Tetramethylpyrazine is an important component of liquor flavor, which endows liquor with certain health effects. In order to increase the production of tetramethylpyrazine by fermentation,the mutant strain E20-ΔbdhA was successfully constructed by knocking out the encoding gene（bdhA） of 2,3-butanediol dehydrogenase（BDH） of Bacillus subtilis E20 as the original strain by homologous recombination. The mutant strain and the original strain were inoculated into soybean fermentation medium to simulate the solid-state fermentation of soy sauce. The results showed that the content of tetramethylpyrazine in the fermentation of the mutant strain was100.45 mg/kg,which was 71.97% higher than that of the original strain,and the Maotai-flavor had no obvious change. The conditions for synthesis of TTMP in solid state were optimized by using mutant strain E20-ΔbdhA,It was determined that when the inoculum amount of seed bacteria was 5%, the fermentation medium was sorghum and wheat 1∶1 medium,and the addition amount of（NH4）2HPO4was 70 g/kg,the content of tetramethylpyazine in the fermentation material was the highest 380.61 mg/kg.

本研究以本实验室曾在酒曲、酒糟、窖泥等样品中分离的具有酱香味的13株菌株作为出发菌株,经脱脂奶粉平板、纤维蛋白双层平板筛选出纤溶酶活性最高的菌株GZHZV-8,结合形态学特征、生理生化特征及16S rDNA序列同源性分析,GZHZV-8菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).以GZHZV-8菌株总DNA为模板扩增出纤溶酶基因的编码区,与pMD18-T载体连接、转化至大肠杆菌(Escherichia coli)DH5α,分析表明纤溶酶基因开放阅读框包含1092 bp碱基,编码363个氨基酸.纤溶酶基因与表达载体pET-22b(+)连接转化至E.coli BL21中表达,表达菌株经IPTG诱导表达分泌活性纤溶酶,培养后测得发酵上清液、菌体细胞破碎上清液和菌体细胞破碎沉淀的纤溶酶活力分别为613.26 IU/mL、407.38 IU/mL、993.12 IU/mL.

应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)指纹图谱技术对比分析贵州4种豆豉粑中的微生物多样性及亲缘关系,确定优势菌群和共有的微生物类群.结果表明:4种贵州豆豉粑中的优势细菌菌群以芽孢杆菌(Bacillus)为主,共有的种群是枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus licheniformis);主要的真菌菌群包含酵母菌属(扣囊复膜孢酵母(Sacharomycopsis fibuligera)、班图酒香酵母(Brettanomyces custersianus)、库德里阿兹氏毕赤酵母(Pichia kudriavzevii)、粉状毕赤酵母(Pichia farinosa)和汉逊德巴利酵母(Debaryomyces hanseni))和霉菌属(曲霉菌(Aspergillus) AlbC-4和沙门柏干酪青霉(Penicillium carnemberti)),没有发现共有的真菌种群.

The exopolysaccharide-producing lactic acid bacteria SR2-2 was isolated from sour meat of the Dong Minority in Guizhou.The optimum exopolysaccharide-producing fermentation conditions and extraction process of exopolysaccharide were researched by single factor and orthogonal experiments.The results showed that optimum fermentation conditions were sucrose 2％,tryptone:peptone 2:1,fermentation temperature 31 ℃,fermentation time 24 h.Under the conditions,exopolysaccharide yield was (565.52±6.81) mg/L,which was 58.32％ higher than that of before optimization.The optimum extraction process of exopolysaccharide was trichloroacetic acid (TCA) final concentration 10％,absolute ethyl alcohol 24 ml,alcohol precipitation time 18 h.Under the conditions,polysaccharide extraction yield was (790.22± 13.00) mg/L,which was 53.31％ higher than that of before optimization.

54 soil samples were collected from Pogang nature reserve in Xingyi city,Qingshui River nature reserve in Xingren county and Qinglong Mountain nature reserve in Anlong county in Karst landform area of southwest of Guizhou province.The Bacillus-like species were isolated by utilizing plate separation method and then identified using morphological observation,Physiological and biochemical experiments and 16SrRNA gene sequence analysis.Results showed that 129 strains isolated from these soil samples belonged to 24 Bacillus-like species and 3 genus (Bacillus,Lysinibacillus and Brevibacillus).The dominant bacteria forming spore are Bacillus thuringiensis(20.16％),Bacillus cereus (14.73％),Bacillus wiedmannii (10.08％) of three reserves and Bacillus is dominant genera(93.80％).Further research in terms of diversity indicated that every index of Bacillus-like in Qingshui River was the highest(3.9377,2.4455,0.9031),but Pogang had the lowest level of what included Shannon-Wiener diversity index and Pielou evenness index (2.2160,0.8183),and the Margalef richness index about Qinglong Mountain was the lowest (3.3581).Pogang was not similar to Qingshui River and Qinglong Mountain(0.2432,0.1875),while Qingshui River and Qinglong Mountain were not either moderately concerning the Jaccard similarity index(0.2571).

Black tea as materials, Bacillus subtilis E20 as fermentation strain,produced Black Tea with Maotai-flavor. Its production process were as follows:2％ of the E20 suspension was inoculated into the fermentation medium,fermentation temperature program increased and cultivated for 6d. Then extracted 100 mL ethanol (0. 0113 mol/L) solution for 12 h and centrifuged 20 min by 4000 r/min, and the fermentation extract was obtained. Added 20％ of the fermentation extract to the black tea, and dryed it at 35℃ 3 days. The Maotai-flavor Black Tea not only has black tea fragrance,but also has a unique flavor of Maotai. Two flavor can organ-ic integration, make the overall flavor mellow. Maotai-flavor of black tea chemical composition compared with contrast, found that the total free amino acid content is a significant difference;tea polyphenols, caffeine, tea red pigment, theaflavins,and tea brown pigment were not significant difference. Through detection, the amino acid in the Maotai-flavor Black Tea was provided by fermentation extract.

对微生物学课程进行教学模式的改革与探索.通过丰富和更新教学内容,改进课堂教学方法和手段,改革实验教学模式和成绩考核方式,以提升学生的兴趣和自主学习能力.近几年的教学实践证明,该教学模式的改革显著提高了学生的科研实践能力和创新思维能力.

The genome difference of E20 strain (tester)with producing Maotai-flavor and 10075 strain (driver)without producing Maotai-flavor was compared by using suppression subtracted hybridization (SSH),and sequence and biological information of the obtained differential fragments were analyzed to preferably develop and utilize microorganism resources of Maotai-flavor wine and to explain the formation mechanism of Maotai-flavor.Results:77 differential fragments are obtained by library gene sequencing.65 metabolins with an accurate annotation are obtained and their average similarity is 98%. 26 of 65 metabolins are enzymes.On the one hand,the enzymes are related to genes of acetoin,alsS,alsD and relative metabolism,but on the other hand related to relevant reactants of Maillard reaction to a certain extent.

With fresh beef as materials,fermented by Bacillus,beef jerky was produced.The flavor,color and texture of products obviously improved.The paper determined the best formula and technological conditions to produce flavored beef by GZJSI-2 Bacillus.The technologcial conditions were as follows:after sterilizing,salt 3.0%,glucose 2.5%,soybean powder 2.5% were added,inoculum was 3.0%;fermentation temperature increased to 50℃,cultivating for 4 d;roasting temperature was 60℃,roasting time was 210 min.The volatile flavor of beef after fermented and roasted had special fragrance of beef jerky and unique soysauce-like aroma.

Lactic acid bacteria are gram-positive, non-spore, anaerobic, a class of bacteria using carbohy-drates to produce lactic acid, which are valuable resource of microorganisms, and are closely related to our health. This paper described the function of lactic acid bacteria on improving immune, antioxidant, decreasing cholesterol, antihypertensive, antibacterial, and improving the flavor functions, which were currently being used in food and medicines. Some applications were also described such as lactic acid bacteria, genetic engi-neering lactic acid, lactic acid producing enzyme, lactobacillus, lactobacillus starch entrapped and Se lactic acid, so as to provide a reference for the further development and utilization.

The correlation between spore generation and temperature,and correlations between amino nitrogen content and browning degree,laccase activity were analyzed by determining laccase activity of bacteria strains isolated and preserved by microbiological laboratory of Guizhou university to explore methyl catechol browning oxidized by laccase and browning path of L-tyrosine oxidation under ABTS condition for study the effect of laccase in bacillus on browning of fermented soybean.The results showed that:1)Spore content and browning degree of the simulation fermented soybean increase with temperature increment during the browning process of the simulation fermented soybean fermented by bacillus strains;2)Laccase activity of bacillus strains is up to maximum at 60℃,which is consistent with accumulation temperature of high temperature Daqu with Maotai-flavor;3 )Pearson's correlation coefficients with significant correlation between browning of the fermented soybean and laccase activity,amino nitrogen content of fermentation products are 0.617 and 0.766;4)Brownness substance can be produced from methyl catechol oxidized by laccase of bacillus strains,and brownness substance can be produced by oxidizing L-tyrosine by laccase of bacillus strains under ABTS condition also.In conclusion,browning caused by laccase of bacillus strains has the certain effect on browning of Daqu with Maotai-flavor during the fermentation process.

在摇瓶实验的基础上,对解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GZJSI-12-7产纤溶酶最佳条件进行19L发酵罐实验,结果表明:GZJSI-12-7在发酵罐中培养6h时开始产纤溶酶,78h后纤溶酶产量基本稳定,达到4 178.39 IU/mL,产酶高峰比摇瓶发酵提前12h左右.将发酵液经离心除菌、硫酸铵分级盐析、透析除盐、SephadexG-75凝胶过滤,得到纤溶酶的纯化倍数为9.84倍,酶活力回收率为42.52％,比活力为48 073.193 IU/mg.对纤溶酶的酶学性质研究表明,该酶的分子质量约为30.2 kD,最适温度和pH值分别为40℃、7.5;在40℃以下酶的稳定性良好,超过50℃后酶活力迅速降低,耐热性较差;金属离子Mg2+、Ca2+、Mn2+对纤溶酶活性均有较强的激活作用,而Cu2+和Fe3+对酶活性具有明显的抑制作用.

应用PCR-DGGE技术研究贵州侗族发酵肉中的细菌群落结构及其品种间的多样性差异.结果表明:酸肉中的优势菌群差异不明显,3个样品中共检测到21种菌,共4个属,分别是葡萄球菌属(Staphylococcus)、乳酸杆菌属(Lactobacillus)、魏斯氏菌属(Weissella)和四联球菌属(Tetraggenococcus).另还发现两种不可培养细菌uncultured 1和uncultured 2.

A mutant named GZJSI-12-7 was achieved from Bacillus amyloliquefaciens GZJSI-12 with combined mutation by ordinal treatment of UV and nitrosoguanidine(NTG).The plasmin activity of GZJS I-12-7 was 2 046.44 IU/mL,which was 1.51 times higher than that of GZJSI-12.Plackett-Burman and response surface analysis methods were applied to optimize conditions of fibrinolytic enzyme fermentation.The results indicated that fibrinolytic enzyme yield was mainly influenced by concentration of initial MgSO 4,NaH2PO4 and temperature.The obtained optimal fermentation conditions were glucose 30 g/L,peptone 10 g/L,Tween-80 5 g/L,CaCl 2 0.4 g/L,MgSO4 0.847 g/L,Na2HPO4 2 g/L,NaH2PO4 6.988 g/L,pH 9.0,inoculation volume of 4%,age of inoculum 21 h,temperature 31.3 ℃,liquid loading 50 mL per 250 mL and rotation speed of 150 r/min.Under these conditions,the fibrinolytic enzyme yield was 4 789.08 IU/mL.

To provide a theoretical reference for developing and utilizing plasmin,the mutant strains with high production of plasmin were screened taking Bacillus amyloliquefaciens GZJSⅠ-12 as the original strain,and the fermentation conditions were optimized.The results showed that a mutant named GZJSⅠ-12-1 was achieved by ordinal treatment of UV and 2.5% of diethyl sulfate.The plasmin activity of GZJSⅠ-12-1 was 1 593.591 IU/mL which was 2.22 times higher than GZJSⅠ-12.The optimized conditions of liquid fermentation were as follows: initial pH of 9.5 and 6% inoculum volume in 250 mL shake flaske containing 30 mL medium.Recover rate of activity was 6.30%,purify multiple was 7.90 and specific activity was 6 383.06 IU/mg.

The Paper studied the releasing potassium from two kind of insoluble potassium-bearing minerals by fermentting feldspathic mineral and glauoonite with silicate bacteria strains Z1,Z9,S5,and analyzed the potassium ion dynamic process among the components "liquidoid-bacteria-mineral" of mineral fermentation.Results revealed that all strains had ability of releasing potassium from insoluble potassium-bearing minerals,strain Z9,S5 got the optimal effect on feldspathic mineral and glauoonite's dissolution respectively,and all strains had no obvious specificity on both minerals.The potassium releasing rate of glauoonite fermentation was higher than feldspathic mineral fermentation.The diversity results of fermentation effect was determined by potassium ion concentration in "liquidoid" of fermentation medium.

Preliminary analysis of 28 samples collected in each production procedure of Maotai Liquor was carried out,and then 104 Bacillus sp.strains were screened out in different step during the preparation of high-temperature Daqu,and wheat starter was used for simulated temperature-rising fermentation to screen aroma-producing strains,and these 25 strains which could produce Maotai flavor were identified as Bacillus licheniformis,Bacillus subtilis,B.licheniformis and Bacillus velezensis,compared with other Bacillus sp.strains not producing Maotai flavor,all the 25 strains could produce diacetyl,acetoin,proteinase and amylase etc.Through the analysis of aroma-producing mechanism of Maotai liquor,we may conclude that those Bacillus sp.strains produce varieties of enzymes and metabolites such as protease and amylase etc.at the first step and then those metabolites further produce characteristic flavoring compositions of Maotai liquor such as pyrazine etc.through maillard reaction under high temperature and acidic circumstances.

The present research status of the diversity of liquor-making microbes in China was reviewed. The difference in liquor-making microflora structure and their growth and decline rules for liquor produced in different regions, of different liquor flavor types, and by different analytic technology was compared. And the existing problems at present were introduced, which could provide reference for further research on the relations between microbial metabolism mechanism and the formation of liquor body and liquor flavor.

Strains of Staphylococcus aureus were isolated and identified from raw milk by traditional taxonomic identification and polymerase chain reaction(PCR),and their toxin producing ability was detected by mini-VIDAS to study the S.aureus contamination in raw milk.The results showed that S.aureus was the main contamination bacteria,9 strains of them were positive which detected by mini-VIDAS,and the detection rate of toxigenic strains was 12.86% in the 70 strains of S.aureus isolated.The PCR designed targeting the primer of thermonuclease(nuc) gene of S.aureus had coincidence rate of 100% with traditional taxonomic identification,and it had higher specificity and sensitivity.