José Vicente Pascual Gil

Mycobiota and mycotoxin producing fungi from cocoa beans

The present study reports on the natural mycobiota occurring in cocoa beans, paying special attention to the incidence of fungal species that are potential producers of mycotoxins. The results show that predominant fungi were different species of the genus Aspergillus belonging to section Flavi and Nigri. Of the 214 strains of Aspergillus section Flavi collected from cocoa beans, 120 were identified as A. flavus and 94 as A. tamarii. Of Aspergillus section Nigri 138 strains were isolated, with 132 belonging to A. niger aggregate and 6 to A. carbonarius species. Potential ability to produce aflatoxins (AFs) B1, B2, G1 and G2, cyclopiazonic acid (CPA) and ochratoxin A (OTA) was studied by isolate culture followed by HPLC analysis of these mycotoxins in the culture extracts. Results indicated that 64.1% and 34.2% of the A. flavus strains produced AFs and CPA, respectively. Most of the A. flavus strains presented moderate toxigenicity with mean levels of AFs ranging from 100 ng g(-1) to 1000 ng g(-1). All the CPA-producing strains of A. flavus were highly toxigenic producing >30 microg g(-1) of CPA. Furthermore, 98% of A. tamarii strains produced CPA and over 50% of them were highly CPA toxigenic. With respect to OTA-producing fungi, a high percentage of black aspergilli strains (49.2%) were able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 microg g(-1) to 100 microg g(-1). These results indicate that there is a possible risk factor posed by AFs, CPA and OTA contamination of cocoa beans, and consequently, cocoa products.

Past and Future of Non-Saccharomyces Yeasts: From Spoilage Microorganisms to Biotechnological Tools for Improving Wine Aroma Complexity

It is well established that non-Saccharomyces wine yeasts, considered in the past as undesired or spoilage yeasts, can enhance the analytical composition, and aroma profile of the wine. The contribution of non-Saccharomyces yeasts, including the ability to secret enzymes and produce secondary metabolites, glycerol and ethanol, release of mannoproteins or contributions to color stability, is species- and strain-specific, pointing out the key importance of a clever strain selection. The use of mixed starters of selected non-Saccharomyces yeasts with strains of Saccharomyces cerevisiae represents an alternative to both spontaneous and inoculated wine fermentations, taking advantage of the potential positive role that non-Saccharomyces wine yeast species play in the organoleptic characteristics of wine. In this context mixed starters can meet the growing demand for new and improved wine yeast strains adapted to different types and styles of wine. With the aim of presenting old and new evidences on the potential of non-Saccharomyces yeasts to address this market trend, we mainly review the studies focused on non-Saccharomyces strain selection and design of mixed starters directed to improve primary and secondary aroma of wines. The ability of non-Saccharomyces wine yeasts to produce enzymes and metabolites of oenological relevance is also discussed.

Autologous fat transfer to the cranio-maxillofacial region: Updates and controversies

BACKGROUND Autologous fat grafts have gained popularity among Cranio-Maxillofacial surgeons within the past years. Most publications report favourable outcomes but lack quantifiable evidence of graft survival. OBJECTIVES To assess autologous fat transfer for facial asymmetry, and review the literature focusing on current indications, techniques, complications, fat survival and patient satisfaction. PATIENTS AND METHODS Nine patients presented facial asymmetry due to onchological resection, congenital anomaly, or craniofacial traumatism. A total of 11 autologous fat transfers were performed. Four procedures followed the Coleman technique; in the other seven procedures, fat centrifugation was obviated. RESULTS No significant complications derived. Progressive volumetric decrease was evidenced for up to 6 months after surgery. A second procedure was performed in two patients. No clinical differences in cosmetic outcomes or graft survival were observed between centrifuged and non-centrifuged grafts. Patients reported high satisfaction. CONCLUSIONS Facial recontouring with autologous fat transfer restores volumetric defects with high patient satisfaction. The scientific literature offers inconsistent results. The authors did not find clinical differences between centrifuged and non-centrifuged grafts. Volume gain may result from induced fibrosis, inflammation and native adipocyte growth or differentiation. The heightened interest in these procedures should instigate further investigation to refine surgical procedures and improve predictability.

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA.

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.

Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions.

Aspergillus carbonarius is an important ochratoxin A producing fungus that is responsible for mycotoxin contamination of grapes and wine. In this study, the proteomes of highly (W04-40) and weakly (W04-46) OTA-producing A. carbonarius strains were compared to identify proteins that may be involved in OTA biosynthesis. Protein samples were extracted from two biological replicates and subjected to two dimensional gel electrophoresis analysis and mass spectrometry. Expression profile comparison (PDQuest software), revealed 21 differential spots that were statistically significant and showed a two-fold change in expression, or greater. Among these, nine protein spots were identified by MALDI-MS/MS and MASCOT database and twelve remain unidentified. Of the identified proteins, seven showed a higher expression in strain W04-40 (high OTA producer) and two in strain W04-46 (low OTA producer). Some of the identified amino acid sequences shared homology with proteins involved in regulation, amino acid metabolism, oxidative stress and sporulation. It is worth noting the presence of a protein with 126.5 fold higher abundance in strain W04-40 showing homology with protein CipC, a protein with unknown function related with pathogenesis and mycotoxin production by some authors. Variations in protein expression were also further investigated at the mRNA level by real-time PCR analysis. The mRNA expression levels from three identified proteins including CipC showed correlation with protein expression levels. This study represents the first proteomic analysis for a comparison of two A. carbonarius strains with different OTA production and will contribute to a better understanding of the molecular events involved in OTA biosynthesis.

The Antarctic yeast Candida sake: Understanding cold metabolism impact on wine

Current winemaking trends include low-temperature fermentations and using non-Saccharomyces yeasts as the most promising tools to produce lower alcohol and increased aromatic complexity wines. Here we explored the oenological attributes of a C. sake strain, H14Cs, isolated in the sub-Antarctic region. As expected, the cold sea water yeast strain showed greater cold growth, Na+-toxicity resistance and freeze tolerance than the S. cerevisiae QA23 strain, which we used as a commercial wine yeast control. C. sake H14Cs was found to be more sensitive to ethanol. The fermentation trials of low-sugar content must demonstrated that C. sake H14Cs allowed the cold-induced lag phase of growth to be eliminated and also notably reduced the ethanol (-30%) and glycerol (-50%) content in wine. Instead C. sake produced sorbitol as a compatible osmolyte. Finally, the inspection of the main wine volatile compounds revealed that C. sake produced more higher alcohols than S. cerevisiae. In conclusion, our work evidences that using the Antarctic C. sake H14Cs yeast improves low-temperature must fermentations and has the potential to provide a wine with less ethanol and also particular attributes.