Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake

Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

产丁二酸工程菌的构建及其厌氧发酵

为了考察过量表达苹果酸酶对于E.coli NZN111(ldhA::Kan pfl::Cam)厌氧发酵产丁二酸的影响, 将连接有苹果酸酶基因sfcA的表达载体pTrc99a-sfcA转化进NZN111中, 构建了重组NZN111(pTrc99a-sfcA)。0.5 mmol/L IPTG诱导8 h后, 测定的苹果酸酶比酶活为30.67 u/mg, 比受体菌提高了140倍。采用两阶段发酵模式, 结果表明: 过量表达的苹果酸酶在NZN111体内催化了从丙酮酸到苹果酸的逆向反应, 丁二酸是发酵过程中积累的主要有机酸, 且当加入0.7 mmol/L IPTG诱导, 初始葡萄糖糖浓度为18.5 g/L时, 选择对数生长期后期的菌种以10%的接种量转入厌氧发酵, 发酵结束时发酵液中丁二酸的浓度为12.84 g/L, 对葡萄糖的收率为69.43%, 乙酸为0.58 g/L, 二者浓度比为22:1, 没有检测到甲酸和乳酸。构建的菌种具有高产丁二酸和副产物极少的优点, 在同类菌种中处于先进水平。     

混合菌群发酵秸秆合成菌体蛋白及其动力学分析

混合菌群发酵秸秆可有效提高秸秆纤维的降解率及菌体蛋白的转化率,对拓广蛋白饲料来源、减少环境污染起到积极的作用。本研究以小麦秸秆为原料,在纤维素酶水解预处理的基础上,以米曲霉作为先导菌,进一步分解残留的粗纤维,为后期发酵提供充足的碳源。根据不同微生物的代谢特征和协同机理,试验确定了发酵阶段混合菌群的组成为:米曲霉、产朊假丝酵母和枯草芽胞杆菌;接种顺序为:先接种米曲霉,再接种产朊假丝酵母,最后接种枯草芽胞杆菌。正交试验表明,影响发酵主要因素的主次顺序为:秸秆与麸皮配比>接种比例>发酵时间>接种量>发酵温度;发酵的最适条件为:米曲霉的接种量2.5%,发酵12h后接入5%的产朊假丝酵母,继续发酵8h后接入2.5%的枯草芽胞杆菌,发酵温度为28℃,秸秆与麸皮的配比为4∶1,尿素添加量为1.2%;结合动力学分析,将混合菌群的发酵时间优化为35h,发酵产物中粗蛋白含量由原来的5.47%提高到25%左右。对最适发酵条件下的动力学过程进行了探讨,建立了以Logistic方程为基础的数学模型和动力学方程。本研究表明,混合菌群发酵秸秆提高了发酵产物中的粗蛋白含量。动力学分析对于了解发酵机理、掌握整个发酵过程中混合菌群生长的动态变化、优化发酵工艺具有重要的指导意义。     

Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis Strains Isolated from Fermented Soybean Foods in Southeast Asia

Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.     

发酵法生产S-腺苷蛋氨酸前体蛋氨酸补加策略

利用酿酒酵母菌株高密度发酵法生产S-腺苷蛋氨酸关键的影响因素之一是前体L-蛋氨酸的补加策略.本研究采用一支经过常规诱变处理的S-腺苷蛋氨酸优势积累菌株酿酒酵母SAM0801,通过5 L发酵罐高密度发酵实验研究,考察了6种补加策略,最终确定了L-蛋氨酸的加入时机为30h左右,当茵体干重达到100g/L时,补加量为每罐40gL-蛋氨酸,发酵58 h左右达到最高生物量干重168 g/L,产量14.48 g/L.     

Predicting vegetative inoculum performance to maximize phytase production in solid-state fermentation using response surface methodology

Microbial phytase is used to reduce the environmental loading of phosphorus from animal production facilities. The limiting factors in the use of this enzyme in animal feeds can be overcome by solid-state fermentation (SSF), which is a promising technology for commercial enzyme production with lower production costs. Inoculum quality and the influence of inoculum quality on phytase production are important factors which need in-depth investigation before scaling-up of high-yielding fermentation process. A full factorial experimental design for 240 h with sampling at every 24 h was used to determine the effects of the treatments, inoculum age (plate and liquid culture), media composition and the duration of SSF on the production of fungal biomass and phytase in SSF systems using Aspergillus niger. The optimal treatment combination for maximal phytase production was determined by statistically comparing all treatments at each sampling time. Both 7- and 14-day plate cultures and M1+ medium composition with 72-h-old liquid inoculum treatments resulted in optimal phytase production at 144 h of SSF, which was the shortest duration observed for maximal phytase production. This resulted in maximal phytase production with a mean of 884±121 U/g substrate, while the maximal phytase production observed at 216 h of SSF (mean phytase activity of 1008±121 U/g substrate), with the same treatment combinations, was not statistically significant from that at 144 h of SSF. Phytase production was strongly growth-associated with younger inocula. The significant treatment variables, age of liquid inoculum and the duration of SSF, were used to predict the system response for phytase production using response surface methodology. From the response surface model, the optimal response of the experiment was predicted and the reliability of the prediction was checked with the verification experiment. Journal of Industrial Microbiology & Biotechnology (2001) 26, 161–170. Received 06 June 2000/ Accepted in revised form 14 October 2000     

The structure and physical properties of glucuronic acid oligomers produced by a Gluconacetobacter hansenii strain using the waste from beer fermentation broth

The aim of this work was to study the chemical structure and physical properties of water-soluble oligosaccharides (WSOS) produced by Gluconacetobacter hansenii PJK using the waste from beer fermentation broth as a basal medium. The analysis of the hydrolyzed products and the spectroscopic studies of the native WSOS showed that it is a mixture of oligomers all having a single sugar -linked glucuronic acid as building blocks with an O-acetyl and O-methyl group, in the terminal unit of the non-reducing end. The thermal studies displayed a progressive degradation of WSOS without a major weight loss throughout a range of temperatures. The melting point and pyrolysis temperature were found to be 130.16 and 275.25 °C, respectively. The optimum concentration of WSOS for a maximum emulsifying ability was found to be 0.10% (w/v). The resulting emulsions, however, did not demonstrate a noteworthy stability.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.