嗜杀酵母的研究进展及其在酿酒工业中的应用

嗜杀酵母能够分泌毒素蛋白抑制或杀死敏感细胞,在发酵生产中利用其特性可以净化环境,提高产品的质量。文中介绍了嗜杀酵母的分布、作用机理以及嗜杀酵母质粒的提取和选育,并概述了嗜杀酵母在酿酒工业中的应用和研究进展。     

Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives

In this work 51 yeasts strains isolated from seasoned green table olives and belonging to the Candida, Debaryomyces, Kluyveromyces, Pichia, and Saccharomyces genera were characterized by their killer activity in different conditions. Killer activity of isolates was analyzed in a medium with different pH's (3.5 to 8.5) and NaCl concentrations (5, 8, and 10%). At every pH tested, all the genera studied had killer strains, although the smallest percentages of killer yeasts were found at the highest pH (8.5). The presence of 5 and 8% NaCl increased the detected killer percentage, but the highest salt concentration (10%) decreased it. The interaction between the reference killer yeasts and yeasts isolated from olives was analyzed. Most isolates were killer-sensitive to one or more killer reference strains. Only 2 of the 51 strains tested were considered killer-neutral. Cross-reaction trials between isolates and spoilage yeasts showed that, of the isolates, nine killer strains, belonging to Debaryomyces hansenii, Kluyveromyces marxianus, Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae, had the broadest spectra of action against yeasts that cause spoilage. These killer yeasts and the toxins that they produce are candidates for further investigation as suppressors of indigenous olive table yeast growth. The results confirmed the highly polymorphic expression of the killing activity, with each strain showing different killer activities. This method may thus be very useful for simple and rapid characterization of yeast strains of industrial interest.     

利用细胞质导入法选育优良葡萄酒嗜杀酵母

将核融合缺陷菌株嗜杀酵母5045(Kar1-1)的嗜杀质粒导入葡萄酒酵母1450(Screujsjae)中,已获成功。利用这种方法,我们选育出了具有1450细胞核和5045细胞质基因的嗜杀酵母R-58。通过萄萄酒酿造实践表明,该融合嗜杀株不仅具有抗野生酵母的能力,而且能酿制出优质葡萄酒。     

Yeasts of the soil – obscure but precious

Pioneering studies performed in the nineteenth century demonstrated that yeasts are present in below‐ground sources. Soils were regarded more as a reservoir for yeasts that reside in habitats above it. Later studies showed that yeast communities in soils are taxonomically diverse and different from those above‐ground. Soil yeasts possess extraordinary adaptations that allow them to survive in a wide range of environmental conditions. A few species are promising sources of yeast oils and have been used in agriculture as potential antagonists of soil‐borne plant pathogens or as plant growth promoters. Yeasts have been studied mainly in managed soils such as vineyards, orchards and agricultural fields, and to a lesser extent under forests and grasslands. Our knowledge of soil yeasts is further biased towards temperate and boreal forests, whereas data from Africa, the Americas and Asia are scarce. Although soil yeast communities are often species‐poor in a single sample, they are more diverse on the biotope level. Soil yeasts display pronounced endemism along with a surprisingly high proportion of currently unidentified species. However, like other soil inhabitants, yeasts are threatened by habitat alterations owing to anthropogenic activities such as agriculture, deforestation and urbanization. In view of the rapid decline of many natural habitats, the study of soil yeasts in undisturbed or low‐managed biotopes is extremely valuable. The purpose of this review is to encourage researchers, both biologists and soil scientists, to include soil yeasts in future studies.     

THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

Occurrence of the killer character in yeasts associated with Spanish wine production

The occurrence of neutral, sensitive or killer phenotypes in 270 oenological yeast strains belonging to 18 species was studied. Strains were isolated from a total of 54 samples representing spontaneous fermentations at 11 wineries of the three Madrid viticultural areas (Arganda, Navalcarnero and San Martı́n de Valdeiglesias). The percentage of strains with the killer phenotype was 42·6% but this percentage varied during the fermentation: 18·8% in must, 50·0% in the middle and 58·8% by late fermentation. The highest frequency of the killer phenotype occurred in the species Saccharomyces cerevisiae (86·9%). Killer activity for this species was shown to result from toxins in the K₂ and K₃ groups.     

FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

Modelling the growth dynamics of interacting mixed cultures: a case of amensalism

In this work, a specific membrane bioreactor was used to perform co-cultures of two Saccharomyces cerevisiae yeast strains: a killer strain and a sensitive strain. Biomass could be segregated into four groups: viable killer yeasts, dead killer yeasts, viable sensitive yeasts and dead sensitive yeasts. An existing mathematical model describing the population dynamics in the mixed killer/sensitive cultures was confronted with the new experimental data. As it gave poor accuracy, some improvements were proposed and tested. In particular, a lag phase before the beginning of the lethal interaction between the two strains was introduced, in correspondence to the experimental observations.     

Isolation of epiphytic yeasts with potential for biocontrol of Aspergillus carbonarius and A. niger on grape

Antagonistic yeasts were isolated from the epiphytic flora associated with grape berries cv. Negroamaro and identified at species level using molecular methods. A total of 144 yeast isolates were tested in a preliminary screening on agar to select isolates showing a killer activity against Aspergillus carbonarius and A. niger, the main species responsible for the accumulation of ochratoxin A in grape. Twenty-eight yeast isolates were selected for their inhibitory effects on the above fungal species and assayed by an in vitro nutritional competition test for their antagonistic capacity towards three selected ochratoxigenic strains. Six yeast isolates belonging to five species, namely 2 isolates of Issatchenkia orientalis and one each of Metschnikowia pulcherrima, Kluyveromyces thermotolerans, Issatchenkia terricola and Candida incommunis, were finally selected and screened on wounded grape berries for their ability to inhibit infection by ochratoxigenic moulds. With the exception of the K. thermotolerans isolate, when inoculated at 10(9) CFU/wound, the other five challenger yeasts reduced the A. carbonarius and A. niger colonization on grape berry (P<0.05). In particular, the best antagonistic activity was shown by the two I. orientalis isolates. Results suggest that antagonist yeasts with the potential to control A. carbonarius and A. niger on grape can be found among the microflora associated with the berries.     

Review: Utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit

Significant losses in harvested fruit can be directly attributable to decay fungi. Some of these pathogenic fungi are also the source of mycotoxins that are harmful to humans. Biological control of postharvest decay of fruits, vegetables and grains using antagonistic yeasts has been explored as one of several promising alternatives to chemical fungicides, the use of which is facing increasingly more stringent regulation. Yeast species have been isolated over the past two decades from a variety of sources, including fruit surfaces, the phyllosphere, soil and sea water, and their potential as postharvest biocontrol agents has been investigated. Several mechanisms have been proposed as responsible for their antagonistic activity, including competition for nutrients and space, parasitism of the pathogen, secretion of antifungal compounds, induction of host resistance, biofilm formation, and most recently, the involvement of reactive oxygen species (ROS) in defense response. It has been recognized that a biocontrol system is composed of a three-way interaction between the host (commodity), the pathogen and the yeast, all of which are affected by environmental factors. Efficacy and consistent performance in controlling postharvest diseases are the hurdles that must be overcome if the use of yeast biocontrol agents and other alternatives are to be widely used commercially. Therefore, attempts have been made to combine alternative treatments in order improve their overall performance. The current review provides a brief overview of the topic of the use of yeasts as postharvest biocontrol agents and includes information on the sources from which yeast antagonists have been isolated, their mode of action, and abiotic stress resistance in yeast as it relates to biocontrol performance. Areas in need of future research are also highlighted.     

Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.     

Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias

This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22′ and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of β-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3′ and 19′) to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with β-glucosidase activity are reported for the first time in autochthonous cider yeasts.     

The ecology and evolution of non‐domesticated Saccharomyces species

Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best‐studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd.     

嗜杀苹果酒酵母的构建

采用核融合缺陷原生质体融合技术 ,将嗜杀质粒供体菌 5 0 45 (α ,his4,kar1— 1[KIL—K1 ]rho+ )中的嗜杀质粒转移到受体菌苹果酒酵母AW中去。用 3 5 %PEG ,3 0℃诱导融合 2 0min ,经嗜杀活性检出、小型酿造实验、遗传稳定性实验、细胞大小及体积的测定、dsRNA质粒的提取及琼脂糖凝胶电泳等实验 ,筛选出 4株遗传性状稳定、具有嗜杀活性且发酵性能良好的融合子F12、F3 0、F5 7、F65。     

Cellular death of two non-Saccharomyces wine-related yeasts during mixed fermentations with Saccharomyces cerevisiae

The early death of two non-Saccharomyces wine strains (H. guilliermondii and H. uvarum) during mixed fermentations with S. cerevisiae was studied under enological growth conditions. Several microvinifications were performed in synthetic grape juice, either with single non-Saccharomyces or with mixed S. cerevisiae/non-Saccharomyces inocula. In all mixed cultures, non-Saccharomyces yeasts grew together with S. cerevisiae during the first 1-3 days (depending on the initial inoculum concentration) and then, suddenly, non-Saccharomyces cells began to die off, regardless of the ethanol concentrations present. Conversely, in both non-Saccharomyces single cultures the number of viable cells remained high (ranging 10(7)-10(8) CFU ml(-1)) even when cultures reached significant ethanol concentrations (up to 60-70 g l(-1)). Thus, at least for these yeast strains, it seems that ethanol is not the main death-inducing factor. Furthermore, mixed cultures performed with different S. cerevisiae/ H. guilliermondii inoculum ratios (3:1; 1:2; 1:10; 1:100) revealed that H. guilliermondii death increases for higher inoculum ratios. In order to investigate if the nature of the yeast-yeast interaction was related or not with a cell-cell contact-mediated mechanism, cell-free supernatants obtained from 3 and 6 day-old mixed cultures were inoculated with H. guilliermondii pure cultures. Under these conditions, cells still died and much higher death rates were found for the 6 days than for the 3 day-old supernatants. This strongly indicates that one or more toxic compounds produced by S. cerevisiae triggers the early death of the H. guilliermondii cells in mixed cultures with S. cerevisiae. Finally, although it has not been yet possible to identify the nature of the toxic compounds involved in this phenomenon we must emphasise that the S. cerevisiae strain used in the present work is killer sensitive with respect to the classical killer toxins, K1, K2 and K28, whereas the H. guilliermondii and H. uvarum strains are killer neutral.     

Genome comparison and evolutionary analysis of different industrial lager yeasts (Saccharomyces pastorianus)

Fermented beverages, especially beer, have accompanied human civilizations throughout our history. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. For hundreds of years, lager yeast (Saccharomyces pastorianus) has been subjected to multiple rounds of domestication owing to artificial selection during beer production. As a result, this species comprises a genetically diverse collection of strains that are used in different breweries. However, the scope of genetic diversity captured during the domesticated evolution of this species remains to be determined. To begin to address this, we collected the genome information of the only four lager strains that had been whole‐genome sequenced. For the first time, genome comparison was conducted between lager yeasts and clear signatures were found that defined each industrial yeast strain. The genetic variation comprises both single nucleotide polymorphisms and insertions and deletions. In addition, the core–pan genome was introduced for the first time to the genomic analysis of lager yeasts, detecting numerous strain‐specific and species‐shared genes. Furthermore, phylogenetic tree and synteny analysis results obtained in this study revealed information regarding the evolutionary relationship and group differentiation of studied strains. Genome comparison of the lager strains will, therefore, enable the characterization of the overall genetic diversity of this species, assist in the identification of genomic loci that play important roles in regulating key industrial phenotypes, and highlight the understanding of the hybrid nature and evolutionary details. Copyright © 2016 The Institute of Brewing & Distilling     

Nectar yeasts: a natural microcosm for ecology

The species of yeasts that colonize floral nectar can modify the mutualistic relationships between plants and pollinators by changing the chemical properties of nectar. Recent evidence supporting this possibility has led to increased interest among ecologists in studying these fungi as well as the bacteria that interact with them in nectar. Although not fully explored, nectar yeasts also constitute a promising natural microcosm that can be used to facilitate development of general ecological theory. We discuss the methodological and conceptual advantages of using nectar yeasts from this perspective, including simplicity of communities, tractability of dispersal, replicability of community assembly, and the ease with which the mechanisms of species interactions can be studied in complementary experiments conducted in the field and the laboratory. To illustrate the power of nectar yeasts as a study system, we discuss several topics in community ecology, including environmental filtering, priority effects, and metacommunity dynamics. An exciting new direction is to integrate metagenomics and comparative genomics into nectar yeast research to address these fundamental ecological topics.     

巨噬细胞识别酿酒酵母孢子的研究

酿酒酵母是一种对人类和动物重要的益生菌,在不同的营养条件下有营养态和孢子态两种形态.为了研究酵母孢子对免疫系统的作用,初步探讨了巨噬细胞对酵母孢子的响应机制.通过比较巨噬细胞对营养态酵母和野生型孢子的内吞效率,发现巨噬细胞对野生型孢子的内吞效率更高.通过Syk或PI3K抑制剂抑制实验,发现野生型酵母孢子和营养态酵母的内...     

从海洋中分离到的酵母菌及其特性初探

从山东沿海近海海域分离到近百株野生酵母菌,对其进行生理生化实验发现,酵母菌菌体的OD值及在YPD平板上形成菌落的大小都随着渗透压或盐浓度的增加而减少,其生长与发酵时的NaCl的平均极限浓度是2.0～2.5mol/L.我们得到一株耐盐性较强的酵母菌和三株能够产丙酮酸的酵母菌,并对一株产丙酮酸较好的酵母菌进行了分类学实验,该菌株被初步鉴定为假丝酵母属.