Characterization, Bio-preservative Potential and Functional Applications of Bacteriocin-Like Inhibitory Substance Produced by Lactobacillus kefiri Isolated from Kefir

INTRODUCTION

There is an increase in demand for safe and naturally preserved foods (Behera et al., 2018). This requires the use of natural bioactive compounds instead of synthetic preservatives (Behera et al., 2018). Among the many antimicrobial compounds, Bacteriocin-Like Inhibitory Substances (BLIS) produced by probiotic bacteria have shown considerable efficiency due to their effectiveness against food-borne pathogens and biodegradability (Todorov, 2009; Das and Goyal, 2014). BLIS also possess antioxidant and anticancer properties making these effective in food, and biotechnology applications (Yap et al., 2022).

Kefir is a traditional fermented dairy product dominated by lactic acid bacteria and yeast (Leite et al., 2013). Lactobacillus kefiri has probiotic potential and the ability to produce bioactive compounds (Carasi et al., 2014). Previous studies have reported probiotic properties and antimicrobial activity (Bourrie et al., 2016). Studies integrating purification, characterization and functional applications of BLIS from L. kefiri are limited (Carasi et al., 2022). Information on the antioxidant, anticancer, cytotoxic activity and food preservation potential of BLIS from L. kefiri in a single study is limited (Bessa et al., 2024).

With a growing interest in natural preservatives, comprehensive characterization of the L. kefiri produced BLIS is necessary (Yap et al., 2022; Carasi et al., 2014). The present study primarily aimed to isolate and identify Lactobacillus kefiri from kefir, study the probiotic potential, produce and partially purify BLIS, characterize the BLIS, evaluate its functional properties and focus on the preservative potential of the BLIS. The results of this study are expected to contribute to the development of BLIS, as a natural preservative and also as a functional bioactive compound for biomedical and biotechnological applications (Yap et al., 2022).

MATERIALS AND METHODS

Isolation and Identification of the BLIS-producing Lactobacillus strain

The sample, kefir was serially diluted using sterile distilled water and 100 μL was spread plated on MRS (de Man, Rogosa and Sharpe) agar plates (De Man et al., 1960). The plates were anaerobically incubated in an anaerobic chamber with a GasPak system at 37ºC for 24 hr. Morphologically distinct colonies were repeatedly streaked to obtain pure cultures. Colony morphology, Gram's staining and biochemical properties were studied. These were performed using standard microbiological procedures (Sharma, 2007). Molecular identification of the Lactobacillus strain was done by 16S rRNA gene sequencing.

Evaluation of Probiotic Characteristics

Autoaggregation, cell-surface hydrophobicity, tolerance to acidic conditions, salt concentration variation, bile salt concentration variation and phenol resistance were evaluated (Meira et al., 2012). Autoaggregation and cell surface hydrophobicity were measured at 600 nm. Acid tolerance was tested at pH 2, 4, 6, 8 and 10, while salt tolerance was tested using MRS broth supplemented with 2-10% NaCl. 0.2-0.4% sodium taurodeoxycholate and 1-4 % phenol was used to check the bile tolerance (Singhal et al., 2021) and phenol resistance. Haemolysis was assessed on blood agar incubated at 37ºC for 24 hr. Antibiotic sensitivity was assessed by Kirby-Bauer disc diffusion method using commercial antibiotic discs on Mueller-Hinton agar plates (Charteris et al., 1998).

Production and Partial Purification of Cell-Free Supernatant

A single L. kefiri colony was inoculated into MRS broth and incubated anaerobically at 37ºC for 24 hr. After incubation, the culture was centrifuged to obtain Cell-Free Supernatant (CFS) which was considered as crude BLIS preparation (Lowry et al., 1951). BLIS was precipitated by gradual addition of ammonium sulphate to 80% saturation under constant stirring (Wu et al., 2022). The proteins were then recovered by dialysis using a 3.5 kDa MWCO membrane against phosphate buffer (pH 7.0±0.25) and concentrated by ultrafiltration using a Vivaspin® 20 centrifugal filter (50 kDa MWCO).

Characterization of Partially Purified BLIS

The concentrated BLIS was subjected to gel filtration chromatography using Sephadex G-25 column (Brand-Williams et al., 1995) and ten fractions were collected. The active fraction was selected for further characterization by selecting the fraction with both highest antimicrobial activity against E. coli (E. coli cultured procured from NCCS, Pune, India) and highest protein concentration determined using Lowry method with Bovine Serum Albumin (BSA) as standard (Mosmann, 1983). Stability of the partially purified BLIS was evaluated under different pH conditions, temperatures, UV exposure, SDS treatment and trypsin digestion. The peptide profile of the active fraction was analysed by High Performance Liquid Chromatography (HPLC) at the National Agro Foundation, Chennai, using a Shimadzu HPLC system equipped with a C18 column and a Photodiode Array (PDA) detector (Brand-Williams et al., 1995). The chromatograms were recorded at 220 nm. Matrix-Assisted Laser Desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed at the IIT, Madras using a Bruker Daltonics MALDI-TOF mass spectrometer. The matrix used was α-cyano-4-Hydroxycinnamic Acid (HCCA), and the spectra were processed using Bruker flex analysis software (Brand-Williams et al., 1995).

Functional evaluation of the partially purified BLIS

Cytotoxicity, anticancer and antioxidant activity assays were carried out at Life Tech Research Centre, Chennai. VERO cells and COLO 320 DM human colon cancer cells were purchased from National Centre for Cell Science, Pune. DPPH free radical scavenging assay was used to determine the antioxidant activity of the partially purified BLIS at different concentrations (Chuah et al., 2019). Cytotoxicity against VERO cells and anticancer activity against COLO 320 DM human colon cancer cells were assessed using MTT assay (Chuah et al., 2024; Kairey et al., 2023). The food preservation potential was tested by treating sterile apple, carrot, cheese and meat samples with the partially purified BLIS (Carasi et al., 2014), followed by storage at room temperature and under refrigeration at 4ºC. The treated samples were monitored for qualitative characters such as changes in colour, texture, visible fungal growth and overall spoilage during the storage period.

RESULTS

Isolation and Identification of Lactobacillus kefiri

The bacterial isolate obtained from kefir using MRS agar produced medium-sized, white, circular colonies with entire margins, smooth surface, convex elevation, creamy consistency and an opaque appearance. Biochemical tests showed negative results for catalase, oxidase, MR-VP, indole, citrate utilization, gelatin liquefaction and TSI. Starch hydrolysis and milk coagulation was observed. Gram staining showed gram-positive rod-shaped cells. The biochemical profile was consistent with the characteristics of the genus Lactobacillus (Table 1). The Lactobacillus strain showed high similarity with Lactobacillus kefiri based on nucleotide homology and phylogenetic analysis.

Probiotic Characteristics

Autoaggregation of the L. kefiri increased throughout the incubation period, reaching 88.78% after 24 hr. This indicates that the isolate has a strong ability to aggregate over time. Cell surface hydrophobicity was 61.22% showing favourable adhesion properties of the isolate. Maximum growth was observed at pH 6 and reduced growth at highly acidic (pH 2) and alkaline pH (pH 8-10). Although growth decreased with increasing salt concentration, L. kefiri strain showed tolerance up to 10% NaCl. Growth was also observed under different concentrations of bile salt (0.2%-0.4% sodium taurodeoxycholate) and phenol (1-4%). This shows the resistance of the strain to simulated gastrointestinal stress. No haemolytic activity was detected and this confirms the safety of the Lactobacillus kefiri. Antibiotic sensitivity results showed sensitivity to doxycycline, tetracycline, azithromycin, ciprofloxacin and clindamycin, whereas resistance was observed against methicillin, cefuroxime and ceftriaxone.

Production, partial purification and characterization of BLIS

The cell-free supernatant obtained from L. kefiri was subjected to ammonium sulphate precipitation, dialysis, ultrafiltration and gel filtration chromatography to obtain partially purified BLIS. Ten fractions were collected. Among the fractions obtained, F1, F6, F9 and F10 exhibited antimicrobial activity against E. coli. F6 produced the largest zone of inhibition (10 mm) and contained the highest protein concentration (1.320 mg/mL). Hence, F6 fraction was selected as active fraction for further characterization (Table 2).

HPLC analysis of the active fraction showed multiple chromatographic peaks indicating the presence of several peptides in the partially purified preparation (Figure 1). Similarly, MALDI-TOF mass spectrometric analysis revealed multiple peptide ion peaks distributed over different molecular masses (Figure 2). This confirms that the BLIS consists of more than one peptide component.

The partially purified BLIS was subjected to different physicochemical conditions to evaluate its stability. The BLIS retained antimicrobial activity at 30ºC but lost activity at 4ºC, 80ºC and 121ºC. The BLIS showed activity over the pH range of 2-10 with maximum activity at pH 6. Activity reduced with the increasing time of UV exposure. Trypsin treatment completely inhibited the antimicrobial activity. SDS-treated BLIS retained inhibitory activity against E. coli. These results indicate that the antimicrobial compound is proteinaceous in nature and exhibits physicochemical stability (Table 2).

Functional Applications of Partially Purified BLIS

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The partially purified BLIS exhibited concentration-dependent increase in antioxidant activity in DPPH assay with radical scavenging activity increasing to 27.35% at the highest 3). concentration. Cytotoxicity studies on VERO cell demonstrated high cell viability across all concentrations, indicating low toxicity towards normal mammalian cells. BLIS reduced the viability of COLO 320 DM human colon cancer cells in a concentration-dependent manner with an estimated IC 50 of 125 μg/mL (Table

The food preservation study demonstrated that the efficiency of the partially purified BLIS was influenced by storage temperatures. Refrigeration significantly improved the food preservation efficiency of BLIS-treated samples. There were no visible changes in the colour and texture of carrot, cheese and meat samples throughout the observation period of 30 days. Only apple exhibited browning on day 2 and delayed fungal growth after day 24 (Figure 3). Under room temperature, spoilage progressed rapidly in apple with browning on day 2 and visible fungal growth from day 10 onwards. There was complete fungal colonization by day 14. There was sliminess observed in cheese and meat samples from day 7 onwards which led to complete deterioration of the samples by day 21. Watery exudate and colour change was observed in carrot by day 21 (Figure 3). This emphasizes that BLIS-treated samples were more stable under refrigeration than at room temperature thereby extending the shelf life of the treated food samples.

DISCUSSION

Lactobacillus kefiri isolated from kefir exhibits probiotic characteristics with the ability to produce active BLIS (Carasi et al., 2014). Strong autoaggregation, cell surface hydrophobicity, tolerance to acid, bile salts, sodium chloride and phenol with the absence of haemolytic activity indicate that the L. kefiri strain is capable of surviving in the conditions similar to gastrointestinal tract (Meira et al., 2012; Singhal et al., 2021). These observations are similar to previous studies that have reported on Lactobacillus species isolated from dairy and fermented products exhibited strong adhesion, aggregation and tolerance to gastrointestinal stress conditions (Bourrie et al., 2016; Carasi, Malamud, and Serradell, 2022). This is an important probiotic character to promote intestinal survival and colonization.

The purification methods used in this study yielded a partially purified yet active fraction with inhibitory effect against E. coli (Das and Goyal, 2014; Yap et al., 2022). Loss of antimicrobial activity after trypsin treatment confirms the proteinaceous nature of the BLIS (Das and Goyal, 2014). In addition, HPLC and MALDI-TOF analysis showed that the BLIS contains multiple peptide components (Wu et al., 2022). The presence of multiple peptide components observed in this study is comparable to previous studies on partially purified BLIS (Wu et al., 2022; Yap et al., 2022). The retention of activity over different pH and temperatures and chemical conditions exhibit that the partially purified BLIS has the potential to be used in food preservation where stability is important for practical use (Yap et al., 2022).

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The partially purified BLIS demonstrated antioxidant activity, low cytotoxicity against VERO cells as well as antiproliferative activity against COLO 320 DM cells (Das and Goyal, 2014; Kairey et al., 2023). Similar antioxidant and anticancer activities have been reported for bacteriocins and BLIS produced by probiotic Lactobacillus strains. These findings indicate that the partially purified BLIS is not only an effective antimicrobial agent but also has the ability to be used in food and biomedical applications (Yap et al., 2022).

The food preservation study emphasized the practical importance of the partially purified BLIS (Carasi et al., 2014). BLIS-treated food samples under refrigeration remained stable longer than those kept at room temperature (Carasi et al., 2014). This shows that BLIS treatment combined with low temperature can delay spoilage of food effectively (Leite et al., 2013). The present findings emphasize the potential of Lactobacillus kefiri-derived BLIS as natural and multifunctional biopreservatives (Behera et al., 2018; Yap et al., 2022). Further purification and peptide sequencing can facilitate its transition into practical applications.

CONCLUSION

The present study demonstrated that Lactobacillus kefiri isolated from kefir is capable of producing Bacteriocin-Like Inhibitory Substances (BLIS) with probiotic properties and has the potential for food and biotechnological applications. The probiotic characteristics along with physicochemical stability and biological activity make the BLIS a natural bioactive compound. The partially purified BLIS showed effective antimicrobial activity against E. coli, antioxidant potential, antiproliferative activity against COLO 320 DM cells and low cytotoxicity against VERO cells. This indicates that the BLIS can be considered an alternative to synthetic preservatives. HPLC and MALDI-TOF confirmed the presence of multiple peptide components. Food preservation studies demonstrated stability of BLIS- treated samples under refrigeration. Overall, these results strengthen the application of Lactobacillus kefiri- derived BLIS as a natural food preservative and a bioactive compound in biological applications. Further purification and structural characterization of the peptides are needed to facilitate future industrial and therapeutic applications.

ACKNOWLEDGEMENT

The authors sincerely acknowledge National Agro Foundation, Research and Development Centre Chennai, for providing the facilities to perform HPLC analysis. The authors are grateful to the Indian Institute of Technology Madras (IIT Madras), Guindy, Chennai, for providing the MALDI-TOF analysis facilities. The authors also express their sincere thanks to Life Tech Research Centre, Chennai, for providing laboratory facilities and technical support for the antioxidant, cytotoxicity and anticancer studies.

ABBREVIATIONS

BLIS

Bacteriocin-Like Inhibitory Substance

MRS

de Man, Rogosa and Sharpe (agar/broth)

CFS

Cell-Free Supernatant

MWCO

Molecular Weight Cut-Off

HPLC

High-Performance Liquid Chromatography

PDA

Photodiode Array

MALDI-TOF MS

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

HCCA

α-Cyano-4-hydroxycinnamic Acid

SDS

Sodium Dodecyl Sulfate

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

DPPH

2,2-Diphenyl-1-Picrylhydrazyl

IC₅₀

Half-Maximal Inhibitory Concentration

TSI

Triple Sugar Iron

BSA

Bovine Serum Albumin

NCCS

National Centre for Cell Science.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

AUTHORS CONTRIBUTION

The first author conceived and designed the study, performed the experiments, collected and analyzed the data, interpreted the results, and prepared the original draft of the manuscript. The corresponding author supervised the research work, provided scientific guidance, critically reviewed the manuscript, and approved the final version of the manuscript. All authors have read and approved the final manuscript.