保加利亚乳杆菌诱导产亚油酸异构酶条件研究

亚油酸异构酶可由保加利亚乳杆菌经诱导产生,可以将亚油酸(LA)转化为共轭亚油酸(CLA)。本实验对诱导保加利亚乳杆菌产亚油酸异构酶的条件进行研究,利用紫外和气质联用仪(GC-MS)检测所生成的CLA。结果表明：在培养基中添加1.5‰(V/V) LA 时所产酶的共轭亚油酸转化率最高;温度为36℃,培养36h 为较适的培养条件;单独添加0.1%(m/V)的乳糖或0.1%(m/V)的氯化钠有利于诱导产酶;在培养基中直接添加LA 的效果优于培养3至12h 后再进行添加。诱导所产酶可将LA 转化为CLA,且含有9c,11t-CLA 异构体。     

植物乳杆菌ZS2058生物转化产物共轭亚油酸的分析方法的研究

研究了紫外分光光度法、GC、Ag~+-HPLC和GC-MS四种分析方法对植物乳杆菌ZS2058生物转化亚油酸(LA)产生的共轭亚油酸(CLA)检测时的异同点。结果表明,这4种分析方法在对CLA进行检测时各有特色,应用范围也有不同。紫外分光光度法检测成本最低,操作最快速,但检测结果为转化产物中各种CLA异构体的总和,而GC、Ag~+-HPLC和GC-MS能将产物中的各类CLA异构体分开,可对复杂的生物转化产物进行分析。其中,GC的最大优点在于可以检测到转化底物LA,Ag~+-HPLC可将转化产物中c9,t11-CLA和t8,c10- CLA很好的分离,而GC-MS可以将各种异构体与其它副产物明确区分开来。总之,在检测生物转化法产生的CLA时,根据不同的实验需求来选择不同的检测方法,并需将这几种方法灵活的结合起来应用。     

瘤胃细菌生物合成共轭亚油酸的累积特性

为了解瘤胃细菌生物合成共轭亚油酸(CLA)的特性,通过毛细管电泳(CE)分析,发现瘤胃细菌生物合成CLA的主要异构体有c9,t11-CLA、t10,c12-CLA和t9,t11-CLA 3种。在厌氧和有氧条件下,瘤胃细菌均能合成CLA,且氧气有利于CLA的累积,随反应时间的延长,CLA的量呈现先增加后减少的变化趋势。结果表明瘤胃细菌参与了反刍动物体内CLA异构体的生物合成与代谢,瘤胃细菌生物合成CLA异构体的特异性还有待更深入的研究。     

植物乳杆菌p-8转化亚油酸为共轭亚油酸的分析

研究植物乳杆菌（Lactobacillus plantarum）p-8的菌体、菌体破碎液和重组亚油酸异构酶系转化亚油酸（linoleic acid,LA）为共轭亚油酸（conjugated linoleic acid,CLA）的能力和机制。结果表明：L. plantarum p-8在含有LA的MRS上清液和菌体破碎液体外催化LA时,都可以低效产生cis9,trans11-CLA（c9,t11-CLA）、trans10,cis12-CLA（t10,c12-CLA）和trans9,trans11-CLA（t9,t11-CLA）,但菌体中只有很少的t10,c12-CLA。实时聚合酶链式反应结果表明,亚油酸异构酶系的表达水平较低可能是CLA产量较低的原因。独立表达的重组亚油酸异构酶系成员、黄素腺嘌呤二核苷酸（flavin denine dinucleotide,FAD）和烟酰胺腺嘌呤二核苷酸都存在才可完成LA转化为c9,t11-CLA、t10,c12-CLA和t9,t11-CLA,转化途径与L. plantarum AUK1009一致。L. plantarum p-8的亚油酸水合酶经同源建模后有3 个结构域,底物结合位点与FAD位点位于3 个结构域连接处的疏水空腔中,M76和Y180是2 个必需基团。     

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.     

Linoleic acid Isomerase Activity in Enzyme Extracts from Lactobacillus Acidophilus and Propionibacterium Freudenreichii ssp. Shermanii

Enzyme extracts from Lactobacillus acidophilus (CCRC14079) and Propionibacterium freudenreichii ssp. shermanii (CCRC11076) were reacted with linoleic acid at 50 °C for 10 min at pH 5, 6, 7, and 8, and the levels of conjugated linoleic acid (CLA) formation were determined by high performance liquid chromatography. CLA formations were observed in all the reactions catalyzed by the retentates using ultrafiltration membranes with 100 kDa nominal molecular weight cutoff, indicating the presence of the activity of linoleic acid isomerase with nominal molecular weight higher than 100 kDa in the retentates from two cultures. More CLA was formed at pH 5 of L. acidophilus treatment and t10c12, c11t13, and c9t11 CLA were 3 major CLA isomers produced. Results demonstrate a potential for CLA production through linoleic acid isomerase.     

乙醇胁迫提高植物乳杆菌p-8的共轭亚油酸(CLA)转化率和相关酶转录水平

该文研究了在MRS培养基中添加0.05 mg/mL LA（Linoleic acid,LA）和不同浓度的乙醇时植物乳杆菌p-8的CLA（Conjugated linoleic acid,CLA）转化率和CLA合成相关酶转录水平的差异情况。结果显示,发酵液中的三种CLA异构体转化率都是在添加0.50%乙醇时最高,其中转化cis9,trans11-CLA（t9,t11-CLA）异构体最高,为2.49%,比不添加乙醇增加2.37倍。添加不同浓度乙醇的发酵液中trans10,cis12-CLA（t10,c12-CLA）转化率都是最低的。菌体中产生的CLA非常少,但规律与发酵液的基本一致。添加0.50%乙醇菌体中t9,t11-CLA转化率最高,其转化率仅为0.05%,比不添加乙醇增加了5倍。当乙醇浓度高于0.50%时,各种不同CLA异构体的转化率却都减少。结果表明CLA是在胞液内产生后再被运转到胞外的,一定浓度范围内的乙醇胁迫通过提高CLA合成相关的酶基因转录水平,进而促进了CLA的转化,可见CLA合成相关酶基因转录水平是造成CLA转化率差异的主要原因。结果为阐明植物乳杆菌p-8产CLA的分子机制和寻找有效提高CLA生成的调控手段奠定了基础。     

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%.     

共轭亚油酸对小鼠肥胖的抑制作用

采用小鼠营养性肥胖模型法,以昆明小鼠为实验动物,设置基础饲料对照组、肥胖模型对照组、共轭亚油酸（conjugated linoleic acid,CLA）高、中、低剂量组,分别连续灌胃6 周,考察小鼠体质量、体内脂肪质量、血脂水平、肝脏脂肪酸合成酶（fatty acid synthase,FAS）含量及脏器的变化,研究CLA对小鼠肥胖的抑制作用。结果表明：CLA各剂量组小鼠的Lee’s指数、脂肪系数和血清甘油三酯（triglyceride,TG）、总胆固醇（totalcholesterol,TC）、低密度脂蛋白胆固醇（low density lipoprotein cholesterol,LDL-C）水平均显著或极显著低于肥胖模型对照组（P＜0.05或P＜0.01）,高密度脂蛋白胆固醇（high density lipoprotein cholesterol,HDL-C）水平均极显著高于肥胖模型对照组（P＜0.01）,各剂量的CLA对小鼠除肝脏以外的其他脏器无显著影响（P＞0.05）,高剂量（0.15 mL/10 g）CLA可使喂食营养饲料小鼠的各项肥胖指标均处于喂食基础饲料小鼠的水平,表明CLA能有效抑制小鼠肥胖,同时对小鼠生长无毒副作用;CLA可极显著降低小鼠肝脏FAS含量（P＜0.01）,降低脂肪酸的合成,从而抑制小鼠肥胖。     

瘤胃中干酪乳杆菌的亚油酸异构酶基因的克隆与表达

以从黄牛瘤胃中分离到的一株具有将亚油酸转化为c9,t11-共轭亚油酸(conjugated linoleic acid,CLA)的干酪乳杆菌(Lactobacillus casei)Fx为出发菌株,提取其基因组DNA,聚合酶链式反应(polymerase chain reaction,PCR)扩增得到1 700 bp大小的亚油酸异构酶(linoleic acid isomerase,LAI)基因片段,将该基因片段纯化后进行TA克隆,得到重组质粒p UCm-T-LAI,将重组质粒p UCm-T-LAI和表达质粒p ET-Dsb A同时进行双酶切,连接得到重组表达载体p ET-Dsb A-LAI,经PCR鉴定和酶切后,将重组表达载体转化到大肠杆菌BL21中,得到具有LAI活性的重组菌株,能将亚油酸转化为c9,t11-CLA,表明从Lactobacillus casei Fx成功克隆LAI,该研究将有助于深入了解不同瘤胃细菌特异性合成不同CLA异构体的LAI基因差异。     

Effectiveness of oils rich in linoleic and linolenic acids to enhance conjugated linoleic acid in milk from dairy cows

Forty Holstein dairy cows were used to determine the effectiveness of linoleic or linolenic-rich oils to enhance C_18:2cis-9, trans-11 conjugated linoleic acid (CLA) and C_18:1trans-11 (vaccenic acid; VA) in milk. The experimental design was a complete randomized design for 9 wk with measurements made during the last 6 wk. Cows were fed a basal diet containing 59% forage (control) or a basal diet supplemented with either 4% soybean oil (SO), 4% flaxseed oil (FO), or 2% soybean oil plus 2% flaxseed oil (SFO) on a dry matter basis. Total fatty acids in the diet were 3.27, 7.47, 7.61, and 7.50 g/100 g in control, SO, FO, and SFO diets, respectively. Feed intake, energy-corrected milk (ECM) yield, and ECM produced/kg of feed intake were similar among treatments. The proportions of VA were increased by 318, 105, and 206% in milk fat from cows in the SO, FO, and SFO groups compared with cows in the control group. Similar increases in C_18:2cis-9, trans-11 CLA were 273, 150, and 183% in SO, FO, and SFO treatments, respectively. Under similar feeding conditions, oils rich in linoleic acid (soybean oil) were more effective in enhancing VA and C_18:2cis-9, trans-11 CLA in milk fat than oils containing linolenic acid (flaxseed oil) in dairy cows fed high-forage diets (59% forage). The effects of mixing linoleic and linolenic acids (50:50) on enhancing VA and C_18:2cis-9, trans-11 CLA were additive, but not greater than when fed separately. Increasing the proportion of healthy fatty acids (VA and CLA) by feeding soybean or flaxseed oil would result in milk with higher nutritive and therapeutic value.     

Bacterial Production of Conjugated Linoleic and Linolenic Acid in Foods: A Technological Challenge

Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers are present in foods derived from ruminants as a result of the respective linoleic acid (LA) and α-linolenic acid (LNA) metabolism by ruminal microorganisms and in animals’ tissues. CLA and CLNA have isomer-specific, health-promoting properties, including anticarcinogenic, antiatherogenic, anti-inflammatory, and antidiabetic activity, as well as the ability to reduce body fat. Besides ruminal microorganisms, such as Butyrivibrio fibrisolvens, many food-grade bacteria, such as bifidobacteria, lactic acid bacteria (LAB), and propionibacteria, are able to convert LA and LNA to CLA and CLNA, respectively. Linoleate isomerase activity, responsible for this conversion, is strain-dependent and probably related to the ability of the producer strain to tolerate the toxic effects of LA and LNA. Since natural concentrations of CLA and CLNA in ruminal food products are relatively low to exert their health benefits, food-grade bacteria with linoleate isomerase activity could be used as starter or adjunct cultures to develop functional fermented dairy and meat products with increased levels of CLA and CLNA or included in fermented products as probiotic cultures. However, results obtained so far are below expectations due to technological bottlenecks. More research is needed to assess if bacterial production kinetics can be increased and can match food processing requirements.     

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).     

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。     

Influence of sodium glycocholate on production of conjugated linoleic acid by cells of Lactobacillus reuteri ATCC 55739

ABSTRACT:  The possible influence of a bile salt on production of conjugated linoleic acid (CLA) by Lactobacillus reuteri ATCC 55739 was evaluated. Cells of the lactobacilli grown in MRS broth with and without linoleic acid (LA, 0.2%) were harvested and washed. The washed cells were added to buffer containing 0.2% LA and incubated 18 h at 37 °C. The cells, which had been grown without LA, transformed LA into CLA (mainly c9t11-C18:2) better than did those cells grown with it. When sodium glycocholate (0.3%) was added to the washed cell suspensions, about the same level of CLA was formed as in its absence regardless of whether or not the cells had been grown in broth supplemented with free LA. Thus, glycocholate that occurs in humans did not influence production of CLA by resting cells of the lactobacilli.     

共轭亚油酸生物合成及其对肠道菌群影响的研究进展

共轭亚油酸(CLA)是一种人和动物自身无法合成但又不可缺少的脂肪酸,它具有抗癌、抗肥胖、抗动脉粥样硬化、抗糖尿病等潜在生理功能,被广泛用作食品营养补充剂。由于天然CLA来源较少、化学合成副产物较多,而利用乳酸菌生物合成的CLA结构单一且转化效率高,是一种有前景的合成方法。目前微生物将亚油酸(LA)转化为CLA的机制主要有2种,即以瘤胃微生物为主的生物氢化合成和乳酸菌为代表的多酶系合成。现有研究表明,CLA具有调节肠道菌群的作用,并指出其潜在生理功能可能与其对肠道微生态的影响密切相关。文章总结了CLA生物合成和影响因素,分析了CLA的生物合成机制以及其对肠道菌群的调节作用,为进一步筛选出高产CLA菌株和CLA的产业化应用提供理论依据。     

共轭亚油酸的营养分配作用及生物合成研究进展

共轭亚油酸 (CLA)最早是从反刍动物瘤胃中分离出来的一种不饱和脂肪酸。因动物试验及癌细胞培养结果显示其具有强烈的抗癌作用、营养分配作用、抗动脉粥样硬化作用及免疫功能而备受瞩目。它可使啮齿类动物、哺乳类动物及人体脂含量明显下降 ,体内的蛋白含量提高 ,而总的体重不变。对它的营养分配机理的研究国外已有不少报道。主要结论是CLA可抑制脂肪细胞的分化并促进脂肪细胞的凋亡 ,另外CLA也通过抑制脂酰CoA脱氢酶活性而抑制脂肪酸的生物合成。反刍动物合成CLA有 2条途径 :1是在瘤胃细菌的作用下 ,亚油酸 (C18∶2 )被异构化为CLA。 2是脂肪组织中在Delta 9脱氢酶的作用下将反 -1 1C18∶1脱氢生成CLA。丙酸细菌、乳酸细菌均有催化亚油酸生成CLA的能力。     

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.     

超临界法制备玉米共轭亚油酸的工艺研究

以玉米胚芽油为原料,采用超临界法制备玉米共轭亚油酸(CLA)。通过正交试验方法确定最佳工艺条件为温度80℃、油:碱为1:0.5、时间4h、油:溶剂为1:2、压力15MPa,此时异构化效果理想,经气相色谱检测CLA转化率97.65%,产品中c9,t11-CLA与t10,c12-CLA含量分别为27.72%和30.88%。     

超临界CO2状态下直接酯化法制备共轭亚油酸甘油酯

在超临界CO2状态下,采用脂肪酶催化共轭亚油酸(conjugated linoleic acid,CLA)与甘油反应制备共轭亚油酸甘油酯,分别应用单因素和正交试验考察分子筛添加量、酶用量、反应压力、温度和时间对CLA酯化率的影响。结果表明,最佳工艺条件为分子筛用量6%、酶用量4%、反应温度60℃、反应时间20h、反应压力11MPa,此条件下CLA的酯化率可达到90.98%。这种CLA甘油酯的脂肪酸组成中,9c,11t-CLA和10t,12c-CLA的含量分别为37.79%和41.66%。