Enological functions of parietal yeast mannoproteins

Parietal yeast mannoproteins play a very important role in the overall vinification process. Their production and release, both during winemaking and aging on lees, depends on the specific yeast strain and the nutritional conditions. The following enological functions of parietal yeast mannoproteins have been described: (a) adsorption of ochratoxin A; (b) combination with phenolic compounds; (c) increased growth of malolactic bacteria; (d) inhibition of tartrate salt crystallization; (e) interaction with flor wines; (f) prevention of haze; (g) reinforcement of aromatic components; (h) wine enrichment during aging on fine lees; (i) yeast flocculation and autolysis in sparkling wines. Further discoveries related to their enological functions are foreseeable. Yeast-derived mannoproteins may well induce chemical, sensorial and health benefits, thus greatly improving wine quality.     

Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection

The presence of the fungal pathogen, Botrytis cinerea, in the vineyard causes reductions in both quality and quantity of grapes and wine. Because proteins are involved in the foam stabilization of sparkling wines, we have undertaken, for the first time, a thorough proteomic analysis of two champagne base wines prepared with either healthy or botrytized Chardonnay grapes, using two-dimensional electrophoresis (2DE) coupled with immunodetection and tandem mass spectrometry. Most of the identified proteins were from grape origin: invertase and pathogenesis-related (PR) proteins. The disappearance of numerous grape proteins was observed in the botrytized wine, suggesting that they were probably degraded or even repressed or the result of a differential expression of grape proteins upon fungal infection. On the other hand, two pectinolytic enzymes secreted by B. cinerea were found in the botrytized wine.     

Preliminary assessment on the use of immobilized yeast cells in sodium alginate for sparkling wine processes

Summary Use of alginate-immobilized yeasts in the production of sparkling wine using the champenois method was investigated. The results indicate that there are no variations in the principal chemical-physical characteristics between sparkling wines obtained through immobilized yeast and traditional sparkling wines.     

Bulk sparkling wine production by external encapsulated yeast bioreactor

Summary A new method for bulk sparkling wine production is proposed. The use of an external bioreactor with high immobilized yeast loading for second fermentation was studied. The new process is much faster than the traditional one and the sparkling wines obtained are perfectly clear without showing quality faults.     

Protein separation by differential drainage from foam

Commercially available beer, which is a dilute solution containing components of yeast, malt, and hop used in the manufacture of the beer, was used as a model system to demonstrate the potential of foam fractionation beyond the primary foaming stage. Most of the components present in the beer concentrated in the initial foam, but they drained differentially in the subsequent collapsed foam collected over a period of 30 min. This resulted in further enrichment, in particular, of components which were present in low concentration in the original beer, Preferential drainage from foam, hence, might provide a novel way of fractionating further the proteins concentrated initially in the liquid films of foam. (c) 1994 John Wiley & Sons, Inc.     

Cell wall polysaccharides: before and after autolysis of brewer’s yeast

Brewer’s yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of β-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.     

"Our Beer": Ethnographic Brands in Postsocialist Georgia

Although Georgia is known for its wines, industrial production of beer far outstrips industrial wine production for local markets: wine consumption occurs in ritual contexts in which new wine, typically purchased from peasant producers, is preferred; bottled, aged wines are primarily for exports. Beer, therefore, is a key area in which industrial production for indigenous consumers has been elaborated. Such goods are packaged and presented as being both ecologically "pure" and following "traditional" methods, often referencing "ethnographic" materials about traditional life in brand images, even as they proclaim their reliance on Western technologies.     

Filtration, haze and foam characteristics of fermented wort mediated by yeast strain

AIMS: To investigate the influence of the choice of yeast strain on the haze, shelf life, filterability and foam quality characteristics of fermented products. METHODS AND RESULTS: Twelve strains were used to ferment a chemically defined wort and hopped ale or stout wort. Fermented products were assessed for foam using the Rudin apparatus, and filterability and haze characteristics using the European Brewing Convention methods, to reveal differences in these parameters as a consequence of the choice of yeast strain and growth medium. CONCLUSIONS: Under the conditions used, the choice of strain of Saccharomyces cerevisiae effecting the primary fermentation has an impact on all of the parameters investigated, most notably when the fermentation medium is devoid of macromolecular material. SIGNIFICANCE AND IMPACT OF THE STUDY: The filtration of fermented products has a large cost implication for many brewers and wine makers, and the haze of the resulting filtrate is a key quality criterion. Also of importance to the quality of beer and some wines is the foaming and head retention of these beverages. The foam characteristics, filterability and potential for haze formation in a fermented product have long been known to be dependant on the raw materials used, as well as other production parameters. The choice of Saccharomyces cerevisiae strain used to ferment has itself been shown here to influence these parameters.     

Probing heat-stable water-soluble proteins from barley to malt and beer

Proteins determine the quality of barley in malting and brewing end-uses. In this regard, water-soluble barley proteins play a major role in the formation, stability, and texture of head foams. Our objective was to survey the barley seed proteins that could be involved in the foaming properties of beer. Therefore, two-dimensional (2-D) electrophoresis and mass spectrometry were combined to highlight the barley proteins that could resist the heating treatments occurring during malting and brewing processes. As expected, from barley to malt and to beer, most of the heat-stable proteins are disulfide-rich proteins, implicated in the defense of plants against their bio-aggressors, e.g., serpin-like chymotrypsin inhibitors (protein Z), amylase and amylase-protease inhibitors, and lipid transfer proteins (LTP1 and LTP2). For LTP1s, the complex pattern displayed in 2-D electrophoresis could be related to some chemical modifications already described elsewhere, such as acylation or glycation through Maillard reactions, which occur on malting. Our proteomics approach allowed the identification of the numerous proteins present in beer in addition to the major ones already described. The involvement of these proteins in the quality of beer foam can now be evaluated.     

Candida albicans adhesins: Biochemical aspects and virulence

The recognition of host cells by the pathogenic yeast, Candida albicans, is probably an essential step in the pathogenesis of disease development. The interaction of yeast and hyphal mannoproteins with host cell receptors has been studied by a number of laboratories. C. albicans recognizes a variety of host cells as well as host cell extracellular matrix proteins. This observation is not unexpected given the number of sites within and on the body which can be colonized and infected by the organism. Indeed, it would appear that C. albicans has evolved a number of ways in which it recognizes the host. This statement is made with the qualification that the organism uses other processes to infect, such as morphogenesis, phenotypic switching and the production of invasive enzymes, including secreted aspartyl proteases and phosholipases. Recognition of epithelial cells is accomplished through cell surface mannoproteins (adhesins) which bind to carbohydrate-containing receptors. The number of such mannoproteins is not known; pro adhesins exist. The organism also binds to keratinocytes, endothelial cells and matrix proteins, such as fibronectin, laminin, collagen and entactin, and, as such, appears to have a integrin-like cell surface adhesin. In most cases, the adhesin for each of these host proteins is a mannoprotein. The biochemistry of the candidal adhesins has been extensively studied. However, molecular analyses of the encoding genes is only now being studied. Thus, until clean, genetic analyses are complete and strains lacking an adhesin function are constructed, a direct role for the adhesins in pathogenesis can only be inferred. At present, spontaneous, non-adhering strains of the organism have been described which are avirulent in animal models of candidiasis. However, these data only suggest a role for adherence; future studies should be directed towards resolving questions about the role of these proteins in pathogenesis.     

Deletion of BCY1 from the Saccharomyces cerevisiae genome is semidominant and induces autolytic phenotypes suitable for improvement of sparkling wines

Autolysis of Saccharomyces cerevisiae is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work the possibility of accelerating this slow process in order to improve the quality of sparkling wines by using genetically engineered wine yeast strains was explored. The effect of partial or total deletion of BCY1 (which encodes a regulatory subunit of cAMP-dependent protein kinase A) in haploid and diploid (heterozygous and homozygous) yeast strains was studied. We proved that heterozygous strains having partial or complete BCY1 deletions have a semidominant phenotype for several of the properties studied, including autolysis under simulated second-fermentation conditions, in contrast to previously published reports describing mutations in BCY1 as recessive. Considering the degree of autolysis, ethanol tolerance, and technical feasibility, we propose that deletion of the 3' end of the open reading frame of a single copy of BCY1 is a way to improve the quality of sparkling wines.     

Development of DNA markers associated with beer foam stability for barley breeding

Traits conferring brewing quality are important objectives in malting barley breeding. Beer foam stability is one of the more difficult traits to evaluate due to the requirement for a relatively large amount of grain to be malted and then the experimental costs for subsequent brewing trials. Consequently, foam stability tends to be evaluated with only advanced lines in the final stages of the breeding process. To simplify the evaluation and selection for this trait, efficient DNA makers were developed in this study. Previous studies have suggested that the level of both of the foam-associated proteins Z4 and Z7 were possible factors that influenced beer foam stability. To confirm the relationship between levels of these proteins in beer and foam stability, 24 beer samples prepared from malt made from 10 barley cultivars, were examined. Regression analyses suggested that beer proteins Z4 and Z7 could be positive and negative markers for beer foam stability, respectively. To develop DNA markers associated with contents of proteins Z4 and Z7 in barley grain, nucleotide sequence polymorphisms in barley cultivars in the upstream region of the translation initiation codon, where the promoter region might be located were compared. As a result, 5 and 23 nucleotide sequence polymorphisms were detected in protein Z4 and protein Z7, respectively. By using these polymorphisms, cleaved amplified polymorphic sequence (CAPS) markers were developed. The CAPS markers for proteins Z4 and Z7 were applied to classify the barley grain content of 23 barley cultivars into two protein Z4 (pZ4-H and pZ4-L) and three protein Z7 (the pZ7-H, pZ7-L and pZ7-L2) haplotypes, respectively. Barley cultivars with pZ4-H showed significantly higher levels of protein Z4 in grain, and those with pZ7-L and pZ7-L2 showed significantly lower levels of protein Z7 in grain. Beer foam stability in the cultivars with pZ4-H and pZ7-L was significantly higher than that with pZ4-L and pZ7-H, respectively. Our results indicate that these CAPS markers provide an efficient selection tool for beer foam stability in barley breeding programs.     

Characterization of AfpA, an alkaline foam protein from cultures of Fusarium culmorum and its identification in infected malt

AIMS: The objective of this study was to evaluate the capability of Fusarium culmorum to produce non-hydrophobin surface-active proteins in vitro, to isolate and characterize such proteins from liquid cultures, to analyse their effect on overfoaming (gushing) of beer and to elucidate their prevalence in pure cultures and infected malt. METHODS AND RESULTS: A 20 kDa protein was isolated from liquid cultures of F. culmorum BBA 62182 upon enrichment by foaming. BLAST search with N-terminal and internal sequences of the protein revealed high homology with a hypothetical protein predicted within the F. graminearum PH1 genome sequence. Oligonucleotide primers designed to bind 30 nt upstream and downstream of the predicted gene were used to amplify a 695 nt PCR fragment from genomic DNA of F. culmorum BBA 62182. Cloning and sequencing of the product revealed a 635 nt open reading frame which had 98% homology to the predicted F. graminearium PH1 gene code. Removal of a 59 nt intron and translation resulted in a 191 amino acid protein of 20.754 kDa with a calculated pI of 9.1. Amino acids obtained by Edman sequencing of fragments within the 20 kDa protein were 100% homologous with the sequence deduced from the DNA sequence. According to its properties, the new protein was termed alkaline foam protein A (AfpA). Sequence comparison revealed some homologies with proteins in Emericella nidulans, which are involved in phialide development and response to antifungal agents. Homologies with other hypothetical fungal proteins suggest a new group of proteins, for which we suggest the name fungispumins. Addition of AfpA to beer showed that overfoaming (gushing) is not induced in stable beer but can significantly enhance this effect in beer showing moderate gushing. Use of a polyclonal anti-AfpA antibody in a Western blot revealed that the protein is produced by various F. culmorum strains and also by F. graminearum, but not by other Fusarium spp. tested. PCR testing of 69 species of Fusarium and Trichoderma reesei with a gene specific primer pair revealed that the gene may be present exclusively in F. culmorum, F. graminearum, F. cerealis, F. lunulosporum and F. oxysporum f. sp . dianthi. Immunochemical detection of AfpA in malts artificially inoculated with F. culmorum and F. graminearum showed that the protein was present in gushing inducing malts (gushing test) but absent in malts which were negative in a gushing test. CONCLUSIONS: AfpA is a member of a new protein class, fugispumins, and can be isolated from pure liquid cultures of F. culmorum. A homologous protein is synthesised by F. graminearum. The protein is produced in contaminated malt and enhances gushing of beer. The gene coding for AfpA is restricted to Fusarium species presumably involved in the induction of beer gushing. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a new class of proteins, fungispumins, the natural function of which remains to be elucidated. Findings add useful information to research on the mechanisms involved in foam stability of beer. AfpA may be useful as a marker for gushing in future quality control applications for the brewing industry.     

A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.     

Deletion of BCY1 from the Saccharomyces cerevisiae Genome Is Semidominant and Induces Autolytic Phenotypes Suitable for Improvement of Sparkling Wines

Autolysis of Saccharomyces cerevisiae is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work the possibility of accelerating this slow process in order to improve the quality of sparkling wines by using genetically engineered wine yeast strains was explored. The effect of partial or total deletion of BCY1 (which encodes a regulatory subunit of cAMP-dependent protein kinase A) in haploid and diploid (heterozygous and homozygous) yeast strains was studied. We proved that heterozygous strains having partial or complete BCY1 deletions have a semidominant phenotype for several of the properties studied, including autolysis under simulated second-fermentation conditions, in contrast to previously published reports describing mutations in BCY1 as recessive. Considering the degree of autolysis, ethanol tolerance, and technical feasibility, we propose that deletion of the 3′ end of the open reading frame of a single copy of BCY1 is a way to improve the quality of sparkling wines.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002     

Enzymatic activity of micro-organisms isolated from cork wine stoppers

The production of enzymes by micro-organisms which are found on vegetal substrates is important due to their ability to decompose cellulose, lignin and other components, which guarantee the integrity of the vegetal cell. The objective of this study was to determine the enzymatic activity of filamentous fungi, yeasts and bacteria, isolated from natural cork stoppers for bottles of still and sparkling wines. Suspensions of fungal conidia, yeasts and bacterial cells of micro-organisms were established in concentrations of 10(6) CFU/ml. The enzymatic activity of these micro-organisms was evaluated by means of the API ZYM system, with which it was possible to determine and semi-quantify nineteen enzymatic activities simultaneously. The enzymes produced by all of the species were esterase (C1), esterase lipase and naphthol-AS-BI-phosphohydrolase. The micro-organisms with the greatest enzymatic activity were Monilia sitophila, Alternaria alternata, Aspergillus niger and Aeromonas sp.     

Expression of GAI gene and disruption of PEP4 gene in an industrial brewer's yeast strain

Aims:  Construction of an industrial brewer's yeast strain, which could improve foam stability and reduce calorific values of beer.
Methods and Results:  An industrial brewer's yeast strain (Ts-10) was constructed by integrating glucoamylase encoding gene GAI amplified from Saccharomycopsis fibuligera by PCR into the locus of proteinase A (PrA) gene ( PEP4 ). The resulting recombinant strain identified by PCR could grow on YNB minimal medium plate with starch as sole carbon source. Its highest GAI activity was 91·69 U ml⁻¹, but it had no PrA activity. The real extract was reduced by 21·07% and the main residual maltotriose content was reduced by 14% in wort fermented with the recombinants strain. Its foam retention in beer was higher 39 s and the contents of potential off-flavour compounds, such as diacetyl, pentanedione and acetaldehyde were lowered by 16%, 13% and 14%, respectively, as compared with the industrial brewer's yeast YSF-5.
Conclusions:  An industrial brewer's yeast strain was constructed by introducing GAI gene and disrupting PEP4 gene.
Significance and Impact of the Study:  The recombinant strain (Ts-10) had better foam performance and mouthfeel in addition to low-calories values. It was free of heterologous DNA sequences and drug-resistance genes and could be safely used in beer production.     

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called "shot-gun" approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.