植物乳杆菌ZS2058生物转化共轭亚油酸的反应动力学

摘要：【目的】对本实验室从泡菜中筛选到的植物乳杆菌ZS2058完整细胞生物转化共轭亚油酸的反应动力学进行研究。【方法】探讨底物浓度、细胞浓度、反应体系pH值等因素对生物转化共轭亚油酸反应速度的影响,并通过双倒数和Hanes-Woolf作图法拟合反应初始阶段的速度方程。【结果】生物转化共轭亚油酸时存在明显的底物抑制现象,当亚油酸浓度为0.4 mg/mL时产c9, t11-共轭亚油酸的反应速度达最大值15.99 μg/(mL?h);反应速度随细胞浓度增加而上升,当细胞浓度为5×1010 cfu/mL时反应速度达到最高;最适pH值和最适反应温度分别为6.5和40 ℃。利用双倒数和Hanes-Woolf作图法求得米氏常数和最大反应速度,在低底物浓度下,反应初始阶段的反应规律与经典的米氏方程相符,而在高底物浓度下,存在明显的底物抑制现象。【结论】通过对植物乳杆菌ZS2058完整细胞催化合成共轭亚油酸各因素的考察,在得到最佳反应条件的同时建立了不同底物浓度范围内的反应速度方程,这对于实现共轭亚油酸的生产和研究其生理功能具有十分重要的理论价值。     

一株产共轭亚油酸乳酸菌的鉴定及其特性

从酸菜汁中分离筛选到一株产共轭亚油酸(CLA)能力较高的乳酸菌。经鉴定,确定为植物乳杆菌Lactobacliius plantarum。微氧条件可提高CLA的产量,催化亚油酸(LA)生成CLA的酶受着LA的诱导。37℃对细胞生长和CLA生成最为有利。对数生长期为6～12h,18h后进入稳定期。在14～22h,CLA生成量快速增加,24h时达到最高值。该菌的培养物经萃取、甲酯化后,进行了气相色谱分离,生成的CLA产物为c9/t9,c11-CLA和t10,c12-CLA异构体的混合物。     

植物乳杆菌ZS2058的亚油酸异构酶基因在乳酸克鲁维酵母中的克隆表达

目的：将植物乳杆菌ZS2058（Lactobacillus plantarum ZS2058）的亚油酸异构酶基因在乳酸克鲁维酵母（Kluyveromyces lactis）中进行克隆表达。方法：根据NCBI中已报道亚油酸异构酶（linoleate isomerase,LAI）基因的序列特征,设计引物对筛得的植物乳杆菌ZS2058进行PCR扩增,得到亚油酸异构酶全基因序列,克隆至乳酸克鲁维酵母表达载体pKLAC1,电转化得重组菌pKLAC1-LAI /Kluyveromyces lactis GG799。结果：SDS-PAGE检测,重组菌进行分泌表达获得目的蛋白,大小约为67 kDa;气相色谱（Gas Chromatogram,GC）检测到共轭亚油酸（conjugated linoleic acids,CLA）典型峰。结论：植物乳杆菌ZS2058中的亚油酸异构酶基因在乳酸克鲁维酵母中得到分泌表达,重组酶转化效率约为26%。     

产共轭亚油酸菌株的筛选及其等离子体诱变

本实验从东北酸菜汁中筛选获得了一株产共轭亚油酸能力较好的菌株SC-05,特别是气相色谱分析结果表明,合成的CLA产物构型为单一构型,仅为c9, t11-18：2-CLA,经鉴定该菌为乳杆菌属Lactobacillus sp.。菌株SC-05经低温等离子体处理后,筛选出突变株A-08,其共轭亚油酸产量达到119.24μg/mL,比出发菌株增加了83.0%,且突变株A-08具有较好的遗传稳定性。     

共轭亚油酸生理功能及微生物合成调控研究进展

共轭亚油酸(conjugated linoleic acid,CLA)是一种新型功能性油脂,顺9,反11-十八碳二烯酸(c9,t11-CLA)和反10,顺12-十八碳二烯酸(t10,c12-CLA)由于具有比其他异构体更强的生理功能得到广泛关注和研究.微生物合成CLA具有安全性高、选择特异性强等特点,研究CLA产量提高...     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。     

一株产共轭亚油酸瘤胃细菌Streptococcus infantarius RB111的筛选与鉴定

从内蒙古鄂尔多斯山羊瘤胃中分离筛选到1株产共轭亚油酸的瘤胃细菌RB111,该菌株的cis9,trans11-CLA和trans10,cis12-CLA总产量为269.2 mg/L,其中cis9,trans11-CLA占52.64%,trans10,cis12-CLA占47.36%。对菌株RB111进行了形态学观察、生理生化鉴定、脂肪酸组成分析以及16S rRNA基因序列分析。16S rRNA基因序列分析结果表明该菌株与婴儿链球菌(Streptococcus infantarius)的模式菌株NCDO 599的序列相似性为99%,该菌株的形态特征及生理生化特性与文献报道的Streptococcus infantarius一致。脂肪酸组成分析结果显示,菌株RB111的细胞脂肪酸主要成分是C16:0、C18:1ω9c和C18:0,3种脂肪酸占脂肪酸总量的60.64%。综合以上结果,菌株RB111被鉴定为婴儿链球菌Streptococcus infantarius。     

植物乳杆菌P8产共轭亚油酸条件的优化

以植物乳杆菌P8菌株为研究对象,系统研究了发酵条件对共轭亚油酸生成的影响。分别研究了培养条件和培养基成分对共轭亚油酸产量的影响。通过单因素和正交试验表明:植物乳杆菌P8产共轭亚油酸的最佳条件为:培养时间为22 h、亚油酸(LA)添加量为0.9 mg/mL、接种量为1.5%、氮源采用2 g/L的胰蛋白胨,碳源采用3 g/L的葡萄糖。     

共轭亚油酸诱导人结肠癌细胞Caco-2凋亡的作用

共轭亚油酸（conjugated linoleic acid,CLA）有着多种异构体以及多种生理活性,如抗癌、减肥、抗动脉粥样硬化等。本研究主要对c9,t11-CLA和t9,t11-CLA两种异构体的混合物诱导人结肠癌细胞Caco-2凋亡的作用及可能机制进行了探讨。采用MTT方法、透射电镜观察、流式细胞术检测和RT—PCR方法,研究了CLA混合物抑制Caco-2细胞增殖,诱导Caco-2细胞凋亡的作用及可能机制。实验结果证明,c9,t11-CLA和t9,t11-CLA混合物能抑制Caco-2细胞的增殖,并可诱导Caco-2的凋亡。随着所用的CLA混合物浓度的增加以及作用时间的延长,细胞凋亡率增加。通过RT—PCR分析,Caco-2细胞中的caspase 3的mRNA的表达随着CLA混合物浓度的增加而上调。这两种CLA的混合物可抑制Caco-2细胞的增殖,诱导Caco-2的凋亡,而caspase 3的表达上调可能与细胞凋亡密切相关。     

基于转录组分析植物乳杆菌AR195在亚油酸胁迫下的生理响应

植物乳杆菌（Lactiplantibacillus plantarum）和共轭亚油酸（CLA,Conjugated linoleic acid）均具有良好的益生特性和保健功能。目前人们已经明确了植物乳杆菌CLA生物合成的途径，但仍然缺乏对底物亚油酸（Linoleic acid,LA）响应机制的研究。文章基于转录组学分析了LA对植物乳杆菌AR195生长和基因表达的影响，结果表明LA能够抑制AR195的生长，添加LA后AR195中157个基因上调，67个基因下调，差异表达基因在代谢、环境信息处理和细胞过程等KEGG通路中富集。LA毒性主要来自于生长抑制和氧化还原失衡，而AR195可通过维持氧化还原平衡、生物转化CLA、增强碳源摄取及代谢、全局转录调控等生理响应过程缓解LA毒性。文章首次提出植物乳杆菌AR195应对LA胁迫的分子机制，为植物乳杆菌对LA的应激响应及CLA合成的内在动因提供了新的见解。     

Bioconversion of linoleic acid into conjugated linoleic acid by immobilized Lactobacillus reuteri

Lactobacillus reuteri was immobilized on silica gel to evaluate the bioconversion of linoleic acid (LA) into conjugated linoleic acid (CLA), consisting of cis-9,trans-11 and trans-10,cis-12 isomers. The amount of cell to carrier, the reaction time, and the substrate concentration, pH, and temperature for CLA production were optimized at 10 mg of cells/(g of carrier), 1 h, 500 mg/L LA, 10.5, and 55 degrees C, respectively. In the presence of 1.0 mM Cu(2+), CLA production increased by 110%. Under the optimal conditions, the immobilized cells produced 175 mg/L CLA from 500 mg/L LA for 1 h with a productivity of 175 mg/(L.h) and accumulated 5.5 times more CLA than that obtained from bioconversion by free washed cells. The CLA-producing ability of reused cells was investigated over five reuse reactions and was maximal at pH 7.5, 25 degrees C, and 1.0 mM Cu(2+). The total amount of CLA by the combined five reuse reactions was 344 mg of CLA/L reaction volume. This was 8.6 times higher than the amount obtained from reuse reactions by free washed cells.     

Antiobesity effect of trans-10,cis-12-conjugated linoleic acid-producing Lactobacillus plantarum PL62 on diet-induced obese mice

AIMS: To observe the antiobesity activity of trans-10,cis-12-conjugated linoleic acid (CLA)-producing lactobacillus in mice. METHODS AND RESULTS: Lactobacillus plantarum PL62, which can grow in the presence of linoleic acid, was selected and studied. The culture supernatant of Lact. plantarum PL62 contained trans-10,cis-12-conjugated linoleic acid (6.4 microg ml(-1)), and the crude enzyme prepared from washed cells produced trans-10,cis-12 CLA (1395 microg mg(-1) protein). Lact. plantarum PL62 reduced the weights of epididymal, inguinal, mesenteric, and perirenal white adipose tissues and significantly reduced the blood levels of total glucose and body weights of mice (P<0.01). CONCLUSIONS: trans-10,cis-12-CLA-producing Lact. plantarum PL62 can exert the same antiobesity activity as trans-10,cis-12-CLA in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: trans-10,cis-12-CLA-producing Lactobacillus can be a replacement for CLA for obesity treatment via the continuous production of trans-10,cis-12-CLA. The results provide a novel opportunity to develop foods with antiobesity activity.     

Antioxidant activity of conjugated linoleic acid isomers, linoleic acid and its methyl ester determined by photoemission and DPPH techniques

The chemiluminescent response of conjugated linoleic acid isomers (CLAs), linoleic acid (LA) and methyl linoleate (LAME) against the prooxidant t-butyl hydroperoxide (tBHP) was analyzed. The c9, t11-CLA and t10, c12-CLA isomers showed significant photoemission at the highest concentration used, while photoemission was not detected at any concentration of LA and LAME analyzed. These results show that CLAs are more susceptible to peroxidation than LA and LAME. Likewise, the effect of CLA, LA and LAME on lipid peroxidation of triglycerides rich in C20:5 omega3 and C22:6 omega3 (Tg omega3-PUFAs) was investigated. For that, chemiluminescence produced by triglycerides in the presence of tBHP, previously incubated with different concentrations of CLAs, LA and LAME (from 1 to 200 mM) was registered for 60 min. Triglycerides in the presence of t-BHP produced a peak of light emission (3151+/-134 RLUs) 5 min after addition. CLAs produced significant inhibition on photoemission, t10, c12-CLA being more effective than the c9, t11-CLA isomer. LA and LAME did not have an effect on lipid peroxidation of Tg omega3-PUFAs. CLA isomers, LA and LAME were also investigated for free radical scavenging properties against the stable radical (DPPH()). Both CLA isomers reacted and quenched DPPH() at all tested levels (from 5 to 25 mM), while LA and LAME did not show radical quenching activity even at the highest concentration tested. These data indicate that CLAs would provide protection against free radicals, but LA and LAME cannot.     

Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

Ricinoleic acid and castor oil as substrates for conjugated linoleic acid production by washed cells of Lactobacillus plantarum

Ricinoleic acid (12-hydroxy-cis-9-octadecaenoic acid) was an effective substrate for conjugated linoleic acid (CLA) production by washed cells of Lactobacillus plantarum AKU 1009a. The CLA produced was a mixture of cis-9,trans-11- and trans-9,trans-11-octadecadienoic acids. Addition of alpha-linolenic acid to the culture medium increased the CLA productivity of the washed cells. In the presence of lipase, castor oil, in which the main fatty acid component is ricinoleic acid, also was a substrate for CLA.     

Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria

Digesta samples from the ovine rumen and pure ruminal bacteria were incubated with linoleic acid (LA) in deuterium oxide-containing buffer to investigate the mechanisms of the formation of conjugated linoleic acids (CLAs). Rumenic acid (RA; cis-9,trans-11-18:2), trans-9,trans-11-18:2, and trans-10,cis-12-18:2 were the major CLA intermediates formed from LA in ruminal digesta, with traces of trans-9,cis-11-18:2, cis-9,cis-11-18:2, and cis-10,cis-12-18:2. Mass spectrometry indicated an increase in the n+1 isotopomers of RA and other 9,11-CLA isomers, as a result of labeling at C-13, whereas 10,12 isomers contained minimal enrichment. In pure culture, Butyrivibrio fibrisolvens and Clostridium proteoclasticum produced mostly RA with minor amounts of other 9,11 isomers, all labeled at C-13. Increasing the deuterium enrichment in water led to an isotope effect, whereby (1)H was incorporated in preference to (2)H. In contrast, the type strain and a ruminal isolate of Propionibacterium acnes produced trans-10,cis-12-18:2 and other 10,12 isomers that were minimally labeled. Incubations with ruminal digesta provided no support for ricinoleic acid (12-OH,cis-9-18:1) as an intermediate of RA synthesis. We conclude that geometric isomers of 10,12-CLA are synthesized by a mechanism that differs from the synthesis of 9,11 isomers, the latter possibly initiated by hydrogen abstraction on C-11 catalyzed by a radical intermediate enzyme.     

Structural analysis of conjugated linoleic acid produced by Lactobacillus plantarum,and factors affecting isomer production

An isomer of the conjugated linoleic acid (CLA) produced from linoleic acid by Lactobacillus plantarum was identified as cis-9,trans-11-octadecadienoic acid by proton nuclear magnetic resonance spectroscopy. Together with earlier results, we concluded that the bacterium produces two CLA isomers, cis-9,trans-11- and trans-9,trans-11-octadecadienoic acid from linoleic acid. The addition of L-serine, glucose, AgNO3, or NaCl to the reaction mixture reduced production of the latter.     

Potent inhibitory effect of trans9, trans11 isomer of conjugated linoleic acid on the growth of human colon cancer cells

This study compared the growth inhibitory effects of pure conjugated linoleic acid (CLA) isomers [cis(c)9,c11-CLA, c9,trans(t)11-CLA, t9,t11-CLA, and t10,c12-CLA] on human colon cancer cell lines (Caco-2, HT-29 and DLD-1). When Caco-2 cells were incubated up to 72 h with 200 μM, each isomer, even in the presence of 10% fetal bovine serum (FBS), cell proliferation was inhibited by all CLA isomers in a time-dependent manner. The strongest inhibitory effect was shown by t9,t11-CLA, followed by t10,c12-CLA, c9,c11-CLA and c9,t11-CLA, respectively. The strongest effect of t9,t11-CLA was also observed in other colon cancer cell lines (HT-29 and DLD-1). The order of the inhibitory effect of CLA isomer was confirmed in the presence of 1% FBS. CLA isomers supplemented in the culture medium were readily incorporated into the cellular lipids of Caco-2 and changed their fatty acid composition. The CLA contents in cellular lipids were 26.2±2.7% for t9,t11-CLA, 35.9±0.3% for c9,t11-CLA and 46.3±0.8% for t10,c12-CLA, respectively. DNA fragmentation was clearly recognized in Caco-2 cells treated with t9,t11-CLA. This apoptotic effect of t9,t11-CLA was dose- and time-dependent. DNA fragmentation was also induced by 9c,11t-CLA and t10,c12-CLA. However, fragmentation levels with both isomers were much lower than that with t9,t11-CLA. t9t11-CLA treatment of Caco-2 cells decreased Bcl-2 levels in association with apoptosis, whereas Bax levels remained unchanged. These results suggest that decreased expression of Bcl-2 by t9t11-CLA might increase the sensitivity of cells to lipid peroxidation and to programmed cell death, apoptosis.     

Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species

Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers have attracted great interest because of their potential health benefits. Formation of CLA and CLNA takes place in the rumen during biohydrogenation. Several studies have indicated that certain types of intestinal bacteria, including bifidobacteria, are able to convert linoleic acid (LA) to CLA. The role of intestinal bacteria in the formation of CLNA isomers is largely unknown. In the present study, a screening of 36 different Bifidobacterium strains for their ability to produce CLA and CLNA from free LA and α-linolenic acid (LNA), respectively, was performed. The strains were grown in MRS broth, to which LA or LNA (0.5 mg ml⁻¹) were added after 7 h of bacterial growth. Cultures were further incubated at 37°C for 72 h. Six strains (four Bifidobacterium breve strains, a Bifidobacterium bifidum strain and a Bifidobacterium pseudolongum strain) were able to produce different CLA and CLNA isomers. Conversion percentages varied from 19.5% to 53.5% for CLA production and from 55.6% to 78.4% for CLNA production among these strains. The CLA isomers produced were further identified with Ag⁺-HPLC. LA was mainly converted to t9t11-CLA and c9t11-CLA. The main CLNA isomers were identified with GC-MS as c9t11c15-CLNA and t9t11c15-CLNA.