Studies on the production of conjugated linoleic acid from linoleic and vaccenic acids by mixed rumen protozoa

The conversion of β-myrcene to the furanoid flavour compound perillene by Pleurotus ostreatus was investigated using trideutero β-myrcene, trideutero α-(Z)-acaridiol and non-labeled 1,2- and 3,10-epoxy-β-myrcene, α,α-acarilactol, and perillene as substrates. Myrcene diols were formed from the cleavage of myrcene epoxides, but only α-(Z)-acaridiol, a 1,4-butanediol derivative most likely generated through a base-catalysed epoxide opening, was a suitable precursor of perillene. Once formed, this key intermediate was rapidly oxidised and the resulting cyclic lactol was dehydrated to yield perillene. Bioconversion of the supplemented perillene to α,α-acariolide indicated that perillene was another intermediate of the pathway and prone to further oxidative degradation. The data suggest that the fungus converted the cytotoxic β-myrcene in its environment into a metabolically useable carbon source along this route.     

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.     

Effect of pH on conjugated linoleic acid (CLA) formation of linolenic acid biohydrogenation by ruminal microorganisms

Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of ¹³C-LNA with different pH conditions (5.5 and 6.5). The ¹³C enrichment was calculated as a ¹³C/¹²C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from ¹³C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.     

Effect of linoleic acid concentration on conjugated linoleic acid production by Butyrivibrio fibrisolvens A38

Butyrivibrio fibrisolvens A38 inocula were inhibited by as little as 15 microM linoleic acid (LA), but growing cultures tolerated 10-fold more LA before growth was inhibited. Growing cultures did not produce significant amounts of cis-9, trans-11 conjugated linoleic acid (CLA) until the LA concentration was high enough to inhibit biohydrogenation, growth was inhibited, and lysis was enhanced. Washed-cell suspensions that were incubated anaerobically with 350 microM LA converted most of the LA to hydrogenated products, and little CLA was detected. When the washed-cell suspensions were incubated aerobically, biohydrogenation was inhibited, CLA production was at least twofold greater, and CLA persisted. The LA isomerase reaction was very rapid, but the LA isomerase did not recycle like a normal enzyme to catalyze more substrate. Cells that were preincubated with CLA lost their ability to produce more CLA from LA, and the CLA accumulation was directly proportional (r(2) = 0.98) to the initial cell density. Growing cells were as sensitive to CLA as LA, the LA isomerase and reductases of biohydrogenation were linked, and free CLA was not released. Because growing cultures of B. fibrisolvens A38 did not produce significant amounts of CLA until the LA concentration was high, biohydrogenation was arrested, and the cell density had declined, the flow of CLA from the rumen may be due to LA-dependent bacterial inactivation, death, or lysis.     

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.     

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid

From the simultaneous accumulation of hydrogenation intermediates and the disappearance of Isotricha prostoma after algae supplementation, we suggested a role of this ciliate and/or its associated bacteria in rumen biohydrogenation of unsaturated fatty acids. The experiments described here evaluated the role of I. prostoma and/or its associated endogenous and exogenous bacteria in rumen biohydrogenation of C18:2n-6 and its main intermediates CLA c9t11 and C18:1t11. Fractions of I. prostoma and associated bacteria, obtained by sedimentation of rumen fluid sampled from a monofaunated sheep, were used untreated, treated with antibiotics or sonicated to discriminate between the activity of I. prostoma and its associated bacteria, the protozoan or the bacteria, respectively. Incubations were performed in triplicate during 6 h with unesterified C18:2n-6, CLA c9t11 or C18:1t11 (400 μg/ml) and 0.1 g glucose/cellobiose (1/1, w/w). I. prostoma did not hydrogenate C18:2n-6 or its intermediates whereas bacteria associated with I. prostoma converted a limited amount of C18:2n-6 and CLA c9t11 to trans monoenes. C18:1t11 was not hydrogenated by either I. prostoma or its associated bacteria but was isomerized to C18:1c9. A phylogenetic analysis of clones originating from Butyrivibrio-specific PCR product was performed. This indicated that 71% of the clones from the endogenous and exogenous community clustered in close relationship with Lachnospira pectinoschiza. Additionally, the biohydrogenation activity of solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was examined and compared with the activity of the non-fractioned I. prostoma monofaunated rumen fluid (LAB + SAB). Both SAB and LAB were involved in rumen biohydrogenation of C18:2n-6. SAB fractions performed the full hydrogenation reaction to C18:0 while C18:1 fatty acids, predominantly C18:1t10 and C18:1t11, accumulated in the LAB fractions. SAB and LAB sequence analyses were mainly related to the genera Butyrivibrio and Pseudobutyrivibrio with 12% of the SAB clones closely related to the C18:0 producing B. proteoclasticus branch. In conclusion, this work suggests that I. prostoma and its associated bacteria play no role in C18:2n-6 biohydrogenation, while LAB convert C18:2n-6 to a wide range of C18:1 fatty acids and SAB produce C18:0, the end product of rumen lipid metabolism.     

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.     

Bioconversion of linoleic acid to conjugated linoleic acid byBifidobacterium breve

The bioconversion of linoleic acid (LA) to conjugated linoleic acid (CLA) was investigated to examine LA-adaptation ofBifidobacterium breve KCTC 3461 to additions of 1 to 5 mg/mL of LA overtime. To induce LA-adaptation,B. breve KCTC 3461 was treated with LA, according to three schemes. For LA-adaptedB. breve the maximum concentration of CLA, 300–350 μg/mL, was obtained in cys-MRS medium containing 1 mg/mL of LA. The CLA production significantly increased with increasing LA concentration, from 1 to 4 mg/mL, but the conversion of LA to CLA gradually decreased. The CLA production capability ofB. breve, and its tolerance, improved significantly with LA-adaptation. The addition of LA (1 mg/mL) into the culture broth after 24 h of cultivation in a 100-mL media bottle was most effective at promoting CLA production. In a 2.5-L stirred-tank bioreactor, the observed conversion and productivity of 56.6% and 35.4 μgml⁻¹h⁻¹, respectively, by LA-adaptedB. breve were approximately 6.6 and 9.8 times higher than those of LA-unadaptedB. breve.     

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。     

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。     

The biohydrogenation of α-linoleic acid and oleic acid by rumen micro-organisms

1. α-[U-¹⁴C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas–liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis–cis–cis-octadecatrienoic acid, non-conjugated trans–cis (cis–trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate α-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.     

Cell-adhered conjugated linoleic acid regulates isomerization of linoleic acid by resting cells of Propionibacterium freudenreichii

The microbiological isomerization of linoleic acid (LA) to conjugated linoleic acid (CLA) was studied in resting cell suspensions of a propionibacterium and micellar LA to identify factors critical in the isomerization efficiency. These suspensions, containing cells 5x10(10) colony-forming units ml(-1) and 510 micro g LA ml(-1), isomerized about 90% of LA to CLA. However, the yield was not improved with higher amounts of micellar LA, suggesting that the cells had a fixed capacity to carry out the isomerization. This was explained by the fact that the CLA formed had a tendency to accumulate in the cell mass rather than in the aqueous micellar phase during the isomerization. Concomitantly, cell viability and isomerization rates were gradually reduced. Upon cessation of the reaction, about 46% of all the CLA formed was in the cell material. This accumulation to the cells was prevented by adding the detergent in excess to that required for micellization of LA. Then the cells remained viable, but the rate of isomerization was drastically lowered, due to impaired availability of LA from the fortified micellar phase to the cells. It was concluded that the phase distribution of substrate and product plays a critical role in the microbiological production of CLA.     

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.