Study of the phenotypic and genotypic biodiversity of potentially ochratoxigenic black aspergilli isolated from grapes.

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, carcinogenic, teratogenic and immunotoxic effects, naturally found in agricultural products including grapes and wine. Black Aspergillus species (Section Nigri) are mainly responsible for OTA accumulation in wine grapes and in particular Aspergillus carbonarius and Aspergillus niger aggregate. The biodiversity of potentially ochratoxigenic strains of black aspergilli from different French vineyards in the southern Mediterranean region of Languedoc-Roussillon was studied. One hundred and eighty nine black strains were isolated from grapes and studied according to harvest year, production zone, grape variety and pre-harvest treatment of grapevines. The strains were identified and classified in two groups according to macroscopic and microscopic characters; these were called the A. carbonarius representative group and the A. niger aggregate representative group. Members of each group were classified in subgroups based on macroscopic morphological colony characters. Strain biodiversity was studied according to phenotypic and genotypic characterization and to the OTA production of selected strains on PDA medium. After identification was confirmed by specific PCR using primer pair ITS1/CAR and ITS1/NIG, 24 potential ochratoxigenic strains belonging to A. carbonarius and A. niger aggregate were discriminated by RAPD-PCR using 8 different OPC primers. The use of specific primers supported the identification based on phenotypic and morphological characters. RAPD-PCR patterns demonstrated a considerable diversity among the strains. Clustering among A. niger aggregate strains was associated with production zone and harvest year, but not grape variety or pre-harvest treatment. Clustering among A. carbonarius strains was not associated with any of the above parameters. OTA production of strains on culture medium seemed to correlate better with morphological characters than with genotypic profiles. No clear relation could be established between phenotypic and genotypic characters of the studied black aspergilli.

OTA-producing fungi isolated from stored cocoa beans.

AIMS: The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates. METHODS AND RESULTS: Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [

Effect of operating conditions on ochratoxin A extraction from roasted coffee.

Operating conditions affect ochratoxin A (OTA) extraction from roasted coffee. The OTA content found in the beverage can thus be greater than that found in the roasted coffee used to prepare it. Three extraction parameters were studied for roasted coffee: type of extraction solvent (alkaline, neutral, acid), temperature (ambient temperature/23 degrees C, 60 degrees C and 85 degrees C), and extraction time (5, 20, 30, 40, 50, 60 and 80 min). The alkaline solvent used in the method recommended by the European Union extracted OTA better, but a maximum content was obtained at 60 degrees C after 50 min. At least a 100% improvement in extraction was obtained when compared with the European Union usual quantification method that is carried out at ambient temperature. It turned out that the OTA extraction parameters for roasted coffee, as defined by that method, were not optimum and needed to be modified. These results were verified in double-extraction experiments showing that OTA is not completely extracted by this method. Confirmation was obtained by comparison of extraction methods on several commercial samples of roasted coffee.

Fate of mucilage cell wall polysaccharides during coffee fermentation.

Effects of a 20-h fermentation on cell wall polysaccharides from the mucilage of pulped coffee beans were examined and compared to those of unfermented beans, on alcohol insoluble residues (AIRs), their hot-water-soluble crude pectic substances (PECTs), and their hot-water-insoluble residues (RESs). Yields and compositions were very similar: AIRs, which consisted of approximately 30% highly methylated pectic substances, approximately 9% cellulose, and approximately 15% neutral noncellulosic polysaccharides, exhibited no apparent degradation. However, PECTs from fermented beans were shown to have undergone a slight reduction of their intrinsic viscosity and weight-average molecular weight by capillary viscosimetry and high-performance size-exclusion chromatography. After fermentation, hot-water-insoluble pectic substances of RES exhibited partial de-esterification. Removal of coffee bean mucilage by natural fermentation seems to result from a restricted pectolysis, the mechanism of which remains to be elucidated.

Microbiological and biochemical study of coffee fermentation.

The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous. The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation, 60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic microorganisms.

Influence of yeast flocculation on the rate of Jerusalem artichoke extract fermentation.

Variations in residual sugar composition have been observed during Jerusalem artichoke extract fermentations by using Saccharomyces diastaticus NCYC 625, a flocculating yeast strain. In batch cultures, these differences were due to the inulin polymer size distribution of the extracts: measurements of enzymatic activities on different polymerized substrates have shown that the hydrolysis and fermentation yield decreased when the fructose/glucose ratio of the extract increased. Inulin hydrolysis appeared to be the limiting factor of the fermentation rate. A comparison of continuous and batch cultures with the same extract showed that fermentability differences were related to the structure and size of the yeast flocs. This led to an hydrolysis selectivity of the inulin polymers according to their size: the chemostat culture in which the floc average size was larger gave longer chained residual sugars.

Enterotoxin production by staphylococci isolated from foods in France.

Two hundred and thirteen Staphylococcus aureus and 51 other staphylococcal strains were isolated from 121 foodstuffs of current consumption and two cutaneous samples. Their ability to produce staphylococcal enterotoxins was tested and S. aureus strains were biotyped. The S. aureus strains (30.5%) produced at least one of the five known staphylococcal enterotoxins whereas coagulase negative staphylococci did not produce any of them. The raw milk cheeses analysed were primarily contaminated by strains belonging to animal or unspecified biovars. Only 15.9% of the S. aureus strains isolated from these products produced enterotoxins whereas 43% were found to be enterotoxigenic amongst the S. aureus strains isolated from the other foodstuffs. The scheme of biotyping used seems to be reliable, allowing the classification of 73.7% of the strains. S. aureus strains of human biovar origin were most often enterotoxigenic and enterotoxin C was the predominant type identified. It was produced by 66% of the enterotoxigenic strains, singly or in combination with other enterotoxins. Approximately 77% of the human strains also produced enterotoxin C, which is an amazing epidemiological distinctive feature of the strains studied. Moreover ELISA tests used in this work exhibit problems of specificity.

Structure and sucrose hydrolysis activity of Saccharomyces cerevisiae aggregates.

The sucrose hydrolysis activity of dense spherical yeast flocs, cultivated on a sucrose medium in a continuous reactor with internal settler, is nearly proportional to the particle surface. From computer simulation, in good agreement with experimental determinations, the calculated sucrose penetration depth is in the range 0.2-0.3 mm, a dimension smaller than the usual diameter of strongly flocculating yeast particles. From specific gravity determinations, the flocs can be considered as homogeneous and cannot exhibit a fractal structure, reported in the literature for a number of microbial aggregates. However, the analysis of the sucrose hydrolysis rates reveals that the cell density may be lower in the outer layer of the flocs.

[Isolation and characterization of a flocculating mutant of Saccharomyces diastaticus]. / Isolement et caractérisation d'un mutant floculant de Saccharomyces diastaticus.

A flocculating strain of Saccharomyces diastaticus NRRL2416 was isolated after ethidium bromide mutagenesis and density gradient separation. Flocculation did not change the general characteristics of the strain. Flocculation was better on maltodextrin-containing media than on others. It was found to be calcium-dependent and sensitive to growth conditions. The deflocculating effects of some sugars suggested that mannose may be involved in the mechanism of flocculation, as for strains of the FLO1 phenotypic group.

Impairment of insulin assimilation and beta-fructosidase activity due to a petite mutation in Kluyveromyces marxianus.

A respiratory deficient mutant of Kluyveromyces fragilis was isolated using ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a + a3 and deficiency in cytochrome b. This petite mutant has brought about modifications in the excretion pattern of beta-fructosidase active on saccharose and inulin. The mutant practically no longer excretes the enzyme, and is incapable of growth and fermentation in the presence of inulin. The study of the activities of different enzyme extracts (culture medium, whole and disrupted cells) on inulin and saccharose suggests the existence of an unique enzyme system capable of taking several forms, and also shows the influence of the growth substrate on the I/S activity ratio.

Isolation of a respiratory-deficient Kluyveromyces fragilis mutant for the production of ethanol from Jerusalem artichoke.

A respiratory-deficient, mutant of Kluyveromyces fragilis was isolated using a ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a + a3 and by an improvement of its inulinase activity. Under anaerobic conditions this mutant was always better than the wild strain for ethanol production especially from Jerusalem artichoke extracts containing large amounts of high polyfructosans ("early" extracts).

Isolation of an inulinase derepressed mutant of Pichia polymorpha for the production of fructose.

Pichia polymorpha has inulinase activity and could be used for the production of fructose syrup from inulin. The application of immobilized P. polymorpha whole cells for the continuous hydrolysis of inulin is, however, limited since the biosynthesis of this enzyme system is repressed by the reaction products, dextrose and fructose. A derepressed mutant hyperproducer of inulinase was isolated after treatment with EMS followed by a selection step with deoxyglucose.

Selection of yeasts for single cell protein production on media based on Jerusalem artichoke extracts.

Several yeast strains can grow with good yield (0.16 to 0.19 mg protein/mg carbohydrate) on nitrogen supplemented Jerusalem artichoke extract. The most promising strain is Lipomyces starkeyi. Including by-products (pulps, proteins of extract), protein production can reach 2 metric tons/ha.

Inulinase of Debaryomyces cantarellii.

Debaryomyces cantarellii excretes into a buffered medium an inulinase of beta-fructofuranosidase type, its synthesis being induced by inulin. The enzyme has a pH optimum at 4 and its optimum temperature is 50 degrees C. Its Km for inulin is 15 mM.

Selection of yeast strains for ethanol production from inulin.

Of the many yeast species capable of fermenting inulin, some can produce sufficient amounts of ethanol from the substrate, in particular Kluyveromyces fragilis and Torulopsis colliculosa. The results indicate the feasibility of producing ethanol from inulin-rich plants, such as Jerusalem artichoke.

Study of inulase from Debaryomyces phaffii Capriotti.

Debaryomyces phaffii possesses an inulinase activity which can be induced by growth on beta-fructosidase and particularly on inulin. The enzyme is located in the cell wall but is easily excreted into the culture medium. Maximum activity on inulin is observed at pH 4 and 50 degrees C. The Km on inulin is 1.2 X 10(-2) M. The enzyme breaks down inulin by splitting off terminal fructosyl units and it is active on sucrose and raffinose.

Inulinase activity of Debaromyces cantarellii.

Debaromyces cantarellii Capriotti contains an inulinase activity which is inducible by growth on inulin but not on other beta-fructosides. The induction is inhibited by glucose and fructose. The system is situated in the cell wall and can be best extracted with a 20 mM phosphate buffer at pH 8.5. The inulinase activity shows pH optima at 4 and 6, suggesting the presence of two enzymes, the latter being more tightly bound to the cell wall. Both enzymes degrade inulin from the nonreducin end. The cells also contain a constitutive beta-fructofuranosidase with a specificity partly overlapping with that of the inulinase(s).