Comprehensive Review of Strategies for Lactic Acid Bacteria Production and Metabolite Enhancement in Probiotic Cultures: Multifunctional Applications in Functional Foods

Abstract

Lactic acid bacteria (LAB) play a crucial role in probiotics, functional foods, and sustainable biotechnologies due to their ability to produce bioactive metabolites such as short-chain fatty acids, bacteriocins, vitamins, and exopolysaccharides. These metabolites aid in gut health, pathogen inhibition, and enhanced productivity in the food, pharmaceutical, and aquaculture industries. However, the high production cost remains a major challenge, necessitating cost-effective media formulations and bioprocess optimization. This review explores strategies for maximizing LAB yields and functionality through the precision control of key cultivation parameters, including temperature, pH, and agitation speed, ensuring probiotic viability in compliance with regulatory standards (≥10⁶ CFU/g or mL). Furthermore, advances in metabolic engineering, synthetic biology, and the utilization of agro-industrial by-products are driving cost-efficient and eco-friendly LAB production. By integrating scalable fermentation technologies with sustainable resource management, LAB have the potential to bridge the gap between food security, environmental sustainability, and biotechnological innovation. This review provides a comprehensive overview of recent advances in LAB cultivation and bioprocess optimization, ensuring high-quality probiotic production for diverse industrial applications.

1. Introduction

The International Scientific Association for Probiotics and Prebiotics (ISAPP) now defines probiotics as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”, a slightly revised definition from the original one given by the WHO in 2001 [1]. Probiotics have various benefits for human health, such as their ability to support gastrointestinal health and strengthen the immune system [2]. Today, the market for probiotics has grown significantly, and they are considered to be in high demand all over the world. Various probiotic bacteria are being investigated to produce goods with great health benefits and yet produced using low-cost fermentation procedures [3]. Probiotics, which are related to lactic acid bacteria (LAB) such as Lactobacillus and Bifidobacterium, are the most common probiotics used in the industry [4]. Lactobacillus species are commonly used as starter cultures for fermenting food products, particularly in the dairy industry. In contrast, Bifidobacterium species are primarily utilized as complementary or probiotic cultures rather than as primary starter cultures for fermentation. The selection of LAB as a starter culture has played a crucial role in shaping the characteristics of fermented products. Different LAB strains impart distinct attributes, influencing a product’s aroma, flavor, texture, and even resistance to bacteriophages [5]. Furthermore, LAB play a significant role in metabolic traits supporting fermentation, including sugar metabolism (glucose, lactose, and fructose), bile tolerance, protein hydrolysis, and antimicrobial activity [6]. They produce beneficial compounds like organic acids, antimicrobials, exopolysaccharides, short-chain fatty acids, amines, bacteriocins, and vitamins, though their metabolic characteristics vary across strains due to genetic makeup, growth conditions, and environmental adaptation [4,6]. For instance, Lactobacillus delbrueckii subsp. bulgaricus, used in yogurt, specializes in lactose metabolism, while Lactiplantibacillus plantarum, found in fermented vegetables, metabolizes diverse sugars [6]. Selecting strains with defined traits and optimizing fermentation parameters are crucial for ensuring product quality. On the other hand, LAB can be used to promote health in multiple applications, such as in aquaculture, by serving as probiotics [7]. Moreover, these bacteria are known for their beneficial effects on the gastrointestinal health of fish, their ability to inhibit pathogenic microorganisms, and their role in enhancing the overall health and productivity of aquaculture systems [7,8]. Moreover, LAB play a crucial role in livestock feed, particularly in ruminant health and production, by enhancing silage fermentation, improving nutrient bioavailability, and inhibiting spoilage microorganisms. The metabolic activities of LAB, including the production of organic acids, bacteriocins, and hydrolytic enzymes, contribute to improved feed preservation and digestibility, ultimately supporting animal growth and productivity [6]. Additionally, LAB supplementation has been shown to enhance rumen microbial balance, leading to improved fiber degradation and feed efficiency [8].

For instance, due to the growing global interest in probiotic functional foods all over the world, regulations and legislation are necessary to ensure these products are both safe and effective. The rising demand has driven the development of specific regulatory frameworks across different countries. A common requirement is that the viable probiotic count must remain at or above 10⁶ colony-forming units (CFU) per milliliter (mL) or gram (g) throughout the product’s shelf life [9]. Mass production of the LAB is a crucial step toward commercialization. To achieve this, it is essential to develop a cost-effective growth medium that supports high yields of LAB and the production of their bioactive metabolites. The cultivation of L. plantarum strains, for instance, has been evaluated for their ability to ferment milk and influence the fermentation characteristics, including the viable count, pH and titratable acidity, texture, aroma, and sensory profile of milk [10]. The comprehensive aim of this review is to examine strategies for optimizing the commercial production of LAB by developing cost-effective media and identifying critical production parameters such as temperature, pH, and agitation speed. This review also highlights the significance of efficient LAB production in driving the growth of the probiotic industry and ensuring the consistent availability of high-quality probiotic products and metabolites for diverse industrial applications.

In this review, a comprehensive literature search was conducted using databases such as PubMed, Scopus, and Web of Science. The search included publications from 2010 to 2024, using keywords such as ‘Lactic Acid Bacteria’, ‘probiotic fermentation’, ‘bioprocess optimization’, ‘metabolite production’, and ‘functional foods’. Only peer-reviewed articles, book chapters, and conference proceedings relevant to LAB cultivation, probiotic stability, and industrial applications were considered. Studies were selected based on their relevance to probiotic viability, cost-effective fermentation strategies, and emerging biotechnological advancements. This systematic approach ensured a broad yet focused synthesis of recent advances in LAB research.

2. Cultivation Mode for Probiotic Bacteria Production

Media conditions need to be maintained periodically, as they serve as a life-sustaining mechanism for probiotic cell proliferation and contribute to most of the high output of biomass production [3]. Traditional fermentation systems frequently result in decreased cell viability and overall probiotic efficiency bacteria due to accumulation of inhibitory by-product such as organic acids and other metabolites, can lower the pH and create unfavorable conditions for bacterial survival and replication [11]. A high concentration of organic acids in the fermentation broth was discovered to enhance the osmotic pressure of the culture medium, inhibiting cell proliferation [11,12]. To mitigate these adverse effects during fermentation, control of pH by adding NaOH was one of the common techniques [11].

In industries, bioreactors are widely used to produce a large scale of probiotics in an efficient method [12,13]. There were several studies on using different types of mode for cultivation of Lactobacillus strains. For instance, the developed bioprocess for Bacillus coagulans, using a modified, chemically defined medium and pH-control, significantly improved the sporulation efficiency, enabling industrial-scale production of probiotics and resulted in a sporulation efficiency of 80–90% in a 10 L bioreactor, facilitating upscaling and application to industrial scale [3]. In the study of Abedin et al., (2023) [4], both Bifidobacterium and Lactobacillus strains were cultivated in a large-scale bioreactor, showing improved efficiency in probiotic yield. Moreover, the membrane bioreactor had also been used to improve the peptide production of Lactobacillus helveticus strain [14]. The membrane bioreactor (MBR) offers significant potential for improving the production of lactic acid bacteria, enabling higher volumetric productivity and achieving elevated cell concentrations compared to traditional batch systems. Kuznetsov et al., (2017) [15], reported 130 cycles of lactic acid biosynthesis from glucose in a membrane reactor, the bioreactor achieved a specific productivity of 46 g of lactic acid per liter per hour, with a near 100% yield from glucose and lactic acid concentrations ranging between 100 and 120 g/L. However, the growth and growth rate during perfusion culture in the membrane reactor can be hindered by challenges such as gas bubble formation and membrane fouling [16].

In addition, Vafajoo et al., (2011) [17], demonstrated that the tanks-in-series model effectively simulates the dynamic performance of airlift bioreactors for lactic acid production, accounting for variables such as substrate concentration, air velocity, and air velocity gradient. However, L. plantarum was a facultative anaerobic bacterium, so the demand for oxygen was not the main concern during cultivation. To date, stirred tank bioreactors are among the most widely used systems for cell culture due to their scalability, efficient fluid mixing, and high oxygen transfer rates. These bioreactors can be customized with various types of impellers to suit different microbial needs [18]. Recently, in a stirred tank bioreactor, in batch experiments, the growth of L. plantarum BG112 with Camellia sinensis as a prebiotic showed an increase in the maximum specific growth rate and the affinity constant [19]. Moreover, the growth of L. plantarum BG24 was optimized in the original MRS (de Man, Rogosa, and Sharp) broth (pH of 5.7), the specific growth rate was 0.416 h⁻¹, the doubling time was 1.67 h, and the biomass productivity was 0.14 gL⁻¹ h⁻¹. However, in MRS broth (pH of 6.5) enriched with 5 g/L yeast extract, higher values were achieved: a specific growth rate of 0.483 h⁻¹, a doubling time of 1.43 h, and a biomass productivity of 0.17 gL⁻¹ h⁻¹ [20].

For instance, batch cultivation in 2L bioreactors under optimal conditions has proven to be an effective approach for the biomass production of probiotic B. bifidum. Moreover, high-cell-density fed-batch strategies combined with precise pH control have emerged as superior alternative strategies to enhance the biomass production of probiotic B. bifidum [21]. Therefore, Tang et al., (2021) [22], reported that fed-batch fermentation using a molasses-based medium coupled with a ramp-feeding strategy demonstrated cost-effective and significant success in producing Enterococcus faecium CW3801, a non-vancomycin resistant strain with promising industrial potential for probiotic applications. In addition, from the study of Beitel et al., (2020) [23], fed-batch bioreactor cultivations with a controlled pH, agitation, and inoculum size were applied, showing increased productivity of L. delbrueckii compared to batch fermentation. The fermentation using molasses and corn steep liquor offers an economical, sustainable approach for industrial-scale production of high-purity D(-) lactic acid. By utilizing cost-effective, agro-industrial by-products, this method reduces production costs and supports circular economy practices. Leveraging the metabolic efficiency of L. delbrueckii, it achieves high yields of D(-) lactic acid, widely used in food, pharmaceuticals, biodegradable polymers, and also aquaculture application. For instance, Lactobacillus rhamnosus GG ATCC 53,103 demonstrates significantly enhanced biomass production in fed-batch cultures compared to traditional batch cultures. Utilizing optimized nutrient feeding strategies, fed-batch cultivation achieves a 2.67-fold increase in biomass yield compared to growth in basal medium. This improvement is attributed to the controlled supply of essential nutrients, which prevents substrate depletion and minimizes the accumulation of inhibitory by-products during the fermentation process [24]. Following biomass formation, the probiotics were separated from the fermentation medium by centrifugation. This process was critical for isolating the probiotic cells and producing a concentrated probiotic solution [3]. The isolated probiotic cells were then processed further, including stabilization, formulation, and packaging. In stabilization, sucrose stabilizer was added to maintain stability of cells, cryoprotectants, and lyoprotectant [25].

3. Physiochemical Condition for Lactic Acid Bacteria Production in Different Types of Medium

Various studies have reported the optimization of the medium to grow LAB to enhance its production of metabolites such as lactic acid, bacteriocin, and exopolysaccharides [26]. Optimizing cultural and environmental factors is critical for mass production since such conditions have helped in achieving the necessary output. Different strains of LAB have different nutritional needs and growth conditions that suit them well. The optimal temperature and pH conditions for lactobacilli growth were 30–40 °C and a pH of 5.5–6.2, respectively. However, the Lactobacillus genus was diverse, and bacteria belonging to it can grow in temperatures ranging from 2 to 53 °C and a pH ranging from 4.5 to 6.5, with some strains growing in even lower pH [27]. Culture conditions and fermentation medium composition greatly affected the growth kinetics of Lactobacillus sp. bacteria, particularly the specific growth rate and lag phase duration. In order to save time and reduce production costs, various statistical tools were studied to optimize the diverse cultural condition of LAB [3]. From the study of Śliżewska and Chlebicz-Wójcik, (2020) [27], the one-factor-at-a-time (OFAT) method was applied to determine the optimum temperature and initial pH of the medium, the optimum temperature would be 37 °C and the initial pH would be 6.0 for a few Lactobacillus strains including Lacticaseibacillus paracasei, Lactiplantibacillus pentosus, Lactiplantibacillus plantarum, Limosilactobacillus reuteri, and Lacticaseibacillus rhamnosus. L. plantarum recorded a maximum production of exopolysaccharide with 27 °C and 100 rpm of agitation speed with 36 h of fermentation. In term of biomass production, the study of Choi et al., (2021) [26], showed that the optimum growth conditions for L. plantarum were 30 °C, a pH of 6.5, and 200 rpm of agitation speed in modified medium. From the results of Matejčeková et al., (2019) [28], the L. plantarum had an optimal growth temperature of 36.6 °C in MRS broth, 34.7 °C in milk, and 34.2 °C in lactose-free milk. In addition to the OFAT method, there are only a few studies using response surface methodology (RSM) to determine the optimum growth conditions of L plantarum. There are three common experiment designs in RSM, Plackett–Burmann Design (PBD), Box–Behnken Design (BBD), and central composite design (CCD) [13,29]. The PBD is mainly used to determine the main effects of the factors, and to screen the significant factors affecting the biomass production of L. plantarum [26]. In the study of Mathiyalagan et al., (2021) [30], cultural conditions such as pH, temperature, and incubation time were evaluated using PBD prior to BBD. The temperature and pH were identified as significant factors with 41.8 °C and a pH of 7.02. For instance, the CCD was considered as a more complex and suitable method to identify the significant effects and the interactions between the factors [29]. In the study by Hemalatha and Subathra Devi, (2022) [31], temperature and pH were identified as significant factors influencing the growth of L. plantarum and analyzed using CCD. The optimal growth conditions were determined to be a temperature of 40 °C, a pH of 6.0, and an inoculum size of 3%. Under these optimized conditions, riboflavin production after 24 h reached 12.33 mg/L, closely aligning with the expected value of 12.29 mg/L predicted by the RSM model. The optimized parameters resulted in a 3.66-fold increase in riboflavin yield within 24 h.

The study of Prema et al., (2024) [32], reported utilizing a Box–Behnken experimental design of RSM employed to determine the optimal conditions for antibacterial production by L. plantarum. The highest concentration of antibacterials was achieved at a temperature of 35 °C, a pH of 6.5, and an incubation time of 48 h. Under these optimized conditions, the antibacterial titer increased more than 10-fold compared to unoptimized conditions. The cultivation conditions of the fermentation medium in which L. plantarum are produced can impact growth kinetics’ characteristics such as specific growth rate and lag phase length, which was the time period during which bacteria adjust to new media but do not multiply [27].

Table 1. Summary of the recent studies on different cultivation condition used while using different types of medium as substrate at different conditions, which were able to increase biomass yield and productivity of metabolite LAB.

Cultivation Mode	Medium	Conditions	Remarks	Reference
Fed-batch fermentation	Molasses-based medium	37 °C, pH 6.5	2.67-fold increase in biomass yield with optimized nutrient feeding strategy of Lacticaseibacillus rhamnosus	[24]
Batch fermentation	Food waste	37 °C, pH 6.0	The highest lactic acid yield (0.46 g/g-TS) and productivity (278.1 mg/L h) include microbial species involved Lactobacillus spp., Enterococcus spp., Bacillus spp., and Clostridium spp.	[33]
Batch fermentation	Fruit and vegetable waste	37 °C, pH 4.0	Acidogenic fermentation of fruit and vegetable wastes can produce 10–20 g/L lactic acid involving Lactobacillus spp., Bacillus spp., and Clostridium spp.	[34]
Batch fermentation	MRS medium	35 °C, uncontrolled pH	Maximum lactic acid produced of 37.39 g/L, productivity 0.79 g/L·h, and yield of lactic acid 0.94 g/g of Lactobacillus spp.	[35]
Batch fermentation	Milk-based medium	37 °C, pH 6.5	The formation of important flavor compounds during the fermentation process and production of short-chain fatty acids in cultivation of Lacticaseibacillus casei	[36]
Batch fermentation	Soybean straw	30 °C, uncontrolled pH	Lactic acid productivity was increased by the use of soybean straw enzymatic hydrolysate on Lacticaseibacillus casei	[37]
Batch fermentation	Corn steep liquor	36 °C, pH 6.5	Lactic acid yield 11 g/L enhanced with optimized process control of Lacticaseibacillus casei	[38]
Fed-batch fermentation	Glycerol	Optimize temperature (35–45 °C), pH 6.5	Maximal lactic acid concentration of 90.4 g/L was obtained after 80 h fermentation, a productivity of 1.13 g/L/h, which is 1.2 times more than constant temperature Lacticaseibacillus casei	[35]
Solid-state fermentation	Agro-industrial waste	temperature, moisture content uncontrolled	Produced bioproduct and lactic acid yield using cost-effective substrates in cultivation of Lacticaseibacillus casei	[39]
Co-culture with yeast	Whey-based medium	30 °C, anaerobic conditions	Enhanced growth and metabolite production due to synergistic interactions, which involved of Lacticaseibacillus casei and Kluyveromyces marxianus	[40]
Microcarrier-based culture	Algal extract	32 °C, optimized agitation	High-density biomass and reduced substrate consumption during cultivation of Bifidobacterium longum and L. Plantarum in combination with Chlorella sorokiniana	[41]
Immobilized cell system	Microalgae hydrolysate	33 °C, continuous substrate feeding	Increased productivity of lactic acid reached 9.93 g/L/h when using microalgae hydrolysate as substrate in cultivation of L. Plantarum	[42]

summarizes recent studies on various cultivation conditions using different types of media as substrates, highlighting conditions that successfully enhanced biomass yield and metabolite productivity of LAB.

In industrial probiotic production, cost was a critical factor influencing the selection of cultivation media. The cultivation media should provide all the essential nutrients and growth conditions for probiotic bacteria to produce viable cells. The MRS medium was the commercial medium that consists of the essential nutrient to grow LAB while inhibiting undesirable bacterial growth [26]. Although the MRS medium had various benefits in terms of LAB biomass production, it was complex and expensive which made it less favorable for the use in large scale and commercial production [43]. Aside from MRS agar, M17 and modified MRS agar were also the alternative media to produce large amounts of LAB. However, these alternative choices were still high in cost as the chemical used should meet with food grading to ensure it was safe for human consumption [44]. While chemical media synthesis has been a conventional strategy for probiotic production, the use of renewable agricultural waste as a medium has gained interest due to its sustainability and cost-effectiveness [39]. However, the incorporation of agricultural waste into probiotic production raises concerns in terms of legislative, economic, and technological aspects. Aside from the concerns, low-cost substrate has been reported by many studies based on Lactobacillus biomass production such as whey, maize starch, cane molasses, and agro-industrial leftovers [3]. For instances, based on Śliżewska and Chlebicz-Wójcik (2020) [27], the cost-effective use of wheat, barley, maize, and rye flours was developed to support the growth and production of cell biomass for various lactic acid bacteria strains while production costs were decreased. Meanwhile, the egg white hydrolysates have been proved to be an effective fermentation medium for probiotic LAB, resulting in increased cell biomass production and lactic acid, with a formulation with 0.5% fructose and 1.0% molasses enhancing growth and cell biomass production in all strains, except L. gasseri CRL1421, which thrived in 1.5% corn syrup [45]. Developing cost-effective culture media with alternative ingredients and by-products reduces production costs while sustaining or enhancing LAB growth and metabolite production, supporting large-scale applications.

Effect of Milk-Based Medium as Substrate on Cultivation Lactobacillus sp.

Due to the increase in health-conscious consumers, probiotic functional foods have gained huge attention from society. Milk serves as a suitable substrate for the cultivation of Lactobacillus species, offering a cost-effective and viable medium for probiotic propagation, especially when optimized for specific strain requirements [46]. A study by Guo et al., (2023) [47], identified Lactobacillus as a core microbiota in bovine milk with beneficial properties. This finding showed its ability to survive under normal milk conditions. In bovine milk, lactose is the primary carbon source which is composed of galactose and glucose. A study from Wang et al., (2023) [48], indicated that L. plantarum can use lactose as a carbon source for growth. In addition to the carbon sources, nitrogen sources are also an important factor for the growth of L. plantarum. In the bovine milk, the primary nitrogen sources are the protein named casein and whey proteins [49]. Both casein and whey proteins have been proved to be able to enhance the growth of various members of the Lactobacillus genera [50,51]. Media composition was important as it could influence functionality, metabolic activity, biomass growth, cell viability, and lactic acid production. Bovine milk is considered as the best source of fats, protein, and micronutrients when compared to other dairy products [52], especially the amino acids that are considered important such as isoleucine, leucine, valine, tyrosine, methionine, and phenylalanine [44]. Among bovine milk, there are several milk types like full cream, low-fat, and skimmed. Each of these milk types had differences in terms of the nutritional content. In terms of both carbohydrates and protein, skimmed milk has the highest amount while full cream milk has the lowest amount [52]. A study of Abdulrazzaq and Khalil, (2022) [53], demonstrated the growth of L. acidophilus with the skimmed milk medium and showed it could support the growth of Lactobacillus species. To date, studies using milk as the main medium are limited and this might be due to the limited studies on the optimization of milk medium as a cultivation medium for probiotic production. Meanwhile, there is another milk type available in the market; fresh milk which has the same nutritional contents as full cream milk but with higher pricing, shorter shelf life, and required chilled storage conditions. Other than nutrient supplements, origin growth conditions are important to ensure cell viability. Nath et al., (2020) [54], reported that fermented milk can serve as a carrier for L. plantarum, ensuring high cell viability, which is crucial when the strain is used as a starter culture. One of the reasons might be because the milk originally had a pH level of about 6.7 which was in the optimum range of growth for L. plantarum. With the evidence shown by several studies, bovine milk was believed to be the potential low-cost alternative medium to grow L. plantarum. Recently, Zhang et al., (2024) [44], reported that the growth of Lactobacillus strains, including L. gasseri and L. plantarum, in milk requires supplementation with essential nutrients such as peptides, amino acids, and yeast extract. L. gasseri, in particular, exhibits optimal growth when peptides are provided as a nitrogen source rather than intact proteins or free amino acids.

4. Metabolism of LAB During Fermentation by Formation of Lactic Acid

Lactic acid is a by-product produced continuously during the fermentation and growth of the LAB. In the food sector, particularly the dairy industry, everything starts with raw milk, and LAB plays a dynamic role in its transformation into dairy products such as yogurt, cheese, and fermented milk. During fermentation, LAB converts lactose into lactic acid. Such a biochemical conversion is desirable as it serves multiple purposes in producing a desirable final product [55]. The demand for lactic acid has been constantly growing, but production has not improved [56]. Lactic acid has been proved not only in improving the nutritional value of food but also directly benefits human health. In the metabolism of LAB, there are two types of fermentation involved, homolactic fermentation and heterolactic fermentation [6]. The type of lactic acid fermentation that is carried out by a particular strain of Lactobacillus sp. is determined by its genetic makeup. Homofermentative LAB is preferred for commercial lactic acid production due to its high yield and optical purity of lactic acid [57]. In contrast, heterofermentative LAB is less suitable for commercial production due to its lower lactic acid production and the production of carbon dioxide during fermentation [58]. For example, in the dairy industry, heterofermentative LAB is not commonly used as a starter culture because the carbon dioxide it produces could cause problems such as bloated packaging and cracks in dairy products [57]. Nowadays, the most common practice in the industry is the use of fermentation strategies to improve the yield and purity of lactic acid.

To produce lactic acid, growth media is the important factor. Milk has been proved to be a reliable growth media for L. plantarum to grow and produce lactic acid. The L. plantarum that was fermented in skimmed milk for 12 h achieved a final pH level of 4.32 and titratable acidity of 0.74% [59]. Based on Fonseca et al., (2020) [60], it was demonstrated that L. plantarum is able to produce lactic acid with the milk media and achieve a titratable acidity of 1.19% at 25 °C when fermented for 60 days. The biggest challenges in the industry in achieving the economic production of lactic acid are due to the raw material costs of the cultivation medium. Therefore, cost-effective alternative media were the main field to be studied, especially utilizing dairy products or waste to optimize the production of lactic acid.

5. Metabolite Formation of LAB in Functional Food as Antimicrobial Agent

Other than organic acid (lactic and acetic acids), LAB could produce metabolites such as aromatic compounds, viscous exopolysaccharides (EPS), and bacteriocins [55]. These metabolites possessed characteristics that influence the nutritional and sensory properties (texture, color, flavor, and aroma) to fermented foods [61]. During production of these metabolites, the bacterial strain used, medium conditions, incubation period, temperature, and initial pH were the crucial factors [62]. Other than lactic acid and bacteriocin, the EPS produced by LAB has gained significant attention from both researchers and manufacturers [63]. EPS produced by LAB naturally could not only improve the rheology of products in the dairy industry, but it also proved to have health benefits [64]. The optimum cultivation conditions to produce EPS identified by the existing studies were a 35.6 °C fermentation temperature, an initial pH of 7.4, and 6.4% of inoculation size [48].

Moreover, LAB was known for its antimicrobial properties, and it was achieved through its metabolites, bacteriocins. Bacteriocins were well-known antibacterial proteins synthesized by bacterial ribosomes, and either killed or inhibited the growth of pathogens [65]. As a result, the use of LAB bacteriocins in foods has expanded significantly especially in fermented products, where it could replace the use of chemical preservatives to improve the shelf-life and safety of food products [66]. Bacteriocins are well-known low-molecular peptides with low oral toxicity in humans and show promising applicability in the food industry as bio-preservatives [6]. There are many studies that have proved that bacteriocins provide protective effects against pathogen in different types of foods such as fermented dairy products, bakery products, and vegetables [65,66]. Bacteriocins could have both a bactericidal or bacteriostatic effect which cause cell death by blocking cell wall production or disrupting the membrane by forming pores [67]. The reason that bacteriocins were widely used in the food industry was because of its properties such as heat stability, wide pH tolerance, and resistance to enzymes. Zangeneh et al., (2020) [65], stated that the bacteriocin produced by L. plantarum could withstand a wide pH range (2–10), high heat process (60–121 °C), and considered resistant to enzymes (pepsin, trypsin, and proteinase K). The activity of the bacteriocin did not show a significant difference compared to the control. Bacteriocins produced by LAB could have various forms and based on their structure and properties, they can be classified into three different categories: Class I (antibiotics), Class II, and Class III [68]. Although there are various types of bacteriocins, nisin and pediocin are the only types that are commercially available and used in the dairy industry [69]. Nisin is especially effective in dairy products but less effective in meat products as the lipids from meat products could affect the efficacy of nisin [70]. Environmental factors also act as important factors that affect not only production but also the efficacy of bacteriocin. For instance, Vajid and Vijaya (2022) [71] stated that the optimal condition to produce bacteriocin by L. plantarum was 36 °C at a pH of 6.5 using 1% inoculum size. While the maximal bacteriocin activity was observed when the temperature was at 30 °C and with an initial pH of 6.0 [72].

The activity of bacteriocins produced by L. plantarum has been proved by several studies through different cultivation media [65,73]. Bacteriocins produced by L. plantarum that isolated from traditional sourdough exerted an inhibitory effect against the growth of S. aureus and E. coli [65]. The L. plantarum strains isolated from sheep milk cheese showed antibacterial activities against pathogens including E. coli, P. aeruginosa, and S. aureus [73]. The study of Lim et al. (2019) [59] demonstrated that a clear inhibition zone was formed by L. plantarum against S. aureus and E. coli. The bacteriocins produced from L. plantarum have been proved to be active against the common pathogens in food products. However, the cost-effective media remained as the major challenge to produce high density and effective bacteriocin which could be used as bio-preservatives. Therefore,

Table 2. The overview of metabolite produced during cultivation for growth of LAB.

Strain	Metabolites	Remarks	References
Lactobacillus spp., Streptococcus thermophilus zlw TM11, Lactococcus lactis, Lactobacillus delbrueckii subsp. bulgaricus	Viscous exopolysaccharides	Strain cultivated at a temperature of 35.6 °C, initial pH of 7.4, and 6.4% of inoculation size applied	[74]
Lactiplantibacillus plantarum, Leuconostoc lactis	Bacteriocins; leucosin	36 °C at a pH of 6.5 using 1% inoculum size	[74]
Lactiplantibacillus plantarum, Leuconostoc lactis	Riboflavin	After 20 h of fermentation at 37 °C, the RYG-YYG-9049-M10 strain LAB was able to enhance the amount of riboflavin in fermented soy milk by ten times.	[75]
Lactobacillus sp., Limosilactobacillus reuteri	3-hydroxypropionic acid	Produced through glycerol metabolism pathway	[76]
Lactiplantibacillus plantarum, Pediococcus acidilactiti, and Streptococcus spp.	Succinic acid	Undertaken in wet and spray-dried fish-based raw material for 3 weeks under room temperature (25 °C)	[77]
Levilactobacillus brevis, Limosilactobacillus fermentum and Lactiplantibacillus plantarum	Phenolic acid	Produced through decarboxylase and reductase	[78]

shows an overview of the different types of metabolites that have been produced during cultivation for the growth of LAB under different conditions.

6. Metabolites in Lactic Acid Bacteria Functional in Food, Pharmaceuticals and Aquaculture Applications

Lactic acid bacteria (LAB) are versatile microorganisms known for their ability to produce a wide range of bioactive compounds with applications in food production, pharmaceuticals, and aquaculture. Their metabolites, including lactic acid, bacteriocins, exopolysaccharides, and short-chain fatty acids, play crucial roles in enhancing food safety, improving human health, and promoting sustainable aquaculture practices. L. casei is a lactic acid-producing strain commonly used in fermentation processes. Similarly, L. lactis produces bacteriocins such as nisin, which are effective in treating Clostridium difficile-associated diarrhea and serve as natural preservatives in food and beverages by inhibiting the growth of harmful bacteria [79]. Pediococcus inopinatus, another bacteriocin-producing strain, has shown significant potential in reducing multidrug-resistant Pseudomonas aeruginosa growth and biofilm formation, as demonstrated in studies involving strains isolated from kimchi [80]. Additionally, B. bifidum produces lactic acid, acetic acid, and bacteriocins, which are valuable in the production of fermented dairy products, infant formulas, and dietary supplements. These LAB-derived postbiotics underline the versatile applications of bioactive compounds in improving food safety, human health, and nutrition.

The LAB are well known for their capacity to synthesize a diverse range of bioactive metabolites with significant industrial relevance. These metabolites contribute to multiple sectors, including food production, pharmaceuticals, and aquaculture, by enhancing product safety, improving quality, and providing functional benefits [81]. Their roles span antimicrobial activity, bio-preservation, probiotic effects, and immune modulation. Among the most notable metabolites produced by LAB are organic acids (such as lactic acid, acetic acid, and propionic acid), which lower pH and inhibit the growth of spoilage microorganisms and foodborne pathogens. Furthermore, LAB synthesize bioactive peptides derived from protein hydrolysis, which exhibit antihypertensive, antioxidant, and antimicrobial activities, making them valuable in functional foods and nutraceuticals [74]. Their ability to metabolize complex carbohydrates and enhance gut microbiota balance has further positioned them as key players in probiotic development, supporting gut health and immune function [82].

Table 3. The overview of metabolites from lactic acid bacteria: functions and applications in food production, pharmaceuticals, and aquaculture.

Metabolite	Function	Application	References
Lactic Acid	Lowers pH, preservative, flavor enhancer	Food preservation, dairy products, beverages	[75,83]
Vitamins (e.g., B12, K2)	Nutritional enhancement	Fortified foods, dietary supplements	[75,83]
Exopolysaccharides	Texture improvement, prebiotic effects	Dairy products, functional foods, aquaculture	[75,84]
Short-Chain Fatty Acids	Gut health, anti-inflammatory	Probiotics, functional foods, pharmaceuticals	[75,84]
Bacteriocins	Antimicrobial activity	Food safety, biopreservation, pharmaceuticals	[83,84]
γ-Aminobutyric Acid (GABA)	Neurotransmitter, stress relief	Functional foods, dietary supplements	[83,84]
Conjugated Linoleic Acid	Anti-carcinogenic, anti-obesity	Functional foods, dietary supplements	[84]
Hydrogen Peroxide	Antimicrobial activity	Food preservation, pharmaceuticals	[85]
Diacetyl	Flavor compound, antimicrobial	Dairy products, food flavoring	[85]
Reuterin	Broad-spectrum antimicrobial	Food preservation, pharmaceuticals, aquaculture	[86]

below provides a comprehensive summary of key metabolites produced by LAB, their biological functions, and their applications across various industries. This growing body of research underscores the importance of LAB as industrially significant microorganisms with far-reaching implications for food security, human health, and sustainable biotechnological advancements.

Furthermore, LAB generate short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, which promote gut health, improve nutrient absorption, and provide energy to gut epithelial cells [87]. Therefore, LAB play a crucial role in improving the digestibility of feed for production animals through the production of enzymes such as proteases, amylases, and cellulases, which enhance the breakdown of complex carbohydrates and proteins, increasing nutrient availability and absorption [88]. Additionally, LAB contribute to gut health by modulating intestinal microbiota, producing organic acids, and inhibiting pathogenic bacteria, thereby improving overall animal growth and productivity [86]. While LAB applications in aquaculture have been widely studied, their benefits in livestock and poultry nutrition should also be emphasized, as they contribute to enhanced feed efficiency and animal performance [85], and also, since LAB produce digestive enzymes, which enhance nutrient breakdown and improve the growth performance of aquatic species [88]. Essential vitamins like B12 and folate synthesized by LAB contribute to the health and immunity of these species [89]. Additionally, metabolites with antioxidant properties protect cells from oxidative stress, enhancing immune response and reducing mortality under stress conditions [90]. The proteolytic activity of certain LAB strains aids food digestion and improves nutrient utilization, particularly in larval stages [91]. These multifaceted functions highlight LAB’s potential in promoting sustainable aquaculture practices in

Table 4. Bioactive metabolites produced by lactic acid bacteria (LAB) and their functional roles in aquaculture.

Metabolites	Function	Application	References
Lactic Acid	The primary metabolite produced by LAB, which lowers the pH of the environment, inhibiting the growth of harmful pathogens.	Reduces the colonization of pathogenic bacteria in the gut of fish and shrimp.	[81]
Bacteriocins	Antimicrobial peptides that specifically target pathogenic bacteria.	Control of harmful bacteria such as Vibrio sp. and Staphylococcus aureus in aquaculture.	[81]
Exopolysaccharides (EPS)	Enhance water quality by forming biofilms and stabilizing the microbial community.	Improve water quality by reducing ammonia and other toxic compounds in water systems.	[83]
Short-Chain Fatty Acids (SCFAs)	LAB produce short-chain fatty acids such as acetate, propionate, and butyrate during fermentation.	Improve gut health, enhance nutrient absorption, and provide energy to gut epithelial cells.	[87]
Enzymes	Produced digestive enzymes such as proteases and amylases that help in the breakdown of nutrients.	Enhance the digestive efficiency of aquatic species, leading to better growth performance.	[88]
Vitamins	Essential vitamins such as B vitamins (e.g., B12, folate).	Improve overall health and immunity of aquatic species by providing essential micronutrients.	[89]
Antioxidants	Antioxidant properties that protect cells from oxidative stress.	Enhance the immune system of aquatic species and reduce mortality during stress conditions.	[90]
Proteolytic activities	LAB strains possess proteolytic activities.	Assist in situ food digestion and improve nutrient utilization in larval gut.	[91]

.

7. Future Perspectives on Lactic Acid Bacteria in the Bio-Economy

The bio-economy presents both opportunities and challenges for utilizing lactic acid bacteria in the production of bioactive metabolites for functional foods. LAB are widely recognized for their ability to synthesize valuable compounds such as exopolysaccharides (EPS), bacteriocins, vitamins, and bioactive peptides, making them essential for the functional food industry.

However, optimizing their productivity and application requires overcoming key challenges. Enhancing metabolite production, particularly EPS and bacteriocins, is critical and necessitates a deeper understanding of their biosynthetic pathways and structure–function relationships in food matrices [92,93]. The EPS, in particular, have gained attention for their pharmacological and nutraceutical potential due to their biocompatibility, non-toxicity, and biodegradability. Advanced metabolic engineering strategies, including CRISPR-Cas9 and synthetic biology, offer promising avenues to enhance LAB traits for producing value-added compounds and improving the nutritional properties of food products [4]. Beyond traditional fermentation, LAB hold significant potential in diverse applications, including the sustainable production of bioactive compounds with health-promoting effects [6]. Future efforts should focus on developing cost-effective and eco-friendly production methods, such as utilizing agro-industrial by-products like lignocellulosic biomass to support a circular bio-economy [94]. Additionally, advances in genomics and high-throughput screening technologies provide new opportunities to optimize LAB strains for industrial applications [95]. The increasing demand for functional foods with enhanced nutraceutical properties presents a substantial market opportunity for LAB-derived products. Addressing challenges related to productivity, metabolic engineering, and sustainability will be crucial for maximizing the potential of LAB in the bio-economy. By integrating cutting-edge biotechnological advancements and focusing on their health benefits, LAB can contribute significantly to sustainable food production and the expanding functional food industry.

8. Conclusions

Lactic acid bacteria (LAB) are integral to the expanding bio-economy, particularly in the production of bioactive metabolites for functional foods, pharmaceuticals, and aquaculture. Their capacity to synthesize valuable compounds such as exopolysaccharides, bacteriocins, and bioactive peptides presents significant industrial opportunities. However, optimizing metabolite production, enhancing strain performance, and ensuring cost-effective, sustainable processes remain critical challenges. Advances in metabolic engineering, synthetic biology, and bioprocess optimization will be essential in maximizing LAB’s industrial potential. By leveraging innovative biotechnological strategies and circular bio-economy principles, LAB-based production systems can drive the development of sustainable, high-value functional foods and nutraceuticals, contributing to global food security and human health.