合成己酸乙酯脂肪酶产生菌的产酶条件优化及其催化生产香酯液研究

从生产现场分离得到l株具有产己酸乙酯脂肪酶能力的红曲霉(Monascus purpureus)I518,对其产脂肪酶条件进行了初步优化.结果表明,该菌株较理想的产酶发酵条件为:麸皮∶豆粕=2∶3、接种量20％、培养基初始pH值为7、培养温度为32℃、培养时间为5d,该条件下所产的粗酶制剂所催化的香酯液中己酸乙酯的含量达0.38 mg/mL,比未优化条件有明显增加.     

浓香型酒已酸乙酯合成的生物学研究——泸型酯化菌M-101分离特性及酯化条件的探讨

己酸乙酯为浓香型酒的主体香成份,作者在对泸酒的微生物与发酵研究工作中,首次分离到一株具有酯化合成己酸乙酯能力的有效菌株,定名泸型酯化菌M—101。本文对该菌的形态、培养特征进行了研究;并着重研讨了该菌对浓香型酒己酸乙酯合成的诸条件。     

漫谈己酸乙酯的酯化

以国内外大量翔实的例证和数据阐释了己酸乙酯的酯化过程及效果，认为除己酸菌外，酵母菌亦有酯化作用，也有学者认为能合成己酸乙酯的菌株多为霉菌。黄水＋麸曲＋酵母＋处理己酸的酯化效果最好。指出酯化过程是可逆的，对己酸乙酯的分解测定是今后的重要研究课题。     

浓香型白酒酿造相关酵母发酵糟醅产己酸乙酯的研究

在分装至三角瓶的入窖粮糟中分别接种分离自浓香型白酒酿造环境的122株酵母,并设立空白对照和窖内对照,发酵结束后检测糟醅中己酸乙酯的含量。结果显示,窖内对照糟醅中己酸乙酯含量显著高于空白对照;73株酵母可使糟醅中己酸乙酯含量高于空白对照,其最高的己酸乙酯含量分别为空白对照的58倍和窖内对照的3倍。对8株促进糟醅己酸乙酯生成能力显著(高于窖内对照)的菌株进行了生理生化鉴定和基于26S rDNA D1/D2区的系统发育分析,发现这些菌株分属于Debaryomyces hansenii(4株)、Issatchenkia orientalis(2株)、Zygosaccharomyces bailii(1株)、Trichosporon coremiiforme(1株)等4种。研究表明,浓香型白酒酿造环境中存在大量的能够促进糟醅产己酸乙酯的酵母。     

浓香型白酒酿造大曲及糟醅中功能芽孢杆菌的筛选

为从白酒酿造中分离筛选出有助于提高浓香型白酒风味成分的芽孢杆菌,本文从浓香型白酒酿造的大曲和糟醅中分离得到了55株芽孢杆菌,通过筛选得到了一株在发酵液中产生较多己酸乙酯和较少正丙醇的菌株,编号为YB-16,该菌株所产己酸乙酯含量符合国标高度优级酒的范围,所产正丙醇含量符合国标特级食用酒精的范围。通过对该菌株进行形态特征观察、生理生化特征分析、分子生物学鉴定和构建系统发育树,发现YB-16与Bacillus cereus strain CCM NBRC 15305聚为一个分支,亲缘关系最近,可将菌株YB-16鉴定为蜡质芽孢杆菌(Bacillus cereus)。若将筛选的该Bacillus cereus应用至浓香型白酒传统酿造工艺,将能够提升浓香型白酒的香味和品质。     

己酸、酒尾对低浓度乙醇体系中己酸乙酯的影响

向0.26g/L己酸乙酯乙醇溶液(15%)中加入己酸、酒尾,定期测定溶液中己酸乙酯含量变化情况。结果表明,己酸和酒尾均可使低浓度乙醇溶液中己酸乙酯含量上升。与酒尾相比,己酸对低浓度乙醇溶液中己酸乙酯含量影响更为显著,更加有益。贮存期内增强环境的酸性,可使低浓度乙醇条件下己酸乙酯含量提高。     

利用内源性插入序列IS1构建thyA缺陷型大肠杆菌

为获得由大肠杆菌内源性IS1序列插入胸腺嘧啶合成酶基因thyA而产生的胸腺嘧啶营养缺陷型的菌株,利用4%的葡萄糖和1.25μg/mL链霉素的培养基提高IS1的插入频率,以三甲氧苄胺嘧啶(TMP)法定向筛选thyA缺陷型菌株。通过PCR扩增与DNA测序分析突变基因的结构;测定thyA缺陷型菌株在含或不含胸腺嘧啶的基础培养基上的生长曲线以研究其生长特性;转化大肠杆菌-乳酸菌穿梭载体衍生的含外源thyA基因的互补质粒pMG36e-thyA,采用抗生素抗性基因与质粒上互补的thyA基因两种方法筛选转化子,检测突变菌株在基础培养基上的恢复生长情况。诱导增加IS1的转座频率后,以含3μg/mL的TMP的GSCTt筛选培养基得到thyA缺陷型菌株,将该菌株命名为D8。PCR扩增结果显示,该突变株的thyA基因比正常基因长约800bp,测序结果显示该基因内部插入了完整的IS1序列。D8菌株需依赖外源胸腺嘧啶才能在基础培养基上生长,当转化入pMG36e-thyA后,可利用质粒上的互补基因恢复在基础培养基上的生长性能。大肠杆菌内源性插入序列IS1可插入thyA内部使该基因沉默,为利用内源性插入序列构建缺陷型菌株提供了科学基础。     

己酸乙酯高产菌的诱变选育

以红曲霉５０３５为出发菌株，通过紫外线诱变处理，采用透明圈法初筛和制成殖曲进行酯化实验，选育到以己酸和乙醇为底物，高效合成己酸乙酯的己酸乙酯高产菌Ｍ５０３５－１３，具有生产应用价值。     

FAS2基因过表达对酿酒酵母风味酯生成能力的影响

研究了共表达脂肪酸合成酶复合体中α亚基的编码基因FAS2和β亚基的编码基因FAS1对酿酒酵母产风味酯的影响。前期实验室构建的过表达FAS1基因的菌株α5F-FAS1和α5O-FAS1可以提高酿酒酵母产中链脂肪酸乙酯的含量,本研究以这两株菌为出发菌株过表达FAS2基因,相比较α5F-FAS1菌株,己酸乙酯、辛酸乙酯、癸酸乙酯分别提升了154.31%,65.22%和23.53%;相比较α5O-FAS1菌株,己酸乙酯、辛酸乙酯、癸酸乙酯、十二酸乙酯分别提升了17.34%,30.71%,27.12%和28.13%。结果显示共表达FAS1和FAS2基因可以更进一步的提升酿酒酵母中链脂肪酸乙酯的产量。同时,本研究还发现,过表达FAS1可以提高乙酸乙酯的生成量。为了探究本现象产生的初步机理,通过RT-PCR测定了合成与利用乙酰辅酶A相关基因的表达量,实验结果表明,过表达FAS1导致的乙酸乙酯生成量上升的现象主要是由于过表达FAS1使得ATF1的表达量上升造成的。     

一株生产固态白酒丢糟菌体饲料的酵母菌筛选

从贵州某白酒企业生产过程中分离得到1株丢糟中繁殖能力强的菌株IS516.将该菌株接种到未灭菌的固态白酒丢糟中培养,结果表明,该菌株能充分利用固态白酒丢糟,5d便可繁殖至×1012cfu/mL,将固态白酒丢糟转化为高蛋白饲料和酵母菌剂,实现清洁生产和资源的循环利用.经26S rDNA分子克隆测定,该菌株为酿酒酵母属(Saccharomyces cerevisiae).     

pH吸附法纯化乳酸乳球菌素Lacticin LLC518

对乳酸乳球菌素Lacticin LLC518在不同pH条件下产生菌的吸附作用进行研究,结果表明,pH 6.0时吸附率达100%,pH 2.0时吸附率为0。利用该特性,可将产生菌细胞从发酵液中分离,制备Lacticin LLC518粗制品。通过与硫酸铵沉淀样品进行对照,表明pH吸附法具有良好的分离效果,Lacticin LLC518的回收率为20%,纯化倍数高达83.02。     

基于低拷贝质粒的高产紫色杆菌素谷氨酸棒杆菌的构建和发酵

该研究以公认安全（Generally Recognized as Safe,GRAS）的谷氨酸棒杆菌（Corynebacterium glutamicum）为宿主,构建高产紫色杆菌素的重组菌株。利用谷氨酸棒杆菌天然大质粒pTET3的复制与分配元件,构建了低拷贝质粒pOK12CG1,该质粒在谷氨酸棒杆菌中的拷贝数约为6拷贝/基因组,且与谷氨酸棒杆菌常用质粒pEC-XK99E和pXMJ19兼容。以低拷贝质粒pOK12CG1为骨架构建了携带紫色杆菌素合成操纵子（vioABCDE）的质粒pCGvio,并分别以谷氨酸棒杆菌标准株ATCC 13032和插入序列（Insertion Sequence,IS）元件删除株为宿主,构建了7株合成紫色杆菌素的重组菌株。通过初步筛选,发现基于低拷贝质粒的重组菌株ATCC 13032/pCGvio,其紫色杆菌素产量（508.24 mg/L）高于基于中高拷贝质粒的重组菌株ATCC 13032/pECvio（376.16 mg/L）,而基于低拷贝质粒的IS元件删除重组菌株ISDM023/pCGvio紫色杆菌素产量达到了610.13 mg/L。进一步采用正交实验设计对重组菌株ISDM023/pCGvio进行培养基体积比（VLB:VBHIS）、诱导时间和IPTG诱导剂浓度这3个因素的发酵条件优化。结果表明,在VLB:VBHIS 为1:2、诱导时间为18 h、IPTG浓度为0.75 mmol/L的优化条件下,紫色杆菌素的摇瓶发酵产量可达了1 007.47 mg/L。该研究成功构建了紫色杆菌素的谷氨酸棒状杆菌重组菌株ISDM023/pCGvio,为谷氨酸棒状杆菌高效合成紫色杆菌素奠定了实验基础,也为其它产物的高效合成提供参考。     

Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON

The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (¹³C₁₅)-deoxynivalenol ((¹³C₁₅)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (¹³C₁₅)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% ± 1.9% (external calibration) or 101% ± 2.4% (internal calibration) were reached, respectively, after sample clean-up.     

脂肪酶氨基酸及其二联体与最适温度的相关性分析

基于34条微生物脂肪酶序列,用多元逐步回归法分析了影响脂肪酶最适温度的氨基酸及其二联体.结果表明:与最适温度呈正相关性的氨基酸是Y,负相关性的有I、S、K;呈正相关性的氨基酸二联体有IR、KS、NY、SA、ST和YR,负相关性的有DK、DY、IS、KA、WS、YS和QI.通过对相关性氨基酸二联体在耐热和不耐热脂肪酶中空间位置的分析发现:与最适温度呈正相关性的氨基酸二联体多数位于α-螺旋区,且分布在酶蛋白表面;而呈负相关性的多数位于β-折叠区和无规则卷曲区,一般分布于酶蛋白内部,并常出现在多肽链的N或C端.     

Comparison of Mycobacterium tuberculosis genomes reveals frequent deletions in a 20 kb variable region in clinical isolates

The Mycobacterium tuberculosis complex is associated with a remarkably low level of structural gene polymorphism. As part of a search for alternative forms of genetic variation that may act as a source of biological diversity in M. tuberculosis, we have identified a region of the genome that is highly variable amongst a panel of unrelated clinical isolates. Fifteen of 24 isolates examined contained one or more copies of the M. tuberculosis-specific IS6110 insertion element within this 20 kb variable region. In nine of the isolates, including the laboratory-passaged strain H37Rv, genomic deletions were identified, resulting in loss of between two and 13 genes. In each case, deletions were associated with the presence of a copy of the IS6110 element. Absence of flanking tri- or tetra-nucleotide repeats identified homologous recombination between adjacent IS6110 elements as the most likely mechanism of the deletion events. IS6110 insertion into hot-spots within the genome of M. tuberculosis provides a mechanism for generation of genetic diversity involving a high frequency of insertions and deletions.     

Effect of traditional processes on phytate and mineral content of pearl millet

Six pearl millet genotypes were used in this study: IS 91333, IS 91666, and IS 89111 for dough fermentation and IS 880004, IS 91777 and YD-X3 genotypes for Damirga flour. Investigation showed that traditional fermentation for 14 h at 37 °C caused a decline in pH with time; a sharp drop was observed at the beginning, which gradually levelled off. Fermentation resulted in significant reduction of starch and phytic acid: 9.5–9.8% and 43–44%, respectively. Protein content was not affected. The Damirga process significantly elevated starch content (by 8–19%) but significantly reduced the protein and phytic acid contents (by 10.9–12.1 and 86–93%, respectively). Damirga flour was found to retain 25–84% of the major minerals (Ca, Mg, P, K and Na) and 52–65% of the minor minerals (Zn, Mn and Fe); losses occurred for all minerals except Cu.     

Insertion sequence elements as mediators of strain diversity in Lactobacillus helveticus

Insertion sequence (IS) elements were found to be associated with the truncation of predicted cellobiose transport, acetaldehyde dehydrogenase and diacetyl reductase genes in the genome of Lactobacillus helveticus DPC 4571. The conservation of the IS elements in these different genomic locations among L. helveticus cheese isolates was determined by amplification with gene-specific and IS element-specific primers. The presence of two of the IS elements was found to follow a genotypic profile of the strains generated by randomly amplified polymorphic DNA (RAPD)-PCR and strains that clustered by RAPD-PCR tended to have the IS element in the same position. However, the IS element that interrupted the cellobiose transport gene was found to be common to all strains tested. This conserved genotype suggests the insertion event occurred early in the evolution of L. helveticus as a separate species.     

Application of IS900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk

A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 10(8) cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosis by DNA sequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.     

Incongruence between the cps type 2 genotype and host-related phenotypes of an Enterococcus faecalis food isolate

Enterococcus faecalis is a nosocomial opportunistic pathogen, but is also found in fermented food products where it plays a fundamental role in the fermentation process. Previously, we have described the non-starter E. faecalis cheese isolate QA29b as harboring virulence genes and proven to be virulent in Galleria mellonella virulence model. In this study, we further characterized this food strain concerning traits relevant for the host-pathogen relationship. QA29b was found to belong to sequence type (ST) 72, a common ST among food isolates, and thus we consider it as a good representative of food E. faecalis strains. It demonstrated high ability to form biofilms, to adhere to epithelial cells and was readily eliminated by J774.A1 macrophage cells. Despite carrying the cps locus associated with the capsular polysaccharide CPS 2 type, cps genes were not expressed, likely due to an IS6770 inserted in the cpsC-cpsK promoter region. This work constitutes the first study of traits important for interaction, colonization and infection in the host performed on a good representative of E. faecalis food isolates. Reported results stress the need for a reliable serotyping assay of E. faecalis, as cps genotyping may not be reliable. Overall, QA29b characterization shows that despite its virulence potential in an insect model, this food strain is readily eliminated by mammalian macrophages. Thus, fine tuned approaches combining cellular and mammalian models are needed to address and elucidate the multifactorial aspect of virulence potential associated with food isolates.     

Impregnation and Infiltration Kinetics of Isotonic Solution in Whole Jalapeño Pepper Using a Vacuum Pulse

ABSTRACT: The kinetics incorporation of an isotonic solution (IS) into the whole jalapeño pepper, as a function of the vacuum pressure (p₁, vacuum application time (t₁), and relaxation time (t₂), is necessary to determining the conditions leading to the highest incorporation of IS. This study was aimed at determining the operation conditions to achieve maximum impregnation (M_in) of whole jalapeño pepper tissue, and a complete infiltration (M_in) of its inner void with an IS, using a vacuum pulse. Impregnation of whole jalapeño peppers was conducted using an IS (a_w= 0.993 ± 0.001), a vacuum pulse, and 5 levels for each process variable (t₁, t₂, p₁), according to a central composite design. The amounts of impregnated and infiltrated IS were measured by following changes in pepper weight. The p₁, t₂ had a significant effect (P < 0.01) on the rate of M_im and M_in (g IS/g pepper). It was found that the structure of whole jalapeño plays an important role in the deformation‐relaxation process, which also affects the impregnation and infiltration kinetics. A high level of p₁ (666 mbar) and t₂ (840 min) allowed to achieve the maximum values of M_im and M_in (0.07 and 0.29 g IS/g pepper, respectively). These results suggest that different driving force acts in promoting the M_im and M_in, during the t₁ and t₂. This information will be of great value in the analysis of the pickling process of whole jalapeño pepper with a hypertonic solution and a vacuum pulse.