Anaerobic flora, Selenomonas ruminis sp. nov., and the bacteriocinogenic Ligilactobacillus salivarius strain MP3 from crossbred-lactating goats

This study aimed to examine the distribution of anaerobic bacteria in the rumen fluid of Thai crossbred goats and to screen potential probiotic strains capable of producing antimicrobial compounds and inhibiting bacteria that cause milk fat depression. Thirty-four strains of bacteria from the rumen fluid were divided into 13 groups within 12 genera based on 16S rRNA gene sequences. The RF1-5 and RF5-12 were identified as Streptococcus luteliensis and Bacillus licheniformis, respectively, and demonstrated non-ropy exopolysaccharide. Furthermore, mPRGC5T was closely related to Selenomonas caprae JCM 33725 T (97.8% similarity) based on 16S rRNA gene sequences. It exhibited low average nucleotide identity, digital DNA–DNA hybridization, and average amino acid identity values with related type strains ranging from 84.9 to 86.0%, 21.3 to 21.8%, and 73.8 to 76.1%, respectively. The genotypic and phenotypic characteristics of mPRGC5T strongly support this strain as a new species of the genus Selenomonas for which the name Selenomonas ruminis mPRGC5T was proposed. The type strain is mPRGC5T (= JCM 33724 T = KCTC 25177 T). Ligilactobacillus salivarius MP3 showed antibacterial activity against Cutibacterium acnes subsp. acnes DSM 1897 T and Kocuria rhizophila MIII. The enterolysin A cluster gene was identified in its genome. The auto-aggregation of L. salivarius MP3 was 93.6 ± 0.2%. Additionally, co-aggregation of L. salivarius MP3 with C. acnes DSM 1897 T and K. rhizophila MIII had 92.2 ± 3.4% and 87.3 ± 4.5%, respectively. The adhesion capacity of strain MP3 was 76.11 ± 2.2%. Probiogenomic analysis revealed that L. salivarius MP3 was nonhazardous to animal supplementation and included acid- and bile-tolerant ability. However, strain MP3 contained three antibiotic resistance genes. Thus, the supplementation of L. salivarius MP3 could increase the milk fat content by suppressing C. acnes DSM 1897 T with antibiotic resistance gene horizontal transfer awareness.

Group VII only contained strain RF2-14.This strain exhibited a rod shape and was Gram-positive.The phenotypes presented the survival at pH 3 and 45 °C.Arginine and nitrate were both negative.Lactate and acetate were the main products of glucose utilization.The strain RF2-14 was identified as Lactobacillus amylovorus using 16S rRNA sequencing, which showed a similarity of 99.8%.
Group VIII contained two cocci strains (RF1-4 and RF2-5).Both strains grew at wide-range temperatures ranging from 15 to 45 °C.The hemolysis results revealed alpha hemolysis.The ability to use the various carbohydrates and end products from glucose fermentation was presented in Table S2.The RF2-5 strain, representative of this group, showed 99.7% similarity with Enterococcus avium ATCC 14025 T .
Group X included two facultative tetracoccal strains (RF6-12 and RF2-1).Two strains survived in the pH range from 3 to 9 and temperature range from 15 to 50 °C.These two strains converted glucose to lactate and acetate.Furthermore, no hemolytic activity was found for either strain.Based on the 16S rRNA gene sequence (99.50-99.83%),these bacteria were identified as Pediococcus acidilactici.
Group XII included strain MP4 and RF6-15.Both strains were strictly anaerobes.These two rods were distinct from the others because they generated gas products from glucose fermentation.Similarly, lactate and acetate were the major products of glucose utilization.The two strains were able to proliferate at pH 3, pH 9, 15-45 °C, and 3% NaCl.No positive hemolysis was found.MP4 and RF6-15 were identified as Sharpea azabuensis based on the 16S rRNA gene sequence (99.7% similarity).
Group XIII included the curved-rod anaerobic bacterium (strain mPRGC5).This strain exhibited different phenotypes from the other strains (Table S2).The strain mPRGC5 was grown in the presence of L-arabinose, D-cellobiose, fructose, D-galactose, D-glucose, lactose, D-mannose, D-maltose, salicin, sucrose, and D-xylose.Lactate, acetate, and propionate were produced from glucose fermentation.Based on the16S rRNA gene sequence (97.88% similarity, 1407 bp) with Selenomonas caprae JCM 33724 T , it was lower than the species delineation value.Hence, this strain could be a novel species of the genus Selenomonas.
The distribution of LAB in the rumen was similar to that in the dairy bovine rumen according to a previous study.According to Hu, et al. 9 , the study isolated potential LAB probiotics from rumen fluid, which included Enterococcus avium, Streptococcus lutetiensis, and Streptococcus equinus.These species were identical to those found in this study.In addition, another study identified the LAB isolated from the rumen fluid and feces of dairy cows as Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Limonilactabacillus fermentum 10 .L. amylovorus, L. rhamnosus, L. salivarius, L. oris, and P. acidilactici were identified as members of the family Lactobacillaceae by the LAB strains in the present study.Therefore, the family Lactobacillaceae was the dominant LAB isolated from the rumen fluid.The feed composition is an additional variable that can alter the rumen microbiome.According to Bo Trabi, et al. 11 , compared with the low-grain TMR diet, the high-grain TMR diet fed to fattening lambs increased the number of simple sugar-fermentative bacteria, such as Dialister, Megasphaera, Parafannyhessea, and Sharpea.Similarly, the donors in this study were fed a high-grain TMR diet (60% concentrate).Additionally, a review of ruminomics data from 1,000 cows revealed the core rumen isolates, including Treponema bryantii for cellulose degradation, Prevotella for hemicellulose degradation, Parafannyhessea umbonata for soluble sugar utilization and Selenomonas ruminantium for secondary fermentation product utilization 12 .The distributions of Parafannyhessea sp. and Selenomonas sp. in the rumen fluid of goats were corroborated by a previous study.Furthermore, Mitsuokella jalaludinii was found primarily in the rumen of Malaysian cattle, and the bacteria produced phytase, an enzyme commonly used in poultry production 13 .In this study, two isolates of M. jalaludinii were isolated from the rumen of goats, confirming the habitat of these bacteria in the rumen.Thus, the rumen of Thai crossbred lactating goats is a valuable isolation source of LAB.However, the bacterial diversity was dependent on the type of donor diet.
The strain mPRGC5 T was found to grow well on PYG agar.This strain was grown under strictly anaerobic conditions.After 2 days of incubation, the colonies were white, circular, convex, and opaque (1.0-3.0 mm in diameter).The cells exhibited curved rods (Fig. S2), which were characterized by their gram-negative and nonspore-forming ability.The dimensions of these cells ranged from 1.6 to 3.8 µm in length and 0.4-0.5 µm in width.Strain mPRGC5 T exhibited the motile peritrichous flagella required for locomotion.The identification of the flagellar assembly proteins MotB, FliM/N/R/E/I, FlaA, FlgB/C/D/H, FleN, and FlhA/B/F also confirmed the presence of flagella.The different phenotypic characteristics of strain mPRGC5 T and closely related type strains are described in Table 1.The strain mPRGC5 T was grown at pH 6.0-10.0,22-39 °C, and 1% (w/v) NaCl.The nitrate reduction, methyl red test, and starch hydrolysis were positive.Contrary, catalase, indole production, hydrogen sulfide production, the Voges-Proskauer test, gelatin liquefaction, and casein hydrolysis were negative.The acid produced from carbohydrate fermentation was shown in Table S4.Similar patterns of carbohydrate utilization were observed between strain mPRGC5 T and closely related type strains.In addition, the carbohydrate utilization was further genomically supported.The genomic data of strain mPRGC5 T contained the several carbohydrate-associated genes, such as L-arabinose utilization (EC: 5.1.17).This strain demonstrated vigorous acid phosphatase activity and weak alkaline phosphatase, esterase, esterase lipase, and naphthol-AS-BI-phosphohydrolase activity.The cellular fatty acid composition of strain mPRGC5 T consisted of C 18:1 ω9c (17.5%),C 16:1 ω9c (9.1%), C 11:0 (7.0%), and C 15:1 ω8c (6.7%) (Table 2).The presence of C 18:1 ω9c and C 16:1 ω9c was commonly observed in strain mPRGC5 T and in the closely related type strains.The fatty acid C 11:0 exhibited similarity primarily with S. ruminantium subsp.lactilytica JCM 6582 T .Furthermore, strain mPRGC5 T contained phosphatidylethanolamine, three unidentified aminophospholipids, an unidentified ninhydrin positive glycolipid, a phospholipid, and two unidentified lipids (Fig. S3).The final products from glucose fermentation were acetate, propionate, valerate, hexanoate, and heptanoate.The strains of S. caprae JCM 33725 T , S. ruminantium subsp.lacticolytica JCM 6582 T , and S. ruminantium subsp.ruminantium DSM 2150 T were found to synthesize acetic acid, propionic acid, and valeric acid.In addition, the ability of the mPRGC5 T strain to produce n-heptanoic acid was similar to that of S. caprae JCM 33725 T .
In addition to the results of previous genotypic analysis, mPRGC5 T could be distinguished from the closely related species S. caprae JCM 33725 T , S. ruminantium subsp.lactilytica JCM 6582 T , and S. ruminantium subsp.ruminantium DSM 2150 T based on its phenotypic characteristics.According to phenotypic characteristics, chemotaxonomic characteristics, in silico G + C content, 16S rRNA gene sequence analysis, ANI, AAI, dDDH, and the genomic annotation findings, the strain mPRGC5 T represents a novel species of the genus Selenomonas, for which the name Selenomonas ruminis sp.nov. is proposed.

Volatile fatty acid production
All the strains produced acetic acid as the main product of glucose fermentation (Table 3).Valeric acid is also a common product from all strains except Ligilactobacillus salivarius RF6-11, Parafannyhessea umbonata MP18, and Parafannyhessea umbonata MP20.The VFA results are shown in Table S2.Propionic acid was produced from the bacterial strains RF4-12, MP3, RF6-11, RF6-5, RF4-11, mPRGC15-3, RF3-23, MP14-2, MP16-1, mPRGC17 and MP29.In addition, glucose fermentation revealed that strains MP3, RF6-11, and RF3-23 synthesized butyric acid as their end products.Several strains, including strains RF5-12, MP3, RF6-11, and RF3-23, produced caproic  A strictly anaerobic or facultative anaerobic rumen microorganism produces end products consumed directly by the host or indirectly by other microorganisms.VFAs were major microbial end products.Some of these VFAs, such as propionate and butyrate, can be absorbed across the rumen wall and serve as an energy supply for ruminants 14 .In addition, microbial VFAs were considered alternatives to petroleum based VFAs in commercial chemical production due to their sustainability.VFAs have also been used as precursors in anaerobic fermentation to produce biogas and flavor enhancers in the food industry 15 .Furthermore, acetate played a role in the production of terephthalic acid and can also be utilized in polyethylene terephthalate production.Valerate is primarily used in the cosmetics and food and beverage industries.Likewise, the WHO considers valeric acid to be a safe food additive, and it is primarily used as an intermediate substance in the production of flavors and fragrances, agricultural chemicals, and pharmaceuticals 16 .In this study, all the strains isolated from the rumen of Thai goats produced acetic acid through the fermentation of simple sugars.Furthermore, thirty-one strains of bacteria from the goat rumen generate valeric acid as a byproduct of glucose fermentation.Therefore, the goat rumen is presumed to be a beneficial source for identifying potential VFA-producing microorganisms for use in commercial industries and livestock.

Spot resistant study for antimicrobial activity and exopolysaccharide production
All the strains were screened for antimicrobial activity against the indicator strains.Ligilactobacillus salivarius MP3 had inhibitory effects on Kocuria rhizophila MIII (55.75 mm in diameter) and C. acnes subsp.acnes DSM 1897 T (30.82 mm in diameter) (Fig. S4).and septicemia in immunodeficient patients, invasive surgery, and catheter placement.Nevertheless, several strains of K. rhizophila widely exhibited antimicrobial resistance 18 .L. salivarius was the potential tool for limiting pathogenic growth in this study.Guerrero Sanchez, et al. 19 reported that L. salivarius could prevent enteropathogenic infection in piglets and calves.In addition, L. salivarius inhibited Staphylococcus aureus by secreting the LysM-containing peptidoglycan-binding protein and protein peptidase M23B 20 .Regarding the milk lipid depression of ruminants caused by C. acnes, no specific L. salivarius target has been reported for C. acnes in animals.Therefore, L. salivarius is a potential bacterium in terms of bactericidal activity, particularly against K. rhizophila and C. acnes.
In the present study, EPSs were produced from S. luteliensis RF1-5 and B. licheniformis RF1-12.The EPS production of Bacillus licheniformis RF5-12 was comparable to that reported in previous studies of Asgher, et al. 21and Petrova, et al. 22 .The antioxidant capacity and antimicrobial activity of S. luteliensis strain RF1-5 and B. licheniformis strain RF1-12 should be investigated in further research.According to these findings, the goat rumen is a possible source of antimicrobial compound-producing bacteria that could be used as alternatives in animal supplements and agricultural industries.

The probiotic characterization of strain MP3
The complete genome of Ligilactobacillus salivarius MP3 encompassed 1,876,541 bp with a GC content of 32.6% (Fig. 3).Microorganisms with lower GC contents were typically characterized by reduced energy consumption and metabolic rates, resulting in enhanced genome stability.Strain MP3 displayed the highest average nucleotide   S5).The survival rates from gastrointestinal conditions, including pH 2, pH 3, 0.3% bile, and 1.0% bile, were 56.00%, 78.59%, 90.63%, and 86.73%, respectively.Additionally, several genes in strain MP3 supported the resistance in acid and bile conditions, as shown in Table S6.Furthermore, strain MP3 demonstrated the ability to thrive under extreme pH conditions.In addition to acid and bile tolerance, the expression of genes associated with probiotic attributes was also altered in strain MP3.Notably, it carried the bsh gene, which is known for its involvement in cholesterol-lowering activity.Furthermore, the genome of strain MP3 harbored putative genes, including clpB, IspA, and tuf, which play vital roles in immune system modulation.The critical regulatory genes involved in bile salt tolerance in L. salivarius, including beta-N-acetylhexosaminidase, PTS mannose transporter subunit IIB, and type 1 glutamine amidotransferase, play crucial roles in peptidoglycan synthesis and the PTS system 23 .MP3 exhibited 23.0 ± 0.6% cell surface hydrophobicity.The auto-aggregation ability of strain MP3 was 93.6 ± 0.2% (Fig. 4A).In addition, the co-aggregation abilities of strain MP3 with K. rhizophila MIII and of strain MP3 with C. acnes subsp.acnes were 87.3 ± 4.5% and 92.2 ± 3.4%, respectively (Fig. 4B).Furthermore, the potential adhesion of strain MP3 to Caco-2 cells was 76.1 ± 2.2%.Adhesion to the host was supported by representative genes such as srtA, dltA, Mub, groS, and glnA (Table S6).
The bacterial strains MP3 and LGG exhibited a hazy to greenish area (α-hemolysis) around their growth on sheep blood agar, indicating their lack of hemolytic activity.Hemolysis is an acknowledged attribute of pathogenic microorganisms that enhances their capacity to induce disease.On the other hand, it is essential to consider the lack of hemolytic activity while selecting probiotics 27 .For the genomic risk assessment, the pathogenicity of L. salivarius MP3 compared to that of L. rhamnosus GG and L. salivarius CGMCC20700 was presented in Table S7.The results demonstrated that MP3, LGG, and LS were non-human pathogens.The antibiotic resistance genes (ARGs) in strain MP3 were VanT, tet(M), fexB, and ErmC, similar to those in the probiotic strain L. salivarius CGMCC20700, in which the VanT gene was found.The antibiotic susceptibility of strain MP3 displayed resistance to amoxicillin, ceftazidime, clindamycin, erythromycin, kanamycin, tetracycline, oxacillin, bacitracin, trimethoprim, amoxiclav, vancomycin and carbenicillin.On the other hand, the sensitivity of the MP3 strain to antibiotic disc appeared in ampicillin, cefotaxime, ciprofloxacin, gentamicin, streptomycin, imipenem, neomycin, and rifampicin.
The ARGs of strain MP3 correlated with the antimicrobial susceptibility phenotype.The genes associated with increased resistance to clindamycin, erythromycin, and tetracycline were ermC and tet(M).Similarly, L. salivarius CGMCC20700 was reported tetracycline (tetM, tetL) and macrolide (ermC) resistance-related genes 28 .Tetracycline and erythromycin are commonly used for livestock treatment.Mobile genetic elements are frequently linked to genetic determinants that contribute to resistance to these antibiotics 29 .Furthermore, strain MP3 displayed a resistance phenotype to trimethoprim and vancomycin.Previous research has shown that Lactobacillus strains demonstrated high intrinsic resistance to trimethoprim and vancomycin 29 .Conversely, another study of L. salivarius revealed that this bacterium was susceptible to ampicillin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.However, this bacterium also exhibited kanamycin and vancomycin resistance.Even if the bovine-derived probiotic L. salivarius strain consisted of antibiotic resistance genes, the study indicated a low potential for horizontal transfer to cows 30 .
According to the BAGEL4 analysis, strain MP3 contained enterolysin A at contig 3.3 (starting at 172,580 and ending at 193,087) (Fig. 3).Contig 3.3 contained enterolysin A as a core protein, one ABC Arginine transport ATP-binding protein, and several open reading frames (ORFs) (Fig. 5).Enterolysin A was a class III bacteriocin that was first discovered in Enterococcus faecalis LMG 2333.Enterolysin A was found in L. salivarius, E. hirae 32 , L. fermentum, and L. paracasei 33 .Additionally, enterolysin A suppressed the growth of Fusobacterium nucleatumand Clostridium difficile 34 .Therefore, the antimicrobial activity of strain MP3 was demonstrated through the addition of lactic acid, acetic acid, hydrogen peroxide, and bacteriocin to K. rhizophila and C. acnes subsp.acnes.The antimicrobial activity of strain MP3 showed its potential to antagonize pathogenic bacteria, including L. monocytogenes ATCC 19115, S. aureus ATCC 25923, E. coli ATCC 25922, B. cereus ATCC 6633 and K. rhizophila MIII (Fig. S7).Raw milk can be contaminated with L. monocytogenes, E. coli, and B. cereus, which can cause foodborne illnesses in humans and illness in dairy cattle [35][36][37] .The inhibitory effects of L. salivarius on S. aureus, E. coli, and L. monocytogenes were similar to those described in previous studies [38][39][40] .The utilization of MP3 could be an effective method for treating listeriosis and gastrointestinal illness by alleviating the potential negative impacts of subclinical carriage of L. monocytogenes, E. coli, and B. cereus on the gut health of animals.The production, induction, and characterization of enterolysin A from MP3 should be further investigated.
In conclusion, L. salivarius MP3 represented a bacteriogenic strain supported by the ability to adhere to host cells, tolerate GIT conditions, and produce antimicrobial substances.The antagonistic effect of strain MP3 on the milk fat depression caused by C. acnes was reported in this study.As a specific host probiotic, strain MP3 should be further evaluated for the risk of lateral antibiotic resistance gene transfer in vitro before supplementation of dairy ruminants.

Animal sample collection and lactic acid bacterial isolation
The animal experiment was evaluated by the ethical authority, received approval from the Chulalongkorn University Animal Care and Use Committee, Thailand (CUA CUC; Approval No. 2231044) and was conducted in accordance with the relevant regulations of the authorities and the ARRIVE guidelines (https:// www.arriv eguid elines.org).
The six rumen fluid samples were obtained from donor lactating goats (Saanen × Thai native).The donors received a total mixed ration diet of 40:60 (roughage: concentrate) using corn silage as a roughage source.The rumen fluid samples were provided by the Veterinary Student-training Center, Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Nakhon Pathom.The rumen fluid samples were collected at 4 h post-feeding.The samples were transferred to the laboratory in an anaerobic container by using AnaeroPack® (MITSUBISHI Gas Chemical America, Inc.).The rumen samples were prepared for bacterial isolation in an anaerobic chamber (Bactron, Sheldon Manufacturing, Inc.).After that, the rumen samples were passed through a 3-layer cheesecloth to remove the feed particles.All the rumen samples were tenfold serially diluted in phosphate buffer solution.The dilutions at 10 -4 to 10 -8 were spread on De Man Rogosa and Sharpe (MRS; Difco®) agar, peptone yeast extract glucose (PYG) agar, and modified peptone yeast extract fructose (mPYF) agar 41 and incubated in an anoxic atmosphere (N 2 90%: H 2 5%: CO 2 5%) at 39 °C for 48 h.The colony was restreaked on MRS agar supplemented with 0.3% (w/v) CaCO 3 for screening of lactic acid bacteria and incubated for 48 h at 39 °C.The lactic acid bacterial isolates were maintained in MRS media.All colonies that exhibited a clear zone were picked and stored at -20 °C in 15% (v/v) glycerol stock.

Identification methods
Phenotypic characterization was performed for all the LAB strains.The cell morphology, colony appearance, and culture characteristics of the LAB strains were determined on MRS agar after incubating for 48 h.The physiological and biochemical characteristics were evaluated at different temperatures (15 °C, 30 °C, and 45 °C), pH (3.0, 6.0 and 9.0), and NaCl concentrations (3%, 6%, and 8%).The acid production from L-arabinose, D-cellobiose, D-fructose, D-galactose, D-lactose, D-maltose, D-mannitol, D-mannose, D-melibiose, D-raffinose, L-rhamnose, D-ribose, salicin, D-sorbitol, D-sucrose, D-trehalose and D-xylose was determined according to previous methods Tanasupawat, et al. 42 .Additionally, nitrate reduction, catalase, gas generation (CO 2 ), and arginine dihydrolase test were determined.All the phenotypes were analyzed in the hierarchical cluster using SPSS v29.The 16S rRNA gene of the LAB strains was prepared and purified following the protocol of the AccuPrep Genomic DNA Extraction Kit (Bioneer Pacific, Australia).DNA sequencing was carried out at Macrogen, Inc. (Korea) with universal primers (27F, 1492R).The sequence similarity values related to the strains were compared on the EzBioCloud server 43 .The phylogenetic tree was constructed using the neighbor-joining (NJ) method via MEGA X 44 .
For novel species, phenotypic characteristics were evaluated.After 48 h of culture on PYG agar, the cell morphology, colony appearance, and colony pigmentation were investigated.Cell morphology was observed under a scanning electron microscope (JEOL, Japan) after cultivation at 39 °C for 48 h.The flagella were stained following the method of Forbes 45 after incubation at 37 °C for 18 h.The ability to grow at different temperatures (20, 22, 30.37, 39, 45, and 48 °C), pH (4.0-10 with 0.5 interval scale), and NaCl concentration (1-10% w/v) were determined in PYG broth.Acid production from carbohydrates was evaluated by peptone yeast extract medium supplemented with 1% (w/v) substrate.The carbohydrate substrates in this experiment consisted of amygdalin, D-adonitol, L-arabinose, D-arabitol, L-arabitol, D-cellobiose, dulcitol, esculin ferric citrate, erythritol, fructose, D-galactose, glycerol, inositol, inulin, D-lactose, D-maltose, D-mannose, D-melezitose, D-melibiose, D-raffinose, L-rhamnose, D-ribose, D-saccharose, salicin, D-sorbitol, L-sorbose, starch, D-tagatose, D-trehalose, xylitol, and D-xylose.The physiological characteristics of mPRGC5 T were observed the starch hydrolysis, casein hydrolysis, nitrate reduction, arginine hydrolysis, gelatin liquefaction, indole production, methyl red, and Voges-Proskauer test and hydrogen sulfide production.The enzymatic activities were determined by using an API® ZYM kit (bioMérieux SA).Chemotaxonomy was analyzed by using freeze-dried cells cultivated under optimal growth conditions.The fatty acid profile was extracted and detected by gas chromatography according to Sasser 46 .Polar lipids were extracted from freeze-dried cells and identified by two-dimensional TLC following the protocol of Minnikin, et al. 47 .For novel taxa, the concentrations of acetate, propionate, butyrate, valerate, iso-butyrate, and iso-valerate were determined using gas chromatography (GC-FID).
The genomic DNA of the 16S rRNA gene was extracted and sequenced.The contig DNA sequence was generated by using BioEdit software version 7.2.5.The contig sequences of the closely related strains were compared with those of the EzBioclound server.The evolutionary relationships among the bacterial strains were represented by phylogenetic trees.The construction of phylogenetic trees was based on the neighbor-joining, maximumlikelihood, and maximum-parsimony algorithms using the MEGA X program.The program was set to calculate by the Kimura-2-parameter model with 1000 replications.Thresholds of 98.7%, 94.5%, and 86.5% 16S rRNA gene sequence identity were indicated for novel species, genera, and families, respectively 48 .The assembled draft genomes of MP3 and mPRGC5 were deposited in the GenBank database under accession numbers JAN-FQI000000000 and VTOY00000000, respectively.The overall genome related index (OGRI) was calculated as the average nucleotide identity (ANI) value, the digital DNA-DNA hybridization (dDDH) value, and the average amino acid identity (AAI) score to evaluate the species distinct from the nearest type strains.ANI and dDDH values were analyzed using the JSpecies WS web-based tool 49 and the Genome-to-Genome Distance Calculator (GGDC 3.0) with the BLAST + method of formula 2 for the draft genome 50 .The cutoff values of ANI and dDDH, considered to indicate similar species, are > 95% and 70%, respectively 51 .The AAI value was calculated via a web-based service 52 .The genome phylogenetic tree was reconstructed by the type (strain) genome server 50 .The functional genes were annotated using rapid annotation using subsystem technology (RAST) 53 , the DDBJ fast annotation and submission tool (DFAST) 54 , and the KBase application 55 .The metabolic pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) with BlastKOALA 56 .Moreover, the circular genome was visualized and explored using Proksee 57 .

Volatile fatty acid and exopolysaccharide production
The supernatants of all the strains inoculated in MRS broth were collected and stored at −20 °C for volatile fatty acid analysis.Gas chromatography with flame-ionization detection (Agilent 7890B) was applied to evaluate the concentrations of volatile fatty acids, including acetate, propionate, butyrate, and valerate.The carrier gas was helium, and the flow rate was 1.0 mL.min −1 .In addition, the flows of hydrogen gas and air zero were set at 30 and 400 mL.min −1 .At the inlet, the sample was split 10:1, and the injection volume was 1 µL.The oven temperature was programmed as described by Darwin, et al. 58 .Standard calibration was performed using a volatile fatty acid mixture analytical standard (Merck®, Germany).
Glucose in the MRS agar medium was replaced with sucrose (2%) for exopolysaccharide (EPS) screening 59 .The LAB strains were streaked on EPS agar and incubated at 37 °C under anaerobic conditions for 48 h.The EPS-producing strain exhibited a ropy phenotype, as detected by touching the colony with an inoculated loop.The colonies showed an unbreakable strand or presented a mucoid non-ropy phenotype (slime formation, glistening, smooth colony) on the plate.

Spot resistant study for antimicrobial activity
The protocol followed the method of Luo, et al. 60 .The 24 h inoculum of bacterial strains in MRS broth was used in this test.Anaerobes were incubated at 37 °C in an anaerobic chamber.All the isolated strains were centrifuged at 7000 × g at 4 °C for 10 min to harvest the cell pellet, after which the cells were resuspended in phosphate buffer solution to adjust the OD600 to 1.0.For screening antimicrobial activity, the target strain was Cutibacterium acnes subsp.acnes DSM 1897 T , which was the milk fat depression bacterium found in dairy ruminants.C. acnes was cultured in PYG broth for 72 h at 37 °C under anaerobic conditions, after which the bacterial cells were collected as described above.The antimicrobial activities of the strains against Lactobacillus sakei subsp.sakei JCM 1157 T , E. coli ATCC 25922 T , Staphylococcus aureus ATCC 25923 T , Streptococcus agalactiae 1611 and K. rhizophila MIII were screened.MRS medium was used for the preculture of Lb. sakei subsp.sakei JCM 1157 T and brain heart infusion medium (BHI; Difco, Leeuwarden, The Netherlands) were used to grow the pathogens.Subsequently, Lb. sakei subsp.sakei JCM 1157 T and the pathogen strains were incubated at 37 °C for 24, 48 and 72 h.First, 100 µl of C. acnes subsp.acnes DSM 1897 T , Lb. sakei subsp.sakei JCM 1157 T and pathogens were swabbed on PYG, MRS, and BHI plates to make up the basal layer.Consequently, 2 µL of bacterial suspension was added to the center of the plate.All the strains were analyzed in triplicate and incubated at 37 °C for 24 h in the anaerobic chamber.The spot inhibition zone was measured as the diameter (mm) of the clear zone around the bacterial spot.All the strains that presented inhibitory activity were selected for further study.

Evaluation of the probiotic properties of strain MP3
Strain MP3 was inoculated in MRS broth at 37 °C for 48 h under anaerobiosis.The samples were collected every 3 h to evaluate the cell concentration via a spectrophotometer at 600 nm, and the colony-forming units per milliliter (CFU/mL) were estimated through plate-serial dilution spotting.The growth curve was plotted to determine the exponential phase (log phase).
The acid and bile tolerance were determined following the modified method of Kingkaew, et al. 61 .The active culture of strain MP3 was inoculated into MRS broth adjusted to pH 2 and pH 3 or into MRS broth supplemented with 0.3% and 1.0% (w/v) bile salt.Subsequently, all the inoculates were incubated at 37 °C for 3 h.The viable cell counts were estimated by plate-serial dilution spotting, and all the plates were incubated at 37 °C for 24 h.The viable cell count was expressed as CFU/mL.The culture in normal MRS was used as control.
The cell surface hydrophobicity of strain MP3 was evaluated according to Cele, et al. 62 .This method was used to investigate hydrophobicity.The strain MP3 was cultured overnight in MRS broth at 37 °C in an anaerobic chamber.The cells were centrifuged (7,300 × g, 4 °C, 10 min) and washed in phosphate buffer solution (PBS).After that, the cell pellet was resuspended in 1.5 mL of PBS, and the optical density at 600 nm (OD 0 ) was determined.The cell suspension was mixed with ethyl acetate at a ratio of 1:1.Then, the solution was vortexed for 2 min and incubated at room temperature for 1 h to separate the aqueous and organic phases.Finally, the aqueous phase sample was collected, and the optical density was measured at 600 nm (OD 1 ).The hydrophobicity was calculated according to the following Eq.(1): The auto-aggregation and co-aggregation assays were performed following the method of Li, et al. 63 .In brief, overnight culture of strain MP3 in MRS medium was used to collect the cells by centrifugation at 7,300 rpm for 10 min.The cells were washed 3 times with phosphate-buffered saline (PBS) and resuspended in 2 mL of PBS.The initial OD 600 (A 0 ) was evaluated.Subsequently, the bacterial solution was incubated at room temperature for 24 h.Afterwards, 100 µL of the upper part of the bacterial solution was taken at 2, 4, 6, 8, 10, and 24 h after standing to measure the optical density at OD 600 (A 1 ).The auto-aggregation rate was calculated according to Eq. 2. The bacterial solutions for co-aggregation were prepared as described above.The 2 mL of strain MP3 was mixed with 2 mL of the indicator strains (C.acnes subsp.acnes DSM 1897 T and K. rhizophila MIII) and incubated at room temperature.The OD 600 was measured after 0, 2, 4, 6, 8, 10, and 24 h of incubation.The percentage of co-aggregation was determined via Eq. 3.
An adhesion assay was performed to investigate the adhesion of strain MP3 to Caco-2 cells 61 .Caco-2 cells (5 × 10 5 cells/mL) were used in this study.Cells of strain MP3 were prepared by overnight culture in MRS broth.The overnight culture was centrifuged at 7000× g and 4 °C for 7 min, after which the cell pellet was resuspended in fresh warm Dulbecco modified Eagle Minimum Essential Medium (DMEM) (N 0 ).After the addition of the suspension of strain MP3 to the plate, three replicates were incubated at 37 °C in a 5% CO 2 incubator for 90 min.Finally, the Caco-2 cells were washed with PBS and then lysed cells with 0.05% of Triton-X100 solution.The MP3 inoculum (N 0 ) and adherent cells with Caco-2 cells (N 1 ) were evaluated via the spot-plate technique.The percentage of adhesion capability was calculated using Eq. ( 4): The hemolytic activity was measured to assess its safety.The MP3 and LGG strains were streaked onto blood agar plates containing 5% (v/v) sheep blood and incubated at 37 °C for 48 h.Hemolysis was detected in surrounding areas of bacterial growth.Beta (β) hemolysis refers to the presence of a clear zone surrounding bacterial growth, indicating positive activity.On the other hand, the presence of a greenish to brownish zone (α-hemolysis) or the absence of a zone (γ-hemolysis) were classified as non-hemolytic activity.

Figure 1 .
Figure 1.The neighbor-joining phylogenetic tree of representative strains of thirteen groups based on the 16S rRNA gene divided by dendrogram analysis.

Figure 2 .
Figure 2. The phylogenomic tree of the mPRGC5 T strain and its closely related strains was reconstructed via TYGS.
identity (ANI) values compared to L. salivarius DSM 20555 T , with ANIb and ANIm values of 96.9% and 97.3%, respectively.These findings strongly support the classification of strain MP3 as a member of L. salivarius species.The growth pattern of L. salivarius MP3 is illustrated in Fig.S6.Strain MP3 entered the log phase approximately 3 h after incubation, maintaining stable bacterial cell growth for up to 15 h post-incubation, with the pH stabilizing at 4.75.Strain MP3 survived at pH 2, pH 3, 0.3% bile salt, and 1.0% bile salt (Table

Figure 3 .
Figure 3. Circular genomic map of Ligilactobacillus salivarius MP3 visualized using a CG viewer.The open reading frame (ORF) is shown.The inner circle shows the genome size (1,876,541 bp).

Figure 4 .
Figure 4.The percentage of auto-aggregation (A) and co-aggregation (B) with Cutibacterium acnes subsp.acnes DSM 1897 T (black triangle) and K. rhizophila MIII (black circle) by strain MP3.The data are presented as the means ± standard deviations.

Table 3 .
Volatile fatty acid produced by isolates cultivated in PYG media at 37°C under anaerobic conditions for 48 h.−; No detection.