新疆传统发酵乳品中乳酸菌与 酵母菌的益生特性

目的观察新疆传统发酵乳品中分离的14种菌株的生长特点及产酸能力,筛选出具有较强耐胆盐能力,并能在人工胃肠液中存活的菌株。方法对10株乳酸菌和4株酵母菌进行生长曲线、pH、耐胆盐能力和耐人工胃肠液检测。结果 10株乳酸菌和4株酵母菌具有良好的生长曲线和产酸能力;马乳酒样乳杆菌具有较强的耐胆盐能力;希氏乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工胃液能力;乳酸乳球菌、哈尔滨乳杆菌、瑞士乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工肠液能力。结论 10株乳酸菌和4株酵母菌具有优良的益生特性,有望成为益生菌制剂的备用菌株。     

烟叶果胶质分解菌的选育

从烟叶叶面分离到产果胶酶真菌 18株 ,在此基础上 ,进行液体培养 ,测定酶活 ,得到一株酶活性较高的黄曲霉DPE - 0 0 5。对该菌株产酶培养基的碳、氮源进行正交法研究 ,正交实验的结果表明 ,影响该菌产酶活性的因素依次为A(麸皮 ) >B(乳糖 ) >C(果胶 ) >D(硫酸铵 ) ,其最佳组合为A2B3C3D1。最适产酶条件为 :麸皮 4 0 % ,果胶 0 3% ,乳糖 1 5 % ,(NH4 ) 2 SO4 0 5 % ,KH2 PO4 0 2 5 % ,MgSO4 ·7H2 O 0 0 5 % ,NaNO30 0 2 % ,FeSO4 ·7H2 O 0 0 0 1% ,起始pH 6 0 ,在 2 8℃摇床培养 7d产酶量达到最高。以玉溪B3F烟叶为材料 ,施加DPE - 0 0 5菌株所产酶液 ,在 5 0℃贮存 12h。经化学成分检测 ,结果表明 ,果胶质降低了 18 15 %。     

福建红曲醋中产酸菌株的分离及鉴定

添加含有高浓度乙醇的红曲酒至福建传统红曲醋醋母中富集产酸菌株,采用高浓度乙醇平板,依据溶解圈指标从福建传统红曲醋液体循环工艺样品中分离出7株产酸菌株。综合菌株形态、生理生化实验以及16S r DNA序列测定等信息,确定这7株菌株分类地位为变形菌门(Proteobacteria)α-变形菌纲(Alphaproteobacteria)红螺菌目(Rhodospirillales)醋酸菌科(Acetobacteraceae)葡糖酸醋杆菌属(Gluconacetobacter),其中菌株Y5052、Y5054、Y5072、Y5092鉴定为斯氏葡糖酸醋杆菌(Gluconacetobacter swingsii);菌株Y5032、Y5033、Y5071鉴定为欧洲葡糖酸醋杆菌(Gluconacetobacter europaeus)。测定这些菌株的产酸能力,菌株Y5052产酸量最低,7 d达15 g/L;菌株Y5054产酸能力最强,7 d产酸量达57.0 g/L。     

山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析

酮古龙酸杆菌Ketogulonigenium vulgare是维生素C二步混菌发酵过程中的产酸菌。山梨酮脱氢酶(L-sorbosone dehydrogenase,缩写为SNDH)作为维生素C直接前体2-酮基-L-古龙酸(2-KGA)合成的关键酶,其作用机制并不十分清楚。借助全基因组测序抽提2个山梨酮脱氢酶基因,分别位于基因组(缩写为sndhg)和质粒(缩写为sndhp)上。通过工程化改造技术在工业产酸菌中构建山梨酮脱氢酶功能模块,比较其对2-KGA产量的影响。研究发现sndhg过表达对菌株产酸影响不明显,sndhp过表达使菌株明显产生副产物。将sndhg和sndhp分别配合辅因子PQQ合成基因pqq A,分别构建sndhg-pqq A和sndhp-pqq A模块,得到的工程菌株产酸情况与之前的结果大致相同。将4株K.vulgare工程菌株分别与内生芽孢杆菌Bacillus endophyticus混合培养传代50 d后,分离菌株进行混菌发酵,其2-KGA的转化率分别提高了15.4%、179%、0.65%和125%。表明混菌适应性进化策略是一种增加功能模块与底盘细胞适配性,进而快速获得优良性状菌种的有效方法。     

热稳定碱性脂肪酶产生菌的筛选及发酵条件的优化

从渤海湾盐碱地被油污染的土样中分离筛选出6株产热稳定碱性脂肪酶菌株。其中菌株1-7产脂肪酶能力较强,其最高酶活为8.67U/mL。根据其16S rDNA序列分析和Biolog生理生化分析,初步鉴定为醋酸钙不动杆菌(Acinetobacter calcoaceticus)。初步酶学性质研究表明该菌所产脂肪酶具有较好的热稳定性,最适作用pH为9.0。摇瓶实验表明,该菌株最适产酶培养基为(g/L):玉米粉10,黄豆饼粉20,K_2HPO_4 1,NaNO_3 5,橄榄油10。最适产酶条件为:初始pH 8.0,培养温度37℃,培养时间29 h。接种量为2%,250 mL摇瓶装液量为30 mL。     

β-葡聚糖酶和木聚糖酶高产菌株的选育及在小麦加工中的应用

以Aspergillus nigerJ5为出发菌株,经Co60γ-射线诱变,筛选到一株β-葡聚糖酶和木聚糖酶活力都较出发菌株高的突变株A-25,其产β-葡聚糖酶和木聚糖酶的合适发酵条件为:大麦粉4%、玉米浆2.5%、NaNO30.4%、Na2HPO40.1%、MgSO4.7H2O0.03%、FeSO4.7H2O0.01%、CaCO30.5%、吐温-800.25%,初始pH6.7,300mL三角瓶的装液量为50mL,在此条件下培养84h,β-葡聚糖酶活力达到1203.9I U/mL,较出发菌株提高35.9%,木聚糖酶活力达到395.2I U/mL,较出发菌株提高27.8%。突变株粗酶液降解工业面粉非淀粉多糖的能力明显高于出发菌株。     

斜卧青霉A10产纤维素酶的酶学性质研究

以斜卧青霉（Penieillium decumbens）A10为出发菌株,经450Gy ^60Coγ-射线诱变处理,选育出一株具有较高纤维素酶活力且传代稳定的正突变株A50,发酵60h后,其CMC酶活和滤纸酶活分别为27．28IU／mL和1．98IU／mL,较出发菌株A10分别提高了33．2％和45．59％。对突变株A50的产酶组分进行研究,通过SDS—PAGE电泳分析,从蛋白质水平上证明突变株A50确实是A10在遗传物质上发生改变的菌株,其CMC酶活最适作用pH值为4．0,最适作用温度为60℃;而滤纸酶活的最适作用pH值为5．2,最适作用温度45℃,二者在一定范围内具有较高的稳定性。     

C．shehatae TZ8耐乙醇菌株的筛选及发酵性能研究

以C.shehataeTZ8为出发茵株,利用1％溶壁酶和1％蜗牛酶酶解1．5h,制备成C.shehataeTZ8原生质体,并对原生质体进行紫外诱变,以含不同浓度乙醇的木糖液体培养基培养进行初筛和复筛,获得一株遗传性能稳定、耐乙醇能力达5．5％（v／v）的蕾株C．shehataeTZ8-4,比初始菌株耐乙醇能力提高了2％。对突变株C．shehataeTZ8-4发酵性能的研究结果表明：C．shehataeTZ8-4发酵糖能力从80g/L（葡糖糖和木糖比为2：1）提高到120g／L,最大乙醇产量从27．41g／L提高到43．12g／L。     

一株油藏嗜热厌氧杆菌的分离、鉴定及代谢产物特征

[目的]了解油藏环境中细菌的生理生化特性及代谢产物.[方法]采用Hungate厌氧操作技术从胜利油田罗801区块油层采出水中分离到一株厌氧杆菌SC-2.采用生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株的系统发育地位,用气相色谱分析其代谢产物.[结果]菌株SC-2为严格厌氧的革兰氏阴性杆菌,菌体大小为0.38 um×1.7um-3.9um,单生、成对或成串生长,产端生芽孢.温度生长范围40℃-75℃(最适温度70℃);pH范围5.5-9.5(最适pH 6.5);NaCl浓度范围0%～5%(最适NaCl浓度0%).能够利用葡萄糖、麦芽糖、甘露糖、木糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、丙酸、H2、CO2及少量的乳酸.菌株SC-2的(G C)mol%含量为30.8%,与Thermoanaerobacter mathranii subsp.mathranii的16S rDNA序列相似性为99.85%.菌株利用葡萄糖产乙酸、乙醇的最佳初始pH为8.0;酵母粉能刺激生长并显著提高发酵葡萄糖的产酸、产醇率;培养基中添加4%(V/V)的乙醇能明显抑制菌体生长.[结论]菌株SC-2是从特殊生境(油层采出水)中分离到的一株嗜热、耐盐的厌氧菌,其发酵葡萄糖产生的代谢产物有利于改善油藏中的微环境.菌株SC-2与T.mathranii subsp.mathranii 11426T的最适pH和最大耐受NaCl浓度有所不同,且二者的(G C)mol%含量差异较大.     

激光诱变微生物的遗传和刺激效应机理及育种研究 Ⅰ.CO_2激光对酒精酵母菌的诱变效应

本文选择甘蔗糖蜜酒精工业性生产用酿酒酵母菌SaccharomycescerevisiaeAS2.1189作为出发菌株,利用CO2激光对其进行辐照处理．制定出该菌最佳致死量的基础上,从A、B……E等5个不同照射剂量中,共挑取辐照菌639株．经糖蜜发酵后,对产乙醇含量进行了气相色谱分析．结果筛选出乙醇含量高于出发菌株5％～11％范围的辐照菌有5株;而低于10％～33％之间的为9株．表明CO2激光对该酵母菌产乙醇含量的高低有刺激效应．亦提示了CO2激光对酵母菌乙醇脱氢酶系的激活有刺激作用．     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.     

Acetic acid tolerance in Drosophila is a prerequisite for ethanol tolerance

Acetic acid tolerance compared with ethanol tolerance of Drosophila simulans and six Drosophila melanogaster strains shows a curvilinear relation with apparent asymptotic hyperbolic profile. The upper limit of acetic acid tolerance is lower than that for ethanol. We compared strains which had pairwise identical alcohol dehydrogenase (ADH) coding regions but different genetic backgrounds. A positive regression existed for ethanol tolerance on ADH activity. Adh-null mutants with very low ethanol tolerances had appreciable acetic acid tolerances and as a consequence did not fit the curve. ADH-F and ADH-S strains selected for high ethanol tolerances had the ability to tolerate high ethanol concentrations even after selection had been relaxed for several years. These selected lines tolerated higher acetic acid concentrations than the non-selected original strains. We propose that intake of high concentrations of ethanol and oxidation into acetic acid induces esterification of ethanol and acetic acid into ethylacetate. This cannot take place after the intake of acetic acid only, which also gives a lower energy yield.     

Isolation and Identification of Acetic Acid Bacteria for Submerged Acetic Acid Fermentation at High Temperature

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.     

Ethanol tolerance and carbohydrate metabolism in lactobacilli

Summary This paper describes the ethanol tolerance and metabolism of 31 strains ofLactobacillus on glucose, xylose, lactose, cellobiose and starch. The purpose of this work was to determine the suitability of the 31 strains as potential host for the ethanol producing genes, pyruvate decarboxylase and aldehyde dehydrogenase, fromZymomonas mobilis. The 31 strains were screened for their ability to grow in 0 to 8% v/v ethanol on all five carbohydrates. Those strains that were able to grow to an OD of 1.0 in 8% ethanol were evaluated at ethanol concentrations up to 16%. v/v. The fermentative products from the five carbohydrates were analyzed to determine the ratios of lactic acid, ethanol, and acetic acid.Published as Paper No. 9786, Journal Series Nebraska Agricultural Experiment Station, Lincoln, NE 68583-0704.     

Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

Long-chain fatty acids and ethanol affect the properties of membranes inDrosophila melanogaster larvae

The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains ofDrosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 1.1.1.1) activity in all of the strains. Dietary ethanol also increased the incorporation of¹⁴C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of¹⁴C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids. We are grateful for the support of National Institutes of Health Grant AA06702 (B.W.G.) and National Science Foundation Grant CHE-891987 (R.G.K.).     

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

High ethanol tolerance of new isolates of Clostridium thermocellum strains SS21 and SS22

Clostridium thermocellum strains SS21 and SS22, producing high yields of ethanol, were tolerant to 4.0 and 5.0% (v/v) ethanol, respectively. This is the highest ethanol tolerance so far reported by wild type strains of C. thermocellum. In the presence of added ethanol, both the strains had extended period of growth arrest. On addition of ethanol at different culture ages increase in ethanol tolerance upto 7.0 and 8.0% (v/v) by strains SS21 and SS22, respectively was observed. The optimum growth temperature for strain SS21 decreased as the concentration of ethanol in the medium increased and remained constant for strain SS22. Both the strains were tolerant to various solvents and acetic acid indicating that high ethanol tolerance of the strains is due to the general solvent tolerance of the organisms.     

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.2 +/- 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH- deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH- deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH- deer mice were 2.0 +/- 0.1 and 2.3 +/- 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH- deer mice in addition to the dehydrogenase systems.