Mycological and Multiple Mycotoxin Surveillance of Sorghum and Pearl Millet Produced by Smallholder Farmers in Namibia.

Mycological (mycotoxigenic Fusarium and aflatoxigenic Aspergillus spp.) and multiple mycotoxins [aflatoxin B1 (AFB1), fumonisin B (FB), deoxynivalenol and zearalenone] surveillance was conducted on raw whole grain sorghum (Sorghum bicolor) and pearl millet (Pennisetum glaucum) produced on smallholder farms, and processed products sold at open markets in northern Namibia. Fungal contamination was determined with morphological methods as well as with quantitative Real-Time PCR (qPCR). The concentrations of multiple mycotoxins in samples were determined with liquid chromatography tandem mass spectrometry. The incidence of mycotoxigenic Fusarium spp., Aspergillus flavus and A. parasiticus, as well as the concentrations of AFB1 and FB were significantly (P < 0.001) higher in the malts as compared to the raw whole grains, with Aspergillus spp. and AFB1 exhibiting the highest contamination (P < 0.001). None of the analysed mycotoxins were detected in the raw whole grains. Aflatoxin B1 above the regulatory maximum level set by the European Commission was detected in sorghum (2 of 10 samples; 20%; 3-11 µg/kg) and pearl millet (6 of 11 samples; 55%; 4-14 µg/kg) malts. Low levels of FB1 (6 of 10 samples; 60%; 15-245 µg/kg) were detected in sorghum malts and no FB was detected in pearl millet malts. Contamination possibly occurred postharvest, during storage, and/or transportation and processing. By critically monitoring the complete production process, the sources of contamination and critical control points could be identified and managed. Mycotoxin awareness and sustainable education will contribute to reducing mycotoxin contamination. This could ultimately contribute to food safety and security in northern Namibia where communities are exposed to carcinogenic mycotoxins in their staple diet.

Differential Modulation of Gene Expression Encoding Hepatic and Renal Xenobiotic Metabolizing Enzymes by an Aspalathin-Enriched Rooibos Extract and Aspalathin.

Modulation of the expression of hepatic and renal genes encoding xenobiotic metabolizing enzymes by an aspalathin-enriched green rooibos (Aspalathus linearis) extract (GRE) was investigated in the liver and kidneys of F344 rats following dietary exposure of 28 d, as well as selected xenobiotic metabolizing genes in rat primary hepatocytes. In the liver, GRE upregulated genes (p < 0.05) encoding aldehyde dehydrogenase, glucose phosphate isomerase, and cytochrome P450 while 17ß-hydroxysteroid dehydrogenase 2 (Hsd17ß2) was downregulated. In primary hepatocytes, GRE lacked any effect, while aspalathin downregulated Hsd17ß2, mimicking the effect of GRE in vivo, and upregulated catechol-O-methyl transferase and marginally (p < 0.1) cytochrome P450 2e1. In the kidneys, GRE upregulated (p < 0.05) genes encoding the phase II xenobiotic metabolism enzymes, glutathione-S-transferase mµ and microsomal glutathione-S-transferase, while downregulating genes encoding the ATP binding cassette transporter, cytochrome P450, gamma glutamyltransferase 1, and N-acetyltransferase 1. Differential modulation of the expression of xenobiotic metabolizing genes in vivo and in vitro by GRE is dose-related, duration of exposure, the tissue type, and interactions between specific polyphenol and/or combinations thereof. Aspalathin is likely to be responsible for the downregulation of estradiol and testosterone catabolism by GRE in the liver. The differential gene expression by GRE in the liver and kidneys could, depending on the duration exposure and dose utilized, determine the safe use of such an extract in humans for specific health and/or disease outcomes.

Autophagy-induced cell death by aqueous and polyphenol-enriched extracts of honeybush (Cyclopia spp.) in liver and colon cancer cells.

The anti-cancer potential of Cyclopia species (honeybush) has been demonstrated in several models. The present study investigated the effects of aqueous and polyphenol-enriched (PE) extracts of C. subternata and C. genistoides, as well as mangiferin and hesperidin, on different cell growth parameters in human liver (HepG2) and colon (HT-29) cancer cells. Mangiferin and hesperidin were most abundant in C. genistoides and C. subternata, respectively. Cyclopia subternata extracts had the highest ferric-reducing antioxidant capacity. Following exposure of the cells to the extracts and compounds, cell viability, proliferation, and death (apoptosis and autophagy) were determined. Cyclopia subternata extracts reduced cell viability and inhibited cell proliferation the most, associated with depletion of ATP. In HepG2 cells, the PE extracts were less effective than the aqueous extracts in reducing cell viability but more effective in inhibiting cell proliferation. Despite disrupting cell growth, none of the extracts induced apoptosis. The aqueous extracts affected autophagy in both cancer cells. Disruption of mitochondrial membrane integrity by the different extracts, presumably via polyphenol/iron interactions, is postulated to be involved; however, mangiferin and hesperidin had no effect, suggesting that other polyphenols and/or complex interactions between compounds are likely responsible for the differential cytotoxic and/or cytoprotective effects of the extracts.

Biocontrol of Fusarium Species Utilizing Indigenous Rooibos and Honeybush Extracts.

Mycotoxins produced by several Fusarium species have a significant effect on reducing maize yield and grain quality and have led to food safety concerns. The antifungal activities of rooibos (Aspalathus linearis) and honeybush (Cyclopia species) tea extracts reduced the growth of plant pathogen Botrytis cinerea, but their efficacy against Fusarium spp. is unknown. In this study, we examined the effects of fermented and unfermented rooibos (A. linearis) and honeybush (Cyclopia subternata) aqueous extracts as well as green tea (Camellia sinensis) against 10 Fusarium species. Conidial viability was assessed by fluorescence microscopy dyes, ATP production was determined using the BacTiter-Glo assay, the mode of action was analyzed by scanning electron microscopy (SEM), and quantification of polyphenols was done using high-performance liquid chromatography with diode array detection (HPLC-DAD). Fermented rooibos extract demonstrated the highest antifungal activity (P < 0.0001) against Fusarium verticillioides MRC 826-E, Fusarium subglutinans MRC 8553, Fusarium proliferatum MRC 8549, and Fusarium globosum MRC 6647, with only 9.53%, 9.26%, 11.0%, and 12.7% ATP production, respectively, followed by antifungal activity of the fermented C. subternata extract against F. subglutinans MRC 8553, F. subglutinans MRC 8554, F. proliferatum MRC 8550, and F. verticillioides MRC 826-E with 3.79%, 6.04%, 6.04%, and 8.40% ATP production, respectively. Extract-treated conidia examined by SEM exhibited disruption of conidial hyphae and collapsed spores. Overall, the fermented rooibos and C. subternata extracts showed higher antifungal activity against the Fusarium species than the unfermented extracts. IMPORTANCE In maize subsistence farming areas in South Africa, daily consumption of maize contaminated by high level of mycotoxins contributes to long-term health effects such as immune deficiency and cancer. Biocontrol methods that are safe and cost-effective are critical to addressing this public health problem. Plant extracts known as biocides or green pesticides are alternatives to chemical pesticides due to their safety and eco-friendly properties. In South Africa, rooibos (Aspalathus linearis) and honeybush (Cyclopia species) contain polyphenols with significant antioxidant and antimicrobial properties. These indigenous herbal teas are widely available and consumed in South Africa and have potential as an innovative approach to reduce mycotoxin levels and, subsequently, human and animal exposure to these toxins. This study evaluates the efficacy of the antifungal activities of several aqueous extracts prepared from fermented and unfermented rooibos (A. linearis), honeybush (Cyclopia subternata), and green tea (Camellia sinensis) on 10 Fusarium strains.

Sulfate-reducing and methanogenic microbial community responses during anaerobic digestion of tannery effluent.

Microbial communities were monitored in terms of structure, function and response to physicochemical variables during anaerobic digestion of tannery and associated slaughterhouse effluent in: (i) 2 L biochemical methane potential batch reactors at different inoculum to substrate ratios (2-5) and initial sulfate concentrations (665-2000 mg/L), and (ii) 20 L anaerobic sequencing batch reactors with different mixing regimes (continuous vs. intermittent). Methanogenic and sulfidogenic community compositions in the 2 L reactors evolved initially, but stabilised after the start of biogas generation, although significant (ANOSIM p < 0.05) changes in the physicochemical parameters indicated continued metabolic activity. Both hydrogenotrophic and acetoclastic archaeal genera were present in high relative abundances. Continuous stirring preferentially selected the metabolically versatile genus Methanosarcina, suggesting that higher specific methane generation in the continuously stirred system (168 vs. 19.5 mL methane per gram volatile solids per week) was related to the metabolic activities of members of this genus.

Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was genetically modified to assemble a minicellulosome on its cell surface by heterologous expression of a chimeric scaffoldin protein from Clostridium cellulolyticum under the regulation of the phosphoglycerate kinase 1 (PGK1) promoter and terminator regulatory elements, together with the beta-xylanase 2 secretion signal of Trichoderma reesei and cell wall protein 2 (Cwp2) of S. cerevisiae. Fluorescent microscopy and Far Western blot analysis confirmed that the Scaf3p is targeted to the yeast cell surface and that the Clostridium thermocellum cohesin domain is functional in yeast. Similarly, functionality of the C. thermocellum dockerin domain in yeast is shown by binding to the Scaf3 protein in Far Western blot analysis. Phenotypic evidence for cohesin-dockerin interaction was also established with the detection of a twofold increase in tethered endoglucanase enzyme activity in S. cerevisiae cells expressing the Scaf3 protein compared with the parent strain. This study highlights the feasibility to future design of enhanced cellulolytic strains of S. cerevisiae through emulation of the cellulosome concept. Potentially, Scaf3p-armed yeast could also be developed into an alternative cell surface display strategy with various tailor-made applications.

The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates.

In Saccharomyces cerevisiae, branched-chain amino acid transaminases (BCAATases) are encoded by the BAT1 and BAT2 genes. BCAATases catalyse the transfer of amino groups between those amino acids and alpha-keto-acids. alpha-Keto-acids are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavour of yeast-derived fermentation products. The objective of this study was to investigate the influence of BAT-gene expression on general yeast physiology, on aroma and flavour compound formation and on the sensory characteristics of wines and distillates. For this purpose, the genes were overexpressed and deleted in a laboratory strain, BY4742, and overexpressed in an industrial wine yeast strain, VIN13. The data show that, with the exception of a slow growth phenotype observed for the BAT1 deletion strain, the fermentation behaviour of the strains was unaffected by the modifications. The chemical and sensory analysis of fermentation products revealed a strong correction between BAT gene expression and the formation of many aroma compounds. The data suggest that the adjustment of BAT gene expression could play an important role in assisting winemakers in their endeavour to produce wines with specific flavour profiles.

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates.

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1- and ATF2-encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1-encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1-encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester-synthesizing and ester-degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2-phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester-producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles.