Conjugated linoleic acid concentration as affected by lactic cultures and added linoleic acid

Six lactic cultures: Lactobacillus acidophilus (CCRC14079), L. delbrueckii subsp. bulgaricus (CCRC14009), L. delbrueckii subsp. lactis (CCRC14078), Lactococcus lactis subsp. cremoris (CCRC12586), Lc. lactis subsp. lactis (CCRC10791), and Streptococcus salivarius subsp. thermophlius (CCRC12257) were tested for the effects of addition of 1000 and 5000 μg/ml linoleic acid, and incubation time from 0 to 48 h. The levels of conjugated linoleic acid (CLA) formation were determined by gas chromatography. A sharp increase in CLA level was observed in linoleic acid added cultures. Incubation of L. acidophilus in 1000 μg/ml linoleic acid added-skim milk medium for 24 h was most effective in promoting CLA formation. Increasing linoleic acid addition from 1000 to 5000 μg/ml and prolonging incubation from 24 to 48 h did not appear to enhance CLA formation.     

Conjugated linoleic acid production by cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus with additions of different fatty acids

The objective of the study was to determine the effect of fatty acid additions to the cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) on CLA production. Washed cells of L. delbrueckii ssp. bulgaricus, obtained by cultivation in a MRS broth, were mixed with BSA and each of the three fatty acids: linoleic, oleic, and linolenic acids in sodium phosphate buffer at pH 6.5. After incubation at 37 °C for 108 h, CLA concentration was analyzed by HPLC. Enzyme extract from the culture was also reacted with each fatty acid at 50 °C for 10 min at pH 5 to test for CLA production. Results showed that linoleic acid addition to the culture improved CLA production, indicating the presence of linoleic acid isomerase activity in the culture. The crude enzyme extract from the culture was observed to be capable of oleic and linolenic acid conversions into CLA, demonstrating the possible presence of desaturase activity in the enzyme extract.     

Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

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。     

Effect of incubation conditions and possible intestinal nutrients on cis-9, trans-11 conjugated linoleic acid production by Lactobacillus acidophilus F0221

The purpose of this study was to optimise the incubation conditions for cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) production by Lactobacillus acidophilus F0221 and evaluate the effect of possible intestinal nutrients on c9, t11 CLA production. Growth in Mann Rogosa Sharp broth supplemented with cysteine and containing 0.5 g L⁻¹ linoleic acid at pH 6.5 with anaerobic incubation at 37 °C for 40 h were found to be the optimal conditions for CLA production. Galactosaccharide, arabinogalactan, galactose and glucose had a higher promoting effect on CLA production (96.19–123.89 μg mL⁻¹) than other carbohydrates. Acetic acid had a higher promoting effect on CLA production (130.98 μg mL⁻¹) than other short chain fatty acids. These results provide detailed parameters for the production of c9, t11 CLA by L. acidophilus F0221 and are valuable for further understanding of c9, t11 CLA formation in the human intestine by this strain.     

c9t11‐Conjugated linoleic acid‐rich oil fails to attenuate wasting in colon‐26 tumor‐induced late‐stage cancer cachexia in male CD2F1 mice

Scope: Cancer cachexia is characterized by muscle and adipose tissue wasting caused partly by chronic, systemic inflammation. Conjugated linoleic acids (CLAs) are a group of fatty acids with various properties including anti‐inflammatory cis9, trans11 (c9t11)‐CLA and lipid‐mobilizing trans10, cis12 (t10c12)‐CLA. The purpose of this study was to test whether dietary supplementation of a c9t11‐CLA‐rich oil (6:1 c9t11:t10c12) could attenuate wasting of muscle and adipose tissue in colon‐26 adenocarcinoma‐induced cachexia in mice. Methods and results: Loss of body weight, muscle and adipose tissue mass caused by tumors were not rescued by supplementation with the c9t11‐CLA‐rich oil. In quadriceps muscle, c9t11‐CLA‐rich oil exacerbated tumor‐induced gene expression of inflammatory markers tumor necrosis factor‐α, IL‐6 receptor and the E3 ligase MuRF‐1 involved in muscle proteolysis. In epididymal adipose tissue, tumor‐driven delipidation and atrophy was aggravated by the c9,t11‐CLA‐rich oil, demonstrated by further reduced adipocyte size and lower adiponectin expression. However, expression of inflammatory cytokines and macrophage markers were not altered by tumors, or CLA supplementation. Conclusion: These data suggest that addition of c9t11‐CLA‐rich oil (0.6% c9t11, 0.1% t10c12) in diet did not ameliorate wasting in mice with cancer cachexia. Instead, it increased expression of inflammatory markers in the muscle and increased adipose delipidation.     

Preparation and properties of probiotic concentrated yoghurt (labneh) fortified with conjugated linoleic acid

Milk of high conjugated linoleic acid (CLA) level (1.25 g per 100 g milk fat) was produced by inclusion of fish oil and rousted soy bean in the ration of Holstein cows as compared to 0.55 g per 100 g milk fat in the milk of animals receiving control diet. Milk of normal (control) and high CLA content (treatment) was spray‐dried. Labneh was made from 20 g L^?1 reconstituted milk using 3 mL per 100 mL yoghurt starter and 2 mL per 100 mL of probiotic cultures of Lactobacillus casei or Lactobacillus acidophilus. The control (C) and high CLA (T) labneh were analysed chemically and microbiologically, and their viscosities were determined during cold storage for 15 days. The fat content of labneh of high CLA was less than that of the control, but the total solids (TS) were unaffected by the CLA level. Labneh made with Lb. acidophilus had lower TS and higher acidity, exopolysaccharides and acetaldehyde contents and viscosity than that made with the use of Lb. casei. Labneh from the different treatments retained high counts of the added probiotic (>10⁸ cfu g^?1) throughout the storage period. The storage period had significant effects on all parameters determined.     

固定化亚油酸异构酶制备及其性质

以海藻酸钠、壳聚糖为载体,分别采用直接包埋、交联-包埋法制备固定化亚油酸异构酶;研究酶的固定化条件和固定化酶的部分性质。结果表明：以海藻酸钠为载体,采用交联-包埋法以戊二醛为交联剂时固定化效果较好;最佳固定化条件为：海藻酸钠质量浓度为3g/100mL,戊二醛质量浓度为0.3g/100mL,CaCl2质量浓度为2g/100mL;固定化酶的最适反应温度为50℃,最适反应pH值为5.0;与游离酶相比,固定化酶的热稳定性显著提高,温度在20～60℃之间较稳定,pH值在2～8之间表现出较好的酸碱耐受性;固定化亚油酸异构酶的Km为0.36mg/mL。连续操作6次固定化相对酶活力仍保持70.6%,与游离酶相比,固定化亚油酸异构酶催化效率约提高了50%。     

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.     

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.     

Effect of a high dose of CLA in finishing pig diets on fat deposition and fatty acid composition in intramuscular fat and other fat depots

Sixteen gilts were fed a control (4% of sunflower oil) or an experimental diet (4% conjugated linoleic acid (CLA) oil). CLA had no effect on intramuscular fat (IMF) content neither in longissimus thoracis (LT) nor in semimembranosus (SM) muscles but increased liver weight, reduced perirenal fat and tended to reduce backfat between the last 3th–4th lumbar vertebrae. Despite the fact that 9c,11t and 10t,12c CLA isomers were included in the same proportion in the diet, the 9c,11t and 9c,11c were the isomers more deposited in all tissues. Addition of CLA in the diet affected fatty acid composition in a tissue specific manner, increasing percentages of SFA in all tissues, reducing percentages of MUFA in LT and LT subcutaneous fat, and of PUFA in LT subcutaneous fat, liver and SM. The FA modification by dietary CLA in LT IMF was reflected in the different lipid fractions, SFA and MUFA mainly in the neutral lipid fraction, and PUFA in the polar fraction.     

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.     

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。     

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%。     

Antioxidative properties of extracts from Aspergillus candidus broth filtrate

This work evaluated the antioxidative properties of ethyl acetate extracts from Aspergillus candidus CCRC 31543 broth filtrate (EAEAC). EAEAC markedly exhibited antioxidative activity in a soybean oil peroxidation system as measured by the Rancimat method. This finding significantly correlated (r=0·9702, P<0·05) with the extent of antioxidative activity in a linoleic acid peroxidation system as measured by the thiocyanate method. EAEAC also displayed good thermal stability when heated at 185°C for 2 h. Although the antioxidative activity of the EAEAC was unstable at the alkali pH value, it was more effective at the neutral and acidic pH values. EAEAC had synergistic effects with α-tocopherol and citric acid in linoleic acid peroxidation system. EAEAC showed no mutagenicity to Salmonella typhimurium TA98 and TA100 as evaluated by Ames test. These properties indicated that Aspergillus candidus CCRC 31543 might be a potential antioxidant for application in food products. ©1997 SCI     

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。     

Probiotic in lamb rennet paste enhances rennet lipolytic activity, and conjugated linoleic acid and linoleic acid content in Pecorino cheese

Cheeses manufactured using traditional lamb rennet paste, lamb rennet paste containing Lactobacillus acidophilus, and lamb rennet paste containing a mix of Bifidobacterium lactis and Bifidobacterium longum were characterized for the lipolytic pattern during ripening. Lipase activity of lamb rennet paste, lamb rennet containing Lb. acidophilus, and lamb rennet containing a mix of bifidobacteria was measured in sheep milk cream substrate. Rennet paste containing probiotics showed a lipase activity 2-fold greater than that displayed by traditional rennet. Total free fatty acid (FFA) in sheep milk cream was lower in lamb rennet paste (981 μg/g of milk cream) than in lamb rennet containing Lb. acidophilus (1,382.4 μg/g of milk cream) and in lamb rennet containing a mix of bifidobacteria (1,227.5 μg/g of milk cream) according to lipase activity of lamb rennet paste. The major increase of FFA in all cheeses occurred during the first 30 d of ripening with the greatest values being observed for C16:0, C18:0 C18:1. At 60 d of ripening all cheeses showed a reduction in the amount of free fatty acids; in particular, total free fatty acids underwent a decrease of more than 30% from 30 to 60 d in cheeses manufactured using traditional lamb rennet paste, whereas the same parameter decreased 10% in cheeses manufactured using lamb rennet paste containing Lb. acidophilus and cheeses manufactured using lamb rennet paste containing a mix of B. lactis and B. longum. Cheese containing Lb. acidophilus was characterized by the greatest levels of total conjugated linoleic acids (CLA) 9-cis, 11-trans CLA and 9-trans, 11-trans CLA, whereas cheese containing bifidobacteria displayed the greatest levels of free linoleic acid. Rennet pastes containing viable cells of Lb. acidophilus and a mix of B. lactis and B. longum were able to influence the amount of FFA and CLA in Pecorino cheese during ripening.     

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。     

C18 : 1, C18 : 2 and C18 : 3 trans and cis fatty acid isomers including conjugated cis δ9,trans δ11 linoleic acid (CLA) as well as total fat composition of German human milk lipids

In particular with respect to infant nutrition knowledge of the current contents of trans fatty acids (TFA) and of conjugated linoleic acid (CLA) in human milk lipids is of interest. After pre-separation by Ag-TLC 11 trans-C18 : 1 isomers could be quantified by GC with a mean total content of 2.40 ± 0.60 wt% in samples from 40 German women. For the positional isomers t4, t5, t6–8, t9, t10, t11, t12, t13, t14, t15 and t16 contents of 0.02, 0.02, 0.21, 0.37, 0.32, 0.68, 0.23, 0.15, 0.18, 0.09 and 0.14 wt% were established, with vaccenic acid being the predominant isomer. Further, small trans-C14 : 1 and trans-C16 : 1 contents of 0.08% and 0.15% on average were found. As the trans-C18 : 1 isomers also the trans-C16 : 1 isomers of human milk lipids could for the first time be baseline-resolved by GC to a great extent. Moreover, besides a mean CLA (c9,t11) content of 0.40 ± 0.09% further 6 cis/trans isomers of linoleic acid with a total content of 1.07 ± 0.56% on average (w/o CLA) were determined. Further, 4 trans isomers of α-linolenic acid could be baseline-resolved exhibiting a total content of 0.11%. Altogether German human milk lipids on average were found to contain 3.81 ± 0.97% TFA with a range of 2.38–6.03%. Direct connections between the dietary intake of trans-C18 : 1 isomers and the composition of human milk lipids could be established. The major fatty acids exhibited the following contents (wt%): C4: 0.16, C6: 0.18, C8: 0.06, C10: 0.58, C12: 3.12, C14: 6.43, C16: 25.28, C18: 7.41, C18 : 1 (total): 33.67, C18 : 2 (total): 10.63 and α-C18 : 3: 0.87.