Foodborne pathogens such as Staphylococcus aureus and Listeria monocytogenes pose serious public health risks due to their antibiotic resistance and biofilm-forming abilities. This study aimed to characterise the antimicrobial activity of the exopolysaccharide (EPS) produced by Lentilactobacillus buchneri KU200793 against S. aureus and L. monocytogenes, as well as its mechanism of action. Purified EPS from L. buchneri KU200793 appeared as a white, water-soluble powder with a yield of 575 mg/L in de Man, Rogosa, and Sharp broth supplemented with 5

Lactic acid bacteria (LAB) harbor diverse enzymes that facilitate carbohydrate metabolism and have immense biotechnological potential. This study examined the genome of a LAB strain isolated from kimchi, Levilactobacillus brevis strain B3A1, for the identification of glycoside hydrolase (GH) enzymes that have the potential to hydrolyze lignocellulose-derived materials. Whole-genome sequencing revealed that B3A1 harbors a genome of 2.47 Mbp and two plasmids. Phylogenetic analysis showed that it is closely related to other food-associated Lv. brevis strains. Survey of the B3A1 genome revealed 28 putative GH genes belonging to 15 families. To examine the potential of B3A1 for saccharification, three GH enzymes were chosen and recombinantly expressed in Escherichia coli: two GH 43 β-xylosidases (LbXyl43A and LbXyl43B) and a GH 3 β-glucosidase (LbBgl3). The recombinant enzymes were purified and characterized. The LbXyl43A and LbXyl43B showed substrate preference towards p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-β-D-arabinofuranoside. The optimal β-xylosidase activities of these enzymes were achieved at pH 7.0 (both enzymes) and 25°C and 40°C, respectively. Both enzymes demonstrated activity towards xylobiose and xylan hemicelluloses substrates. LbBgl3 exhibited specificity toward p-nitrophenyl-β-D-glucopyranoside, with optimal activity at pH 9.0 and 45°C. The LbBgl3 demonstrated thermal stability at up to 45°C. It showed high activity towards cellobiose. Structural analysis of the three GH enzymes confirmed the presence of hallmark domains in their respective GH families and conserved catalytic residues. This study aims to contribute to the understanding of LAB-derived GH enzymes through genomic surveys and recombinant protein expression and their potential use in industrial applications.

Introduction Environmentally friendly pork production is crucial to the pig industry, where the enhancement of growth performance and feed efficiency with reduced environmental impacts is favored. This study aimed to evaluate the effect that protease supplementation in a low crude protein diet has on the growth performance, nutrient digestibility, nitrogen retention, and gut microbiome in growing pigs.Methods Eighty pigs (Landrace & times; Yorkshire & times; Duroc; 24.72 kg) were selected, and based on initial body weight and sex, randomly allocated to one of the following dietary treatments: H, 16% crude protein (CP) diet; L, 14% CP diet; L+E1, low CP diet + 0.1% protease; and L+E2, low CP diet + 0.2% protease. Each treatment comprised four replicates with five pigs per pen.Results Pigs fed a low CP diet with protease supplementation showed a significantly higher body weight, average daily gain, and feed conversion ratio than those fed a high CP diet. In addition, ammonia emissions were lower in the L+E2 group than in the L group. Based on microbiome analysis, the L+E1 and L+E2 groups showed an increased Firmicutes-to-Bacteroidota ratio and elevated expression of pathways related to carbohydrate metabolism, coinciding with higher concentrations of short-chain fatty acids, such as butyrate and propionate, which support intestinal health. Additionally, the predicted function of the microbiota of pigs fed protease exhibited reduced nitrogen and sulfur metabolism, suggesting a potential reduction in excreted odorous compounds.Discussion These findings highlight the role of protease in enhancing growth performance and feed efficiency by modulating gut microbial composition and metabolic functions and reducing noxious gas emissions. Also, potential feed-cost savings are inferred from lower CP formulation.

High stocking density in pig production is associated with stress and reduced growth, but its effects on nasal and fecal bacterial communities, gut metabolites, and predicted functions are not fully understood. We hypothesized that high stocking density disrupts host–microbiota interactions, contributing to stress and impaired growth. In this study, 98 finishing pigs [(Landrace × Yorkshire) × Duroc; 21 weeks old] were assigned to high stocking density (HD: 5 pigs per pen, 0.58 m2 per pig, k = 0.029) or control (CON: 3 pigs per pen, 0.97 m2 per pig, k = 0.049) in a completely randomized block design. Growth, blood profile, and coughing index were monitored over 4 weeks. Nasal and fecal microbiota were characterized using full-length 16S rRNA sequencing, fecal metabolites were quantified by high-performance liquid chromatography, and correlations with physiological parameters were analyzed. After 4 weeks, pigs in HD group had lower final body weight (P = 0.048), average daily gain (P = 0.035), and average daily feed intake (P = 0.048), alongside elevated cortisol (P = 0.037) and coughing index (P < 0.001). Alpha-diversity of the nasal microbiota was reduced (Shannon Entropy, P = 0.009), while the fecal microbiota showed non-significant decrease. Beta-diversity revealed distinct clustering between CON and HD in nasal (P = 0.003; R2 = 0.021) and fecal (P = 0.005; R2 = 0.042) bacterial communities. Taxonomic profiling of the HD group revealed enrichment of Rothia nasimurium, Psychrobacter faecalis, and Globicatella sp. in nasal microbiota, and increased Clostridium sensu stricto 1 and Terrisporobacter, with marked decline in Lactobacillus, particularly L. amylovorus and L. johnsonii, in fecal microbiota (P < 0.05). Metabolism-related functional pathways were altered in both bacterial communities. Fecal lactate was decreased (P = 0.005) and was positively correlated with tumor necrosis factor-α (TNF-α) (P = 0.048). Biomarkers in the HD group were negatively correlated with growth performance, whereas those in the CON group showed positive correlations (P < 0.05). High stocking density impaired growth, increased blood cortisol and stress-related coughing, and altered nasal and fecal bacterial communities in finishing pigs, highlighting potential impacts of crowding on pig productivity.

Noxious odors from swine farms pose significant health and environmental concerns. Hydrogen sulfide (H2S) is one of the major compounds causing noxious odors. Using biological inoculants offers a promising, efficient, and cost-effective method to reduce H2S emissions from these farms. Several studies have found that Priestia megaterium (formerly Bacillus megaterium) can help mitigate the production of H2S. However, detailed knowledge regarding its mechanisms of action is still limited. In this study, the potential of novel strain of P. megaterium, strain S188, for the reduction of H2S in swine manure has been demonstrated. The artificial manure-based assay revealed that P. megaterium S188 significantly reduced the headspace concentration of H2S. Moreover, transcriptomic analysis revealed that exposure to H2S upregulated the expression of several cysteine and methionine biosynthesis-related genes in P. megaterium S188. Furthermore, treatment of swine manure with P. megaterium S188 was associated with significant modulation of the microbiota in the manure. Overall, this study provides insights into the putative mechanisms underlying the reduction in H2S levels mediated by P. megaterium S188 and its potential as a biological inoculant for H2S mitigation in swine farms. • Priestia megaterium S188 has shown the ability to lower H2S from swine manure. • Transcriptomic analysis revealed upregulation of H2S-related biosynthesis genes. • P. megaterium S188 treatment is associated with changes in the manure microbiota.

ABSTRACTThe coronavirus disease 2019 (COVID‐19) is a fatal disease caused by severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). To date, several vaccines have been developed to combat the spread of this virus. Mucosal vaccines using food‐grade bacteria, such as Lactobacillus spp., are promising strategies for developing safe and effective vaccines against SARS‐CoV‐2. In this study, we designed a non‐GMO surface‐displayed SARS‐CoV‐2 spike S1 epitope on Limosilactobacillus fermentum‐derived bacteria‐like particles (BLPs). After that, we evaluated its efficacy to induce immune responses in immunocompetent mice. Moreover, we examined the influence of oral immunisation on the gut microbiome and microbiota metabolites. Twenty‐eight 6‐week‐old male C57BL/6 mice were orally immunised with the following: PBS (control), Lm. fermentum‐derived BLPs only, BLPs displaying SARS‐CoV‐2 spike S1‐2, or BLPs displaying SARS‐CoV‐2 spike S1‐3 epitopes. Our results showed that mucosal immunisation of mice with surface‐displayed SARS‐CoV‐2 spike epitopes provoked high‐level secretory IgA and systemic IgG production. Moreover, the immunisation exhibited a Th1‐like immune response, characterised by an elevated IgG2a‐to‐IgG1 ratio and high antiviral IFN‐γ production. In addition, we observed gut microbiome modulation and increased butyrate production in immunised mice. Overall, the use of Lm. fermentum‐derived BLPs and the anchor CshA to display SARS‐CoV‐2 spike S1epitopes is a promising novel strategy in developing a cost‐effective, non‐GMO mucosal vaccine alternative against SARS‐CoV‐2.

The display of enzymes on bacterial surfaces is an interesting approach for immobilising industrially important biocatalysts. In recent years, non-recombinant surface display using food-grade bacteria, such as lactic acid bacteria (LAB), have gained interest because of their safety, simplicity, and cost-effectiveness. β-Xylosidase is one of the many biocatalytic enzymes targeted for immobilisation due to its key role in the complete saccharification of lignocellulosic biomass, including xylan hemicellulose. Recently, the xylose-tolerant β-xylosidase, LfXyl43, was identified in Limosilactobacillus fermentum. LfXyl43 is capable of producing xylose from the degradation of xylo-oligosaccharides (XOS) and beechwood xylan. This study aimed to immobilise this new biocatalyst on the surface of LAB-derived bacteria-like particles (BLP) and investigate its applicability and reusability in the degradation of xylan hemicellulose. Additionally, the influence of the anchor position and the presence of linker peptides on the display and activity of the β-xylosidase was investigated. Four expression vectors were constructed to express different anchor-xylosidase fusion proteins. Upon expression and purification, all anchor-xylosidase fusion proteins were active towards the artificial substrate p-nitrophenyl-β-D-xylopyranoside. In addition, all anchor-xylosidase fusion proteins were successfully displayed on the surface of BLP. However, only the β-xylosidases with linker peptide showed hydrolytic activity after immobilisation on BLP. BLP displaying β-xylosidases demonstrated high activity against XOS and beechwood xylan, thereby producing high amounts of xylose. Moreover, the immobilised enzyme demonstrated reusability across several bioconversion cycles. Overall, this study highlights the potential industrial application of surface-displayed β-xylosidase for the effective degradation of lignocellulosic biomass.

The degradation of hemicellulose, including xylan, is an important industrial process as it provides cheap and sustainable source of economically valuable monosaccharides. β-xylosidases are key enzymes required for complete degradation of xylan and are used in the production of monosaccharides, such as xylose. In this study, we characterized a novel, xylose-tolerant β-xylosidase isolated from Limosilactobacillus fermentum SK152. Sequence analysis and protein structure prediction revealed that the putative β-xylosidase belongs to the glycoside hydrolase (GH) family 43 subfamily 11 and exhibits high homology with other characterised GH43 β-xylosidases from fungal and bacterial sources. The putative β-xylosidase was named LfXyl43. The catalytic residues of LfXyl43, which are highly conserved among GH 43 β-xylosidases, were predicted. To fully characterise LfXyl43, the gene encoding it was heterologously expressed in Escherichia coli. Biochemical characterisation revealed that the recombinant LfXyl43 (rLfXyl43) was active against artificial and natural substrates containing β-1,4-xylanopyranosyl residues, such as p-nitrophenyl-β-D-xylopyranoside (pNPX) and oNPX. Moreover, it demonstrated weak α-L-arabinofuranosidase activity. The optimal activity of rLfXyl43 was obtained at pH 7.0 at 35°C. rLfXyl43 could degrade xylo-oligosaccharides, such as xylobiose, xylotriose, and xylotetraose, and showed hydrolysing activity towards beechwood xylan. Moreover, rLfXyl43 demonstrated synergy with a commercial xylanase in degrading rye and wheat arabinoxylan. The activity of rLfXyl43 was not affected by the addition of metal ions, chemical reagents, or high concentrations of NaCl. Notably, rLfXyl43 exhibited tolerance to high xylose concentrations, with a Ki value of 100.1, comparable to that of other xylose-tolerant GH 43 β-xylosidases. To our knowledge, this is the first β-xylosidase identified from a lactic acid bacterium with high tolerance to salt and xylose. Overall, rLfXyl43 exhibits great potential as a novel β-xylosidase for use in the degradation of lignocellulosic material, especially xylan hemicellulose. Its high activity against xylo-oligosaccharides, mild catalytic conditions, and tolerance to high xylose concentrations makes it a suitable enzyme for industrial applications.

Ginseng (Panax) is a perennial herb with medicinal properties found in Asia and North America. Ginseng extracts contain several compounds, such as ginsenosides, which have therapeutic properties and have been extensively studied. Because of their deglycosylated nature, minor ginsenosides exhibit more potent bioactive properties than their parent ginsenosides. However, untreated ginseng extracts contain low levels of bioactive minor ginsenosides. Thus, converting major ginsenosides to minor ginsenosides using various methods, including microbial bioconversion, is required. Probiotic bacteria such as lactic acid bacteria and bifidobacteria are safe and excellent agents for bioconverting ginsenosides. Numerous studies have demonstrated the application of probiotic bacteria to produce minor ginsenosides; however, a comprehensive discussion focusing on using probiotics in ginsenoside bioconversion has been lacking. Therefore, this review investigates the application of probiotic bacteria to produce minor ginsenosides. Moreover, improving the health-promoting properties of ginseng with the help of probiotics is also reviewed.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, characterized by vomiting and watery diarrhea, and has a major economic burden on the global pork industry. The objective of this study was to develop a new surface display system for PEDV antigens fused with a cell wall-anchoring domain, using Lactiplantibacillus plantarum as a host. The B-cell epitopes of the PEDV membrane (M) protein epitopes, designated as M1, M2, and M3, generated by online prediction tools, were stably expressed and displayed in Lp. plantarum SK156 and verified by immunofluorescence microscopy. Stimulation of porcine intestinal epithelial cells (IPEC-J2) with the surface displayed M epitopes resulted in elevated production of interferon (IFN)-γ and interleukin (IL)-10. To investigate the immunogenicity of the M epitopes, 30 female BALB/c mice (n = 6 per group) were orally administered Lp. plantarum displaying M1, M2, or M3 epitopes and wild-type Lp. plantarum, or phosphate buffered saline (PBS). On days 21 and 35, mice immunized with the M1 epitope showed consistently high levels of antigen-specific secretory immunoglobulin (Ig)-A and serum IgG, demonstrating the induction of both mucosal and humoral immune responses. However, no changes were observed in the cytokine profiles of the immunized mice. To the best of our knowledge, this is the first report of PEDV M epitopes on the surface of lactic acid bacteria (LAB). Our findings highlight the immunogenic potential of the PEDV M protein and the possibility of further research on the development of a Lactobacillus-based oral vaccine against PEDV infection.

Exopolysaccharides (EPSs) have recently emerged as significant substances due to their remarkable biological properties. This research aimed to analyze EPS B151 extracted from Levilactobacillus brevis B151 and evaluate its biological activities. EPS B151 was obtained at a yield of 438 mg/L. Structural analysis using the Congo red method and Fourier transform infrared spectroscopy (FTIR) revealed a random coil structure and characteristic typical carbohydrate peaks (O-H, C-H, C = O, and C-O-C), along with a pyranose ring containing both α- and β-glycosidic bonds. Scanning electron microscopy (SEM) revealed a porous, irregular, and loosely compact morphology. High-pressure liquid chromatography (HPLC) analysis indicated that EPS B151 is a heteropolysaccharide composed of mannose, ribose, glucosamine, glucuronic acid, glucose, galactose, and the rare sugar fucose. This unique composition is the first reported for an L. brevis-derived EPS. EPS B151 demonstrated significant antimicrobial activity against Staphylococcus aureus KFRI00188 and Listeria monocytogenes ATCC3596, inhibiting growth and cell viability in a dose-dependent manner, with bactericidal effects confirmed by propidium iodide staining and SEM analysis. It also inhibited biofilm formation (up to 58.09 ± 2.97

The postweaning period is stressful for pigs due to changes in their environment and diet. The occurrence of diarrhea at this stage is high. Growth promoters such as antibiotics and zinc oxide (ZnO) have been used not only to reduce postweaning diarrhea but also to improve the growth performance of weaning pigs. It has also been shown that the growth performance of pigs is negatively associated with bile salt hydrolase (BSH) in the gut. Antibiotic growth promoters (AGP) and ZnO administration have demonstrated effective inhibition of BSH, which is linked to enhanced growth performance in pigs. Therefore, this study evaluated the effects of a plant-based supplement, green tea extract, with butyric acid, and vitamin K (GBK), on growth performance, nutrient digestibility, fecal score, blood profile, and BSH activity of the gut microbiota of weaning pigs. Here, 192 crossbred weaning pigs [(Yorkshire × Landrace) × Duroc], aged 21 d, were subjected to a 4-wk-long feeding experiment. Pigs were divided into six treatments (n = 32 per treatment, 8 pens per treatment). After feeding, the average daily gain (ADG) and gain-to-feed (G:F) ratio improved linearly with GBK supplementation. During the feeding period, diarrhea was not observed in the treatment groups, and the fecal scores of the weaned pigs were not affected. Supplementation had no negative impact on the blood profile parameters of weaned pigs, including white blood cell count, red blood cell count, lymphocyte percentage, and blood urea nitrogen. Moreover, supplementation of GBK decreased the TNF-α and IL-6, while immunoglobulins (IgA, IgG) increased. In addition, GBK reduced the abundance of gut microbiota with BSH activity, including Clostridium sensu stricto 6, the Clostridia vadinBB60 group, Marvinbryantia, Muribaculaceae, and Enterococcus. Correlation analysis revealed that gut microbiota function related to secondary bile acid biosynthesis had a strong negative correlation with ADG, average daily feed intake, and G:F ratio of the pigs. The combination of green tea, butyric acid, and vitamin K is an effective alternative to AGP and ZnO for improving growth performance, feed efficiency, and diarrhea score of weaned pigs. In addition, this feeding strategy had a modulatory effect on the gut microbiome, altering BSH activity associated with improved growth performance in weaning pigs.

Exopolysaccharides (EPSs) are large-molecular-weight, complex carbohydrate molecules and extracellularly secreted bio-polymers released by many microorganisms, including lactic acid bacteria (LAB). LAB are well known for their ability to produce a wide range of EPSs, which has received major attention. LAB-EPSs have the potential to improve health, and their applications are in the food and pharmaceutical industries. Several methods have been developed and optimized in recent years for producing, extracting, purifying, and characterizing LAB-produced EPSs. The simplest method of evaluating the production of EPSs is to observe morphological features, such as ropy and mucoid appearances of colonies. Ethanol precipitation is widely used to extract the EPSs from the cell-free supernatant and is generally purified using dialysis. The most commonly used method to quantify the carbohydrate content is phenol–sulfuric acid. The structural characteristics of EPSs are identified via Fourier transform infrared, nuclear magnetic resonance, and X-ray diffraction spectroscopy. The molecular weight and composition of monosaccharides are determined through size-exclusion chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography. The surface morphology of EPSs is observed via scanning electron microscopy and atomic force microscopy, whereas thermal characteristics are determined through thermogravimetry analysis, derivative thermogravimetry, and differential scanning calorimetry. In the present review, we discuss the different existing methods used for the detailed study of LAB-produced EPSs, which provide a comprehensive guide on LAB-EPS preparation, critically evaluating methods, addressing knowledge gaps and key challenges, and offering solutions to enhance reproducibility, scalability, and support for both research and industrial applications.

Abstract Antibiotic growth promotor (AGP) and zinc oxide (ZnO) has been shown to inhibit bile salt hydrolase (BSH) activity, resulting in improved animal growth performance and reduced post-weaning diarrhea but, their usage should be minimized at pig farms. The objective of this study was to evaluate the effects of green tea and butyric acid (hereafter called Gutluk) as alternative BSH inhibitors on the growth performance, nutrient digestibility, fecal score, blood profile, and gut microbiota of weaning pigs. A total of 192 weaned pigs [(Landrace × Yorkshire) × Duroc)] were divided into six groups. Dietary treatments were NC, Basal diet; PC1, Basal diet + 0.2% antibiotics (colistin); PC2, Basal diet + 2,000ppm ZnO; TRT1, Basal diet + 0.05% Gutluk; TRT2, Basal diet + 0.10% Gutluk; TRT3, Basal diet + 0.20% Gutluk. After 4 wk of the trial, fresh fecal samples were collected from the rectum for microbiome analysis. TRT3 improved average daily gain (ADG) and gain to feed ratio (G:F) at wk 3 and 4. The digestibility, white blood cell, red blood cell, lymphocyte percentage, blood urea nitrogen, fecal score and diarrhea score of weaned pigs were not affected by the addition of Gutluk. The treatment groups showed decreased TNF-α and IL-6 (P < 0.05) while immunoglobulin (IgA, IgG) increased (P < 0.05). GutLuk significantly enriched Bifidobacterium (P < 0.01). Conversely, GutLuk reduced the abundance of Clostridium sensu stricto 6, Marvinbryantia, and Clostridia vadinBB60 group, and marginally reducing Muribaculaceae, Enterococcus, and Staphylococcus. The correlation analysis also revealed that the abundance of secondary bile acid biosynthesis had a strong positive correlation with Marvinbryantia, Enterococcus, Clostridium sensu stricto 6, Lactobacillus, and Clostridia vadinBB60 (P < 0.001). The ADG of the pigs also exhibited a slight negative correlation with secondary bile acid biosynthesis. These results indicated that the supplementation of GutLuk had an inhibitory effect on the BSH activity of certain gut microbiota. Consequently, A combination of green tea and butyric acid can be effective alternatives of AGP and ZnO in early weaning pig diet to improve growth performance, immune response, oxidative stress, diarrhea score and gut microbiota.

Lactic acid bacteria (LAB) expressing foreign antigens have great potential as mucosal vaccines. Our previous study reported that recombinant Lactiplantibacillus plantarum SK156 displaying SARS-CoV-2 spike S1 epitopes elicited humoral and cell-mediated immune responses in mice. Here, we further examined the effect of the LAB-based mucosal vaccine on gut microbiome composition and function, and gut microbiota-derived metabolites. Forty-nine (49) female BALB/c mice were orally administered L. plantarum SK156-displaying SARS-CoV-2 spike S1 epitopes thrice (at 14-day intervals). Mucosal immunization considerably altered the gut microbiome of mice by enriching the abundance of beneficial gut bacteria, such as Muribaculaceae, Mucispirillum, Ruminococcaceae, Alistipes, Roseburia, and Clostridia vadinBB60. Moreover, the predicted function of the gut microbiome showed increased metabolic pathways for amino acids, energy, carbohydrates, cofactors, and vitamins. The fecal concentration of short-chain fatty acids, especially butyrate, was also altered by mucosal immunization. Notably, alterations in gut microbiome composition, function, and butyrate levels were positively associated with the immune response to the vaccine. Our results suggest that the gut microbiome and its metabolites may have influenced the immunogenicity of the LAB-based SARS-CoV-2 vaccine.

Among various biological agents, bacteriocins are important candidates to control Listeria monocytogenes which is a foodborne pathogen.In this study, a novel bacteriocin, named agilicin C7, was isolated from Ligilactobacillus agilis C7 showing inhibitory activity against L. monocytogenes.Agilicin C7 biosynthesis gene was characterized by bioinformatics analyses and heterologously expressed in Escherichia coli for further study.The anti-listeria activity of recombinant agilicin C7 (r-agilicin C7) was lost by proteases and α-amylase, suggesting that agilicin C7 is a glycoprotein.r-Agilicin C7 has wide pH and thermal stability and is also stable in various organic solvents.It destroyed L. monocytogenes by damaging the integrity of the cell envelope.These properties of r-agilicin C7 indicate that agilicin C7 is a novel amylase-sensitive anti-listerial Class IId bacteriocin.Physicochemical stability and inhibitory activity against L. monocytogenes of r-agilicin C7 suggest that it can be applied to control L. monocytogenes in the food industry, including dairy and meat products.

Characterization of novel amylase-sensitive, anti-listerial Class IId bacteriocin, agilicin C7 1 produced by Ligilactobacillus agilis C7 2 3 4 Jeong Min Yoo, Ji Hoon Song, Robie Vasquez, In-Chan Hwang, Jae Seung Lee, and Dae-Kyung Kang 5 Department of Animal Biotechnology, Dankook University, Cheonan 31116, Republic of Korea 6 7 8 Running title: Characterization of anti-listerial agilicin C7 9 10 11 * Corresponding author 12 Name: Dae-Kyung Kang 13 Postal address: Department of Animal Biotechnology, College of Biotechnology and Bioengineering, 14 Dankook University, Cheonan 31116, Republic of Korea 15 E-mail: dkkang@dankook.ac.kr 16 Phone: +82-41-550-3655 17 Fax: +82-41-550-3655 18 19 20

Both crude protein (CP) and probiotics can modulate the gut microbiome of the host, thus conferring beneficial effects. However, the benefits of low CP diet supplemented with multispecies probiotics on gut microbiome and its metabolites have not been investigated in pigs. Thus, we investigated the combinatory effects of low CP diet supplemented with multispecies probiotics on gut microbiome composition, function, and microbial metabolites in growing pigs. In total, 140 6 week-old piglets (Landrace × Yorkshire × Duroc) were used in this study. The pigs were divided into four groups with a 2 × 2 factorial design based on their diets: normal-level protein diet (16% CP; NP), low-level protein diet (14% CP; LP), NP with multispecies probiotics (NP-P), and LP with multispecies probiotics (LP-P). After the feeding trial, the fecal samples of the pigs were analyzed. The fecal scores were improved by the probiotic supplementation, especially in LP-P group. We also observed a probiotic-mediated alteration in the gut microbiome of pigs. In addition, LP-P group showed higher species richness and diversity compared with other groups. The addition of multispecies probiotics in low CP diet also enhanced gut microbiota metabolites production, such as short-chain fatty acids (SCFAs) and polyamines. Correlation analysis revealed that Oscillospiraceae UCG-002, Eubacterium coprostanoligenes, Lachnospiraceae NK4A136 group, and Muribaculaceae were positively associated with SCFAs; and Prevotella, Eubacterium ruminantium, Catenibacterium, Alloprevotella, Prevotellaceae NK3B31 group, Roseburia, Butyrivibrio, and Dialister were positively correlated with polyamines. Supplementation with multispecies probiotics modulated the function of the gut microbiome by upregulating the pathways for protein digestion and utilization, potentially contributing to enriched metabolite production in the gut. The results of this study demonstrate that supplementation with multispecies probiotics may complement the beneficial effects of low CP levels in pig feed. These findings may help formulate sustainable feeding strategies for swine production.

Limosilactobacillus mucosae LM1 (LM1) is previously isolated from the intestine of piglets, but its potential as a probiotic supplement has not yet been assessed in growing pigs. In this study, we analyzed the probiotic effect of LM1 on the growth performance, apparent total tract digestibility (ATTD) of nutrients, immune properties, intestinal morphology, and gut microbiota and their metabolites in growing pigs. The experiment included 145 Duroc × (Landrace × Yorkshire) pigs (average body weight: 21.21 ± 1.14 kg) distributed into five treatment groups. The pigs were fed either a control diet (CON), or the control diet supplemented with incremental doses of LM1, namely low-dose LM1 (LL, 8.3 × 108 CFU/kg), moderate-low dose LM1 (ML, 4.2 × 109 CFU/kg), moderate-high dose LM1 (MH, 8.3 × 109 CFU/kg), and high-dose LM1 (HH, 2.1 × 1010 CFU/kg) for 42 d. On d 42, 12 pigs from each of the CON and MH groups were slaughtered. The results indicated that the ATTD of nitrogen (N, P = 0.038) was improved with MH supplementation. In addition, increasing dose of LM1 improved the immune response in pigs by reducing serum pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-alpha) and increasing anti-inflammatory cytokines (interleukin-10). Pigs fed with MH LM1 also had higher jejunal villus height and ileal villus height: crypt depth ratio, demonstrating improved intestinal morphology. Moreover, moderate-high LM1 supplementation enriched SCFA-producing taxa such as Lactobacillus, Holdemanella, Peptococcus, Bifidobacterium, Eubacterium_hallii_group, and Lachnospiraceae_AC2044_group, which correlated positively with increased fecal levels of butyrate and iso-valerate. These results strongly suggest the probiotic potential of LM1 on growing pigs. Overall, the current study provides insights on the use of L. mucosae LM1 as a novel livestock probiotic to improve pig gut health.

Recent studies have demonstrated the potential of surface display technology in therapeutic development and enzyme immobilization. Utilization of lactic acid bacteria in non-GMO surface display applications is advantageous due to its GRAS status. This study aimed to develop a novel, non-GMO cell wall anchoring system for lactic acid bacteria using a cell-surface hydrolase (CshA) from Lactiplantibacillus plantarum SK156 for potential industrial and biomedical applications. Analysis of the CshA revealed that it does not contain any known classical anchor domains. Although CshA lacks a classical anchor domain, it successfully displayed the reporter protein superfolder GFP on the surface of several lactic acid bacteria in host dependent manner. CshA-sfGFP fusion protein was displayed greatest on Limosilactobacillus fermentum SK152. Pretreatment with trichloroacetic acid further enhanced the binding of CshA to Lm. fermentum. The binding conditions of CshA on pretreated Lm. fermentum (NaCl, pH, time, and temperature) were also optimized, resulting in a maximum binding of up to 106 CshA molecules per pretreated Lm. fermentum cell. Finally, this study demonstrated that CshA-decorated pretreated Lm. fermentum cells tolerates gastrointestinal stress, such as low pH and presence of bile acid. To our knowledge, this study is the first to characterize and demonstrate the cell-surface display ability of CshA. The potential application of CshA in non-GMO antigen delivery system and enzyme immobilization remains to be tested.