氩激光对啤酒酵母菌的生物学效应

本文应用全谱线氩离子激光辐照啤酒酵母菌,然后进行细胞培养啤酒发酵试验,並做多项生物效应的测定。结果表明:酵母细胞的出芽率、生长速度、代谢产物乙醇的产量、付产物双乙酰的含量以及有关酶类的活力均发生了变化。对于提高啤酒发酵的产量和改善啤酒的风味极为有利。     

啤酒生产中双乙酰形成的分子遗传学及其控制

双乙酰是啤酒中的重要风味物质,也是影响啤酒成熟和质量的关键因素之一。双乙酰是由酵母缬氨酸生物合成的中间产物α－乙酰乳酸经氧化脱羧产生的。目前,可以通过改良传统发酵工艺,添加酶制剂以及利用基因工程手段选育酵母工程菌等方法降低双乙酰在啤酒中的含量,缩短啤酒后酵期,以加速啤酒成熟。     

LD和YAG激光对极大螺旋藻生理特性的影响

采用Nd:YAG和LD激光辐射极大螺旋藻。Nd:YAG激光波长λ=532nm,功率P＝500mW,功率密度S＝160mW/cm^2,辐射10min,λ=1060nm,功率P＝7W,功率密度S＝35.84mW/cm^2,辐照20s:Nd:YAG激光（λ=532nm）辐照10min,再用LD激光（波长λ=650nm,功率P＝40mW功率密度S＝13mW/cm^2)辐照20min。实验结果表明,所设计的三种实验方 均能促进极大螺旋藻生长、β－胡萝卜素积累和胞外多糖含量的增加,且波长532nm的Nd:YAG激光的促进作用最明显,它可使β－胡萝卜素提高14＝．31％,胞多糖含量增加19．12％,红外光谱分析表明,经激光辐照后的藻细胞官能基团没有发生变化。     

α-乙酰乳酸脱羧酶在啤酒生产上的应用

双乙酰是啤酒发酵的副产物,它赋予啤酒特殊风味,但当含量超过一定阈值会使啤酒产生不愉快的馊饭味。因此,在发酵过程中尽可能地减少双乙酰含量,以缩短发酵周期、提高啤酒的产量,成为工艺上必须迫切解决的问题。     

ILV2基因缺失对啤酒酵母生长与双乙酰代谢的影响

【目的】旨在应用分子生物学方法降低啤酒发酵液中双乙酰含量,改善啤酒感官质量。【方法】以酿酒酵母S2(Saccharomyces cerevisiae)为出发菌株,通过同源重组敲除四倍体啤酒酵母α-乙酰乳酸合成酶部分基因(ILV2),构建缺失一个和两个ILV2等位基因的突变株QI2-1和QI2-2,并进行啤酒发酵实验。【结果】ILV2基因的缺失,会导致菌株初始生长速率的降低。其中QI2-2较为明显,12 h时,突变株与出发菌株的生长速率达到一致。啤酒发酵结果表明,与出发菌株相比,突变株QI2-1双乙酰峰值与双乙酰最终含量分别降低17.50%和17.83%,而QI2-2分别降低51.67%和45.65%。其他啤酒指标如酒精度、发酵度、残糖和风味物质等略有变化,但都在优质啤酒指标范围内,符合啤酒发酵的质量要求。【结论】通过同源重组敲除部分ILV2基因和选育低产双乙酰菌株是降低啤酒双乙酰含量、提高啤酒质量的有效方法,具有一定的实际应用价值。     

Ar+激光、Nd:YAG激光辐照亚心形扁藻生物学效应初探

采用Ar^＋激光（488 nm,照射时间分别为10 min、20 min、30 min）、Nd：YAG（1064 nm,照射时间为1 min、2 min、3 min）辐照扁藻,通过细胞计数以及扫描色素的吸收光谱研究激光辐照扁藻的生物效应.研究结果表明：非色素吸收峰的激光可以产生激光生物效应;不同波长,不同剂量的激光辐照扁藻可以表现出相类似的生物效应;Ar＋辐照20 min、Nd：YAG辐照2 min可以刺激扁藻生长,明显提高色素吸收光密度值,促进光合作用的进行.文中对不同激光辐照扁藻所产生的生物效应进行了比较和探讨.     

固定化酵母细胞流化床反应器的 操作与嫩啤酒双乙酰水平

利用海藻酸钠固定化酿酒酵母细胞和流化床生物反应器进行介质循环发酵。反应器中固定化胶体最适体积分数(φ)为O．40(v／v)。在给定固定化胶体体积分数(φ=O．40),给定发酵温度(10℃)和循环比(n=5)的条件下,研究了循环流速对主发酵周期和对嫩啤酒双乙酰水平的影响。结果表明,发酵周期随流速的增加而减少,而双乙酰浓度则随流速的增加而提高。当停留时间τt=2．8h,发酵周期T(=nτ r)为14h,嫩啤酒中双乙酰浓度为0．5ppm。     

固定化酵母细胞流化床反应器的 操作与嫩啤酒双乙酰水平

利用海藻酸钠固定化酿酒酵母细胞和流化床生物反应器进行介质循环发酵。反应器中固定化胶体最适体积分数(φ)为O．40(v／v)。在给定固定化胶体体积分数(φ=O．40),给定发酵温度(10℃)和循环比(n=5)的条件下,研究了循环流速对主发酵周期和对嫩啤酒双乙酰水平的影响。结果表明,发酵周期随流速的增加而减少,而双乙酰浓度则随流速的增加而提高。当停留时间τt=2．8h,发酵周期T(=nτ r)为14h,嫩啤酒中双乙酰浓度为0．5ppm。     

Nd:YAP激光辐照对雨生红球藻生理效应的荧光分析

目的：Nd：YAP激光辐照对雨生红球藻生理效应的荧光分析j方法：利用Nd：YAG激光（功率10W,辐照剂量为15s、35s、55s）辐照雨生红球藻,在对辐照细胞进行生长测定以及色素吸收光谱分析的基础上,进一步利用激光共聚焦扫描显微镜（LSCM）,在波长488nm的Ar^＋激发光条件下,对细胞的自体荧光以及吖啶橙染色的细胞核荧光物质进行定位定量分析,获得细胞自发荧光光谱和细胞核荧光物质的荧光光谱。结果：①低剂量的Nd：YAG激光辐照（15s左右）对藻细胞有促长作用,生长速率提高20．3％,色素吸收峰的峰值提高25．3％。较高剂量的Nd：YAG激光辐照（35s、55s）对藻细胞生长有抑制作用,并有明显的致死、致突效应。②对自体荧光的分析结果表明,与对照组相比,低剂量激光辐照组的细胞,在荧光光谱的峰型及峰值上变化不大,在682nm处均有较强的荧光发射,而高剂量激光辐照组的细胞多无明显的荧光发射。③对细胞核荧光物质的分析,雨生红球藻细胞在530nm（DNA）和640nm（RNA）处均有荧光发射峰。与对照组相比,低剂量激光辐照组的DNA荧光发射有所增强,但RNA的荧光发射则有所减弱;高剂量激光辐照组的核物质荧光发射谱,在峰型上与对照组存在差异,荧光强度也明显下降。结论：低剂量的Nd：YAP激光辐照对细胞核的DNA合成与复制有一定的刺激作用,可促进光合色素的合成并提高其对光能的吸收效率,从而增强光合活性,促进细胞的增殖与生长;高剂量激光辐照则对细胞的DNA有损伤作用,是致死率上升并发生突变的可能原因。     

基于激光共聚焦扫描显微技术分析激光对扁藻的影响

本文以亚心形扁藻为样品,波长1 341 nm的Nd∶YAP激光为光源,通过激光共聚焦扫描显微技术,研究Nd∶YAP激光辐照亚心形扁藻对亚心形扁藻叶绿体自体荧光强度和叶绿体面积大小的影响。Nd∶YAP激光辐照后的亚心形扁藻通过488 nm Ar^+激光激发获得亚心形扁藻自体荧光图像及其荧光光谱。结果表明,试验中除(10 W,60 s)辐照剂量组外,其余辐照剂量组均提高了亚心形扁藻的自体荧光强度,且所有的辐照剂量组均增大了亚心形扁藻的叶绿体面积。Nd∶YAP激光可刺激亚心形扁藻的叶绿体发育,促进藻细胞的生长,改善叶绿体光合作用的活性。     

Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

Isolation and characterization of brewer's yeast variants with improved fermentation performance under high-gravity conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

高温高浓发酵工业啤酒酵母菌种的构建

高温高浓发酵技术作为一项新兴的啤酒生产技术,它为啤酒生产带来诸多利益的同时,也存在着发酵结束后酵母絮凝性下降、高级醇生成量过高等系列问题。为提高高温高浓发酵条件下酿酒酵母的絮凝性同时降低高级醇的合成能力,首先构建了以酿酒酵母BAT2基因为整合位点过表达FLO5基因的菌株,重组菌株S6-BF的絮凝性达到67.67%,比出发菌株S6提高了29%,而高级醇生成量仅降低5.9%;进一步构建以BAT2基因为整合位点再次过表达FLO5基因的菌株,与出发菌株S6相比,重组菌株S6-BF2的絮凝性提高了63%,达到85.44%,高级醇生成量下降至159.58 mg/L,降低了9.0%;通过弱化线粒体支链氨基酸转氨酶(BAT1)的表达,高级醇的生成量得到进一步的降低,达到142.13 mg/L,比原始菌株S6降低了18.4%,同时重组菌株S6-BF2B1的絮凝性没有受到影响;风味物质的测定结果表明啤酒中醇酯比例较为合理。研究结果对工业啤酒酵母发酵后的沉降分离和提高啤酒风味品质有着重要的意义。     

Accumulation and cellular distribution of zinc by brewing yeast

This study focused on the interactions between yeast and zinc in relation to beer fermentations. Yeast accumulation of zinc from growth media, including malt wort, was found to be rapid following inoculation with a brewing strain of Saccharomyces carlsbergensis. In contrast, at the onset of the fermentation, the uptake of other divalent cations such as magnesium and calcium was not as pronounced compared with zinc. At the end of fermentation, both growth media and yeast cells became zinc-depleted, the latter due to dilution of zinc to daughter cells following growth and cell division. In addition, in brewing fermenters, the levels of intracellular zinc were much higher in suspended yeast cells compared with cells that sedimented in the yeast cone at the end of fermentation. This may result in impaired yeast performance in subsequent fermentations if yeast is recycled into low zinc media and if the sub-population is composed by zinc-depleted daughter cells. Cellular uptake of zinc was mediated by a metabolism-dependent mechanism as evidenced by impaired uptake following heat shock. Zinc was thereafter localised in the yeast cell vacuole. As industrial fermentation processes may occasionally be suppressed due to zinc deficiencies, the findings of this study are pertinent for several yeast-based industries, especially beer production.     

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.     

Farm-scale production of fuel ethanol and wet grain from corn in a batch process

The batch production of fuel grade ethanol and distillers' wet grain (wet solids) in a farm-scale process (1240-15,580 L/batch) is described. The employs yeast fermentation of amylase-treated corn mash and a two-stage distillation. Primary emphasis in this study was on the cooking, fermentation, and centrifugation steps. Without recycling, fermentation of the mash yield beers with 10.0-10.5% ethanol. Recycling of stillage supernatant at full, 75, or 50% strengths produced enriched mashes that after 48-h fermentation yielded beers with 5-;14% more ethanol. Recycling twice with full-strength supernatant at pH 7.0 increased the ethanol yield in the final beer 16.5%; however, the time to complete the final fermentation was extended form 48 to 72 h and salt buildup occurred. By recycling at pH 5.4, it was possible to avoid rapids salt buildup and obtain beers with 10.3-10.5% ethanol. Recycling resulted in increased levels of glucose, starch, crude protein, and fat in the beer and a reduced moisture content while the wet solids showed an increased starch content. Centrifugation after cooking or fermentation yield in the subsequently produced beer. Fermentation of a volume-resorted mash supernatant gave a beer with only 9.25% ethanol. Mash wet solids varied somewhat chemically from beer and stillage solids. An economic and energy balance analysis of various modes of plant operation are provided and plant considerations are suggested.     

产糖化酶黑曲霉的固定化研究

采用多孔聚酯材料作为固定化载体,考察并比较了载体吸附固定化黑曲霉菌丝细胞的条件,当菌丝体细胞与载体预培养的条件为pH值5.0、孢子浓度为105个/ml、固液比为1/75时,有利于菌丝体的生长、吸附固定及发酵产酶.在产糖化酶的发酵过程中,与游离菌丝体细胞相比,发酵过程持续产酶时间有一定程度的延长,产糖化酶活力始终高于游离菌丝体.     

Influence of aeration during propagation of pitching yeast on fermentation and beer flavor

The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.     

Reactors for continuous primary beer fermentation using immobilised yeast

A continuous multistage column bioreactor with fluidised beds and continuous gas-lift bioreactor system with immobilised yeast Saccharomyces cerevisiae was developed for the first step of wort fermentation. The system of gas-lift reactor with yeast entrapped in calcium pectate beads was stable for 5 weeks by the optimal residence time of 12.75h and produced beer with a composition and flavour profile similar to that of beer produced by batch fermentation. Concentration of diacetyl was less than 0.1mg/l.