Contribution of the fermenting yeast strain to ethyl carbamate generation in stone fruit spirits

Fermented fruit and beverages frequently contain ethyl carbamate (EC), a potentially carcinogenic compound that can be formed by the reaction of urea with ethanol. Both are produced by the yeast Saccharomyces cerevisiae with ethanol as the major end product of hexose fermentation and urea as a by-product in arginine catabolism. In spirit production, EC can also be derived from cyanide introduced by stone fruit. To determine the relative contribution of yeast metabolism to EC production, we genetically engineered a diploid laboratory strain to reduce the arginase activity, thus blocking the pathway to urea production. For this purpose, strains with either a heterozygous CAR1/car1 deletion or a homozygous defect (car1/car1) were constructed. These strains were compared to the parental wild type and to an industrial yeast strain in cherry mash fermentations and spirit production. The strain with the homozygous car1 deletion showed a significant reduction of EC in the final spirits in comparison to the non-engineered controls. Nevertheless, using this strain for fermentation of stoneless cherry mashes did not completely impede EC formation. This indicates another, as yet unidentified, source for this compound.     

Chemopreventive effect of N-homocysteine thiolactonyl retinamido cobalamin on carcinogenesis by ethyl carbamate in mice

Because of abnormalities of metabolism of homocysteine thiolactone and methionine in malignant cells, and because of the chemopreventive activity of N-homocysteine thiolactonyl retinamide against chemical carcinogenesis by ethyl carbamate in mice, the cobalamin derivative of this retinamide was prepared and tested for chemopreventive activity. The substance, N-homocysteine thiolactonyl retinamido cobalamin, was found to have a different UV-visible absorption spectrum from that of 5'-deoxyadenosyl cobalamin or N-homocysteine thiolactonyl retinamide. Spectral analysis suggests a ratio of 2 mol of retinamide/mol of cobalamin within the molecule. To demonstrate chemopreventive activity, ethyl carbamate was given in a dose of 2 mg/animal to A/J mice (15-18 g) weekly over a period of 10 weeks to induce pulmonary tumors. A total dose of N-homocysteine thiolactonyl retinamido cobalamin of 60 mg/kg, given for a total of 16 weeks, decreased by one fourth (P less than 0.05) the number of pulmonary tumors induced by ethyl carbamate. An equimolar dose of 5'-deoxyadenosyl cobalamin (40 mg/kg) increased the number of tumors by one third (P less than 0.001), and an equimolar dose of N-homocysteine thiolactonyl retinamide (20 mg/kg) had no effect on the number of pulmonary tumors. No mortality was observed in the experiment. When the ethyl carbamate was given in a single dose of 20 mg/animal, all three substances produced significant mortality in doses of 0.75-30 mg/kg. In the survivors of this experiment, doses of 0.75-30 mg/kg of N-homocysteine thiolactonyl retinamido cobalamin decreased the number of pulmonary tumors induced by ethyl carbamate to 52-82% of controls (P less than 0.01). The results show that N-homocysteine thiolactonyl retinamido cobalamin has chemopreventive activity against chemical carcinogenesis by ethyl carbamate in mice.     

高通量筛选诱变菌株降低黄酒发酵氨基甲酸乙酯前体积累

【目的】氨基甲酸乙酯是发酵食品中普遍存在的一种潜在危害物。黄酒中氨基甲酸乙酯的前体主要是尿素和乙醇。本研究通过高通量筛选策略降低黄酒发酵过程中尿素的积累,从而降低氨基甲酸乙酯积累。【方法】以一株黄酒生产工业菌株酿酒酵母XZ-11为研究对象,采用ARTP诱变和高通量筛选策略,获得尿素积累量较低菌株。使用实时定量PCR技术检测氮代谢中尿素代谢和转运相关基因(DUR1,2和DUR3)的变化。【结果】筛选得到一株尿素高效稳定性利用菌株5-11C。其尿素积累量比酿酒酵母XZ-11降低了50.6%。实时定量荧光PCR结果表明,与尿素代谢和转运相关的基因(DUR1,2和DUR3)表达量分别提高了3.3和2.2倍。【结论】高通量筛选策略可以用于降低黄酒生产过程中氨基甲酸乙酯前体尿素的含量。由于未采用基因工程手段,避免了可能的法规问题,消费者易于接受,在发酵食品行业具有较好的应用前景。     

Possible use of Biolog methodology for monitoring yeast presence in alcoholic fermentation for wine‐making

Aims: A research was undertaken to explore the possibility to use Biolog system of microbial metabolic characterization for the monitoring of yeast population evolution during alcoholic fermentation for wine production. Methods and Results: An application of Biolog system was employed for the characterization of yeasts of oenological interest, in pure cultures and mixed consortia, in various cell concentrations. The system’s capacity to discriminate among different cell concentrations of the same yeast strain was ascertained, along with the capacity to discriminate between mixed and pure populations. Conclusions: The tested application of Biolog system resulted suitable for a quick recognition (24 h) of the presence of starter cultures within mixed populations of autochthonous yeasts. Such discrimination was confirmed with the one resulting from molecular techniques. Significance and Impact of the Study: The study suggests the possibility to employ Biolog system for an early monitoring of yeast evolution in modern wine‐making fermentations, where specialized yeasts are more and more frequently used as starters and their ability to overcome autochthonous yeast populations is crucial.     

本土酵母NX11424对赤霞珠葡萄酒发酵中酵母菌多样性及香气成分的影响

【背景】NX11424酵母是一株具有良好发酵特性且能赋予赤霞珠葡萄酒浓郁果香的宁夏本土酿酒酵母。【目的】解析NX11424酵母发酵的赤霞珠葡萄酒中的水果香气特征。【方法】以赤霞珠为材料,设置3个发酵处理：自然发酵、灭菌接种NX11424的发酵和直接接种NX11424的发酵,利用26S rDNA D1/D2区测序分析法鉴定发酵过程中酵母菌的种类,并通过顶空固相微萃取和气相色谱-质谱联用技术定量测定不同发酵处理下赤霞珠葡萄酒的香气成分及含量。【结果】三个发酵处理下赤霞珠葡萄酒各理化指标无显著性差异。所分离到的酵母菌鉴定为2属3种：萄葡汁有孢汉逊酵母(Hanseniaspora uvarum)、酿酒酵母(Saccharomyces cerevisiae)和布拉迪酵母(Saccharomyces boulardii,S. boulardii);这2属3种均存在于自然发酵中,接种发酵中仅存在H. uvarum和S. cerevisiae两种酵母。三个发酵处理下的赤霞珠葡萄酒中香气物质种类无差异,均为69种;其中,酯类28种,醇类25种,有机酸5种,萜烯类2种和其他类化合物9种;但各发酵处理下香气物质的含量存在显著差异。聚类分析表明,69种香气成分被聚为3类。第1类香气物质包括香茅醇、丙醇等9种成分,其中7种香气物质的含量在灭菌接种发酵中较高;第2类香气物质包括己酸乙酯、棕榈酸乙酯等31种成分,其含量均在直接接种发酵中较高;第3类香气物质包括丁酸乙酯、乳酸乙酯等29种成分,其中27种香气物质的含量在自然发酵中较高。虽然直接接种发酵处理中酵母菌的多样性低于自然发酵处理,但是该处理的赤霞珠葡萄酒中酯类香气物质含量较多,水果香气浓郁,对赤霞珠葡萄酒香气的改善更明显。【结论】NX11424与发酵中的其他本土酵母间的相互作用可以改善葡萄酒的质量,为宁夏本土酵母NX11424在赤霞珠葡萄酒酿造过程中改善葡萄酒质量奠定了坚实的基础。     

The binding of ethyl carbamate to liver DNA in gonadectomised mice

Castration, with or without subsequent hormone administration resulted in different levels of binding of a metabolite of ethyl carbamate to mouse liver DNA. In ovariectomised and ovariectomy plus testosterone-treated females, the binding persisted for longer than in intact and ovariectomy plus estradiol-treated females, although the maximum levels of binding were similar in all four groups. In males, orchidectomy dramatically lowered the level of binding compared with that in intact males. Twelve hours after dosing, the levels of binding were 276 and 9.8 nanomoles g. DNA^?1 respectively in the two groups. Estradiol treatment did not restore the level of binding whereas testosterone treatment did.     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

The effect of start wort on the initial period of Baker's yeast fermentation

В лабораторных ферментационных танках, в условиях, как можно больше отвечающих производственным, исследовалось влияние субстрата на период взбраживания при дрожжевом брожении. Исследовались влияние концентрации инвертного сахара на ход\(Q_{CO_2 }^{N_2 } \) на мальтозе и глюкозе, а также динамика содержания РНК и ДНК. Далее исследовалась также динамика фосфатных фракций и некоторых других составных частей клеток (трегелёза, полисахариды, белки, свободные аминокислоты). Было установлено, что субстрат быстро метаболизируется и немедленно протекает синтез РНК, всех углеводов и белков, что сопровождается регулярным понижением содержания 7-минутного фосфора. Синтез ДНК осущест-вляется позднее. Изменения составных частей клетки находятся в корреляции с морфологическими изменениями клетки, с процессом роста и деления дрожжей. Из результатов опытов очевидно, что процессы, протекающие в этот период брожения, можно сравнить с кратким циклом роста. С точки зрения технологии дрожжевого брожения констатируется, что 1-часовый период взбраживания слишком долог и что клетки в указанных усповиях страдают от недостатка сахарного субстрата и бывают принуждены с углеводного субстрата перейти на алкоголь.     

Reduced production of ethyl carbamate for wine fermentation by deleting <Emphasis Type="Italic">CAR1</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.     

The effect of neutral resins on the fermentation production of rubradirin

Summary Rubradirin is an antibiotic of complex chemical structure which is activevs. methicillin resistant staphylococci. Its development has been limited due to inadequate production yields. The incorporation of neutral resins into fermentations ofStreptomyces achromogenes v.rubradiris, UC^® 8051 resulted in the enhanced production of rubradirin. Resins HP-20, HP-21, XAD-2, XAD-7 and XAD-16 were employed in flask and tank fermentations. The incorporation of these resins promoted 2- to 4-fold enhancements of the rubradirin activity produced in flask fermentations, and the incorporation of XAD-16 and HP-21 into tank fermentations promoted production titer increases >5 fold.     

The production of caproic and butyric acids by the methane fermentation of ethyl alcohol

Summary A fermentation of ethyl alcohol under the influence of Methanobacterium Omelianskii and an associated spore-forming bacterium is described in which the alcohol is converted into acetic, butyric and caproic acids. At the same time there occurs a reduction of carbon dioxide to methane. The data provide direct evidence for the formation of butyric and caproic acids by a condensation of a C₂-compound which probably is acetaldehyde.Division of Plant Nutrition, University of California, Berkeley, California. The author takes great pleasure in expressing his indebtedness to Prof. A. J. Kluyver for extending the hospitality of the Delft laboratory and for his interest, encouragement and advice during the progress of the work.     

The use of a cyanine dye in measuring membrane potential in yeast

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.