Comparison of fatty acid composition of bacterial and protozoal fractions in rumen fluid of sheep fed diet supplemented with sunflower, rapeseed and linseed oils

The objective of this study was to investigate effects of oil supplements on the composition of fatty acids (FA), especially of trans11-C18:1 (vaccenic acid, TVA) and cis9, trans11-C18:2 conjugated linoleic acid (c9,t11-CLA), in bacterial (BF) and protozoal (PF) fractions of rumen fluid of sheep that was fractionated centrifugation. Four sheep were fed a diet consisting of meadow hay (960 g dry matter (DM)/day) and of barley grain (240 g DM/day), with sunflower oil (SO), rapeseed oil (RO) or linseed oil (LO) as supplements (60 g/day) in a Latin square design. The oils were used as they are rich in linoleic acid (SO, 533 g/kg of FA), oleic acid (RO, 605 g/kg of FA) and α-linolenic acid (LO, 504 g/kg of FA). Compared to the control (i.e., without oils), oil supplements influenced the concentration of unsaturated (UFA) and saturated fatty acids (SFA). In both BF and PF, the main fatty acids were palmitic and stearic, but PF contained a higher proportion of TVA and c9,t11-CLA than BF. In PF, TVA concentrations, ranked by oil supplement, were SO > RO > LO > Control (174, 150, 118, 74 g/kg of FA, respectively) and the c9,t11-CLA concentrations were RO > SO > LO > Control (59, 51, 27 and 15 g/kg of FA, respectively). Concentrations of c9,t11-CLA in PF were two to three times higher than in BF with all the oil supplements versus the control. Oil treatments impacted the c9,t11-CLA concentration in the fractions, especially SO and RO. The protozoal fraction contained a higher proportion of TVA and c9,t11-CLA than did the bacterial fraction, and dietary addition of SO, RO and LO resulted in a higher incorporation of TVA into both bacterial and protozoal microbial fractions, which probably positively affected TVA flow from the rumen.     

Bioconversion of linoleic acid into conjugated linoleic acid during fermentation and by washed cells of Lactobacillus reuteri

Conjugated linoleic acid (CLA) was produced at 300 mg l^–1 after 24 h culture of Lactobacillus reuteri in de Man–Rogosa–Sharpe medium containing 0.9 g linoleic acid (LA) l^–1 and 1.67% (v/v) Tween 80. CLA was mainly located in the extracellular space of the cells. Washed cells previously grown on LA were less active than unadapted washed cells in converting LA into CLA. Most of the CLA transformed by washed L. reuteri cells was located in cells or associated with cells. CLA production by washed L. reuteri cells was most efficient in conversion with 0.45 g LA l^–1 at pH 9.5 and 37°C for 1 h.     

Production of conjugated linoleic acid by isolated Bifidobacterium strains

Two isolates from Korean faecal samples converted linoleic acid (LA) into conjugated linoleic acid (CLA), and were identified as Bifidobacterium breve and Bifidobacterium pseudocatenulatum by analysis of 16S rRNA sequences. In both cases, the major CLA was the cis-9, trans-11 isomer and CLA production paralleled the increase in cell biomass with both bacteria and was maximal at 30 h. The quantities of supernatant CLA produced by B. breve and B. pseudocatenulatum were 160 and 135 mg l^–1, from 500 mg LA l^–1, respectively. In the presence of 0.05% LA, the growth of B. breve was weakly inhibited but that of B. pseudocatenulatum was not affected over 6 days fermentation. During fermentation, the majority of CLA isomers were in the culture supernatant, but with washed cells obtained at the early stationary phase, 36 h, about 40% was detected in the cellular lipid. The optimal culture age with equal concentrations of washed cells for CLA production by the two bifidobacteria was determined to be 36 h. At this culture age, total CLA conversion of supernatant and cell pellets was 78% in B. breve and 69% in B. pseudocatenulatum from 0.01% LA.     

Kinetics of microbial hydrogenation of free linoleic acid to conjugated linoleic acids

Aims: To investigate the ability of selected probiotic bacterial strains to produce conjugated linoleic acid (CLA) and also to estimate the biohydrogenation kinetics of Lactobacillus acidophilus on the production of CLA from free linoleic acid (LA). Methods and Results: Six probiotic bacteria, Lact. paracasei, Lact. rhamnosus GG, Lact. acidophilus ADH, and Bifidobacterium longum B6, Lact. brevis, and Lact. casei, were used to examine their ability to convert LA to CLA. LA tolerance was evaluated by addition of different LA concentrations in MRS broth. Lact. acidophilus showed the major tolerant to LA and the greatest CLA‐producing ability (36–48 μg ml^?1 of CLA). The rate‐controlling steps were k₂ and k₁ for the addition of 1 and 3 mg ml^?1 of LA, respectively. The percentage of CLA conversion was higher in MRS broth supplemented with 1 mg ml^?1 (65%) than 3 mg ml^?1 (26%). Conclusion: The results provide useful information and new approach for understanding the biohydrogenation mechanisms of CLA production. Significance and Impact of the Study: This study would help elucidate the pathway from LA to stearic acid (SA), known as biohydrogenation. In addition, the use of selected probiotic bacteria might lead to a significant improvement in food safety.     

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.     

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.     

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。     

Development and potential of starter lactobacilli resulting from exploration of the sourdough ecosystem

Lactic acid bacteria are widely used as starter organisms in food fermentations. The development of such cultures as organisms fulfilling all metabolic, technical and handling requirements is the result of a multidisciplinary approach, i.e. to analyse, follow and direct the microbial ecology in food fermentations by molecular biology tools, gene cloning, biochemical and physiological analyses, pilot trials and modelling of behaviour and metabolism. The possibilities and restrictions of such an approach is given for cereal fermentations, namely sourdoughs. In this environment highly adapted lactobacilli are predominant, sharing the environment with yeasts present in traditional preparations. The competitiveness of these lactobacilli and their contribution to flavour, machineability and prebiosis of doughs and bread relies on their maltose and amino acid metabolism, use of electron acceptors and EPS formation. Their reactions on environmental stresses can be used to embed these recalcitrant organisms into starter culture preparations. Beyond the cereal environment the described strategy can be generally applied to understand ecosystems in food fermentations and finally control them. This revised version was published online in August 2006 with corrections to the Cover Date.     

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。     

Contrasting apoptotic responses of conjugated linoleic acid in the liver of obese Zucker rats fed palm oil or ovine fat

We hypothesized that reducing weight properties of conjugated linoleic acid (CLA) are due to adipocyte apoptosis and that CLA differentially modulates the apoptotic responses in hepatic lipotoxicity from rats fed saturated fat diets. Obese Zucker rats were fed atherogenic diets (2% w/w of cholesterol) formulated with high (15% w/w) saturated fat, from vegetable or animal origin, supplemented or not with 1% of a mixture (1:1) of cis-9, trans-11 and trans-10, cis-12 CLA isomers for 14 weeks. CLA induced no changes on retroperitoneal fat depot weight, which was in line with similar levels of apoptosis. Interestingly, CLA had a contrasting effect on cell death in the liver according to the dietary fat. CLA increased hepatocyte apoptosis, associated with upregulation of Fas protein in rats fed palm oil, compared to rats receiving palm oil alone. However, rats fed ovine fat alone displayed the highest levels of hepatic cell death, which were decreased in rats fed ovine fat plus CLA. This reducing effect of CLA was related to positively restoring endoplasmic reticulum (ER) ATF-6α, BiP and CHOP protein levels and increasing phosphorylated c-Jun NH₂-terminal kinase (JNK) and c-Jun, thus suggesting an adaptive response of cell survival. These findings reinforce the role of CLA as regulator of apoptosis in the liver. Moreover, the dietary fat composition is a key factor in activation of apoptosis.     

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.     

Combination of conjugated linoleic acid with fish oil prevents age-associated bone marrow adiposity in C57Bl/6J mice

The inverse relationship between fat in bone marrow and bone mass in the skeleton of aging subjects is well known. However, there is no precise therapy for the treatment of bone marrow adiposity. We investigated the ability of conjugated linoleic acid (CLA) and fish oil (FO), alone or in combination, to modulate bone loss using 12 months old C57Bl/6J mice fed 10% corn oil diet as control or supplemented with 0.5% CLA or 5% FO or 0.5% CLA+5% FO for 6 months. We found, CLA-fed mice exhibited reduced body weight, body fat mass (BFM) and enhanced hind leg lean mass (HLLM) and bone mineral density (BMD) in different regions measured by dual energy x-ray absorptiometry (DXA); however, associated with fatty liver and increased insulin resistance; whereas, FO fed mice exhibited enhanced BMD, improved insulin sensitivity, with no changes in BFM and HLLM. Interestingly, CLA+FO fed mice exhibited reduced body weight, BFM, peroxisome proliferator-activated receptor gamma and cathepsin K expression in bone marrow with enhanced BMD and HLLM. Moreover, CLA+FO supplementation reduced liver hypertrophy and improved insulin sensitivity with remarkable attenuation of bone marrow adiposity, inflammation and oxidative stress in aging mice. Therefore, CLA with FO combination might be a novel dietary supplement to reduce fat mass and improve BMD.     

The effect of covalently bonded conjugated linoleic acid on the reduction of oxidative stress and blood coagulation for polysulfone hemodialyzer membrane

Conjugated linoleic acid (CLA) was covalently bonded to a layer of poly(acrylic acid) (PAA) grafted onto the surface of polysulfone (PSF) membranes. The effect of CLA-bonding on oxidative stress and blood coagulation was then evaluated. The surface was characterized with contact angle measurement and FTIR spectroscopy. Blood coagulation, platelet aggregation, and oxidative stress were evaluated using human blood. The complete blood count (CBC) and coagulation time (CT) were evaluated in vitro for hemocompatibility. The production of reactive oxygen species (ROS) was measured by the chemiluminescence (CL) method to evaluate the oxidative stress. The results showed that the CLA-bonding PSF membrane exhibited more stable CBC values, longer CT, and less adsorption of plasma proteins than the unmodified PSF membrane. In addition, the CL counts of hydrogen peroxide and superoxide values for CLA-bonding PSF membrane were more stable than for unmodified PSF membrane. These results demonstrate that CLA-bonding can improve the blood compatibility of PSF membrane. The CLA-bonding PSF membrane could offer protection for patients against oxidative stress and could also reduce the dosage of anticoagulant required during hemodialysis.     

Optimization and oxidative stability of the microencapsulated conjugated linoleic acid

We used response surface methodology to optimize the preparation conditions of conjugated linoleic acid (CLA) microcapsules for maximum entrapment efficiency. Three independent variables were used: the ratio of CLA core material to agar and waxy corn starch wall material (X₁), the temperature of dispersion fluid (X₂), and the concentration of emulsifier (X₃). The optimized values of X₁, X₂, and X₃ were found to be 3.82:6.18, 19.97 °C, and 0.34%, respectively. The CLA oxidation stability was significantly protected by microencapsulation. These results suggest that CLA-loaded microcapsules can be used as a means to enhance not only the entrapment efficiency but also the oxidative stability of CLA.     

Fed-batch fermentation of Lactobacillus lactis for hyper-production of l-lactic acid

A fed-batch fermentation of Lactobacillus lactis to produce l-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy. Up to 210 g l-lactic acid l^–1 (97% yield) was obtained. The maximal dry cell was 2.7 g l^–1 and the average l-lactic acid productivity was 2.2 g l^–1 h^–1.     

Double enrichment of chicken eggs with conjugated linoleic acid and n-3 fatty acids through dietary fat supplementation

Yolk fat fatty acid (FA) concentrations, sensory quality and firmness of eggs and laying hen performance were evaluated with respect to the combined inclusion in the diet of conjugated linoleic acid (CLA), high n-3 oil sources and high-oleic sunflower oil (HOSO). Nine diets were arranged factorially, with three levels of n-3 FA supplementation (2.9, 3.7 and 4.5 g/kg) from three different sources (two fish oils highly concentrated in eicosapentanoic (EPA) or docosahexanoic acid (DHA) and one algae oil with a very high-DHA content) in diets added with fixed amounts of CLA (2.5 g/kg) and HOSO (30 g/kg). A commercial feed with no CLA, n-3 or HOSO added, and another one containing 4.5 g/kg of high-DHA fish oil but not CLA or HOSO were also formulated. An increase in n-3 FA supplementation had little effect on proportions of CLA, monounsaturated FA, saturated FA or total polyunsaturated FA in yolk fat, but increased (P<0.005) long-chain n-3 FA and decreased (P<0.001) long-chain n-6 FA. An increment of dietary n-3 FA also impaired linearly (P<0.001) egg acceptability by consumers. An increment in the proportion of DHA with respect to total n-3 FA from 0.28 to 0.96 increased yolk concentrations of DHA (P<0.001) and total n-3 FA (P<0.01), but decreased (P<0.001) concentrations of EPA and docosapentanoic acid FA. Current data indicate that addition of HOSO to diets supplemented with moderate amounts of CLA and n-3 FA allows the production of double enriched eggs while maintaining sensory quality for consumers at acceptable levels.     

Effect of conjugated linoleic acid on dietary lipids utilization, liver morphology and selected immune parameters in sea bass juveniles (Dicentrarchus labrax)

Increased energy content in fish feeds has led to an enhanced fat deposition, particularly in European sea bass, concerning fish farmers. Inclusion of conjugated linoleic acid (CLA) could reduce fat deposition as in other vertebrates. To determine if dietary CLA affects fat deposition, lipid metabolism, lipid composition and morphology of different tissues, growth and selected immune parameters, European sea bass juveniles were fed 4 graded levels of CLA (0, 0.5, 1 and 2%). Growth and feed conversion were not affected by CLA, whereas feed intake was reduced (P < 0.05) by feeding 2% CLA. In these fish perivisceral fat was also reduced (P < 0.05), particularly reducing (P < 0.05) monounsaturated fatty acids. CLA has not affected tissue proximal composition, but reduced (P < 0.05) saturated and monounsaturated fatty acids and increased (P < 0.05) the n−3 and n−3 highly unsaturated fatty acids in muscle and increase (P < 0.05) CLA content in muscle, liver and perivisceral fat. A progressive reduction in lipid vacuolization of hepatocytes cytoplasm and regular-shaped morphology was found in fish fed increased CLA levels, together with a progressive increase in malic enzyme activity (only significant in fish fed 1% CLA). Finally, inclusion of CLA up to 1% increased (P < 0.05) plasma lysozyme activity and was positively correlated with alternative complement pathway.