Evaluation of the efficacy of MONTANIDE™ GR01, a new adjuvant for feed-based vaccines, on the immune response and protection against Streptococcus agalactiae in oral vaccinated Nile tilapia (Oreochromis niloticus) under laboratory and on-farm conditions.

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDE™ GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDE™ GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDE™ GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feed-based adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDE™ GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

Exploring environmental exposomes and the gut-brain nexus: Unveiling the impact of pesticide exposure.

This review delves into the escalating concern of environmental pollutants and their profound impact on human health in the context of the modern surge in global diseases. The utilisation of chemicals in food production, which results in residues in food, has emerged as a major concern nowadays. By exploring the intricate relationship between environmental pollutants and gut microbiota, the study reveals a dynamic bidirectional interplay, as modifying microbiota profile influences metabolic pathways and subsequent brain functions. This review will first provide an overview of potential exposomes and their effect to gut health. This paper is then emphasis the connection of gut brain function by analysing microbiome markers with neurotoxicity responses. We then take pesticide as example of exposome to elucidate their influence to biomarkers biosynthesis pathways and subsequent brain functions. The interconnection between neuroendocrine and neuromodulators elements and the gut-brain axis emerges as a pivotal factor in regulating mental health and brain development. Thus, manipulation of gut microbiota function at the onset of stress may offer a potential avenue for the prevention and treatment for mental disorder and other neurodegenerative illness.

Effects of Dietary Tuber Ethanolic Extract of Nut Grass (Cyperus rotundus Linn.) on Growth, Immune Response, and Disease Resistance in Nile Tilapia (Oreochromis niloticus).

Nut grass (Cyperus rotundus Linn.) is a weed that grows in all tropical, subtropical and temperate regions of the world, including areas where it grows on saline soil. This research was conducted to evaluate the effect of C. rotundus tuber extract in the diet on the growth performance and disease resistance of Nile tilapia. Various components of phytochemical importance of nut grass, including sugars/carbohydrates, terpenoids, tannins, and flavonoids were found in C. rotundus. Tilapia (n = 25 fish/group in triplicate) were fed with different levels of nut grass extract including 0 (control; T1), 0.4 (T2), 0.8 (T3), and 1.6 (T4) g/kg for 60 days in a completely randomized design (CRD) experiment. After the feeding trial, the highest weight gain and average daily gain (ADG) were observed in the T4 group, but it was not significantly different from T3 (Nile tilapia fed with a 0.8 g/kg) (p > 0.05). The lowest feed conversion ratio (FCR) was observed in the T3 group. Moreover, the fillet, crud lipid content, and blood chemical profiles (aspartate aminotransferase (AST), cholesterol, and malondialdehyde (MDA)) in fish fed with 1.6 g/kg were highest when compared in all groups. In addition, the T3 group presented with the immune response parameter found in red blood cells (RBC), lysozyme activity, and antioxidant (superoxide dismutase activity (SOD)) being higher than those of the control group (p < 0.05). The highest survival (93.33%) was observed in fish fed with 0.8 g/kg (T3) after a 14 day challenge with Streptococcus agalactiae. Thus, it was concluded that nut grass extract at 0.8 g/kg can be used to improve the growth performance and the tendency for resistance to S. agalactiae in Nile tilapia.

Dietary Administration Effects of Exopolysaccharide Produced by Bacillus tequilensis PS21 Using Riceberry Broken Rice, and Soybean Meal on Growth Performance, Immunity, and Resistance to Streptococcus agalactiae of Nile tilapia (Oreochromis niloticus).

Overuse of antibiotics in aquaculture has generated bacterial resistance and altered the ecology. Aquacultural disease control requires an environmentally sustainable approach. Bacterial exopolysaccharides (EPSs) as bioimmunostimulants have not been extensively explored in aquaculture. This study investigated EPS produced from 5% w/v riceberry broken rice as a carbon source and 1% w/v soybean meal as a nitrogen source by Bacillus tequilensis PS21 from milk kefir grain for its immunomodulatory, antioxidant activities and resistance to pathogenic Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus). The FTIR spectrum of EPS confirmed the characteristic bonds of polysaccharides, while the HPLC chromatogram of EPS displayed only the glucose monomer subunit, indicating its homopolysaccharide feature. This EPS (20 mg/mL) exhibited DPPH scavenging activity of 65.50 ± 0.31%, an FRAP value of 2.07 ± 0.04 mg FeSO4/g DW, and antimicrobial activity (14.17 ± 0.76 mm inhibition zone diameter) against S. agalactiae EW1 using the agar disc diffusion method. Five groups of Nile tilapia were fed diets (T1 (Control) = 0.0, T2 = 0.1, T3 = 0.2, T4 = 1.0, and T5 = 2.0 g EPS/kg diet) for 90 days. Results showed that EPS did not affect growth performances or body composition, but EPS (T4 + T5) significantly stimulated neutrophil levels and serum lysozyme activity. EPS (T5) significantly induced myeloperoxidase activity, catalase activity, and liver superoxide dismutase activity. EPS (T5) also significantly increased the survival of fish at 80.00 ± 5.77% at 14 days post-challenge with S. agalactiae EW1 compared to the control (T1) at 53.33 ± 10.00%. This study presents an efficient method for utilizing agro-industrial biowaste as a prospective source of value-added EPS via a microbial factory to produce a bio-circular green economy model that preserves a healthy environment while also promoting sustainable aquaculture.

Data on exopolysaccharides produced by Bacillus spp. from cassava pulp with antioxidant and antimicrobial properties.

This data evaluated the capacity of Bacillus spp. isolated from Thai milk kefir to produce exopolysaccharide (EPS) on cassava pulp and tested its antioxidant and antibacterial properties. Thailand's starch industry generates million tons of cassava pulp, which is underutilized or bio-transformed into higher-value bioproducts. Antioxidant and antibacterial bacterial exopolysaccharides are beneficial in the food, feed, pharmaceutical, and cosmetic industries. Moisture, ash, fat, protein, fiber, starch, sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed from cassava pulp as an EPS substrate. After 3 days of bacterial fermentation, EPS generation, culture pH, reducing sugar amount, and bacterial count were recorded. Antioxidant activities and bioactive content including hydroxyl radical scavenging activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), total phenolic and flavonoid content (TPC and TFC), and antimicrobial activity against two Nile tilapia pathogens (Streptococcus agalactiae and Staphylococcus aureus) from different Bacillus species were evaluated. Proximate analysis, dinitrosalicylic acid assay, pH value record, bacterial count using spread plate method, antioxidant activity and bioactive content assays via spectrophotometry, and agar disk diffusion were the main approaches. This study used microbial cell factories to convert agro-biowaste, such as cassava pulp, into EPS bioproducts which accords with a bio-circular green economy model.

Exploring the Bioactive Potential of Calostoma insigne, an Endangered Culinary Puffball Mushroom, from Northeastern Thailand.

Calostoma insigne puffball mushrooms are only found in forests with rich biodiversity in very few countries including Thailand, and their biofunctions remain largely unexplored. This study used the agar disk diffusion assay, the anti-glucosidase assay, and the 3, 4, 5-dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide (MTT) assay to evaluate the bioactive potential of these endangered puffball mushrooms. Internal transcribed spacer (ITS) gene analysis identified C. insigne, a puffball mushroom with green, globose, and spiny spores. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the polysaccharide structure while scanning electron microscopy (SEM) revealed a fiber-like network. The ethanolic gelatinous fruiting body extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging capacity (57.96%), a ferric ion-reducing antioxidant power (FRAP) value of 1.73 mg FeSO4/g, and α-glucosidase inhibition (73.18%). C. insigne cytotoxicity was effective towards HT-29 colon cancer cells using the MTT assay (IC50 of 770.6 µg/mL at 72 h) and also showed antiproliferative capacity (IC50 of 297.1 µg/mL). This puffball mushroom stimulated apoptotic genes and proteins (caspase-3, Bax, and p21) via an intrinsic apoptotic pathway in HT-29 cells. In the laboratory, the medium formula consisting of 20% potato, 2% sucrose, and 0.2% peptone was optimal to increase fungal mycelial biomass (2.74 g DW/100 mL), with propagation at pH 5.0 and 30 °C. Puffball mushrooms are consumed as local foods and also confer several potential health benefits, making them worthy of conservation for sustainable utilization.

Application of antimicrobial, potential hazard and mitigation plans.

The tremendous rise in the consumption of antimicrobial products had aroused global concerns, especially in the midst of pandemic COVID-19. Antimicrobial resistance has been accelerated by widespread usage of antimicrobial products in response to the COVID-19 pandemic. Furthermore, the widespread use of antimicrobial products releases biohazardous substances into the environment, endangering the ecology and ecosystem. Therefore, several strategies or measurements are needed to tackle this problem. In this review, types of antimicrobial available, emerging nanotechnology in antimicrobial production and their advanced application have been discussed. The problem of antimicrobial resistance (AMR) due to antibiotic-resistant bacteria (ARB)and antimicrobial resistance genes (AMG) had become the biggest threat to public health. To deal with this problem, an in-depth discussion of the challenges faced in antimicrobial mitigations and potential alternatives was reviewed.

Valorisation of biomass and diaper waste into a sustainable production of the medical mushroom Lingzhi Ganoderma lucidum.

Global solid waste is expected to increase by at least 70% annually until year 2050. The mixture of solid waste including food waste from food industry and domestic diaper waste in landfills is causing environmental and human health issues. Nevertheless, food and diaper waste containing high lignocellulose can easily degrade using lignocellulolytic enzymes thereby converted into energy for the development and growth of mushroom. Therefore, this study explores the potential of recycling biomass waste from coffee ground, banana, eggshell, tea waste, sugarcane bagasse and sawdust and diaper waste as raw material for Lingzhi mushroom (Ganoderma lucidum) cultivation. Using 2% of diaper core with sawdust biowaste leading to the fastest 100% mushroom mycelium spreading completed in one month. The highest production yield is 71.45 g mushroom; this represents about 36% production biological efficiency compared to only 21% as in commercial substrate. The high mushroom substrate reduction of 73% reflect the valorisation of landfill waste. The metabolomics profiling showed that the Lingzhi mushroom produced is of high quality with a high content of triterpene being the bioactive compounds that are medically important for treating assorted disease and used as health supplement. In conclusion, our study proposed a potential resource management towards zero-waste and circular bioeconomy for high profitable mushroom cultivation.

Omics technologies used in pesticide residue detection and mitigation in crop.

In agriculture, the convenience and efficacy of chemical pesticides have become inevitable to manage cultivated crop production. Here, we review the worldwide use of pesticides based on their categories, mode of actions and toxicity. Excessive use of pesticides may lead to hazardous pesticide residues in crops, causing adverse effects on human health and the environment. A wide range of high-tech-analytical methods are available to analyse pesticide residues. However, they are mostly time-consuming and inconvenient for on-site detection, calling for the development of biosensors that detect cellular changes in crops. Such new detection methods that combine biological and physicochemical knowledge may overcome the shortage in current farming to develop sustainable systems that support environmental and human health. This review also comprehensively compiles domestic pesticide residues removal tips from vegetables and fruits. Synthetic pesticide alternatives such as biopesticide and nanopesticide are greener to the environment. However, its safety assessment for large-scale application needs careful evaluation. Lastly, we strongly call for reversions of pesticide application trends based on the changing climate, which is lacking in the current scenario.

Screening of Probiotic Lactic Acid Bacteria Isolated from Fermented Pak-Sian for Use as a Starter Culture.

The objective of this research was to evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from fermented Pak-Sian, a traditional Thai food in the northeastern region of Thailand (Kalasin, Sakon Nakhon, Maha Sarakham, and Khon Kaen Provinces). A total of 61 LAB isolates isolated from selective MRS agar were screened by SDS-PAGE and identified by 16S rDNA analysis. Seventeen bacterial strains were found consisting of Pediococcus pentosaceus (6 strains) Lactiplantibacillus plantarum (5 strains), Levilactobacillus brevis (1 strain), Lactobacillus fermentum (3 strains), and Weissella cibaria (2 strains). A PH tree grouped 17 LAB strains into 5 clusters with three clusters only recorded from Sakon Nakhon Province. All strains were tested for probiotic properties. Results showed that 14 strains had the ability to resist pH 2.5 as resistant to bile salts and survive in gastric juices and the intestinal tract. LAB demonstrated antimicrobial activity against 4 pathogens: Staphylococcus aureus TISTR746, Salmonella typhimurium TISTR1472, Escherichia coli ATCC25922, and Bacillus cereus TISTR1449. Most LAB strains were resistant to all antibiotics tested, while some Lactobacillus strains were moderately susceptible to chloramphenicol, rifampicin, and ampicillin. None of the 14 strains produced biogenic amine and eight showed no hemolysis activity, indicating the safety of these strains. These 8 strains were selected to determine mucin adhesion capacity. Lactobacillus fermentum SK324 and Levilactobacillus brevis SK335 showed the highest adhesion capacity of 2.39 and 2.34%, respectively. These two strains showed promise as alternative starter cultures to improve probiotic health benefits of local fermented Pak-Sian products as a functional food.

Effect of cold plasma and elicitors on bioactive contents, antioxidant activity and cytotoxicity of Thai rat-tailed radish microgreens.

BACKGROUND: Raphanus sativus var. caudatus or Thai rat-tailed radish (RTR) contains glucosinolates and isothiocyanates with chemopreventive effects; however, only mature plants have been investigated to date. Thus, the present study aimed to determine isothiocyanates, phenolic compounds and flavonoid compounds, antioxidant activity, cytotoxicity, and antiproliferative activity of RTR microgreens grown from seeds treated with cold plasma (21 kV for 5 min), organic elicitor (160 mmol L-1 NaCl, 10 mmol L-1 CaCl2 or 176 mmol L-1 sucrose) or both in combination. Seeds were germinated on vermiculite and sprayed with deionized water or elicitor for 7 days before harvest. RESULTS: Cold plasma had insignificant effect on growth, whereas NaCl and CaCl2 increased fresh weight. Plasma with CaCl2 led to the highest total isothiocyanate (ITC) content [1.99 g kg-1 dry weight (DW)] in RTR microgreens containing raphasatin as the only ITC detected. Plasma treatment gave the highest total phenolic content (7.56 mg gallic acid equivalents g-1 DW), antioxidant activity from a 2,2-diphenyl-1-picrylhydrazyl assay (7.70 mg trolox equivalents g-1 DW) and ferric reducing antioxidant power assay (21.72 mg Fe2+ g-1 DW). Microgreen extracts from plasma showed an IC50 value of 29.28 and 13.83 µg mL-1 towards MCF-7 and HepG2, respectively, with inhibitory properties on matrix metalloproteinase (MMP)-2 and MMP-9 proteins. Plasma enhanced Bax and Caspase-3 gene expression but reduced Bcl-2 and MMP-9 expression, indicating activation of apoptosis. CONCLUSION: Cold plasma shows promise as an innovative tool to enhance bioactive compounds with chemopreventive benefits in microgreens. © 2020 Society of Chemical Industry.

Psychobiotic Effects of Multi-Strain Probiotics Originated from Thai Fermented Foods in a Rat Model.

This work aimed to investigate the psychobiotic effects of six bacterial strains on the mind and behavior of male Wistar rats. The probiotic (PRO) group (n=7) were rats pre-treated with antibiotics for 7 days followed by 14-day probiotic administration, antibiotics (ANT) group (n=7) were rats treated with antibiotics for 21 days without probiotics. The control (CON) group (n=7) were rats that received sham treatment for 21 days. The six bacterial strains with probiotic properties were mostly isolated from Thai fermented foods; Pedicoccus pentosaceus WS11, Lactobacillus plantarum SK321, L. fermentum SK324, L. brevis TRBC 3003, Bifidobacterium adolescentis TBRC 7154 and Lactococcus lactis subsp. lactis TBRC 375. The probiotics were freeze-dried into powder (6×109 CFU/5 g) and administered to the PRO group via oral gavage. Behavioral tests were performed. The PRO group displayed significantly reduced anxiety level and increased locomotor function using a marble burying test and open field test, respectively and significantly improved short-term memory performance using a novel object recognition test. Antibiotics significantly reduced microbial counts in rat feces in the ANT group by 100 fold compared to the PRO group. Probiotics significantly enhanced antioxidant enzymatic and non-enzymatic defenses in rat brains as assessed using catalase activity and ferric reducing antioxidant power assay, respectively. Probiotics also showed neuroprotective effects with less pyknotic cells and lower frequency of vacuolization in cerebral cortex. This multi-strain probiotic formulation from Thai fermented foods may offer a potential to develop psychobiotic-rich functional foods to modulate human mind and behaviors.

Formation of Sulforaphane and Iberin Products from Thai Cabbage Fermented by Myrosinase-Positive Bacteria.

Myrosinase-positive bacteria from local fermented foods and beverages in Thailand with the capacity to metabolize glucosinolate and produce isothiocyanates (ITCs) were isolated and used as selected strains for Thai cabbage fermentation. Enterobacter xiangfangensis 4A-2A3.1 (EX) from fermented fish and Enterococcus casseliflavus SB2X2 (EC) from fermented cabbage were the two highest ITC producers among seventeen strains identified by 16S rRNA technique. EC and EX were used to ferment Thai cabbage (Brassica oleracea L. var. capitata) containing glucoiberin, glucoraphanin and 4-hydroxyglucobrassicin at 430.5, 615.1 and 108.5 µmol/100 g DW, respectively for 3 days at 25 °C. Different amounts of iberin nitrile, iberin, sulforaphane and indole 3-acetonitrile were produced by spontaneous, EX- and EC-induced cabbage fermentations, and significantly higher ITCs were detected (p < 0.01) with increased antioxidant activities. Iberin and sulforaphane production in EX-induced treatment peaked on day 2 at 117.4 and 294.1 µmol/100 g DW, respectively, significantly higher than iberin at 51.7 µmol/100 g DW but not significantly higher than sulforaphane at 242.6 µmol/100 g DW in EC-induced treatment at day 2. Maximum health-promoting benefits from this functional food can be obtained from consumption of a liquid portion of the fermented cabbage with higher ITC level along with a solid portion.

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.

Glucosinolate and Desulfo-glucosinolate Metabolism by a Selection of Human Gut Bacteria.

Glucosinolate (GSL) hydrolysis is mediated by the enzyme myrosinase which together with specifier proteins can give rise to isothiocyanates (ITCs), thiocyanates, and nitriles (NITs) in cruciferous plants. However, little is known about the metabolism of GSLs by the human gut flora. The aim of the work was to investigate the metabolic fates of sinigrin (SNG), glucotropaeolin (GTP), gluconasturtiin (GNT), and their corresponding desulfo-GSLs (DS-GSLs). Three human gut bacterial strains, Enterococcus casseliflavus CP1, Lactobacillus agilis R16, and Escherichia coli VL8, were chosen for this study. GNT was metabolized to completion within 24 h to phenethyl ITC and phenethyl NIT (PNIT) by all bacteria, except for L. agilis R16 which produced only PNIT. At least 80 % of GTP and SNG were metabolized by all bacteria within 24 h to the corresponding ITCs and NITs. The pH of media over time gradually became acidic for both L. agilis R16 and E. coli VL8, while for E. casseliflavus CP1 the media became slightly alkaline with NIT and ITC production occurring between pH 3.0 and 7.5. ITC production peaked between 4 and 10 h in most cases and gradually declined while NIT production increased and remained relatively constant over time. The total percentage products accounted for 3-53 % of the initial GSL. NITs were produced from DS-GSLs suggesting an alternative metabolism via desulfation for the food based GSLs. The metal ion dependency for NIT production for GNT and its DS form was investigated where it was shown that Fe(2+) increased NIT production, while Mg(2+) stimulated the formation of ITC.

Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) ß-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil.

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.

Identification of Proteins Possibly Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8.

This study was aimed to identify sinigrin-induced bacterial proteins potentially involved in the metabolism of glucosinolate in two glucosinolate-metabolising bacteria Lactobacillus agilis R16 and Escherichia coli VL8. Sinigrin (2 mM) was used to induce the proteins in both bacteria under anaerobic incubation for 8 h at 30 °C for L. agilis R16 and 37 °C for E. coli VL8 and the controls without sinigrin were performed. Allyl isothiocyanate and allyl nitrile as two degradation products of sinigrin were detected in sinigrin-induced cultures of L. agilis R16 (27% total products) and E. coli VL8 (38% total products) from a complete sinigrin degradation in 8 h for both bacteria. 2D gel electrophoresis was conducted to identify induced proteins with at least twofold increased abundance. Sinigrin-induced L. agilis R16 and the control produced 1561 and 1543 protein spots, respectively. For E. coli VL8, 1363 spots were detected in sinigrin-induced and 1354 spots in the control. A combination of distinct proteins and upregulated proteins of 32 and 35 spots in L. agilis R16 and E. coli VL8, respectively were detected upon sinigrin induction. Of these, 12 and 16 spots from each bacterium respectively were identified by LC-MS/MS. In both bacteria most of the identified proteins are involved in carbohydrate metabolism, oxidoreduction system and sugar transport while the minority belong to purine metabolism, hydrolysis, and proteolysis. This indicated that sinigrin induction led to the expressions of proteins with similar functions in both bacteria and these proteins may play a role in bacterial glucosinolate metabolism.

The metabolism of methylsulfinylalkyl- and methylthioalkyl-glucosinolates by a selection of human gut bacteria.

SCOPE: Certain myrosinase-positive human gut bacteria can metabolize glucosinolates (GSLs) to produce isothiocyanates (ITC) as chemopreventive agents. We investigated glucoerucin, glucoiberin, and glucoraphanin (present in broccoli) metabolism by human gut strains. METHODS AND RESULTS: All tested bacteria metabolized glucoerucin to completion within 16 h to erucin and erucin nitrile (NIT). Lactobacillus agilis R16 metabolized only 10% of glucoiberin and glucoraphanin with no detectable products. Enterococcus casseliflavus CP1, however, metabolized 40-50% of glucoiberin and glucoraphanin producing relatively low concentrations of iberin and sulforaphane. Interestingly, Escherichia coli VL8 metabolized 80-90% of glucoiberin and glucoraphanin and also bioconverted glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively, producing erucin, erucin NIT, iberverin, and iberverin NIT from the two GSLs. The putative reductase enzyme in the cell-free extracts of this bacterium required both Mg(2+) and NAD(P)H as cofactors for bioconversion. The cell-free extract of E. coli VL8 containing the reductase enzyme was able to reduce both the GSL glucoraphanin and its hydrolysis product sulforaphane to glucoerucin and erucin/erucin NIT, respectively. CONCLUSION: The composition and metabolic activity of the human gut bacteria can indirectly impact on the potential chemopreventive effects of GSL-derived metabolites.

A trio of viral proteins tunes aphid-plant interactions in Arabidopsis thaliana.

BACKGROUND: Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: 'peach-potato aphid', 'green peach aphid'). METHODOLOGY/PRINCIPAL FINDINGS: Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. CONCLUSIONS/SIGNIFICANCE: Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.