Rapid characterization of mucin oligosaccharides from rat small intestine with gas chromatography-mass spectrometry

Mucin glycopeptides were isolated from rat small intestinal mucosa after reduction/alkylation, trypsin digestion and gel chromatography. The oligosaccharides were released by using alkaline-NaBH4, separated into neutral and acidic species and permethylated. The derivatized mixtures were analysed with fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry using thin film columns. Permethylated neutral oligosaccharides with up to seven sugars could be chromatographed and detected with mass spectrometry. The complex mixture revealed was partly due to the linkage GalNAc being substituted at both position 3 and 6. The approach will be very useful when analysing small amounts of mucins and mucin fragments.     

Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts

Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice.     

A Recombinant Saccharomyces cerevisiae Strain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

Stabilization against protein haze was one of the first positive properties attributed to yeast mannoproteins in winemaking. In previous work we demonstrated that deletion of KNR4 leads to increased mannoprotein release in laboratory Saccharomyces cerevisiae strains. We have now constructed strains with KNR4 deleted in two different industrial wine yeast backgrounds. This required replacement of two and three alleles of KNR4 for the EC1118 and T73-4 backgrounds, respectively, and the use of three different selection markers for yeast genetic transformation. The actual effect of the genetic modification was dependent on both the genetic background and the culture conditions. The fermentation performance of T73-4 derivatives was clearly impaired, and these derivatives did not contribute to the protein stability of the wine, even though they showed increased mannoprotein release in vitro. In contrast, the EC1118 derivative with both alleles of KNR4 deleted released increased amounts of mannoproteins both in vitro and during wine fermentation assays, and the resulting wines were consistently less susceptible to protein haze. The fermentation performance of this strain was slightly impaired, but only with must with a very high sugar content. These results pave the way for the development of new commercial strains with the potential to improve several mannoprotein-related quality and technological parameters of wine.     

Factors in acid treated bagasse inhibiting ethanol production from d-xylose by Pachysolen tannophilus

The fermentation of d-xylose, the major sugar-cane bagasse hemicellulose component, to ethanol by Pachysolen tannophilus is inhibited by various factors produced or released during the acid hydrolysis of the bagasse or during the fermentation process. These include ethanol, iron, chromium, copper, nickel, acetic acid and furfural. Ethanol production by P. tannophilus is inhibited by ethanol fconcentrations >24 g l^?1. Furfural and acetic acid concentrations as low as 0.3 and 7 g l^?1, respectively, and iron, chromium, nickel and copper at concentrations of 0.07, 0.01, 0.01 and 0.004 g l^?1, respectively. Similar concentrations may be found in acid-hydrolysed bagasse. The removal of these factors by treatment with ion-exchange resin resulted in the fermentation of the sugars to ethanol. The d-glucose was used rapidly and completely whereas d-xylose utilization was slow and incomplete. An ethanol concentration of 4.1 g l^?1 was produced and an ethanol yield of 0.32 was obtained. Xylitol in significant amounts was produced.     

Molecular analysis of red wine yeast diversity in the Ribera del Duero D.O. (Spain) area

Molecular characterization of wine yeast population during spontaneous fermentation in biodynamic wines from Ribera del Duero D.O. located at northern plateau of Spain has been carried out during two consecutive years. A total of 829 yeast strains were isolated from the samples and characterized by electrophoretic karyotype. The results show the presence of three population of yeast differentiated by their electrophoretic karyotypes, (1) non-Saccharomyces yeast dominant in the initial phase of the fermentations (NS); (2) Saccharomyces bayanus var uvarum detected mainly mid-way through the fermentation process at 20–25 °C; and (3) Saccharomyces cerevisiae which remained dominant until the end of the fermentation. This is the first study showing the population dynamic of S. bayanus var. uvarum in red wines produced in Ribera del Duero that could represent an important source of autochthonous wine yeasts with novel oenological properties.     

Contribution by Saccharomyces cerevisiae yeast to fermentative flavour compounds in wines from cv. Albariño

A comparative study was made of the fermentation products of Spanish Albariño wines produced with spontaneous yeast flora and an indigenous selected Saccharomyces cerevisiae strain (Alb16). The content of fermentative volatile compounds was determined by gas-chromatography-FID. Fifteen compounds (5 alcohols, 7 esters and 3 acetates) were identified in the two Albariño wines studied. Higher alcohols, ethyl esters (except ethyl hexanoate and ethyl octanoate) and acetates were in greater concentration in the spontaneous fermentation wine than in that with selected Alb16 strain. Principal components analysis showed good separation between the different wines.     

Alcohol fermentation in olive oil extraction effluents

Eight culture-collection yeast strains of various species and five newly isolated strains were tested for both growth in olive oil extraction effluents and fermentation of the sugars in the same media. The culture-collection yeast strains did not grow in an effluent containing 2·86% sugar (w/v), 8 g/litre phenolic substances, 4·58 g/litre titratable acidity and pH 4·96, whereas the newly isolated strains of Torulopsis sp. MK-1, Saccharomyces norbensis MC-1, S. oleaceus MC-2 and S. oleaginosus grew well and fermented the sugars. In the medium mentioned above, they produced alcohol in amounts of 1·63 to 1·38%, respectively. None of the yeasts grew in an olive oil extraction effluent vacuum-concentrated to over 13–14% of dry matter. The strain of T. sp. MK-1 showed a hogher stability.     

Integrated continuous winemaking process involving sequential alcoholic and malolactic fermentations with immobilized cells

An integrated winemaking process – including sequential alcoholic and malolactic fermentations operated continuously – was developed. For the continuous alcoholic fermentation, yeast cells (Saccharomyces cerevisiae) were immobilized either on grape stems or on grape skins, while bacterial cells (Oenococcus oeni) used for conducting continuous malolactic fermentation were immobilized on grape skins only. The produced wines were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds). The final proposed integrated continuous process permitted the production of 960 mL/d of a dry white wine, with an alcoholic strength of about 13 vol%, by using two 1.5 L tower bed reactors packed with 260 g of grape skins. The produced wines revealed a good physicochemical quality. Moreover, 67% of the malic acid concentration could be reduced in the second reactor. Both fermentative processes proved to be much more efficient than those conducted traditionally with free cells or even with immobilized cells, but in the batch mode of operation.     

Yeast Population Dynamics during the Fermentation and Biological Aging of Sherry Wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of “fino” sherry wine making. The four races of “flor” Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

微生物发酵-膜分离法制备褐藻胶寡糖及其产物分析

比较褐藻胶裂解酶产生菌Alteromonassp .在摇瓶和发酵罐培养过程中生物量、褐藻胶寡糖含量以及褐藻胶裂解酶活性的变化 ,根据其变化确立了通过微生物发酵 膜分离技术结合制备褐藻胶寡糖的条件 ,并对寡糖进行凝胶过滤色谱和薄层色谱分析。用组成为每升含酵母粉 5g、蛋白胨 10g、FeSO4 0 1g、褐藻酸钠 12g、NaCl 1 5g ,pH为7 5的培养基 ,在 2 8℃培养褐藻胶裂解酶产生菌 ,结果表明 ,发酵罐培养 30h ,发酵液寡糖含量达到最大。发酵液通过超滤 纳滤两级膜分离 ,得到褐藻胶寡糖 ,寡糖的回收率和脱盐率分别为 94 0 %和 93 3%。通过凝胶柱分离和TLC分析 ,得到 5个褐藻胶寡糖组分。     

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.     

Rice wine brewing with sprouting rice and barley malt

Though alcoholic beverages are widely made with barley malt in Western countries, as well as in Asiatic countries today, alcoholic beverages are rarely made with sprouting rice. Rice wines were obtained from cooked nonglutinous rice using sprouting rice and barley malt as saccharifying agents with compressed baker's yeast and Kyokai no. 9 yeast, and a comparative study was conducted of the resulting rice wines. The saccharifying activity of barley malt was higher than that of sprouting rice. The amounts of ethyl alcohol, volatile aromatic components, and reducing sugars in the rice wine made with barley malt were higher than those in the wine made with sprouting rice. The rice wine made with barley malt was faintly brownish in color and had heavy, complicated and vulgar characteristics. By contrast, sprouting rice wine was colorless and had light, simple and refined characteristics in terms of both aroma and taste. Sprouting rice wine made with Kyokai no. 9 yeast contained about 8% ethanol with an acidity of around 4.1. Sprouting rice was found to be applicable as a saccharifying agent for ethanol fermentation, as is barley malt. The quality of the sprouting rice wine was further improved through the use of Kyokai no. 9 yeast.     

Study of yeast populations and their enological properties in Guijoso Appellation of Origin (Spain)

The aim of the present study was to select strains of yeast with good enological qualities which were adapted to the ecological surroundings of Guijoso Appellation of Origin (A.O). For this, 11 white and red vats from different grape varieties and stages of fermentation were studied, making a total of 28 samples, with a selection of 370 isolated yeasts. Yeast cells of the Saccharomyces genus were analysed by DNA mitochondrial restriction for discrimination at the strain level, obtaining a total of 23 different molecular patterns. The pattern most frequently found was of G04, with 56 % of the isolated yeasts, followed by G02 with 15 % and G07 with 9 %. Other patterns found showed percentages close to 3 %, such as G01, G03, G05 and G06, while the remaining patterns were limited one or two isolated yeasts. Microfermentations at 25 and 15 °C were performed using a synthetic must, and the rate of fermentation, SH₂ and foam production and the capacity to consume sugars from the medium were studied. Furthermore, the killer phenotype, flocculation capacity and phenolic off-flavour (POF) characteristics were also analysed. Natural musts from Chardonnay and Cabernet Sauvignon varieties were fermented using preselected strains and the wines obtained were analysed and tasted. Two strains were selected (G01 and G04) to be used as starters in Guijoso A.O.     

Structure and location of asparagine-linked oligosaccharides in the Fc region of a human immunoglobulin D

Seven kinds of asparagine-linked oligosaccharides were bound to the Fc region of a human immunoglobulin D(NIG-65). The oligosaccharides quantitatively released from four species of glycopeptides by digestion with almond glycopeptidase, were separated by Bio-Gel p-4 column chromatography and were purified further by thin-layer chromatography. The sugars were identified with GC-MS following the permethylation of respective oligosaccharide. To Asn-68(NIG-65 Fc numbering (1)), two kinds of high-mannose-type oligosaccharides were bonded. To Asn-159, a kind of hybride-type and two kinds of bisected complex-type oligosaccharides were attached. From Asn-210, four kinds of bisected complex-type oligosaccharides were isolated.     

Structure determination of five sulfated oligosaccharides derived from tracheobronchial mucus glycoproteins

The structure of five sulfated oligosaccharide units of highly anionic tracheobronchial mucous glycoproteins, isolated from a cystic fibrosis patient's sputum, were established. Reduced oligosaccharides (84%) were released under alkaline borohydride conditions, and the acidic oligosaccharides (63%) were isolated by Dowex 1-X2 chromatography. Following the removal of acidic oligosaccharides possessing N-acetylneuraminic acid and L-fucose by lectin affinity chromatography a heterogeneous mixture of sulfated oligosaccharides was obtained. From this fraction, five short chain monosulfated oligosaccharides (S-I to S-V) were purified by sequential separation by SynChroprep AX300 anion exchange high pressure liquid chromatography, gel filtration on Bio-Gel P-2, and high voltage paper electrophoresis. Based on the results of carbohydrate composition, sequential exoglycosidase degradation, permethylation analysis, lectin affinity chromatography, and periodate oxidation, the following structures (where GalNAcol is N-acetylgalactosaminitol) were proposed for these oligosaccharides. (formula; see text)     

Occurrence of Lactic Acid Bacteria During the Different Stages of Vinification and Conservation of Wines

We showed that the growth of lactic acid bacteria during alcoholic fermentation depends on the composition of the must. We illustrated how the addition of sulfur dioxide to the must before fermentation and the temperature of storage both affect the growth of these bacteria in the wine. Whereas species of Lactobacillus and Leuconostoc mesenteroides were isolated from grapes and must, Leuconostoc oenos was the only species isolated after alcoholic fermentation. This organism was responsible for the malolactic fermentation. Isolates of this species varied in their ability to ferment pentoses and hexoses. The survival of Leuconostoc oenos in wines after malolactic fermentation depended on wine pH, alcohol concentration, SO₂ concentration, and temperature of storage.     

Nonenzymatic Glycosylation of Lepidopteran-Active Bacillus thuringiensis Protein Crystals

We used high-pH anion-exchange chromatography with pulsed amperometric detection to quantify the monosaccharides covalently attached to Bacillus thuringiensis HD-1 (Dipel) crystals. The crystals contained 0.54% sugars, including, in decreasing order of prevalence, glucose, fucose, arabinose/rhamnose, galactose, galactosamine, glucosamine, xylose, and mannose. Three lines of evidence indicated that these sugars arose from nonenzymatic glycosylation: (i) the sugars could not be removed by N- or O-glycanases; (ii) the sugars attached were influenced both by the medium in which the bacteria had been grown and by the time at which the crystals were harvested; and (iii) the chemical identity and stoichiometry of the sugars detected did not fit any known glycoprotein models. Thus, the sugars detected were the product of fermentation conditions rather than bacterial genetics. The implications of these findings are discussed in terms of crystal chemistry, fermentation technology, and the efficacy of B. thuringiensis as a microbial insecticide.     

Harnessing Genetic Diversity in Saccharomyces cerevisiae for Fermentation of Xylose in Hydrolysates of Alkaline Hydrogen Peroxide-Pretreated Biomass

The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na⁺, acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.     

Continuous ethanol production from raw starch using a reversibly soluble-autoprecipitating amylase and flocculating yeast cells

Direct ethanol production from raw starch was performed continuously using a combination of a reversibly soluble-autoprecipitating amylase (D-AS) in which Dabiase K-27 was immobilized covalently on an enteric coating polymer (hydroxypropyl methylcellulose acetate succinate, AS) as a carrier, and a flocculating yeast. Continuous production was carried out using a reactor equipped with a mixing vessel and a separation vessel. D-AS and the yeast were separated continuously from the product solution by self-sedimentation in the separation vessel and they were utilized repeatedly. In the continuous saccharification of raw starch by D-AS alone, the glucose productivity was about 3.6 g/l/h at a dilution rate (D) of 0.1 h⁻¹. In the continuous ethanol production from raw starch by a combination of D-AS and flocculating yeast cells, high ethanol productivity up to 2.0 g/l/h was achieved at D=0.1 h⁻¹. Although the enzymatic activity of D-AS is inactivated due to insolubilization of the enzyme by the accumulation of NaCl produced in controlling the pH in the reactor, it is possible to recover the D-AS enzymatic activity by removing the NaCl. This continuous fermentation system suggests a potential for effective ethanol production from raw starch, and it may be widely applicable in heterogeneous culture systems using solid substrates other than raw starch.