Use of Enterocins-Producing Lactic Acid Bacteria for Food Biopreservation: Diversity and Functional Insights

Antimicrobial peptides are class of small, positively charged peptides known for their broad‐spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin‐YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100‐fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide‐binding protein A and LPcin‐YK3 peptide. Given that degradation related to the LPcin‐YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin‐YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide‐binding protein A in respect of LPcin‐YK3 peptide.

Functional insights into microbial communities of Korean traditional rice wine (makgeolli) during fermentation

Oenococcus oeni is the lactic acid bacterium (LAB) that most commonly drives malolactic fermentation in wine. Although oenococcal prophages are highly prevalent, their implications on bacterial fitness have remained unexplored and more research is required in this field. An important step toward achieving this goal is the ability to produce isogenic pairs of strains that differ only by the lysogenic presence of a given prophage, allowing further comparisons of different phenotypic traits. A novel protocol for the rapid isolation of lysogens is presented. Bacteria were first picked from the center of turbid plaques produced by temperate oenophages on a sensitive nonlysogenic host. When streaked onto an agar medium containing red grape juice (RGJ), cells segregated into white and red colonies. PCR amplifications with phage-specific primers demonstrated that only lysogens underwent white-red morphotypic switching. The method proved successful for various oenophages irrespective of their genomic content and attachment site used for site-specific recombination in the bacterial chromosome. The color switch was also observed when a sensitive nonlysogenic strain was infected with an exogenously provided lytic phage, suggesting that intracolonial lysis triggers the change. Last, lysogens also produced red colonies on white grape juice agar supplemented with polyphenolic compounds. We posit that spontaneous prophage excision produces cell lysis events in lysogenic colonies growing on RGJ agar, which, in turn, foster interactions between lysed materials and polyphenolic compounds to yield colonies easily distinguishable by their red color. Furthermore, the technique was used successfully with other species of LAB.IMPORTANCE The presence of white and red colonies on red grape juice (RGJ) agar during enumeration of Oenococcus oeni in wine samples is frequently observed by stakeholders in the wine industry. Our study brings an explanation for this intriguing phenomenon and establishes a link between the white-red color switch and the lysogenic state of O. oeni It also provides a simple and inexpensive method to distinguish between lysogenic and nonlysogenic derivatives in O. oeni with a minimum of expended time and effort. Noteworthy, the protocol could be adapted to two other species of LAB, namely, Leuconostoc citreum and Lactobacillus plantarum It could be an effective tool to provide genetic, ecological, and functional insights into lysogeny and aid in improving biotechnological processes involving members of the lactic acid bacterium (LAB) family.

Weissella koreensis is a Gram-positive, rod-shaped, nonmotile, and facultative anaerobic species belonging to the lactic acid bacteria (LAB). The members of this species have been repeatedly isolated from kimchi (a traditional Korean fermented food) and are known for their beneficial effects on human and animal intestinal microflora through producing various clinically important amino acids such as γ-aminobutyric acid and ornithine. Here we report the genome sequence of the type strain of W. koreensis (KCTC 3621(T)) to provide taxonomic and functional insights into the species.

Gir cow (Bos indicus) milk is widely valued for its nutritional quality, digestibility, and A2 β-casein content; however, the microbial determinants contributing to these attributes remain insufficiently characterized. In this study, dominant lactic acid bacteria (LAB) associated with Gir cow milk were isolated and evaluated using an integrated phenotypic and genome-based approach to elucidate their functional relevance. Raw milk samples were analyzed to isolate LAB, which were characterized through morphological, biochemical, and molecular analyses. Predominant isolates were identified as Weissella cibaria and Weissella confusa. Selected isolates exhibited strain-dependent tolerance to acidic pH, bile salts, and phenolic stress, along with auto-aggregation ability, cholesterol assimilation, and antagonistic activity against enteric pathogens. Survival under simulated gastrointestinal conditions was further validated by viable cell count analysis. Whole-genome sequencing of W. cibaria revealed a high-quality draft genome of approximately 2.43Mb with a GC content of 44.97%, assembled into 21 contigs, encoding 2,282 protein-coding sequences, 11 rRNA genes, and 72 tRNA genes. Functional annotation indicated enrichment of genes associated with carbohydrate metabolism, stress adaptation, and membrane transport, while screening confirmed the absence of known virulence determinants and transferable antibiotic resistance genes, supporting its safety profile. The consistent predominance of Weissella spp. in Gir cow milk suggests a breed-associated microbial signature that may contribute to the functional characteristics of this indigenous dairy system. Collectively, these findings position Weissella as a promising functional component of the Gir cow milk microbiome and provide a genomic framework supporting its potential application in probiotic and functional dairy research.

IntroductionLactiplantibacillus plantarum is a versatile lactic acid bacterium (LAB) recognized for its probiotic potential, with key traits such as adhesion to intestinal epithelial cells and tolerance to bile salts and gastric acid being essential for its efficacy. In this study, we isolated and characterized L. plantarum strain UTNGt3 from Chrysophyllum oliviforme (Caimitillo) fruits collected in the Ecuadorian Amazon.MethodsWhole-genome sequencing, gene annotation, and in silico analyses were performed to explore genomic architecture, identify probiotic gene markers (PGMs), and assess safety features, including bacteriocin gene clusters (BGCs). In vitro assays evaluating bile salt and acid tolerance, cell surface hydrophobicity, auto-aggregation, and adhesion to Caco-2 intestinal epithelial cells were conducted to characterize probiotic traits. Additionally, the biocompatibility of UTNGt3 external metabolites was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and LDH (lactate dehydrogenase) release assay on intestinal cells.ResultsUTNGt3 genome spans 3,569,352 bp with 43.95% GC content. EggNOG analysis showed enrichment in genes related to general function prediction (11.89%), carbohydrate metabolism (8.97%), and transcription (8.45%), with 25.92% annotated as hypothetical proteins. No acquired antibiotic resistance or virulence genes were detected. Genome mining revealed three BGCs, plantaricin_N, enterolysin_A, and plantaricin_W-beta, associated with antimicrobial functions. Diverse PGMs involved in stress tolerance, adhesion, and vitamin biosynthesis were also identified. Phenotypic assays confirmed strong acid and bile tolerance, high auto-aggregation, surface hydrophobicity, and superior adhesion to Caco-2 cells compared to E. coli. Biocompatibility assays confirmed over 85% cell viability and minimal membrane damage, supporting their safety.ConclusionThese findings establish UTNGt3 as a safe, multifunctional probiotic candidate with potential for functional food applications and future gut health studies.

Bekasam is one of the Indonesian fish fermented products that contain lactic acid bacteria (LAB). Previous research has obtained four LAB strains which are considered as bacteriocin producers, have antibacterial activity against pathogenic bacteria from food. In addition to a bacteriocins, LAB produce another compounds, such as organic acids. This study aimed to determine the produced organic acids and their antibacterial activity during LAB growth against of Lactobacillus monocytogenes, Staphylococcus typhimurium, Escherichia coli, Bacillus cereus and Staphylococcus aureus, to determine the organic acid contents using HPLC and to identify LAB isolates using molecular method based on 16S rDNA sequences. The four LAB strains (BP (3), BP (20), BI (3) and SK (5)) were grown in MRS broth medium, then incubated at 37 °C for 48 hours and every 4 hours incubation was tested of titratable acid total (TAT), pH, LAB growth and antibacterial activity. The result showed that the exponential phase of bacteria growth occurred at 16-20 hours incubation and at the end of the exponential phase produced the highest antibacterial activity in the four LAB strains. The highest growth, TAT and antibacterial activity produced by the SK (5) strain. The highest organic acids content were lactic acid in the SK(5) strain and acetic acid in the BI(3) strain. Identification result indicated that BI(3), BP(3), and BP(20) strains were Pediococcus pentosaceus IE 3 with similarity of 98%, 97%, and 98%, respectively. While SK(5) strain showed 93% similarity to Lactobacillus plantarum subsp. plantarum NC 8. The four LAB strains produced organic acids and had antibacterial activity, so they can be developed as food biopreservatives.

India's diverse culinary heritage includes a wide spectrum of traditional fermented foods that harbour complex microbial communities essential for flavour development, preservation, and nutritional enhancement. These microorganisms-primarily lactic acid bacteria, yeasts, and molds-contribute functional properties that extend beyond food transformation to confer health benefits, including probiotic potential and metabolic regulation. This review integrates classical microbiological studies with modern molecular approaches such as metagenomics, metatranscriptomics, and metabolomics to elucidate the microbial diversity of Indian fermented foods. It highlights how geography, substrates, and ethnic traditions shape region-specific microbial consortia sustained through long-standing ethno-microbiological practices. Special focus is given to the glycemic modulation achieved through microbial fermentation, wherein organic acid production and resistant starch formation lower glycemic index and improve glucose metabolism. These processes, along with enhanced nutrient bioavailability, vitamin synthesis, and immunomodulation, illustrate the broader functional potential of fermentation. The review also examines interactions between food-borne microbes and the human gut microbiota, underscoring implications for personalized nutrition. Finally, it discusses modernization and commercialization strategies and outlines future directions involving multi-omics integration, indigenous starter cultures, and microbiome-based innovations to harness India's microbial heritage for improved health and sustainable food development.

The quality of bread directly depends on several physical and chemical indicators of the quality of flour, which is used in the technology of making products. Various types of flour, phyto-additives, oils, and leavening microorganisms are added to its recipe to improve the quality and increase the biological value of bread. Pure cultures of lactic acid and propionic acid bacteria are among the fermenting microbiota most often used in the technological process of bread production. The work aimed to determine the effect of adding pure cultures of lactic acid and propionic acid bacteria on the technological process of bread production. Three samples of dough and dough were prepared: the first with lactic acid bacteria and yeast; the second – propionic and lactic acid bacteria and yeast; the third only on a yeast base. Then, samples of rye-wheat dough were mixed in these ovens, and bread was baked. The symbiotic effect of propionic acid bacteria on the acidification of the environment, that is, on the development of yeast and lactic acid bacteria, was established because it was sample № 2 in which this entire microbial landscape was present that had the highest acidity of the dough. An interdependent relationship between added bacteria to the wort and reduction in fermentation time was revealed. In test № 2, the time required to reach the final fermentation process was about 77 min, about 30 min faster than in the control test. In dough № 2 with added microbiota, the lifting force time was the lowest. Therefore, the addition of propionic and lactic acid bacteria to the dough at the same time as yeast contributes to the active gas-forming ability of the dough. Thus, introducing this microbiota into the steam will increase semi-finished products in a short production time.

The optical purity of lactic acid is a critical parameter for producing high-performance polylactic acid (PLA). To investigate the genetic basis of stereospecific lactic acid biosynthesis, the present study aimed to functionally characterize the roles of ldh1 and ldh2 in Lacticaseibacillus paracasei NC4 through targeted gene disruption. A CRISPR/Cas9 nickase-based system was employed to construct three mutant strains (Δldh1, Δldh2, and Δldh1Δldh2). Fermentation experiments were conducted under identical conditions, and the concentrations of D- and L-lactic acid were quantified using HPLC. Three mutant strains, Δldh1, Δldh2, and Δldh1Δldh2, were successfully constructed from the wild-type NC4 using the CRISPR-Cas9 system. Compared with the wild-type NC4, which produced 89.31 ± 0.21g/L L-lactic acid and 10.74 ± 0.19g/L D-lactic acid, the Δldh1 mutant produced 76.31 ± 2.22g/L L-lactic acid and 7.72 ± 0.36g/L D-lactic acid, while the Δldh2 mutant yielded 81.73 ± 0.46g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant generated 75.57 ± 2.96g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant similarly exhibited no detectable D-lactic acid formation, supporting the role of ldh2 in D-lactate biosynthesis. These results indicate that targeted deletion of ldh genes significantly alters the stereospecificity of lactic acid biosynthesis. In particular, deletion of ldh2 was sufficient to eliminate detectable D-lactic acid formation, whereas deletion of ldh1 alone did not completely abolish D-lactate production. Overall, this study provides functional genetic insight into the roles of ldh1 and ldh2 in controlling lactic acid stereospecificity in L. paracasei NC4 and establishes a genetic basis for future metabolic and process-oriented optimization of optically pure lactic acid production.

This chapter provides an overview of the biologically based preservation technologies termed “biopreservation.” Acid production by lactic acid bacteria (LAB) in temperature-abused foods (controlled acidification) is covered in the chapter. Some LAB produce antimicrobial proteins, called bacteriocins, which inhibit spoilage and pathogenic bacteria without changing (e.g., through acidification, protein denaturation, and other processes) the physicochemical nature of the food. While organic acids are usually added to foods, LAB can produce lactic acid in situ. The controlled production of acid in situ is an important form of biopreservation. There are many different ways to use bacteriocins in foods. The first is to add bacteriocins directly to the food for the purpose of inhibiting spoilage or pathogenic bacteria. The second way to use bacteriocins is to add bacteriocinogenic cultures to the food or use them as starter cultures that produce the bacteriocin in situ. A third way to use bacteriocins is to facilitate the use of defined starter cultures in fermented foods. Emulsifiers such as Tween 80 or the entrapment of the pediocin in multilamellar vesicles increases pediocin effectiveness in fatty foods. Nisin is the only bacteriocin approved internationally for use in foods. The biological methods of food preservation covered here mark only the beginning of the biopreservation era in the food industry.

Biopreservation of food using bacteriocin from lactic acid bacteria (LAB) was an innovative breakthrough. Lactic acid bacteria can protect against food spoilage and pathogen bacteria by producing bacteriocin. The purpose of this study was to characterize the bacteriocin produced by LAB isolated from solid waste of soymilk that had probiotics properties. The LAB having antibacterial activities was evaluated their growth, and identified by using 16S rRNA gene sequence analysis. Its bacteriocin activities was tested on various pHs (2, 3, 4, 5, 6, 7, 8, and 9) and temperature (60-100 ° C). Its activities was evaluated againts pathogenic bacteria (Staphylococcus aureus ATCC 25923 and Listeria. monocytogenes CFSAN004330), enzymes (trypsin, catalase and protease-K), and antibiotics (penicillin and ampicillin). The results showed that LAB A23.4 isolates, which had 16S rRNA gene sequence were L. plantarum strain TMW 1.1623. Its Bacteriocin had antimicrobial activity against S. aureus ATCC 25923 and L. monocytogenes CFSAN004330 at pH 2-7, at temperatures of 60, 70, 80, 90, 100 ° C for 60 minutes and lysed by the enzymes trypsin and protease-K. Bacteriosin activity was stronger than that of the antibiotics of penicillin and ampicillin against S. aureus and L. monocytogenes. The inhibition zone of supernatant bacteriocin was 10 and 20 mm for S. aureus and L. monocytogenes. On the other hand, penicillin and ampicillin inhibition zones were 0 and 3 mm, respectively. From these results, it can be concluded that the antimicrobial produced by L. plantarum strain TMW 1.1623 was a bacteriocin used as food preservation that its processing using relatively wide range temperature (60-100) with pH 2-7.

Lactiplantibacillus plantarum is a lactic acid bacterium (LAB) capable of producing antimicrobial compounds such as lactic acid, hydrogen peroxide, exopolysaccharides, and a bacteriocin known as plantaricin. Plantaricin exhibits strong inhibitory effects against both Gram‐positive and Gram‐negative pathogenic and spoilage bacteria, making it a promising natural preservative for food products. This review synthesizes current knowledge on the environmental and nutritional factors influencing plantaricin production, such as culture media composition, pH, temperature, growth phase, and salinity, as well as evaluates the challenges and future prospects of its application in biopreservation. Liquid MRS medium has been shown to be effective in stimulating plantaricin production, particularly under acidic conditions (pH 3 and pH 4.9). Plantaricin 423 production increased under acidic conditions (pH 4.9) by 25.03% (2961 AU/mL/OD max), compared to pH 5.8 (2368 AU/mL/OD max), and at temperatures ranging from 30°C to 37°C. Media salinity also affects plantaricin synthesis by altering lactic acid metabolism. The effectiveness of plantaricin can be enhanced through techniques such as freeze‐drying, microencapsulation, and direct surface application on food products. Major challenges in its application include degradation by protease enzymes, pathogen resistance, and relatively high production costs. Strategies such as encapsulation and application of hurdle technology offer potential solutions. This review identifies key parameters influencing plantaricin synthesis and suggests strategies to overcome its application barriers in food biopreservation.

An experiment was conducted to investigate the effects of citric acid residue (CAR) and lactic acid bacteria (LAB) on fermentation quality and aerobic stability of alfalfa silage. Alfalfa was treated as follows: (1) control without additive (CON); (2) LAB (L); (3) 12 g/kg CAR (C); (4) 12 g/kg CAR + LAB (CL) and fermented for 3, 6, 9, 15, 30 and 45 days. The residual silages ensiled for 45 days were evaluated for aerobic stability. The results showed that addition of CAR with or without LAB inoculant decreased pH, acetic acid, ammonia nitrogen , neutral detergent fibre, hemicellulose contents and Enterobacteriaceae counts (p < .05) and increased dry matter and lactic acid contents (p < .05) after 45 days of ensiling. Whereas, the lower lactic acid and higher acetic acid contents were observed in L silages (p < .05) after 45 days of ensiling. During aerobic exposure, L and CL silages remained stable in pH, lactic and acetic acid contents, while the control and C silages showed higher pH, yeast and mould counts and lower lactic and acetic acid contents. Compared with the control, L and CL improved aerobic stability, whereas the poorer aerobic stability was shown in C silages (p < .05). In conclusion, applying a combination of CAR and LAB inoculant improved both fermentation quality and aerobic stability of alfalfa silage. Highlights Citric acid residue (CAR) was compared with a lactic acid bacteria (LAB) inoculant in ensiling alfalfa. The CAR improved fermentation quality compared to control and LAB treated silages. Aerobic stability improved when CAR was used in combination with LAB but not when used alone.

Rumen liquid is a waste product of slaughterhouse that has the potential to be a pollutant, contains lactic acid bacteria which can be used as bio preservatives in food. The purpose of this study was to identify the antimicrobial activity of lactic acid bacteria (LAB) isolates from rumen fluid against Gram positive and Gram negative bacteria using well diffusion and disc diffusion methods and using lactic acid bacteria isolates (supernatant) and non-filtrate from rumen fluid. The main research materials used were LAB rumen fluid isolates, MRSA media (Mann Rogosa Sharpe Agar), MRSB media (Mann Rogosa Sharpe Broth), MHA media (Muller Hinton Agar), and pathogenic bacteria Bacillus cereus, Staphylococcus aureus, Escherichia coli and Salmonella Enteritidis. The results of the study showed that the LAB of rumen fluid carried out as an active LAB with Gram positive characteristics, round shape, negative catalase and non motile. Based on the results of testing the antimicrobial activity of lactic acid bacteria from rumen fluid isolates against pathogenic Gram positive (B. cereus and S. aureus) and Gram negative bacteria (Escherichia coli and Salmonella Enteritidis) using well and disc diffusion methods showed that Gram negative bacteria were more sensitive to antimicrobial of LAB compared to Gram positive bacteria. The diameter of the larger inhibition zone is produced using the disc method with the inhibition zone diameter range of 13.66-28.3 mm, while the well method ranges from 0-24.2 mm. The antimicrobial activity of LAB using non filtrate BAL produce inhibition zone diameter size range of 0-26.1 mm, while the filtrate BAL produce inhibition zone diameter range of 0-28.3 mm with the optimum time to produce antimicrobial activity 48 hours compared to 24 hours after incubation.