Yeast population dynamics in five spontaneous fermentations of Malvasia must

The dynamics of the wine yeast strains presented in five spontaneous Malvasia wine fermentations have been studied. Samples were analysed for their microbiological characteristics and chemical substances. All 937 isolates were characterized using electrophoretic karyotyping and tested for their killer activity. The non- Saccharomyces population was identified using a combination of PCR-RFLP analysis of the rDNA spacer region and physiological testing. The total yeast population level in the must after sedimentation was 10⁵cfu ml⁻¹and included the following genera:Candida, Metschnikowia, Hanseniaspora, Rhodotorula, Issatchenkia and Debaryomyces. However, Saccharomyces sp. was not detected in fresh must samples plated on YEPD medium. Based on the chromosome length polymorphism among 649 isolates from the subsequent phases of fermentation, 46 different electrophoretic patterns of Saccharomyces cerevisiae were distinguished. The most abundant karyotypes were L₁, L₄, L₁₂, P₆. A sequential substitution of S. cerevisiae strains occurred during the different phases of fermentation. At the slow fermentation rate, karyotype L₄was most abundant in almost all fermenters. At the beginning of the tumultuous fermentation phase, the most frequent karyotype became L₁followed by karyotype L₄. Finally, during the fermentation process, pattern L₄was clearly replaced by karyotype L₁followed by pattern L₁₂. Despite the same fermentation source (grape must), differences among five spontaneous fermentations were observed. The population dynamics of S. cerevisiae yeasts, especially the dynamics of the major S. cerevisiae strains (L₁, L₄, and L₁₂) were quite similar in all five fermenters in opposite to the minor strains of S. cerevisiae.     

Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.     

Permanence of yeast inocula in the winery ecosystem and presence in spontaneous fermentations

The oenological practice of systematic inoculation with active dried yeasts is commonly used by many wineries around the world. However, the use of these yeasts is not free from controversy, since this practice has occasionally been described as having a negative effect on the biodiversity of natural yeast present in the wineries. The purpose of this study is to analyse the presence of commercial yeasts used as inocula in the ecosystem of three D.O.Ca. (“Qualified” Designation of Origin) Rioja wineries. It studies the permanence of these yeasts in winery equipment and their participation in spontaneous fermentations where they have not been used for inoculation. The results indicate that the presence of the active dry yeasts used in the wineries was scarce or non-existent, both in the ecosystem of each winery and in the spontaneous fermentations where they had not been added. So, repeated inoculation with active dry yeasts allowed a high presence and development of autochthonous (Saccharomyces and non-Saccharomyces) yeasts, both in equipment and in the spontaneous fermentations carried out.     

Characterization of the yeast ecosystem in grape must and wine using real-time PCR

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.     

Application of PCR-DGGE to analyse the yeast population dynamics in slurry reactors during degradation of polycyclic aromatic hydrocarbons in weathered oil

Slurry-phase reactors have been used to investigate the biodegradation feasibility of polycyclic aromatic hydrocarbons (PAHs) in weathered crude oil, by mixed culture containing five PAHs-degrading yeast strains. Yeasts were isolated from the oily soil by enrichment culture, using phenanthrene as a sole carbon source, and identified based on the 26S ribosomal DNA (rDNA) sequence. Yeast strains belonged to the genera Candida, Pichia, Rhodotorula and Sporidiobolus. The experiment was carried out for a period of 6 weeks at room temperature with a solid : liquid ratio of 50% w/w. The results showed that high removal efficiency was obtained for all PAHs, including low molecular weight (LMW) and high molecular weight (HMW) compounds (89.3-98.6% and 66.3-89.4% within 6 weeks, respectively). The higher removal efficiency for HMW-PAHs obtained in this work suggested that yeast strains mixture could play an important role to reclaim oil-contaminated sites. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S rRNA genes was used to follow the changes of yeast populations during the slurry reactor process. The results of DGGE indicated that Candida maltosa-like and Pichia guilliermondii were the dominant species but Rhodotorula dairenensis appeared as a weak band and Sporidiobolus salmonicolor and Pichia anomala disappeared during the study. Moreover, the results showed that all of the five strains, including the two belonging to the same genus, could be differentiated from each other in the DGGE profile.     

Combining microsatellite markers and capillary gel electrophoresis with laser-induced fluorescence to identify the grape (Vitis vinifera) variety of musts

In this work, a new method that combines the use of microsatellite markers (VVMD5 and ZAG79) together with capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) is developed and applied to the identification of Albariño and Moscatel Grano Menudo musts. The GCE-LIF method uses commercially available products including polymers, DNA-intercalating dyes and bare fused silica capillaries to provide reproducible and sensitive separations of DNA fragments for grapevine characterization. The CGE–LIF procedure offers highly resolved separations of DNA fragments from 48 to 1031 bp in ca. 30 min with efficiencies up to 1.8×10⁶ plates/m allowing the separation of fragments that differ in 4 bp. The use of different DNA standards (i.e., 100 bp ladder, Ф×174 and pBR322) and their effect on size assignment of the amplified DNA is also investigated. It is demonstrated that the microsatellite markers (VVMD5 and ZAG79) provide DNA amplification patterns specific for Albariño and Moscatel Grano Menudo grapes that can be adequately differentiated by using CGE–LIF. Moreover, the DNA sizes determined by this CGE–LIF method are corroborated using a more standard procedure (i.e., an automatic genetic analyzer with a commercial kit) demonstrating the usefulness of this new methodology.     

Effect of variety, vintage and winery on the prediction by visible and near infrared spectroscopy of the concentration of glycosylated compounds (G-G) in white grape juice

The use of visible (Vis) and near infrared (NIR) spectroscopy was explored as a rapid, simple and low cost measurement of the concentration of total glycosylated compounds in white grape juice. The effects of variety (Chardonnay, Riesling and Sauvignon Blanc), winery and vintage (2004 to 2006) on the Vis-NIR spectra were also examined. Juice samples from South Australian wineries were scanned in transmittance mode on a FOSS NIRSystems6500 instrument and subjected to laboratory analyses for the measurement of the concentration of total glycosylated compounds (G-G), total soluble solids (TSS), pH and total phenolics (TP). Partial least squares (PLS) regression method was used to relate the G-G reference data to the Vis-NIR spectra. For all samples, PLS regression resulted in a coefficient of determination in calibration ( R ²_cal) and standard error of cross validation (SECV) of 0.82 and 49.15 μM, respectively. Splitting the sample set by variety, winery or vintage improved the PLS calibrations for the variety sets. The results show that Vis-NIR spectroscopy has potential for use as a rapid, semi-quantitative technique to predict G-G concentration in white grape juices as 'low', 'medium' or 'high'. This method will be valuable when taking decisions at the winery during vintage to allocate juices according to their aroma potential. Further studies are in progress to validate the robustness and accuracy of the calibration models.     

烟蚜及天敌种群动态的模糊聚类分析

本文应用模糊聚类方法研究了烟蚜及其天敌的种群动态。揭示了春烟田中烟蚜及其天敌的发生规律和天敌对烟蚜的控制作用。烟蚜的种群消长与烟蚜天敌的种群数量密切相关,呈显著的双峰型。天敌是造成烟田前期蚜量下降的主要因素。     

叶分析在葡萄营养诊断及施肥中的应用

本文评述了葡萄叶分析营养诊断技术及其在葡萄施肥中的具体应用。要成功地运用叶分析技术进行葡萄营养诊断，就必须建立统一的取样、制样、标准分析方法及各品种叶矿质元素含量的标准值。解释叶分析结果的主要方法有临界值法、适宜值法和比值法。叶分析诊断技术主要应用在确定葡萄施肥种类、施肥时期、施用量及测定葡萄植株对肥料反应的研究等方面。     

Analysis of yeast and archaeal population dynamics in kimchi using denaturing gradient gel electrophoresis

Kimchi is a traditional Korean food that is fermented from vegetables such as Chinese cabbage and radish. Many bacteria are involved in kimchi fermentation and lactic acid bacteria are known to perform significant roles. Although kimchi fermentation presents a range of environmental conditions that could support many different archaea and yeasts, their molecular diversity within this process has not been studied. Here, we use PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 26S rRNA genes, to characterize bacterial, archaeal and yeast dynamics during various types of kimchi fermentation. The DGGE analysis of archaea expressed a change of DGGE banding patterns during kimchi fermentation, however, no significant change was observed in the yeast DGGE banding patterns during kimchi fermentation. No significant difference was indicated in the archaeal DGGE profile among different types of kimchi. In the case of yeasts, the clusters linked to the manufacturing corporation. Haloarchaea such as Halococcus spp., Natronococcus spp., Natrialba spp. and Haloterrigena spp., were detected as the predominant archaea and Lodderomyces spp., Trichosporon spp., Candida spp., Saccharomyces spp., Pichia spp., Sporisorium spp. and Kluyveromyces spp. were the most common yeasts.     

Influence of pre-fermentative treatment on the fatty acid content of Saccharomyces cerevisiae (M(3)30-9) during alcoholic fermentation of grape must

The effect of skin maceration and must filtration on the growth and fermentative capacity of one strain of Saccharomyces cerevisiae was studied. Skin maceration increased the fatty acid concentration of the must, thus affecting the lipid composition of the yeast cell membranes. Sterile filtration resulted in a reduction of the fatty acid content of the must extracted during maceration, but increased the fatty acid content of the yeast membranes, thus increasing the fermentative capacity without affecting either growth or cell viability.     

Application of sequential injection analysis (SIA) to food analysis

For effective control of food quality, a laboratory should be able to analyse a large number of samples in an accurate, reproducible and quick way. As sequential injection analysis (SIA) is becoming an important tool for the automation of chemical procedures, this paper presents an overview of the applications of this emergent methodology to food analysis. SIA systems developed to date offer good characteristics for routine use and the analytical methods proposed show adequate precision and accuracy. In addition, the results obtained are in good agreement with those furnished by the application of the reference methods.     

Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation

To determine the grape or winery origin of the Saccharomyces cerevisiae involved in spontaneous fermentation, musts were collected at different stages of wine-making process and fermented. First, grapes were collected in two different vineyards and crushed at the laboratory. Second, musts were collected after crushing and clarification in the cellar. Third, musts collected in the cellar were sterilized and inoculated with tartar deposit collected in the vats. The fourth fermentation was in the cellar. For the two vineyards, two hundred of S. cerevisiae clones were isolated for each of the four fermentations, driving to a library of 1600 clones. All the library was analysed by inter-delta PCR with a basic set of primers and about 20% of the library was further analysed by inter-delta PCR with an improved set of primers. Six, and more than 30 different PCR patterns were obtained from basic- and improved-PCR analysis, respectively. The amounts of each family were analysed at the different stages of wine making. Our study demonstrates that the two vineyards present different S. cerevisiae populations. Moreover the S. cerevisiae strains involved in spontaneous fermentation in the cellar originate partly from the vineyard and partly from the winery, in amounts varying with the must.     

安琪牌葡萄酒活性干酵母在草莓酒酿造中的应用

研究了安琪葡萄酒活性干酵母在草莓汁中的发酵条件。表明发酵温度为20℃,初始pH值为3．3,接种量为0．2％.SO2添加量为100mg／L,果胶酶量添加量为0．3g／L,发酵出的酒质较好。采用安琪葡萄酒活性干酵母酿制得干型草莓酒具有典型的草莓香和酒香。