La nueva Biotecnología apuesta por la vinificación

La OTRI del Centro Tecnológico Nacional de la Conserva y Alimentación junto con la OTT del Consejo Superior de Investigaciones Científicas, colaboran en el Proyecto AGROCSIC, el cual fue aprobado por el Ministerio de Ciencia y Tecnología y financiado por el Ministerio de Educación y Ciencia. El objetivo principal de esta nueva actuación es estudiar las distintas líneas de trabajo de los Centros del CSIC relacionadas con la alimentación, para transferir sus resultados al sector industrial.Desde el punto de vista microbiológico, la transformación del mosto de uva en vino o vinificación es un proceso complejo en el que intervienen distintos microorganismos, y donde las levaduras, principalmente Saccharomyces cerevisiae, juegan el papel más destacado. Durante la vinificación las levaduras utilizan los azúcares y otros componentes del mosto para su crecimiento, produciendo etanol, anhídrido carbónico, y en menor medida otros compuestos responsables de la composición química y las cualidades sensoriales del vino. Desde un punto de vista bioquímico, el vino puede considerarse como un producto de la transformación enzimática del mosto de uva.En las últimas décadas, coincidiendo con el avance de la Biotecnología, se han desarrollado nuevas técnicas de vinificación, que incluyen, entre otras, la utilización de cepas seleccionadas de S. cerevisiae (para normalizar la microbiota inicial y dar lugar a fermentaciones homogéneas año tras año), y el empleo de enzimas (para solucionar problemas puntuales del proceso y mejorar la calidad del producto final). El uso de levaduras seleccionadas, capaces de conducir la fermentación alcohólica e imponerse al resto de levaduras presentes, abre la posibilidad de aplicar técnicas de ingeniería genética a la levadura vínica para obtener nuevas cepas capaces de producir, a lo largo de la fermentación, las enzimas de interés en enología cuya adición se realiza de forma regular. Asimismo, las técnicas de ADN recombinante se están aplicando a la vid, donde se están produciendo importantes avances (como por ejemplo la producción de plantas resistentes a ciertas enfermedades víricas y fúngicas) y a los microorganismos productores de los preparados enzimáticos comerciales de uso en enología (por ejemplo para conseguir mayores rendimientos y preparados más específicos).La información que las autoras aportan a esta revisión es fruto del desarrollo de varios proyectos financiados por la Comisión Interministerial de Ciencia y Tecnología (CICYT) entre los que se incluyen los proyectos en curso AGL2002-01906, AGL2003-01295/ALI y AGL2004-00978/ALI.Peer reviewe

Yeast species and genetic heterogeneity within Debaryomyces hansenii along the ripening process of traditional ewes' and goats' cheeses

The yeasts present during the ripening process of ewes' and goats' cheeses produced in a small traditional dairy in Mediterranean Spain were isolated and identified. Five hundred and thirty strains pertaining to eleven yeast species representing eight genera were identified using molecular methods. Debaryomyces hansenii was the yeast species most frequently isolated in all cheeses. Other yeast species commonly found in dairy products were present at the first maturing weeks. Two yeast species Trichosporon coremiiforme and Trichosporon domesticum have been reported in cheeses for the first time, and Meyerozyma guilliermondii has been newly isolated from goats' cheeses. Yeast species composition changed greatly along the process; although, D. hansenii dominated at the end of ripening in all cheeses. Most yeast isolates were able to hydrolyze casein and fatty acid esters. One hundred and eighty seven D. hansenii isolates were genotyped by PCR amplification of M13 satellites and an UPGMA dendrogram was constructed. The majority of isolates were grouped in 5 clusters while 7 profiles were represented by 1–3 isolates. These results demonstrate the genetic heterogeneity present in D. hansenii strains isolated from raw milk cheeses.Authors thank grants Consolider Ingenio Fun-C-Food, CSD2007-00063 and INIA RM2007-00010 for financial support. BP thanks CSIC for a JAE Predoc contract. Authors are grateful to Cheese Company “Los Corrales” for kindly supplying the cheese samples.Peer reviewe

The role of non-Saccharomyces yeasts in industrial winemaking

The fermentation of grape juice into wine is a complex microbiological process, in which yeasts play a central role. Traditionally, identification and characterization of yeast species have been based on morphological and physiological characteristics. However, the application of molecular biology techniques represents an alternative to the traditional methods of yeast identification and are becoming an important tool in solving industrial problems. Although Saccharomyces cerevisiae is responsible for the alcoholic fermentation, the presence of non-Saccharomyces species could be important since they produce secondary metabolites, which can contribute to the final taste and flavor of wines

Ethanol Production from Sugarcane Bagasse Pretreated by Steam Explosion

Bioethanol is an alternative renewable fuel that can be produced from cellulosic biomass through hydrolysis and fermentation based processes. Sugarcane bagasse constitutes a potential lignocellulosic substrate for bioethanol production, since it has high sugar content and is a renewable, cheap and readily available feedstock. In this work, steam explosion pretreatment at different temperatures (200°C, 215°C and 230°C) for 5 min was evaluated on sugarcane bagasse for ethanol production by simultaneous saccharification and fermentation (SSF). Sugar recovery in the solid and liquid fraction obtained after pretreatment and the enzymatic digestibility of the pretreated material were used to determine the optimum temperature, 215°C for 5 min, which resulted in an overall glucose yield of 86.8% of the content in raw material. The solid fraction of sugarcane bagasse pretreated at 215°C was submitted to SSF at increasing solid loading and the effect of xylanase supplementation and a presaccharification step was assessed. The highest final ethanol concentration (56.3 g L-1) was achieved with SSF supplemented with xylanase enzyme at 20% of solid concentration. Ethanol yields exceeded 0.30 g per gram of glucose in all the conconditions tested. The results obtained demonstrate the effectiveness of steam explosion in the treatment of sugarcane bagasse fiber

L-Rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake

<p>Abstract</p> <p>Background</p> <p>Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus <it>Aspergillus nidulans </it>growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (<it>rhaE</it>) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second α-L-rhamnosidase gene, (AN10277/<it>rhaA</it>).</p> <p>Results</p> <p>Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (<it>rhaE</it>). The cDNA of <it>rhaE </it>was expressed in <it>Saccharomyces cerevisiae </it>and yielded <it>p</it>NP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse <it>A. nidulans </it>strains cultivated under different growth conditions indicate that <it>rhaA </it>and <it>rhaE </it>are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these <it>rha </it>genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression.</p> <p>Conclusions</p> <p>The <it>A. nidulans rhaE </it>gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of <it>rhaE </it>and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, <it>A. nidulans </it>α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR.</p

Potential impact of dairy yeasts on the typical flavour of traditional ewes' and goats' cheeses

The contribution of Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus strains to the typical flavour of traditional ewes' and goats' cheeses was assessed. Fourteen yeast strains were grown in liquid medium mimicking cheese composition and volatile compounds were identified by gas chromatography-mass spectrometry. Yeasts were able to produce key volatile compounds characteristic of the cheeses from which they were isolated. Inter-species and inter-strain variations were observed. Under the conditions tested, D. hansenii produced the lowest levels of volatile compounds, with large intra-strain variations. Kluyveromyces strains primarily produced esters and alcohols. K. marxianus strains were associated with the production of acids, ethyl decanoate, 1-propanol and benzaldehyde, whereas K. lactis was correlated with the presence of ketones, ethyl acetate and secondary alcohols. In conclusion, this study shows the heterogeneous potential of dairy yeasts to contribute to final cheese flavour.The authors thank grants Consolider Ingenio Fun-C-Food, CSD2007-00063, INIARM2007-00010 and AGL2012-38884-C02-01 for financial support. BP thanks CSIC for a JAE Predoc contract. The authors are grateful to the Cheese Company “Los Corrales” for kindly supplying the cheese samples.Peer reviewe

Dairy Debaryomyces hansenii strains produce the antihypertensive casein-derived peptides LHLPLP and HLPLP

The ability of dairy Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus strains to release the casein-derived antihypertensive sequences RYLGY, AYFYPEL, LHLPLP, HLPLP, VPP and/or IPP was examined. Yeast strains were grown in medium with casein as sole nitrogen source and the yeast casein hydrolysates (CSHs) were analysed by HPLC-MS/MS to search for the six antihypertensive sequences. Only LHLPLP and HLPLP were identified in CSHs and exclusively in D. hansenii Dh1 and Dh14 hydrolysates in which both antihypertensive sequences represented approximately 6 (CSH Dh1) and 10% (CSH Dh14) of total peptide content. In addition, a complete analysis of selected CSHs by HPLC-MS/MS allowed the identification of 35 (Dh1) and 46 (Dh14) additional peptides, which could also contribute to the observed in vitro ACE inhibitory potency of both hydrolysates (Dh1, IC50 = 13.6 ± 1.8 μg/mL; Dh14, IC50 = 17.5 ± 2.1 μg/mL) and might confer them multifunctional properties. Finally casein zymography revealed the presence of at least one extracellular protease with a molecular mass of about 50 kDa. In conclusion, the present study contributes to a better insight into bioactive compounds produced by dairy yeasts and shows the feasibility of D. hansenii strains to produce antihypertensive casein-derived peptides.This work was supported by grants AGL2010-21009 and AGL2011-24643 from ‘Ministerio de Educación y Ciencia – FEDER’, Consolider Ingenio 2010, Fun-C-Food, CSD2007-00063 and RETICS INVICTUS RD12/0014/0004 from ‘Instituto de Salud Carlos III’. A. García-Tejedor and L. Sánchez-Rivera are recipients of predoctoral fellowships from ‘Ministerio de Educación y Ciencia’ (BES-2011-044424) and CSIC (JAE-PreDoc), respectively.Peer reviewe

Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum

Antifungal proteins (AFPs) from Ascomycetes are small cysteine-rich proteins that are abundantly secreted and show antifungal activity against non-producer fungi. A gene coding for a class B AFP (AfpB) was previously identified in the genome of the plant pathogen Penicillium digitatum. However, previous attempts to detect the AfpB protein were not successful despite the high expression of the corresponding afpB gene. In this work, the structure of the putative AfpB was modeled. Based on this model, four synthetic cysteine-containing peptides, PAF109, PAF112, PAF118, and PAF119, were designed and their antimicrobial activity was tested and characterized. PAF109 that corresponds to the gamma-core motif present in defensin-like antimicrobial proteins did not show antimicrobial activity. On the contrary, PAF112 and PAF118, which are cationic peptides derived from two surface-exposed loops in AfpB, showed moderate antifungal activity against P. digitatum and other filamentous fungi. It was also confirmed that cyclization through a disulfide bridge prevented peptide degradation. PAF116, which is a peptide analogous to PAF112 but derived from the Penicillium chrysogenum antifungal protein PAF, showed activity against P. digitatum similar to PAF112, but was less active than the native PAF protein. The two AfpB-derived antifungal peptides PAF112 and PAF118 showed positive synergistic interaction when combined against P. digitatum. Furthermore, the synthetic hexapeptide PAF26 previously described in our laboratory also exhibited synergistic interaction with the peptides PAF112, PAF118, and PAF116, as well as with the PAF protein. This study is an important contribution to the mapping of antifungal motifs within the AfpB and other AFPs, and opens up new strategies for the rational design and application of antifungal peptides and proteins

In vivo antihypertensive mechanism of lactoferrin-derived peptides: Reversion of angiotensin I- and angiotensin II-induced hypertension in Wistar rats

Novel peptides with antihypertensive effects in SHR rats have previously been identified in lactoferrin (LF) hydrolysates. To investigate their in vivo antihypertensive mechanism, we have assessed the blood pressure lowering effects of two of these LF-derived peptides (RPYL and DPYKLRP) in Wistar rats subjected to either angiotensin I- or angiotensin II-induced hypertension. Blood pressure was measured by the tail-cuff method, hypertension was induced by subcutaneous infusion of angiotensins, and then captopril, valsartan or LF-derived peptides orally administered. Angiotensin I- and angiotensin II-induced hypertension were reversed by captopril and valsartan, respectively. RPYL and DPYKLRP reversed angiotensin I-induced hypertension, while DPYKLRP but not RPYL produced a modest reversion of angiotensin II-elicited hypertension. Neither RPYL nor DPYKLRP modified normotension. Thus, in vivo ACE inhibition is involved in the antihypertensive effects of LF-derived peptides like RPYL and DPYKLRP, while inhibition of AT1 receptor-mediated vasoconstriction plays a less relevant role.This work was supported by grant AGL2010-21009 from ‘Ministerio de Educación y Ciencia – FEDER’, Consolider Ingenio 2010, Fun-C-Food, CSD2007-00063 and RETICS INVICTUS RD12/0014/0004 from ‘Instituto de Salud Carlos III’. A. García-Tejedor is recipient of a predoctoral fellowship from ‘Ministerio de Educación y Ciencia’ (BES-2011-044424).Peer reviewe