Understanding Ligilactobacillus salivarius from Probiotic Properties to Omics Technology: A Review

Ligilactobacillus salivarius (basonym: Lactobacillus salivarius, L. salivarius) is a type of lactic acid bacteria (LAB) commonly found in the oropharyngeal-gastrointestinal tract (OGT). It has gained significant attention due to its probiotic and functional properties as well as its various health-promoting roles. L. salivarius strains exhibit strong resistance and adhesion in the OGT along with outstanding antioxidant and antimicrobial properties. Additionally, numerous L. salivarius strains have the ability to produce bacteriocins with antagonistic activity. These probiotic characteristics of L. salivarius indicate its remarkable potential in promoting favorable effects on human health. It has also been observed that L. salivarius has a positive effect on the composition of intestinal microbiota, thereby improving the metabolic profiling of intestinal microbiota, promoting a healthy and balanced internal environment. In recent years, multi-omics technologies such as genomics, transcriptomics, proteomics and metabolomics have been employed to gain a deeper understanding of the roles and mechanisms of L. salivarius associated with its functional properties. This review aims to provide an overview of the probiotic characteristics of L. salivarius, containing its specific interactions with the host microflora, as well as insights from omics studies.


Introduction
Ligilactobacillus salivarius, formerly named Lactobacillus salivarius, has been referred to as this species name for almost 70 years.It was initially described by Rogosa et al. in 1953 as an obligatory homo-fermentative lactic acid bacteria [1,2].Over time, L. salivarius has undergone several reclassifications and is now recognized as a Gram-positive bacterium capable of both homotypic and parthenogenic heterotypic fermentation [3].In the past three years, L. salivarius strains have been reclassified and moved from the Lactobacillus genus to a new genus called Ligilactobacillus, which consists of 16 species.The name Ligilactobacillus implies a combination, with a host-adapted lifestyle, specifically referring to the vertebrate host of L. salivarius [4].
L. salivarius is a non-motile, non-sporulating, oxidase-and catalase-negative, rodshaped microorganism with a general size of 0.6-1.9µm × 1.5-5 µm.L. salivarius exists in different niches and currently isolates are primarily derived from the intestines or feces of birds and mammals [5], e.g., geese, chickens, turkeys, pigeons, ducks, pigs and cattle.Moreover, it has also been shown to exist in the oral cavity, vagina and breast milk of humans, and in honeybee guts, and in foods such as grape wine, meat and St. Ivel cheese [5].Recently, L. salivarius has received increasing attention as a potential probiotic, and various applications of L. salivarius strains have been explored.
Based on available data as of April 2023, the average genome size of L. salivarius strains that have been sequenced is 1.99 ± 0.14 Mbp, containing 1946.70 ± 382.15 genes with a GC content of 32.79 ± 0.16%.In addition to chromosomes, the genome typically includes a repA-type megaplasmid ranging from 100 to 380 kbp and small plasmids.Studies have shown significant differences in both chromosomal and plasmid sequences among L. salivarius strains, particularly in genes encoding glycoside hydrolases (GH), bacteriocins, proteases and EPS synthesis.
In terms of functional characteristics, most L. salivarius strains have a strong tolerance to acidic pH, resistance to the OGT conditions and adhesion to the intestinal mucosa, enabling them to effectively colonize in the host.In addition, excellent antioxidant and antibacterial properties have also demonstrated that this ligilactobacillus can have favorable effects on the host health.L. salivarius strains possess antagonistic properties against bacterial pathogens such as Salmonella enterica (S. enterica), Clostridium perfringens (C.perfringens), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K.pneumoniae), Pasteurella multocida (P.multocida), Riemerella anatipestifer (R. anatipestifer) and Campylobacter sp.This ability is attributed to the production of lactic acid, H 2 O 2 and bacteriocins, as well as the capacity to colonize the gut for an extended period, leading to the exclusion of unfavorable microflora [6].Moreover, previous studies have shown that most L. salivarius strains can improve the composition of the intestinal microflora.Indeed, the correlation between L. salivarius strains and indigenous gut microbiota has emerged as a popular area in scientific research.On the other hand, studies have shown that L. salivarius strains possess several functional properties that are beneficial to the food industry.These properties include improving nutritional quality, enhancing flavor properties, exhibiting antioxidant and antimicrobial activities, increasing the shelf-life of foods, and reducing undesirable compounds [7].Based on these favorable functional characteristics mentioned above, L. salivarius has been awarded the "generally recognized as safe" (GRAS) status by the United States Food and Drug Administration (FDA), and listed as a recommended biological agent intentionally added to food by the European Food Safety Authority (EFSA) and the Ministry of Public Health of China [8].While studying the functional properties of L. salivarius, the multi-omics technologies including genomics, transcriptomics, proteomics and metabolomics have achieved rapid development.Among them, genomics and transcriptomics provide a strong guarantee for genetic information analysis and the functional gene identification of probiotics.Proteomics and metabolomics are very effective methods to study the adaptive mechanism of probiotics to physiological and environmental changes.In a recent study, Lugli et al. proposed a novel concept of "probiotic genomics", which undoubtedly provides a proprietary identity card for each probiotic [9].Therefore, omics technologies are growing in significance as their ability to elucidate the molecular mechanisms underlying the functional and probiotic properties of LAB.This review summarized the probiotics properties of L. salivarius as well as the application of omics approaches in L. salivarius strains.It provides a reference for subsequent research applications of L. salivarius strains.

Probiotic Properties and Roles of Ligilactobacillus salivarius
L. salivarius is an important member of the LAB family and it has been widely used as a probiotic due to its excellent characteristics (Table 1).Although early studies focused on the isolation of probiotic strains of L. salivarius and their bioactive metabolites (mainly bacteriocins), more research is now available to better understand the role of L. salivarius strains and their metabolites in various fields and their adaptability to environmental stresses.

Strain and Origin
Probiotic Properties Potential Application Reference F14 1.

3.
Antimicrobial activity against Gram-positive and -negative bacteria.
Good resistance to acid and bile salt.

2.
Good auto-aggregation and co-aggregation potential.
Resistance to gastrointestinal conditions.2.
Adhesion to both vaginal and intestinal cells.
Resistance to osmotic changes and acid (pH 2.0).
Resistance to acid and bile.

3.
Produces a small heat-stable bacteriocin Abp-118 and has a broad-spectrum antimicrobial activity.

1.
Good resistance to gastric acid, intestinal fluid and bile.

2.
High affinity to intestinal mucus and epithelial cells.

3.
Effectively inhibited the growth of Helicobacter strain.

4.
Supplementation of L. salivarius Ren or its metabolites could effectively inhibit oral and colon cancer.
Strong antimicrobial activity against enteropathogens.

2.
Tolerance to acid and bile stress.

3.
Had good effects in the prevention and treatment of liver failure.
Good gastrointestinal tolerance and good adhesion to both Caco-2 and HT-29 cells.

4.
Enhances both natural and acquired immune responses.
High survival rate in gastrointestinal tract (GIT).
Has adhesion capacity to porcine mucins and porcine intestinal epithelial (PIE).

2.
Functionally modulates the innate immune responses triggered by TLR3 and TLR4 activation in PIE cells and effectively adheres to these cells.

3.
Reduction of rotavirus replication levels in PIE cells.

Strain and Origin Probiotic Properties Potential Application Reference
Ls-33 1.
Tolerance to bile salts.
Possesses potent antibacterial activities.

1.
Good resistance to bile salt and NaCl.

4.
Good adhesion to mucins and PIE.
Displays strong antimicrobial activity against otopathogens.

2.
Good tolerance to GIT conditions.
Produces bacteriocin, L-lactate and acetate, does not produce H 2 O 2 and D-lactate.
Antibacterial and antiviral activities.

3.
Has a broad-spectrum antibiotic resistance.
High degree of adhesiveness to chicken intestinal epithelial cells (IEC).

2.
Antagonistic activity against Salmonella enteritidis and E. coli.
Antagonistic activity against Salmonella, Clostridium, and E. coli.

2.
Good resistance to acidic pH.
Antagonistic activity against enteric pathogens.
Antimicrobial activity against adhesion to S. aureus.
Good antagonistic activity against Streptococcus mutans.
Resistance to acidic condition and bile salts.

2.
Has a good inhibitory effect on pathogens, such as E. coli, Salmonella, S. aureus and C. perfringens.

.1. Resistance to Oropharyngeal-Gastrointestinal Conditions
A requisite condition for any microorganism to be a probiotic is the ability to survive or pass the harsh conditions of the GT.The stomach is a very unfriendly environment, with its internal gastric juice consisting mainly of pepsin and hydrochloric acid (HCl).This directly contributes to the low pH (1.5-4.5) of the gastric environment.Therefore, acidic pH tolerance is considered to be the main criterion for probiotic screening.In this unfavorable environment, L. salivarius strains are potential probiotics by maintaining intracellular pH balancing, ATR signaling pathway and macromolecule protection and repair, among other strategies to achieve pH and bile tolerance [42].In addition, the acid resistance of some LAB can also be improved by exposing themselves to non-lethal acidic conditions through acid resistance reactions [43].It is known that different strains of the same LAB species have highly variable acid resistance and this is also true for L. salivarius strains.A study shows that L. salivarius IBB3154 showed good resistance and survived at low pH (value is 3.5) conditions [44].Similarly, Sajedinejad et al. demonstrated that L. salivarius NK02 displayed significant tolerance to low pH conditions.Specifically, this strain exhibited survival rates (7 logs CFU/mL) when exposed to simulated gastric juice (pH 2.2).Furthermore, L. salivarius NK02 was also found to be significantly tolerant to bile, lysozyme, and 1-5% NaCl [37].
In addition to gastric acid, probiotics must also tolerate exposure to bile salts in the small intestine.Bile salt is a sodium or potassium salt formed by the combination of bile acids secreted by hepatocytes with glycine or taurine.It is the main component of bile involved in fat digestion and absorption.Cell membrane disruption, DNA damage, intracellular acidification, oxidative stress and metabolic changes triggered by bile salts, pose a serious threat to the colonization and survival of probiotics [45].Therefore, bile tolerance is another major criterion for screening potential probiotic strains.L. salivarius strains, an OGT natural flora, have been exposed to bile salt stress for a long time and have evolved various mechanisms to cope with bile salt toxicity, mainly including the production of bile salt hydrolases (BSHs), alteration of cell membrane composition and structure, and the use of a transport system to transfer bile salts [46].BSH is considered to be the main component of bile tolerance in LAB.It is responsible for catalyzing the depolymerization of glycine and taurine residues in cholesterol.Although the exact nature of how bile salt depolymerization limits negative effects on cellular homeostasis is unknown, many literatures have reported that bile salt hydrolase expression is associated with bile resistance in many lactobacilli species [47].However, Fang et al. demonstrated that BSH is not the primary determinant of bile resistance in L. salivarius strains, and may have additional biological importance because of its varying effects upon bile as a signaling molecule in the host [48].Pan et al. also showed that there was no association between bile salt hydrolase and bile salt tolerance in L. salivarius strains and indicated that bile salt tolerance in L. salivarius strains was associated with processes such as peptidoglycan synthesis, the phosphotransferase system (PTS) and DNA damage repair [49].This is consistent with the results of Wang et al. [50] and Lv et al. [51].
In addition to gastrointestinal microbiota, oral microbiota is also part of the human microbiome.In the daily diet process, it is essential to add salt, vinegar and other food ingredients to various diets, and these high osmotic pressure foods will inevitably affect the survival and growth of lactic acid bacteria in the oral tract.Therefore, probiotics must also tolerate a variety of complex conditions.L. salivarius AR809 was isolated from a healthy adult oral cavity with good resistance to acidic pH, bile, lysozyme and H 2 O 2 [35,42].
It can be seen that most of the L. salivarius have the characteristics of acid and bile salt tolerance.Certainly, tolerance to various conditions could impact the probiotic potential of different strains of L. salivarius.Nevertheless, on the other hand, it is essential to recognize that resistance to low pH and bile is crucial for probiotics, but it does not guarantee that acid pH-tolerant and bile-tolerant strains will exhibit probiotic properties.

Adherence to the Intestinal Mucosa and Extracellular Matrix Components
Adhesion is an important prerequisite for the colonization and function of LAB strains in the OGT, which is directly related to cell wall components such as adhesins, polysaccharides and proteins (Figure 1).It includes two steps, non-specific adhesion and specific adhesion.Through adhesion, LAB can be permanently colonized in the IEC membrane, enhance the signal exchange between cells, promote the stability of intestinal flora, regulate the body's immunity, form a biological barrier, and resist pathogenic bacteria [52].Therefore, adhesion is also one of the important criteria for screening probiotic lactic acid bacteria in vitro.In this case, many researchers have begun to study the adhesion ability of L. salivarius strains.A study conducted by Jia et al. [35] demonstrated that in a cell adhesion assay simulating the human oral environment, the extent of adhesion to FaDu cells of L. salivarius AR809 (31.1%) was significantly higher than that of L. plantarum AR113 (4.44%) and L. plantarum AR195 (8.28%).In addition, L. salivarius AR809 also significantly reduced the adhesion effect of S. aureus to FaDu cells through exclusion, competition and displacement.In a similar way, Dash et al. demonstrated that the L. salivarius F14 strain had good adhesion to the Caco-2 cells through the co-culture model in vitro, and it also could significantly inhibit the adhesion process of S. typhimurium ST-Xen 33 on Caco-2 cells [10].For their part, Zhang et al. [53] and Ren et al. [54] showed that L. salivarius strains had good adhesion to the Caco-2 cells which could be associated with the presence of genes that encode different proteins attributed to adhesion to different extracellular matrices and intestinal mucus [46].Of course, we should also list more in vivo results to confirm that L. salivarius strains can adhere to the intestinal mucosa/extracellular matrix components and persist.

Antioxidant Activity
Oxidative stress is when the balance between anti-oxidants and prooxidants in the cell is disturbed, resulting in DNA hydroxylation, protein denaturation, lipid peroxidation and ultimately cell apoptosis.It is the fundamental reason for aging and aging-related diseases, which can induce diabetes, atherosclerosis, arthritis, hyperlipidemia, cardiovascular diseases and many other diseases [55].In recent years, a large number of studies have shown that lactic acid bacteria can remove the active oxygen molecules in the intestine, so that the active oxygen molecules in the body remain relatively stable, thereby re-

Antioxidant Activity
Oxidative stress is when the balance between anti-oxidants and prooxidants in the cell is disturbed, resulting in DNA hydroxylation, protein denaturation, lipid peroxidation and ultimately cell apoptosis.It is the fundamental reason for aging and aging-related diseases, which can induce diabetes, atherosclerosis, arthritis, hyperlipidemia, cardiovascular diseases and many other diseases [55].In recent years, a large number of studies have shown that lactic acid bacteria can remove the active oxygen molecules in the intestine, so that the active oxygen molecules in the body remain relatively stable, thereby reducing the body damage caused by the oxidation reaction.These lactobacilli exert antioxidant effects mainly through scavenging reactive oxygen radicals in and around cells, chelating metal ions, alleviating lipid peroxidation, modulating their antioxidant defense system, regulating the host cell antioxidant defense system, and modulating host cell antioxidantrelated signaling pathways [56].With the gradual increase in reports on the antioxidant effect of lactobacillus, the understanding of the antioxidant function of lactobacillus is also increasing.Zhang et al. identified a strain of Limosilactobacillus fermentum YLF016, which contained various antioxidant enzyme encoding genes, as well as having a strong ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals (-OH) scavenging ability [57].Zhai et al. evaluated the antioxidant activity of 10 lactobacillus strains and found that L. plantarum CCFM 8661 exhibited the strongest lipid peroxidation inhibitory activity [58].
Currently, there have been studies on the in vivo/vitro antioxidant function evaluation of L. salivarius strains at home and abroad.Zhang et al. studied the protective mechanism of L. salivarius by establishing an alcohol injury model [59].Specifically, L. salivarius M18-6 protected mouse hepatocytes from alcohol-induced oxidative stress damage by downregulating serum alanine transaminase (ALT) levels, upregulating superoxide dismutase (SOD) levels and activating the keap1-Nrf2 signaling pathway.This research provided a scientific basis for the clinical application and product development of the L. salivarius strain for alcohol injury.Wang et al. revealed the molecular mechanism of L. salivarius Ren in response to bile-induced oxidative stress by transcriptomics and proteomics [60].L. salivarius MG242 isolated from the human vagina by Kang et al. exhibited good antioxidant properties with DPPH radical scavenging rate and 2, 2 ′ -azino-bis (3-ethylbenzothiazo line-6-sulfonic acid) (ABTS) of 56.9% and 97.1%, respectively [61].Chooruk et al. investigated the antioxidant activities of 201 LAB strains in vitro [62].Among them, the antioxidant capacity of L. salivarius differed significantly from other lactobacillus, which also indicates the specificity of antioxidant properties of different species and strains.The supplementation of L. salivarius AP-32 in Parkinsonian rats modulates short-chain fatty acid (SCFA) production, increases antioxidant enzyme activity and protects mitochondria from reactive oxygen species (ROS)-induced damage [63].Therefore, the antioxidant potential of L. salivarius can provide ideas for anti-aging related research.

Active Metabolites and Its Antimicrobial Activity
Although the traditional view is that only live probiotics can exert their probiotic effects, more and more studies have shown that some of the probiotic properties of probiotics are closely related to the active products produced by their metabolism.At present, the active metabolites of probiotics mainly include EPS, bacteriocins, organic acids, SCFAs, vitamins and some bioactive enzymes and small molecules (Figure 2).These active metabolites have been proven to have anti-inflammatory, antimicrobial, antioxidation, immune regulating properties useful for the prevention or treatment of a variety of metabolic diseases.Antimicrobial properties are one of the most notable characteristics of probiotics.This can be achieved through competition for nutrients and adhesion space, inducing environments that are harmful to pathogens, and that produce antimicrobial metabolites and regulate immune responses [46].This subsection is devoted to the antimicrobial activity of L. salivarius strains.
diseases.Antimicrobial properties are one of the most notable characteristics of pro This can be achieved through competition for nutrients and adhesion space, induc vironments that are harmful to pathogens, and that produce antimicrobial meta and regulate immune responses [46].This subsection is devoted to the antimicro tivity of L. salivarius strains.L. salivarius strains can produce several antimicrobial active compounds that antagonistic activity against pathogenic organisms.First, many L. salivarius stra good producers of many small proteins such as antimicrobial peptides and bacte This allows them to compete with other bacteria in the environment and disrupt walls and cell membranes of pathogenic bacteria, causing the intracellular mat leach out, thus producing an antibacterial effect [64][65][66][67].L. salivarius PS7 showed L. salivarius strains can produce several antimicrobial active compounds that exhibit antagonistic activity against pathogenic organisms.First, many L. salivarius strains are good producers of many small proteins such as antimicrobial peptides and bacteriocins.This allows them to compete with other bacteria in the environment and disrupt the cell walls and cell membranes of pathogenic bacteria, causing the intracellular material to leach out, thus producing an antibacterial effect [64][65][66][67].L. salivarius PS7 showed strong antagonistic activity against ten acute otitis media (AOM) related pathogens (Streptococcus pneumoniae MP07, Streptococcus pyogenes MP03, S. aureus MP29, Staphylococcus epidermidis MP33, Alloiococcus otitidis MP02, Enterococcus faecalis MP64, Haemophilus influenzae MP04, Moraxella catarrhalis MP08, P. aeruginosa MP24 and E. coli MP69) [28].Similarly, Martín et al. found that L. salivarius CECT5713 inhibited the growth of Gram-negative bacteria (including, E. coli CECT4076, K. pneumoniae CECT 142, K. oxytoca CECT 860T and Proteus vulgaris CECT484), Gram-positive bacteria (E.faecium P21, E. faecalis TAB28, Listeria monocytogenes ScottA, L. monocytogenes Ohio, L. innocua RdC, S. aureus CECT5191, S. epidermidis CECT 231, Lactococcus lactis MG1614 and Latilactobacillus sakei NCFB2714) and the yeasts Rhodotorula mucilaginosa CECT10359, this effect being greater in L. monocytogenes Ohio and K. oxytoca CECT 860 T [68].The antimicrobial effect of both strains of L. salivarius was attributed not only to the presence of bacteriocins but it was also related to the formation of organic acids (such as common lactic acid and SCFAs) and H 2 O 2 that leads to a change in the medium, which can alter the development of indicator organisms [69].Furthermore, L. salivarius strains can also produce EPS with natural antibacterial activity, which is usually attributed to its anti-biofilm effect [70].Bikric et al. found that EPSBIS312 and EPSBIS722 derived from L. salivarius BIS312 and L. salivarius BIS722, respectively, could significantly inhibit the biofilm formation of Enter.faecalis 29212, Staph.aureus EB1 and E. coli ATCC 11229, and were significantly higher than commercial inulin [71].This indicates that these two EPS may become substitutes for plant prebiotics (such as inulin) in poultry.
Moreover, many L. salivarius strains also possesses antiviral activity.L. salivarius YM33 isolated from the feces of nursing piglets has good activity against pig epidemic diarrhea virus (PEDV) and can significantly down-regulate proinflammatory cytokine levels [72].This research was the first demonstration of the antiviral activity of L. salivarius against PEDV.Similarly, Shojadoost et al. reported that L. salivarius JTBo9 enhanced the antiviral activity of macrophages of chicken against avian influenza virus infection via virus titer reduction and increased the expression of IL-1β and IFN-γ virus titer in an in vitro cell model [73].It is also because of the antimicrobial activity of L. salivarius strains that it is widely used in the breeding industry.In conclusion, the antimicrobial activity of L. salivarius provides favorable conditions for its application in various fields.

Host OGT Micro-Ecosystem Modulation
The host gut is a complex micro-ecosystem consisting of communities of bacteria, viruses, archaea, fungi and protozoa that live in the GT [74], known as the intestinal flora.Gut microbiota performs useful functions including fermenting unused energy substrates, training the immune system with metabolic end products, maintaining the intestinal epithelium, synthesizing host vitamins [75], producing hormones that induce host fat accumulation, metabolizing dietary and pharmaceutical compounds and controlling immune function, and even affects behavior via the gut-brain-axis [76].
Given these facts, intestinal microflora plays a key role in the maintenance of host health and the pathogenesis of many diseases [77].Therefore, gut microbiota eubiosis is essential for the prevention of infectious and non-infectious diseases and for preventing disturbances (also known as dysbiosis) in the balance of the microbial community equilibrium [78].However, the gut microbiota is an open micro-ecosystem whose composition and/or activity can be influenced by many elements, including the mode of birth, sex, host inheritance, immune system and host health or disease status, geographic location, social economic factors, diet, the use of therapeutic drugs, etc. [79].In point of fact, the intestinal microbiota is constantly exposed to transient exogenous microorganisms transmitted through food, as demonstrated by Veiga et al. [80].In this regard, probiotics can regulate the composition of gut microbiota and correct the abnormal response of the immune system [81], thereby exerting different favorable effects on host health (Figure 3).In summary, probiotics may be a therapeutic strategy to regulate intestinal flora and improve human disease [60,82].In this sense, several studies have found that different strains of L. salivarius have a significant regulatory effect on intestinal flora (Table 2).Increased Firmicutes, Bacteroidota and Actinobacteriota phylum.

3.
Intestinal microflora was decreased and overall microbial diversity increased.4.
Reduced Aeromonas and increased Bifidobacterium.
Relative abundance of L. salivarius and Ligilactobacillus agilis were significantly improved in the co-feeding group of L. salivarius + L. agilis and L. salivarius + Ligilactobacillus aviaries.
Restored temozolomide-induced reduction in bacterial abundance and diversity.

3.
Increased the abundance of Bacteroidota and Firmicutes, and increased the ratio of Firmicutes/Bacteroidota (F/B ratio).4.
Partially limited the effects of Campylobacter on Anaerotruncus sp.decrease and Subdoligranulum sp.increase.
Increased the abundance of beneficial bacteria such as Lactobacilli and Bifidobacteria.

2.
Decreased the abundance of harmful bacteria such as E. coli and total aerobes.

1.
Increased the number of Lactobacillus sp.

2.
Decreased plasma ammonia content, the number of E. coli in ceca and fecal ammonia emission.
Reduced Firmicutes and increased Bacteroidetes phylum.

2.
Reduced the metabolisms of lipids and acetate.

3.
Enlarged the number of SCFA-producing bacteria.

4.
Increased the relative abundance of the microbes involved in GDP-mannose biosynthesis.
Increased the abundance of Firmicutes.
Altered the cecal microbiome composition.

2.
Prevented bacterial translocation in liver injury.

2.
Increased the F/B ratio.

4.
Decreased bacterial species related to pro-inflammatory cytokines and profibrogenic genes.
Higher abundance of Alloprevotella.
Increased the number of faecal Lactobacilli.

2.
Maintained the dynamic balance of Bifidobacterium.

4.
Reversed the decrease in acetic acid, isobutyric acid, valeric acid and total SCFA levels caused by LPS.

L. salivarius Strains Main Effects on Gut Microbiota Reference
HK LS 189 1.
Changed the gut microbiota diversity.

3.
Regulated the metabolic functions of the intestinal microflora.4.
Increased the metabolisms of lipids.
Corrected the reduced species abundance and species diversity of the intestinal flora caused by E. coli K88 infection.

2.
Improved the levels of beneficial bacteria such as Lactobacillus and Bifidobacterium.

3.
Reduced the levels of harmful bacteria such as Ralstonia and Helicobacter. [41] For instance, Moturi et al. explored the fecal microflora composition in piglets fed two different L. salivarius strains during the lactation period [94].Specifically, significantly fewer OTUs and a lower phylogenetic diversity index and Chao index of bacteria were observed after supplementation with L. salivarius from normal piglets compared to the control group, suggesting that probiotics may inhibit bacterial growth.Similarly to these results, Riboulet-Bisso et al. stated that the application of L. salivarius UCC118 wild type reduced the number of Gram-negative bacteria present in the intestine of pigs [95].In addition, L. salivarius 144 (from fast-growing pigs)-treated piglets showed a significant increase in the abundance of beneficial bacteria and a decrease in the abundance of C. perfringens, which may be related to their antimicrobial activity, and these changes in microbial communities may reduce the susceptibility of weaned piglets to pathogenic infections at weaning.Furthermore, L. salivarius (LS144 and LS160) supplementation could promote the growth of villus in all of the intestinal segments.On the contrary, in a recent study, Wei et al. found that the ingestion of L. salivarius WZ1 corrected the reduced species abundance and species diversity of the intestinal flora caused by E. coli K88 infection and increased the abundance of beneficial bacteria (Lactobacillus and Bifidobacterium), as well as decreased the abundance of harmful bacteria (Ralstonia and Helicobacter) [41].Similar results were obtained by Xin et al. who found that the Shannon, Chao1 and Ace indices of the Sinocyclocheilus grahami gut microbiome were all found to increase after feeding supplemented with L. salivarius S01, suggesting that L. salivarius S01 can promote gut microbial diversity and abundance in the host [83].These results show that the probiotic function of different L. salivarius strains also varies greatly.
Xu et al. found that the supplementation of L. salivarius CML352 in late phase improved the gut microflora composition of laying hens [88].Specifically, this strain significantly reduced the relative abundance of the phylum Firmicutes and increased the relative abundance of the phylum Bacteroidetes, thus resulting in a significant decrease in F/B ratio.Many human studies have consistently shown that the F/B ratio is positively correlated with the degree of obesity [96].Moreover, the gene expression levels related to methanogenesis from acetate in the L. salivarius CML352 group were significantly lower than in the control group, which has been presumed to reduce fat deposition and obesity [97].Thus, L. salivarius strains may have the potential to reduce obesity.Additionally, the increased abundance of Bacteroidetes may promote gut health in chickens.Simply speaking, the majority of the species in the Bacteroidetes phylum are producers of SCFAs [98], which play a significant role in innate immunity, are an important source of energy for IEC, and maintain epithelial barrier function and inhibit pro-inflammatory cytokines.In addition, eggshell strength and thickness increased significantly (p < 0.05) after feeding L. salivarius CML352.This may be due to the fact that feeding L. salivarius CML352 increases the vitamin content of the gut and promotes the absorption of mineral elements [99].
For their part, Lv et al. investigated the influence of five LAB on dysbiosis produced in a rat model with acute liver injury induced by D-galactosamine [19].They discovered that L. salivarius LI01 and Pediococcus pentosaceus LI05 were beneficial in preventing acute liver failure.Specifically, L. salivarius LI01 significantly decreased alanine aminotransferase and aspartate aminotransferase levels, inhibited total bilirubin accumulation, reduced the histological abnormalities of both the liver and the terminal ileum, prevented bacterial translocation, increased the serum interleukin 10 (IL-10) and/or interferon-γ (IFN-γ) levels, and resulted in a cecal microbiome that differed from that of the liver injury control.In addition, L. salivarius LI01 also attenuated liver fibrosis by increasing microbial abundance, improving the integrity of the intestinal barrier, and reducing plasma endotoxin levels and regulating Toll-like receptors (TLR) gene expression, among other mechanisms [90].Furthermore, it also synergized with Bifidobacterium to significantly improve the symptoms of D-galactosamine-induced liver failure in rats [91].It can be seen that in the complex OGT environment, the regulation of intestinal flora may be attributed to the synergistic effect of multiple probiotics.In conclusion, the above studies provide strong evidence for the prevention and treatment of liver injury and demonstrate that gut microbiome homeostasis contributes to the improvement of liver disease.

Multi-Omics Approach to Understanding the Role of Ligilactobacillus salivarius
Omics technology belongs to the concept of systems biology, mainly including genomics, transcriptomics, proteomics and metabolomics, etc.With the continuous improvement and evolution of omics technology research, combined with chemometric tools, people have a comprehensive and in-depth understanding of the mechanism behind the functional and specific interactions between probiotics and hosts (Figure 4).Researchers have successfully revealed the genetic information of different microorganisms with various phenotypes using these methods.Therefore, these are very useful tools to bridge the gap between genetic information and cell-specific metabolites.Among these, genomics allows for the identification of functional genes contained in the target strain and transcriptomics, proteomics and metabolomics allow for the quantification of mRNA, proteins and metabolites (<1500 Daltons), respectively, under specific physiological conditions.Therefore, it is worth noting that all of these methods are completely different from traditional characterization methods.Considering the complexity and uniqueness of organisms, a single method is not enough to characterize organisms.Most lactobacillus has been widely used in food and pharmaceutical fields.It is only recently, however, that advances in technology and methods have revealed the mechanisms that explain the beneficial effects of these bacteria on the host [100].To date, many scholars have integrated various "omics" approaches to understanding the functional role of LAB, including L. salivarius [51].
Foods 2024, 13, x FOR PEER REVIEW 13 of 22 traditional characterization methods.Considering the complexity and uniqueness of organisms, a single method is not enough to characterize organisms.Most lactobacillus has been widely used in food and pharmaceutical fields.It is only recently, however, that advances in technology and methods have revealed the mechanisms that explain the beneficial effects of these bacteria on the host [100].To date, many scholars have integrated various "omics" approaches to understanding the functional role of LAB, including L. salivarius [51].

Genomics Transcriptomics
Proteomics Metabolomics DNA: High-throughput DNA sequencing and bioinformatics assembly and analysis of the function and structure of the whole genome.
RNA: Better understanding genes functionality and utility (e.g.antimicrobial mechanism) through studying the expression of genes in organisms under different conditions.
Proteins: Evaluate its beneficial properties (e.g.adhesion ability, bile resistance) that are supported by certain alterations in proteins.

Figure 4.
Understanding probiotic functional properties using an omics approach.

Genomics Approach to Comprehend the Role of Ligilactobacillus salivarius
With the rise of genomics, LAB have important research value for human production and life, and gradually become the focus of research.It is expected that this will reveal the diversity and evolution of LAB at the molecular level, enable analysis of their physiological and metabolic mechanisms, and uncover functional genes related to important traits in order to accelerate the breeding and transformation of excellent strains, and provide a basis for the efficient utilization of LAB and improvement in the industrial-level control

Genomics Approach to Comprehend the Role of Ligilactobacillus salivarius
With the rise of genomics, LAB have important research value for human production and life, and gradually become the focus of research.It is expected that this will reveal the diversity and evolution of LAB at the molecular level, enable analysis of their physiological and metabolic mechanisms, and uncover functional genes related to important traits in order to accelerate the breeding and transformation of excellent strains, and provide a basis for the efficient utilization of LAB and improvement in the industrial-level control of fermentation.
Based on the genome database of NCBI (https://www.ncbi.nlm.nih.gov/genome,accessed on 11 May 2023), United States, 22 complete genome maps of L. salivarius have been constructed as of May 2023.Yang et al. determined the whole genome of L. salivarius AR809 and it contains 1967 genes, of which a total of 1593 genes encoding proteins, 79 tRNA genes and 22 rRNA genes were found on the circular chromosome, and 240, 28, 3 and 2 genes encoding proteins were found on plasmid pA-pD, respectively [42].In addition, genomic analysis also screened a series of genes related to beneficial properties, such as carbohydrate metabolism, environmental stress and adhesion [42], which provide valuable guidance for the oral colonization of L. salivarius AR809.Sun et al. reported the whole genome sequence of L. salivarius Ren, which contained a 1,751,565 bp circular chromosome and two plasmids [101].Bioinformatics analysis identified several genes important for gastrointestinal tolerance and adhesion, such as genes involved in acid and bile salt tolerance responses, genes encoding S-adenosylmethionine synthase, and fibronectin-binding protein genes [101].In addition, it found that the absence of an α-glycerophosphate oxidase coding gene in L. salivarius ATCC11741, which fails to degrade 4-hydroxyaminoquinoline 1-oxide (4-HAQO), suggested it may be one of the reasons for the significant differences between L. salivarius Ren and ATCC11741 in the secretion of H 2 O 2 and degradation of 4-HAQO [101].

Transcriptomics Approach to Comprehend the Role of Ligilactobacillus salivarius
Microarray and RNA sequencing (RNA-Seq) are two main techniques currently used for transcriptomic research [102].In the probiotics field, the transcriptome has been used to study the molecular mechanisms involved in environmental stress responses, e.g., tolerance to the stress of acid and bile salts in the GIT [51].In addition, transcriptomics help to reveal molecular strategies for probiotic interactions with the host or other microbiota, including changes in metabolic profiles, signaling pathways regulation, cell growth and communication (intercellular signaling) [103].Song et al. used RNA sequencing analysis to identify the first well-characterized endogenous constitutive promoter library from L. salivarius, providing a useful toolbox for the subsequent metabolic engineering and synthetic biology of L. salivarius and other prokaryotes [104].Xia et al. fused a multi-omics strategy of transcriptomics, metabolomics and cytokine arrays to explore the effects of colonization with L. salivarius LI01 on growth, immunity and metabolism in germ-free rats [105].Similarly, Lv et al. used transcriptome sequencing combined with proteome, and proposed the first model of a bile stress response mechanism for L. salivarius LI01, which provides a reference for the subsequent bile salt resistance mechanism of L. salivarius [51].The combination of transcriptomics and other omics is one of the most common methods to solve experimental problems.Considering the specific environment, the integrated study of transcriptome combined with other technologies can be used in many fields, e.g., studying the role of LAB in food spoilage, potential probiotic properties of LAB strains, etc.

Proteomics Approach to Comprehend the Role of Ligilactobacillus salivarius
Proteomics mainly studies the dynamic changes in proteins during development and their responses to internal or external stimuli [106].Based on the complexity of protein structure, researchers have developed a comprehensive proteomics technique to deeply analyze all of the proteins present in a sample [107].In recent years, the proteomics research on L. salivarius has been increasing (Table 3).Kang et al. used 2D gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-ToF/ToF MS) analysis and identified potential secreted proteins that may be responsible for the antimicrobial activity [108].A total of 21 secreted proteins were identified, of which five were produced by L. salivarius.The LysM domain protein was a peptidoglycan binding protein that may cause the lysis of S. aureus upon binding to the cell wall but does not affect lactobacillus.Kelly et al. identified three proteins, DnaK, Ef-Ts and pyruvate kinase, in the cell wall of L. salivarius UCC118 by combining proteomics analysis with enzymatic techniques [109].These proteins may play an important role in adhesion and promoting host immune perception.In addition, the proteome can also be used for strain identification.Hamza et al. used MALDI-TOF spectroscopy and 16S rDNA sequencing to identify 67 isolates [110].All identified isolates were L. salivarius and L. plantarum.Proteomic analysis can help to understand the different characteristics of L. salivarius and identify the proteomic profile of individual characteristics, which can be used as biomarkers for the initial selection of potential probiotic strains.Helping the cells to maintain surface homeostasis.

2.
Enhancing the hydrophobicity of the cell surface.

3.
May be helpful for tolerating bile exposure.

4.
May help to promote attachment of secondary cell wall polymers with discrete linkage units to peptidoglycan, maintaining the structure of the cell wall. 5.
The bile reaction mechanism was studied for the first time in L. salivarius using transcriptomics. [51] RT-qPCR and microarray assay ND 1.
Feasibility of finding specific molecules as new targets responsive to oral L. salivarius PS2 in mastitis-infected human mammary glands. [111] RNA-sequencing and RT-qPCR 2112 1.
Native constitutive promoters with various strengths were identified in L. salivarius. [104] High-throughput transcriptomic sequencing and qRT-PCR Not specified 1. Upon L. salivarius + L. agilis feeding, in crypt, genes involved in oxidative phosphorylation, biosynthesis of amino acids, and ribosome-related pathways were activated. [84] RNA-sequencing and RT-qPCR >1941  Addition of probiotics enriched some potentially beneficial microbes and small molecular compounds with antimicrobial activity, and inhibited potential pathogens in fermented feed. [117] 1 H-NMR More than 50 molecules 1.
Possible presence of bacteriocins in the identified amino acids, resulting in antimicrobial activity of the supernatant.
Compounds related to tryptophan metabolism and primary BA biosynthesis were identified.[84] Untargeted GC-MS 348 1.

Metabolomics Approach to Comprehend the Role of Ligilactobacillus salivarius
The metabolome is believed to be the result of the genome, transcriptome and proteome, and directly influences the molecular phenotype of microbial cells.Its application in probiotics research has been developed rapidly in the past few years.Metabolomics has been used to map metabolic pathways and reveal microbial metabolic networks, e.g., studying types and changes of metabolites in food, evaluating the effect of probiotics on the metabolic activity of resident microflora and characterizing microbial molecules secreted during the industrial production of probiotics.Zhu et al. used genomics and metabolomics to explore the alleviation mechanism of the L. salivarius strain on nonalcoholic fatty liver disease (NAFLD) [112].Specifically, most of the 250 metabolites from L. salivarius SNK-6 are involved in multiple metabolic pathways, including amino acid and lipid metabolism.Studies have reported that the L. salivarius strain can effectively alleviate liver injury by regulating liver lipid metabolism.In addition, a further analysis of the metabolomics data showed that butyric acid, acetic acid and propionic acid were the main SCFAs produced by L. salivarius strain, while cholic acid (CA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and tauroursodeoxycholic acid (TCA) were the four most abundant bile acids in the metabolites of L. salivarius SNK-6.Studies have shown that they are important signaling molecules that regulate lipid metabolism genes and can alleviate lipid accumulation and inflammation in NAFLD rats.Bile acids play an important role in regulating lipid, glucose and energy metabolism, and are essential for protecting hepatocytes from cholesterol and bile acid toxicity.CA shows a specific affinity for bile acid receptors, and as a signal molecule, it has a unique role in regulating liver lipid metabolism.UDCA is used to treat primary biliary cirrhosis, activate PKC and MAPK signaling and anti-inflammatory hepatocyte pathways, and promote bile HCO 3 − secretion to reduce cholestasis and liver damage.CDCA can reduce body weight and improve glucose tolerance.TCA can reduce hepatic steatosis, intestinal inflammation and insulin resistance in mice.
In summary, we found that genomics and transcriptomics can analyze the genetic information and functional genes of L. salivarius, and proteomics and metabolomics can delve into the adaptive mechanism of L. salivarius in response to physiological and environmental changes.Therefore, when we study L. salivarius, the combined use of the above four histological approaches can comprehensively reveal the complex mechanisms behind the functions and beneficial properties of L. salivarius from multiple perspectives, including genetics, expression and metabolism.

Conclusions
In recent years, L. salivarius has gained increasing attention from researchers due to its tolerance to the oral cavity and gastrointestinal environment, as well as its ability to adhere to human cells.Studies have shown that L. salivarius strains have various physiological functions, such as anti-oxidation, immune regulation, and maintaining the balance of intestinal flora, providing a theoretical and experimental basis for the development of functional lactic acid bacteria products and healthy foods with the potential to lower blood fat and prevent cardiovascular diseases.Despite the recognized functional properties of different L. salivarius strains, further research is needed to fully understand the role this microbe plays in host health.Omics approaches are becoming increasingly important in this field of research as they enhance the understanding of the functions and mechanisms behind the probiotic properties of beneficial bacteria such as L. salivarius.Genomics and transcriptomics can reveal the genetic information of L. salivarius strains, analyze their physiological and metabolic mechanisms, and discover important functional genes.Proteomics and metabolomics can study the effect of L. salivarius strains on the mechanisms of adaptation to physiological and environmental changes, such as adhesion, biofilm formation, antagonistic capacity, and tolerance.However, there are still many questions that need to be clarified, emphasizing the importance of further research in the field of multi-omics techniques to increase our understanding of how L. salivarius exerts its probiotic activity.

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13,  x FOR PEER REVIEW 7 of 22 confirm that L. salivarius strains can adhere to the intestinal mucosa/extracellular matrix components and persist.

Figure 1 .
Figure 1.LAB cell wall structure.LTA represents lipoteichoic acids, WTA represents wall teichoic acids, EPS represents exopolysaccharides, WAP represents wall-associated proteins, SLP represents surface layer proteins, SP represents secreted proteins, MP represents membrane proteins, LP represents lipoproteins and MAP represents membrane-associated proteins.

Figure 1 .
Figure 1.LAB cell wall structure.LTA represents lipoteichoic acids, WTA represents wall teichoic acids, EPS represents exopolysaccharides, WAP represents wall-associated proteins, SLP represents surface layer proteins, SP represents secreted proteins, MP represents membrane proteins, LP represents lipoproteins and MAP represents membrane-associated proteins.

Figure 2 .
Figure 2. Active metabolites of probiotics, their functions and applications.

Figure 2 .
Figure 2. Active metabolites of probiotics, their functions and applications.

Figure 3 .
Figure 3. Effects on the gut microbiota following administration of lactobacillus.

Figure 4 .
Figure 4. Understanding probiotic functional properties using an omics approach.

Table 1 .
Probiotic properties and applications of Ligilactobacillus salivarius.

Table 2 .
Effect of different Ligilactobacillus salivarius strains on the intestinal microbiota.

Table 3 .
Potential functional properties of Ligilactobacillus salivarius revealed using omics techniques.