Understanding of the interaction of Lactobacillus in fermented food and beverages: targeting food safety and new foods

1 Introduction

Lactic acid bacteria (LAB) are Gram-positive, non-sporulating, and non-motile microorganisms of the order Lactobacillales (Firmicutes), occurring as either rods or cocci. They are metabolically adapted to both aerobic and anaerobic environments and are principally recognized for their fermentative capabilities, underpinning their extensive applications in food systems as well as emerging roles in agriculture and human health (Virdis et al., 2021).

LAB encompasses members from multiple taxonomic lineages and is ubiquitous. They inhabit a broad range of ecological environments, occurring on plant materials, in milk and soil, and as resident microbiota of the gastrointestinal tract and mucosal surfaces of humans and animals (Stabnikov et al., 2025). LAB has gained increasing attention in the food and beverage industry due to their ability to enhance food safety, product quality, and nutritional attributes. Their use as starter cultures, probiotic organisms, and sources of natural antimicrobial substances enables the production of foods and drinks that cater to the increasing demand for functional, health-supporting, and minimally processed products.

LAB such as Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), Levilactobacillus brevis, Leuconostoc mesenteroides, Lpb. pentosus, Ligilactobacillus salivarius, Latilactobacillus sakei, and Limosilactobacillus fermentum confer food safety (Lee et al., 2021; Wang et al., 2022). They do so by inhibiting spoilage microorganisms such as Salmonella, Listeria, Escherichia, Aspergillus, Fusarium and Penicillium via organic acid and bacteriocin (nisin and pediocin) production. They also aid in bio-preservation of food through selective targeting of spoilage microorganisms without disturbing the consumer’s intestinal microbiota through the production of antimicrobial compounds (Anumudu et al., 2024). Additionally, bacteriocins secreted by LAB also help in biopreservation more effectively than chemical preservatives. They have high efficacy due to their stability at varied pH ranges and temperatures. Bacteriocins can enhance food quality and sensory characteristics by limiting spoilage, while also promoting desirable biochemical changes like improved proteolysis during ripening (Putri et al., 2024).

Additionally, biogenic amines are a class of non- volatile nitrogenous compounds produced by decarboxylation of amino acids. They act as food intoxicants in spoilage of the food. Certain L. plantarum strains possess amine oxidase activity or generate metabolites capable of breaking down or suppressing biogenic amines, thereby, reducing the risk of food toxicity (Shao et al., 2022). In this context, this review provides critical insights of the beneficial impacts of LAB on fermented foods and human health, emphasizing their role in enhancing digestive function, gut microbiota composition, and overall well-being.

Recent advances in fermented food biotechnology and probiotic research have shifted safety evaluation from conventional species-level assumptions toward strain-level assessment supported by genomic tools. While several studies have traditionally relied on phenotypic characteristics such as antimicrobial activity, gastrointestinal tolerance, and adhesion properties to evaluate probiotic functionality (Bisht et al., 2024; Hu et al., 2023), these approaches may not sufficiently identify risks associated with virulence factors, transferable antimicrobial resistance genes, or mobile genetic elements. Consequently, genome-based safety assessment is increasingly being recognized as an essential component of modern regulatory frameworks for probiotic and fermented food applications (Merenstein et al., 2023). In this context, the present review discusses recent developments in LAB taxonomy, metabolic functionality, food safety, and emerging fermented food systems, while also highlighting the growing importance of genomic safety evaluation and regulatory considerations in the development of next-generation probiotic products.

1.1 Literature search strategy and review methodology

A structured literature search was conducted using major scientific databases including PubMed, Scopus, Web of Science, ScienceDirect, Wiley Online Library, and Google Scholar. The search focused on peer-reviewed articles published primarily between 2020 and 2025, while selected older foundational studies were included where scientifically relevant (Figure 1).

The search strategy combined keywords and Boolean operators such as: (“Lactobacillus” OR “lactic acid bacteria” OR “LAB”) AND (“fermented foods” OR “fermentation”) AND (“food safety” OR “probiotics” OR “functional foods” OR “next-generation foods” OR “metabolites” OR “regulatory safety”). Additional searches were performed using combinations of terms related to bacteriocins, exopolysaccharides, omics technologies, strain-level safety evaluation, and fermented beverages.

Studies were included if they focused on LAB applications in fermented food systems, addressed metabolic, technological, probiotic, safety, or regulatory aspects, were published in peer-reviewed journals, and provided experimentally supported or review-based scientific evidence.

Studies were excluded if they lacked scientific relevance to fermented food systems, were non-peer-reviewed sources, contained insufficient methodological detail, or focused exclusively on non-food industrial applications. The selected literature was critically analyzed and organized into thematic categories including taxonomy, metabolic pathways, food safety, fermented food applications, functional benefits, industrial advancements, and regulatory considerations.

2 Evolutionary adaptation to diverse functionalities

From a historical perspective, LAB has been integral to human civilization for millennia through fermented foods. A novel angle involves detailing the ​​evolution of LAB strains in specific fermentation niches, along with their evolutionary adaptations to diverse functionalities in agriculture, food and health sector​​ (Murata et al., 2025). This includes tracing the genetic divergence, selective pressures and screening that led to the specialization of various LAB species for particular food matrices such as meat, dairy and plant based fermentations. For example, thermophilic bacteria such as Streptococcus thermophilus and L. delbrueckii subsp. bulgaricus widely started being employed in yogurt manufacture. These microorganisms produced the enzyme β-D-galactosidase, which hydrolyzed lactose, thereby, facilitating lactose digestion and improving tolerance in lactose-intolerant individuals (Agagunduz et al., 2022). Similarly, in a study by Albano et al. (2020), LAB species such as L. plantarum, L. paracasei subsp. paracasei, L. casei, Enterococcus faecium, and Enterococcus lactis were screened for folate synthesis. Among these, folate levels increased by 54% on day 30 and by 108% on day 60, primarily due to the activity of L. casei VC199 and L. paracasei SE160. In another study by Abouloifa et al. (2024), LAB isolated from fermented olives (Lpb. plantarum, Lpb. pentosus, Lvb. brevis, and Pediococcus pentosaceus) exhibited strong acidification, reaching a final pH below 4 (pH 3.8). In contrast, those isolated from raw goat’s milk (Lactococcus lactis subsp. lactis var. diacetylactis and Lactococcus lactis subsp. Lactis) exhibited weak acidification.

Figure 2 presents an integrated conceptual framework summarizing the interconnected roles of LAB in fermented food systems. The framework highlights how strain selection, substrate characteristics, metabolic pathways, functional metabolite production, safety assessment, and regulatory validation collectively influence the development of next-generation fermented foods and beverages. This systems-level perspective emphasizes the transition from traditional empirical fermentation approaches toward precision-guided and functionally tailored food biotechnology applications.

2.1 Taxonomic diversification

Traditionally, LAB, particularly members of the genus Lactobacillus, were classified based on phenotypic and metabolic traits such as cellular morphology, growth temperature, carbohydrate utilization patterns and homo/heterofermentative metabolism (Mokoena, 2017; Salvetti et al., 2012). However, inconsistencies in these characteristics among closely related species often resulted in unreliable classification, highlighting the limitations of phenotype-based taxonomy (Hammes and Hertel, 2009).

Advancements in genome sequencing technologies, comparative genomic studies, and phylogenetic analyses have significantly transformed the classification framework of LAB. To address this taxonomic complexity and establish a more phylogenetically coherent system, Zheng et al. (2020) introduced a comprehensive reorganization based on genomic relationships, conserved protein sequences, evolutionary divergence, ecological distribution and adaptation and functional diversity of these microorganisms.

Under this revised framework, the previously large genus Lactobacillus was divided into 25 genera, consisting of a revised genus Lactobacillus along with 23 newly designated genera, including Lactiplantibacillus, Lacticaseibacillus, Levilactobacillus, Latilactobacillus, Limosilactobacillus, and Ligilactobacillus.

Consequently, several commercially and technologically important species were reassigned to newly established genera. For instance, Lactobacillus plantarum is now classified as Lactiplantibacillus plantarum, Lactobacillus casei as Lacticaseibacillus casei, Lactobacillus brevis as Levilactobacillus brevis, and Lactobacillus fermentum as Limosilactobacillus fermentum. In contrast, certain well-characterized species such as Lactobacillus acidophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii remained within the revised genus Lactobacillus without nomenclature changes.

Besides members of Lactobacillaceae, several additional bacterial genera are routinely included within the broader LAB group, including Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Weissella, Oenococcus, and Enterococcus.

Accordingly, the present review follows the updated nomenclature system proposed by Zheng et al. (2020) throughout the manuscript. Earlier taxonomic names have been retained only at their initial mention where required to maintain consistency with previously published literature.

3 Global consumption trends

Fermented foods are firmly rooted in the cultural traditions of diverse societies, with their origins tracing back nearly 8, 000 years. Each region has developed fermentation methods based on the raw materials accessible, as well as social, cultural, and religious preferences (Arora et al., 2023; Arora and Chandel, 2023). Tamang et al. (2020) reported that East Asian countries (Japan and Korea), largely employed their fermentation practices on plant-based substrates (rice, soybeans, and sprouts) resulting in products like miso, soy sauce, and kimchi, while fermented meat products are comparatively rare. In contrast, India and many European countries, developed a diverse range of fermented milk products, like yogurt, curd, cheese, and other dairy-based foods. Scandinavian regions (coastal communities) focused on fermented fish products due to their available marine resources. Additionally, Valentino et al. (2024) reported that in African countries, the fermented cereal staples such as sorghum, maize, and wheat, are exploited for the production of fermented porridges, beverages, and breads. Despite differences in raw materials and processing techniques, all these fermented foods harbor complex and dynamic microbial communities.

Together, these region-specific fermentation practices highlight a globally shared food tradition shaped by local resources yet connected by common microbial processes.

4 Metabolic synthesis and fermentation mechanism

During fermentation, LAB transform lactose and other sugars into lactic acid, causing acidification that improves food stability, nutritional quality, and associated health benefits. This metabolic activity proceeds through two distinct pathways - homolactic and heterolactic fermentation, determined by the genetic characteristics of individual Lactobacillus strains. Table 1 shows the metabolites produced by different LAB strains via different fermentation pathways.

Table 1. Metabolites produced by different LAB strains via different fermentation pathways.

Homofermentative LAB predominantly produce lactic acid with high yield, making them well suited for industrial lactic acid production (Loo et al., 2025). Conversely, heterofermentative LAB follow a mixed metabolic pathway that yields lactic acid together with carbon dioxide and ethanol, resulting in reduced lactic acid efficiency. The release of gas during fermentation limits their application in dairy processing, as carbon dioxide accumulation can lead to packaging expansion and the formation of cracks or textural defects in fermented dairy products (Zhang et al., 2022).

4.1 Organic acids

LAB is characterized by their ability to produce multiple organic acids during carbohydrate fermentation, with lactic acid as the main product alongside acetic, formic, succinic, and other acids (Wang et al., 2021). Additionally, they are also the key producers of bacteriocins, exopolysaccharides and vitamins.

Utilizing glucose as their primary substrate, homolactic fermentation proceeds with formation of two molecules of pyruvate via the EMP glycolytic pathway, yielding 2 ATP and 2 NADH per glucose. Pyruvate is subsequently reduced to lactic acid by lactate dehydrogenase (LDH), regenerating NAD⁺, sustaining continuous glycolytic flux (Aguirre-Garcia et al., 2025). The type of lactate formed (L or D) during fermentation is determined by the specific LDH isoforms expressed by the specific microbial strain. This predominance not only influences acidity, but also flavor development and nutritional quality of the final product (Farhana and Lappin, 2025).

In contrast, heterofermentative LAB follow the pentose phosphate and phosphoketolase routes, generating lactic acid together with ethanol or acetic acid and carbon dioxide during fermentation. This adaptability is especially relevant in cereal and vegetable-based substrates rich in pentoses (Okoye et al., 2025). Additionally, LAB also transform citrate and related organic acids into metabolites such as acetate, succinate, and formate through enzymes like fumarate reductase, pyruvate-formate lyase and fumarate contributing to the development of complex flavors in fermented dairy products.

4.2 Bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by LAB that selectively inhibit closely related microorganisms and several foodborne pathogens. Unlike conventional antibiotics, bacteriocins are generally inactivated by gastrointestinal proteases such as trypsin and pepsin, thereby, limiting their persistence in the gut and reducing disruption of the resident microbiota. Although their antimicrobial spectrum is relatively narrow, LAB bacteriocins may exert activity against Gram-negative bacteria through synergistic interactions with environmental factors including organic acids, low temperature, and membrane disrupting agents. Imade et al. (2021) recognized that, bacteriocins are typically designated according to their producing genus or species, with representative examples including plantaricin (Lactiplantibacillus plantarum), nisin and lacticin (Lactococcus spp.), pediocin (Pediococcus spp.), enterocin (Enterococcus spp.), leucocin (Leuconostoc spp.), and carnocin (Carnobacterium spp.).

Bacteriocin production is a genetically encoded process in which LAB synthesize an inactive precursor peptide that undergoes post translational modification and is exported via dedicated secretion systems (Chen et al., 2025). Further Perez et al. (2022) described, bacteriocin synthesis places a considerable metabolic burden on the producing strain, as the genes involved are organized in operons as it requires tightly coordinated expression of multiple genes responsible for precursor processing, export, self-protection, and regulatory control of production. Expression is typically controlled by a quorum-sensing three-component system comprising an inducing peptide, histidine kinase, and response regulator. Okoye et al. (2025) reported that recent advances in omics and synthetic biology have enabled improved yields and expanded antimicrobial spectra, enhancing the relevance of bacteriocins in clean-label and minimally processed foods.

4.3 Exopolysaccharides

Exopolysaccharides (EPS) are extracellular, high molecular weight biopolymers synthesized by microorganisms, including LAB and Bifidobacteria, during fermentation. These naturally derived polymers are biobased, biodegradable, and biocompatible, forming an integral component of the microbial extracellular matrix together with proteins, nucleic acids, and lipids. EPS biosynthesis is frequently upregulated in response to environmental and biotic stresses, such as fluctuations in pH, temperature, light exposure, and osmotic pressure, thereby, enhancing microbial resilience and survival under adverse conditions (Yin et al., 2019).

EPS biosynthesis in LAB relies on intracellular pools of monomeric sugars, such as glucose, galactose, mannose, rhamnose, fructose, and pentoses, as well as sugar derivatives including uronic acids and amino sugars. Abarquero et al. (2022), described the process to include four coordinated steps: carbohydrate uptake, formation of activated sugar nucleotides, assembly of repeating units in the cytoplasm, and polymerization with subsequent export. Further, Raj et al. (2022) added that, polymerization is catalyzed by EPS specific glycosyl and acetyltransferases, which link activated monomers into polysaccharide chains and mediate their translocation across the cell envelope. The genes encoding these biosynthetic enzymes and transport proteins are typically organized in strain specific EPS gene clusters and co transcribed as operons, enabling tightly regulated and efficient EPS production in LAB.

4.4 Vitamins

Fermentation driven metabolic activity of LAB enhances the synthesis and bioavailability of several vitamins such as, vitamin C, riboflavin (B₂), and folate. The vitamin producing potential of LAB is highly strain and species specific, reflecting differences in their metabolic pathways, as discussed in the Table 2 in detail. Among these, Lactiplantibacillus plantarum has been reported to exhibit superior folate biosynthetic capacity relative to other LAB strains. In addition, Lactococcus lactis and S. thermophilus are widely employed as starter cultures in yogurt fermentation owing to their consistent ability to synthesize folate, thereby, contributing to the nutritional enrichment of fermented dairy products (Abdul et al., 2023).

Table 2. Vitamin production by different LAB strains under different fermentation conditions.

*GTP, guanosine triphosphate; DHPPP, 6-hydroxymethyl-7, 8-dihydropterin pyrophosphate; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; pABA, para-aminobenzoic acid (synthesized via pabA/pabB genes).

For example, Averianova et al. (2020) explained the biosynthesis of riboflavin (vitamin B₂), an important precursor for the formation of cofactors flavin mono nucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential for cellular redox reactions. In LAB, riboflavin is synthesized via a dedicated pathway encoded by the rib gene cluster, where guanosine triphosphate and ribose-5-phosphate are converted into riboflavin through the action of RibA, RibB, RibH, and RibC operon genes and the enzyme riboflavin synthase.

These biosynthetic properties underpin the use of selected LAB in food biofortification strategies aimed at enhancing the nutritional value of fermented foods, effectively transforming them into sources of naturally synthesized vitamins for human consumption.

5 Application in fermented foods and beverages

LAB species are extensively used in fermented foods due to their robust metabolic properties. Several LAB species, including Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, Lactococcus lactis, and Leuconostoc mesenteroides, are widely employed as starter and adjunct cultures in fermented foods owing to their diverse metabolic and technological functionalities. LAB strains are typically used in production of probiotics and traditional fermented foods. These microorganisms contribute significantly to fermented processes through acidification, proteolysis, exopolysaccharide production, and flavor development, thereby, enhancing the sensory and functional quality of fermented products (Yang et al., 2025).

LAB-mediated fermentation also improves the nutritional and functional properties of foods through enzymatic biotransformation of complex substrates. In particular, LAB drives enzymatic and metabolic transformations that convert raw substrates into more easily assimilable forms, thereby, facilitating nutrient uptake. These biochemical conversions increase the production of beneficial metabolites while simultaneously improving the bioaccessibility and bioavailability of nutrients, ultimately maximizing their physiological utilization (Anumudu et al., 2024). Certain LAB strains additionally contribute to phytate degradation, synthesis of bioactive peptides, vitamin production, and generation of postbiotic compounds with potential health-promoting effects.

LAB is crucial to biopreservation because they produce a variety of antibacterial compounds during the fermentation and development stages. Species such as Lactococcus lactis, Pediococcus acidilactici, and Lactilactobacillus sakei produce antimicrobial metabolites including organic acids, hydrogen peroxide, diacetyl, and bacteriocins that enhance microbial safety and shelf stability of fermented foods. Among the most commonly utilized antimicrobials are the bacteriocins, which can eradicate undesirable bacterial strains without endangering themselves. Remarkably, some LAB bacteriocins have shown efficacy against Listeria monocytogenes, a significant problem in traditional raw-milk cheeses (Ahansaz et al., 2023).

Several studies show that the involvement of LAB could successfully impart desirable flavor and aroma compounds during food ripening and fermentation. LAB generally contributes to texture development and preservation throughout the fermentation process, preventing spoiling and enhancing the sensory quality of meats and fish, such as salami, sausages, or fish sauces (Kröckel, 2013). Additionally, in cereal-based fermentations such as sourdough bread, lactic acid bacteria play a pivotal role in improving sensory and nutritional attributes. Through the degradation of anti-nutritional compounds, these microorganisms refine flavor and texture while increasing the accessibility and absorption of essential nutrients (Jain et al., 2025).

The metabolic versatility and stain-specific functionality of LAB highlight their industrial significance in improving food safety, shelf stability, sensory attributes, and nutritional quality across diverse fermented food system (Anumudu et al., 2024).

6 Food safety and new foods

Recent studies increasingly demonstrate the use of whole-genome sequencing (WGS) to confirm the safety of Lactobacillus strains before their application in fermented foods. For example, Lactiplantibacillus plantarum TWK10 underwent comprehensive genomic analysis that revealed no virulence factors, antimicrobial resistance genes, or genes involved in biogenic amine synthesis, confirming its suitability as a safe probiotic strain for food applications and human consumption (Hsu et al., 2022). Similarly, genomic characterization of Lactiplantibacillus plantarum LVS14, confirmed the absence of safety-related risk genes and identified functional genes related to stress tolerance and carbohydrate metabolism. Following this genomic validation, the strain was successfully incorporated as a starter culture in fermented milk, improving sensory quality while maintaining product safety and probiotic functionality (El Halfawy et al., 2025). Another investigation identified Lactiplantibacillus plantarum Z-5 from traditional fermented foods, where WGS confirmed its safety profile and revealed plantaricin biosynthesis genes responsible for antimicrobial activity; the strain demonstrated strong inhibition of Listeria monocytogenes and potential application as a biopreservative in dairy systems (Qiao et al., 2025). These examples illustrate how genome-based safety evaluation is increasingly being used to validate Lactobacillus strains for safe use in modern fermented food products.

The development of novel fermented foods and beverages has expanded rapidly in recent years, driven by consumer demand for plant-based, functional, and microbiome-supporting products. Recent reviews highlight that fermentation is increasingly applied to non-dairy substrates such as cereals, legumes, nuts, and seeds, enabling the production of products including fermented soy yogurt, oat-based probiotic beverages, almond yogurt, and cereal-based fermented drinks (Dhiman et al., 2025).

6.1 Dairy products fermentation

Dairy products consist of one of the most well-known and well-established matrices for LAB driven fermentation, owing to their favorable nutrient composition and long history of consumption. Fermented dairy foods are increasingly recognized not merely as carriers of viable LAB, but as complex biological systems delivering a consortium of beneficial microorganisms together with a diverse spectrum of fermentation-derived metabolites. These metabolites highlight the technological relevance, influencing product texture, flavor development, stability, and overall quality. Consequently, LAB associated with dairy fermentations is now being explored beyond their traditional acidification role, with growing emphasis on their functional and application-oriented attributes (Zhang et al., 2025).

LAB causes a significant metabolic shift during fermentation by acting on lactose (homofermentative route). This specific biochemical change has several uses, including eliminating harmful or anti-nutritional elements to prevent lactose intolerance, increasing the bioavailability of minerals (like calcium), improving texture, reducing product syneresis, etc (Gänzle, 2015). The metabolites and secretory molecules such as exopolysaccharides, adhesion factors, bacteriocins, etc. produced during fermentation depend on the raw material source, the fermentation medium, and the processing conditions like incubation time, temperature, inoculum level, etc.

The structural properties of the matrix of fermented dairy products, such yogurt, are greatly influenced by starter culture and its mixture. In addition, the concentration and composition of the inoculum or starter culture are critical determinants of textural development in fermented dairy products. This becomes particularly important when dairy matrices are combined with non-dairy components such as spices, fruits, or cereals, where appropriate starter selection and dosage markedly influence structural integrity, consistency, and overall product quality (Sharma et al., 2023).

During yogurt making, LAB ferment the lactose in milk and produce lactic acid. The pH is lowered from 6.8 to 4.5 during the fermentation process. This pH shift, commonly reported in both laboratory and commercial processes, plays a central role in yogurt structure development (Wang and Zhao, 2022). As acidification progresses, colloidal calcium phosphate dissociates and casein micelles lose stability, promoting their aggregation into a continuous gel matrix. According to Wang and Zhao (2022), fermentation conditions such as incubation temperature (37–45 °C) and processing time markedly influence the strength and consistency of this gel, with faster acidification at higher temperatures often resulting in comparatively weaker protein networks. In addition, differences between starter strains, particularly L. delbrueckii subsp. bulgaricus and S. thermophilus, have been associated with distinct acidification rates and variations in viscosity and firmness of the final product. Beyond acid-induced changes, certain LAB strains synthesize exopolysaccharides during fermentation, with reported levels varying widely (approximately 20–200 mg L^-¹) depending on strain and growth conditions. These polymers interact with milk proteins and water, contributing to improved viscosity and water-holding capacity. Together, available studies suggest that yogurt texture is shaped by interplay between fermentation-driven protein modifications and strain-specific metabolic activity, underlining the importance of starter culture selection and process control.

Various LAB strains like Lactobacillus and Bifidobacterium, used in yogurt-making are regarded as probiotics, because they may help the gut microbiome and overall health (Bhatia et al., 2024). Several studies have demonstrated that regular consumption of yogurt containing these strains can positively modulate gut microbiota composition, enhance intestinal barrier function, and support host metabolic and immune responses. For instance, Lactobacillus- and Bifidobacterium-enriched yogurts have been reported to improve microbial diversity and increase beneficial short-chain fatty acid–producing populations in the gut. This has resulted in an increase in consumption and demand for probiotic dairy products in recent years (Liang et al., 2021). In addition to this, some studies state that LAB is used as a starter culture in cheese fermentation. During maturation, LAB-mediated hydrolysis of milk proteins, especially caseins, leads to the release of bioactive peptides with documented antihypertensive, antioxidant, and antimicrobial properties. Mixed-strain starter cultures are often preferred, as synergistic interactions among LAB strains can enhance acid production, aroma formation, and peptide release compared to single-strain systems. As a result, the function of LAB in cheese extends beyond acid production, contributing to both sensory development and functional value, thereby, distinguishing cheese as a more complex probiotic carrier compared to fermented dairy products such as yogurt (Liang et al., 2021).

Beyond solid and semi-solid dairy matrices as discussed above, LAB also play a central role in fermenting dairy beverages like kefir, buttermilk, yogurt drinks, where differences in substrate availability, microbial interactions, and processing conditions influence fermentation dynamics and product functionality (Al-Kharousi, 2025). Moreover, a comparative evaluation of LAB performance across dairy, mixed dairy-oat and plant-based beverages has reported that substrate composition strongly governs fermentation kinetics, physicochemical stability, and sensory outcomes. The study showed that LAB cultures exhibit substrate-dependent fermentation dynamics in dairy and cereal-based beverages. Dairy matrices support rapid acidification to a pH of approximately 4.4–4.6, whereas oat-based systems show slower acidification and higher final pH (~4.8–5.2) due to limited fermentable sugars. Nevertheless, LAB maintain high viability (>10⁷ CFU/mL) across matrices, while improving texture and stability, demonstrating their adaptability in both dairy and plant-based fermented beverages (Huang et al., 2020).

6.2 Vegetable and cereal fermentations

Vegetable and cereal fermentation such as pickles, sauerkraut, and sourdough are characterised by a rapid succession of LAB that transform the raw matrix into a nutritionally superior product. During the early stage of pickle and sauerkraut fermentations, facultative hetero-fermentative Lactobacillus spp. dominates, producing lactic acid and acetic acids that lowers pH, inhibit spoilage microorganism and create characteristic flavor (Knez et al., 2023). This research group further highlighted that LAB activity during vegetable fermentation contributes to improved mineral bioavailability and partial degradation of plant anti-nutritional compounds, supporting the role of LAB not only as preservation agents but also as key contributors to the nutritional upgrading of fermented plant-based foods. As the fermentation progress, the succession of LAB plays a decisive role in determining product stability and quality through sustained acidification and metabolic activity. In vegetable fermentations, LAB dominance has been associated with the rapid pH decline to < 4.0 within 48–72 h during vegetable fermentations, which has been documented in studies of LAB-driven pickle fermentations, for instance, Knez et al. (2023) also reported that LAB dominance drives pH to 3.8–3.9 after 2 days, promoting microbial stability, controlled tissue softening, and formation of organic-acid-derived aroma compounds.

In cereal-based systems such as sourdough, LAB further interacts with native yeasts, creating a coordinated metabolic network that governs carbohydrate utilization and organic acid production (Khanna et al., 2025). This increases lactic and acetic acid concentrations by approximately 30%–45% compared to single-culture fermentations, alongside accelerated maltose depletion and improve gas retention capacity. These metabolic shifts translated into measurable improvements in dough rheology, including increased elasticity and loaf volume, ultimately enhancing bread texture and structural uniformity. These findings demonstrate how LAB adaptability to plant-based matrices supports both technological functionality and process standardization in fermented cereal products. Extension LAB driven sourdough process enables partial degradation of FODMAP components, improving dough performance and product suitability for sensitive consumers. This highlights the technological relevance of LAB in cereal-based fermentations beyond conventional acidification (Arora et al., 2023).

6.3 Meat fermentations

In fermented meat systems, LAB further contribute to product safety and stability through the production of antibacterial metabolites such as organic acids and bacteriocins. These compounds suppress spoilage and pathogenic microorganisms during fermentation and storage, thereby, enhancing shelf life without reliance on chemical preservations. Such bio-preservation functions reinforce the technological importance of LAB in traditional and modern fermented meat products (Kaveh et al., 2023). Beyond microbial safety, LAB play a central role in shaping the sensory and quality attributes of fermentable meat products. LAB-driven proteolysis and liquid metabolism during fermentation contribute to the formation of characteristic flavor compounds, improved texture, and color stability. These metabolic activities underline the importance of LAB not only as starter cultures but as key determinants of overall product quality in fermented meats (Wang and Zhao, 2022). In dry-cured meat products, LAB contributes to visual quality and oxidative stability during fermentation. Stadnik et al. (2022) reported that LAB activity during ripening was associated with improved color retention and a reduced extent of lipid and protein oxidation, factors directly linked to consumer acceptance and shelf life. These observations highlight the multifunctional contribution of LAB to fermented meat systems, extending their role beyond acid production to quality preservation.

6.4 Alcoholic and non-alcoholic fermented beverages

In fermented beverage systems, LAB play a multifunctional role by simultaneously supporting fermentation stability, improving product quality, and contributing to nutritional enhancement across both alcoholic and non-alcoholic beverages (Anumudu et al., 2024). In non-alcoholic fermented beverages, LAB can be strategically applied to modulate fermentation outcomes and product characteristics. Tong et al. (2025) demonstrated that re-fermentation of sterilized kombucha using Acetobacter sp. alone or in combination with Lactiplantibacillus plantarum effectively eliminated ethanol, reducing its concentration from 0.66% to undetectable levels through ethanol oxidation by Acetobacter. Co-fermentation with L. plantarum further improved sensory quality, yielding beverages with higher overall acceptability, which was attributed to the production of lactic acid with milder acidity compared to acetic acid alone. In addition to sensory enhancement, the combined fermentation markedly increased functional attributes, with total polyphenol and flavonoid contents raised from 215.32 to 336.33 mg GAE/L and from 163.38 to 196.35 mg CE/L, respectively, alongside enhanced antioxidant activity. These results show the potential of LAB-assisted re-fermentation as a viable strategy for producing non-alcoholic fermented beverages with improved sensory appeal and functional value. Similarly, in another study by Ferreira et al. (2022) the benefits of LAB and yeast co fermentation have been reported in plant-based beverage systems. It has been demonstrated that co culturing Lactiplantibacillus plantarum with potential probiotic yeast enhances fermentation performance while maintaining microbial viability and sensory acceptability in beverages. This study explains that LAB-yeast interactions supported stable acidification, improved flavor balance, and retention of probiotic potential, reinforcing the applicability of mixed microbial cultures for developing non-dairy fermented beverages with added functional value (Ferreira et al., 2022).

In contrast, recent advances in fermented alcoholic beverages have emphasized the selective incorporation of probiotic lactic acid bacteria alongside conventional alcoholic fermentation processes to enhance functional properties while maintaining acceptable sensory characteristics. Carboni et al. (2023) examined a range of traditional Latin American fermented alcoholic beverages and reported that LAB frequently coexist with yeasts during fermentation, contributing measurably to both functional and sensory attributes of the final products. The authors highlighted that LAB activity during alcoholic fermentation influences organic acid profiles, microbial stability, and the formation of bioactive metabolites, while maintaining desirable flavor characteristics typical of traditional beverages. In several systems, LAB presence was associated with improved product safety through competitive exclusion of undesirable microorganisms and modulation of fermentation dynamics. These observations support the controlled use of LAB as adjunct cultures in alcoholic fermentations to enhance functionality without disrupting the sensory identity of traditionally fermented beverages.

7 Health and functional benefits

Fermented food involves not only undergo microbial transformation of substrates but also contribute to human health through bioactive components (Goyal et al., 2026). LAB -based fermentations influence gut microbiota composition, gut brain axis, support immune function, and can inhibit pathogens through competitive interactions and metabolite production (Figure 3). As a result, regular consumption of LAB fermented foods has been linked with enhanced digestive health and broader nutritional benefits, positioning these products as valuable functional foods within the modern diet (Ibrahim et al., 2023) Moreover, fermentation enhances the nutrient bioavailability of functional foods by modifying their biochemical composition. Through enzymatic activity, LAB reduces antinutritional factors such as phytates and tannins while enhancing mineral bioavailability and protein digestibility. These nutritional improvements contribute to the health-promoting potential of LAB-fermented foods, particularly in cereal- and legume-based diets (Adebo et al., 2022).

In addition to improving nutrient bioavailability, certain LAB strains contribute to health benefits through the production of extracellular metabolites like exopolysaccharides. In recent study, LAB- driven exopolysaccharides have been associated with enhanced probiotic functionality, including improved gut colonization, immunomodulatory potential and protective effects against environmental stresses during gastrointestinal transit (Srinivash et al., 2023). These functional characteristics further strengthen the role of fermented foods with health promoting properties. Beyond nutritional and metabolic effects, LAB fermented foods exert pronounced immunological benefits through their capacity to modulate gut microbial composition and host immune signaling pathways. Consumption of functional fermented foods has been shown to restore microbial balance and promote immune homeostasis by influencing T-cell differentiation and regulatory mechanisms. A research study indicates that LAB intake can enhance regulatory T-cell (Treg) responses while modulating Th1/Th2/Th17 balance, thereby, contributing to controlled inflammatory responses and improved mucosal immunity (Oh et al., 2022). It further reported that certain LAB-mediated immune modulation is closely associated with alterations in cytokine profiles, including increased anti-inflammatory cytokines such as IL-10 and TGF-β, alongside suppression of pro-inflammatory mediators. These host–microbe interactions underline the broader functional relevance of LAB-fermented foods in maintaining intestinal integrity and supporting systemic immune health.

8 Safety considerations and regulatory status

The safety of LAB intended for use in fermented foods is a fundamental requirement of both regulatory approval and consumer acceptance. LAB used in fermented foods are generally regarded as safe due to their long history of consumption and well documented use in traditional and industrial fermentations (Ayivi et al., 2020). Many LAB species have been granted regulatory recognition, such as Generally Recognized as Safe (GRAS) status, based on taxonomic identity and consistent performance in food systems (Min et al., 2022). This regulatory acceptance provides a foundation for the continued use of LAB, while highlighting the need for further evaluation at the strain level.

Recent studies of food driven LAB strains emphasize the importance of evaluating technological suitability along with safety attributes such as absence of virulence factors, hemolytic activity, transfer of antibiotic resistance and biogenic amine production, as well as a low potential for horizontal transfer of antibiotic resistance genes. Zhu et al. (2024) highlighted that such multifactorial evaluation is critical to distinguish technologically promising strains from those unsuitable for food applications, particularly in the context of probiotic and functional food development. This integrated approach not only ensures consumer safety but also supports regulatory compliance and facilitates the responsible incorporation of LAB into modern fermented food systems.

Despite the general recognition of safety for many LAB species, regulatory frameworks increasingly promote structured safety assessment systems for microbial starter cultures. Heo et al. (2022) outlined an integrated safety assessment framework combining phenotypic screening such as hemolytic activity, biogenic amine formation, and antibiotic susceptibility with whole-genome analysis to verify taxonomic identity and detect virulence determinants or transferable antibiotic resistance genes. Their analysis of starter candidates isolated from fermented foods demonstrated that strains passing both phenotypic and genomic criteria exhibited stable technological performance while lacking safety-associated risk factors. Such multi-tiered assessment systems complement existing GRAS concept by shifting safety evaluation from species-based assumptions to strain-specific validation. The adoption of these standardized safety frameworks enhances regulatory confidence and supports the consistent and responsible use of LAB in modern fermented food production.

9 Advances in research and industrial applications

The application of LAB, particularly Lactobacillus spp., in food fermentation has undergone a substantial shift over the past decade (Ayivi et al., 2020). Traditional empirically driven fermentation practices are increasingly being replaced by knowledge-based, precision-guided systems that integrate molecular biology, bioinformatics and advanced fermentation engineering (Abd-Elsalam and Lim, 2021). This transition has enabled a more comprehensive understanding of strain-specific functionality, ecological adaptation and performance under industrial conditions (Sun et al., 2025). As a result, Lactobacillus-based fermentations are now being optimized not only for preservation and sensory quality but also for nutritional enhancement, functional efficacy and process sustainability (Katu et al., 2025).

The convergence of systems biology with industrial biotechnology has also facilitated the rational design of fermentation processes tailored to specific food matrices, including dairy, cereals, vegetables and emerging plant-based alternatives. These advances have significantly improved fermentation predictability, product consistency and scalability, while expanding the scope of fermented functional foods (Singh and Kumar, 2025). Recent advances in Lactobacillus research and industrial applications are summarized in Table 3.

Table 3. Recent advances in Lactobacillus research and industrial applications.

9.1 Omics-based technologies for strain selection

The integration of multi-omics technologies, encompassing genomics, transcriptomics, proteomics and metabolomics, has revolutionized contemporary Lactobacillus research and industrial strain development (Liu et al., 2025a). The major challenges associated with Lactobacillus-based fermentation and the corresponding emerging solutions are summarized in Table 4. Whole-genome sequencing is now routinely employed to elucidate strain-level diversity, providing detailed insights into genetic determinants of carbohydrate metabolism, acid tolerance, stress response and biosynthetic pathways for bacteriocins, vitamins, exopolysaccharides and other bioactive compounds (Verma et al., 2025).

Table 4. Major challenges and emerging solutions in Lactobacillus-based fermentation.

Genomic data enable the early identification of technologically desirable traits while simultaneously supporting safety assessment through the detection of virulence-associated genes or transferable antibiotic resistance elements (Fahy et al., 2024). Transcriptomic analyses further refine this understanding by revealing dynamic gene expression patterns during fermentation, highlighting how Lactobacillus strains adapt to fluctuating pH, osmotic pressure, oxygen exposure and nutrient availability (Liu et al., 2025b). These studies have been particularly informative in explaining strain-dependent differences in fermentation kinetics and robustness.

Proteomic approaches complement genomic and transcriptomic data by identifying enzymes directly responsible for proteolysis, aroma compound formation, antimicrobial activity and stress adaptation (Mbye et al., 2020). In parallel, metabolomics provides a detailed biochemical profile of fermentation end-products, including organic acids, volatile compounds and health-associated metabolites (Liu et al., 2025b). This integrative approach establishes a direct link between microbial metabolism and food quality attributes, enabling predictive modeling of fermentation outcomes. Together, these omics tools support rational strain selection, replacing traditional trial-and-error approaches with predictive models that improve fermentation efficiency, product consistency, and functional performance (Jiang et al., 2025).

9.2 Co-culture fermentation strategies

Although single-strain fermentations offer a high degree of control, they often fail to replicate the metabolic complexity and sensory richness characteristic of traditional fermented foods. Consequently, co-culture and multi-species fermentation systems have gained increasing attention in both research and industrial applications (An et al., 2023). In these systems, Lactobacillus strains interact synergistically with yeasts, Bifidobacteria, or other LAB, resulting in more balanced acidification, improved substrate utilization and enhanced microbial stability (Cheng et al., 2025). Metabolic interactions in co-cultures frequently involve cross-feeding mechanisms, where one microorganism supplies essential nutrients such as amino acids, peptides, vitamins, or growth factors that support the growth and metabolic activity of another (Li et al., 2025). In cereal- and plant-based fermentations, such interactions have been shown to improve mineral bioavailability, accelerate phytate degradation and enhance the formation of desirable flavor and aroma compounds (Thivya et al., 2025).

Co-culture strategies are increasingly being optimized for sourdough fermentation, fermented beverages and novel plant-based dairy alternatives, where microbial synergy plays a critical role in texture development, sensory complexity and functional value (Dhiman et al., 2025). Advances in microbial ecology and modeling allow better prediction and control of co-culture dynamics, improving reproducibility and industrial applicability.

9.3 Starter culture development and strain improvement

Modern starter culture development places strong emphasis on strain robustness, adaptability and functional specificity. Rather than relying on genetically modified organisms, the food industry increasingly employs adaptive laboratory evolution, stress preconditioning and selective breeding to enhance desirable technological traits (Yadav et al., 2024). These approaches improve tolerance to acid stress, bile salts, osmotic pressure, freeze-drying and oxygen exposure, the key parameters influencing industrial processing, storage stability and product shelf life (Wang and Zhong, 2024).

Functional starter cultures are also being designed to deliver targeted outcomes, such as enhanced proteolysis for flavor development, increased vitamin synthesis for nutritional enrichment, or elevated exopolysaccharide production to improve texture and mouthfeel (Anumudu et al., 2024). Mixed-strain starters that combine complementary metabolic capabilities have demonstrated superior performance under variable processing conditions, offering greater resilience and fermentation reliability (Zhao et al., 2025). The growing demands for clean-label and minimally a processed food have further accelerated the adoption of naturally optimized starter cultures that align with consumer expectations while maintaining industrial efficiency (Inguglia et al., 2023).

9.4 Encapsulation and advanced delivery systems

Maintaining Lactobacillus viability throughout processing, storage and gastrointestinal transit remains a critical challenge, particularly for functional and probiotic fermented foods. Encapsulation and advanced delivery systems have emerged as effective strategies to protect bacterial cells from thermal, oxidative and acidic stresses encountered during food manufacturing and digestion (Singh et al., 2022). Encapsulation matrices such as alginate, chitosan, starch derivatives, whey proteins and lipid-based carriers provide physical protection and enhance microbial stability without adversely affecting sensory properties. These systems also enable controlled release of viable cells in the intestinal environment, thereby, improving probiotic efficacy and functional reliability (Zabot et al., 2022).

Recent developments focus on multilayer and nano-encapsulation technologies, which offer improved barrier properties, higher encapsulation efficiency and greater stability during long-term storage. As these technologies continue to mature, they are expected to play a pivotal role in the next generation of functional fermented foods and beverages (Kumari et al., 2025).

10 Challenges and regulatory considerations

Despite substantial advances in microbial characterization, fermentation control and functional food development, the industrial application of Lactobacillus in fermented foods continues to face a complex set of scientific, technological and regulatory challenges (Uhegwu and Anumudu, 2025). These challenges arise from the biological variability inherent to live microorganisms, the increasing demand for standardized and scalable production systems, and the evolving regulatory expectations surrounding safety, efficacy and consumer transparency (Gupta et al., 2024). Addressing these constraints is essential to ensure the long-term sustainability, credibility and global acceptance of Lactobacillus-driven fermented foods.

10.1 Consistency, stability and scale-up issues

Achieving reproducible fermentation outcomes during industrial scale-up remains one of the most persistent challenges in Lactobacillus-based food production. Variability in raw materials particularly in cereal, vegetable and plant-based substrates can significantly influence microbial growth dynamics, metabolic activity and end-product quality (Qiao et al., 2025). Factors such as carbohydrate composition, mineral availability, buffering capacity and the presence of inhibitory compounds often vary between batches, affecting fermentation kinetics and acidification patterns (Sokra et al., 2026).

Moreover, environmental parameters including temperature gradients, oxygen exposure and shear stress differ substantially between laboratory, pilot and industrial scales (Liu et al., 2026). Even well-characterized Lactobacillus strains may exhibit altered metabolic behavior under these conditions, resulting in deviations in organic acid production, flavor compound formation, texture development and microbial stability (Petka and Walczycka, 2026). In mixed or spontaneous fermentation, microbial competition further complicates process control, as indigenous microbiota may out compete starter cultures or introduce undesirable metabolic activities (Sawant et al., 2025).

To mitigate these issues, the industry increasingly relies on precision starter culture formulations, real-time process monitoring and automated fermentation control systems. The integration of predictive modeling and digital fermentation platforms is emerging as a promising approach to anticipate microbial behavior, reduce batch-to-batch variability and enhance overall process robustness (Yee et al., 2025).

10.2 Safety assessment and antibiotic resistance

Although Lactobacillus species are widely recognized for their long history of safe use in foods, heightened regulatory scrutiny has emerged in recent years due to growing concerns surrounding antibiotic resistance (Muteeb et al., 2023). Advances in whole-genome sequencing have revealed that certain strains harbor intrinsic or acquired resistance genes, some of which may be transferable under specific conditions (Matsumura et al., 2025). This has prompted regulatory authorities to adopt a more conservative, science-driven approach to safety evaluation.

Current safety assessments increasingly emphasize strain-level characterization rather than species-level classification. This includes genomic confirmation of the absence of virulence factors, mobile genetic elements and transferable antibiotic resistance determinants, as well as phenotypic validation of antimicrobial susceptibility profiles (Elbehiry and Marzouk, 2025). While this approach represents a significant advancement in risk assessment and consumer protection, it also introduces additional costs, technical requirements and regulatory complexity for product developers and manufacturers (Gupta et al., 2024). The need for transparent safety documentation is particularly critical for products marketed with probiotic or health-related claims, where consumer exposure is frequent and long-term (Abbasi et al., 2025).

10.3 Regulatory approval and labelling frameworks

Regulatory frameworks governing fermented foods and probiotics remain highly fragmented across global markets, creating challenges for international product development and commercialization (Mukherjee et al., 2022). Differences in safety classification systems, approval pathways and labelling requirements can result in inconsistent regulatory outcomes for the same Lactobacillus strain or product formulation. For example, strains considered acceptable in one jurisdiction may require extensive additional evaluation in another (Yunes et al., 2022).

In parallel, regulatory authorities are imposing stricter requirements for health and functional claims associated with fermented foods. Claims related to probiotic efficacy, immune modulation, digestive health, or metabolic benefits increasingly demand robust clinical, mechanistic, or biomarker-based evidence (Yuan et al., 2025). This has raised the scientific threshold for substantiating functional claims, necessitating greater investment in interdisciplinary research, human intervention studies and standardized evaluation protocols (Zhang et al., 2025).

Clear and accurate labelling has also become a focal point of regulatory oversight, particularly regarding strain identification, viable cell counts at the end of shelf life and the distinction between live cultures and probiotic claims (Liang et al., 2024).

10.4 Personalized nutrition and next-generation fermented foods

Emerging evidence indicates that the health effects of Lactobacillus-based fermented foods are strongly influenced by individual-specific factors, including gut microbiota composition, dietary habits, age and metabolic status (Pandey and Yadav, 2025). This recognition has accelerated interest in personalized nutrition, where fermented foods are designed or selected to match specific consumer profiles or health objectives (Ajayeoba and Ijabadeniyi, 2025).

While this concept holds significant promise, its translation into commercially viable products faces substantial obstacles. Challenges related to scalability, cost-effectiveness, regulatory approval, data privacy and consumer education currently limit widespread adoption (Nascimento and Barros, 2025). Moreover, regulatory frameworks have yet to fully accommodate personalized or microbiome-informed food products, creating uncertainty for developers. Bridging the gap between personalized microbial science and mass-scale food production represents a critical frontier in food biotechnology (Siddiqui et al., 2023). Future progress will depend on the development of flexible production systems, evidence-based personalization strategies and harmonized regulatory guidelines that balance innovation with safety and consumer trust.

11 Conclusion

LAB, with Lactobacillus spp. at the forefront, remains indispensable microorganisms in food fermentation due to their multifaceted roles in preservation, flavor development, nutritional enhancement and promotion of human health. Recent progress in omics technologies, metabolic engineering and fermentation science has significantly expanded the understanding and application of LAB in functional and therapeutic foods. Despite these advances, several scientific and industrial challenges still limit their broader application and commercialization. Future research should emphasize strain-level validation to accurately determine probiotic efficacy, genetic stability and host-specific responses. The establishment of harmonized international safety and regulatory frameworks is also necessary to ensure standardized evaluation, quality control and reliable health claims for LAB-based products. Moreover, greater clinical evidence derived from large-scale, long-term human studies is required to substantiate the therapeutic potential of fermented foods and probiotics. Another important area of focus is the development of scalable, cost-effective and sustainable plant-based fermentation systems capable of meeting increasing consumer demand for non-dairy functional products. In addition, advanced process control strategies involving precision fermentation, biosensors and real-time monitoring technologies should be explored to improve product consistency, microbial viability and industrial reproducibility. Collectively, continued interdisciplinary research integrating microbiology, biotechnology, food engineering and clinical sciences will be essential for developing next-generation LAB-fermented foods that support human health, sustainability and future global food security.

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Footnotes

Edited by: Teresa Semedo-Lemsaddek, University of Lisbon, Portugal

Reviewed by: Catarina Prista, University of Lisbon, Portugal

Juliana Aparecida Correia Bento, Federal University of Mato Grosso, Brazil