Enzyme from Padina arborescens Holmes Synthesizes Parinaric Acid, a Conjugated Tetraenoic Fatty Acid, from α-Linolenic Acid.

Conjugated fatty acids have anticancer effects. Therefore, the establishment of a synthetic method for conjugated fatty acids is important for overcoming cancer. Here, we attempted to synthesize conjugated fatty acids using enzymes extracted from seaweeds containing these fatty acids. Lipids from 12 species of seaweeds from the seas around Japan were analyzed, and Padina arborescens Holmes was found to contain conjugated fatty acids. Then, we synthesized parinaric acid, a conjugated tetraenoic acid, from α-linolenic acid using the enzyme of P. arborescens. This method is expected to have a variety of potential applications for overcoming cancer.

Effect of pomegranate seed oil on egg production, egg quality and yolk fatty acid deposition in laying hen.

BACKGROUND: Pomegranate seed oil (PSO) contains punicic acid as well as conjugated linolenic acid isomers, including α-eleostearic and catalpic acids, along with phytosterols, especially ß-sitosterol, campesterol and stigmasterol, with lipotropic impact and egg fortifying effect in laying hens. OBJECTIVES: The present experiment was designed to examine the effects of PSO on egg production, egg quality, blood lipids and yolk fatty acid deposition in laying hens. METHODS: A total of 360 Hy-line laying hens (w-80), at 25 weeks of age, were randomly allotted to five dietary treatments in a completely randomized design during a 10-week period. Experimental treatments consisted of a basal diet or supplementation of 1, 2, 3 and 4 g PSO/kg to basal diet. Performance indicators and fatty acid composition of egg yolk were evaluated during different experimental periods. Blood lipid attributes were evaluated at the end of the experiment. RESULTS: Dietary supplementation of 4 g PSO/kg feed increased daily feed intake and egg production rate of laying hens (p < 0.05). There was not any significant influence of experimental treatments on egg quality, whereas an increasing trend observed in egg yolk colour of hens received dietary graded levels of PSO. Dietary supplemental 4 g/kg PSO increased the proportion of yolk poly-unsaturated fatty acid (PUFA) concentration (p < 0.05). Furthermore, the PUFA to saturated fatty acid ratio increased after dietary supplementation of 1 or 4 g/kg PSO (p < 0.05). Serum concentration of cholesterol, triacylglycerol and low-density lipoprotein decreased in response to the supplementation of more than 3 g PSO/kg in the feed (p < 0.05). CONCLUSIONS: In conclusion, dietary supplementation with 4 g/kg PSO improved production rate and decreased blood lipids in laying hens. Moreover, dietary supplemental PSO modified yolk fatty acid deposition without detrimental effects on the egg quality.

Health-Promoting Ingredients in Goat's Milk and Fermented Goat's Milk Drinks.

The present study aimed to determine the content of health-promoting compounds, and fatty acids, with particular emphasis on the content of cis9trans11 C18:2 (CLA) acid, selected minerals, folates in organic and commercial goat's milk and fermented goat's milk drinks. The analyzed milk and yoghurts had various contents of particular groups of fatty acids, CLA, minerals, and folates. Raw organic goat's milk had a significantly (p < 0.05) higher content of CLA (3.26 mg/g fat) compared to commercial milk (2.88 mg/g fat and 2.54 mg/g fat). Among the analyzed fermented goat's milk drinks, the highest CLA content (4.39 mg/g fat) was determined in commercial natural yoghurts, while the lowest one was in organic natural yoghurts (3.28 mg/g fat). The highest levels of calcium (1322.9-2324.4 µg/g), phosphorus (8148.1-11,309.9 µg/g), and copper (0.072-0.104 µg/g) were found in all commercial products and those of manganese (0.067-0.209 µg/g) in organic products. The contents of the other assayed elements (magnesium, sodium, potassium, iron, and zinc) did not depend on the production method, but only on the product type, i.e., the degree of goat's milk processing. The highest folate content in the analyzed milks was found in the organic sample (3.16 µg/100 g). Organic Greek yoghurts had a several times higher content of folates, reaching 9.18 µg/100 g, compared to the other analyzed fermented products.

Determination of triacylglycerol and fatty acid compositions of Impatiens seed oils using reverse phase high performance liquid chromatography.

In the present work, triacylglycerol and fatty acid compositions of Impatiens balsamina L. and Impatiens walleriana Hook.f. seed oils were determined using reverse phase high performance liquid chromatography with both refractive index and spectrophotometric detections. The presence of conjugated octadecatetraenoic moieties was confirmed by UV and IR spectroscopy. Triacylglycerol (TAG) compositions were performed using an incremental approach and confirmed by the results of MS and electronic spectra. The quantitative analysis of TAG was achieved by careful calibration, introducing correction factors for the sensitivity of each compound. The results showed that both Impatiens seed oils contain the same 23 TAGs. The mole fraction of 15 TAGs containing conjugated moieties was more significant than 88% (for Impatiens balsamina L.) and 81% (for Impatiens walleriana Hook.f.). Seed oils of Impatiens balsamina and Impatiens walleriana contain 43.44% and 36.12% mole of conjugated octadecatetraenoic fatty acids, respectively. These conjugated fatty acids were determined to be α-parinaric (C18:49Z,11E,13E,15Z) and ß-parinaric (C18:49Z,11E,13E,15Z), in which isomer ß-parinaric represents 23.21% and 26.27% of conjugated octadecatetraenoic acids for I. balsamina and I. walleriana seed oils, respectively. In addition, the mole fraction of α-linolenic acid in both seed oils was also abundant at 24.5% and 28.2% for I. balsamina and I. walleriana. Therefore, I. balsamina L.and I. walleriana seed oils are potential sources of polyunsaturated fatty acids, especially conjugated octadecatetraenoic acids.

Improving the Production of Punicic Acid in Baker's Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply.

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, feeding, and oleochemical industries. Since the production of PuA is severely limited by the fact that its natural source (pomegranate seed oil) is not readily available on a large scale, there is considerable interest in understanding the biosynthesis and accumulation of this plant-based unusual fatty acid in transgenic microorganisms to support the rational design of biotechnological approaches for PuA production via fermentation. Here, we tested the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created an effective "push-pull" approach to increase PuA content, which could prove valuable in future efforts to produce PuA in industrial yeast and other microorganisms via fermentation.

Production of conjugated fatty acids: A review of recent advances.

Conjugated fatty acids (CFAs) have received a deal of attention due to the increasing understanding of their beneficial physiological effects, especially the anti-cancer effects and metabolism-regulation activities. However, the production of CFAs is generally difficult. Several challenges are the low CFAs content in natural sources, the difficulty to chemically synthesize target CFA isomers in high purity, and the sensitive characteristics of CFAs. In this article, the current technologies to produce CFAs, including physical, chemical, and biotechnical approaches were summarized, with a focus on the conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) which are the most common investigated CFAs. CFAs usually demonstrate stronger physiological effects than other non-conjugated fatty acids; however, they are more sensitive to heat and oxidation. Consequently, the quality control throughout the entire production process of CFAs is significant. Special attention was given to the micro- or nano-encapsulation which presented as an emerging technique to improve the bioavailability and storage stability of CFAs. The current applications of CFAs and the potential research directions were also discussed.

Response of high leaf-oil Arabidopsis thaliana plant lines to biotic or abiotic stress.

Recent studies have shown that it is possible to engineer substantial increases in triacylglycerol (TAG) content in plant vegetative biomass, which offers a novel approach for increasing the energy density of food, feed, and bioenergy crops or for creating a sink for the accumulation of unusual, high-value fatty acids. However, whether or not these changes in lipid metabolism affect plant responses to biotic and/or abiotic stresses is an open question. Here we show that transgenic Arabidopsis thaliana plant lines engineered for elevated leaf oil content, as well as lines engineered for accumulation of unusual conjugated fatty acids in leaf oil, had similar short-term responses to heat stress (e.g., 3 days at 37°C) as wild-type plants, including a reduction in polyunsaturated fatty acid (PUFA)-containing polar lipids and an increase in PUFA-containing neutral lipids. At extended time periods (e.g., 14 days at 37°C), however, plant lines containing accumulated conjugated fatty acids displayed earlier senescence and plant death. Further, no-choice feeding studies demonstrated that plants with the highest leaf oil content generated cabbage looper (Trichoplusia ni) insects with significantly heavier body weights. Taken together, these results suggest that biotic and abiotic responses will be important considerations when developing and deploying high-oil-biomass crops in the field.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 µM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 µM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.

Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2.

There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, ß-eleostearic (9E,11E,13E-18:3) and ß-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49nM and ~125nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1µM, respectively. By contrast, COX-2 was ≤30% inhibited at 10µM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data.

Research on food and nutrition characteristics of conjugated fatty acids.

In this study, the physiological effects of fatty acids with conjugated double bonds were widely examined in vitro and in vivo. Initially, a method for determination of conjugated fatty acids in food and biological samples was established. I then clarified that the oxidative stability of conjugated fatty acids was improved by the form of triacylglycerol and addition of an antioxidant, and the influence of this effect on the metabolism and pharmacokinetics of conjugated fatty acids was clarified in vivo. In addition, antitumor, anti-angiogenesis, and antiobesity effects of conjugated fatty acids were found for the first time, thus demonstrating the usefulness of conjugated fatty acids. This communication mainly outlines the data obtained for conjugated linolenic acid. In addition, this review summarizes my research on conjugated fatty acid.

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria.

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

Quality traits and modeling of coagulation, curd firming, and syneresis of sheep milk of Alpine breeds fed diets supplemented with rumen-protected conjugated fatty acid.

The aim of this study was to test the modeling of curd-firming (CF) measures and to compare the sheep milk of 3 Alpine breeds supplemented with or without rumen-protected conjugated linoleic acid (rpCLA). Twenty-four ewes of the Brogna, Foza, and Lamon breeds were allotted to 6 pens (2 pens/breed) and fed a diet composed of corn grain, corn silage, dried sugar beet pulp, soybean meal, wheat bran, wheat straw, and a vitamin-mineral mixture. The rpCLA supplement (12 g/d per ewe plus 4 g/d for each lamb older than 30 d) was mixed into the diet of 1 pen per sheep breed (3 pens/treatment) to provide an average of 0.945 and 0.915 g/d per ewe of the cis-9,trans-11 C18:2 and trans-10,cis-12 C18:2 conjugated linoleic acid isomers, respectively. The trial started at 38 ± 23 d after parturition, and individual morning milk samples were collected on d 16, 23, 37, 44, and 59 of the trial. Milk samples were analyzed for composition, and duplicate samples were assessed for milk coagulation properties (MCP). A total of 180 CF measures for each sample (1 every 15s) were recorded. Model parameters were the rennet coagulation time, the asymptotic potential CF, the CF instant rate constant, the syneresis instant rate constant, the maximum CF achieved within 45 min (CFmax), and the time at achievement of CFmax. The data were analyzed using a hierarchical model that considered the fixed effects of breed, diet, lamb birth, and initial days in milk, which were tested on individual ewe (random) variance; the fixed effect of sampling day, which was tested on the within-ewe sample (random) variance; and the fixed effect of instrument or cuvette position (only for MCP), which was tested on the residual (replicates within samples) variance. The local Alpine sheep breeds displayed similar milk compositions, traditional MCP, and CF modeling parameters. Supplementation with rpCLA triggered changes in milk composition and worsened MCP (e.g., delayed rennet coagulation time, slower CF instant rate constant, and a doubling of syneresis instant rate constant), but did not influence potential CF. Overall, our results indicate that rpCLA supplementation reduced the actual maximum CF (CFmax) but did not modify the interval between rennet addition and CFmax or time to CFmax.

Avaliação em ratos do efeito do óleo da semente de romã ( Punica granatum L. ) sobre o perfil lipídico tecidual e sua influência sobre parâmetros bioquímicos em processos oxidativos / Evaluation of the effects of pomegranate seed oil (Punica granatum L. ) on tissue lipid profile and its influence on biochemical parameters in oxidative processes of rats

O objetivo geral deste estudo foi avaliar, em ratos, o efeito do óleo da semente de romã (PSO) sobre o perfil lipídico tecidual e sua influência sobre parâmetros bioquímicos em processos oxidativos. Foi realizada a caracterização do PSO, confirmando a presença do ácido punícico (PA; 55%) como ácido graxo majoritário e a alta concentração de fitosteróis (539mg/100g), bem como a presença de vitamina E (175mg/100g). O PSO apresentou-se dentro dos padrões de qualidade e a sua estabilidade oxidativa foi melhor em comparação ao óleo de linhaça. A suplementação de ratos saudáveis com o PSO, por via intragástrica durante 40 dias, não afetou o ganho de peso total e o peso dos tecidos muscular (gastrocnêmio) e adiposos (epididimal e retroperitonial). No entanto o PA foi metabolizado e incorporado na forma de ácido linoléico conjugado, sendo dose-dependete nos tecidos hepático, muscular, cardíaco, renal e adiposos. No cérebro, não foram observados ácidos graxos conjugados, mas as substâncias reativas ao ácido tiobarbitúrico (TBARS) apresentaram-se significativamente reduzidas nos animais suplementados com PSO, em relação ao controle. De modo geral, os resultados mostram que o PSO não provoca alterações no metabolismo lipídico e não participa do processo de inibição da oxidação em animais saudáveis. Em ratos submetidos ao estresse oxidativo hepático pelo tetracloreto de carbono (CCl4), a suplementação com PSO durante 21 dias não foi capaz de prevenir o quadro de estresse oxidativo, indicando que este óleo não tem efeito antioxidante utilizando esse modelo animal; embora a análise histológica tenha mostrado menores áreas lesionadas no parênquima hepático nos grupos tratados. Os resultados obtidos neste trabalho contribui com a literatura fornecendo mais informações a respeito do uso dos ácidos graxos conjugados, bem como do PSO em organismos saudáveis e submetidos à estresse oxidativo

The aim of this study was to evaluate the effect of pomegranate seed oil (PSO) on tissue lipid profile and its influence on biochemical parameters in oxidative processes of Wistar rats. Characterization of PSO was carried out, confirming the presence of the punicic acid (PA, 55%) as the major fatty acid present in the oil and high concentrations of phytosterols (539mg/100g) were also observed, as well as the presence of vitamin E (175mg/100g). The PSO was within quality standards and it presented a higher oxidative stability as compared to flaxseed oil. The supplementation of healthy rats with the PSO via gavage during 40 days did not affect weight gain and total weight of muscle (gastrocnemius) and adipose (epididymal and retroperitoneal) tissues. However, PA was metabolized and incorporated as CLAs in a dose-dependent manner in the liver, muscle, heart, kidney and adipocytes. In the brain, conjugated fatty acids were not detected, but the values of thiobarbituric acid reactive substances were significantly reduced in animals supplemented with PSO as compared to the control group. Overall, the results showed that the PSO caused no changes in the lipid metabolism and did not inhibit tne oxidation in healthy animals. In rats that underwent hepatic oxidative stress by carbon tetrachloride (CCl4), the PSO supplementation for 21 days was not able to prevent the oxidative stress, indicating that this oil has no antioxidant effect using this animal model; although histological analysis has shown less injured areas in the liver parenchyma in the test groups. The results obtained in this study are a good addition to the literature once it provided more information about the use of conjugated fatty acids as well as garnered useful information about the effects of consumption of PSO in oxidative stress-induced rats