Yeast biodiversity from oleic ecosystems: study of their biotechnological properties.

The aim of this study was to know the yeast biodiversity from fresh olive (Olea europaea L.) fruits, olive paste (crush olives) and olive pomace (solid waste) from Arbequina and Cornicabra varieties. Yeasts were isolated from fruits randomly harvested at various olive groves in the region of Castilla La Mancha (Spain). Olive paste and pomace, a byproduct of the processing of this raw material, were also collected in sterile flasks from different oil mills. Molecular identification methodology used included comparison of polymerase chain reaction (PCR) amplicons of their 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2 followed by restriction pattern analysis (RFLP). For some species, sequence analysis of the 5.8S rDNA gene was necessary. The results were compared to sequences held in public databases (BLAST). These techniques allowed to identify fourteen different species of yeasts, belonging to seven different genera (Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces, Saccharomyces, Candida, Torulaspora) from the 108 yeast isolates. Species diversity was thus considerable: Pichia caribbica, Zygosaccharomyces fermentati (Lachancea fermentati) and Pichia holstii (Nakazawaea holstii) were the most commonly isolated species, followed by Pichia mississippiensis, Lachancea sp., Kluyveromyces thermotolerans and Saccharomyces rosinii. The biotechnological properties of these isolates, was also studied. For this purpose, the activity of various enzymes (beta-glucosidase, beta-glucanase, carboxymethylcellulase, polygalacturonase, peroxidase and lipase) was evaluated. It was important that none of species showed lipase activity, a few had cellulase and polygalacturonase activities and the majority of them presented beta-glucanase, beta-glucosidase and peroxidase activities.

Relationship between Debaryomyces pseudopolymorphus enzymatic extracts and release of terpenes in wine.

As a result of the interest that exists in the liberation of aromas in young wines, we obtained some different enzymatic extracts (purified extract, P; lyophilized purified extract, LP; immobilized purified extract, IP; and immobilized lyophilized purified extract, ILP) with beta-glucosidase activity from Debaryomyces pseudopolymorphus, which excreted the enzyme in the growth medium. The extracts were added to natural glycosides isolated from different grape varieties. The results were compared with the effect of seven commercial enzyme preparations (CEP), obtained from molds used in wine making. It was shown that some yeast extracts had effects similar to those of the CEP, and the next step was to use them on wine samples elaborated in the laboratory. The effect was studied at 9 and 16 days of contact, quantifying both the precursors that were retained and the liberated terpenes. The results were compared with a control wine (without any extract) and with wine treated with a commercial enzyme preparation specially indicated for the liberation of aromas. It was observed that the enzymatic extracts from Db. pseudopolymorphus hydrolyzed the precursors in wine and that they could compete with the commercial preparations since the liberation was produced in even less time.

Fungi isolated from olive ecosystems and screening of their potential biotechnological use.

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for ß-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for ß-glucosidase, CMCase and lipase); Rhizopus oryzae (for ß-glucosidase and lipase); Rhizomucor variabilis (for ß-glucosidase, CMCase and polygalacturonase); Mucor fragilis (ß-glucosidase, CMCase and lipase); Galactomyces geotrichum (for ß-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, ß-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and ß-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.

Extracellular beta-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology.

beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.