产共轭亚油酸植物乳杆菌的筛选、鉴定与诱变

从传统泡菜中筛选得到1 株乳酸菌lp15 能够合成共轭亚油酸(CLA),经16S rRNA 全序列分析法和API 系统鉴定法鉴定为植物乳杆菌(Lactobacillus plantarum)。利用人工诱变方法,以lp15 为出发菌株,采用紫外线、硫酸二乙酯(DES)依次诱变处理,经进一步液体发酵复筛获得多株突变菌株,CLA合成能力较出发菌株提高了29.3%～52.2%。其中lp15-2-1 突变菌株为CLA 生成能力最高菌株,MRS 培养液中添加0.2mg/mL LA 培养48h,CLA 产量达30.13μg/mL。经气相色谱(GC)检测分析,产物中cis9, trans11- 共轭亚油酸(c9, t11-CLA)占76.5%,trans10, cis12-共轭亚油酸(t10,c12-CLA)占23.5%。     

内蒙古锡林郭勒盟地区传统奶油制品中产共轭亚油酸乳酸菌的分离筛选与鉴定

本实验从内蒙古锡林郭勒盟地区传统奶油制品中分离筛选共轭亚油酸（conjugated linoleic acid,CLA）高产乳酸菌。采用紫外分光光度法检测菌株发酵液中CLA含量,筛选出高产菌株,分析其菌落特征、细胞形态及生理生化特性,并结合16S rDNA全序列分析以及系统发育树,鉴定其种属。结果表明,从样品中共筛出20 株产CLA菌株,当亚油酸（linoleic acid,LA）添加量为0.06%,接种量为2%时,分离自镶黄旗酸油的11 株产CLA菌株中,菌株HS4产量最高,为23.586 μg/mL,转化率为7.86%,经鉴定其为干酪乳杆菌（Lactobacillus casei）;分离自正镶蓝旗稀奶油的9 株产CLA菌株中,菌株LX5产量最高,为20.508 μg/mL,转化率为6.84%,经鉴定其为副干酪乳杆菌（Lactobacillus paracasei）。     

产共轭亚油酸丙酸菌的筛选及转化条件初探

通过富集培养、梯度密度稀释和液体培养发酵,采用紫外分光光度法测定共轭亚油酸(CLA),从市售奶酪中筛选获得1株转化生成CLA能力相对较高的菌株P9:考察了菌体接种量、亚油酸(LA)的添加量、反应温度、反应体系pH值以及乳化剂种类等因素对菌株P9 CLA生成量的影响.通过对菌株P9的菌落和形态特征、产丙酸特性及生理生化特性分析,初步鉴定该菌株为费氏丙酸杆菌(Propionibacterium frudenreichii).研究结果显示,菌株P9在菌体接种量10%(v/v),LA添加量0.75mg/mL,pH值为6.8,反应温度30℃,Tween-80作为乳化剂的条件下,CLA生成量为63.371μg/mL,亚油酸转化率为8.45%,表现出较好的产共轭亚油酸能力.     

一株产共轭亚油酸乳酸菌的筛选和鉴定

从酸菜汁中分离到一株具有较高产共轭亚油酸(CLA)能力的乳酸菌菌株9#,在小麦胚芽培养基中发酵CLA产量达到64.28μg/mL.通过菌株菌落形态学特征、菌体形态学特征、菌株生化特征和细菌16S rDNA进行的同源性比对,利用MEGA4软件构建系统发育树,表明菌株9#属于干酪乳杆菌(Lactobacillus casei).     

牛乳中高产共轭亚油酸乳酸杆菌的研究

以云南7个地区的牛乳、水牛乳样品中分离的40株乳酸杆菌为基础,以亚油酸为发酵底物,利用紫外分光光度法对共轭亚油酸(conjugated linoleic acid,CLA)产生菌进行筛选;利用气相色谱法检测高产菌株中CLA的主要异构体及各组分所占比例。本研究得到5株CLA产生菌:M2、M9、M16、M25、M28,产量分别为:25.54、34.56、15.48、20.46、14.79μg/mL,其中菌株M9为CLA高产菌株;气相结果显示菌株M9中CLA的主要异构体为:C18∶2 9c,11t;与对照组相比,油酸、亚油酸和C18∶2 9c,11t的含量变化表明菌株产生C18∶2 9c,11t与油酸和亚油酸有关。本研究为产CLA乳酸菌的筛选和CLA的产生原因提供了科学依据。     

一株嗜酸乳杆菌产共轭亚油酸条件的优化

共轭亚油酸(conjugated linoleic acid,CLA)是具有抗癌、减肥等许多生理功能的天然脂肪酸.生物法合成共轭亚油酸无毒且异构体单一,但目前产量较低.为提高CLA产量,以嗜酸乳杆菌(Lactobacillus acidophilus)为出发菌株,优化其培养条件.结果表明:发酵液初始pH为6.5,红花油添加量为0.15%,接种量5%,发酵温度为37°C,发酵时间为60h时最有利于共轭亚油酸的合成,此时CLA的合成最为94.7μg/mL.     

南瓜籽油中高产CLA菌株的筛选鉴定及培养条件的优化

从泡菜中筛选到一株高产共轭亚油酸(Conjugated Linoleic Acid,CLA)的菌株.经生理生化鉴定及16S rDNA序列分析,鉴定为嗜酸乳杆菌,命名为L44.为了提高南瓜籽油中共轭亚油酸的产量,对嗜酸乳杆菌的培养条件进行优化,采用5因素进行单因素试验设计,然后通过二次旋转组合设计进行优化.确定最优组合为培养时间24h,培养温度37℃,pH值7,接种量10％,装液量150mL,同时所得CLA产量从95.1425μg/mL提高为107.0989μg/mL.     

产CLA嗜酸乳杆菌的筛选诱变与鉴定

从传统泡菜中选育高产共轭亚油酸(Conjugated Linoleic Acid,CLA)的菌株L28,利用人工诱变方法,以L28为出发菌株,采用紫外线、亚硝基胍(NTG)依次诱变及紫外、亚硝基胍复合诱变处理,经进一步液体发酵复筛获得多株突变菌株,CLA的最优产量为95.1425μg/mL.其中L44突变菌株为CLA生成能力最高菌株.经16S rDNA全序列分析法和生理生化试验确定菌株的种属,然后经过Gene Bank基因比对确定同源基因,最后经鉴定为嗜酸乳杆菌(Lactobacillus acidophilus).     

植物乳杆菌转化生成CLA的研究

从酸菜汁中分离出的一株植物乳杆菌LactobacillusplantarumLT2-6能将亚油酸转化成共轭亚油酸。该菌株在MRS培养基中经0.03%(w/v)的亚油酸诱导培养后,所获得的洗涤细胞具有很强的转化能力。在厌氧环境下利用L.plantarumLT2-6的洗涤细胞进行转化生成CLA的反应。结果表明,适宜反应条件为:温度30℃、pH7.0(0.1mol/L的磷酸盐缓冲系统)、细胞浓度20%(w/v)、游离亚油酸浓度1.5%(w/v)、反应时间64h,获得CLA最高产量为7.87mg/mL,产物为c9、t11/t9、c11-CLA。     

乳酸菌UV_3-9包埋固定化技术发酵产共轭亚油酸

采用包埋法将乳酸菌UV3-9固定化处理后,对该固定化乳酸菌小球进行了发酵生产共轭亚油酸(CLA)的研究。结果显示,固定化小球的最佳直径为2～3 mm,固定化小球在氯化钙和硼酸混合液中需浸泡过夜;固定后的小球发酵产CLA最佳条件为:发酵温度37℃,初始pH 6.5,发酵时间28 h,底物LA添加量为0.3%,CLA最大产量达到92.061μg/mL。该固定化乳酸菌小球已完成了7批次的共轭亚油酸发酵,其产量均在80.476μg/mL以上。而且固定化UV3-9的CLA发酵产量是游离UV3-9的1.28倍。     

Identification of Lactic Acid Bacterial Strains with High Conjugated Linoleic Acid-Producing Ability from Natural Sauerkraut Fermentations

ABSTRACT:  Natural sauerkraut fermentations contain a great number of lactic acid bacteria. The aim of this study was to identify lactic acid bacterial strains with high conjugated linoleic acid (CLA)-producing ability from natural sauerkraut fermentations. Fifteen CLA-producing lactic acid bacterial strains were isolated in the study. One of these strains, designated as NCUL005, showed the highest CLA-producing ability (0.623 mg/mL). The transformation efficiency of converting linoleic acid into CLA by NCUL005 was 26.67%. The CLA produced by NCUL005 comprised a mixture of 32.2% cis9, trans11-C18:2 isomer and 67.8% trans10, cis12-C18:2 isomer. NCUL005 was identified as Lactobacillus plantarum , based on its cell morphology, characteristics of lactic acid production, and analysis result from Biolog Microbial Identification System (BMIS).     

产共轭亚油酸乳酸菌的筛选及生产条件的研究

从分离到的乳酸菌中筛选到1株共轭亚油酸高产菌株,乳杆菌L1(Lactobacillus sp.),在MRS培养基上,乳杆菌L1产生共轭亚油酸的最适亚油酸的浓度是0.1%(v/v),最适培养时间是42h,参与共轭亚油酸形成的酶是胞内酶,并且可能是由多种酶共同作用的结果.建立了共轭亚油酸的紫外光谱扫描和高效液相色谱检测方法.     

生物合成共轭亚油酸菌种的筛选与鉴定

从传统泡菜和生牛乳中筛选出一株乳酸菌ZS2058能生物合成共轭亚油酸,经API系统鉴定为植物乳杆菌(Lactobacillusplantarum).该菌株在MRS培养基中将质量分数11.6%的亚油酸(1.024mg/mL)转化为共轭亚油酸,经气相色谱分析证实c9,t11 18∶2占75.9%,t10,c12 18∶2占24.1%.     

Production of free conjugated linoleic acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal origin

A gas chromatographic procedure was used for analysis of conjugated linoleic acid (CLA) isomers cis-9, trans-11-octadecadienoic; trans-10, cis-12 octadecadienoic; and trans-9, trans-11-octadecadienoic (c9t11, t10c12, t9t11) produced by lactobacilli. Four different cultures, two strains each of Lactobacillus acidophilus and Lactobacillus casei were tested for their ability to produce CLA from free linoleic acid in MRS broth supplemented with linoleic acid. Different concentrations of linoleic acid (0, 0.05, 0.1, 0.2 and 0.5 mg/ml) were added to MRS broth, inoculated with the lactobacilli, and incubated at 37 degrees C. Viable counts and amounts of individual isomers of CLA (c9t11, t10c12, t9t11) were measured at 0, 24, 48, and 72 h. All the cultures were able to produce free CLA in media supplemented with linoleic acid. Maximum production of CLA (80.14 to 131.63 microg/ml) was observed at 24 h of incubation in broth containing 0.02% of free linoleic acid. No significant (P > 0.05) increases in total CLA levels were observed after 24 h of incubation. The ability of the cultures to produce CLA in skim milk supplemented with 0.02% free linoleic acid also was studied. In this medium, the total amounts of free CLA after 24 h of incubation ranged from 54.31 to 116.53 microg/ml. The use of lactic acid bacteria able to form free CLA in cultured dairy products may have potential health or nutritional benefits. Free CLA in the products likely would be more readily available for absorption from the digestive tract than if it were incorporated into the cells of the starter culture.     

定向进化植物乳杆菌和痤疮丙酸杆菌高产共轭亚油酸

为获得高产共轭亚油酸（conjugated linoleic acid，CLA）的菌株，对亚油酸异构酶系植物乳杆菌的肌球蛋白交叉反应抗原（myosin-cross-reactive antigen，MCRA）基因mcra和痤疮丙酸杆菌的亚油酸异构酶基因lai-p进行克隆，克隆成功后连接到骨架质粒pCold-SUMO上构建两种含mcra基因和lai-p基因的大肠杆菌重组菌株，利用异丙基-β-D-硫代半乳糖苷低温诱导阳性重组菌蛋白表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳验证得到目的蛋白表达条带，说明两种基因表达成功。采用易错聚合酶链式反应定向进化mcra基因和lai-p基因，构建重组菌突变体库，利用流式细胞术分选获得吸光度提高和CLA产量提高的重组菌株，其中pCold-ycmcra-gfp重组菌243 株，吸光度最高的为155号菌1.050 6±0.000 4，提高了5.02 倍；pCold-yclai-p-gfp重组菌156 株，产量最高的为39号菌（11.62±0.003 6）μg/mL，提高了2.99 倍。本研究为后期深入了解突变菌株CLA产量提高的原因，并获得两种亚油酸异构酶的产酶机制奠定了一定基础。     

响应面法优化植物乳杆菌lp15-2-1产共轭亚油酸发酵条件

对植物乳杆菌(Lactobacillus plantarum)lp15-2-1 转化亚油酸生成共轭亚油酸(CLA)发酵工艺进行研究。通过单因素试验确定最佳接种量、培养温度、初始pH 值、培养时间、亚油酸添加时间、亚油酸质量浓度。采用响应面Box-Benhnken 试验设计,建立初始pH 值、培养温度和亚油酸质量浓度的二次多项式回归方程模型,所得植物乳杆菌发酵产共轭亚油酸的最佳参数为：温度30℃、亚油酸质量浓度0.21mg/mL、初始pH6.3,此条件下,CLA得率为44.77μg/mL,CLA 理论得率为45.326μg/mL,转化率为21.32%,与优化前转化率7.78% 相比,有了很大提高。     

生物转化共轭亚油酸乳杆菌的筛选

以选定的乳杆菌为对象,系统性地探究其生物转化共轭亚油酸(CLA)的规律,并筛选得到高产菌株。首先用酶标仪快速检测法,对5个种属76株乳杆菌生物转化CLA的能力进行了分析,然后利用GC-MS进一步分析高产菌株生物转化CLA产物异构体组成。结果显示:植物乳杆菌大部分可生物转化CLA,但菌株间转化能力存在较大差异;副干酪乳杆菌、干酪乳杆菌和卷曲乳杆菌普遍具备生物转化CLA的能力,但转化能力不高;唾液乳杆菌普遍不具备生物转化CLA的能力。筛选得到3株高产菌株,其中植物乳杆菌CCFM241 CLA的转化能力最高,CLA转化率为16.35%,转化产物存在3种CLA异构体,以t9,t11-CLA为主。     

Conjugated linoleic acid conversion by six Lactobacillus plantarum strains cultured in MRS broth supplemented with sunflower oil and soymilk

Six strains of Lactobacillus plantarum, isolated from traditional dairy products of minority nationalities, were evaluated for their ability to produce conjugated linoleic acid (CLA) from free linoleic acid in vitro. All the 6 strains were found to be capable of converting linoleic acid to CLA when using sunflower oil as substrate or during soymilk fermentation. The inhibitory effect of linoleic acid on the growth of the L. plantarum was also discussed. The production of CLA was increased with adding high concentration of substrate in sunflower oil and IMAU60042 produced the highest CLA both in sunflower oil and soymilk. The CLA was composted by 2 isomers: cis9, trans11-CLA and tran10, cis12-CLA, and cis9, tran s11-CLA covered the most part of the total CLA formed except for L. plantarum P8. The production of CLA was decreased during the storage of fermented soymilk. The CLA contents decreased significantly in the first week, also more quickly in 2 wk. Especially, tran10, cis12-CLA decreased more rapidly than cis9, tran11-CLA. No dramatic change was observed among other 8 fatty acids in soymilk. The proportion of unsaturated fatty acids varied after fermentation with different L. plantrum strains, but all decreased the during storage. The research on the ability of converting CLA of L. plantrum strains could be basis for the future research and development of fermented soymilk products. PRACTICAL APPLICATION: Desirable probiotic traits, such as acid and bile tolerance, aggregation activity, and antibacterial activity, have been proved for the 6 Lactobacillus plantarum strains. The 6 L. plantarum strains might be used in the fermentation of soymilk to produce multifunctional probiotic soymilk products, especially the rich CLA contents.