Genes encoding bile salt hydrolase differentially affect adhesion of Lactiplantibacillus plantarum AR113.

BACKGROUND: Adhesion is considered important for Lactiplantibacillus to persist in the human gut and for it to exert probiotic effects. Lactiplantibacillus plantarum contains a considerable number and variety of genes encoding bile salt hydrolases (bsh), but their effects on microbial adhesion remain poorly understood. To clarify the effects of four bsh on adhesion, we tried to knock out bsh (Δbsh) of L. plantarum AR113 using the CRISPR-Cas9 method, and compared the growth, auto-aggregation (RAA ), co-aggregation (RCA ), surface hydrophobicity (AHC ) of AR113 wild-type and Δbsh strains and their adhesion abilities to HT29 cells. RESULTS: We first obtained the AR113 Δbsh1,3,2,4 strain with four bsh knocked out. Their growth was significantly slower than the wild-type strain cultured in De Man, Rogosa, and Sharpe medium (MRS) with 3.0 g L-1 glyco- or tauro-conjugated bile acid. Bsh had no significant effect on the growth of ten strains cultured in MRS, but Δbsh1 inhibited their growth when cultured in MRS containing 3.0 g L-1 sodium glycocholate, whereas Δbsh4 instead promoted their growth in MRS with 3.0 g L-1 sodium glycocholate and sodium taurocholate. RCA and RAA were linearly positive for all strains except AR113 Δbsh2,4, and AHC and RAA were negatively correlated for most strains excluding AR113 Δbsh2, with RAA  = 6.38-25.05%, RCA  = 5.17-9.22%, and ACH  = 3.22-47.71%. The adhesion ability of ten strains cultured in MRS was higher than that of strains cultured in MRS with 3.0 g L-1 bovine bile, and it was related to bsh2. CONCLUSION: Bsh differentially affected the adhesion of AR113 series strains. This adds to the available information about substrate-gene-performance, and provides new information to enable engineering to regulate the colonization of Lactiplantibacillus. © 2021 Society of Chemical Industry.

Structure characterization of a pyruvated exopolysaccharide from Lactobacillus plantarum AR307.

A pyruvated exopolysaccharide designated as LPE-1 was isolated and purified from the fermentation broth of Lactobacillus plantarum AR307 and characterized for its chemical structure. The results indicated that LPE-1 contained galactopyranose (Galp) and glucopyranose (Glcp) at a molar ratio of 2: 1. The weight-averaged molecular weight (Mw) of LPE-1 was 605 kDa, with a polydispersity index (PDI) of 1.57, intrinsic viscosity ([ƞ]) of 3.28 dL/g, Mark-Houwink-Sakurada exponent α of 0.65 and gyration of radius (Rg) of 36.10 nm. The results of GC-MS and NMR revealed that pyruvate (Pyr) was found to form cyclic ketals at O-4 and O-6 position of terminal galactopyranose (T-Galp). The backbone of LPE-1 was identified to be consisted of 1,4-ß-D-Glcp (23.19%), 1,4-α-D-Glcp (11.38%) and 1,4,6-ß-D-Galp (12.05%), branched by 1,6-ß-D-Galp (38.88%) at O-6 position of 1,4,6-ß-D-Galp residue and terminated by T-ß-D-Galp (5.60%) or T-ß-D-(4,6-Pyr)-Galp (8.90%). A possible structural unit was proposed for LPE-1 as follows: where Galp* is either T-ß-D-(4,6-Pyr)-Galp or T-ß-D-Galp. The presence of pyruvate group in LPE-1 would play an important role in improving the viscosity and plasticity of dairy products.

Fractionation, chemical characterization and immunostimulatory activity of ß-glucan and galactoglucan from Russula vinosa Lindblad.

Water-extracted polysaccharides from Russula vinosa Lindblad (WRP) were separated into three fractions (WRP-1, WRP-2 and WRP-3) by gradient ethanol precipitation and gel chromatography. Structural characterization indicated that WRP-1 was a branched ß-(1→3)-glucan and exhibited rigid helical conformation in aqueous solution with Mw of 2,180 kDa and radius of gyration (Rg) of 123.4 nm. The galactoglucan of WRP-2 and WRP-3 were mainly composed of →6)-Galp-(1→ and →4)-Glcp-(1→ terminated by glucose and mannose, presenting much lower Mw (392 and 93.6 kDa) and Rg (57.6 and 42.6 nm), and more incompact flexible conformation than WRP-1. All fractions showed potential immunostimulatory activity by promoting macrophage proliferation, phagocytosis, as well as the release of nitric oxide and cytokines (TNF-α and IL-1ß). WRP-1 with unique structure and conformation showed the best immunostimulatory effects among them. This study suggests that WRP could be explored as natural immunostimulator used in the food and pharmaceutical industry.

Effects of soluble dietary fibers on the viscosity property and digestion kinetics of corn starch digesta.

Four soluble dietary fibers (SDFs) were fortified with corn starch (CS) at different concentrations to match the same viscosity equivalents. The mixtures were subjected to a simulated digestion procedure to study the effects of SDFs on viscosity properties and digestion kinetics of CS. Results showed that SDFs increased the hydration property and decreased the water mobility of digesta. During digestion process, SDFs increased the apparent viscosity of digesta to some extent, and showed significant difference to delay the decay of digesta viscosity (kv). The amylolysis inhibitory ability was similar when each SDF was present at the same viscosity equivalent, however, significant differences were found on the digestion rate constant of k2. Linear correlations between kv and k2 were established for 1 and 2 equivalent groups. These results demonstrated that SDFs could delay the digestion process as chemistry differences, which related to their ability on delaying the change of digesta viscosity.

An increase in cell membrane permeability in the in situ extractive fermentation improves the production of antroquinonol from Antrodia camphorata S-29.

The goals of this study were to increase the production of antroquinonol (AQ) and to elucidate the response mechanism of the cell membrane during the in situ extractive fermentation (ISEF) of Antrodia camphorata S-29. Through ISEF, the concentration of AQ reached a maximum of 146.1 ± 2.8 mg/L, which was approximately (7.4 ± 0.1)-fold that of the control (coenzyme Q0-induced fermentation). Transcriptome sequencing showed that four genes (FAD2, fabG, SCD, and FAS1) related to fatty acid biosynthesis were upregulated. FAD2 and SCD may regulate the increase in oleic acid (C18:1) and linoleic acid (C18:2) in the cell membrane of A. camphorata S-29, resulting in an increase in cell membrane permeability. AQ was successfully transferred to the n-tetradecane phase through the cell membrane, reducing product feedback inhibition and improving the production of AQ from A. camphorata S-29.

Lactobacillus casei LC2W can inhibit the colonization of Escherichia coli O157:H7 in vivo and reduce the severity of colitis.

Enterohemorrhagic Escherichia coli (EHEC) is a strain of human pathogenic E. coli bacteria that can cause serious foodborne diseases. Many probiotics have antagonistic effects on EHEC, but few studies have examined the interactions between probiotics and EHEC in vivo. To investigate the colonization of Lactobacillus casei LC2W and its inhibitory effect on E. coli O157:H7 in vivo, these strains labelled with different fluorescent proteins were monitored in the intestinal tracts of live mice using an in vivo imaging system. The results showed that L. casei LC2W inhibited the colonization of O157:H7 in mice. Further research found that LC2W had both prevention and treatment effects on the colitis severity of mice infected by O157:H7, where the prevention effect dominated over the treatment one. This study demonstrates a feasible method for studying the interactions between probiotics and pathogens, and the mechanisms by which probiotics reduce colitis induced by O157:H7.

Exopolysaccharide from Streptococcus thermophilus as stabilizer in fermented dairy: Binding kinetics and interactions with casein of milk.

Exopolysaccharides (EPSs) from lactic acid bacteria have great effect on the quality of fermented milk products. However, the mechanism for the quality improvement has not been well described. This study aimed to investigate the molecular binding kinetics and interactions between EPS obtained from Streptococcus thermophilus AR333 (EPS333) and casein of milk (CM) in a simulated acidifying process. The results indicated that EPS333 had a significant effect on the stability of casein micelles at acidic pH (6.0-4.5) according to the turbidity, ζ-potential, particle size and distribution analysis. The adsorption-desorption study by bio-layer interferometry identified the direct affinity binding between EPS333 and CM, the interactive moiety of casein was α-casein, rather than ß- or κ-casein. Fluorescence quenching analysis revealed that the force types of interaction between EPS333 and CM were dynamically changeable during the acidifying process, mainly from electrostatic interaction at pH 7.0-6.5, to hydrophobic or hydrogen bonding at pH 6.5-5.5, and then transferred to electrostatic interaction again at pH 5.5-5.0. Conclusively, EPS333 could bind with CM directly via different binding forces during acidifying process to stabilize the properties of casein micelles.

Adhesion to pharyngeal epithelium and modulation of immune response: Lactobacillus salivarius AR809, a potential probiotic strain isolated from the human oral cavity.

Microbiome modulators such as probiotics are known to modulate oral diseases. Very few probiotics are commercially available for use in the oral cavity. In this context, we selected human-origin Lactobacillus salivarius AR809 as a promising oropharyngeal probiotic and characterized its functional and immunomodulatory properties. Results demonstrated that AR809 could efficiently adhere to pharyngeal epithelial FaDu cells, antagonize Staphylococcus aureus, adapt to the oral environment, and modulate host innate immunity by inducing potentially protective effects. Particularly, AR809 diminished proinflammatory activity by enhancing the production of IL10 and inhibiting the expression of tumor necrosis factor-α, IL1B, inducible nitric oxide synthase, and RELA. Finally, we observed that AR809 grew efficiently when cultured in milk, suggesting that the preparation of a fermented milk product containing AR809 could be a practical way to administer this probiotic to humans. In conclusion, AR809 has high potential to adhere to the pharyngeal mucosa and could be applied in novel milk-based probiotic fermented food products.

Genomic and phenotypic analyses of exopolysaccharide biosynthesis in Streptococcus thermophilus S-3.

Streptococcus thermophilus, one of the most important industrial lactic acid bacteria, is widely used as a starter culture in the dairy industry. Streptococcus thermophilus S-3 isolated from Chinese traditional dairy products has shown great potential for the production of larger amounts of exopolysaccharides (EPS), which significantly affect the organoleptic properties of fermented milk products. To understand the relationship between the genotype and phenotype of S. thermophilus S-3 in terms of EPS biosynthesis, its genome of strain S-3 was sequenced and the genes related to carbohydrate utilization, nucleotide sugars synthesis, and EPS biosynthesis were investigated. The genomic analysis revealed that S. thermophilus S-3 can use sucrose, mannose, glucose, galactose, and lactose. Phenotypic analysis showed that S-3 prefers fermenting lactose to fermenting glucose or galactose. The genetic analysis of nucleotide sugars and EPS biosynthesis revealed that S-3 can synthesize uridine diphosphate (UDP)-glucose, deoxythymidine diphosphate-glucose, deoxythymidine diphosphate-rhamnose, UDP-galactose, UDP-N-acetylgalactosamine, and UDP-N-acetylglucosamine. A high yield of EPS from S-3 cultivated with lactose rather than glucose as the carbon source was correlated with high transcriptional levels of the genes associated with metabolism of these nucleotide sugars and EPS biosynthesis. Our results provide a better understanding of EPS biosynthesis in S. thermophilus and can facilitate enhanced EPS production by lactic acid bacteria fermentation via genetic and metabolic engineering approaches.

Rheological properties of Prunus persica exudate: Potential effects of proteins and polyphenols.

This study was to investigate the rheological properties of graded Prunus persica exudates (peach gums). Graded peach gums were extracted by hot water and precipitated by ethanol to obtain white peach gum polysaccharide (WPGP) and yellow peach gum polysaccharide (YPGP). Chemical analysis showed that YPGP had 0.11% proteins and 0.23% polyphenols, which were not detected in WPGP. Molecular weight of WPGP and YPGP were determined as 6.09 × 103 kDa and 5.82 × 103 kDa, respectively, by using HPGPC. Monosaccharide analysis revealed that WPGP and YPGP were mainly composed of arabinose and galactose in an approximate molar ratio of 2: 1. These results suggested that WPGP and YPGP might have the same polysaccharide composition. Rheological analysis revealed that peach gum aqueous solutions (10% w/w) exhibited the non-Newtonian shear-thinning behavior and gel structure. Apparent viscosity and viscoelasticity of peach gums were slightly decreased in alkaline solution (pH = 11), high temperature (40 °C) and the presence of salt ions. YPGP solution possessed higher viscosity and storage modulus than WPGP solution at all the tested conditions, which suggested that conjugates of proteins and polyphenols might be involved in the gel network of YPGP solution, resulting in an increase in apparent viscosity.

Composition and Rheological Properties of Polysaccharide Extracted from Tamarind (Tamarindus indica L.) Seed.

A polysaccharide was extracted in high yield from tamarind (Tamarindus indica L.) seed (TSP) by acidic hot water extraction and ethanol precipitation. It was composed of 86.2% neutral polysaccharide, 5.4% uronic acid and 1.3% protein. The molecular weight of TSP was estimated to be about 1735 kDa, with glucose, xylose, and galactose in a molar ratio of 2.9:1.8:1.0 as the major monosaccharides. The steady shear and viscoelastic properties of TSP aqueous solutions were investigated by dynamic rheometry. Results revealed that TSP aqueous solution at a concentration above 0.5% (w/v) exhibited non-Newtonian shear-thinning behavior. Dynamic oscillatory analysis revealed that 10% (w/v) TSP showed as a "weak gel" structure. Apparent viscosities and viscoelastic parameters of TSP solutions decreased drastically in an alkaline solution of pH > 10, but slightly influenced by acidic solution, high temperature and the presence of salt ions and sucrose. These results indicated that TSP possessed excellent pH-resistance and thermo-stability, which might be suitable for applications in acidic beverages and high-temperature processed foodstuffs.

The ameliorative effect of a Lactobacillus strain with good adhesion ability against dextran sulfate sodium-induced murine colitis.

The objective of this study was to effectively screen out a Lactobacillus strain with excellent adhesion ability and ameliorative effect on the disease symptoms of a murine ulcerative colitis model. The auto-aggregation rate (RAA), co-aggregation rate with Escherichia coli O157 (RCA), and cell surface hydrophobicity (AHC) of 17 Lactobacillus strains were measured for primary selection. The results indicate that Lactobacillus plantarum AR326 displayed the topmost overall aggregation performance among all strains examined. A positive and linear relationship between RCA and RAA values was observed for 15 Lactobacillus strains with RAA = 16-46% and RCA = 15-27%. For adhesion ability to human adenocarcinoma HT-29 cells, five representative Lactobacillus strains showed a good, positive dependence on RAA, where L. plantarum AR326 and L. fermentum AR184 showed a higher adhesion ability than the others. Using the 5 (6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA SE)-labeling technique, L. plantarum AR326 was confirmed to adhere to and colonize well in the intestinal mucosa of mice mainly in the ileum and colon. Finally, L. plantarum AR326 at the dosage applied (daily 2 × 109 cfu per mouse) could attenuate murine dextran sulfate sodium (DSS)-induced colitis by effectively decreasing the body weight loss, disease activity index, colon length shortening, myeloperoxidase activity, and colon epithelial damage of experimental animals. The protective effects involved the restoration of the tight junction protein expression and reduction of the abnormal expression of pro-inflammatory cytokines. In conclusion, L. plantarum AR326 can be used as promising probiotics to ameliorate DSS-induced colitis.

A Surface Protein From Lactobacillus plantarum Increases the Adhesion of Lactobacillus Strains to Human Epithelial Cells.

Adhesion to epithelial cells is considered important for Lactobacillus to exert probiotic effects. In this study, we found that trypsin treatment decreased the adhesion ability of Lactobacillus plantarum AR326 and AR269, which exhibit good adhesion ability, and surface proteins extracts increased the adhesion of the strains with poor adhesion ability. By SDS-polyacrylamide gel electrophoresis and mass spectrometry analysis, the main component of the surface proteins was detected and identified as a protein of approximately 37 kDa. It was 100% homologous with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from L. plantarum WCFS1. The adhesion of AR326 and AR269 was decreased significantly by blocking with the anti-GAPDH antibody, and GAPDH restored the adhesion of AR326 and AR269 treated with trypsin. In addition, purified GAPDH significantly increased the adhesion of the strains with poor adhesion ability. These results indicated that GAPDH mediates the adhesion of these highly adhesive lactobacilli to epithelial cells and can be used to improve the adhesion ability of probiotics or other bacteria of interest.

Carrageenan polysaccharides and oligosaccharides with distinct immunomodulatory activities in murine microglia BV-2 cells.

This study was to investigate the anti-inflammatory activities in vitro of various carrageenans (Car) fractions (κ-, ι-, and λ-types) with well characterized molecular properties, using murine microglia BV-2 cell line treated with lipopolysaccharide (LPS) as model. It is indicated that pretreatments with the oligosaccharide fractions from κ- or ι-carrageenan acid hydrolysates (κ- and ι-CarAOS, respectively) at 125-500 µg/mL significantly and dose-dependently decreased the levels of tumor necrosis factor α (TNF-α) secreted from LPS-treated BV-2 cells, showing promisingly anti-inflammatory effects. Differently, pretreatments of most of polymeric carrageenans at 250-500 µg/mL significantly increased the TNF-α level, implying the co-inflammatory effects with LPS. The co-inflammatory effectiveness of pure carrageenans at 125 µg/mL was notable for λ-Car, followed by ι-Car, and insignificantly for κ-Car. Generally, cytokine TNF-α was a more sensitive biomarker to the presence of carrageenans than was the IL-6. The TNF-α level varied greatly at a low carrageenan concentration (125 µg/mL) and high polymer percentage (e.g. purified κ- and ι-Car). Conclusively, the anti-inflammatory effects on LPS-treated BV-2 cells could be attenuated by pretreatments with κ- and ι-CarAOS at 125-500 µg/mL.