Exopolysaccharide Research Articles

Lactobacillus-isolated exopolysaccharide as emulsifier ensure extended stability of eugenol encapsulation, potent anti-microbial activity, and application on fresh produce

In this study, we employed a novel encapsulation approach to develop a stable eugenol oil-in-water (O/W) emulsion by using Lactobacillus plantarum ATCC 8014 isolated exopolysaccharide (EPS) as a natural emulsifier. The emulsion was optimized and analyzed for physical stability, microstructure, rheology, and chemical behavior using fourier-transform infrared analysis (FTIR). The eugenol emulsion consisting of a 40:60 O/W ratio with 3 % w/v EPS exhibited a desired droplet size of 192 ± 1.89 nm with multimodal size distribution, polydispersity index of 0.362 ± 0.01 and zeta-potential of −32 ± 1.90 mV. Scanning electron microscopy (SEM) revealed porous and granulated structures with profound consistency. EPS maintained its emulsifying activity across processing conditions (−20 to 70 °C, pH 3–9, salinity 1 %–30 %), with stability over 90 days. Both EPS and eugenol synergistically enhanced the antimicrobial activity of emulsion against Listeria monocytogenes B600 and Salmonella Poona 36. The practical application of optimized emulsion was assessed by demonstrating a reduction in microbial load on the lettuce surface. Microbial load was determined by immersion and sprayed treatment methods which resulted in more than 3.0 log reduction. This potential application of lactobacillus–derived EPS as a natural emulsifier for encapsulating bioactive essential oils will enable its future applications for food safety.

Complementary roles of EPS, T3SS and Expansin for virulence of Erwinia tracheiphila, the causative agent of cucurbit wilt.

Erwinia tracheiphila (Smith) is a recently emerged plant pathogen that causes severe economic losses in cucurbit crops in temperate Eastern North America. E. tracheiphila is xylem restricted, and virulence is thought to be related to Exopolysaccharides (EPS) and biofilm formation, which occlude the passage of sap in xylem vessels and causes systemic wilt. However, the role of EPS and biofilm formation, and their contribution to disease in relation to other virulence loci are unknown. Here, we use deletion mutants to explore the roles of EPS, Hrp Type III secretion system (Hrp T3SS) and Expansin in plant colonization and virulence. Then, we quantify the expression of the genes encoding these factors during infection. Our results show that Exopolysaccharides are essential for E. tracheiphila survival in host plants, while Hrp T3SS and Expansin are dispensable for survival but needed for systemic wilt symptom development. EPS and Hrp T3SS display contrasting expression patterns in the plant, reflecting their relevance in different stages of the infection. Finally, we show that expression of the eps and hrpT3SS operons is downregulated in mildly increased temperatures, suggesting a link between expression of these virulence factors and geographic restriction of E. tracheiphila to temperate regions. Our work highlights how E. tracheiphila virulence is a complex trait where several loci are coordinated during infection. These results further shed light into the relationship between virulence factors and the ecology of this pathosystem, which will be essential for developing sustainable management strategies for this emerging pathogen.

Acclimated green microalgae consortium to treat sewage in an alternative urban WWTP in a coastal area of Central Italy

This study exposed a microalgal consortium formed by Auxenochlorella protothecoides, Tetradesmus obliquus, and Chlamydomonas reinhardtii to six mixed wastewater media containing different proportions of primary (P) or secondary (S) effluents diluted in centrate (C). Algae could grow at centrate concentrations up to 50 %, showing no significant differences between effluents. After acclimation, microalgae cultivated in 50%P-50%C and 50%S-50%C grew at a rate similar to that of control cultures (0.59–0.66 d−1). These results suggest that the consortium acclimated to both sewage streams by modulating the proportion of the species and their metabolism. Acclimation also altered the photosynthetic activity of wastewater-grown samples compared to the control, probably due to partial photoinhibition, changes in consortium composition, and changes in metabolic activity. No major differences were observed between the two streams with respect to biochemical composition, biomass yield, or bioremediation capacity of the cultivated algae but algae grown in the secondary effluent showed qualitatively higher exopolysaccharides (EPS) production than algae grown in primary. Regarding wastewater remediation, microalgae grown in both WW media showed proficient nutrient removal efficiencies (close to 100 %); however, the final pH value (close to 11) would be controversial if the system were upscaled as it is over the legal limit and would cause phosphorus precipitation, so that CO2 addition would be required. The theoretical scale-up of the microalgae system could achieve water treatment costs of 0.109 €·m−3, which was significantly lower than the costs of typical activated sludge systems.

Limosilactobacillus fermentum strains MC1 and D12: Functional properties and exopolysaccharides characterization

Lactic acid bacteria (LAB) produce a broad spectrum of exopolysaccharides (EPSs), commonly used as texturizers in food products. Due to their potential contribution to LAB probiotic properties, like adhesion to human epithelial cells and competitive exclusion of pathogens from human intestinal epithelial cells, this study was focussed on the structural and functional characterization of the EPSs produced by two Limosilactobacillus fermentum strains – MC1, originating from mother's milk, and D12, autochthonous from Croatian smoked fresh cheese. Whole-genome sequencing and functional annotation of both L. fermentum strains by RAST server revealed the genes involved in EPS production and transport, with some differences in functionally related genes. EPSs were extracted from the cell surface of both bacterial strains and purified by size-exclusion chromatography. Structural characterization of the EPSs, achieved by chemical analyses and 1D and 2D NMR spectroscopy, showed that both strains produce an identical mixture of three different EPSs containing galactofuranose and glucopyranose residues. However, a comparison of the functional properties showed that the MC1 strain adhered better to the Caco-2 cell line and exhibited stronger antimicrobial effect against Salmonella enterica serovar Typhimurium FP1 than the D12 strain, which may be attributed to the potential bacteriocin activity of the MC1 strain.

Isolation of biocrust cyanobacteria and evaluation of Cu, Pb, and Zn immobilisation potential for soil restoration and sustainable agriculture

Soil contamination by heavy metals represents an important environmental and public health problem of global concern. Biocrust-forming cyanobacteria offer promise for heavy metal immobilisation in contaminated soils due to their unique characteristics, including their ability to grow in contaminated soils and produce exopolysaccharides (EPS). However, limited research has analysed the representativeness of cyanobacteria in metal-contaminated soils. Additionally, there is a lack of studies examining how cyanobacteria adaptation to specific environments can impact their metal-binding capacity. To address this research gap, we conducted a study analysing the bacterial communities of cyanobacteria-dominated biocrusts in a contaminated area from South Sardinia (Italy). Additionally, by using two distinct approaches, we isolated three Nostoc commune strains from cyanobacteria-dominated biocrust and we also evaluated their potential to immobilise heavy metals. The first isolation method involved acclimatizing biocrust samples in liquid medium while, in the second method, biocrust samples were directly seeded onto agar plates. The microbial community analysis revealed Cyanobacteria, Bacteroidota, Proteobacteria, and Actinobacteria as the predominant groups, with cyanobacteria representing between 13.3 % and 26.0 % of the total community. Despite belonging to the same species, these strains exhibited different growth rates (1.1–2.2 g L−1 of biomass) and capacities for EPS production (400–1786 mg L−1). The three strains demonstrated a notable ability for metal immobilisation, removing up to 88.9 % of Cu, 86.2 % of Pb, and 45.3 % of Zn from liquid medium. Cyanobacteria EPS production showed a strong correlation with the removal of Cu, indicating its role in facilitating metal immobilisation. Furthermore, differences in Pb immobilisation (40–86.2 %) suggest possible environmental adaptation mechanisms of the strains. This study highlights the promising application of N. commune strains for metal immobilisation in soils, offering a potential bioremediation tool to combat the adverse effects of soil contamination and promote environmental sustainability.

NaCl Modifies Biochemical Traits in Bacterial Endophytes Isolated from Halophytes: Towards Salinity Stress Mitigation Using Consortia.

Bacterial endophytes (120) were isolated from six halophytes (Distichlis spicata, Cynodon dactylon, Eragrostis obtusiflora, Suaeda torreyana, Kochia scoparia, and Baccharis salicifolia). These halophiles were molecularly identified and characterized with or without NaCl conditions. Characterization was based on tests such as indole acetic acid (IAA), exopolysaccharides (EPS), and siderophores (SID) production; solubilization of phosphate (P), potassium (K), zinc (Zn), and manganese (Mn); mineralization of phytate; enzymatic activity (acid and alkaline phosphatase, phytases, xylanases, and chitinases) and the mineralization/solubilization mechanisms involved (organic acids and sugars). Moreover, compatibility among bacteria was assessed. Eleven halophiles were characterized as highly tolerant to NaCl (2.5 M). The bacteria isolated were all different from each other. Two belonged to Bacillus velezensis and one to B. pumilus while the rest of bacteria were identified up to the genus level as belonging to Bacillus, Halobacillus, Halomonas, Pseudomonas, Nesterenkonia, and three strains of Oceanobacillus. The biochemical responses of nutrient solubilization and enzymatic activity were different between bacteria and were influenced by the presence of NaCl. Organic acids were involved in P mineralization and nutrient solubilization. Tartaric acid was common in the solubilization of P, Zn, and K. Maleic and vanillic acid were only detected in Zn and K solubilization, respectively. Furthermore, sugars appeared to be involved in the solubilization of nutrients; fructose was detected in the solubilization tests. Therefore, these biochemical bacterial characteristics should be corroborated in vivo and tested as a consortium to mitigate saline stress in glycophytes under a global climate change scheme that threatens to exacerbate soil salinity.

Identification of Oil-Loving Cupriavidus necator BM3-1 for Polyhydroxyalkanoate Production and Assessing Contribution of Exopolysaccharide for Vegetable Oil Utilization.

Polyhydroxyalkanoates (PHA) have received attention owing to their biodegradability and biocompatibility, with studies exploring PHA-producing bacterial strains. As vegetable oil provides carbon and monomer precursors for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), oil-utilizing strains may facilitate PHA production. Herein, Cupriavidus necator BM3-1, which produces 11.1 g/L of PHB with 5% vegetable oil, was selected among various novel Cupriavidus necator strains. This strain exhibited higher preference for vegetable oils over sugars, with soybean oil and tryptone determined to be optimal sources for PHA production. BM3-1 produced 33.9 g/L of exopolysaccharides (EPS), which was three-fold higher than the amount produced by H16 (10.1 g/L). EPS exhibited 59.7% of emulsification activity (EI24), higher than that of SDS and of EPS from H16 with soybean oil. To evaluate P(3HB-co-3HHx) production from soybean oil, BM3-1 was engineered with P(3HB-co-3HHx) biosynthetic genes (phaCRa, phaARe, and phaJPa). BM3-1/pPhaCJ produced 3.5 mol% of 3HHx and 37.1 g/L PHA. BM3-1/pCB81 (phaCAJ) produced 32.8 g/L PHA, including 5.9 mol% 3HHx. Physical and thermal analyses revealed that P(3HB-co-5.9 mol% 3HHx) was better than PHB. Collectively, we identified a novel strain with high vegetable oil utilization capacity for the production of EPS, with the option to engineer the strain for P(3HB-co-3HHx).

Bio-Prospecting Xylose-Utilizing, Exopolysaccharide (EPS)-Producing Bacteria and EPS Quantification through Submerged Fermentation using Xylose as the Major Carbon Source

Study’s Novelty/ Excerpt This study identified bacterial isolates from diverse environments capable of utilizing xylose for exo-polysaccharide (EPS) production, an area with limited prior research. Potential EPS producers were isolated and identified as Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia species using a salt-based medium with xylose as the primary carbon source. The highest EPS production was observed in Enterobacter cloacae D1, E. cloacae D2, Klebsiella oxytoca D2, and K. oxytoca G1, ranging from 0.04 g/L to 2.0 g/L, highlighting these isolates' potential for optimization and application in various industries. Full Abstract Many microorganisms are capable of producing Exo-polysaccharides (EPS) while utilizing simple sugars and hexoses. These EPS found applications in various fields, such as agricultural biotechnology, pharmaceuticals, textiles, and food industries. However, there is a lack of studies on EPS-elaborating bacteria that can utilize pentoses like xylose. Therefore, the utilization of alternative carbon sources for EPS production has become a focus of recent research. This study aimed to prospect bacteria that can utilize xylose for EPS production. Samples from agricultural soil, dump sites, saline soil, cement-contaminated soil, fresh cow milk, cow dung, and yogurt were serially diluted and cultured in a salt-based medium with xylose as the primary carbon source. Slimy and mucoid colonies were selected as potential EPS-producing isolates and identified morphologically and biochemically using the VITEK 2 Automated identification system. The quantification of EPS production by these isolates was conducted through submerged fermentation with xylose as the sole carbon source. The mean heterotrophic bacterial count of xylose-utilizing bacteria ranged from 2.1x106 CFU to 3.5x108 CFU per gram of the samples analyzed. The slimy and mucoid colonies were identified as members of the genera Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia. The quantities of EPS produced by the isolates ranged from 0.04 g/L to 2.0 g/L, with E. cloacae D1, E. cloacae D2, K. oxytoca D2, and K. oxytoca G1 elaborating the highest amount of EPS. Bacterial isolates capable of utilizing xylose for EPS production were obtained from various sources, showing potential for further optimization

Inter-species interaction of bradyrhizobia affects their colonization and plant growth promotion in Arachis hypogaea.

Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.

EPS Production by Lacticaseibacillus casei Using Glycerol, Glucose, and Molasses as Carbon Sources.

This study demonstrates that Lactobacillus can produce exopolysaccharides (EPSs) using alternative carbon sources, such as sugarcane molasses and glycerol. After screening 22 strains of Lactobacillus to determine which achieved the highest production of EPS based on dry weight at 37 °C, the strain Ke8 (L. casei) was selected for new experiments. The EPS obtained using glycerol and glucose as carbon sources was classified as a heteropolysaccharide composed of glucose and mannose, containing 1730 g.mol-1, consisting of 39.4% carbohydrates and 18% proteins. The EPS obtained using molasses as the carbon source was characterized as a heteropolysaccharide composed of glucose, galactose, and arabinose, containing 1182 g.mol-1, consisting of 52.9% carbohydrates and 11.69% proteins. This molecule was characterized using Size Exclusion Chromatography (HPLC), Gas chromatography-mass spectrometry (GC-MS), Fourier-transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance spectroscopy (1H-NMR). The existence of polysaccharides was confirmed via FT-IR and NMR analyses. The results obtained suggest that Lacticaseibacillus casei can grow in media that use alternative carbon sources such as glycerol and molasses. These agro-industry residues are inexpensive, and their use contributes to sustainability. The lack of studies regarding the use of Lacticaseibacillus casei for the production of EPS using renewable carbon sources from agroindustry should be noted.

Effects of elicitors from culture filtrate of Fusarium solani CL105 on flavonoid production of Scutellaria baicalensis calli.

Endophytic fungi can promote secondary metabolite accumulation in medicinal plants. Previously, we observed that the culture filtrate of Fusarium solani CL105 promoted flavonoid production in Scutellaria baicalensis calli. However, the active ingredients and mechanisms associated with this secondary metabolite accumulation remain unclear. This study evaluates the effects of different elicitors from the culture filtrate of F. solani CL105 namely, exopolysaccharide (EPS), exoprotein (EP), and other parts (OP), on the flavonoid production in S. baicalensis calli by HPLC. Subsequently, the underlying mechanism of EPS induced flavonoid production in S. baicalensis calli was revealed by transcriptomics and RT-PCR. The results indicated a significant increase in flavonoid production in S. baicalensis calli following treatment with EPS. Baicalin (1.40 fold), wogonoside (1.91 fold), and wogonin (2.76 fold) were most significantly up-regulated compared with the control. Transcriptome analysis further revealed up-regulation of key enzyme genes (CHS, CHI, FNS, and F6H) involved in flavonoid synthesis after 5 days of EPS treatment. Moreover, the expression of GA2ox and CYP707A-genes involved in gibberellin acid (GA) and abscisic acid biosynthesis (ABA), respectively-were significantly up-regulated. The expression levels of certain transcription factors, including MYB3, MYB8, and MYB13, were also significantly higher than in controls. Our results indicated that EPS was a main active elicitor involved in promoting flavonoid production in S. baicalensis calli. We postulated that EPS might stimulate the expression of MYB3, MYB8, MYB13, GA2ox, and CYP707A, leading to markedly upregulated CHS, CHI, FNS, and F6H expression levels, ultimately promoting flavonoid synthesis. This study provides a novel avenue for large-scale in vitro production of flavonoids in S. baicalensis.

Characterization and antioxidant properties of three exopolysaccharides produced by the Cyclocarya paliurus endophytic fungus

In recent years, the considerable potential of endophytic bacteria and fungi as prolific producers of exopolysaccharides (EPSs) have attracted interest. In this study, 56 endophytes were isolated from Cyclocarya paliurus, and the secondary metabolites of EPSs were extracted from Monascus purpureus, Penicillium citrinum and Aspergillus versicolor, screened, and named MPE, PCE and AVE, respectively. In this work, the physicochemical properties and antioxidant activities of three EPSs, their cell proliferation activity on IEC-6 and RAW264.7 were investigated. The three EPSs were mainly composed of neutral sugar and differ in microstructure. However, MPE had a loose structure, and PCE exhibited a dense and sheet-like structure. In addition, the three EPSs performed ordinary antioxidant activity in vitro but showed excellent cell proliferation activity on IEC-6 and RAW264.7. The cell proliferation activity of PCE was 1.4-fold that of the controls at a concentration of 800 μg/mL on IEC-6, and MPE exhibited 1.3-fold increase on RAW264.7. This study provided scientific evidence and insights into the application of endophytes as a novel plant resource possessing huge application potential.

Improving the Antioxidant and Anti-Inflammatory Activity of Fermented Milks with Exopolysaccharides-Producing Lactiplantibacillus plantarum Strains.

Exopolysaccharides (EPSs) producing lactic acid bacteria have been claimed to confer various health benefits to the host, including the ability to face oxidative and inflammatory-related stress. This study investigated the ability of food-borne Lactiplantibacillus (Lpb.) plantarum to improve the antioxidant activity of fermented milks by producing EPSs. Two Lpb. plantarum strains, selected as lower and higher EPSs producers, have been applied in lab-scale fermented milk production, in combination with conventional starters. Antioxidant activity was investigated in vitro using DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and FRAP (ferric reducing antioxidant power) assays while the ability to modulate reactive oxygen species (ROS) level was evaluated in an intestinal healthy model, subjected to both oxidative and inflammatory stress. Furthermore, to verify whether digestion affects functionality, fermented milks were evaluated before and after in vitro-simulated INFOGEST digestion. The results showed an improved antioxidant activity of fermented milk enriched with Lpb. plantarum LT100, the highest EPSs producer. Furthermore, the data showed a different ROS modulation with a protective anti-inflammatory effect of samples enriched with Lpb. plantarum strains. Our data suggest the use of selected EPS-producing strains of Lpb. plantarum as a natural strategy to enrich the functionality of fermented milks in terms of ROS modulation and inflammatory-related stress.

The halotolerant exopolysaccharide-producing Rhizobium azibense increases the salt tolerance mechanism in Phaseolus vulgaris (L.) by improving growth, ion homeostasis, and antioxidant defensive enzymes

Globally, agricultural productivity is facing a serious problem due to soil salinity which often causes osmotic, ionic, and redox imbalances in plants. Applying halotolerant rhizobacterial inoculants having multifarious growth-regulating traits is thought to be an effective and advantageous approach to overcome salinity stress. Here, salt-tolerant (tolerating 300 mM NaCl), exopolysaccharide (EPS) producing Rhizobium azibense SR-26 (accession no. MG063740) was assessed for salt alleviation potential by inoculating Phaseolus vulgaris (L.) plants raised under varying NaCl regimes. The metabolically active cells of strain SR-26 produced a significant amount of phytohormones (indole-3-acetic acid, gibberellic acid, and cytokinin), ACC deaminase, ammonia, and siderophore under salt stress. Increasing NaCl concentration variably affected the EPS produced by SR-26. The P-solubilization activity of the SR-26 strain was positively impacted by NaCl, as demonstrated by OD shift in NaCl-treated/untreated NBRIP medium. The detrimental effect of NaCl on plants was lowered by inoculation of halotolerant strain SR-26. Following soil inoculation, R. azibense significantly (p ≤ 0.05) enhanced seed germination (10%), root (19%) shoot (23%) biomass, leaf area (18%), total chlorophyll (21%), and carotenoid content (32%) of P. vulgaris raised in soil added with 40 mM NaCl concentration. Furthermore, strain SR-26 modulated the relative leaf water content (RLWC), proline, total soluble protein (TSP), and sugar (TSS) of salt-exposed plants. Moreover, R. azibense inoculation lowered the concentrations of oxidative stress biomarkers; MDA (29%), H2O2 content (24%), electrolyte leakage (31%), membrane stability (36%) and Na+ ion uptake (28%) when applied to 40 mM NaCl-treated plants. Further, R. azibense increases the salt tolerance mechanism of P. vulgaris by upregulating the antioxidant defensive responses. Summarily, it is reasonable to propose that EPS-synthesizing halotolerant R. azibense SR-26 should be applied as the most cost-effective option for increasing the yields of legume crops specifically P. vulgaris in salinity-challenged soil systems.

Evaluation of functional properties of lactic acid bacteria isolated from traditional cheese

The objective of this research was to study a total of 10 strains of lactic acid bacteria previously isolated from traditional cheese, explore the effects of various in vitro tests, and evaluate their functional and potential probiotic properties. We tested the strains for cholesterol assimilation, antimicrobial activity, enzyme and exopolysaccharides (EPS) production, cell surface traits, antibiofilm, and tolerance to lysozyme and phenol. E. faecalis S135 and E. durans S1121 were characterized by strong antibacterial activities against C. albicans and S. aureus. The strains E. durans S092 and E. faecalis S135 were differentiated by phytase-producing and coagulation activity, respectively. Moreover, E. faecalis S135 had a greater cholesterol reduction percentage (66.6 ± 0.00 %) than the others. Separately, E. durans S104 and L. coryniformis subsp. torquens S232 were found to possess stronger cell surface properties, e.g., auto-aggregation (94.3 ± 0.30 %) and cell surface hydrophobicity (65.1 ± 0.11 %). All LAB 10 strains were able to handle 0.4 % phenol and 2 mg/mL lysozyme well. They also had some antibiofilm activity, but not much EPS production. Industrially important strains with antimicrobial activity, such as E. durans (S1121, S092, and S104) and E. faecalis S135, could serve as bio-protective agents. These four strains can therefore find application in various fields, including the production of fermented and/or functional foods and enzymes.

New insight into protective effect against oxidative stress and biosynthesis of exopolysaccharides produced by Lacticaseibacillus paracasei NC4 from fermented eggplant.

Fermented eggplant is a traditional fermented food, however lactic acid bacteria capable of producing exopolysaccharide (EPS) have not yet been exploited. The present study focused on the production and protective effects against oxidative stress of an EPS produced by Lacticaseibacillus paracasei NC4 (NC4-EPS), in addition to deciphering its genomic features and EPS biosynthesis pathway. Among 54 isolates tested, strain NC4 showed the highest EPS yield and antioxidant activity. The maximum EPS production (2.04 ± 0.11g/L) was achieved by culturing in MRS medium containing 60g/L sucrose at 37°C for 48h. Under 2mM H2O2 stress, the survival of a yeast model Saccharomyces cerevisiae treated with 0.4mg/mL NC4-EPS was 2.4-fold better than non-treated cells, which was in agreement with the catalase and superoxide dismutase activities measured from cell lysates. The complete genome of NC4 composed of a circular chromosome of 2,888,896bp and 3 circular plasmids. The NC4 genome comprises more genes with annotated function in nitrogen metabolism, phosphorus metabolism, cell division and cell cycle, and iron acquisition and metabolism as compared to other reported L. paracasei. Of note, the eps gene cluster is not conserved across L. paracasei. Pathways of sugar metabolism for EPS biosynthesis were proposed for the first time, in which gdp pathway only present in few plant-derived bacteria was identified. These findings shed new light on the cell-protective activity and biosynthesis of EPS produced by L. paracasei, paving the way for future efforts to enhance yield and tailor-made EPS production for food and pharmaceutical industries.

Role of Bacillus subtilis exopolymeric genes in modulating rhizosphere microbiome assembly

BackgroundBacillus subtilis is well known for promoting plant growth and reducing abiotic and biotic stresses. Mutant gene-defective models can be created to understand important traits associated with rhizosphere fitness. This study aimed to analyze the role of exopolymeric genes in modulating tomato rhizosphere microbiome assembly under a gradient of soil microbiome diversities using the B. subtilis wild-type strain UD1022 and its corresponding mutant strain UD1022eps−TasA, which is defective in exopolysaccharide (EPS) and TasA protein production.ResultsqPCR revealed that the B. subtilis UD1022eps−TasA− strain has a diminished capacity to colonize tomato roots in soils with diluted microbial diversity. The analysis of bacterial β-diversity revealed significant differences in bacterial and fungal community structures following inoculation with either the wild-type or mutant B. subtilis strains. The Verrucomicrobiota, Patescibacteria, and Nitrospirota phyla were more enriched with the wild-type strain inoculation than with the mutant inoculation. Co-occurrence analysis revealed that when the mutant was inoculated in tomato, the rhizosphere microbial community exhibited a lower level of modularity, fewer nodes, and fewer communities compared to communities inoculated with wild-type B. subtilis.ConclusionThis study advances our understanding of the EPS and TasA genes, which are not only important for root colonization but also play a significant role in shaping rhizosphere microbiome assembly. Future research should concentrate on specific microbiome genetic traits and their implications for rhizosphere colonization, coupled with rhizosphere microbiome modulation. These efforts will be crucial for optimizing PGPR-based approaches in agriculture.

Drought stress tolerance and metabolomics of Medicago sativa induced by Bacillus amyloliquefaciens DGL1.

This study used Bacillus amyloliquefaciens DGL1 isolated from the arid sandy land of the Qinghai-Tibetan Plateau as the research strain and investigated the effects of DGL1 on the biomass, physiology, and metabolites of Medicago sativa under different intensities of drought stress to provide a high-quality bacterial source and a theoretical basis for the research and development of biological fertilizer suitable for arid areas. The exopolysaccharides (EPS), 1-Aminocyclopropane-1-carboxylate deaminase (ACC), and phosphorus solubilizing capacity of DGL1 were determined. The effects of a DGL1 suspension on alfalfa biomass, physiological indexes, degree of peroxidation of cell membranes, and activity of antioxidant enzymes were determined after irrigating roots under drought stress. The effects on soil physicochemical properties were also evaluated, and metabolomics analysis was performed to explore the effect of DGL1 on the metabolites of alfalfa under drought stress. Strain DGL1 produced extracellular polysaccharide EPS and ACC deaminase and was capable of phosphorus solubilization. Treatment with DGL1 increased the biomass of alfalfa under different degrees of drought stress, significantly increased the activities of alfalfa antioxidant enzymes Super Oxide Dismutase (SOD), Peroxidase (POD), and catalase (CAT), reduced the content of MDA and H2O2, and increased the content of quick-acting phosphorus, quick-acting potassium, ammonium nitrogen, and nitrate nitrogen in the soil, thus improving soil fertility. Through metabolomics analysis, DGL1 was shown to affect amino acid metabolic pathways, such as arginine, leucine, glutamate, and tyrosine, as well as the levels of energy-providing polysaccharides and lipids, in alfalfa under 15% PEG-6000 drought stress, enhancing alfalfa's capacity to resist drought stress. Strain DGL1 enhances the drought suitability of alfalfa and has the potential for dryland development as a biological agent.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Screening of Lactic Acid Bacteria Strains for Potential Sourdough and Bread Applications: Enzyme Expression and Exopolysaccharide Production.

Twenty-eight strains of lactic acid bacteria (LAB) were characterized for the ability to express enzymes of interest (including protease, xylanase, α-amylase, laccase, and glucose oxidase) as well as the ability to produce exopolysaccharide (EPS). The screening of enzyme capability for all LAB strains proceeded in a progressive 3-stage manner that helps to profile the efficiency of LAB strains in expressing chosen enzymes (Stage 1), highlights the strains with affinity for flour as the substrate (Stage 2), and discerns strains that can adapt well in a simulated starter environment (Stage 3). The theoretical ability of LAB to express these enzymes was also assessed using Basic Local Alignment Search Tool (BLAST) analysis to identify the underlying genes in the whole genome sequence. By consolidating both experimental data and information obtained from BLAST, three LAB strains were deemed optimal in expressing enzymes, namely, Lb. delbrueckii subsp. bulgaricus (RBL 52), Lb. rhamnosus (RBL 102), and Lb. plantarum (ATCC 10241). Meanwhile, EPS-producing capabilities were observed for 10 out of 28 LAB strains, among which, Lactococcus lactis subsp. diacetylactis (RBL 37) had the highest total EPS yield (274.15mg polysaccharide/L culture) and produced 46.2% polysaccharide with a molecular mass of more than 100kDa.