacilli. The bile salt and low-pH (acid) tolerance were tested at an interval of 24, 48 and 72 h, respectively, following the protocol of Liong and Shah [29], and to sodium chloride (NaCl), by using the protocol of Chowdhury et al. [30], with slight modifications as mentioned elsewhere [23]. Briefly, the isolated lactobacilli were grown (for 24 h at 37 °C), in sodium chloride containing (of 2, 4, 6% and 8%) MRS broth, and then, the growth of lactobacilli, following subculture of the MRS broth cultures, on MRS agar (for 24 h at 37 °C), showed their tolerance to sodium chloride.

Antibacterial activity

The antibacterial activity of eight LAB isolated from fresh and fermented idli batter was determined by agar-well diffusion method, against the indicator strains of pathogenic bacteria (procured from clinical samples including urine, threat swab and pus), both Gram negative: Pseudomonas aeruginosa (n = 3; strain code PA1, PA2 and PA3), Escherichia coli (n = 2; strain code EC1, EC2) and Gram positive: Staphylococcus aureus strain SA1, and food borne bacteria, Gram positive: Bacillus cereus (n = 2; strain code: BC1, BC2) along with two standard strains of Gram negative: E. coli ATCC 25922 and Gram positive: Listeria monocytogenes MTCC 657. The bacterial strains are maintained in cystine tryptone agar (Hi-Media, India) stabs in room temperature.

Agar-well diffusion method

On the surface of nutrient agar plate swabbed with indicator bacterial broth culture, wells (of 6 mm diameter) were prepared, and isolated LAB culture filtrates of increasing concentrations (25, 50 and 75 µL/well) were loaded in the wells followed by the protocol of Tagg and McGiven [31]. After 24-h incubation at 37 °C (in occurrence of atmospheric CO₂), ZDI (zone diameter of inhibition) values (nearest whole) were recorded, and interpreted as less active, moderately active and highly active with ZDIs ≤ 10 mm, 11–14 mm and ≥ 15 mm, respectively [15].

The antibacterial activity of the test LAB (at minimum concentration in which inhibition observed) in arbitrary unit per mL (AU/mL) was determined as a measure of production of bioactive components using the formula mentioned elsewhere [32]. The “R” (width of clear zone) values of all the isolates were also calculated (at maximum concentration of culture filtrate: 75 µl/well) as per the formula specified previously [33]. The scores of antagonism of indicator bacteria were measured as zero inhibition capacity when “R” was < 2 mm; low or intermediate inhibition capacity with “R” values of 2–5 mm, and high inhibition capacity with “R” values ≥ 5.5 mm [34, 35].

Safety profiling

The safety profile of the idli isolates (LMEM1001, LMEM1002, LMEM1003, LMEM1004, LMEM1005, LMEM1006, LMEM1007 and LMEM1008) was determined by their gelatine liquefaction test, DNase test and antibiotic susceptibility.

Gelatine liquefaction test

Gelatine liquefaction test was performed using nutrient gelatin media (following 24-h incubation at 37 °C then freezing at 4 °C in alternative manner up to 7 days and checking the liquefaction of gelatin media) followed by the protocol of Dela Cruz and Torres [36] with slight modifications, to confirm the capacity of isolated LAB to hydrolyse gelatine by producing gelatinase.

Deoxyribonuclease (DNase) test

The DNase test was done using DNase agar (Hi-Media, India) for all the isolated LAB in order to determine the ability of an organism to produce the DNase enzyme followed by protocol of Bergey’s manual [25].

Antibiotic susceptibility test

All the LAB isolates were tested against antibiotics (Hi-Media, India): amikacin (Ak: 30-μg/disc), ampicillin (Am: 10-μg/disc), cefoxitin (Cx: 30-μg/disc), chloramphenicol (C: 30-μg/disc), ciprofloxacin (Cp: 5-μg/disc), gentamycin (Gn: 10-μg/disc), nalidixic acid (Na: 10-μg/disc), piperacillin (Pi; 100-μg/disc), tetracyclines (Te: 30-μg/disc) and vancomycin (V: 30-μg/disc), using the disc diffusion method [37], as per the Clinical and Laboratory Standards Institute (CLSI) criteria [38], as described earlier [39].

Cumulative probiotic potential (CPP)

The probiotic potential of the LAB isolates was evaluated using 8-point scores, and the CPP was calculated as per the formula mentioned elsewhere [15, 40] and slightly modified as per Wadoum et al. [41]. We had considered probiotic characters (n = 4), safety aspects (n = 3) and antagonistic activity (n = 1) in one scale and that was the main modification in terms of CPP value determination.

Results

Identification of lactic acid bacteria

Among eight lactic acid bacteria (LAB), four (LMEM1001, LMEM1002, LMEM1003 and LMEM1004) from fresh idli batter and remaining four isolates (LMEM1005, LMEM1006, LMEM1007 and LMEM1008) from fermented idli batter sample were isolated. All isolates were Gram positive (Fig. 1), non-spore forming, non-motile rod shaped and were negative to catalase and oxidase tests, and thus recognized as Lactobacillus. After interpretation of morphological, cultural, biochemical tests (Table 1), sugar fermentation profile (Table 2) and 16S rRNA sequencing results (Figs. 2, 3 and 4), the isolates were identified as Lactobacillus pentosus LMEM1001 (currently Lactiplantibacillus pentosus), Lactobacillus plantarum LMEM1002 (currently Lactiplantibacillus plantarum), Lactobacillus sp. LMEM1003 and Lactobacillus sp. LMEM1004, Lactobacillus sp. LMEM1005, L. plantarum LMEM1006, Lactobacillus sp. LMEM1007 and Lactobacillus fermentum LMEM1008 (currently Limosilactobacillus fermentum). The sequences of four LAB strains subjected to molecular identity through 16S rRNA gene sequencing have been submitted to the NCBI (National Centre for Biotechnology Information) GenBank with specific accession numbers: Lactiplantibacillus pentosus LMEM1001 (https://www.ncbi.nlm.nih.gov/nuccore/MT783707), Lactiplantibacillus plantarum LMEM1002 (https://www.ncbi.nlm.nih.gov/nuccore/MW364384), Lactiplantibacillus plantarum LMEM1006 (https://www.ncbi.nlm.nih.gov/nuccore/OR096233) and Limosilactobacillus fermentum LMEM1008 (https://www.ncbi.nlm.nih.gov/nuccore/OR096236).

Fig. 1

Photographs showing morphological characteristics of selected isolated bacteria. All isolates were gram-positive, non-spore forming, non-motile rod-shaped

Table 1 Biochemical test results of isolated lactic acid bacteria

Table 2 Sugar fermentation profile (24–72-h incubation) of idli lactobacilli

Fig. 2

Agarose gel electrophoresis of 16S rRNA gene PCR amplification showing ~ 1.5-kb amplified fragment. Lane L: ladder (500 bp), Lane 1: LMEM1001, 2: LMEM1002, 3: LMEM1006, 4: LMEM1008

Fig. 3

The 16S rRNA gene sequence-based phylogenetic tree for lactobacilli isolated from fresh idli batter. a LMEM1001 showed maximum (96.81%) similarity with Lactobacillus pentosus (strain 124-2), b LMEM1002 showed maximum (95.20%) similarity with Lactobacillus plantarum (strain JCM 1149)

Fig. 4

The 16S rRNA gene sequence-based phylogenetic tree for isolated from fermented idli batter. a LMEM1006 showed maximum (96.11%) similarity with Lactobacillus plantarum (strain CIP 103151), b LMEM1008 maximum (98.40%) similarity with Lactobacillus fermentum (strain NBRC 15885)

Probiotic property

The probiotic property determining tolerance test results to different stresses (sodium chloride, low-pH, wide range of temperature and bile salts) for the isolated LAB is represented in Table 3.

Table 3 Physiological stress tolerance test results (24–72-h incubation) for idli lactobacilli

Antibacterial activity

The idli lactobacilli isolates displayed good antibacterial activity, following agar well diffusion method, against all indicator bacteria (Fig. 5). The L. pentosus LMEM1006 isolate showed maximum growth inhibitory activity at highest concentration tested against Listeria monocytogenes MTCC 657, Bacillus cereus BC2, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PA3 having ZDIs 23, 20, 20 and 19 mm, respectively, while the Staphylococcus aureus had highest sensitivity to L. fermentum LMEM1008 isolate (ZDI: 18 mm); the Lactobacillus sp. LMEM1004 isolate exhibited poor activity against all indicator bacteria tested with ZDIs 8–12 mm (Fig. 6).

Fig. 5

Through agar well diffusion method, idli lactobacilli isolates showed sensitivity against all test bacteria. a activity of L. pentosus LMEM1001 against BC2 (upper part) and LM (lower part), b against SA (upper part) and BC1 (lower part), c activity of Lactobacillus sp. LMEM1003 and Lactobacillus sp. LMEM1004 against EC3, d activity of Lactobacillus sp. LMEM1005 and L. plantarum LMEM1006 against PA1, e activity of Lactobacillus sp. LMEM1005 and L. plantarum LMEM1006 against PA3, f activity of Lactobacillus sp. LMEM1007 and L. fermentum LMEM1008 against SA. BC1: Bacillus cereus 1, BC2: B. cereus 2, SA: Staphylococcus aureus, LM: Listeria monocytogenes MTCC 657, PA1: Pseudomonas aeruginosa 1, PA3: P. aeruginosa 3, EC3: Escherichia coli 3. A: 25 µl, B: 50 µl and C: 75 µl

Fig. 6

The L. pentosus LMEM1006 isolate exhibited top growth inhibitory activity at highest concentration against indicator bacteria. a–c Antibacterial activity of isolated strains (a: 25, b: 50 and c: 75 µl/well) against test gram-negative bacteria, d–f Antibacterial activity of isolated bacteria (d: 25, e: 50 and f: 75 µl/well) against test gram-positive bacteria. PA1: Pseudomonas aeruginosa 1, PA 2: P. aeruginosa 2, PA3: P. aeruginosa 3, EC1: Escherichia coli 1, EC2: E. coli 2, EC3: E. coli 3, BC1: Bacillus cereus 1, BC2: B. cereus 2, SA: Staphylococcus aureus, LM: Listeria monocytogenes MTCC 657

The “R” values and arbitrary unit per mL (AU/mL) of the isolated lactobacilli against indicator bacteria are represented in Fig. 7a, b, respectively. The lowest “R” values (1–3 mm) were documented due to the action of Lactobacillus sp. LMEM1004, while L. pentosus LMEM1006 and L. fermentum LMEM1008 had “R” values 4.5–8.5 mm and 2.5–6 mm, respectively. Against indicator bacteria, Lactobacillus sp. LMEM1004 showed least antagonistic activity (106.66–320 AU/mL); on the other hand, the highest level of growth inhibitory components formed as 640 AU/mL, by L. plantarum LMEM1006, and the values ranged from 240 to 480 AU/mL for both Lactobacillus sp. LMEM1007 and L. fermentum LMEM1008.

Fig. 7

The lactobacilli from idli fresh and fermented samples displayed antibacterial activity with “R” values 1–8.5, while growth inhibitory capacity of lactobacilli was recorded as 106.67-640 AU/mL at highest concentration, against the test bacterial pathogens. a: “R” values (mm) showed by lactobacilli against the test bacteria, b: Growth inhibitory capacity of lactobacilli expressed in “AU/mL” for indicator bacteria

Safety profiling

All the isolated idli lactobacilli showed no activity in the production of gelatinase and DNase enzyme except Lactobacillus sp. LMEM1004 and had gelatine liquefaction capacity in order to synthesis gelatinase. The antibiotic susceptibility test results of all the isolated bacteria are represented in Fig. 8. All the Lactobacillus isolates had resistance to Cx, and L. plantarum LMEM1002 had Gn resistance, in addition to the Cx. All the isolated LAB showed mixed level of sensitivity to Ak, C and Te, while sensitivity to Cp and Va was exhibited by L. plantarum LMEM1002 and Lactobacillus sp. LMEM1004. High level of resistance pattern showed by Lactobacillus sp. LMEM1005 to six antibiotics among ten tested.

Fig. 8

Heatmap represents antibiotic susceptibility test results of isolated idli lactobacilli. Ak: Amikacin, Am: Ampicillin, C: Chloramphenicol, Cp: Ciprofloxacin, Cx: Cefoxitin, Gn: Gentamicin, Na: Nalidixic acid, Pi: Piperacillin, Te: Tetracyclines, Va: Vancomycin. ZDI: zone diameter of inhibition. Green gradient: antibiotic sensitive (greater ZDI values), yellow gradient: intermediately sensitive to antibiotics (ZDI values intermediate between sensitivity and resistance), red gradient: resistant (lesser ZDI values)

Cumulative probiotic potential (CPP)

The individual CPP for the Lactobacillus isolates was 62.5% for Lactobacillus sp. LMEM1004 and 100% for the rest seven isolates: L. pentosus LMEM1001, L. plantarum LMEM1002, Lactobacillus sp. LMEM1003, Lactobacillus sp. LMEM1005, L. plantarum LMEM1006, Lactobacillus sp. LMEM1007 and L. fermentum LMEM1008 (Table 4).

Table 4 Cumulative probiotic potential (CPP) score for the isolated idli lactobacilli

Discussion

As per European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), group of bacteria, viz. Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae (except Shigella and Salmonella), Pseudomonas aeruginosa and Acinetobacter spp., are responsible for nosocomial infections in terms of multidrug resistance (MDR). The increasing number of MDR strains can eventually occupy higher resistance capacity with recognition of extensively drug-resistant (XDR) and pandrug-resistant (PDR), respectively [42]. From the global perspective, acceleration of basic and applied researches is warranted to discover new therapeutics against ESKAPE as well as twelve species of bacteria with critical, high, and medium antibiotic resistance (AR) specified by WHO [43, 44]. To handle the situation of AR, bacteriocins producing probiotic lactic acid bacteria (LAB) strains might be a noble choice for administration against MDR bacteria [45]. As like as dairy-based fermented products, non-dairy-based fermented foods can also be used as the source of isolation of probiotic microorganism, and lactobacilli is the most preferred one among them [46]. Several investigations approved that the presence of different yeasts and LAB strains in idli batter might be responsible for the beneficial nutritive as well as bioactive values including antimicrobial properties [47, 48]. Sharma et al. [49] studied microbiological dynamics of blends of buttermilk and pearl millet flour using number of techniques, viz. yeast and mould count, aerobic plate count, Escherichia coli count, and LAB count. Two probiotic bacteria L. plantarum and Lactococcus lactis have been isolated from idli batter and identified earlier by physiological and biochemical characterization, and both showed antibacterial activity against gram-positive (B. cereus MTCC 1272) and gram-negative (E. coli NRRL 3008) bacteria [50]. Total of 354 bacterial isolates identified by Mandhania et al. [51] on the basis of culture-dependent method through spread plate technique and colony characters were obtained from 3 fully fermented idli batter samples. Kadirvelu [52] noticed new antibacterial compound, 2-hydroxy indole propanamide formed by L. plantarum isolated from idli batter using phenotypic characterization.

For probiotic characterization of lactobacilli, acid tolerance (low pH), wide range of temperature tolerance, survivability in presence of NaCl, bile salt are important criteria, and the pH value of 3.0 has been considered standard for such investigation of probiotic strains [15, 53]. In previous investigations on fermented food products, as have been conducted by Haldar et al. [15], Thakkar et al. [54] and Agaliya and Jeevaratnamthe [22] LAB tolerated and survived in MRS broth at pH ranged between 3–4, 3–6.5 and 3.5–9.5, respectively. Balasingham et al.’s [55] study showed significant growth at pH ≥ 3 of LAB isolated from swine intestine, while reduction in viability has been seen at pH 2. Two strains of L. plantarum subsp. argentoratensis (LQC 2520 and LQC 2320) isolated from spontaneously fermented Greek wheat sourdoughs showed survivability in the presence of NaCl (6.5%), indicating their high sodium chloride tolerance [56]. The lactobacilli isolated from fermented idli batter tested in the presence of 4, 6.5 and 10% of NaCl, whereas survived at 4 and 6.5% concentration of NaCl [22]. In the human intestine, the presence of bile salt is approximately 0.3% that is the reason good probiotic strain should reflect tolerance level greater than this physiological concentration of bile [57]. Iyer et al. [50] found two strains L. plantarum IB-1 and Lactococcus lactis IB-2 from idli batter, and both the strains showed good viability (58.11% and 60.84%, respectively) at high bile salt concentration (2%). As per the research of Mandhania et al. [51], total of seventy two isolates of LAB from fermented idli batter had tolerance to 2% bile salt, whereas some other researchers reported that total of thirty-four probiotic strains showed survivability and growth at the bile salt concentrations of 0.3–1% [21]. Like our study, temperature tolerance tests of isolated lactobacilli from fermented idli batter were executed and confirmed by Agaliya and Jeevaratnamthe [22] determining the growth at temperature ranged between 15 and 45 °C. The diverse range of tolerance to NaCl, bile salts, low pH (pH: 2–4) and temperature have been confirmed in the seven lactobacilli of the current study: L. pentosus LMEM1001, L. plantarum LMEM1002, Lactobacillus sp. LMEM1003, Lactobacillus sp. LMEM1005, L. plantarum LMEM1006, Lactobacillus sp. LMEM1007 and L. fermentum LMEM1008.

Due to the capacity to produce antimicrobials, probiotic is a potential source to substitute the synthetic antibiotics. The Lactobacillus isolates (L. animalis LMEM6, L. plantarum LMEM7, L. acidophilus LMEM8 and L. rhamnosus LMEM9) from curd samples had broad antibacterial spectrum with ZDI ranged between 11.33 ± 0.58 and 35.67 ± 2.52 mm (ZDI in mm ± standard deviation), against gram-negative bacterial pathogens, as has been informed by Haldar et al. [15]. According to Katepogu et al. [58], fermented idli batter contains potential LAB Pediococcus spp. and has maximum growth inhibition property against E. coli (ZDI: 14 mm) and Bacillus subtilis (ZDI: 21 mm) at cell-free suspension of 100 µl. Nine strains of Bacillus and one Leuconostoc strain procured from idli batter showed varied antibacterial activity against food-borne bacteria (ZDI: 6–22 mm) [59]. Two strains L. plantarum IB-1 and Lac. lactis IB-2 also isolated from idli batter by other researcher in other part of the globe, had good antibacterial activity against E. coli NRRL 3008 and B. cereus MTCC 1272 with ZDIs > 9 mm, respectively [50]. The probiotic Pediococcus pentosaceus strains (n = 6), isolated from idli batter, showed growth inhibitory activity against S. aureus MTCC 737, Listeria monocytogenes MTCC 657, Bacillus cereus MTCC 1272, Aeromonas hydrophila MTCC 1739, Vibrio parahaemolyticus MTCC 451 and Escherichia coli MTCC 728 species having ZDIs 11–22 mm [60]. Dubey and Jeevaratnam [61] study revealed two L. pentosus isolates (AJ7 and AJ82), from uttapam fermented batter, which was supplemented with Piper betle L. leaves displayed in situ growth inhibitory activity against Listeria monocytogenes MTCC657 determined using CFU count. The lactobacilli from idli fresh and fermented samples herein had good antibacterial activity with ZDI ranged between 6–16, 6–21 and 8–23 mm at increasing concentration (25, 50 and 75 µl), with “R” values 1 to 8.5 at highest concentration. As has been described by Haldar et al. [15], the bacteriocin production, in terms of antagonistic activity, for the test lactobacilli ranged 410.4–649.2 AU/mL. Five LAB isolates exhibited strong bacteriocin activity ranged between 800 to 1600 AU/mL against Klebsiella pneumoniae ATCC 12296 and E. coli [62]. In the current assay, the growth inhibitory activity of lactobacilli was recorded as 106.67–640 AU/mL, against the test bacterial pathogens at highest concentration.

Probiotic bacteria considered as safe if they show less antibiotic resistance. That is the reason, every strain of lactobacilli should be tested for antibiogram in assessing the safety profile to qualify as safe probiotics [63]. Two L. pentosus strains were reported to be sensitive to cephalexin (Cfx), cephradine (Ced), cloxacillin (Clox), co-trimazine, co-trimoxazole, nitrofurantoin (Nfn) and norfloxacin (Nor), while both strains showed resistance to cefuroxime (Cxm), mecillinam (Mec), nalidixic acid (Na) [61]. The L. plantarum isolated from idli batter showed resistance to Gn, Cp, Na and Nor [52]. Kandasamy et al. [21] found 34 probiotic microflora from fermented idli batter in which one strain was sensitive to Am and rifampicin (Rfm), two strains were sensitive to sulphadiazinee and five strains displayed sensitivity to sulphamethizole. In the current study, isolates of idli lactobacilli were sensitive and intermediately sensitive to most of the antibiotics tested with a common resistance to Cx.

To validate probiotic, the cumulative probiotic potential (CPP) is a developed criterion for the native lactobacilli [40]. The CPP of L. animalis LMEM6 was 80%, and 100% showed by three isolates: L. plantarum LMEM7, L. acidophilus LMEM8 and L. rhamnosus LMEM9 isolated from curd samples [15]. Wadoum et al.’s [41] study revealed Lactobacillus isolated from faecal samples of chickens and ducks and had good CPP values (82% for L. paracasei MW-38CGZ, L. plantarum MW-48CGZ and 100% for L. paracasei MW-37CGZ, L. plantarum MW-18CGZ) as a potential probiotic fulfilling different probiotic properties. L. brevis UN isolated from Dhulliachar (which is a powdered mixture of seeds of Cucurbita pepo and Sesamum indicum) exhibited good CPP value of about 95.83% [64]. In the current study, all the idli lactobacilli strains had CPP value 100% except Lactobacillus sp. LMEM1004 and that means seven lactobacilli among eight, were eligible for the criteria [65], in defining the grade of a safe probiotic.

Conclusion

To overcome global bacterial antibiotic resistance, treatment or therapeutic measures depend on the antibiotic alternatives. Probiotic formulations could be a standard bio-weapon against bacterial infection. However, the isolation and screening of lactobacilli from various locally available non-dairy-based natural sources plausibly be a better choice to develop non-antibiotic antibacterials of medical relevance. However, further investigations are recommended in dose determination and particular efficacy of individual LAB strains.