Acidovorax avenae subsp. avenae (Aaa) is the causal agent of red stripe in sugarcane, a disease characterized by two forms: leaf stripe and top rot. Despite the importance of this disease, little is known about Aaa virulence factors (VFs) and their function in the infection process. Among the different array of VFs exerted by phytopathogenic bacteria, exopolysaccharides (EPSs) often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. They are also main components of the extracellular matrix involved in cell-cell recognition, surface adhesion, and biofilm formation. EPS composition and properties have been well studied for some plant pathogenic bacteria; nevertheless, there is no knowledge about Aaa-EPS. In this work, we describe a simple and reliable method for EPS production, precipitation, and quantification based on cold precipitation after ethanol addition, which will allow to study EPS characteristics of different Aaa strains and to evaluate the association among EPS (e.g., amount, composition, viscosity) and Aaa pathogenicity.

Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

BACKGROUND: Carbohydrates are known as the main natural products of life activities.
RESULTS: Streptomyces rochie strain OF1 isolated from a mangrove tree produced exopolysaccharide S5 (EPSS5) (14.2 gl-1) containing uronic acid 21.98% sulfate content of 11.65 mg/ml, and a viscosity of 1.35 mm2/s. while total hexose amine content was 24.72%. The high performance liquid chromatography (HPLC) analysis of mono sugars revealed that EPS was composed of manouronic acid, glucuronic acid, xylose, and fructose at a molar ratio of 1.0:0.5:1.0:2.0, respectively. It showed that the whole antioxidant activity was 92.06%. It showed antibacterial activity against Staphylococcus aureus, and E. coli, MRSA and Klebsiella pneumoniae. But, EPSS5 displayed low antifungal activity against Candida albicans. While no antifungal activity has been detected against Aspergillus niger. EPSS5 has antibiofilm action that is noticeable toward S. aureus with an inhibition ratio of biofilm up to 50%. Effect of EPS on serum levels of TNF-α and COX2 by 2 fold and 1.9 fold of EPS reduced serum levels of Tumor necrosis factor-α (TNF-α) by 38%, 12%, 49%, and Cyclooxygenase-2 (COX2) by 61%, 34%, and 62%, respectively. By affected of EPSS5 on arthritis in rats stimulated by carrageenan.
CONCLUSIONS: Administration of EPS ameliorated carrageen-induced elevation in inflammatory mediators; TNF-α/COX and suppressed the expressions of metalloproteinase 9 (MMP9) by 68%, 86%, and 75% correspondingly in comparison to the group of carrageenans. Then again, therapy involving a high dose only reduced MMP9 level by 57%, compared to free drug suggesting that EPSS5 is a good inhibitor of the MMP9, as it brought MMP9 back to normal levels via the signaling pathway.

Rhizobium adhering proteins or \'Raps\' are secreted proteins identified in a very restricted group of rhizobial strains, specifically those belonging to R. leguminosarum and R. etli. The distinctive feature of members of the Rap family is the presence of one or two cadherin-like domains or CHDLs that are also present in numerous extracellular bacterial and archaeal proteins and were proposed to confer carbohydrate binding ability. We have previously made an in-depth characterization of RapA2, a calcium-binding lectin, composed by two CHDLs, involved in biofilm matrix remodelling in R. leguminosarum bv. viciae 3841. In this study, CHDLs derived from RapA2 were analysed in detail, finding significant structural and functional differences despite their considerable sequence similarity. Only the carboxy-terminal CHDL retained properties similar to those displayed by RapA2. Our findings were used to obtain a novel fluorescent probe to study biofilm matrix development by confocal laser scanning microscopy, and also to shed some light on the role of the ubiquitous CHDL domains in bacterial secreted proteins.

The current research aimed to solve the environmental pollution of mature coconut water by Lipomyces starkeyi and provide a study of its high value utilization. The innovation firstly investigated the rheological properties and interface behavior of a crude exopolysaccharide and provided a technical support for its application in food. A response surface methodology was performed to ameliorate the fermentation factors of the new exopolysaccharide with mature coconut water as a substrate, and the consequences suggested that the maximum yield was 7.76 g/L under optimal conditions. Rotary shear measurements were used to study the influence of four variables on the viscosity of the exopolysaccharide solution. The results illustrated that the exopolysaccharide solution demonstrated a shear-thinning behavior and satisfactory thermal stability within the test range. The viscosity of the exopolysaccharide solution was significantly affected by ionic strength and pH; it reached the peak viscosity when the concentration of NaCl was 0.1 mol/L and the pH was neutral. The adsorption behavior of the exopolysaccharide at the medium chain triglyceride-water interface was investigated by a quartz crystal microbalance with a dissipation detector. The results demonstrated that the exopolysaccharide might form a multilayer adsorption layer, and the thickness of the adsorption layer was at its maximum at a concentration of 1.0%, while the interfacial film was the most rigid at a concentration of 0.4%. Overall, these results suggest that the exopolysaccharide produced by Lipomyces starkeyi is an excellent biomaterial for usage in drink, makeup and drug fabrications as a thickening and stabilizing agent.

Dietary supplementation with carotenoids can have beneficial health effects, but carotenoids are poorly absorbed.
We aimed to evaluate how milk fermented by lactic acid bacteria affects dietary carotenoid bioavailability in humans and rats and to investigate mechanisms by which active components in milk fermented by Lactobacilli enhance dietary carotenoid absorption.
Male rats (n = 8/group) were administered β-carotene or β-carotene + fermented milk. Rats (n = 6/group) were also pretreated with ezetimibe, a cholesterol absorption inhibitor, to investigate β-carotene transport mechanisms. In humans, 3 studies were conducted using a randomized crossover method. Subjects (n = 16/study) consumed a vegetable (carrot, tomato, or spinach) drink alone or with a fermented milk drink. Blood samples were collected at various time points after consumption.
In rats, the serum β-carotene area under the concentration-time curve (AUC) was significantly higher for the β-carotene + fermented milk than for β-carotene only. A significant correlation (r = 0.83, P < 0.001) between the exopolysaccharide (EPS) content of fermented milk and serum β-carotene AUC was observed. Ezetimibe treatment did not suppress elevations in serum β-carotene concentrations induced by fermented milk ingestion. In humans, the incremental area under the concentration-time curve (iAUC) for β-carotene in the plasma triacylglycerol-rich lipoprotein (TRL) fraction was significantly (1.8-fold, range: 0.6-3.9) higher when carrot + fermented milk was consumed compared with carrot drink alone. A significantly (6.5-fold, range: 0.04-7.7) higher iAUC for lycopene in the plasma TRL fraction was observed for subjects who consumed tomato + fermented milk compared with tomato drink alone. A significant increase in plasma lutein in all fractions was observed after consumption of spinach + fermented milk, but not with spinach drink alone.
Co-ingestion of β-carotene and fermented milk significantly increased dietary β-carotene bioavailability in humans and rats. EPSs could affect the physical properties of fermented milk to enhance dietary β-carotene absorption mediated by simple diffusion mechanisms. These findings may be relevant for methods to increase dietary carotenoid bioavailability.This trial was registered at umin.ac.jp/ctr as UMIN000034838, UMIN000034839, and UMIN000034840.

The aim of this study was to identify the phenotypic and genotypic profiles of Staphylococcus spp. isolated from mastitis milk and cheese processing plant.To evaluate the biofilm production of wild-type strains on contact surfaces by testing different factors through adhered cells and biofilm quantifications, finally, these biofilms were observed by Scanning Electron Microscopy (SEM). Congo red agar (CRA) plate method was used to identify slime production by strains. Screening of genes encoding adhesion factors and biofilm formation was carried out using PCR. After strains selection, adhesion and biofilm assays were designed testing different times (12, 48, 96 h), strains (n = 13), contact surfaces (stainless steel and polypropylene), and temperatures (5 °C and 25 °C); and then, bacterial count and crystal violet staining were conducted. Relative frequencies of positive on CRA and genes presence were determined, and Friedman test was applied for bacterial counts and OD values. Additionally, significant factors (P ≤ .05) were subjected to multiple comparisons using the Nemenyi test. The slime production in CRA was observed by visual inspection in 38.7% of strains. A large distribution of genes was described among strains, implying a high variability of genotypic profiles. Moreover, relative frequencies of CRA positive and gene presence were described. The developed assay showed that the strain, temperature, contact surface, were significant for both variables. The SEM corroborated the findings, showing greater biofilm formation on stainless steel at 25 °C. Thus, it is essential to highlight the importance of temperature control and material with low superficial energy to avoid biofilm formation by staphylococci.

Chemotherapeutic medications, including 5 - fluorouracil (5FU), are the same old technique to most cancers and are associated with numerous peripheral toxicities. We investigated exopolysaccharide (EPSST) produced from the isolated streptomycete of the Mediterranean Sea for the capability to lower the severity of mucositis in vivo. The streptomycete was isolated from Mediterranean Sea sediment from the beaches of Port Said Governorates, Egypt and identified morphologically, physiologically, and biochemically and confirmed by molecularly 16S rDNA analysis. The EPSST was extracted from the supernatant of streptomycete by using 4 volumes chilled ethanol and then the functional groups, MW, and chemical evaluation have been detected via Fourier-transform infrared (FTIR), and high-performance liquid chromatography (HPLC). In addition, antioxidant activity was measured through the usage of 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Thirty-two male rats (180-200 g) were randomly divided into a control group (normal saline), intraperitoneal injection of 5-fluorouracil (5-FU, 150 mg/kg), normal rats were treated with EPSST and 5-FU + EPSST group. These groups were continued up to the day of sacrifice (28 days post treatments). The isolated strain became recognized based totally on 16S rDNA sequence as Streptomyce sp. with accession number SAMN08349905. The chemical evaluations of EPSST were galacturonic, glucose, galactose, mannose, and arabinose with a relative ratio of 2.1: 1: 5.37: 1.62: 1.29 individually, with an average molecular weight (Mw) 9.687 × 103 g/mol. Also, the EPSST contained uronic acid (16%) and sulfate (12.149%) and no protein was detected. EPSST inhibited the DPPH radical activity. The findings of this study propose that EPSST inhibits 5-FU-induced mucositis through adjustment of oxidative stress, apoptosis, inflammatory factors, activation of antioxidant enzymes. The clinical administration of EPSST may recover the chemotherapy-induced intestinal dysfunction, consequently increasing the clinical efficiency of chemotherapy. In addition, the administration of EPSST reduced 5-FU-induced histopathological incongruities such as neutrophil infiltration, loss of cellular integrity, and villus and crypt distortion. The clinical administration of EPSST may recover the chemotherapy-induced intestinal dysfunction, consequently increasing the clinical efficiency of chemotherapy.

Polysaccharides have attracted much attention due to their interesting physico-chemical and also biological properties that are explored in food, cosmetic and pharmaceutical industries. GY785 exopolysaccharide (EPS) presenting an unusual structure is secreted by the deep-sea hydrothermal bacterium, Alteromonas infernus. Low-molecular weight (LMW) derivatives obtained by chemical depolymerization of the native high molecular weight (HMW) EPS were previously shown to exhibit biological properties similar to glycosaminoglycans (GAG). In the present study, in order to generate well defined derivatives with a better control of the depolymerization, an enzymatic approach was applied for the first time. Various commercially available enzymes were firstly screened for their depolymerizing activities, however none of them was able to degrade the polysaccharide. Enzymatic assays performed with A. infernus protein extracts have shown that the bacterium produces by itself endogenous enzymes able to depolymerize its own EPS. The oligosaccharides released by the enzymes were analyzed and their structures allowed to assess that the protein extract contains several depolymerizing activities.

An extracellular layer of exopolysaccharides (EPS) covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW)-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter). Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the interaction with the host.