植物乳杆菌中胆盐水解酶基因的原核表达及纯化

目的克隆植物乳杆菌中胆盐水解酶(BSH)基因,原核表达并纯化重组蛋白。方法利用PCR技术扩增植物乳杆菌BSH基因,克隆至表达载体pET-30a,转化大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行纯化及Western blot鉴定。结果重组表达质粒pET-30a-BSH经双酶切鉴定,可见961bp的目的基因条带。表达的重组蛋白相对分子质量约为40000(含6个His标签),主要以包涵体形式表达,表达量约占菌体总蛋白的43%,纯化后,纯度达90%以上,且可与植物乳杆菌多克隆抗血清发生特异性反应。结论已成功克隆了植物乳杆菌中BSH基因,并在大肠杆菌中获得高效表达。纯化的重组蛋白纯度较高,为高效降解胆固醇的基因工程菌株的研究奠定了基础。     

抑制性消减杂交（SSH）技术及其应用

抑制性消减杂交技术(SSH)是一种高效检测差异表达基因的方法.详细论述了抑制性消减杂交的基本原理及过程,并简要介绍了其在工业生产菌种改良中的应用.     

植物乳杆菌共轭亚油酸异构酶基因的克隆、序列分析及重组表达

利用MRS培养基培养植物乳杆菌AS1.555,收集菌体后提取总DNA,用PCR技术扩增其亚油酸异构酶基因,克隆到pQE30质粒载体上,并进行测序.测序结果表明,扩增DNA全长1710 bp,编码569个氨基酸,分子量为64.7 ku.经同源性比较,此核酸序列与罗伊氏乳杆菌、短双歧杆菌、疮疱丙酸杆菌亚油酸异构酶基因核苷酸序列差别较大,推测此基因的来源与上述菌种不同.     

复合诱变选育新型高产细菌素植物乳杆菌

目的从东北传统发酵肉制品中分离和再诱变选育广谱高产细菌素的植物乳酸菌,并对其进行鉴定。方法四轮复合诱变[微波诱变、亚硝基胍(nitroso-guanidin,NTG)诱变、常压室温等离子体(atmospheric room temperatureplasma,ARTP)诱变、紫外诱变突]具有降胆固醇能力的的植物乳杆菌M1株,获得还可产生广谱细菌素的植物乳杆菌。对其进行稳定性、微生物形态特征、生理生化特征及PCR鉴定。结果原始菌株经四轮复合诱变后获得1株高产细菌素菌株,该菌株兼具遗传稳定性、优良发酵特性和抑菌功效。鉴定该菌株为植物乳杆菌,命名为M1-UVs300,其分泌的细菌素命名为M1-UVs301;菌株保藏号为CGMCC No. 7972。结论通过四轮复合诱变获得的植物乳杆菌M1-UVs300抑菌谱较宽,且兼具降胆固醇和防腐的优良加工特性,工业化推广前景广泛。     

植物乳杆菌组氨酸脱羧酶基因的克隆及序列分析

目的克隆植物乳杆菌组氨酸脱羧酶(Histidine decarboxylase,HDC)基因,并进行序列分析。方法根据GenBank中登录的乳酸菌HDC基因序列设计引物,提取植物乳杆菌Uvs44基因组DNA进行PCR扩增及克隆,并与其他乳杆菌HDC基因的核苷酸序列进行同源性分析。结果克隆的植物乳杆菌HDC基因大小约为900bp,其核苷酸序列与Lactobacillus sakei同源性最高,为99.9%;与Lactobacillus buchneri的同源性最低,为89.1%。32～489bp为高变区序列,T和C发生的替换较多。结论为产组胺乳酸菌的检测提供了理论依据,也为构建HDC基因缺失工程菌奠定了基础。     

植物乳杆菌凝集素的纯化及其体外抗HIV活性

目的纯化植物乳杆菌(L.plantarum PCL)凝集素,并检测其体外抗HIV活性。方法采用Sephadex G-25凝胶脱盐层析和HITrap Q Sepharose XL离子交换层析纯化L.plantarum PCL凝集素;测定纯化产物的兔红细胞凝集素活性、纯度及荧光光谱;采用MTT法检测其对C8166淋巴细胞的毒性作用,以光镜检查其对HIV-1ⅢB诱导C8166细胞形成合胞体的抑制作用,并计算TI值。结果纯化的L.plantarum PCL凝集素经尿素-SDS-PAGE,可见相对分子质量约85000的单一条带;荧光光谱可见在380nm附近有一个发射强度较大的凝集素特征荧光发射峰;L.plantarum PCL凝集素对C8166细胞的IC50为2.239μg/ml,对HIV-1ⅢB诱导C8166细胞合胞体形成的EC50为0.079μg/ml,TI值为28.34。结论已成功纯化了L.plantarum PCL凝集素,并具有抗HIV活性。     

植物乳杆菌M1-UVs29体外降解胆固醇的作用

目的探讨植物乳杆菌M1-UVs29在不同条件下降解胆固醇的能力。方法将M1-UVs29干菌粉活化后,分别接种至MRS液体培养基、MRS-胆盐培养基、MRS-胆固醇培养基和MRS-胆盐-胆固醇培养基中培养,绘制M1-UVs29在不同培养基中的生长曲线;以胆固醇降解率为指标,考察M1-UVs29在不同条件下(胆固醇、胆盐添加量及菌种数量)降解胆固醇的能力。结果胆盐具有抑制菌体生长的作用,胆固醇具有缓解胆盐对菌体生长抑制的作用。随着培养基中胆固醇含量的逐渐增加,胆固醇降解率也随之升高,趋势相对稳定,培养基中胆盐含量为0.2%,胆固醇含量为400μg/ml时,胆固醇降解率可达34.76%;随着培养基中胆盐含量的逐渐增加,胆固醇降解率也增加,但不呈规律性变化,培养基中胆固醇含量为100μg/ml,胆盐含量为0.4%时,胆固醇降解率可达32.85%;培养基中胆盐含量大于0.5%,胆固醇降解率极剧下降;培养基中胆固醇含量为100μg/ml,胆盐含量为0.2%,菌种数量为2×108cfu/ml时,胆固醇降解率达最高,为37.30%。结论植物乳杆菌M1-UVs29具有稳定的降解胆固醇的能力,本实验为其进一步应用提供了参考。     

血管紧张素转换酶基因rs4340和rs4343多态性与心房颤动的相关性

目的探讨血管紧张素转换酶(Angiotensin converting enzyme,ACE)基因rs4340和rs4343多态性与心房颤动(简称房颤)的相关性。方法选择重庆地区4家三甲医院就诊的102例房颤患者及同期住院的无房颤病史患者100例,抽取患者静脉血,分别提取基因组DNA,采用单核苷酸多态性-限制性片段长度多态性(Single nucleotide polymorphism,restriction fragmentlength polymorphism,SNP-RFLP)法及基因测序检测ACE基因rs4340和rs4343的基因型。结果房颤组ACE基因rs4340多态性的基因型及等位基因分布与对照组相比,差异无统计学意义(P>0.05),房颤组rs4343的基因型和等位基因分布与对照组间差异有统计学意义(P<0.001或P=0.001)。与对照组相比,房颤组GG+AG基因型频率明显高于AA基因型频率(P<0.001)。II/AA基因型在房颤组中出现的频率明显少于对照组(P=0.001),而II/AG基因型在房颤组中出现的频率明显高于对照组(P=0.002)。房颤组中rs4340和rs4343各基因型左房前后径与右房横径差异均无统计学意义(P>0.05);而将两位点联合分析发现,携带II/AA基因型房颤患者的左房前后径和右房横径均明显小于其他基因型(P<0.001),同时,携带II/AG基因型房颤患者的左房前后径和右房横径均明显大于其他基因型(P<0.001)。结论 ACE基因rs4343多态性与房颤显著相关,II/AA基因型是房颤发生发展的保护因子,而II/AG基因型是预测房颤发生发展的危险因子。     

基因工程改造植物乳杆菌生产光学纯D-乳酸

为获得高产高光学纯D-乳酸生产菌,本研究从筛选得到的一株耐高温耐高糖的植物乳杆菌出发,利用基因工程技术,敲除其中与L-乳酸合成相关的基因,引入或增强D-乳酸脱氢酶活性,构建了一株基因工程菌Lp-DA。该菌株可45℃发酵,产乳酸近200 g/L,D-乳酸光学纯度达到99.9%,为光学纯D-乳酸工业生产提供了新的菌株选择。     

植物乳杆菌XL-02发酵产γ-氨基丁酸条件的优化

为提高γ-氨基丁酸(GABA)产量,对植物乳杆菌XL-02进行发酵条件的优化.首先,采用单因素方法对底物添加量、发酵温度、发酵时间、接种量和pH值等因素进行考察,在此基础上进行4因素3水平的响应面实验优化.结果表明:在L谷氨酸钠浓度为2.0 g/L,发酵温度30℃,接种量11％和pH值6.0的条件下,发酵60 h,γ-...     

植物乳杆菌来源的α-葡萄糖苷酶抑制剂的分离纯化

通过薄板层析、硅胶柱层析等方法对植物乳杆菌ST-III发酵豆浆中的α-葡萄糖苷酶抑制剂进行了分离纯化,通过质谱鉴定确定其分子量。结果表明,经分离纯化,得到两种α-葡萄糖苷酶抑制剂组分F1和F2,其分子量分别为271.1和255.1,对α-葡萄糖苷酶抑制作用的IC50分别为0.41 mg/m L和0.65 mg/m L。结果显示植物乳杆菌ST-III发酵豆浆是一种优良的α-葡萄糖苷酶抑制剂来源,具有潜在的抗高血糖作用。     

牛病毒性腹泻病毒感染MDBK细胞后差异表达基因分析

目的利用高通量测序技术(Illumina平台)对感染牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的MDBK细胞进行转录组学分析,并对筛选出的差异表达基因进行功能归类及相关分子机制研究。方法用BVDV感染MDBK细胞,分别在12、24、48和72 h收集细胞,提取总RNA进行转录组学分析。通过信号通路数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)及基因功能(Gene Ontology,GO)对差异基因进行功能的注释和富集分析。结果与未接毒的细胞相比,在0～12、13～24、25～48、49～72 h分别共有差异基因56、390、487、164个。感染病毒后12与24 h、24与48 h、48与72 h分别有9、55、270个相同差异基因;经KEGG和GO分析,在感染病毒24、48、72 h后,差异基因主要富集到P53、FoxO、NF-kB和PI3K-AKT信号通路等;筛选出感染后与免疫等相关的差异基因P21、FOXO1、GABARAPL1等,主要集中在细胞周期复制、凋亡和免疫等相关信号通路上。结论本研究通过对筛选出的差异表达基因初步归类分析,为后续BVDV感染MDBK细胞并在细胞内复制的相关分子机制研究奠定了基础。     

植物乳杆菌来源的α-葡萄糖苷酶抑制剂的分离纯化

通过薄板层析、硅胶柱层析等方法对植物乳杆菌ST-III发酵豆浆中的α-葡萄糖苷酶抑制剂进行了分离纯化,通过质谱鉴定确定其分子量。结果表明,经分离纯化,得到两种α-葡萄糖苷酶抑制剂组分F1和F2,其分子量分别为271.1和255.1,对α-葡萄糖苷酶抑制作用的IC50分别为0.41 mg/m L和0.65 mg/m L。结果显示植物乳杆菌ST-III发酵豆浆是一种优良的α-葡萄糖苷酶抑制剂来源,具有潜在的抗高血糖作用。     

Extraction of coconut oil with Lactobacillus plantarum 1041 IAM

Extraction of coconut oil with a pure culture of Lactobacillus plantarum 1041 IAM was investigated. Grated coconut meat and water at 30, 50, and 70°C were mixed in various ratios (1:1, 1:2, and 1:3) and allowed to settle for 2–6 h. The most efficient coconut cream separation was obtained at the 1:1 ratio of grated coconut meat to water at 70°C, followed by 6 h settling time. Fermentation was then conducted on coconut cream emulsion with the sample from 1:1 ratio, 70°C, and 6-h settling time. Oil yield from the fermentation process with 5% inoculum of L. plantarum 1041 IAM after 10 h at 40°C was 95.06% Quality characteristics of the extracted oil were as follows: moisture content, 0.04%; peroxide value, 5.8 meq oxygen/kg; anisidine value, 2.10; free fatty acid, 2.45%; iodine value, 4.9; and color, 0.6 (Y + 5R). Extraction of coconut oil from coconut meat with L. plantarum 1041 IAM was significantly improved in both oil yield and quality over the traditional wet process.     

Yogurt Starter Obtained from Lactobacillus plantarum by Spray Drying

Probiotics have been known to mankind for many centuries and the quest for its health benefits is increasing in this century through focused research on various food products. Researchers have a growing interest in probiotics starter cultures for fermented milk products. Lactobacillus plantarum MA2 was isolated with good fermentation characteristics from Tibet kefir and identified by morphological, physiological, biochemical, and 16S rDNA sequence analysis. This work focused on the preparation of MA2 starter cultures by spray drying and compared the physiological activity of starter cultures to that of freeze-dried powder. The experimental results showed that the best spray-drying conditions were as follows: inlet air temperature of 148.9°C, raw material flow rate of 61.5 mL/h, and a protectant : cell ratio of 3:1. The viable cells of spray-dried starter cultures was 1.82 × 10⁹ colony-forming units (cfu)/g. The in vivo experiments revealed that after the spray-drying process, starter cultures of MA2 had good milk curdling and cholesterol removal ability (45.5%), which was very close to the fresh bacteria broth (46.8%) and freeze-dried starter cultures (46.1%). Furthermore, the spray-dried starter cultures had good stability during storage at 4°C. These results showed that the modified spray-drying process achieved the intended purpose of efficient preparation of the yogurt starter cultures.     

Spray Drying of Lactobacillus plantarum WCFS1 Guided by Predictive Modeling

Shelf life of probiotic microorganisms can be retained by drying. Spray drying is an economically interesting alternative to freeze drying with that respect. However, the viability can decrease due to the drying process and testing it is laborious and expensive. This research shows that the viability of Lactobacillus plantarum WCFS1 during pilot scale drying can be predicted with kinetics gathered at a single droplet level. Using this approach, it could be demonstrated that the viability of L. plantarum WCFS1 during spray drying is mainly determined by the combination of temperature and moisture content during the first 0.5 seconds after atomization. The combination of a high moisture content and a high temperature appeared most detrimental to the residual viability. Moreover, it was found to be important to take into account the particle size distribution during atomization when predicting viability, since this has a large effect on the moisture content during this first 0.5 seconds. Finally, it was observed that shelf life during storage was mainly determined by the moisture content of the powder. A lower moisture content resulted in a higher viability. Above a moisture content of 6%, shelf life stability rapidly decreased in the applied maltodextrin (DE = 16) matrix.     

Viability of Lactobacillus plantarum TISTR 2075 in Different Protectants during Spray Drying and Storage

Spray drying was applied for the production of Lactobacillus plantarum TISTR 2075 powder using maltodextrin as the carrier. A survival rate of 0.85% was achieved for this probiotic bacteria after spray drying. To improve the survival of this strain during the spray-drying process and storage, various protectants were added before drying. These included protein, trehalose, fibersol, ascorbic acid, isomalt, palatinose, and gum acacia. The results indicated that trehalose and protein (a combination of soy protein isolate and milk protein concentrate) significantly (P < 0.05) enhanced the viability during spray drying, with survival rates of 57.70 and 25.31%, respectively. Survival of the dried strain was also monitored over a period of 12 months’ storage at 4 and 25°C. Higher temperature induced lower viability of the strain in all protectants during this long-term storage. Accelerated storage tests using temperatures of 37, 45, 60, and 80°C were also applied to the spray-dried powders. A temperature-dependent prediction model was developed to determine the viability of the spray-dried L. plantarum TISTR 2075 in different protectants for long-term storage.