Review

Comprehensive Review of Strategies for Lactic Acid Bacteria

Production and Metabolite Enhancement in Probiotic Cultures:
Multifunctional Applications in Functional Foods
Jiun Shen Loo 1 , Siti Nur Hazwani Oslan 1,2, * , Nur Anis Safiah Mokshin 1 , Rafidah Othman 3 , Zarina Amin 4 ,
Wipawee Dejtisakdi 5 , Asep Awaludin Prihanto 6 and Joo Shun Tan 7

1 Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, Jalan UMS, Kota Kinabalu 88400, Sabah,
Malaysia; [email protected] (J.S.L.); [email protected] (N.A.S.M.)
2 Food Security Research Laboratory, Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, Jalan
UMS, Kota Kinabalu 88400, Sabah, Malaysia
3 Borneo Marine Research Institute, Higher Institution Centers of Excellence, Universiti Malaysia Sabah, Jalan
UMS, Kota Kinabalu 88400, Sabah, Malaysia; [email protected]
4 Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, Kota Kinabalu 88400, Sabah,
Malaysia; [email protected]
5 Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, 1 Chalong
Krung Rd., Ladkrabang, Bangkok 10250, Thailand; [email protected]
6 Department Fishery Product Technology, Faculty of Fisheries and Marine Science, Brawijaya University,
Malang 65145, East Java, Indonesia; [email protected]
7 School of Industrial Technology, Universiti Sains Malaysia, Gelugor 11800, Pulau Pinang, Malaysia;
[email protected]
* Correspondence: [email protected]

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, pharma-
Academic Editor: Nikos
ceutical, and aquaculture industries. However, the high production cost remains a major
G. Chorianopoulos challenge, necessitating cost-effective media formulations and bioprocess optimization.
Received: 14 February 2025
This review explores strategies for maximizing LAB yields and functionality through the
Revised: 27 March 2025 precision control of key cultivation parameters, including temperature, pH, and agitation
Accepted: 2 April 2025 speed, ensuring probiotic viability in compliance with regulatory standards (≥106 CFU/g
Published: 24 April 2025 or mL). Furthermore, advances in metabolic engineering, synthetic biology, and the uti-
Citation: Loo, J.S.; Oslan, S.N.H.; lization of agro-industrial by-products are driving cost-efficient and eco-friendly LAB
Mokshin, N.A.S.; Othman, R.; Amin, production. By integrating scalable fermentation technologies with sustainable resource
Z.; Dejtisakdi, W.; Prihanto, A.A.; Tan,
management, LAB have the potential to bridge the gap between food security, environmen-
J.S. Comprehensive Review of
tal sustainability, and biotechnological innovation. This review provides a comprehensive
Strategies for Lactic Acid Bacteria
Production and Metabolite
overview of recent advances in LAB cultivation and bioprocess optimization, ensuring
Enhancement in Probiotic Cultures: high-quality probiotic production for diverse industrial applications.
Multifunctional Applications in
Functional Foods. Fermentation 2025, Keywords: lactic acid bacteria; probiotics; metabolite; production; functional food industries
11, 241. https://doi.org/10.3390/
fermentation11050241

Copyright: © 2025 by the authors.

Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article
The International Scientific Association for Probiotics and Prebiotics (ISAPP) now
distributed under the terms and
defines probiotics as “live microorganisms that, when administered in adequate amounts,
conditions of the Creative Commons
Attribution (CC BY) license
confer a health benefit on the host”, a slightly revised definition from the original one given
(https://creativecommons.org/ by the WHO in 2001 [1]. Probiotics have various benefits for human health, such as their
licenses/by/4.0/). ability to support gastrointestinal health and strengthen the immune system [2]. Today,

Fermentation 2025, 11, 241 https://doi.org/10.3390/fermentation11050241

Fermentation 2025, 11, 241 2 of 18

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 proce-
dures [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 comple-
mentary 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 antimi-
crobial 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 aqua-
culture 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
Fermentation 2025, 11, 241 3 of 18

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 produc-
tion, 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,
Fermentation 2025, 11, 241 4 of 18

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 tem-
perature 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.
Fermentation 2025, 11, 241 5 of 18

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 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.

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

2.67-fold increase in biomass yield
Molasses-based ◦ C, with optimized nutrient feeding
Fed-batch fermentation 37 pH 6.5 [24]
medium strategy of Lacticaseibacillus
rhamnosus
The highest lactic acid yield (0.46
g/g-TS) and productivity (278.1
mg/L h) include microbial species
Batch fermentation Food waste 37 ◦ C, pH 6.0 [33]
involved
Lactobacillus spp., Enterococcus spp.,
Bacillus spp., and Clostridium spp.
Fermentation 2025, 11, 241 6 of 18

Table 1. Cont.

Cultivation Mode Medium Conditions Remarks Reference

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

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
Fermentation 2025, 11, 241 7 of 18

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.
Fermentation 2025, 11, 241 8 of 18

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,
Fermentation 2025, 11, 241 9 of 18

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 pro-
teins 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 bac-
teriocins, 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 shows an overview
of the different types of metabolites that have been produced during cultivation for the
growth of LAB under different conditions.
Fermentation 2025, 11, 241 10 of 18

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

Strain Metabolites Remarks References

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

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 aerugi-
nosa 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 multi-
ple 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 microorgan-
Fermentation 2025, 11, 241 11 of 18

isms 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 im-
mune function [82]. Table 3 below provides a comprehensive summary of key metabolites
produced by LAB, their biological functions, and their applications across various indus-
tries. 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.

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

Metabolite Function Application References

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

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
Fermentation 2025, 11, 241 12 of 18

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.

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

Metabolites Function Application References

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

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 exopolysaccha-
rides (EPS), bacteriocins, vitamins, and bioactive peptides, making them essential for the
functional food industry.
However, optimizing their productivity and application requires overcoming key chal-
lenges. Enhancing metabolite production, particularly EPS and bacteriocins, is critical and
necessitates a deeper understanding of their biosynthetic pathways and structure–function
Fermentation 2025, 11, 241 13 of 18

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, includ-
ing the sustainable production of bioactive compounds with health-promoting effects [6].
Future efforts should focus on developing cost-effective and eco-friendly production meth-
ods, 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. Address-
ing 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 exopolysaccha-
rides, 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 circu-
lar 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.

Author Contributions: Conceptualization, S.N.H.O., J.S.L. and J.S.T.; resources, J.S.L., N.A.S.M. and
Z.A.; writing—original draft preparation, J.S.L., N.A.S.M. and S.N.H.O.; writing—review and editing,
W.D., A.A.P. and S.N.H.O.; visualization, R.O. and J.S.T. All authors have read and agreed to the
published version of the manuscript.

Funding: This work received financial support from the Malaysian Ministry of Higher Education
(FRGC031-2024).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The original contributions presented in this study are included in the
article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest: The authors declare no conflicts of interest.

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