A novel, non-GMO surface display in Limosilactobacillus fermentum mediated by cell surface hydrolase without anchor motif.

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.

Alterations in the gut microbiome and its metabolites are associated with the immune response to mucosal immunization with Lactiplantibacillus plantarum-displaying recombinant SARS-CoV-2 spike epitopes in mice.

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.

Characterization of Novel Amylase-Sensitive, Anti-Listerial Class IId Bacteriocin, Agilicin C7 Produced by Ligilactobacillus agilis C7.

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.

Multispecies probiotic supplementation in diet with reduced crude protein levels altered the composition and function of gut microbiome and restored microbiome-derived metabolites in growing pigs.

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.

Bioinformatics and its role in the study of the evolution and probiotic potential of lactic acid bacteria.

Due to their numerous well-established applications in the food industry, there have been many studies regarding the adaptation and evolution of lactic acid bacteria (LAB) in a wide variety of hosts and environments. Progress in sequencing technology and continual decreases in its costs have led to the availability of LAB genome sequence data. Bioinformatics has been central to the extraction of valuable information from these raw genome sequence data. This paper presents the roles of bioinformatics tools and databases in understanding the adaptation and evolution of LAB, as well as the bioinformatics methods used in the initial screening of LAB for probiotic potential. Moreover, the advantages, challenges, and limitations of employing bioinformatics for these purposes are discussed.

Gut microbiome-produced metabolites in pigs: a review on their biological functions and the influence of probiotics.

The gastrointestinal tract is a complex ecosystem that contains a large number of microorganisms with different metabolic capacities. Modulation of the gut microbiome can improve the growth and promote health in pigs. Crosstalk between the host, diet, and the gut microbiome can influence the health of the host, potentially through the production of several metabolites with various functions. Short-chain and branched-chain fatty acids, secondary bile acids, polyamines, indoles, and phenolic compounds are metabolites produced by the gut microbiome. The gut microbiome can also produce neurotransmitters (such as γ-aminobutyric acid, catecholamines, and serotonin), their precursors, and vitamins. Several studies in pigs have demonstrated the importance of the gut microbiome and its metabolites in improving growth performance and feed efficiency, alleviating stress, and providing protection from pathogens. The use of probiotics is one of the strategies employed to target the gut microbiome of pigs. Promising results have been published on the use of probiotics in optimizing pig production. This review focuses on the role of gut microbiome-derived metabolites in the performance of pigs and the effects of probiotics on altering the levels of these metabolites.

Dietary Supplementation of Limosilactobacillus mucosae LM1 Enhances Immune Functions and Modulates Gut Microbiota Without Affecting the Growth Performance of Growing Pigs.

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-1ß 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.

Cudrania tricuspidata Combined with Lacticaseibacillus rhamnosus Modulate Gut Microbiota and Alleviate Obesity-Associated Metabolic Parameters in Obese Mice.

The aim of the presented study was to investigate the synbiotic effects of L. rhamnosus 4B15 and C. tricuspidata extract administration on the gut microbiota and obesity-associated metabolic parameters in diet-induced obese mice. Thirty-one 6-week-old male C57BL/N6 mice were divided into five diet groups: normal diet (ND, n = 7) group; high-fat diet (HFD, n = 6) group; probiotic (PRO, n = 5) group; prebiotic (PRE, n = 7) group; and synbiotic (SYN, n = 6) group. After 10 weeks, the percent of fat mass, serum triglyceride, and ALT levels were significantly reduced in SYN-fed obese mice, compared with other treatments. SYN treatment also modulated the abundance of Desulfovibrio, Dorea, Adlercreutzia, Allobaculum, Coprococcus, unclassified Clostridiaceae, Lactobacillus, Helicobacter, Flexispira, Odoribacter, Ruminococcus, unclassified Erysipelotrichaceae, and unclassified Desulfovibrionaceae. These taxa showed a strong correlation with obesity-associated indices. Lastly, the SYN-supplemented diet upregulated metabolic pathways known to improve metabolic health. Further investigations are needed to understand the mechanisms driving the synbiotic effect of C. tricuspidata and L. rhamnosus 4B15.

Multispecies probiotics alter fecal short-chain fatty acids and lactate levels in weaned pigs by modulating gut microbiota.

Short-chain fatty acids (SCFAs) are metabolic products produced during the microbial fermentation of non-digestible fibers and play an important role in metabolic homeostasis and overall gut health. In this study, we investigated the effects of supplementation with multispecies probiotics (MSPs) containing Bacillus amyloliquefaciens, Limosilactobacillus reuteri, and Levilactobacillus brevis on the gut microbiota, and fecal SCFAs and lactate levels of weaned pigs. A total of 38 pigs weaned at 4 weeks of age were fed either a basal diet or a diet supplemented with MSPs for 6 weeks. MSP administration significantly increased the fecal concentrations of lactate (2.3-fold; p < 0.01), acetate (1.8-fold; p < 0.05), and formate (1.4-fold; p < 0.05). Moreover, MSP supplementation altered the gut microbiota of the pigs by significantly increasing the population of potentially beneficial bacteria such as Olsenella, Catonella, Catenibacterium, Acidaminococcus, and Ruminococcaceae. MSP supplementation also decreased the abundance of pathogenic bacteria such as Escherichia and Chlamydia. The modulation of the gut microbiota was observed to be strongly correlated with the changes in fecal SCFAs and lactate levels. Furthermore, we found changes in the functional pathways present within the gut, which supports our findings that MSP modulates the gut microbiota and SCFAs levels in pigs. The results support the potential use of MSPs to improve the gut health of animals by modulating SCFAs production.