In 2002, the joint FAO/WTO Expert Committee introduced a scientifically grounded definition of probiotics, describing them as living microorganisms that confer health benefits upon the host when ingested in adequate quantities (FAO/WHO, 2002). The most widely used probiotic strains belong to Lactobacillus spp., Bifidobacterium spp., Lactococcus spp., Leuconostoc spp., Streptococcus spp., and various species, with Lactobacillus spp being the most extensively studied. As a representative of probiotics, some Lactobacillus have good probiotic properties, including lowering intestinal pH, regulating intestinal flora, preventing lactose intolerance, and inhibiting the growth of harmful microbes in the dairy products (Garnier et al., 2020), meat products (Danielski et al., 2020), aquatic products (Aymerich et al., 2019), and fruit and vegetable products (Ma et al., 2019), among others. Furthermore, these bacteria play an important role in the fermentation processes of common foods, such as yogurt and kimchi (Mathur et al., 2020; Wang et al., 2021). To demonstrate efficient biological activities, a daily dose of probiotics of about 10⁸–10⁹ colony-forming units (CFU) is required during their passage through the gastrointestinal tract (Liu et al., 2019). Over time, methods to improve the stress resistance have evolved. These approaches include the use of improved strain protective agent (Sánchez et al., 2017), the addition of oligosaccharides (Dong et al., 2023), gene recombination technology (Wang et al., 2013a) and the encapsulation of probiotics in biofilms, referred to as fourth-generation probiotics (Salas-Jara et al., 2016). Both domestically and internationally, especially within the fermentation industry (Rosche et al., 2009), bioreactors-based biofilms are widely used in sewage treatment (Bao and Dai, 2013), biological fermentation (Cheng et al., 2011), and microbial fuel cells (Raganati et al., 2016).

Probiotics can form complex communities known as biofilms, which offer several advantages for microbial populations when facing various abiotic or biotic stress factors (Salas-Jara et al., 2016). The formation of biofilms allows bacteria to preferentially adhere to specific epithelium surfaces, such as the intestinal mucosa, prolonging and stabilizing their presence in the epithelium and helping to exclude pathogenic bacteria by competitive inhibition or steric hindrance (Saxelin et al., 2005). A key characteristic of biofilms is the formation of extracellular polymers, which provide mechanical stability and promote the formation of a microenvironment, which triggers quorum sensing and further regulates the maturation of the biofilm (Allison, 2003). A mature biofilm has stronger antibacterial activity and stress resistance (Lewis, 2001).

The formation of Lactobacillus biofilms is beneficial to the environmental adaptability and probiotic properties of Lactobacillus. Therefore, understanding the regulatory mechanisms of Lactobacillus biofilm formation and the biofilm’s characteristics contributes to the potential role of Lactobacillus on food fermentation industry and promoting human health. However, the relevant knowledge on this topic remains fragmented, highlighting the need for a review on the Lactobacillus biofilm and their practical applications.

The formation of bacterial biofilms is dependent on the regulation of signaling systems, including quorum sensing (QS), second messenger signaling system and cyclic adenosine phosphate-receptor protein (cAMP-CRP) signaling system (Liu et al., 2020; Li and Tian, 2012) (Figure 1). In the existing reports, the QS system is mainly involved in the formation and regulation of Lactobacillus biofilm. QS, also known as “autoinduction,” refers to the induction phenomenon in which bacteria employ self-inducing molecules to communicate and coordinate their group behavior (Papenfort and Bassler, 2016). As bacteria grow, they secrete a signal molecule sensing the surrounding environment. By detecting variations in the concentration of these signal molecules, bacteria can modulate the expression of related genes, thereby regulating associated behaviors. Consequently, interfering in bacterial QS signaling to either promote or inhibit the development of lactic acid bacteria (LAB) biofilms holds substantial significance in terms of enhancing intestinal immunity, promoting health, and elevating the quality of fermented food.

The adhesion of Lactobacillus to the surface is the first step for Lactobacillus to play a probiotic role. At the early stage of adhesion, whether or not the bacteria can attach to the cell surface is determined by the physicochemical properties of the bacterial surface, mainly by the properties of surface proteins. Bacterial initial adhesion and biofilm formation are controlled by different genes, and bacterial initial adhesion is an important prerequisite for biofilm formation (van Merode et al., 2006). For instance, the addition of mucin to MRS medium without glucose increased the biofilm formation ability of L. rhamnosus by 20% (Lebeer et al., 2007). At the later stage of adhesion, more extracellular matrix is secreted to the outside of the cell with the continuous proliferation of bacteria, so that the bacteria accumulate in large numbers, and finally form biofilm. The formation of Lactobacillus biofilm can greatly improve the adhesion ability of the bacteria. The extracellular matrix plays a key role in the later stage, especially exopolysaccharides. It had been reported in comparison to the wild-type L. rhamnosus, the mutant strain containing genes related to extracellular polysaccharide synthesis exhibited stronger biofilm formation ability (Lebeer et al., 2009).

The adhesion ability of Lactobacillus is related to its surface polysaccharides and membrane proteins. It had been found that L. plantarum WCFS1 had a similar QS system lamBDCA to Staphylococcus aureus. The adhesion of mutant lamA to glass surface is lower than that of wild-type strains, which is the first time that non-pathogenic agr systems have been found to encode self-inducible peptides and participate in the regulation of adhesion (Sturme et al., 2005). Because biofilm formation is closely related to the QS system mediated by the signaling molecule AI-2, it has been found that the signaling molecules of the QS system can influence the adhesion of LAB. The AI-2 signaling molecules produced by L. acidophilus NCFM can enhance the formation of biofilms and ultimately increase the adhesion to intestinal epithelial cells (Buck et al., 2009). Zhang et al. (2022) compared the effects of endogenous and synthetic AI-2 on the growth of L. sanfranciscensis. The synthetic AI-2 increased the cell density and cohesive force of L. sanfranciscensis. The adherence of luxS mutant L. acidophilus to Caco-2 decreased by 58% compared with the wild type (Buck et al., 2009). The ability of L. plantarum KLDS1.0391 to adhere to Caco-2 cells was drastically decreased by the luxS gene knockout, speculating that luxS/AI-2 mediated QS may affect the adhesion of LAB and regulate biofilm (Jia et al., 2018). Therefore, it is of great significance to utilize QS system to regulate the formation of Lactobacillus biofilms, thereby improving the adhesion ability of Lactobacillus and enhancing the probiotic effect of Lactobacillus.

Biofilm is a self-protective mechanism formed by microorganisms in an adverse environment. They exhibit a natural capacity for self-production of extracellular polymeric substances (EPS), which include exopolysaccharides, proteins, and extracellular DNA. These substances enhance their adhesion to surfaces, forming a thick protective barrier. In addition to providing a structural barrier, Lactobacillus also tend to survive in the form of biofilm when faced with adverse environmental stress, thus enhancing the stress resistance of the bacteria. The stress resistance of Lactobacillus biofilm to different environments is shown in Table 1. The protective effect of biofilm is thought to be mainly due to the fact that the extracellular polymers of biofilm shield the damage of toxic compounds, antibiotics and enzymes to the embedded bacteria, and the diffusion of harmful substances can be hindered by the extracellular polymers (Nguyen et al., 2022). Therefore, strains located deeper in the biofilm can be better protected. In addition, L. plantarum biofilms have been shown to withstand harsh conditions in fermented foods due to their robust EPS matrix, which protects against desiccation and nutrient depletion (Wang et al., 2021).

Recent research shows that when exposed to acidic environment, Lactobacillus activate a complex stress response that leads to significant physiological adaptations. This stress response often involves upregulation of acid tolerance mechanisms, including the F0F1-ATPase system and the production of chaperones that stabilize cellular proteins on low pH conditions (Zhang et al., 2022; Wang et al., 2021). This adaptation not only enhances survival but also improves intercellular communication and resource sharing through quorum sensing, particularly via the AI-2 signaling pathway (Papadimitriou et al., 2016). Furthermore, the physiological changes triggered by acidic stress responses in Lactobacillus, such as modifications in the cell surface properties and membrane liquid composition have been shown to provide cross-protection against additional stresses, like oxidative and osmotic pressures (Papadimitriou et al., 2016).

As an important regulatory mechanism involved in bacterial metabolism, it is of utmost importance to study the stress mechanism of LAB under different environmental stresses from the LuxS/AI-2 mediated QS system. At present, some progress has been made in this aspect. A total of 72 genes showed differential expression in L. reuteri after being treated in the acidic environment, among which the expression level was luxS gene up-regulated by approximately 3–4 times (Wall et al., 2007). The luxS gene expression was significantly up-regulated in all three strains of L. plantarum F, Lactobacillus sakei L4 and L. plantarum R, in which transcription level was positively associated with salt concentration under high nitrate stress, but differentially among the three strains (Lin et al., 2015). Transcription level of the luxS gene also appear to be upregulated under nutrient deficient conditions (Gu et al., 2018). Given that the luxS gene regulates the synthesis of the signaling molecule AI-2, the influence of AI-2 on the stress resistance of LAB exhibits a certain strain specificity and concentration dependence. Adding the exogenous signaling molecule AI-2 can improve the bile salt tolerance of L. sanfranciscensis (Zhang et al., 2022). The exogenous addition of AI-2 by Gu et al. (2021) significantly improved the tolerance of L. plantarum to bile salts but did not improve the tolerance to acid. Overexpression of luxS gene promoted AI-2 synthesis and enhanced the heat tolerance of L. paraplantarum L-ZS9 (Liu et al., 2018). Meanwhile, the deletion of luxS gene will have some effect on the stress resistance and normal physiological function of LAB. Bove et al. (2012) knocked out the luxS gene of L. plantarum KLDS1.0391 and showed no significant difference in the number of viable cells compared to the wild-type strain under normal culture conditions, but the acid and bile salt tolerance of the strain with the deletion of luxS gene was significantly reduced under high acid and high bile salt environments.

In addition to the luxS gene, other genes related to the QS system were also associated with the stress resistance of strains, such as the gene ftsH encoding FtsH protein in the AAA (ATPase associated with different cellular activities) family, a ubiquitous membrane-bound, ATP-dependent metalloproteinase. Some studies have shown that the mutation of ftsH caused the loss of FtsH protease in L. plantarum WCFS1, which resulted in poor heat and salt tolerance of the mutant and reduced the ability to form biofilm. On the contrary, the overexpression of FtsH enhanced the heat tolerance, salt tolerance and biofilm formation ability of L. plantarum (Del Pozo, 2018). In the L. sanfranciscensis, the addition of artificial AI-2, can up-regulate the expression of ftsH gene, promoting biofilm formation and improving the bile salt tolerance (Zhang et al., 2022). In addition, in the mixed-species biofilms formed by Streptococcus thermophilus with Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus equinosus and L. paracasei, have been shown to offer better microbial protection due to the increased biofilm mass (Yao et al., 2022).

A large number of studies have proved that Lactobacillus biofilms exhibit strong tolerance to the environment. Studies on Lactobacillus stress responses have been relatively comprehensive (Baig et al., 2022), especially regarding acid resistance mechanism (Wang X. et al., 2022). However, studies specifically examining the link between stress responses and biofilm formation in Lactobacillus remains limited. Therefore, future research on stress resistance of Lactobacillus biofilms should focus on exploring the specific stress responses induced by various environmental factors and elucidating the mechanisms behind biofilms’ enhanced resistance. This mechanistic insight will support strategies to improve the resilience of Lactobacillus through biofilm-based approaches.

Historically, Lactobacillus has been mostly used in food industry in the form of planktonic or cell-free metabolites, such as food starter culture and oral bacterial solution. Although most Lactobacillus can play a probiotic role in food, the strain will form a biofilm due to changes of environment, which leads to food spoilage. It has been reported that many Lactobacillus can form biofilms that can affect the quality of meat, cheese, sake, beer and salads. For example, Lactobacillus fructivorans can cause mayonnaise and miso to deteriorate. Lactobacillus acetotolerans and Lactobacillus brevis can cause the deterioration of vinegar, and L. plantarum subsp. plantarum may cause the contamination of common food production plants as well as spoilage of pickles (Somers et al., 2001; Kubota et al., 2009). However, Lactobacillus biofilms also exhibit unique features in terms of stress resistance and antibacterial, which have been used for food production and human health. The application of Lactobacillus biofilm in food mainly involves three aspects, prevent adhesion of pathogens, control spoilage organisms and improve food safety (Jara et al., 2020). Studies have shown that Lactobacillus biofilm can control the growth of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7 (Silva et al., 2020). In addition, pathogen inhibition analysis of Escherichia coli, Staphylococcus aureus, and L. monocytogenes suggested a significant distinction between the planktonic and biofilm of L. reuteri DSM 17938 (Hu et al., 2022a). In addition to inhibiting food spoilage bacteria, it has also been found to remove common toxins in foods. L. rhamnosus biofilm was found to be able to remove aflatoxin M1 from milk by binding it to its cell surface components, reducing the toxin’s bioavailability and thereby potentially enhancing the safety of dairy products (Assaf et al., 2019). The tremendous benefits of Lactobacillus biofilms have not only been noticed against bacterial pathogens but also against fungi, especially in clinical environments. Therefore, the antifungal activities of Lactobacillus biofilms are also important in order to expand the therapeutical approaches to fungi diseases.

Biofilms formed by probiotics are considered an effective strategy against biofilms of pathogenic bacteria in certain disease intervention. They can compete with pathogenic bacteria for nutrients and space through different mechanisms of action, such as producing bacteriostatic substances, playing an immunomodulatory role, and competing with pathogenic bacteria for adhesion sites. Many researchers are trying to use probiotics as an alternative to control infection and introduce promising treatment alternative to infections (Guerrieri et al., 2009). Table 2 shows some examples of lactic acid bacteria biofilm inhibiting foodborne or pathogenic microorganisms.

The use of Lactobacillus biofilms can extend the shelf life of microbial starters, reduce fermentation times, and enhance the characteristic flavors of fermented food. Hu et al. (2019) cultivated the biofilm of L. plantarum on electrospun nanofiber membranes, which had excellent resistance to gastrointestinal fluids. Yogurt produced using nanofiber membranes containing L. plantarum biofilm as a starter culture has shown excellent fermentation properties, which shortens the fermentation time and makes the survival rate of probiotics higher during the shelf life of yogurt. In the production of traditional cheeses, Italian Ragusano cheese and French Salers cheese are made from raw milk, while oak barrels used for fermentation and maturation are rich in microbial biofilms, which are mainly composed of LAB and have an important influence on the unique flavor of cheese (Carpino et al., 2017; Furukawa, 2015).

Lactobacillus biofilm is used to produce metabolites, which is conducive to improving the production efficiency. Bastarrachea et al. (2022) use the biofilm formed by L. paracasei on the chitosan-modified polypropylene material to ferment lactic acid. The findings revealed that the production rate of lactic acid has been significantly improved. The fermentation system has the characteristics of high yield, good continuity and stability, which can maintain a high yield for a long time. Similarly, Zhao et al. (2016) found that a dopamine-modified polypropylene fiber membrane can promote the biofilm formation of L. paracasei, and the biofilm had excellent stress resistance. The purity of L-lactic acid produced by fermentation in the biofilm reactor was maintained at more than 99%. In contrast, the reactor had no fermentation delay period, effectively improving biological fermentation efficiency.

The fermentation process often involves multiple microbial species including Lactobacillus, yeast, and acetic acid bacteria. These microbial communities always exist and play a role in the form of mixed-species biofilm. The application of biofilms formed by Lactobacillus and other species of microorganisms in fermented food production are shown in Table 3. A better understanding of the formation, spatial distribution, systematic succession and function of multi-species biofilms can help researchers better control and utilize biofilms during fermentation and help improve the yield, quality, and safety of fermented products.

To have any biological effect, a daily dose of probiotics of about 10⁸–10⁹ colony-forming units (CFU) is required prior and during passage through the gastrointestinal tract (Liu et al., 2019). Strategies for preserving the viability of probiotic strains have changed over time, leading to the development of the fourth-generation probiotics. Fourth-generation probiotics are represented by encapsulated probiotics, whereby bacterial cells exist in the form of biofilms. Encapsulation of LAB in the form of a biofilm involves embedding the bacteria within a protective matrix of edible colloidal substances like alginate, chitosan, or gelatin. Lactobacillus biofilm encapsulation protects LAB from the external environment and achieves release at the target site under conditions of active metabolism (Salas-Jara et al., 2016). Previous studies have verified that Lactobacillus can be encapsulated and form biofilms within capsules. These encapsulated biofilms show improved resistance to heat, acid, and storage environments. Cheow and Hadinoto (2013) encapsulated L. rhamnosus biofilm with a double-layer coating, which found stronger resistance than the planktonic strains encapsulated with a double-layer coating. Heumann et al. (2020) successfully prepared calcium pectin beads in which encapsulated Lactobacillus paracasei ATCC334 cells showed higher resistance to acid stress, freezing-drying stress, and combined stress. He et al. (2021) encapsulated LAB with low-methoxyl pectin, stimulated the biofilm formation of encapsulated lactobacilli upon in situ cultivation on microcapsules. The findings revealed that the microcapsules formed by biofilm-forming bacteria are more resistant to thermal shock, freeze-drying, gastrointestinal digestion, and drugs than those formed by planktonic bacteria. Vega-Sagardía et al. (2018) encapsulated Lactobacillus fermentum UCO-979C with alginate, xanthan gum, and vegetable oil, and allowed the strain to form biofilm within the microcapsules, and subjected to pH 3.0 maintained the anti-H. pylori inhibitory activity.

Probiotic encapsulation mainly depends on the choice of wall materials, such as emulsion, gel, nanocoating, and liposomes. Each encapsulating material has its advantages and disadvantages with respect to chemical, biological, mechanical, and physical properties (Razavi et al., 2021). Generally, the wall material of microcapsules should not affect the growth and metabolism of probiotics or human health. In addition, it should also have the advantages of being widely available, affordable, edible, and able to ensure targeted release in the colon while simultaneously resisting various adverse environments. Electrospun nanofibrous scaffolds have demonstrated excellent properties in facilitating the biofilm formation of probiotics (Hu et al., 2022b; Hu et al., 2019). However, the output of electrospun nanofibrous scaffolds is limited by the existing production method. Recent studies have explored using natural materials such as polysaccharides and proteins as encapsulation materials (Table 4). Seeking green, inexpensive, and sustainable encapsulation materials that support biofilm formation is important for the successful cultivation and large-scale production of biofilm-based probiotics.

In conclusion, research on the Lactobacillus biofilms has grown significantly in recent years, highlighting their beneficial properties, such as promoting bacterial adhesion, enhancing stress resistance and preventing colonization by pathogens. Despite this progress, substantial doubts remain to be resolved. Existing studies of LAB biofilms have focused mainly on strains of the genus Lactobacillus, whereas reports on biofilm in strains of the genera Bifidobacterium, Streptococcus, Leuconostoc, and Pediococcus are rare. Species-specific biofilm formation mechanisms and biofilm function should be investigated. Meanwhile, the applications of Lactobacillus biofilm is limited by the low cell viability of planktonic cells, the detachment of surface cells, the cell release after biofilm maturation, the incomplete regulatory mechanism and the limited material diffusion within biofilms. Another issue is determining the optimal encapsulation materials and parameters for developing fourth-generation probiotics. Survival of fourth-generation probiotics has been assessed only in vitro and thus requires further studies. These limitations also present opportunities for advancement. The rapid development of emerging technologies, such as multi-omics, gene-editing systems, electrostatic spinning and electrostatic spraying, has provided more options for studying the Lactobacillus biofilms. Continuous increases in the understanding of the Lactobacillus biofilm will undoubtedly help to enhance resistance to stress, inhibit harmful microorganisms, and enable more remarkable breakthroughs in improving product quality and flavor, and producing high-value products.