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。     

c9t11- 和t10c12- 共轭亚油酸抗癌和影响脂质代谢的异同

共轭亚油酸(CLA)是一组位置和构象异构体的总称,异构体c9t11-CLA 和t10c12-CLA 或二者的协同作用赋予了CLA 的许多生理功能,比如抗癌、降低体脂含量、预防糖尿病的发生、降低血脂抑制动脉粥样硬化等;异构体c9t11-CLA 和t10c12-CLA 在结构及来源上存在一定差别--由于双键位置的不同,t10c12-CLA 异构体比c9t11-CLA 异构体更容易氧化;而在生理功能上二者也有差异-- c9t11-CLA 异构体的主要作用是抗癌,而 t10c12-CLA 则是降低体脂、血脂等,大量单一异构体的体外实验研究表明二者在抗癌及脂肪代谢的调节上作用机制也不尽相同。     

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.     

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。     

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。     

Evidences and perspectives in the utilization of CLNA isomers as bioactive compounds in foods

Conjugated alpha linolenic acid (CLNA) isomers are promising lipids owing to their similarities with conjugated linoleic acid (CLA) but exerting their bioactivity at lower doses; some isomers also belong to omega 3 family. This review aims to summarize the state of the art about the utilization of CLNA as a functional ingredient. Indeed, in vitro and in vivo studies reported that CLNA exerted anticancer, anti-inflammatory, anti-obese, and antioxidant activities. However, CLNA has not been tested in humans. These compounds are naturally present in meat and milk fat from ruminants but the highest concentrations are found in vegetable oils. Their incorporation in foodstuffs is one of the most effective strategies to elaborate CLNA-enriched products together with the microbiological production. Lactobacilli, propionibacteria, and bifidobacteria strains have been assayed to produce CLNA isomers but at the current moment there are not high CLNA concentration products elaborated using these strains. Furthermore, it is known that CLNA isomers are highly prone to oxidation when compared with linoleic acid and CLA, but the possible effects of elaboration and storage on high CLNA productsare unknown.The utilization of CLNA as a functional compound still remains a challenge and requires more research to address all of its technological and bioactivity aspects.     

植物乳杆菌ZS2058生物转化共轭亚油酸反应条件的优化研究

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条件进行了系统研究。在1mL磷酸钾缓冲液反应体系中,通过单因素实验和响应面分析,确定最合适的反应条件为:亚油酸底物浓度为0.8mg/mL,细胞浓度为4×1010cfu/mL,反应温度为37℃,缓冲液pH为6.7。在此反应条件下,cis9,trans11-CLA的浓度为374.24μg/mL,转化率高达46.78%,这对于实现共轭亚油酸的高效生产和研究其生理功能具有重要的现实意义和理论价值。      

Effects of Linoleic Acid on Conjugated Linoleic Acid Production by Planktonic Rumen Bacteria from Grain-fed Cows

Ruminal conjugated linoleic acid (CLA) production from linoleic acid (LA) was characterized in vitro. Rumen bacteria from grain‐fed cows were more active in BH than those from hay‐fed cows. Particleassociated bacteria produced more hydrogenated products leaving less CLA than the planktonic bacteria (P < 0.05). CLA production by planktonic bacteria did not always correlate to LA given; longer incubations generally decreased CLA concentration and increased c9, t11/t10, c12 ratio, especially at higher LA concentrations. The preincubated cells to LA produced more CLA than the unexposed ones and the increase was more evident with c9, t11 CLA (P < 0.05). This study provides insight into how cattle diet and LA feedings affect ruminal CLA production.     

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.     

Occurrence of conjugated linoleic acid in longissimus dorsi muscle of water buffalo (Bubalus bubalis) and zebu-type cattle raised under savannah conditions

Lipid extracts from longissimus dorsi muscles of 64 water buffaloes and 68 zebu-type cattle were used to quantify the amount (mg/g of lipids) of total conjugated linoleic acid (CLA), CLA isomers c9, t11 and t10, c12 and linoleic acid (LA), according to species (buffaloes and cattle), age (slaughter groups at 7, 17, 19 or 24 months of age) and gender (bulls and steers). The effects of gender and age were significant (P<0.05) but marginal. Comparisons of lipid extracts from buffaloes vs. cattle showed that total CLA (1.83 vs. 1.47 mg/g), CLA c9, t11 (1.27 vs. 1.01 mg/g) and CLA t10, c12 (0.56 vs. 0.47 mg/g) isomers as well as the CLA/LA ratio (0.10 vs. 0.07) were higher (P<0.05) in buffalo lipids. Considering the sparingly low lipid concentrations (<2 g/100 g of fresh muscle) none of the meat species should be considered a significant source of CLA.     

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。     

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。     

L.helveticus L7生物转化的共轭亚油酸异构体结构分析

利用HPLC分离出L.helveticusL7生物转化的2种CLA异构体单体,通过紫外光谱、傅立叶变换红外光谱、气相色谱-质谱联用分析,确定了L.helveticusL7生物转化形成的两种CLA异构体是t9t11-CLA和c/t-911-CLA。     

Synthesis of the suspected trans-11,cis-13 conjugated linoleic acid isomer in ruminant mammary tissue by FADS3-catalyzed Δ13-desaturation of vaccenic acid

The octadecadienoic conjugated linoleic acid (CLA) isomer with trans-11 and cis-13 double bonds (trans-11,cis-13 CLA) has been described in ruminant milk. For now, this specific CLA is suspected to derive exclusively from ruminal biohydrogenation of dietary α-linolenic acid. However, in rodents, the fatty acid desaturase 3 (FADS3) gene was recently shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of vaccenic acid, producing a Δ11,13-CLA with all the structural characteristics of the trans-11,cis-13 isomer, although no commercial standard exists for complete conclusive identification. Because the FADS3 gene has already been reported in bovine animals, we hypothesized in the present study that an alternative direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue may therefore co-exist with α-linolenic acid biohydrogenation to explain the final ruminant milk trans-11,cis-13 CLA presence. Here, we first confirm that the FADS3 gene is present in ruminant mammal genomic sequence databases. Second, we demonstrate that the Δ11,13-CLA found in milk fat and the highly probable trans-11,cis-13 CLA isomer produced by rodent FADS3 possess exactly the same structural characteristics. Then, we show that bovine mammary MAC-T and BME-UV epithelial cells express both FADS3 and stearoyl-CoA desaturase 1 (SCD1) mRNA and are able to synthesize both the suspected trans-11,cis-13 CLA and cis-9,trans-11CLA (rumenic acid) isomers when incubated with vaccenic acid. Finally, the concomitant presence of the suspected trans-11,cis-13 CLA isomer with FADS3 mRNA was shown in goat mammary tissue, whereas both were conversely very low or even absent in goat liver. Therefore, this study provides several lines of evidence that, by analogy with rumenic acid, trans-11,cis-13 CLA may originate both from ruminal biohydrogenation and from direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue.     

Influence of slaughter season and muscle type on fatty acid composition, conjugated linoleic acid isomeric distribution and nutritional quality of intramuscular fat in Arouquesa-PDO veal

The effects of the slaughter season and muscle type on lipid and conjugated linoleic acid (CLA) contents, fatty acid composition and isomeric profile of CLA in Arouquesa veal, from calves reared according to the specifications of the protected designation of origin (PDO), were assessed. Arouquesa purebred calves (n = 31) were raised in a traditional production system, slaughtered in early autumn (October) or late spring (June), and the longissimus dorsi and semitendinosus muscles were sampled for analysis. Arouquesa-PDO veal only showed seasonal differences in the levels of some minor fatty acids (16:1c9, 17:1c9, 18:1t, 18:3n − 3, 20:0 and 22:4n − 6) and CLA isomers (t12,t14, t9,t11 and c11,t13). Furthermore, significant interactions between the slaughter season and muscle type were obtained for several fatty acids and CLA isomers, total lipids and CLA, and the PUFA/SFA ratio. In both seasons, veal-PDO depicts values of pasture-fed cattle. From a human nutritional perspective, veal-PDO in both slaughter seasons has relatively high CLA contents and percentages of the c9,t11 CLA isomer, which is favourable, while the n − 6/n − 3 ratios are within the recommended values for the human diet. In conclusion, the results suggest that intramuscular fat in Arouquesa-PDO veal has high nutritional value throughout the year.     

共轭亚油酸生理功能及其合成方法

共轭亚油酸（CLA）为亚油酸（LA）的异构体,具有抗癌、降脂、调节免疫、抗粥样硬化等重要的生理功能,目前共轭亚油酸（CLA）的生产大都是利用碱催化亚油酸异构而成,但其异构体繁多。利用微生物转化生产的共轭亚油酸（CLA）,异构体较少,与天然食品中的CLA异构体组成相似,具有很好的商业前景。     

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.     

Conjugated linoleic acid isomer concentrations in milk from high‐ and low‐input management dairy systems

BACKGROUND: Different conjugated linoleic acid (CLA) isomers are known to have contrasting physiology or health effects and there is growing evidence that the profile of natural isomers in milk is influenced by the production system. This survey is the first to compare feeding regimes and concentrations of 14 CLA isomers in milk from three production systems in the UK. RESULTS: Total CLA and seven isomers (including C18:2 c9t11 which comprised > 80% of total) were significantly higher in milk from both organically certified and non‐certified low input (LI) systems compared with milk from conventional high input farms. Sampling date also affected concentrations of total CLA and nine isomers; being lowest in March and highest in August. Seasonal differences were greater in milk from LI herds, thought to be due to changes in herbage and/or stage of lactation. Multivariate analysis showed a strong positive relationship between several CLA isomers and increasing levels of fresh forage in the diet. CONCLUSIONS: These results add to the evidence on how management adjustment may improve the profile of CLA isomers in milk fat, although animal or human intervention studies are required to identify the effects of consuming milk with different CLA levels and isomer profiles on human health. Copyright © 2009 Society of Chemical Industry     

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%。