Genome mining of Lactiplantibacillus plantarum PA21: insights into its antimicrobial potential.

BACKGROUND: The dramatic increase of antimicrobial resistance in the healthcare realm has become inexorably linked to the abuse of antibiotics over the years. Therefore, this study seeks to identify potential postbiotic metabolites derived from lactic acid bacteria such as Lactiplantibacillus plantarum that could exhibit antimicrobial properties against multi-drug resistant pathogens. RESULTS: In the present work, the genome sequence of Lactiplantibacillus plantarum PA21 consisting of three contigs was assembled to a size of 3,218,706 bp. Phylogenomic analysis and average nucleotide identity (ANI) revealed L. plantarum PA21 is closely related to genomes isolated from diverse niches such as dairy products, food, and animals. Genome mining through the BAGEL4 and antiSMASH database revealed four bacteriocins in a single cluster and four regions of biosynthetic gene clusters responsible for the production of bioactive compounds. The potential probiotic genes indirectly responsible for postbiotic metabolites production were also identified. Additionally, in vitro studies showed that the L. plantarum PA21 cell-free supernatant exhibited antimicrobial activity against all nine methicillin-resistant Staphylococcus aureus (MRSA) and three out of 13 Klebsiella pneumoniae clinical isolates tested. CONCLUSION: Results in this study demonstrates that L. plantarum PA21 postbiotic metabolites is a prolific source of antimicrobials against multi-drug resistant pathogens with potential antimicrobial properties.

Revealing the dynamics and mechanisms of bacterial interactions in cheese production with metabolic modelling.

Cheese taste and flavour properties result from complex metabolic processes occurring in microbial communities. A deeper understanding of such mechanisms makes it possible to improve both industrial production processes and end-product quality through the design of microbial consortia. In this work, we caracterise the metabolism of a three-species community consisting of Lactococcus lactis, Lactobacillus plantarum and Propionibacterium freudenreichii during a seven-week cheese production process. Using genome-scale metabolic models and omics data integration, we modeled and calibrated individual dynamics using monoculture experiments, and coupled these models to capture the metabolism of the community. This model accurately predicts the dynamics of the community, enlightening the contribution of each microbial species to organoleptic compound production. Further metabolic exploration revealed additional possible interactions between the bacterial species. This work provides a methodological framework for the prediction of community-wide metabolism and highlights the added value of dynamic metabolic modeling for the comprehension of fermented food processes.

pH-dependent regulation of an acidophilic O-acetylhomoserine sulfhydrylase from Lactobacillus plantarum.

Bacteria have two routes for the l-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated l-homoserine is directly converted into l-homocysteine. The reaction using H2S as the second substrate is catalyzed by a pyridoxal 5'-phosphate-dependent enzyme, O-acetylhomoserine sulfhydrylase (OAHS). In the present study, we determined the enzymatic functions and the structures of OAHS from Lactobacillus plantarum (LpOAHS). The LpOAHS enzyme exhibited the highest catalytic activity under the weak acidic pH condition. In addition, crystallographic analysis revealed that the enzyme takes two distinct structures, open and closed forms. In the closed form, two acidic residues are sterically clustered. The proximity may cause the electrostatic repulsion, inhibiting the formation of the closed form under the neutral to the basic pH conditions. We concluded that the pH-dependent regulation mechanism using the two acidic residues contributes to the acidophilic feature of the enzyme. IMPORTANCE: In the present study, we can elucidate the pH-dependent regulation mechanism of the acidophilic OAHS. The acidophilic feature of the enzyme is caused by the introduction of an acidic residue to the neighborhood of the key acidic residue acting as a switch for the structural interconversion. The strategy may be useful in the field of protein engineering to change the optimal pH of the enzymes. In addition, this study may be useful for the development of antibacterial drugs because the l-methionine synthesis essential for bacteria is inhibited by the OAHS inhibitors. The compounds that can inhibit the interconversion between the open and closed forms of OAHS may become antibacterial drugs.

Discovery of a high-performance phage-derived promoter/repressor system for probiotic lactobacillus engineering.

BACKGROUND: The Lactobacillaceae family comprises many species of great importance for the food and healthcare industries, with numerous strains identified as beneficial for humans and used as probiotics. Hence, there is a growing interest in engineering these probiotic bacteria as live biotherapeutics for animals and humans. However, the genetic parts needed to regulate gene expression in these bacteria remain limited compared to model bacteria like E. coli or B. subtilis. To address this deficit, in this study, we selected and tested several bacteriophage-derived genetic parts with the potential to regulate transcription in lactobacilli. RESULTS: We screened genetic parts from 6 different lactobacilli-infecting phages and identified one promoter/repressor system with unprecedented functionality in Lactiplantibacillus plantarum WCFS1. The phage-derived promoter was found to achieve expression levels nearly 9-fold higher than the previously reported strongest promoter in this strain and the repressor was able to almost completely repress this expression by reducing it nearly 500-fold. CONCLUSIONS: The new parts and insights gained from their engineering will enhance the genetic programmability of lactobacilli for healthcare and industrial applications.

Mechanistic study of the differences in lactic acid bacteria resistance to freeze- or spray-drying and storage.

Lactobacillus delbrueckii subsp. bulgaricus and Lactiplantibacillus plantarum are two lactic acid bacteria (LAB) widely used in the food industry. The objective of this work was to assess the resistance of these bacteria to freeze- and spray-drying and study the mechanisms involved in their loss of activity. The culturability and acidifying activity were measured to determine the specific acidifying activity, while membrane integrity was studied by flow cytometry. The glass transitions temperature and the water activity of the dried bacterial suspensions were also determined. Fourier transform infrared (FTIR) micro-spectroscopy was used to study the biochemical composition of cells in an aqueous environment. All experiments were performed after freezing, drying and storage at 4, 23 and 37 °C. The results showed that Lb. bulgaricus CFL1 was sensitive to osmotic, mechanical, and thermal stresses, while Lpb. plantarum WCFS1 tolerated better the first two types of stress but was more sensitive to thermal stress. Moreover, FTIR results suggested that the sensitivity of Lb. bulgaricus CFL1 to freeze-drying could be attributed to membrane and cell wall degradation, whereas changes in nucleic acids and proteins would be responsible of heat inactivation of both strains associated with spray-drying. According to the activation energy values (47-85 kJ/mol), the functionality loss during storage is a chemically limited reaction. Still, the physical properties of the glassy matrix played a fundamental role in the rates of loss of activity and showed that a glass transition temperature 40 °C above the storage temperature is needed to reach good preservation during storage. KEY POINTS: • Specific FTIR bands are proposed as markers of osmotic, mechanic and thermal stress • Lb. bulgaricus CFL1 was sensitive to all three stresses, Lpb. plantarum WCFS1 to thermal stress only • Activation energy revealed chemically limited reactions ruled the activity loss in storage.

Effects of community ecological network construction on physicochemical, microbial, and quality characteristics of inoculated northeast sauerkraut: A new insight in food fermentation processes.

The enhancement of the quality of northeast sauerkraut can be achieved by inoculation with lactic acid bacteria. However, a comprehensive ecological understanding of the intricate dynamic processes involved is currently lacking, which could yield valuable insights for regulating sauerkraut fermentation. This study compares spontaneously sauerkrauts with the sauerkrauts inoculated with autochthonous Lactiplantibacillus plantarum SC-MDJ and commercial L. plantarum, respectively. We examine their physicochemical properties, quality characteristics, bacterial community dynamics, and ecological network interactions. Inoculation with L. plantarum leads to reduced bacterial community richness and niche breadth, but an increase in robustness, interactions, and assembly processes. Notably, there appears to be a potential correlation between bacterial community structure and quality characteristics. Particularly, sauerkraut inoculated with L. plantarum SC-MDJ may produce a sourness more quickly, possibly attributed to the enhanced ecological role of L. plantarum SC-MDJ. This study establishes a foundation for the targeted regulation of sauerkraut fermentation.

Changes of microbial communities and metabolites in the fermentation of persimmon vinegar by bioaugmentation fermentation.

To evaluate the effects of bioaugmentation fermentation inoculated with one ester-producing strain (Wickerhamomyces anomalus ZX-1) and two strains of lactic acid bacteria (Lactobacillus plantarum CGMCC 24035 and Lactobacillus acidophilus R2) for improving the flavor of persimmon vinegar, microbial community, flavor compounds and metabolites were analyzed. The results of microbial diversity analysis showed that bioaugmentation fermentation significantly increased the abundance of Lactobacillus, Saccharomyces, Pichia and Wickerhamomyces, while the abundance of Acetobacter, Apiotrichum, Delftia, Komagataeibacter, Kregervanrija and Aspergillus significantly decreased. After bioaugmentation fermentation, the taste was softer, and the sensory irritancy of acetic acid was significantly reduced. The analysis of HS-SPME-GC-MS and untargeted metabolomics based on LC-MS/MS showed that the contents of citric acid, lactic acid, malic acid, ethyl lactate, methyl acetate, isocitrate, acetoin and 2,3-butanediol were significantly increased. By multivariate analysis, 33 differential metabolites were screened out to construct the correlation between the differential metabolites and microorganisms. Pearson correlation analysis showed that methyl acetate, ethyl lactate, betaine, aconitic acid, acetoin, 2,3-butanediol and isocitrate positively associated with Wickerhamomyces and Lactobacillus. The results confirmed that the quality of persimmon vinegar was improved by bioaugmentation fermentation.

Enhancing the antioxidant capacity and quality attributes of fermented goat milk through the synergistic action of Limosilactobacillus fermentum WXZ 2-1 with a starter culture.

This study evaluated 75 strains of lactic acid bacteria (LAB) isolated from traditional dairy products in western China for their probiotic properties. Among them, Limosilactobacillus fermentum WXZ 2-1, Lactiplantibacillus plantarum TXZ 2-35, Companilactobacillus crustorum QHS 9, and Companilactobacillus crustorum QHS 10 demonstrated potential probiotic characteristics. The antioxidant capacity of these 4 strains was assessed, revealing that L. fermentum WXZ 2-1 exhibited the highest antioxidant capacity. Furthermore, when cocultured with Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, L. fermentum WXZ 2-1 demonstrated a synergistic effect in growth medium and goat milk. To explore its effect on goat milk fermentation, different amounts of L. fermentum WXZ 2-1 were added to goat milk, and its physicochemical properties, antioxidant activity, flavor substances, and metabolomics were analyzed. The study found that the incorporation of L. fermentum WXZ 2-1 in goat milk fermentation significantly improved the texture characteristics, antioxidant capacity, and flavor of fermented goat milk. These findings highlight the potential of L. fermentum WXZ 2-1 as a valuable probiotic strain for enhancing the functionality and desirability of fermented goat milk, contributing to the development of novel functional foods with improved health benefits and enhanced quality attributes.

Lactiplantibacillus plantarum OL5 biosurfactants as alternative to chemical surfactants for application in eco-friendly cosmetics and skincare products.

Cosmetics have been extremely popular throughout history and continue to be so today. Cosmetic and personal care products, including toothpaste, shampoo, lotions, and makeup, are typically made with petroleum-based surfactants. Currently, there is an increasing demand to enhance the sustainability of surface-active compounds in dermal formulations. Biosurfactants, derived from living cells, are considered more environmentally friendly than synthetic surfactants. Thus, the use of biosurfactants is a promising strategy for formulating more environmentally friendly and sustainable dermal products. Biosurfactants have the potential to replace chemical surface-active agents in the cosmetic sector due to their multifunctional qualities, such as foaming, emulsifying, and skin-moisturizing activities.In this study, two glycolipopeptide biosurfactants derived from Lactiplantibacillus plantarum OL5 were used as stabilizing factors in oil-in-water emulsions in the presence of coconut oils. Both biosurfactants increased emulsion stability, particularly in the 1:3 ratio, dispersion, and droplet size. Moreover, the cytotoxicity of the two Lactiplantibacillus plantarum biosurfactants was assessed on B lymphocytes and MCF-7 cells. Overall, the results gathered herein are very promising for the development of new green cosmetic formulations.

Encapsulated Mn-Saturated Lactoferrin as a Safe Source of Manganese Ions for Restoring Probiotic Lactobacillus plantarum.

The growth of Lactobacillus plantarum, a member of the Lactobacillus genus, which plays a crucial role in the bacterial microbiome of the gut, is significantly influenced by manganese ions. They can be safely delivered to the intestines by exploiting the chelating abilities of lactoferrin. The aim of this work was to encapsulate lactoferrin saturated with manganese ions (MnLf) in a system based on the Eudragit® RS polymer to protect protein from degradation and manganese release in the gastric environment. The entrapment efficiency was satisfactory, reaching about 95%, and most importantly, manganese ions were not released during microparticles (MPs) formation. The release profile of the protein from the freshly prepared MPs was sustained, with less than 15% of the protein released within the first hour. To achieve similar protein release efficiency, freeze-drying was carried out in the presence of 10% (w/v) mannitol as a cryoprotectant for MPs frozen at -20 °C. MPs with encapsulated MnLf exhibited prebiotic activity towards Lactobacillus plantarum. More importantly, the presence of equivalent levels of manganese ions in free form in the medium, as well as chelating by lactoferrin encapsulated in MPs, had a similar impact on stimulating bacterial growth. This indicates that the bioavailability of manganese ions in our prepared system is very good.

A New Culture Medium Rich in Phenols Used for Screening Bitter Degrading Strains of Lactic Acid Bacteria to Employ in Table Olive Production.

The olive oil industry recently introduced a novel multi-phase decanter with the "Leopard DMF" series, which gives a by-product called pâté, made up of pulp and olive wastewater with a high content of phenolic substances and without pits. This study aims to create a new culture medium, the Olive Juice Broth (OJB), from DMF pâté, and apply it to select bacteria strains able to survive and degrade the bitter substances normally present in the olive fruit. Thirty-five different bacterial strains of Lactiplantibacillus plantarum from the CREA-IT.PE Collection of Microorganisms were tested. Seven strains characterized by ≥50% growth in OJB (B31, B137, B28, B39, B124, B130, and B51) showed a degradation of the total phenolic content of OJB ≥ 30%. From this set, L. plantarum B51 strain was selected as a starter for table olive production vs. spontaneous fermentation. The selected inoculant effectively reduced the debittering time compared to spontaneous fermentation. Hydroxytyrosol, derived from oleuropein and verbascoside degradation, and tyrosol, derived from ligstroside degradation, were produced faster than during spontaneous fermentation. The OJB medium is confirmed to be useful in selecting bacterial strains resistant to the complex phenolic environment of the olive fruit.

Non-volatile and volatile compound changes in blueberry juice inoculated with different lactic acid bacteria strains.

BACKGROUND: Lactic acid bacteria (LABs) are widely present in foods and affect the flavour of fermented cultures. This study investigates the effects of fermentation with Lactobacillus acidophilus JYLA-16 (La), Lactobacillus plantarum JYLP-375 (Lp), and Lactobacillus rhamnosus JYLR-005 (Lr) on the flavour profile of blueberry juice. RESULTS: This study showed that all LABs strains preferentially used glucose rather than fructose as the carbon source during fermentation. Lactic acid was the main fermentation product, reaching 7.76 g L-1 in La-fermented blueberry juice, 5.86 g L-1 in Lp-fermented blueberry juice, and 6.41 g L-1 in Lr-fermented blueberry juice. These strains extensively metabolized quinic acid, whereas oxalic acid metabolism was almost unaffected. Sixty-four volatile compounds were identified using gas chromatography-ion mobility spectrometry (GC-IMS). All fermented blueberry juices exhibited decreased aldehyde levels. Furthermore, fermentation with La was dominated by alcohols, Lp was dominated by esters, and Lr was dominated by ketones. Linear discriminant analysis of the electronic nose and principal component analysis of the GC-IMS data effectively differentiated between unfermented and fermented blueberry juices. CONCLUSION: This study informs LABs selection for producing desirable flavours in fermented blueberry juice and provides a theoretical framework for flavour detection. © 2023 Society of Chemical Industry.

A comparative study on flaxseed lignan biotransformation through resting cell catalysis and microbial fermentation by ß-glucosidase production Lactiplantibacillus plantarum.

BACKGROUND: Flax lignan has attracted much attention because of its potential bioactivities. However, the bioavailability of secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, depends on the bioconversion by the colon bacteria. Lactic acid bacteria (LAB) with ß-glucosidase activity has found wide application in preparing bioactive aglycone. RESULTS: LAB strains with good ß-glucosidase activity were isolated from fermented tofu. Their bioconversion of flax lignan extract was investigated by resting cell catalysis and microbial fermentation, and the metabolism of SDG by Lactiplantibacillus plantarum C5 following fermentation was characterized by widely targeted metabolomics. Five L. plantarum strains producing ß-glucosidase with broad substrate specificity were isolated and identified, and they all can transform SDG into secoisolariciresinol (SECO). L. plantarum C5 resting cell reached a maximum SDG conversion of 49.19 ± 3.75%, and SECO generation of 21.49 ± 1.32% (0.215 ± 0.013 mm) at an SDG substrate concentration of 1 mM and 0.477 ± 0.003 mm SECO was produced at 4 mm within 24 h. Although sixteen flax lignan metabolites were identified following the fermentation of SDG extract by L. plantarum C5, among them, four were produced following the fermentation: SECO, demethyl-SECO, demethyl-dehydroxy-SECO and isolariciresinol. Moreover, seven lignans increased significantly. CONCLUSION: Fermentation significantly increased the profile and level of flax lignan metabolites, and the resting cell catalysis benefits from higher bioconversion efficiency and more straightforward product separation. Resting cell catalysis and microbial fermentation of flax lignan extract by the isolated ß-glucosidase production L. plantarum could be potentially applied in preparing flax lignan ingredients and fermented flaxseed. © 2024 Society of Chemical Industry.

Quorum sensing effect of chiral d-glutamine on the modulation of the intestinal microbiota of mice by Lactiplantibacillus plantarum A3.

BACKGROUND: Amino acids (AAs) are the building blocks of proteins, but they also serve as biological compounds in biochemical processes, and d-AA isomers are increasingly being recognized as important signaling molecules. As the main organic substrate used by cells in the intestinal tract, the role of the chiral specificity of glutamine is still largely ignored. RESULTS: In a previous study, we found that d-glutamine affected the quorum sensing of Lactiplantibacillus plantarum A3, promoted the release of signaling molecule AI-2 and up-regulated the expression of the LuxS gene. The results showed that when d-glutamine and L. plantarum A3 were simultaneously applied to a mouse model, the diversity and abundance of intestinal flora in both male and female mice were increased. Interestingly, the simultaneous effect of d-glutamine and L. plantarum A3 on the bacterial diversity and abundance of male mice was significantly higher than that of female mice. In addition, the combination of d-glutamine and L. plantarum A3 can improve the host microecology by enhancing the population of Firmicutes such as Lactobacillus and Lachnospiraceae, reducing the population of Fusobacterium and Bacteroides and affecting metabolic pathways such as AA metabolism and transporter transport. CONCLUSION: d-Glutamine, as a signaling molecule, can better stimulate the endogenous d-glutamine synthesis in mice and be utilized by L. plantarum A3. Furthermore, sex differences in the changes of intestinal microflora are also found in this research. This research sheds some light on the adoption of d-AAs combined with lactic acid bacteria in intestinal tract health treatment. © 2024 Society of Chemical Industry.

Lactobacillus plantarum postbiotics trigger AMPK-dependent autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome activation.

We previously found that Lactobacillus plantarum (LP)-derived postbiotics protected animals against Salmonella infection, but the molecular mechanism remains obscure. This study clarified the mechanisms from the perspective of autophagy. Intestinal porcine epithelial cells (IPEC-J2) were pretreated with LP-derived postbiotics (the culture supernatant, LPC; or heat-killed bacteria, LPB), and then challenged with Salmonella enterica Typhimurium (ST). Results showed that LP postbiotics markedly triggered autophagy under ST infection, as indicated by the increased LC3 and Beclin1 and the decreased p62 levels. Meanwhile, LP postbiotics (particularly LPC) exhibited a strong capacity of inhibiting ST adhesion, invasion and replication. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) led to a significant decrease of autophagy and the aggravated infection, indicating the importance of autophagy in LP postbiotics-mediated Salmonella elimination. LP postbiotics (especially LPB) significantly suppressed ST-induced inflammation by modulating inflammatory cytokines (the increased interleukin (IL)-4 and IL-10, and decreased tumor necrosis factor-α (TNF), IL-1ß, IL-6 and IL-18). Furthermore, LP postbiotics inhibited NOD-like receptor protein 3 (NLRP3) inflammasome activation, as evidenced by the decreased levels of NLRP3, Caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Deficits in autophagy resulted in an increase of inflammatory response and inflammasome activation. Finally, we found that both LPC and LPB triggered AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy, and this was further confirmed by AMPK RNA interference. The intracellular infection and NLRP3 inflammasome were aggravated after AMPK knockdown. In summary, LP postbiotics trigger AMPK-mediated autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome in IPEC-J2 cells. Our findings highlight the effectiveness of postbiotics, and provide a new strategy for preventing Salmonella infection.

Investigating the effect of the probiotic Lactobacillus plantarum and the prebiotic fructooligosaccharides on Pseudomonas aeruginosa metabolome, virulence factors and biofilm formation as potential quorum sensing inhibitors.

Pseudomonas aeruginosa (P. aeruginosa) uses quorum sensing signaling (QS) molecules to control the expression of virulence factors and biofilm formation. In this study, the effects of the probiotic's (Lactobacillus plantarum (L. plantarum)) lysate and cell-free supernatant and the prebiotic (Fructooligosaccharides (FOS)) on the levels of P. aeruginosa QS molecules, virulence factors, biofilm density and metabolites were observed. These effects were investigated using exofactor assays, crystal violet and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach. Results showed that in comparison to untreated P. aeruginosa, the L. plantarum cell-free supernatant (5%) and FOS (2%) significantly reduced the levels of the virulence factor pyoverdine (PVD) and several metabolites in the QS pathway including Pseudomonas autoinducer-2 (PAI-2). Metabolomics study revealed that the level of different secondary metabolites involved in the biosynthesis of vitamins, amino acids and the tricarboxylic acid (TCA) cycle were also affected. L. Plantarum was found to have a higher impact on the metabolomics profile of P. aeruginosa and its QS molecules compared to FOS. Lastly, a decrease in the formation of the P. aeruginosa biofilm was observed in a time-dependent pattern upon treatment with either cell-free supernatant of L. plantarum (5%), FOS (2%) or a combination of both treatments (5% + 2%). The latter showed the highest effect with 83% reduction in biofilm density at 72 h incubation. This work highlighted the important role probiotics and prebiotics play as potential QS inhibitors for P. aeruginosa. Moreover, it demonstrated the significant role of LC-MS metabolomics for investigating the altered biochemical and QS pathways in P. aeruginosa.

Lactobacillus Plantarum NC8 and its metabolite acetate alleviate type 1 diabetes via inhibiting NLRP3.

A healthy organism is the result of host-microbiome co-evolution. Microbial metabolites can also stimulate immune cells to reduce intestinal inflammation and permeability. Gut dysbiosis will lead to a variety of autoimmune diseases, such as Type 1 diabetes (T1D). Most of probiotics, such as Lactobacillus casei, Lactobacillus reuteri, Bifidobacterium bifidium, and Streptococcus thermophiles, can improve the intestinal flora structure of the host, reduce intestinal permeability, and relieve symptoms of T1D patients if ingested above probiotics in sufficient amounts. Lactobacillus Plantarum NC8, a kind of Lactobacillus, whether it has an effect on T1D, and the mechanism of it regulating T1D is still unclear. As a member of the inflammatory family, NLRP3 inflammasome can enhance inflammatory responses by promoting the production and secretion of proinflammatory cytokines. Many previous studies had shown that NLRP3 also plays an important role in the development of T1D. When the NLRP3 gene is deleted, the disease progression of T1D will be delayed. Therefore, this study investigated whether Lactobacillus Plantarum NC8 can alleviate T1D by regulating NLRP3. The results demonstrated that Lactobacillus Plantarum NC8 and its metabolites acetate play a role in T1D by co-modulating NLRP3. Lactobacillus Plantarum NC8 and acetate can reduce the damage of T1D in the model mice, even if orally administered them in the early stage of T1D. The number of Th1/Th17 cells in the spleen and pancreatic lymph nodes (PLNs) of T1D mice were significantly reduced by oral Lactobacillus Plantarum NC8 or acetate. The expression of NLRP3 in the pancreas of T1D mice or murine macrophages of inflammatory model were significantly inhibited by treatment with Lactobacillus Plantarum NC8 or acetate. In addition, the number of macrophages in the pancreas were significantly reduced by the treatment with Lactobacillus Plantarum NC8 or acetate. In summary, this study indicated that the regulatory mechanism of Lactobacillus Plantarum NC8 and its metabolite acetate to T1D maybe via inhibiting NLRP3 and provides a novel insights into the mechanism of the alleviated role of probiotics to T1D.

Limosilactobacillus fermentum NCU003089 and Lactiplantibacillus plantarum NCU001261, two probiotics with inhibition of Escherichia coli and Cronobacter sakazakii translocation in vitro.

The subject of this study was to screen lactic acid bacteria (LAB) with pathogen translocation inhibition and investigate the potential inhibition mechanism of it. Pathogens colonized in the intestine could cross the intestinal barrier to access blood circulation, causing severe complications. This study aimed to screen LAB with favorable inhibitory effects on the translocation of enterinvasive Escherichia coli CMCC44305 (E. coli) and Cronobacter sakazakii CMCC45401 (C. sakazakii), which were two common intestinal opportunistic pathogens. After an elaborate screening procedure including adhesion, antibacterial, and translocation assay, Limosilactobacillus fermentum NCU003089 (L. fermentum NCU3089) and Lactiplantibacillus plantarum NCU0011261 (L. plantarum NCU1261) were found to inhibit 58.38% and 66.85% of pathogen translocation, respectively. Subsequently, LAB pre-treatment suppressed the decline in TEER of Caco-2 monolayers caused by pathogens. Meanwhile, L. fermentum NCU3089 significantly inhibited claudin-1, ZO-1, and JAM-1 degradation caused by E. coli, and L. plantarum NCU1261 markedly reduced claudin-1 degradation caused by C. sakazakii. Also, the two LAB strains significantly decreased TNF-α level. In addition, L. fermentum NCU3089 but not L. plantarum NCU1261 tolerated well in the gastrointestinal fluids, and they were both sensitive or intermediate to nine common clinical antibiotics without hemolytic activity. In short, the two LAB strains could inhibit pathogen translocation by competing for adhesion sites, secreting antibacterial substances, reducing inflammatory cytokines levels, and maintaining intestinal barrier integrity. This study provided a feasible solution to prevent pathogen infection and translocation, and the two LAB strains were safe and had potential in food and pharmaceutical applications.

In-silico functional analysis of hypothetical proteins from Lactiplantibacillus plantarum plasmids reveals enrichment of cell envelope proteins.

Lactiplantibacillus plantarum is one of the important species of lactic acid bacterium (LAB) found in diverse environments, with many strains exhibiting probiotic properties. In our previous study, 41.6% of protein families (PFs) encoded by 395 plasmids from several L. plantarum strains were found to be hypothetical proteins with no predicted function. This study aimed at predicting the functions of these 647 hypothetical proteins using 21 different bioinformatics methods. As a result, 160 PFs could be newly annotated. A lower proportion of plasmid-specific functions was annotated as compared to the functions shared between plasmids and chromosomes. Also, hypothetical proteins were less conserved than the annotated proteins across L.plantarum plasmids. Based on the subcellular localization, cell envelope proteins represented the biggest category in the newly annotated proteins. Transporters (112 PFs) which was a part of cell envelop proteins represented the largest functional group. Additionally, 40 and 25 other PFs were predicted to contain signal peptides and transmembrane helices, respectively. We speculate that such hypothetical proteins might be involved in the transport of various chemicals and environmental interactions in L. plantarum. In the future, functional characterization of these proteins through wet-lab experimental approach can provide novel insights into their contribution to the physiology, probiotic properties, and industrial utility of these bacteria.

Lactiplantibacillus plantarum: a new example of inclusion body producing bacteria.

BACKGROUND: Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. RESULTS: To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250-300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. CONCLUSIONS: These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.