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。     

微生物源α-葡萄糖苷酶抑制剂506157的分离纯化及结构鉴定

采用HD-1大孔酚醛系弱酸性阳离子交换树脂柱层析、HZ806大孔吸附树脂柱层析、高效液相制备色谱等对506157菌株次级代谢产物的活性成分进行分离纯化,所得样品呈白色粉末状,体外活性约为药物拜唐苹的10倍.通过HPLC、紫外光谱、MS、NMR分析,确定该活性成分的结构是4',7-二羟基异黄酮,分子式为C15H10O4.     

海洋微生物来源的α-葡萄糖苷酶抑制剂的筛选及性质研究

建立了α-葡萄糖苷酶抑制剂的体外筛选模型。10株海洋微生物经摇床培养得到发酵液,经大孔树脂吸附、甲醇洗脱得到粗提物;以阿卡波糖为阳性对照,用比色法（PNPG法）对粗提物进行α-葡萄糖苷酶抑制活性测定,并对产生活性代谢产物的菌株做进一步研究。结果发现：有6株菌株的代谢产物均具有不同程度的α-葡萄糖苷酶抑制作用,其中菌株N-1的代谢产物对α-葡萄糖苷酶的抑制作用最强,每毫升N-1样品液中含有的活性代谢产物对α-葡萄糖苷酶的抑制作用相当于5mg的阿卡波糖对α-葡萄糖苷酶的抑制作用;该活性产物具有较强的酸碱耐受性及温度耐受性,在pH值1～14、50～100℃时稳定性较好;初步分析活性代谢产物的成分并非蛋白质、氨基酸、糖类及皂苷类物质。     

纸色谱法筛选知母中α-葡萄糖苷酶抑制剂

采用酶反应、纸色谱和比色相结合法筛选知母中α-葡萄糖苷酶抑制剂。实验条件：1）酶反应：底物浓度10 mg/mL、酶用量为100μL、反应时间8 h;2）纸色谱测定微量葡萄糖：点样量70μL、展开体系正丁醇－丙酮－水（2︰7︰1）、展开方向为径向;3）比色：色谱后苯胺-邻苯二甲酸盐显色,蒸馏水洗脱,比色波长为365 nm。实验结果表明知母水粗提物及其多糖、皂甙、黄酮组分均为α-萄糖苷酶抑制剂,而中草药知母确为优良的降糖植物资源。     

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。     

α-葡萄糖苷酶的研究进展

综述了α-葡萄糖苷酶的主要性质以及当前已知的该酶的提取、纯化及活性测定方法,以便对其今后的应用研究起到导向作用。     

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。     

槐花α-葡萄糖苷酶抑制活性研究

用体外α-葡萄糖苷酶抑制模型,对槐花乙酸乙酯、石油醚、正丁醇、丙酮水总提取物进行活性筛选,对提取物浓度与抑制活性关系进行了研究,并对强活性的3种浸膏进行了抑制动力学研究。槐花总提取物(IC50=50.56 mg.L-1)、乙酸乙酯部位(IC50=1.25 mg.L-1)、正丁醇部位(IC50=16.14 mg.L-1)。活性远高于对照Acarbose(IC50=1 081.27 mg.L-1);并全部属于非竞争性抑制类型(Ki值分别为4.53、652.5 mg.L-1和62.38mg.L-1);从乙酸乙酯部位得到活性成分槲皮素(IC50=8.86 mg.L-1)和山奈酚(IC50=73.69 mg.L-1)。结果表明,槐花乙酸乙酯部位可作为降血糖活性部位进行体内研究。该文报道的新颖性已为河南大学图书馆2009年6月24日出具的第CX200906242号《科技查新报告》所证实。     

金盏银盘提取物对α-葡萄糖苷酶的抑制作用研究

对金盏银盘醇提取物用石油醚、乙酸乙酯和正丁醇进行萃取,测定各部分的酶抑制活性,并初步对金盏银盘酶抑制的动力学进行研究。结果表明:乙酸乙酯部位具有较高的α-葡萄糖苷酶抑制活性,其抑制成分在35～55℃具有较高的稳定性,最佳作用浓度为45μg/mL,且抑制作用迅速。     

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.     

植物乳杆菌差异表达基因的筛选

目的筛选具有高血管紧张素转换酶(angiotensin-converting enzyme,ACE)抑制活性的植物乳杆菌的差异表达基因片段。方法利用抑制性消减杂交(suppression subtractive hybridization,SSH)技术筛选植物乳杆菌差异表达基因片段,构建双向消减文库,用DNAMAN软件比较差异序列,确定差异序列数目及其同源性,用NCBI BLAST软件分析单个差异序列。结果筛选出112个阳性克隆,最终通过对52个阳性菌株测序,获得10条不同序列的差异片段,与已知Lactobacillus plantarum基因组序列高度同源,分别与酶蛋白、膜蛋白及能量运输有关。结论构建了植物乳杆菌ACE抑制酶活消减文库,为进一步研究植物乳杆菌产ACE抑制肽通路机制奠定了理论基础。     

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.     

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。     

Lactobacillus plantarum LY-78芳香族氨基酸转氨酶基因的克隆及序列分析

目的克隆Lactobacillus plantarum LY-78芳香族氨基酸转氨酶基因arat,并对其基因序列进行生物信息学分析。方法利用PCR技术克隆得到芳香族氨基酸转氨酶基因arat的完整序列,测序后运用生物信息学软件预测arat基因对应蛋白的理化性质和结构特征。结果克隆得到了Lactobacillus plantarum LY-78的arat基因,分别命名为arat1和arat2。arat1基因全长为1 188 bp,编码395个氨基酸,蛋白相对分子质量为43 888.9,pI 5.23;arat2基因全长为1 173 bp,编码390个氨基酸,蛋白相对分子质量为43 042.6,pI 5.64;两者均为亲水、热稳定及非分泌型蛋白,具有高度保守性,均属于天冬氨酸转氨酶家族。结论通过基因克隆技术获得了2个arat基因完整序列,为今后Lactobacillus plantarum LY-78 arat基因改造及PLA合成提供了理论支持。     

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.     

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。