The Contribution of 4‐Hydroxyfuranone Derivatives to the Aroma of Commercial Beers and Malts

Analysis of hot water extracts from a range of commercial malts showed that 2,5‐dimethyl‐4‐hydroxy‐3(2H)furanone (DMHF) varied from undetectable (lager malt) to 4.2 mg/litre (crystal malt), a concentration twenty‐six times the flavour threshold in water. 5‐Methyl‐4‐hydroxy‐3 (2H)furanone (MHF) was detected in all samples except one but was always well under its flavour threshold value. 2(or 5)‐Ethyl‐5(or 2)‐methyl‐4‐hydroxy‐3(2H)furanone (EMHF) was not detected in any of the samples. Fermentation of lager, ale and crystal malt extracts with an ale strain of yeast led to the appearance of EMHF in all cases as well as additional DMHF. The greatest increases in both compounds was with the ale malt. Both increases and decreases occurred in MHF concentration as a result of fermentation but final levels were always well below the flavour threshold value. Analysis of ten commercial beers found DMHF in all the samples and in five cases levels exceeded twice the flavour threshold value in beer with flavour units from 2.4 to 9.1. A flavour panel noted that in four of these cases the beer had a distinctly sweet/caramel aroma which is typical of DMHF. EMHF was undetectable in six samples, detectable, but unquantifiable, in three cases but at about 80% of the flavour threshold value in the remaining sample. MHF was found in all samples but at insignificant levels. The results show that DMHF is an important flavour compound in British ales and EMHF may make a contribution in a limited number of situations. The contribution of malt type, brewery processing and yeast strain in determining the concentration of the two 4‐hydroxyfuranones in beer remains uncertain.     

Theasaponin E1 destroys the salt tolerance of yeasts

Cells of Zygosaccharomyces rouxii in a medium containing a high concentration of NaCl were killed during incubation for 2-4 h with a low concentration of a mixture of saponins from tea seeds (TSS). The higher the concentration of NaCl in the medium, the higher the inhibitory effect of TSS on the growth of the yeast. The above inhibitory effect of TSS on the growth of the yeast was not observed when cells were incubated in hypertonic media composed of nonionic substances such as sugars. The ATPase activity of plasma membrane preparations from the yeast cells was slightly affected by the addition of TSS. It is shown that TSS facilitates leakage of glycerol from the yeast cells under NaCl-hypertonic conditions. The major inhibitor in the mixture of saponins was isolated and identified as theasaponin E1. Its isomer, theasaponin E2, did not have any effect on the salt tolerance of Z. rouxii or Saccharomyces cerevisiae.     

硫胺素对鲁氏接合酵母高盐适应性的影响

采用同位素内标-超高效液相色谱串联三重四极杆质谱（UPLC-MS/MS）法测定金沙江攀枝花江段6种常见鱼中卡巴氧代谢物喹恶啉-2-羧酸（QCA）和喹乙醇代谢物3-甲基-喹恶啉-2-羧酸（MQCA）的残留量。样品经过预处理,采用ZORBAX SB-C18色谱柱分离,以0.1%甲酸水-乙腈为流动相进行梯度洗脱;采用电喷雾-正离子多反应监测模式（MRM）检测,同位素内标法定量。结果表明,QCA和MQCA的质量浓度为2.0～100.0 ng/mL时,线性关系良好（R＞0.999）,回收率为91.4%～100.4%,精密度试验结果相对标准偏差（RSD）为6.3%～11.5%,定量限为0.2 μg/kg,检出限为0.05 μg/kg。该方法灵敏度及准确度良好,能满足6种常见鱼的组织中卡巴氧及喹乙醇代谢物残留分析的要求。     

Formation of 4‐Hydroxyfuranones and their Precursors during Production of Worts and Beers

Laboratory beers and samples taken at each stage of production, were analysed for the flavour active 4‐hydroxyfuranones; 5‐methyl‐4‐hydroxy‐3(2H)‐furanone (MHF), 2,5‐dimethyl‐4‐hydroxy‐3(2H)‐furanone (DMHF) and 5‐(or 2)‐ethyl‐2(or 5)‐methyl‐4‐hydroxy‐3(2H)‐furanone (EMHF). The length and temperature of mashing, the length of boiling, the rate of cooling the worts and the effects of grist composition were investigated to identify the 4‐hydroxyfuranone content of worts and subsequent beers. Fermentation temperature and the use of the stabilising agents, PVPP and Lucilite PC5, on the 4‐hydroxyfuranone content of the beer was also investigated. The results demonstrated that several aspects of beer production procedures affect the furanone content of the beer, but in practice the important factors are grist composition, the rate at which the boiled wort is cooled and fermentation temperature. Fermentation has a major effect on final furanone content as yeast produces both DMHF and EMHF. The results suggest that malt levels of precursor compounds, which can be converted to 4‐hydroxyfuranones by the Maillard reaction or by yeast, may prove to be more important than the quantities of the furanones found in malt in determining the final furanone content of beer. A clearer understanding of the nature of the precursors should allow manipulation of their production and beer furanone content.     

Functional analysis of the Zygosaccharomyces rouxii Fps1p homologue

The osmotolerant yeast Zygosaccharomyces rouxii accumulates the polyols glycerol and D-arabitol intracellularly in response to hyperosmotic stress, but the membrane transport proteins regulating polyol accumulation have not been studied. We have cloned and characterized a FPS1 homologue in Z. rouxii NRRL Y2547, and its sequence revealed a 2709 bp open reading frame encoding a peptide of 692 deduced amino acids with 56.9% identity to the Saccharomyces cerevisiae Fps1p. The role of this putative membrane channel protein in polyol accumulation and release during osmoregulation was investigated. The Z. rouxii FPS1 (ZrFPS1) complemented the S. cerevisiae fps1Delta growth defect and glycerol release upon hypo-osmotic shock. Deletion of ZrFPS1 did not affect growth on glycerol as sole carbon source, suggesting that other transport proteins are involved in the uptake of glycerol. However, mutants lacking ZrFPS1 exhibited a significant decrease in glycerol and D-arabitol efflux and poor growth during hypo-osmotic conditions, suggesting that ZrFPS1 might be involved in D-arabitol transport in addition to glycerol. This is the first demonstration of a yeast gene that affects D-arabitol transport. The full-length ZrFPS1 gene sequence including upstream promoter has been deposited in the public database under Accession No. AY488133.     

Concomitant extracellular accumulation of alpha-keto acids and higher alcohols by Zygosaccharomyces rouxii

Alpha-keto acids are key intermediates in the formation of higher alcohols, important flavor components in soy sauce, and produced by the salt-tolerant yeast Zygosaccharomyces rouxii. Unlike most of the higher alcohols, the alpha-keto acids are usually not extracellularly accumulated by Z. rouxii when it is cultivated with ammonium as the sole nitrogen source. To facilitate extracellular accumulation of the alpha-keto acids from aspartate-derived amino acid metabolism, the amino acids valine, leucine, threonine and methionine were exogenously supplied during batch and A-star cultivations of (routants of) Z. rouxii. It was shown that all alpha-keto acids from the aspartate-derived amino acid metabolism, except alpha-ketobutyrate, could be extracellularly accumulated. In addition, it appeared from the concomitant extracellular accumulation of alpha-keto acids and higher alcohols that in Z. rouxii, valine, leucine and methionine were converted via Ehrlich pathways similar to those in Saccharomyces cerevisiae. Unlike these amino acids, threonine was converted via both the Ehrlich and amino acid biosynthetic pathways in Z. rouxii.     

一株耐盐产香鲁氏接合酵母FA-1的鉴定及其在酱油酿造中的应用

为提高酱油风味与品质,从酱油醪液中筛选耐盐产香酵母菌株并应用于酱油酿造。对筛选菌株进行鉴定,并采用顶空固相微萃取-气质联用法（HS-SPME-GC-MS）分别检测其在不同NaCl浓度的发酵液及酱油成品中的挥发性香气成分。结果表明,筛选出1株耐盐产香菌株FA-1,经鉴定为鲁氏接合酵母（Zygosaccharomyces rouxii）,可耐受22%的食盐浓度。在不同NaCl浓度的酱油发酵液中共检出56种挥发性风味物质,且当NaCl质量分数为18%时,更有利于醇类化合物的富集。在酱油生产试验中,添加鲁氏接合酵母FA-1的实验组中醇类化合物占比最大（72.03%）,其中苯乙醇、3-甲基-1-丁醇、乙醇的含量较高,且醇类和酯类化合物的含量分别较添加酵母As 1.039的对照组提高131.05%和189.65%。因此,菌株FA-1具有良好的耐盐产香性能,可作为潜在的酱油发酵菌株。     

Pentadiene production from potassium sorbate by osmotolerant yeasts

Some yeast strains belonging to the species Zygosaccharomyces rouxii and Debaryomyces hansenii are capable of spoiling sorbate containing high-sugar foods by producing pentadiene, a volatile compound reported to have 'petroleum-like' odour. Quantification of the diminution of sorbate and the subsequent increase of pentadiene was performed by growing the yeasts in experimental media containing 600 g/l sucrose and different sorbate concentrations. Final sorbate concentrations were notably lower than their corresponding initial ones, and it was found that the higher the initial concentration of sorbate in the media, the higher the amount of pentadiene produced. In all cases, Z. rouxii was able to produce more pentadiene than D. hansenii when expressing pentadiene concentration as a function of cell biomass. These results suggest that pentadiene is a metabolite of sorbate.     

鲁氏酵母菌QOR6产呋喃酮发酵条件优化及代谢特征分析

为明确鲁氏酵母菌代谢合成呋喃酮工艺条件与代谢情况,以鲁氏酵母(Zygsoaccharomyces rouxii)QOR6为出发菌株,通过单因素实验和正交试验优化QOR6代谢合成呋喃酮的发酵条件,并采用气相色谱-质谱联用技术测定其挥发性香气物质的种类和相对含量。结果表明,当QOR6发酵温度28℃、发酵初始pH4.2、培养转速为180 r/min和接种量为20×10⁷ CFU/mL时,呋喃酮的产量为2.90 mg/L,比优化前提高了1.09倍。发酵液中挥发性成分共有41种,主要包括醇类、醛类、酯类、芳香族和烃类等。其中,醇类含量最高,占总挥发物的81.13%。其他相对含量较高的为乙酸苯乙酯(3.48%)、甲醛(0.34%)、2,6-二叔丁基苯酚(8.77%)、氯甲氧基甲烷(0.20%)和3-乙基-2,5-甲基吡嗪(0.70%)。本文旨在为鲁氏酵母菌在食品中的应用提供了技术支持。     

啤酒废酵母制备1,6-二磷酸果糖的研究

啤酒废酵母生产1,6-二磷酸果糖发酵工艺进行了试验性探讨。正交试验结果表明,啤酒废酵母发酵生产FDP的最佳工艺参数为:发酵体系中葡萄糖的加量为0.5mol/L、无机磷的浓度为0.4mol/L、pH6.5、转化时间6h。     

生香酵母筛选鉴定及其对辣椒酱发酵的影响

为了获得适用于辣椒酱发酵的生香酵母,从自然发酵辣椒酱中筛选优良菌种,并对菌种形态特征、生理生化特性、菌种发酵辣椒基料等进行了研究。经筛选,获得一株生香能力较强的菌株LA-Y09,该菌株被鉴定为鲁氏酵母（Saccharomyces rouxii）。菌种LA-Y09的最适生长温度为28 ℃,在NaCl质量分数为12%的培养基中仍有较高菌数,说明其具有良好的耐盐特性,在质量分数为8%的辣椒基料中能够生成多种呈现蜜香和果香的香气成分,使辣椒发酵醪香气得到明显提升。     

Development of internal transcribed spacer regions amplification restriction fragment length polymorphism method and its application in monitoring the population of Zygosaccharomyces rouxii M2 in miso fermentation

Recently, the use of the dry yeast of Zygosaccharomyces rouxii M2 for miso (soybean paste) fermentation has been established. A molecular monitoring method was developed and validated in this study to analyze the population of Z. rouxii M2 during the fermentation. The method was based on the restriction patterns of internal transcribed spacer (ITS) regions of the rDNA using HaeIII and HhaI. Among the homologous ITS regions of Z. rouxii strains, Z. rouxii M2 produced diagnostic bands by which it can be differentiated from the other strains used. The specific restriction bands were due to the difference in nucleotide sequence of two different copies of ITS of Z. rouxii M2. Both ITS copies showed 94% sequence similarity but a 13-bp nucleotide substitution and a 19-bp deletion were found in the ITS1 region. Phylogenetic trees were constructed based on ITS and 18S rDNA sequences and it was found that the ITS sequences provide better resolution for the classification of Z. rouxii M2. Since Z. rouxii M2 is a promising strain for use in miso fermentation as a dry starter, the method developed is significant in terms of industrial application in monitoring the growth of Z. rouxii M2 in miso fermentation.     

4-羟基-2,5-二甲基-3(2H)呋喃酮对单增李斯特菌AI-2类群体感应的干扰效应

利用群体感应抑制剂靶向干扰病源菌已成为有效控制致病性危害的突破口。本研究通过测定菌体浓度、哈氏弧菌BB170报告菌的发光值,研究不同浓度4-羟基-2,5-二甲基-3(2H)呋喃酮(DMHF)作用下单增李斯特菌(L.m)的生长与信号分子AI-2活性;通过半固体穿刺法、结晶紫法及溶血平板法检测L.m的动力、生物膜及溶血性,来评价DMHF对L.m AI-2类QS的干扰效应。结果表明,≤ 200 μg/mL的DMHF能推迟L.m的生长,且明显抑制了L.m AI-2的活性,实验组的AI-2活性值均低于阴性对照组的40%,由此可判定DMHF是AI-2类QS的抑制剂;当DMHF浓度为100 μg/mL时能明显抑制L.m的运动能力;50、100和200 μg/mL的DMHF对L.m生物膜的形成抑制率分别为29.72%、44.88%和75.27%;200 μg/mL的DMHF完全抑制溶血环的产生,本研究为利用DMHF作AI-2类QS的抑制剂提供依据。     

甘油与鲁氏酵母共固定在高盐稀态酱油酿造中的应用

本文旨在研究相容性溶质之一的甘油对高渗透压环境下鲁氏结合酵母的保护作用,以及将甘油与鲁氏结合酵母共固定对高盐稀态酱油发酵的影响。添加不同浓度甘油至含3 mol/L氯化钠的YPD培养基中,观察酵母菌生长情况。同时,共固定甘油与鲁氏结合酵母,应用于高盐稀态酱醪发酵。结果显示,1.37 mmol/L甘油即可有效缓解高盐环境对鲁氏结合酵母的生长抑制作用,生长延滞期缩短达4~6 h。高盐稀态酱油发酵中试结果显示,与空白对照组相比,试验组蛋白质转化率提高幅度达2.23~5.64%,头油中各理化指标平均提前15 d达到空白组相同水平。综上所述,甘油作为一种相容性溶质可有效保护高盐环境下鲁氏结合酵母的生长繁殖,及其在高盐稀态酱醪发酵中的代谢性能,甘油与鲁氏接合酵母共固定技术能够提高酱油发酵的原料利用率、缩短发酵周期,在酱油酿造领域具有一定的实际应用价值。     

固定化酵母细胞增加酱油香气的研究

在酱油生产中添加酵母可以有效的增加酱油的香气,实验选择不同种的酵母细胞,将其固定化后应用于酱油酿造过程中,应用单标法以2-乙基-4-羟基-5甲基-3(2H)呋喃酮(HEMF)为标准品进行气相色谱检测,结果表明固定化的莫格球拟酵母在酿造过程中产生对酱油香气有较大影响的HEMF.     

Salt shock enhances the expression of ZrATP2, the gene for the mitochondrial ATPase beta subunit of Zygosaccharomyces rouxii

In the course of a study of cell wall proteins from the salt-tolerant yeast Zygosaccharomyces rouxii, a protein that increased its expression as the NaCl concentration of the culture medium increased was identified. Several degenerate primers were constructed based on partial amino acid sequences of this protein and were used in PCR amplification of a gene termed ZrATP2. The amino acid sequence deduced from nucleotide sequence of the gene revealed that ZrATP2 encodes the beta subunit of mitochondrial F1 ATPase. Northern blot analysis demonstrated that NaCl shock induced an elevation in ZrATP2 expression, which corresponded with the resumption of Z. rouxii cell growth after salt shock.     

Molecular cloning and sequence analysis of the Zygosaccharomyces rouxiiLEU2 gene encoding a beta-isopropylmalate dehydrogenase.

A DNA fragment carrying the LEU2 gene of osmotolerant yeast Zygosaccharomyces rouxii was isolated. The sequenced DNA fragment (2630 bp) contained two ORFs; one of them (1086 bp long, predicting a protein of 362 amino acids) shared a high degree of similarity with LEU2 genes of other yeast species. The cloned DNA fragment fully complemented the leu2 mutations of Saccharomyces cerevisiae and Z. rouxii.     

强化嗜盐四联球菌对模拟低盐酱油发酵的影响

通过模拟低盐酱油快速发酵,在添加鲁氏接合酵母（Zygosaccharomyces rouxii）的基础上,考察嗜盐四联球菌（Tetragenococcus halophilus）不同添加量对酱醪理化指标、氨基酸和有机酸含量、挥发性风味物质的影响,揭示嗜盐四联球菌在提高低盐酱油品质方面的应用潜力。结果表明,嗜盐四联球菌对酱油中酵母菌的生长有一定的抑制作用,会使酱醪pH略有下降,总酸和氨基酸态氮含量提高。最适宜提高酱油品质的菌株添加方式是发酵开始接入鲁氏接合酵母,发酵10 d后添加108 CFU/g嗜盐四联球菌,酱油样品中氨基酸和有机酸总量比单独添加酵母菌分别提高43.8%、55.6%,其中鲜味和甜味氨基酸分别提高33.7%、29.9%;挥发性风味物质种类增加43.2%,总量提高2.4倍,其中香气物质愈创木酚和1-辛烯-3-醇含量分别提高11.1倍、8.9倍。     

Regulation of lactate production in Streptococcus bovis: A spiraling effect that contributes to rumen acidosis

When Streptococcus bovis was grown in batch culture with 6 g/L glucose at pH 6.7, maximum specific growth rate was 1.47 h(-1), and lactate was the primary fermentation product. In continuous culture at pH 6.7 and growth rate equal to .10 h(-1), little lactate was formed, and formate, acetate, and ethanol accounted for most of the product. When extra-cellular pH decreased to 4.7, intra-cellular pH declined to 5.4, and organisms switched back to lactate production. Intracellular concentration of fructose 1,6-diphosphate of batch culture cells was greater than 12 mM, a concentration that allowed maximal lactate dehydrogenase activity. When Streptococcus bovis was grown in continuous culture at pH 6.7, intracellular fructose-l,6-diphosphate declined to .4 mM, a concentration which gave little lactate dehydrogenase activity at pH 6.5 or greater. Decreasing pH of continuous culture to 4.7 increased intracellular fructose-1,6-diphosphate concentration to .8 mM. This concentration was still limiting if lactate dehydrogenase was assayed at pH 6.5, but nearly maximal activity was obtained when enzyme was assayed at pH 5.5. The small increase in fructose-l,6-diphosphate and decreased requirement of lactate dehydrogenase for fructose-l,6-diphosphate under acidic assay conditions, accounted for increased lactate production during low pH (4.7) continuous culture. These and other aspects of lactate regulation by Streptococcus bovis are discussed as factors leading to rumen acidosis. This pattern of regulation also helps to explain why rumen acidosis is difficult to reverse.     

高盐稀态酱油发酵S3—2酵母基础发酵性能的研究

鲁氏酵母（S酵母）的耐盐能力抑制了其对高盐稀态酱油风味的改善作用。S3-2酵母是本试验室以S酵母为出发菌株,利用基因组重组技术构建的一株具有高耐盐度的酵母菌株。在YPD发酵培养基中对S3-2酵母进行基础发酵性能研究的结果表明,S3-2比S酵母具有更高的耐盐能力,S酵母能生长的最大盐度为19％,而S3-2酵母在含盐量高达21％的培养基中仍能够良好生长。在含盐量为18％的YPD发酵培养基中,S3-2酵母在提高醇类物质的种类和含量,对提高高盐稀态酱油风味具有重要的意义。