酿酒酵母和酒类酒球菌原生质体制备与再生的条件优化

应用正交设计，研究酶解时间、酶浓度和酶解温度对酿酒酵母和酒类酒球菌原生质体制备和再生的影响。实验结果表明，酶解浓度对原生质体制备和再生的影响最大。最优组织条件为当蜗牛酶浓度为20g/L，酶解温度为37℃和酶解时间为20min时，酿酒酵母原生质体的制备率为88.4%。再生率为32.5%。当溶菌酶浓度为1mg/mL、酶解温度为37℃和酶解时间为20min时，酒类酒球菌原生质体的制备率为87.5%，再生率为31.2%。     

原生质体融合法构建增香型苹果酒酿造酵母的研究

以具有强发酵能力的酿酒酵母1605和产香能力好的苹果酒酵母E2为亲本,通过对原生质体融合条件的研究,得出以下结论:根据两亲本的生长曲线图确定两亲本在制备原生质体时的前培养时间为4h;在酶解温度35℃、蜗牛酶终质量分数为1%的条件下,最佳酶解时间为80min,此时,亲本菌株1605的原生质体形成率和再生率分别为92.4%和29.5%,亲本菌株E2的原生质体形成率和再生率分别为93.5%和30.7%;在60℃时,对亲本菌株1605的原生质体水浴灭活15min,即可完全抑制或钝化其原生质体活性。通过对融合子酿造苹果酒的感官和主要香气成分的分析,利用模糊综合评判法优选出8#菌株,该菌株具有产香能力好、发酵能力强的双亲优点,为优良的增香型苹果酒酵母菌株。     

乳酒酵母紫外诱变原生质体制备研究

在乳酒酵母原生质体形成率和再生率影响单因素研究的基础上,对菌龄、酶解温度、酶解时间、酶浓度、稳渗剂采用L16(4)5正交试验,评价各因素的影响程度,得出适宜该菌原生质体制备的条件.结果表明,菌龄 12 h、酶浓度1.0%、酶解时间1 h、酶解温度28℃、稳渗剂选用KCl的条件下,原生质体形成率76%,再生率62%.     

耐高糖酿酒酵母原生质体制备与再生过程研究

对1株耐高糖酿酒酵母的原生质体制备和再生条件进行了研究.结果表明,发酵7h后为对数生长中期,适宜原生质体化;L16(5)确定制备原生质体的最佳条件为蜗牛酶浓度(1.0%)、KCI高渗缓冲液(O.7mol/L)、酶解时间(1.5h)、预处理剂(0.1%B-巯基乙醇)和酶解温度(26℃),在此条件下原生质体形成率和再生率分别为80.84%和36.03%;原生质体形成后在7%蔗糖高渗培养基上夹层培养再生率较高(39.78%).     

乳酸菌原生质体制备与再生研究

从市售酸乳中分离嗜热链球菌和保加利亚乳杆菌,并对其进行原生质体制备和再生。采用溶菌酶对嗜热链球菌和保加利亚乳杆菌进行处理,脱去细胞壁以探讨原生质体形成和恢复与菌龄、酶浓度、酶解时间及酶解温度之间的关系,利用四因素三水平的正交试验选出原生质体形成和恢复较适宜的条件。试验结果显示嗜热链球菌原生质体制备和恢复的较优条件为,菌龄16h,酶浓度1mg/ml,酶解时间40min,酶解温度42℃,此时原生质体形成率为98.75%,再生率为23.8%;而保加利亚乳杆菌原生质体制备和恢复的较优条件为,菌龄16h,酶浓度10mg/ml,酶解时间30min,酶解温度36℃,此时原生质体形成率为82.05%,再生率为27.1%。本文研究为这两种乳酸菌原生质体的制备、再生条件及其基因操作和相关研究提供技术思路和实验条件。     

安琪超级酿酒酵母原生质体制备与再生条件研究

应用正交试验研究预处理剂、酶种类和浓度、渗透压稳定剂和酶解时间对安琪超级酿酒酵母(Saccharimyces cerevisiae)原生质体形成率和再生的影响.结果表明,渗透压稳定剂在上述4种因素中对原生质体的形成和再生影响最大.最优组合为:使用β-巯基乙醇作预处理剂,0.4%蜗牛酶加0.4%纤维素酶,使用0.53 M蔗糖做渗透压稳定剂,28℃酶解1 h.安琪超级酿酒酵母原生质体的形成率为98.6223%,再生率为19.9904%.     

青稞酒曲中酿酒酵母与假丝酵母原生质体形成与再生条件的研究

本实验通过研究酶种类、菌龄、酶浓度及酶的作用时间、菌体预处理及渗透压稳定剂等不同因素对原生质体形成与再生的影响，得出了青稞酒曲酵母ZY2，ZY8原生质体制备和再生的最佳条件：酵母ZY2在菌龄为10h，0.1mol/LEDTA＋0.1%β-巯基乙醇为预处理剂作用10min；30℃下用1.5%的蜗牛酶酶解1h；酵母ZY8在菌龄为10h，0.1mol/LEDTA＋20mmol/L2-硫苏糖醇混合于蜗牛酶1.5%＋溶壁酶1%的酶解体系中，30℃下酶解1h；配制酶解液时渗透压稳定剂采用0.7mol/LKC1，在稀释及配制再生培养基时采用17%蔗糖的条件下，酵母ZY2形成率达94.15%，再生率达13.85%；酵母ZY8形成率达88.78%，再生率达23.02%。     

南阳酵母2.577原生质体制备和再生条件的研究

南阳酵母2.577是生产燃料乙醇的一种重要的酵母菌.为了培养出一种性能更好的酵母菌,以南阳酵母2.577为出发菌,研究菌龄、酶解温度、酶解时间、酶浓度4个因素对其原生质体形成率和再生率的影响.结果表明:当菌龄为12 h、酶浓度为2.O%、酶解时间为2 h、酶解温度为28℃时,南阳酵母2.577原生质体的形成率和再生率能分别达到最高值.     

纤溶酶产生菌原生质体形成与再生条件的探索

本文研究了菌龄、酶浓度、酶解时间、酶解pH、温度、渗透压稳定剂及青霉素对枯草芽孢杆菌DC-12原生质体形成和再生的影响。确定了DC-12原生质体最佳的形成和再生条件是以改良HM高渗溶液作稳渗系统,在pH7.5、酶浓度为0.2mg/ml（活力单位为50000U/g）和35℃的条件下酶解60min;此条件下所得原生质体形成率为96.1%;以0.6mol/LNaCl为渗透压稳定剂的DM3再生培养基中再生率为21.6%。     

出芽短梗霉原生质体制备及再生的研究

研究不同的酶解液、茵龄、酶处理的时间和温度、培养方法及其他因素对出芽短梗霉原生质体的形成率和再生率的影响.结果表明:出芽短梗霉原生质体制备所需的最佳条件是酶浓度5 mg/mL(蜗牛酶、纤维素酶)、菌龄24 h、酶解温度37℃、酶解时间1 h,在此条件下原生质体形成率达100%.再生率为64.6%.     

The species-specific ratios of 2,3-butanediol and acetoin isomers as a tool to evaluate wine yeast performance

The isomers of 2,3-butanediol [R,R; S,S; R,S (meso-form)] and of acetoin (R,S) were determined in laboratory wine fermentations carried out by 50 yeast strains, 10 for each of the following species, Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata, Metschnikowia pulcherrima and Brettanomyces bruxellensis, in order to evaluate if such parameters might be used to differentiate wines obtained with different yeast species. According to analysis of variance (ANOVA), the strains of the same species behaved similarly, whereas the five yeast species behaved differently so that species-specific profiles were recognized. Moreover, the discriminant analysis grouped the wines into five groups, each including the 10 wines obtained by the 10 yeast strains of the same species. Trials were also included where musts partially fermented by non-Saccharomyces species were inoculated with a selected strain of S. cerevisiae to complete fermentation, and the content in 2,3-butanediol and acetoin isomers was again determined and statistical analysis was performed. Although the final values of these parameters resembled those obtained in pure fermentation with S. cerevisiae, statistical analysis discriminated wines according to the yeast species performing the first fermentation phase.     

Sequencing and functional analysis of the Hansenula polymorpha genomic fragment containing the YPT1 and PMI40 genes

A 6.0 kb genomic DNA segment was isolated by its ability to rescue the temperature-sensitive growth defect and the hypersensitivity to sodium deoxycholate of a spontaneous vanadate-resistant mutant derived from Hansenula polymorpha DL-1. The genomic fragment contains four open reading frames homologous to the Saccharomyces cerevisiae genes YPT1 (which codes for a GTP-binding protein; 75% amino acid identity), PMI40 (encoding phosphomannose isomerase; 61% identity), YLR065c (30% identity) and CST13 (28% identity). The H. polymorpha YPT1 homologue (HpYPT1) was found to be responsible for the complementation of the temperature-sensitive phenotype and the sodium deoxycholate sensitivity of the mutant strain. Disruption of the H. polymorpha PMI40 homologue (HpPMI40) resulted in the auxotrophic requirement for D-mannose. The heterologous expressions of HpYPT1 and HpPMI40 were able to complement the temperature-sensitive phenotype of S. cerevisiae ypt1-1 mutant and the mannose auxotrophy of S. cerevisiae pmi40 null mutant, respectively, indicating that the H. polymorpha genes encode the functional homologues of S. cerevisiae YPT1 and PMI40 proteins. The nucleotide sequence has been submitted to GenBank under Accession No. AF454544.     

海藻糖与酿酒酵母乙醇耐受性相关性的研究进展

为了得到具有高富锌能力的酵母菌,该研究以面包来源的酵母菌DLY28为出发菌株,重复驯化后筛选获得一株优良耐锌酵母,并通过单因素试验及响应面试验对其富锌培养基进行优化。结果表明,通过驯化筛选得到一株优良耐锌酵母S7,其富锌的最优培养基组成为蔗糖含量82 g/L、胰蛋白胨含量26 g/L、锌含量404 mg/L。在此最优条件下,富锌酵母S7的锌吸附量为18.79 mg/g,生物量（OD600 nm值）为1.49。该研究为富锌酵母的生产以及食品有机锌的开发应用提供理论依据。     

Rhizopus oligosporus and yeast co-cultivation during barley tempeh fermentation--nutritional impact and real-time PCR quantification of fungal growth dynamics

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold storage of tempeh (P<0.01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents and compositions, and did not reduce phytate contents. Slight increases of vitamins B(6) and niacinamide, and slight decreases of B(1) and biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh. The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus, real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous substrate such as barley tempeh.     

冰梨酒酿酒酵母的筛选

以鸭梨为原料,榨汁后经冷冻浓缩制成浓缩梨汁（即冰梨汁）,接入酿酒酵母（Saccharomyces cerevisiae）,在15 ℃下进行低温酒精发酵酿制成冰梨酒。通过比较8株酿酒酵母的发酵性能,及其所酿冰梨酒的理化指标、有机酸含量和感官品质,最终确定酿酒酵母R2为酿制冰梨酒的适宜菌种。该酵母生长繁殖快、降糖和产酒精能力强、发酵性能优良;酿出的冰梨酒酒香和谐,果香纯正,口感甜美醇厚、柔和爽口,具有冰梨酒特有的风味。     

酵母菌FW-sc-08的生长及发酵特性研究

通过对影响菌体生长因素、发酵指标和香气成分的测定,研究了从自然发酵黑莓酒中筛选出的酵母菌FW-sc-08的生长和发酵特性。结果显示,酵母菌FM-sc-08对数生长期为3～9 h,对SO2最大耐受质量浓度为800 mg/L;酵母菌FM-sc-08适宜发酵的可发酵糖最大含量为30%,对糖最大耐受量为40%,可发酵酒精度最高16.3%vol。通过测定不同温度对发酵指标的影响,最终确定酵母菌FM-sc-08的最适发酵温度为25 ℃;对比20 ℃和25 ℃发酵的黑莓果酒酒样中的香气成分,可知酵母菌FM-sc-08在20 ℃发酵黑莓酒产香气成分较多（35种）。     

高糖胁迫对酿酒酵母抗氧化活性及代谢的影响

本文以酿酒酵母(Saccharomy cescerevisiae)为模式生物,研究了高糖培养条件对酿酒酵母的生长、抗氧化酶活性及海藻糖、甘油代谢的影响。结果表明:当葡萄糖浓度达到40%时,酿酒酵母生长的对数期延长,对酿酒酵母的生长产生了抑制作用。酿酒酵母在培养4~10 h范围内四组酿酒酵母细胞(20 g/L葡萄糖组、40 g/L葡萄糖组、60 g/L葡萄糖组和80 g/L葡萄糖组)内海藻糖的积累随着胁迫时间的增加发生显著变化(P<0.05),海藻糖的积累量呈先升高后下降的趋势,在8 h时高糖组海藻糖积累量均达到一个最高点,胞内海藻糖的浓度最高达到0.0955 mg/mL。在不同葡萄糖浓度胁迫下,酵母细胞胞内外甘油的积累随着时间增加呈现出先上升后下降的趋势,但是胞内甘油的积累量在80 g/L葡萄糖浓度时达到最高,而胞外甘油的积累量在60 g/L葡萄糖浓度时达到最高。这些结果说明在高糖胁迫下甘油和海藻糖是酿酒酵母的主要相容性溶质。另外,高糖处理后,与对照组相比,高糖组酿酒酵母胞内超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性均显著升高(P<0.05),说明这些抗氧化物酶活性物质对维持有机体胞内正常渗透压起到关键作用。该研究结果将为今后研究酿酒酵母耐高糖渗透压方面提供理论依据。     

Interactions between Lactobacillus kefiranofaciens and Saccharomyces cerevisiae in mixed culture for kefiran production

Since a positive effect on the growth and kefiran production of Lactobacillus kefiranofaciens was observed in a mixed culture with Saccharomyces cerevisiae, the elucidation of the interactions between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, the microbial interaction of each strain was investigated. Apart from the positive effect of a reduction in the amount of lactic acid by S. cerevisiae, a positive effect of S. cerevisiae on the growth and kefiran production of L. kefiranofaciens in a mixed culture was observed. Various experiments were carried out to study this effect. In this study, the observed increase in capsular kefiran in a mixed culture with inactivated S. cerevisiae correlated well to that in an anaerobic mixed culture. Differences in capsular kefiran production were observed for different initial S. cerevisiae concentrations under anaerobic conditions. From these fermentation results, it was concluded that the physical contact with S. cerevisiae mainly enhanced the capsular kefiran production of L. kefiranofaciens in a mixed culture. Therefore, in an anaerobic mixed culture, this direct contact resulted in higher capsular kefiran production than that in pure culture.     

AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering

Using nine primer pairs, amplified fragment length polymorphism (AFLP) analysis was conducted to characterize industrial, laboratory and type strains of Saccharomyces sensu stricto. S. cerevisiae, S. bayanus, S. carlsbergensis and S. paradoxus had species-specific AFLP profiles, with some variations among the strains. Nineteen wine, ale, bakery, whisky and laboratory strains of S. cerevisiae were differentiated by two primer pairs, while out of 19 strains of sake yeast, two groups consisting of two and eight strains were not differentiated using nine primer pairs. A phenogram of 41 strains of S. cerevisiae, two strains of S. bayanus, the type strain of S. pastorianus, three strains of S. carlsbergensis, one hybrid strain of S. cerevisiae and S. bayanus and the type strain of S. paradoxus was obtained by the unweighted pair group method, using arithmetic averages (UPGMA) based on the percentage of shared AFLP fragments of each sample pair. This phenogram demonstrated clear separations of S. cerevisiae, S. bayanus, S. carlsbergensis and S. paradoxus. However, S. pastorianus ATCC 12752(T) showed the highest percentages of shared fragments with the strains of S. bayanus, and formed a cluster with them. Except for the type strain of S. pastorianus, the percentages of shared fragments showed a similar tendency with reported data of DNA relatedness. The cluster of S. cerevisiae separated into three subclusters: one consisting of sake and shochu strains and a whisky strain; another consisting of bakery, wine, ale and whisky strains; and a third consisting of laboratory strains.     

Behavior of Lactobacillus plantarum and Saccharomyces cerevisiae in fresh and thermally processed orange juice

Lactobacillus plantarum and Saccharomyces cerevisiae are acid-tolerant microorganisms that are able to spoil citrus juices before and after pasteurization. The growth of these microorganisms in orange juice with and without pasteurization was investigated. Two samples of orange juice were inoculated with ca. 10(5) CFU/ml of each microorganism. Others were inoculated with ca. 10(7) CFU/ml of each microorganism and then thermally treated. L. plantarum populations were reduced by 2.5 and <1 log10 CFU/ml at 60 degrees C for 40 s and at 55 degrees C for 40 s, respectively. For the same treatments, S. cerevisiae populations were reduced by >6 and 2 log10 CFU/ml, respectively. Samples of heated and nonheated juice were incubated at 15 degrees C for 20 days. Injured populations of L. plantarum decreased by ca. 2 log10 CFU/ml during the first 70 h of storage, but those of S. cerevisiae did not decrease. The length of the lag phase after pasteurization increased 6.2-fold for L. plantarum and 1.9-fold for S. cerevisiae, and generation times increased by 41 and 86%, respectively. The results of this study demonstrate the differences in the capabilities of intact and injured cells of spoilage microorganisms to spoil citrus juice and the different thermal resistance levels of cells. While L. plantarum was more resistant to heat treatment than S. cerevisiae was, growth recovery after pasteurization was faster for the latter microorganism.