Rapid screening method for analyzing the conjugated linoleic acid production capabilities of bacterial cultures

In this paper we describe a rapid method for identifying bacteria which convert free linoleic acid to conjugated linoleic acid (CLA). This method is based on spectrophotometric detection of CLA and compares well with the standard gas-liquid chromatography method. This method should facilitate high-throughput screening of bacterial isolates for the ability to produce conjugated fatty acids.     

酶法生产共轭亚油酸的研究进展

共轭亚油酸(Conjugated linoleic acid,CLA)具有抗癌、抗动脉粥样硬化、减肥和免疫调节等生理活性。共轭亚油酸可以通过酶法异构化获得,将底物亚油酸异构形成具有生物活性物质-共轭亚油酸的异构酶称为亚油酸异构酶。因此,通过介绍亚油酸异构酶的来源、作用机制、酶学性质和基因工程菌生产等方面的研究进展,结合不断发展的基因工程技术,旨在提高亚油酸异构酶的活性、产量和异构化效率,以扩大反应底物范围,降低生产成本,从而推进共轭亚油酸的规模化、可持续性的工业生产。     

Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species

AIMS: To assess strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium for their ability to produce the health-promoting fatty acid conjugated linoleic acid (CLA) from free linoleic acid. METHODS AND RESULTS: In this study, strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium were grown in medium containing free linoleic acid. Growth of the bacteria in linoleic acid and conversion of the linoleic acid to CLA was assessed. Of the bacteria assessed, nine strains of Bifidobacterium produced the c9, t11 CLA isomer from free linoleic acid. The t9, t11 CLA isomer was also produced by some strains, but at much lower concentrations. CONCLUSIONS: The production of CLA by bifidobacteria exhibited considerable interspecies variation. Bifidobacterium breve and B. dentium were the most efficient CLA producers among the range of strains tested, with B. breve converting up to 65% linoleic acid to c9, t11 CLA when grown in 0.55 mg ml(-1) linoleic acid. Strains also varied considerably with respect to their sensitivity to linoleic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of CLA by probiotic bifidobacteria offers a possible mechanism for some health-enhancing properties of bifidobacteria and provides novel opportunities for the development of functional foods.     

Production of conjugated linoleic acid by dairy starter cultures

Nineteen different strains of lactobacilli, lactococci, streptococci and propionibacteria commonly used as dairy starter cultures were tested for their ability to produce conjugated linoleic acid (CLA) from free linoleic acid in vitro. Two strains of Propionibacterium freudenreichii ssp. freudenreichii and one strain of P. freudenreichii ssp. sheramnii were found to be capable of converting free linoleic acid to extracellular CLA. The highest level of CLA formed in the media was 265 μg ml⁻¹. Of the different isomers, cis- and trans-9,11-octadecadienoic acid represented more than 70% of the total CLA formed. The inhibitory effect of linoleic acid on the growth of the bacteria and its conversion to CLA in different media by propionibacteria are discussed.     

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.     

Lactobacillus growth and membrane composition in the presence of linoleic or conjugated linoleic acid

Five Lactobacillus strains of intestinal and food origins were grown in MRS broth or milk containing various concentrations of linoleic acid or conjugated linoleic acid (CLA). The fatty acids had bacteriostatic, bacteriocidal, or no effect depending on bacterial strain, fatty acid concentration, fatty acid type, and growth medium. Both fatty acids displayed dose-dependent inhibition. All strains were inhibited to a greater extent by the fatty acids in broth than in milk. The CLA isomer mixture was less inhibitory than linoleic acid. Lactobacillus reuteri ATCC 55739, a strain capable of isomerizing linoleic acid to CLA, was the most inhibited strain by the presence of linoleic acid in broth or milk. In contrast, a member of the same species, L. reuteri ATCC 23272, was the least inhibited strain by linoleic acid and CLA. All strains increased membrane linoleic acid or CLA levels when grown with exogenous fatty acid. Lactobacillus reuteri ATCC 55739 had substantial CLA in the membrane when the growth medium was supplemented with linoleic acid. No association between level of fatty acid incorporation into the membrane and inhibition by that fatty acid was observed.     

The effects of dietary conjugated nonadecadienoic acid on body composition in mice.

A 19-carbon conjugated diene, conjugated nonadecadienoic acid (CNA), inhibited heparin-releasable lipoprotein lipase and reduced lipid stores in 3T3-L1 adipocytes similarly to conjugated linoleic acid (CLA). When fed to growing mice (0.3% of diet) CNA reduced body fat by 81% whereas CLA reduced body fat by 25%. CLA and CNA differ in length by one carbon atom so they are unlikely to share a common metabolite to account for these observations.     

Conjugated linoleic acid metabolism

Conjugated linoleic acid (CLA) is a naturally occurring fatty acid that is produced by a bio-hydrogenation process in the rumen, and thus is present in dairy products and ruminant meat. In this case the predominant isomer formed is 9cis,11trans. However, CLA includes 28 positional and geometrical isomers, of which only 9cis,11trans and 10trans,12cis have thus far been proven to possess biological activities. Both of these CLA isomers have been shown to undergo elongation and desaturation processes similar to those that occur with linoleic acid, maintaining the conjugated diene structure. There are evidences supporting the hypothesis that CLA metabolism may interfere with eicosanoid formation. Other metabolites with 16 carbon atoms (conjugated 16:2 and 16:3, which are probably derived from peroxisomal beta-oxidation of CLA and its metabolites, respectively) have been detected. This suggests an efficient metabolism of CLA and its metabolites in peroxisomes, which might be linked to their capacity to activate peroxisome proliferator-activated receptors.     

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.     

Activity and mRNA levels of enzymes involved in hepatic fatty acid synthesis and oxidation in mice fed conjugated linoleic acid

The effects of dietary conjugated linoleic acid (CLA) on the activity and mRNA levels of hepatic enzymes involved in fatty acid synthesis and oxidation were examined in mice. In the first experiment, male ICR and C57BL/6J mice were fed diets containing either a 1.5% fatty acid preparation rich in CLA or a preparation rich in linoleic acid. In the second experiment, male ICR mice were fed diets containing either 1.5% linoleic acid, palmitic acid or the CLA preparation. After 21 days, CLA relative to linoleic acid greatly decreased white adipose tissue mass but caused hepatomegaly accompanying an approximate 10-fold increase in the tissue triacylglycerol content irrespective of mouse strain. CLA compared to linoleic acid greatly increased the activity and mRNA levels of various lipogenic enzymes in both experiments. Moreover, CLA increased the mRNA expression of Delta6- and Delta5-desaturases, and sterol regulatory element binding protein-1 (SREBP-1). The mitochondrial and peroxisomal palmitoyl-CoA oxidation rate was about 2.5-fold higher in mice fed CLA than in those fed linoleic acid in both experiments. The increase was associated with the up-regulation of the activity and mRNA expression of various fatty acid oxidation enzymes. The palmitic acid diet compared to the linoleic acid diet was rather ineffective in modulating the hepatic lipid levels or activity and mRNA levels of enzymes in fatty acid metabolism. It is apparent that dietary CLA concomitantly increases the activity and mRNA levels of enzymes involved in fatty acid synthesis and oxidation, and desaturation of polyunsaturated fatty acid in the mouse liver. Both the activation of peroxisomal proliferator alpha and up-regulation of SREBP-1 may be responsible for this.     

Isolation of a novel strain of Butyrivibrio fibrisolvens that isomerizes linoleic acid to conjugated linoleic acid without hydrogenation, and its utilization as a probiotic for animals

AIM: Isolation of a new strain of Butyrivibrio fibrisolvens possessing great capacity to produce conjugated linoleic acid (CLA) in order to utilize as a probiotic for animals. METHODS AND RESULTS: A novel strain (MDT-5) was isolated from the goat rumen, which exclusively converted linoleic acid (LA) to CLA, because of its high LA isomerase activity with virtually no CLA reductase activity. MDT-5 also converted linolenic acid to conjugated linolenic acid that may be more bioactive than CLA. The oral administration of MDT-5 every other day to mice for 2 weeks resulted in increased amounts of CLA in the contents of the large intestine (2.5-fold), as well as in adipose tissue (threefold). Feeding a high-LA diet, as well as prolonging the period of MDT-5 administration, further increased the CLA content in body fat. CONCLUSIONS: MDT-5 has by far greater ability to produce CLA than any other known bacteria. Administration of MDT-5 to mice increases CLA production in the large intestine, which results in increased CLA absorption. SIGNIFICANCE AND IMPACT OF THE STUDY: MDT-5 may be useful in pet animals as a probiotic to provide CLA continuously.     

Reduction of linoleic acid inhibition in production of conjugated linoleic acid by Propionibacterium freudenreichii ssp. shermanii

A method for the production of conjugated linoleic acid (CLA) from linoleic acid (LA) using growing cultures of Propionibacterium freudenreichii ssp. shermanii JS was developed. The growth inhibitory effect of LA was eliminated by dispersing it in a sufficient concentration of polyoxyethylene sorbitan monooleate detergent. For the whey permeate medium used, the optimum LA:detergent ratio was 1:15 (w/w). As a result, the cultures tolerated at least 1000 microg x mL(-1) LA, which was converted to CLA with 57%-87% efficiency. The cis-9, trans-11 and trans-9, cis-11 isomers constituted 85%-90% of the CLA produced. The feasibility of the method was demonstrated also in de Man Rogosa-Sharpe (MRS) broth.     

Accumulation of conjugated linoleic acid in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WU-P19 is enhanced by induction with linoleic acid and chitosan treatment

Production of conjugated linoleic acid (CLA) by the potential probiotic bacterium Lactobacillus plantarum WU-P19 was investigated with the aim of enhancing production. CLA produced using this bacterium may be used to supplement dietary intake. Cultures were fed linoleic acid for conversion to CLA and the CLA produced was measured. In some cases, chitosan was added to cultures to improve cellular uptake of linoleic acid. Under static conditions at 37 °C, the bacterium grew and produced CLA in the pH range of 5.5–6.5. At pH 6.0, a 36-h incubation period maximized the concentration of the dry biomass (0.82 g/L), the CLA content in the biomass (4.1 mg/g), and linoleic acid in the biomass (1.2 mg/g). In comparison with cultures grown without linoleic acid in the medium, supplementing the medium with linoleic acid at 600 μg/mL slowed the production of CLA, but the CLA content in the dry biomass increased to 12–14 mg/g and the linoleic acid content increased to 8–11 mg/g. Supplementing the culture medium with chitosan and linoleic acid enhanced production of CLA in the dry biomass to 21 mg/g within 36 h. Nearly 50% of the CLA was cis-9, trans-11-CLA, and the remainder was trans-10, cis-12-CLA. Linoleic acid content of the dry biomass was increased to 37 mg/g. Accumulation of CLA in the cells was enhanced by feeding linoleic acid. Supplementing the culture with linoleic acid and chitosan further increased accumulation of CLA.     

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...     

植物乳杆菌ZS2058在磷酸盐缓冲液体系中生物转化共轭亚油酸

植物乳杆菌ZS2058是从泡菜中筛选到一株具有转化共轭亚油酸能力的乳酸菌。该菌株在MRS培养基中经0.5mg/mL的亚油酸诱导培养后,所获得的菌体细胞具有较强的转化能力。文中就植物乳杆菌ZS2058水洗细胞在磷酸盐缓冲液体系中生物转化共轭亚油酸进行了深入研究。在非厌氧条件下,植物乳杆菌ZS2058在亚油酸浓度为1mg/mL,湿细胞质量浓度约为150mg/mL,120r/min、37℃的条件下反应24h后,能将亚油酸转化为共轭亚油酸和羟基脂肪酸,其中c9,t11-CLA占所产生的CLA总量的96.4%,产量可高达312.4μg/mL,说明该菌株有很强的专一性。随着反应进一步进行,反应至36h时,c9,t11-CLA含量逐渐减少,伴随着大量羟基脂肪酸的产生;并且,以CLA(c9,t11-CLA和t10,c12-CLA的混合样品)为底物进行反应时,c9,t11-CLA被转化为羟基脂肪酸。由此可知,c9,t11-CLA可能是该菌株生物转化LA过程中的一个中间产物。     

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.     

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.     

Biological activities of conjugated fatty acids: conjugated eicosadienoic (conj. 20:2delta(c11,t13/t12,c14)), eicosatrienoic (conj. 20:3delta(c8,t12,c14)), and heneicosadienoic (conj. 21:2delta(c12,t14/c13,t15)) acids and other metabolites of conjugated linoleic acid

The elongated form of conjugated linoleic acid (CLA), conjugated eicosadienoic acid (CEA, conj. 20:2delta(c11,t13/t12,c14)), was generated from CLA by liver microsomal fractions. Subsequent testing showed that dietary CEA significantly reduced body fat, and increased lean mass similar to CLA when compared to controls. CEA also decreased lipoprotein lipase activity and triacylglyceride, and increased glycerol release in 3T3-L1 adipocytes, correlated with the trans-12,cis-14 isomer, but CEA required a longer incubation period than cells treated with CLA. Based on the fact that CEA fed animals had CLA in tissue, we suggest that the effect of CEA is due to the CLA converted from CEA in the system. The delta-6 desaturated and elongated form of trans-10,cis-12 CLA (conjugated eicosatrienoic acid, CETA, conj. 20:3delta(c8,t12,c14)) inhibited LPL activity and increased glycerol release but was less active than trans-10,cis-12 CLA or CEA. The 21-carbon conjugated fatty acid, conjugated heneicosadienoic acid (CHDA, conj. 21:2delta(c12,t14/c13,t15)), was not active on LPL inhibition, triacylglyceride, or glycerol release in 3T3-L1 adipocytes. We also provide evidence that CLA was metabolized to conjugated dodecadienoic acid (conj. 12:2delta(c3,t5/t4,c6)). In addition, there were indications of the presence of conjugated tetradecadienoic acid (conj. 14:2delta(c5,t7/t6,c8)), suggesting that CLA can be metabolized through fatty acid beta-oxidation. This is the first work to report the presence of conjugated 12 and 14 carbon fatty acids, originated from CLA, and the biological activities of CEA, CETA and CHDA.     

Conjugated linoleic acid and atherosclerosis: studies in animal models

Conjugated linoleic acids (CLA) are isomeric forms of linoleic acid (LA) containing two conjugated sites of unsaturation. The most abundant dietary form of CLA is the cis-9,trans-11 (c-9,t-11) isomer that is found in the fatty tissues and milk of ruminant animals. CLA can also be acquired by ingestion of supplements, which are usually equimolar mixtures of the c-9,t-11 and t-10,c-12 CLA. For more than a decade, the potential for CLA to modify atherosclerosis in animal models has been examined. However, to date, the studies have failed to reach consensus on whether CLA can be effective in reducing the incidence or severity of atherosclerotic lesions, or whether or not plasma lipid and lipoprotein levels can be improved with CLA supplementation. This review will examine the evidence for and against a role for CLA in atherosclerosis, with a focus on the rabbit, the hamster, and the apoE-deficient mouse.     

Detection of conjugated dienoic fatty acids in human vascular smooth muscle cells treated with conjugated linoleic acid

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recent studies demonstrated that conjugated metabolites of CLA are found in tissues of CLA-fed animals and cultured cells treated with CLA. This observation has gained in importance since it has recently been shown that these metabolites of CLA exert specific biological activities. Therefore, the present study aimed to explore the potential formation of metabolites of cis-9, trans-11 CLA, trans-10, cis-12 CLA and trans-9, trans-11 CLA in cells of the vascular wall, which has not yet been shown. Examination of fatty acid composition of total cell lipids using Ag+-HPLC, GC-FID and GC-MS analysis revealed a significant isomer-specific formation of conjugated metabolites of CLA such as CD16:2, CD20:2 and CD22:2 in human coronary artery smooth muscle cells treated with various CLA isomers. Different CD16:2/CLA ratios between various CLA isomers as observed in the present study indicate that fatty acid metabolism is differently affected by the configuration of the double bonds. In conclusion, the observation from the present study suggests that the effects of CLA in vascular cells might not only be mediated by CLA itself but also by its conjugated metabolites. Future studies using highly purified conjugated metabolites of CLA are necessary to study their role in mediating biological effects of CLA in cell culture systems.