低产杂醇高产酯酵母菌株的选育和共酵对黄酒品质的影响及机制分析

杂醇和酯是影响黄酒品质的关键化合物,寻找发酵性能优良的酵母菌株,开发低产杂醇高产酯的发酵工艺具有重要的理论价值和应用前景。以酿酒酵母jiangnan1#作为出发菌株,通过紫外诱变、常压室温等离子体诱变技术,以实验室模拟发酵制得的黄酒中杂醇、酯类化合物质量浓度为评价指标,筛选出1株优良的突变菌株,命名为YAR28。对YAR28进行了生孢纯化筛选,获得1株产醇酯较佳的酿酒酵母,命名为酿酒酵母Y28-23。将酿酒酵母Y28-23连续5次传代培养,进行稳定性验证, 结果显示,该酿酒酵母的酒精产量没有显著性差异(P＞0.05),但产杂醇的质量浓度[(378.16± 28.76)mg/L]比出发菌株降低了21.41%,且产酯的质量浓度[(152.86±19.73)mg/L]增加了35.57%。将酿酒酵母Y28-23与52株具备潜在产酯性能的非酿酒酵母共酵,以低产杂醇高产酯为评价指标,最终确定了异常威克汉姆酵母Y34为较佳的非酿酒酵母。进一步优化酿酒酵母Y28-23与异常威克汉姆酵母Y34的共酵条件,发现在主发酵阶段的温度为20℃的条件下以 1∶10的添加比例共酵黄酒,对于黄酒醇酯比的优化效果较佳,杂醇的质量浓度为(281.20±5.73)mg/L, 酯类的质量浓度为(240.02±2.47)mg/L。相比于单菌发酵,黄酒中杂醇的质量浓度降低了25.63%,而酯类的质量浓度增加了57.13%。基于代谢组学分析,获得多条与黄酒发酵过程中底物利用及风味形成密切相关的差异代谢物和代谢通路,阐明了黄酒共酵过程中异常威克汉姆酵母Y34对酿酒酵母Y28-23的代谢调节作用。研究可为利用发酵菌株控制黄酒酿造过程中的醇酯含量提供技术支持,对黄酒发酵菌种资源的开发利用以及黄酒品质的提升具有参考价值。     

Changes in the Lipid Composition of Saccharomyces cerevisiae Race Capensis (G-1) During Alcoholic Fermentation and Flor Film Formation

The cellular lipid composition of one flor-forming strain of Saccharomyces cerevisiae during fermentation and the subsequent period of film formation with different oxygen levels was studied. Irrespective of fermentation conditions, only those yeasts which came into contact with oxygen after fermentation formed a flor film. After the fermentation, these yeasts entered an adaptation phase in which the percentage of oleic acid increased considerably at the expense of other long-chain fatty acids. Their phospholipid contents remained high, as well as the unsaturation index of their fatty acids and the ergosterol/phospholipids ratio was maintained below 1. These changes allowed an increased viability of yeasts in the wine of up to 80% and the acquisition of sufficient hydrophobicity and floatability to reach the surface and form flor film.     

Influence of indigenous yeasts on the fermentation and volatile profile of plum brandies

The aim of this study was to determine the influence of different yeasts isolated from fresh blue plum fruits (Aureobasidium sp.) and spontaneously fermenting plum musts (Kloeckera apiculata and Saccharomyces cerevisiae), as well as commercial wine and distillery strains, on the fermentation and chemical composition of plum brandies. Gas chromatography methods were used to detect major volatile components. The most rapid fermentation occurred in musts inoculated with S. cerevisiae. However, the highest concentration of ethanol was detected in samples after spontaneous fermentation (8.40% v/v). Plum brandies obtained after distillation contained from 66.3 (K. apiculata) up to 74.3% v/v ethanol (spontaneous fermentation). The samples after spontaneous fermentation were distinguished by a high content of acetoin, ethyl acetate and total esters, accompanied by a low level of methanol and fusel alcohols. Non-Saccharomyces yeasts were responsible for higher concentrations of esters and methanol, while S. cerevisiae strains resulted in increased levels of higher alcohols. It was also found that isolated indigenous strains of S. cerevisiae synthesized relatively low amounts of higher alcohols compared to commercial cultures. Samples obtained using the distillery strain of S. cerevisiae received the highest score (18.2) during sensory analysis and were characterized by a well-harmonised taste and aroma.     

Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M_r) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M_r of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50 °C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.     

Construction of a novel beer proteome map and its use in beer quality control

Beer proteins were analysed by two-dimensional gel electrophoresis (2DE). The protein species associated with major spots on 2DE gels were identified by mass spectrometry followed by a database search to construct a comprehensive beer proteome map. As a result, 85 out of 199 protein spots examined were positively identified and categorised into 12 protein species. A total of 11 beer samples were brewed from the malt of eight cultivars having different levels of protein modification. This experiment was designed to demonstrate the influences of barley cultivar and malt modification on beer protein composition and beer quality characters. The beers produced from these brewing trails were subsequently analysed by 2DE and their proteomes were compared. Cultivars and malt modification affected the concentration of several proteins in beer. Beer protein concentration was associated with differences in the desirable beer quality trait, foam stability. In addition, expression of yeast derived proteins were observed that may also influence beer quality. Overall, the application of a comprehensive beer proteome map provides a strong platform for detection and potential manipulation of beer quality related proteins.     

Meta-analysis of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk production of ruminants

The effects of yeast supplementation on intake, production, and rumen fermentation characteristics have been widely studied, but results are inconsistent between different studies. A quantitative meta-analysis was applied to 110 papers, 157 experiments, and 376 treatments dealing with yeast supplementation in ruminants. The objective was first to highlight the major quantitative effects of live yeast supplementation on intake, rumen fermentation, and milk production, and second, to identify major differences in experimental conditions between studies that can affect the response to treatment. Some of these experimental conditions are referred to as interfering factors. Yeast supplementation increased rumen pH (+0.03 on average) and rumen volatile fatty acid concentration (+2.17 mM on average), tended to decrease rumen lactic acid concentration (−0.9 mM on average), and had no influence on acetate-to-propionate ratio. Total-tract organic matter digestibility was also increased by yeast supplementation (+0.8% on average). Yeast supplementation increased dry matter intake (DMI; +0.44 g/kg of body weight; BW), milk yield (+1.2 g/kg of BW), and tended to increase milk fat content (+0.05%), but had no influence on milk protein content. Dose effects of yeast supplementation, expressed as log₁₀ [1+(cfu per 100 kg of BW)], globally confirmed the qualitative effects observed in the first analysis. The positive effect of yeast supplementation on rumen pH increased with the percentage of concentrate in the diet and with the DMI level. It was negatively correlated with the level of dietary neutral detergent fiber (NDF). The positive effect of yeast supplementation on rumen volatile fatty acid concentration increased with DMI and crude protein levels. The positive effect of yeast supplementation on organic matter digestibility increased with the percentage of concentrate and NDF in the diet. The negative effect of yeast supplementation on lactic acid concentration tended to decrease when the DMI level and the percentage of concentrate in the diet increased. The effects of interfering factors were globally similar when either dose effect or qualitative effect of yeast was taken into account. Although rumen fermentation efficiency per se was not measured, these results suggest an improvement in rumen fermentation by yeast supplementation. This effect could, however, be modulated by several different factors such as DMI, percentage of concentrate or NDF in the diet, or species.     

Influence of living and autoclaved yeasts of Saccharomyces boulardii on in vitro ruminal microbial metabolism

Experimental data on the effects of Saccharomyces boulardii on rumen microbial metabolism are scarce. The aim of this study was to examine whether S. boulardii had an effect on parameters of rumen microbial metabolism at different dosages and whether the yeast would be suitable as a probiotic agent for ruminants. To test whether the potential positive effects of S. boulardii could be attributed to the yeast's viability or to its content of nutrients, living and autoclaved yeasts were tested simultaneously. For this purpose, incubation trials were carried out using the long-term rumen simulation technique. Living and autoclaved yeasts were added to fermentation vessels at a concentration of 0.5 or 1.5 g/d. The addition of living and autoclaved yeasts stimulated microbial metabolism, with no major differences between the treatments. It was concluded that ruminal microbes digested the supplied yeast of S. boulardii as an additional substrate and that S. boulardii, at least in ruminants, is utilized as a prebiotic rather than as a probiotic agent.     

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.     

Diversity of Saccharomyces cerevisiae yeasts associated to spontaneously fermenting grapes from an Italian "heroic vine-growing area"

The main aim of this work was to analyse the diversity of wild Saccharomyces cerevisiae isolated from spontaneous fermentations of grapes collected from heroic vine-growing area.     

Evaluation of different Saccharomyces cerevisiae strains for red winemaking. Influence on the anthocyanin,pyranoanthocyanin and non-anthocyanin phenolic content and colour characteristics of wines

The possible industrial use of three previously-selected Saccharomyces cerevisiae strains (1EV, 2EV and 7EV) has been studied in musts derived from Tempranillo and Cabernet Sauvignon. The anthocyanin, pyranoanthocyanin and non-anthocyanin phenolic content, and colour characteristics of the resulting wines have been compared to those of a commercial strain. Anthocyanins were the compounds most influenced by the yeast strain. Independently of the grape variety, wines derived from 2EV presented significantly higher anthocyanin concentrations than those derived from 1EV and 7EV, which presented similar contents. With the exception of hydroxycinnamic acids and derivatives, no particular influence of the yeast strain was observed on the remaining non-anthocyanin phenolic compounds (i.e, hydroxybenzoic acids and flavanols). Pyranoanthocyanins and metabolites resulting from the alcoholic fermentation such as tyrosol and tryptophol, seemed to be more influenced by the must composition and pH, and thus, by the grape variety, than by the yeast strain.     

Fermentative behavior of Saccharomyces strains during microvinification of raspberry juice (Rubus idaeus L.)

Sixteen different strains of Saccharomyces cerevisiae and Saccharomyces bayanus were evaluated in the production of raspberry fruit wine. Raspberry juice sugar concentrations were adjusted to 16°Brix with a sucrose solution, and batch fermentations were performed at 22 °C. Various kinetic parameters, such as the conversion factors of the substrates into ethanol (Y_p/s), biomass (Y_x/s), glycerol (Y_g/s) and acetic acid (Y_ac/s), the volumetric productivity of ethanol (Q_p), the biomass productivity (P_x), and the fermentation efficiency (E_f) were calculated. Volatile compounds (alcohols, ethyl esters, acetates of higher alcohols and volatile fatty acids) were determined by gas chromatography (GC-FID). The highest values for the E_f, Y_p/s, Y_g/s, and Y_x/s parameters were obtained when strains commonly used in the fuel ethanol industry (S. cerevisiae PE-2, BG, SA, CAT-1, and VR-1) were used to ferment raspberry juice. S. cerevisiae strain UFLA FW 15, isolated from fruit, displayed similar results. Twenty-one volatile compounds were identified in raspberry wines. The highest concentrations of total volatile compounds were found in wines produced with S. cerevisiae strains UFLA FW 15 (87,435 μg/L), CAT-1 (80,317.01 μg/L), VR-1 (67,573.99 μg/L) and S. bayanus CBS 1505 (71,660.32 μg/L). The highest concentrations of ethyl esters were 454.33 μg/L, 440.33 μg/L and 438 μg/L for S. cerevisiae strains UFLA FW 15, VR-1 and BG, respectively. Similar to concentrations of ethyl esters, the highest concentrations of acetates (1927.67 μg/L) and higher alcohols (83,996.33 μg/L) were produced in raspberry wine from S. cerevisiae UFLA FW 15. The maximum concentration of volatile fatty acids was found in raspberry wine produced by S. cerevisiae strain VR-1. We conclude that S. cerevisiae strain UFLA FW 15 fermented raspberry juice and produced a fruit wine with low concentrations of acids and high concentrations of acetates, higher alcohols and ethyl esters.     

Low and high temperatures for the control of cowpea beetle, callosobruchus maculatus (F.) (coleoptera: Bruchidae) in chickpeas

Chickpea (Cicer arietinum L.; Leguminasae) is an important pulse crop grown, around the world. The whole grain of chickpea is damaged by the cowpea seed beetle, Callosobruchus maculatus (Coleoptera: Bruchidae), which is the most important field-carry-over storage pest of pulses. The management of this insect in storage using chemicals leads to insecticide residues in grains and insecticide resistance development in insects. Thermal disinfestation is one of the means of physical insect control. Eggs, larvae, pupae and adults were held at 42 or 0 °C for varying durations. Pupae and adults were equally heat tolerant. The lethal time to reduce survival by 50% (LT₅₀) at 42 °C for eggs, larvae, pupae and adults were 18, 57, 78 and 71 h, respectively. Pupa was the most cold-tolerant stage. The LT₅₀ at 0 °C for eggs, larvae, pupae and adults were 3, 8, 10 and 4 d, respectively. The LT₅₀ for pupae were 4907, 4262, 336, 36 and 13 min at the grain temperature of 42, 45, 50, 55 and 60 °C, respectively. The LT₅₀ of pupae at 0,−5,−10 and −15 °C were 274, 122, 7 and 2 h, respectively.     

Effect of oxygen and lipid supplementation on the volatile composition of chemically defined medium and Chardonnay wine fermented with Saccharomyces cerevisiae

Oxygen or lipids are required to complete stressful alcoholic fermentation. Lack of these nutrients can inhibit sugar uptake and growth, which leads to incomplete or ‘stuck’ fermentation. Oxygen or lipids supplementation not only restores yeast fermentative activity and also affects formation of yeast volatile metabolites. To clarify the effect of oxygen and lipid supplementation on the formation of flavour active metabolites during wine fermentation, we evaluated the addition of these two nutrients to chemically defined grape juice and filter clarified Chardonnay must. Lipid addition increased the concentration of esters, higher alcohols and volatile acids, whereas oxygen increased the concentration of higher alcohols and altered the proportion of acetate to ethyl esters and the proportion of branch-chain acids to medium-chain fatty acids. Combined addition of lipids and oxygen showed an additive effect on concentration of higher alcohols whereas oxygen suppressed the enhancing effect of lipids on formation of esters and volatile acids. Our results demonstrate the potential of lipid and oxygen supplementation for the manipulation of wine aroma in white wine fermentation.     

Determination of the antigenotoxic potencies of some luteolin derivatives by using a eukaryotic cell system, Saccharomyces cerevisiae

In this study, we aimed to examine the mutagenic and antimutagenic potencies of three luteolin derivatives (luteolin-7-O-glucoside, luteolin-7-O-rutinoside and luteolin-7-O-glucuronide) by using a eukaryotic cell system, Saccharomyces cerevisiae (RS112).     

Active dry Saccharomyces cerevisiae can alleviate the effect of subacute ruminal acidosis in lactating dairy cows

The objective of the study was to determine the effect of active dry Saccharomyces cerevisiae (ADSC) supplementation on dry matter intake, milk yield, milk components, ruminal pH, and microbial community during a dietary regimen that leads to subacute ruminal acidosis (SARA). Sixteen multiparous, rumen-cannulated lactating Holstein cows were randomly assigned to 1 of 2 dietary treatments that included ADSC (Biomate; AB Vista, Marlborough, UK; 8 × 10¹⁰ cfu/head per day) or control. During wk 1 to 6, all cows received a high-forage (HF) diet (77:23, forage:concentrate). Cows were then abruptly switched during wk 7 to a high-grain (HG) diet (49:51, forage:concentrate) and remained on the HG until the end of wk 10. Feed intake and milk yields were recorded daily. Ruminal pH was recorded continuously using an indwelling system for 1 to 2 d per week during the pre-experimental phase, and wk 6, 7, and 10. Ruminal digesta samples were collected at the end of the experiment and analyzed for relative change in microbial communities using real-time quantitative PCR. Cows were considered to have SARA if the duration below pH 5.6 was ≥300 min/d. Ruminal pH during wk 6 (HF plateau) was not different across treatments (15 ± 46 min/d at pH <5.6). The dietary regimen successfully induced SARA during wk 7 (transition from HF to HG diet), and ruminal pH (551 ± 46 min/d at pH <5.6) was not different across treatments. However, cows receiving ADSC had an improved ruminal pH (122 ± 57 vs. 321 ± 53 min/d at pH <5.6) during wk 10 (HG plateau) compared with control. Additionally, cows receiving ADSC had a better dry matter intake (23.3 ± 0.66 vs. 21.6 ± 0.61 kg/d) and 4% fat-corrected milk yield (29.6 ± 1.2 vs. 26.5 ± 1.2 kg/d) than control cows during the HG phase (wk 8 to 10). During HG feeding, cows receiving ADSC had greater total volatile fatty acid and propionate concentrations (175 ± 7.5 vs. 154 ± 7.5 and 117 ± 6.1 vs. 94 ± 5.7 mM for ADSC and control, respectively) and lower acetate:propionate ratio (0.26 ± 0.5 vs. 0.36 ± 0.05 for ADSC and control, respectively). Microbial analyses conducted on samples collected during wk 10 showed that cows supplemented with S. cerevisiae had a 9-fold, 2-fold, 6-fold, 1.3-fold, and 8-fold increase in S. cerevisiae, Fibrobacter succinogenes, Anaerovibrio lipolytica, Ruminococcus albus, and anaerobic fungi, respectively, which suggested an increase in cellulolytic microbes within the rumen. Cows supplemented with ADSC had 2.2-fold reduction in Prevotella albensis, which is a gram-negative bacterium predominant during SARA. Prevotella spp. are suggested to be an important source of lipopolysaccharide responsible for inflammation within the rumen. Cows supplemented with ADSC had a 2.3-fold increase in Streptococcus bovis and a 12-fold reduction in Megasphaera elsdenii. The reduction in M. elsdenii may reflect lower concentration of lactic acid within the rumen for ADSC cows. In conclusion, ADSC supplementation to dairy cows was demonstrated to alleviate the condition of SARA caused by abrupt dietary changes from HF to HG, and can potentially improve rumen function, as indicated by greater numbers of cellulolytic microorganisms within the rumen.