SYNTHESIS OF HIGHER ALCOHOLS IN THE GENUS ZYMOMONAS

Several strains of bacteria of the genus Zymomonas were examined with respect to their potential for higher alcohol synthesis. All strains studied were able to produce higher alcohols during growth in a simple medium containing glucose and yeast extract. The higher alcohols produced were mainly n-propanol and iso-amyl alcohol. In contrast to Saccharomyces cerevisiae, only trace amounts of higher alcohols were produced from glucose by resting cells. When amino acids or other precursors were added to the fermentation medium, the resting cells formed higher alcohols. The stimulation of n-propanol synthesis by precursors was the most pronounced. The results obtained indicate that, with minor differences, the mechanisms of higher alcohol synthesis are comparable to these used by yeasts.     

WORT COMPOSITION AND BEER FLAVOUR. I. THE INFLUENCE OF SOME AMINO ACIDS ON THE FORMATION OF HIGHER ALIPHATIC ALCOHOLS AND ESTERS

Wort, to which was added various amounts of glucose solution containing either (NH₄)₂SO₄ or one of the amino acids alanine, leucine, isoleucine, valine or proline, was fermented, and in the resulting beers the concentrations of ethyl acetate, isoamyl acetate, n-propanol, isobutanol, and amyl alcohols were determined by gas chromatography. The nitrogen source had some effect on the formation of higher alcohols, in addition to the effect due to conversion of some of the amino acids to the corresponding alcohols via the Ehrlich pathway. Ethyl acetate formation was not significantly affected by the nitrogen source, and iso-amyl acetate formation increased when amyl alcohol formation increased.     

Synthesis of higher alcohols during cider processing

Ciders from two apple cultivars Gloster and Gala, were prepared. Apples were crushed and juice was extracted, immediately after crushing or after 24 h of maceration. Juice was allowed to ferment at 15°C and analysed for higher alcohols and sugars during fermentation. All higher alcohols analysed increased during fermentation as a result of yeast activity. More higher alcohols were synthesised in cider made from Gala cultivar than in cider from Gloster cultivar. Maceration increased the synthesis of iso-amyl alcohol, 2-phenyl ethanol, n-propanol, n-butanol, iso-butanol and methanol. Sensory evaluation classified cider made from non-macerated Gloster cultivar as the best followed by non-macerated Gala cultivar and macerated Gala cultivar. Sensory evaluation score correlated negatively to the sum of higher alcohols.     

不同干酵母对甘蔗汁酿酒特性的影响

为研究不同酵母对甘蔗果酒的发酵影响,以新鲜的甘蔗压榨汁为原料,常温下选用葡萄酒高活性干酵母、耐高温高活性干酵母和发利干酵母三种商业活性干酵母进行液态发酵酿制甘蔗果酒。研究探讨了三种酵母菌的生长曲线、凝聚性、发酵度、发酵力、酸化力和产酒精能力等酿酒发酵特性。结果表明,三种酵母菌作用甘蔗汁的发酵过程中,酵母菌生长曲线趋势基本一致,三种酵母菌的凝聚值F<20%,均为非凝聚性酵母。三种酵母菌的发酵力基本相当的,最终产酒精的能力差距不大,其中葡萄酒高活性干酵母最终产酒精为12.2% vol,耐高温高活性干酵母和发利干酵母均为12.0% vol。从三种商用酵母菌对甘蔗汁酿酒的各发酵特性来看,葡萄酒高活性干酵母略优于耐高温高活性干酵母和发利干酵母。研究结果为甘蔗汁发酵酿制风味甘蔗果酒提供了技术参考。     

两种酿酒酵母发酵对桑葚米酒理化品质及香气成分的影响

实验以清酒酵母和果酒活性干酵母为发酵菌种,分别采用单独接种、两种酵母按不同顺序接种和两种酵母同时接种共5种发酵方式发酵桑葚米酒,利用气相色谱-质谱法(Gas Chromatography-Mass Spectrometry,GC-MS)分析5种发酵方式发酵桑葚米酒中香气物质,同时对米酒中主要香气成分进行主成分分析。结果表明:接种清酒酵母发酵7 d后再接入果酒活性干酵母发酵的桑葚米酒品质较好,米酒理化指标为酒精度(10.10% vol±0.20% vol)、残糖含量(30.70±0.10 g/L)、总酸(6.85±0.08 g/L)、挥发酸(0.31±0.05 g/L)、感官评分(85.60±0.19)。5组酒样中共检测出175种香气成分,其中酯类62种、醇类30种、酸类8种、醛类11种、酮类19种、烃类29种、其它物质16种;接种清酒酵母发酵7 d后再接入果酒活性干酵母发酵的桑葚米酒中主体风味物质达14种,典型风味成分β-苯乙醇的相对含量达17.88%,较其他4种发酵方式高出2.63~3.96倍,米酒的香气浓郁,果香花香和奶香味突出。     

黄酒固定化发酵过程及香气物质形成的研究

将黄酒酵母用海藻酸钙包埋制成固定化酵母,研究游离酵母与固定化酵母的黄酒发酵过程理化指标和香气物质生成量的差异,结果表明:经过主发酵5 d(28℃),后发酵15 d(15℃),固定化酵母发酵过程的酒精度、总糖、总酸变化曲线与游离发酵过程曲线差异不大,固定化酵母发酵性能稳定,杂菌污染可控。用气相色谱(GC)分析乙酸乙酯等9种黄酒特征性香气物质,发现游离发酵黄酒正丙醇、异丁醇、异戊醇、β-苯乙醇的总高级醇含量为680.91 mg/L,固定化发酵第1,2,3,4,5批黄酒的总高级醇含量分别为633.63,649.79,658.17,637.63,614.68 mg/L,说明固定化发酵有利于控制黄酒中的高级醇含量。固定化发酵酯类总量高于游离酵母,且随着固定化酵母发酵批次的增加而递增,即:固定化第5批(115.90 mg/L)>第4批(90.81 mg/L)>第1~3批(80.10~81.81 mg/L)>游离酵母(55.66 mg/L),表明固定化发酵使黄酒的酯类香气愈加浓郁。根据香气阈值计算的固定化发酵酿得的黄酒的香气不比游离酵母酿得的黄酒差,固定化酵母发酵生产黄酒有商业化应用的前景。     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

WORT COMPOSITION AND BEER FLAVOUR. II. THE INFLUENCE OF DIFFERENT CARBOHYDRATES ON THE FORMATION OF SOME FLAVOUR COMPONENTS DURING FERMENTATION

Wort, to which was added various amounts of solutions of glucose, fructose, sucrose or maltose, was fermented, and in the resulting beers the concentrations of the following flavour components were determined by gas chromatography: ethyl acetate, isobutyl acetate, iso-amyl acetate, 2-phenyl ethyl acetate, ethyl caproate, ethyl caprylate, n-propanol, isobutanol, amyl alcohols, 2-phenyl ethanol, caprylic acid and capric acid. The concentrations of these compounds were affected in different ways by the various amounts of sugar added, and some differences were observed etween the different carbohydrates.     

一株低产高级醇桑椹果酒酵母的分离、鉴定

以桑椹为分离源,从其自然发酵醪液中分离得到35株酵母菌,采用杜氏小管发酵法初筛,产乙醇能力复筛,耐乙醇和SO₂能力三级筛选,再与葡萄酒活性干酵母的发酵性能和高级醇产生量进行比较,得到1株发酵性能优良且高级醇产生量低的桑椹果酒酵母—S-2。对S-2菌种进行的研究表明,该菌在12 h开始产气,48 h后产生的气体充满杜氏小管,在14%乙醇和120 mg/L SO₂的胁迫下仍有较好的发酵表现,该菌在正常情况下发酵产生的乙醇含量高达10.56%,且高级醇产生量为312.41 mg/L。采用18S rDNA对其进行序列鉴定,确定其与酿酒酵母(Saccharomyces cerevisiae)同源性最高,相似度达到100%。该菌完全可以取代葡萄酒酵母专门用于桑椹果酒的发酵。     

The Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

ORGANOLEPTIC THRESHOLD VALUES OF SOME ALCOHOLS AND ESTERS IN BEER

In a Norwegian all-malt pilsener beer the organoleptic threshold values were determined for the following alcohols and esters: n-propanol, isobutanol, amyl alcohols (2-methyl butanol + 3-methyl butanol), 2-phenyl ethanol, ethyl acetate, isobutyl acetate, iso-amyl acetate, 2-phenyl ethyl acetate, ethyl caproate and ethyl caprylate. In addition, threshold values for a number of combinations of these alcohols and esters were determined, and in each case an additive effect was found.     

枣酒酿造用酵母的筛选及发酵性能研究

以枣庄长红枣发酵液和枣园土壤为酵母菌株的分离源,经过富集培养和多次划线分离纯化,来获得优良的枣酒酿造酵母菌株。经过酵母菌株的产气性能和产酒精性能比较,以及耐酒精、耐酸、耐糖、耐SO2等耐性试验,得到了发酵性能较强的酵母菌株Z03、Z13、Z34。经过染色,镜检观察其细胞形态和芽殖方式,确定为酵母菌,经WL培养基培养后观察,初步鉴定其属于酵母属的酿酒酵母（Saccharomyces cerevisiae）。将这三株酵母与活性干酵母按照枣酒发酵工艺程序进行发酵,发酵出来的枣酒经感官评定和理化检测,最终筛选出适合枣酒发酵酵母菌株Z13和Z34,其酒精度为9.5%vol和10.8%vol。     

玫瑰香干红葡萄酒自然发酵过程中优势酵母分离鉴定及其应用潜力分析

为酿造特色玫瑰香干红葡萄酒,开发本土发酵剂是目前重要发展方向。从昌黎产区玫瑰香干红葡萄酒自然发酵醪中共分离得到337株酵母,通过WL(Wallerstein laboratory)鉴别培养基对其进行分类鉴定、计数,将其鉴别为7大类,根据计数结果选取优势酵母接种至BIGGY(bismuth sulphite glucose glycine yeast)固体培养基,从中进一步筛选不产H2S酵母,进行分子鉴定,结果显示HBKS-Y1、HBKS-Y3为酿酒酵母,HBKS-Y2为葡萄汁有孢汉逊酵母。考察3株本土酵母酿造因子耐性,发现3株酵母对乙醇、SO2、糖度均具有较高耐性,满足葡萄酒酿造要求;以商用活性干酵母FX10葡萄汁发酵为对照,考察3株酵母对玫瑰香葡萄汁单菌、混菌发酵理化指标和香气成分的影响,3株本土酵母单菌和混菌发酵各指标(例如:还原糖、酒精度、总酸、挥发酸、甘油和乙醛含量)均符合干红葡萄酒酿造标准。同时发现,与HBKS-Y2单菌发酵相比,3株酵母混菌发酵总酸和挥发酸含量降低,对葡萄酒有益,且混菌发酵对葡萄酒其他理化指标没有不利影响。本土酵母发酵尤其是混菌发酵,表征玫瑰香品种香气的萜烯醇类物质含量明显高于活性干酵母发酵。以上结果表明,3株本土酵母可以应用于玫瑰香干红葡萄酒的酿造,且3株酵母混菌发酵对突出玫瑰香干红葡萄酒品种香气具有一定的应用潜力。     

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: Potential use of Hanseniaspora uvarum as a starter culture

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 wine.     

Effects of yeast fermentation on transforming the volatile compounds of unsalted pork hydrolysate

This study investigated the possibility of transforming unsalted pork hydrolysate into a liquid seasoning by studying volatile compound production through yeast fermentation. The yeasts used included a typical soya sauce yeast and three wine yeasts. The yeasts inoculated in the glucose supplementary pork hydrolysate increased by approximately 2.0 log of CFU mL⁻¹ within 24 hours at 30°C without adding salt. Yeast fermentation largely depleted the abundant aldehydes (hexenal) present in unfermented pork hydrolysate to trace levels with the formation of ethanol, corresponding alcohols and carboxylic acids. The wine yeasts showed higher production of fruity esters such as ethyl acetate and isoamyl acetate, whereas the soya sauce yeast produced more ketones such as acetone and 2-heptanone. This study revealed a potential method of producing a value-added meat hydrolysate from pork by-product.     

固态法发酵白酒中正丙醇产生的机理研究

采用酿酒车间现场取样实测与实验室同步模拟发酵试验方法,测定并分析了"白云边"大曲酒醅中酵母菌和细菌总数与正丙醇含量的关系。结果表明,酒醅发酵过程中酵母菌总数与正丙醇含量成正相关,而与细菌总数成负相关;酒醅的酒精度与正丙醇含量基本呈正相关。小曲酒醅中正丙醇含量在装瓶发酵后24h内激增,4d时正丙醇含量达到最大值。与接种酿酒酵母和根霉的纯种接种剂相比,两者发酵的酒精度和正丙醇含量均接近,表明酵母菌是白酒中正丙醇的主要产生菌。因此推断,固态法发酵白酒中正丙醇主要是在酵母菌的糖厌氧发酵过程中作为酒精的副产物产生的,而不是通过细菌对蛋白质分解导致的。     

甘蔗酒酿造酵母的筛选

选择常用商业果酒酵母菌株KD、DV10、Q23、EC1118、安琪和H7Y7作为供试酵母菌株,以川蔗17为原料发酵生产甘蔗酒,通过分析发酵液中糖含量变化情况及酒精度、澄清度,比较不同菌株发酵特性,结果发现EC1118菌株发酵彻底,产酒能力最强,发酵后甘蔗酒的高级醇含量最低;H7Y7菌株发酵的甘蔗酒中酯类物质最高,为其他菌株的3～6倍;安琪酵母发酵液最易澄清,但其发酵后的甘蔗酒中甲醇含量为其他菌株的5倍以上。感官评分结果显示,不同酵母菌株发酵的甘蔗酒感官评分依次为H7Y7EC1118DV10安琪酵母Q23KD。研究结果表明,供试酵母菌株中EC1118和H7Y7更适宜用于甘蔗酒酿造。     

REDUCTION OF HIGHER ALCOHOLS BY FERMENTATION WITH A LEUCINE-AUXOTROPHIC MUTANT OF WINE YEAST

Several auxotrophic mutants requiring branched chain amino acids (valine, leucine, or isoleucine) were isolated in a strain of Montrachet wine yeast. They were tested for their ability to produce lowered amounts of higher alcohols (‘fusel oil’: isobutyl, active amyl, and isoamyl alcohols) in grape juice fermentations. One strain which required leucine was especially good in this respect. This mutation is recessive and is the result of a deficiency for the enzyme α-isopropylamate dehydratase. In trial fermentations with this mutant, the resulting wines contained up to 20% less total fusel oil and 50% less isoamyl alcohol compared to the parent Montrachet strain. An experienced taste panel did not discern any gross degradation of taste quality in wine made with the mutant strain compared to that made with the parent strain. The mutant strain could be of commercial importance in preparation of distilling material for alcoholic beverages since the reduced fusel oil content would not require any special distillation procedures which are normally used to avoid the unpleasant flavour associated with concentrated higher alcohols. Reduction of the isoamyl alcohol content is particularly significant since this fusel oil component is usually present in the highest amount.     

红提葡萄酒发酵酵母菌的分离及耐受性研究

该实验从红提葡萄发酵过程中分离出酵母菌株,并对其耐受性进行研究,旨在提高红提葡萄酒酿造品质。通过培养不同发酵时期菌株,并用肉眼和显微镜分别观察其菌落和细胞形态,再测定其发酵能力、耐糖性、耐酸性、耐酒精度。结果表明,共分离出酵母菌140株,经菌落形状和细胞形态鉴定后,获得典型的三类共6株酵母菌;在发酵过程中,加入分离菌株比自然发酵可多发酵1.6%葡萄糖,多产生0.78% CO2和0.82%vol的酒精,且发酵趋势和安琪酵母、自然发酵过程一致;以安琪酵母为对照,对6株菌进行耐糖性、耐酸性、耐酒精度试验,得出综合耐受性较好的酵母菌株为A3、B7和C1。     

甘薯酒发酵工艺优化及香气成分分析

以甘薯为原料,经去皮、打浆、液化、麦芽糖化后制得甘薯浆,并以甘薯浆为原料酿酒。在单因素优化基础上,选取发酵温度、酵母添加量和发酵初始pH值为影响因素,以酒精体积分数和总酯含量为响应值,应用BoxBehnken试验设计建立数学模型并进行响应面分析。并用气相色谱-质谱联用技术对甘薯酒中的香气成分进行检测分析。结果表明:甘薯酒的最佳发酵工艺条件为黄酒专用高活性干酵母添加量0.15%、发酵初始糖含量2 4%、发酵温度21.5℃、发酵初始pH 4.0。在此条件下,甘薯酒的酒精体积分数为14.25%,总酯含量为372.41 mg/L;甘薯酒中共检测出54种香气物质,以醇类和酯类为主;酒体呈均匀的金黄色,具有甘薯的薯香味和麦芽的麦香味。