高耐受性醋酸菌的筛选及发酵特性研究

从工业醋醅中分离出5株耐乙醇、耐高温的产醋酸菌株,利用生理生化试验和16S rDNA同源序列分析,初步认定其为巴氏醋杆菌（Acetobacter pasteurianus）。通过对其耐乙醇、耐高温发酵特性的研究,发现菌株FY4可耐受体积分数为12%的乙醇和43 ℃高温,在30 ℃和体积分数10%乙醇条件下产酸量达到最高,为61.2 g/L。在10 L发酵罐试验中,菌株FY4的产酸量始终高于醋酸菌AS1.41,在37 ℃和体积分数10%乙醇条件下,菌株FY4产酸量达到42.2 g/L,而在此条件下工业菌株AS1.41几乎停止产酸。结果表明菌株FY4具有高耐受性,可产生大量的醋酸,在工业生产中具有潜在应用价值。     

耐受乙醇巴氏醋杆菌ZJ-25培养基的优化

以中国传统固态发酵醋醅中分离得到的1株耐受12%vol乙醇的巴氏醋杆菌（Acetobacter pasteurianus）ZJ-25为出发菌株,以产酸量为评价指标,采用单因素试验和正交试验设计对培养基配方进行了优化。结果表明,最佳培养基组合为葡萄糖3%、酵母粉3%、乙醇5.5%、乙酸0.12%。在此最佳培养基条件下,菌株ZJ-25的产酸量由常规培养基条件下的32.5 g/L提高至44.3 g/L。     

一株有氧条件下高产乙醇酵母的筛选、鉴定及其生物学特性研究

采用平板涂布法从老白干香型大曲中获得一株有氧条件下高产乙醇酵母菌株,命名为YF1914。通过菌落形态、细胞显微结构、生理生化特性和26S rDNA D1/D2区方法对其进行鉴定,借助液体培养方式考察其乙醇耐受性、葡萄糖耐受性、乙酸耐受性、生长温度和pH等生物学特性。结果表明,该菌株为酿酒酵母(Saccharomyces cerevisiae)属于乙醇高耐受性酵母,耐受乙醇最高体积分数为18%,同时还具有较高的NaCl和葡萄糖耐受性,最高耐受质量分数分别为15%和80%,在温度为20~50 ℃和pH1~10能够生长,具有宽广的温度和pH适应性。综上,该酵母这些优良特性有利于其在未来白酒酿造方式变化中发挥作用。     

小球藻Chlorella sp.的小分子有机酸/醇培养研究

该研究选择一株对水解酸化液耐受性强、高产油脂的优良藻株Chlorella sp.作为出发藻株,向BG11培养基中分别添加不同质量浓度小分子有机酸/醇（乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸和乙醇）进行小球藻纯培养试验。在单因素试验的基础上,混合乙酸、丙酸、丁酸和乙醇4种小分子有机物质进行响应面的试验,考察混合小分子有机酸对小球藻Chlorella sp.生长影响,研究表明,当乙酸、丙酸、丁酸和乙醇的质量浓度分别为1.02 g/L、0.42 g/L、0.40 g/L和0.18 g/L时,小球藻的单位体积细胞数含量最高,生长状态最好,能获得较高的生物量（1.18×108 CFU/mL）。     

乙酰辅酶A羧化酶对乳酸片球菌耐酸性能的影响

通过对来源于山西老陈醋固态酿造过程中的乳酸菌分离株的耐醋酸性能分析,筛选得到了醋酸耐受性较优的乳酸片球菌（Pediococcus acidilactici）AAF3-3,该菌株对其他种类的酸压力（乳酸和盐酸）也表现出一定的耐受性。利用定量实时聚合酶链反应（qRT-PCR）对该菌株中乙酰辅酶A羧化酶基因（acc）的转录水平进行分析发现,其在醋酸、乳酸和盐酸下的转录水平分别是无压力条件下的4.32、2.02和1.13倍,均出现了明显上调。进一步构建该基因的过表达菌株并进行耐酸性能分析。结果表明,与对照菌株相比,acc过表达菌株在3种酸压力下的生长性能和存活性能均有所提高,其中,3%（V/V）醋酸条件下培养24 h时过表达菌株的相对活菌数是对照菌株的24倍。这表明乙酰辅酶A羧化酶在乳酸片球菌AAF3-3耐受不同种类的酸压力中发挥着重要作用。     

Genome shuffling to improve fermentation properties of top-fermenting yeast by the improvement of stress tolerance

Fermentation properties of top-fermenting yeast are under the control of multiple genes difficult to manipulate directly by classical breeding, metabolic engineering, or other genetic methods with specific genes or pathways as target. Here, genome shuffling is introduced to improve fermentation performance (such as the viability of the yeast, flavor of beer, and the fermentation time) by improving wort and ethanol tolerance of top-fermenting yeast. The strategy was performed not based on polyethylene glycol (PEG)-mediated protoplast fusion but using yeast sexual and asexual reproduction by itself. The best performing strain W3-8 was selected on the selective plates after 3 rounds of genome shuffling. The fermentation time of W3-8 was not only markedly shortened, but also, most flavor compounds were distinctly improved. In particular, ethanol yield was increased by up to 67% after the 3^rd pitching compared with the control. Furthermore, W3-8 promoted desired amounts of esters and higher alcohols, in accordance with specific consumer preferences. Significant improvement in the fermentation traits of the top-fermenting yeast was achieved using genome shuffling.     

诺丽自然发酵汁中醋酸菌的分离鉴定及发酵特性

为分析诺丽自然发酵汁中醋酸菌的多样性,丰富诺丽中醋酸菌种属信息,采用传统分离培养和16S rRNA基因序列分析相结合的方法,对诺丽自然发酵汁中的醋酸菌进行分离鉴定和发酵特性研究。结果表明,从不同发酵阶段诺丽自然发酵汁的67 个分离菌株中鉴定出了24 株醋酸菌,分别为Acetobacter fabarum（9 株）、Acetobacter syzygii（7 株）、Acetobacter pasteurianus（2 株）、Acetobacter tropicalis（1 株）、Acetobacter lambici（1 株）和Gluconobacter japonicus（4 株）,其中A. syzygii和A. fabarum为诺丽自然发酵过程中的优势菌种。在发酵性能方面,A. tropicalis N21性能最优,产酸量可达28.92 g/L,在40 ℃高温中仍能良好生长且产酸量为14.85 g/L,乙醇体积分数为7%时,产酸量略有下降,但依然可达23.60 g/L,其耐高温和耐乙醇能力均高于其他菌株。     

GIS1基因对酿酒酵母耐受性的研究

该研究主要通过过表达GIS1基因来提高酿酒酵母的耐受性。在GIS1基因的N端加入强启动子PGK1p来实现GIS1基因的过表达,然后通过稀释点板实验来对比其对热激、乙醇、渗透压以及乙酸的耐受性,同时通过高温生长曲线和高温浓醪发酵测定其高温耐受性。结果表明,在相同的稀释倍数下,55 ℃热击4 min之后菌株AY12a-gis1的生长能力明显优于出发菌株,在含有5%（V/V）乙酸的平板上改造菌和出发菌耐受性相差不大,同时通过38 ℃浓醪发酵实验发现菌株AY12a-gis1的酒精度提高了3.79%,残糖略有下降,且发酵时间与亲本菌株相一致。因此通过过表达GIS1基因得到菌株AY12a-gis1,是对工业乙醇发酵有一定应用价值的优良耐逆性菌株。     

一株发酵乳杆菌(Lactobacillus fermentium)对柠檬酸的发酵特性研究

本文讨论了一株能高效转化利用高酸性水果(如青梅)中柠檬酸的发酵乳杆菌菌株的发酵特性。结果表明,该菌株能在以柠檬酸为主要碳源和能源的基础培养液中进行发酵生长,在转化利用柠檬酸的过程中仅仅产生少量的乙酸和乳酸,因此能将培养液中可滴定酸含量降低50%以上。菌株对乙醇具有较高的耐受性,当培养液中乙醇浓度达到9%(V/V)以上时,菌株的生长和柠檬酸代谢也就受到明显的抑制;菌株对亚硫酸盐非常敏感,培养液中仅仅添加0.5 m M的亚硫酸钠后,菌株的生长和柠檬酸代谢将会被完全抑制。培养液中蔗糖的添加能促进菌株的生长和柠檬酸代谢,但在柠檬酸存在的情况下,该菌株对蔗糖的转化利用较弱,因此,发酵期间培养液中糖的总含量并没有出现明显的下降。     

Genome shuffling of Zygosaccharomyces rouxii to accelerate and enhance the flavour formation of soy sauce

BACKGROUND: The purpose of this study was to achieve rapid improvement of the flavour of soy sauce by increasing the salt stress resistance of Zygosaccharomyces rouxii. Here, we describe genome shuffling to improve the salt tolerance of Z. rouxii while simultaneously enhancing and accelerating flavour formation of soy sauce. RESULTS: A mutant, S3‐2, with a stronger resistance to salt, was selected after three rounds of genome shuffling. S3‐2 not only grew well in peptone/yeast extract/dextrose medium containing a high salt content with wide range of pH, but also exhibited stronger stress resistance to potassium chloride and lithium chloride. In high‐salt liquid fermentation, S3‐2 obviously accelerated flavour formation of soy sauce, thus decreasing the total time required for development of the aroma. In addition, S3‐2 gave high amino acid nitrogen and good flavour. In particular, the ethyl acetate content was 2.38 times that in the control. S3‐2 distinctly improved the formation of 4‐hydroxy‐2 (or 5) ‐ethyl‐5 (or 2) ‐methyl‐3 (2H) ‐furanone by up to 75%. Another important flavour component, 4‐ethylguaiacol, was also detected. CONCLUSIONS: Genome shuffling was successfully used to achieve significant improvements in flavour formation. The selected strain improved the main flavour components and amino acid nitrogen, thereby enhancing the quality of soy sauce. Copyright © 2009 Society of Chemical Industry     

山西老陈醋醋醅中产酸菌的分离、鉴定及醇酸耐受分析

以山西老陈醋醋醅为研究对象,从中筛选得到耐醇耐酸的产酸菌。结果表明,从山西老陈醋醋醅中筛选得到7株产酸菌,经鉴定,菌株SAV-1、SAV-2、SAV-5和SAV-9为醋酸菌,菌株SAV-6、SAV-7和SAV-8为乳酸菌。筛选获得3株具有高产酸、高醇酸耐受性的优良菌株,分别为巴氏醋杆菌（Acetobacter pasteurianus）SAV-1、植物乳杆菌（Lactobacillus plantarum）SAV-7和SAV-8。其中菌株SAV-1摇瓶发酵96 h后产酸水平为3.06 g/100 mL,且能够耐受体积分数为13%的乙醇和体积分数为3%的乙酸;菌株SAV-7和SAV-8菌株摇瓶发酵96 h后产酸水平分别为2.05 g/100 mL、1.73 g/100 mL,且均能够耐受9%的乙醇和2%的乙酸,是潜在的食醋生产优良菌株。     

Fermentation properties of a wine yeast over-expressing the Saccharomyces cerevisiae glycerol 3-phosphate dehydrogenase gene (GPD2)

Wine yeasts efficiently convert sugar into ethanol. The possibility of diverting some of the sugar into compounds other than ethanol by using molecular genetic methods was tested. Over-expression of the yeast glycerol 3-phosphate dehydrogenase gene ( GPD2 ) in a laboratory strain of Saccharomyces cerevisiae led to an approximate two-fold increase in the extracellular glycerol concentration. In the medium fermented with the modified strain, acetic acid concentration also increased approximately two-fold when respiration was blocked. A strain deleted for the GPD2 gene had the opposite phenotype, producing lower amounts of glycerol and acetic acid, with the latter compound only reduced during non-respiratory growth. A commercial wine yeast over-expressing GPD2 produced 16.5 g/L glycerol in a wine fermentation, compared to 7.9 g/L obtained with the parent strain. As seen for the laboratory strain, acetic acid concentrations were also increased when using the genetically modified wine yeast. A panel of wine judges confirmed the increase in volatile acidity of these wines. The altered glycerol biosynthetic pathway sequestered carbon from glycolysis and reduced the production of ethanol by 6 g/L.     

一株高产乳酸菌株的鉴定及其发酵特性研究

采用MRS培养基从果醋醋醅中分离具有强产酸能力的乳酸菌,通过形态观察、生理生化试验及分子生物学技术对其进行鉴定,并研究其乙醇耐受能力、产酸能力和抗生素敏感性。结果表明,从果醋醋醅中分离得到一株产酸能力较高的菌株,编号为D2,并鉴定其为鼠李糖乳杆菌（Lactobacillus rhamnosus）,该菌株在体积分数4%乙醇条件下能够正常生长,在体积分数14%乙醇条件下能够存活,具有较强的乙醇耐受力;在MRS肉汤培养基中发酵48 h时,总酸含量达到20.03 g/L,其中乳酸含量增加最多,其次是苹果酸和琥珀酸;对四环素、氯霉素、红霉素和吉他霉素敏感,对链霉素、卡那霉素、青霉素、苯唑西林和复方新诺明具有耐药性。综上,该菌株具有较强的乙醇耐受能力和产乳酸能力,安全性高,有作为发酵菌剂改善果醋风味和口感的潜在应用价值。     

宋河大曲中醋酸菌的分离鉴定及产酸特性

对宋河大曲中的醋酸菌进行分离鉴定并研究耐酒精和产酸特性.共分离得到7株醋酸菌,经形态特征观察和生理生化试验,初步鉴定归2个属3个种,1株未知.2个属分别为葡糖杆菌属和醋杆菌属,3个种分别为氧化葡糖杆菌(Gluconobacter oxydans),巴氏醋杆菌(Acetobacter pasteurianus)和醋化醋杆菌(Acetobacter aceti).氧化葡糖杆菌和醋化醋杆菌是宋河大曲中的主要醋酸菌.醋杆菌在产酸过程中可耐受含量为5%vol～11%vol的酒精,氧化葡糖杆菌的产酸性能受含量为9%vol～11%vol酒精.醋杆菌和氧化葡糖杆菌产酸快,30℃培养20h后,可迅速产酸,44h后趋近稳定,最低pH值可达4.2以下.     

Improvement of soy-sauce flavour by genome shuffling in Candida versatilis to improve salt stress resistance

Genome shuffling of Candida versatilis was applied to improve the soy-sauce flavour by further increasing salt stress resistance. A mutant S3-5, with a stronger resistance to salt, was isolated after three rounds of genome shuffling. It is found that S3-5 improved stress tolerance to higher potassium chloride and lithium chloride. S3-5 not only grew well in the YPD (peptone, yeast extract and dextrose) medium containing high concentrations of sodium chloride with various pH values, but also exhibited improvement of growth ability in soy-sauce medium. The main aroma compounds in soy sauce were distinctly improved. Notably, S3-5 produced about 2.78 times as much ethanol as the control strain. Another important aroma compound, 4-hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3 (2H)-furanone (HEMF) was enhanced by up to 80%. Meanwhile, S3-5 accelerated flavour formation of soy sauce thus decreasing the total time required for the aroma development.     

巴氏醋杆菌Ap2012的生长及耐受性研究

考察巴氏醋杆菌Ap2012在不同碳源酵母膏平板的生长情况及其对乙醇、糖、酸、NaCl的耐受性。结果表明,在酵母膏平板上,Ap2012能利用葡萄糖、乙醇作为碳源生长并产酸,能较好地利用甘油、果糖、蔗糖、山梨糖,对甘露醇的利用较差。乙醇体积分数为12.4%时巴氏醋杆菌Ap2012的生长受明显抑制,达到15.8%时转酸能力受明显抑制。葡萄糖的质量浓度达到300 g/L,NaCl的质量浓度为15 g/L时,巴氏醋杆菌Ap2012的生长和转酸能力受到明显抑制。乙酸质量浓度达到42 g/L时,巴氏醋杆菌Ap2012的生长受到明显抑制。结果表明,巴氏醋杆菌Ap2012对高浓度糖及乙醇有较好的耐受性,对乙酸和NaCl的耐受性较差。     

高产酸本土非酿酒酵母菌株的筛选及发酵性能研究

以25株本土非酿酒酵母菌为研究对象,采用酵母浸出粉胨葡萄糖（YPD）10培养基及Triple M改良模拟汁初筛,并进行耐受性（乙醇、SO2、糖及pH）测定及葡萄汁发酵,筛选能够有效增加葡萄酒酸度的优良本土非酿酒酵母菌。结果表明,非酿酒酵母菌株LT1及HU4产酸性能较好,具有较好的乙醇、SO2、糖和pH耐受性,其中菌株LT1能耐受乙醇体积分数12%、糖400 g/L及pH 2.75,菌株HU4能耐受乙醇体积分数6%vol、糖250 g/L及pH 2.75。菌株LT1和HU4在葡萄汁中启酵时间较短,发酵旺盛期CO2质量损失速率均＞0.8 g/（L·h）,乳酸产量分别为0.93 g/L、1.14 g/L,乙酸产量分别为0.38 g/L、0.42 g/L,具备酿造增酸葡萄酒的潜力。     

A Note — Production of Vinegar from Whey

Cheese whey supplemented with lactose was employed to produce vinegar. Whey was first transformed into an alcoholic beverage via fermentation with the yeast Kluyveromyces fragilis, and then the alcoholic product was employed as a substrate for an acetic acid fermentation. The bacteria used for the acetic acid production process were isolated from a starter culture provided by a vinegar factory. The bacteria employed were classified as Acetobacter pasteurianus. The vinegar obtained had a concentration of acetic acid between 5 and 6% (v/v). Ethyl acetate and fusel alcohols (isobutanol, 2‐methyl‐1‐butanol and 3‐methyl‐1‐butanol) were detected in the final product. The process and the substrates employed satisfied FAO requirements that the product was acceptable for human consumption. The efficiency of bio‐transformation of ethanol into acetic acid was 84%.     

Characterization of acetic acid bacteria in "traditional balsamic vinegar"

This study evaluated the glucose tolerance of acetic acid bacteria strains isolated from Traditional Balsamic Vinegar. The results showed that the greatest hurdle to acetic acid bacteria growth is the high sugar concentration, since the majority of the isolated strains are inhibited by 25% of glucose. Sugar tolerance is an important technological trait because Traditional Balsamic Vinegar is made with concentrated cooked must. On the contrary, ethanol concentration of the cooked and fermented must is less significant for acetic acid bacteria growth. A tentative identification of the isolated strains was done by 16S-23S-5S rDNA PCR/RFLP technique and the isolated strains were clustered: 32 strains belong to Gluconacetobacter xylinus group, two strains to Acetobacter pasteurianus group and one to Acetobacter aceti.     

高耐受性醋酸菌的研究和应用进展

酿醋工业的核心微生物是醋酸菌,其中最常用的菌种属于醋酸杆菌（Acetobacter）。研究表明,具有高耐受性的醋酸菌菌株在高浓度乙醇和高温条件下的产酸量仍可达到较高水平,对高耐受性醋酸菌菌株的发现为现代酿造工业带来了新的发展,有利于对不同生长与代谢特征醋酸菌菌种的挖掘以及新型酿造工艺的开发。该文综述了目前已知的高耐受性醋酸菌的特性及其在酿造工业生产中的应用进展,对更多高耐受性醋酸菌菌株的筛选以及酿造工艺的改进都具有重要的现实意义。