Preparation of Highly Pure n-3 PUFA-Enriched Triacylglycerols by Two-Step Enzymatic Reactions Combined with Molecular Distillation

Highly pure n-3 polyunsaturated fatty acids (PUFA)-enriched triacylglycerols (TAG) have attracted considerable attention due to their nutritional benefits and pharmacological effects. In this study, an alternative approach to the conventional method for the synthesis of highly pure n-3 PUFA-enriched TAG by using a multi-step process was reported. First, glyceride mixtures were synthesized through Novozym 435 [Novozymes A/S (Bagsvaerd, Denmark)] catalyzed esterification of n-3 PUFA-enriched FA and glycerol. Second, partial glycerides in the resulting glyceride mixtures were hydrolyzed to FA by immobilized partial glycerides-selective lipase from Malassezia globosa. The purity of TAG reached 99.84% under the optimized conditions: buffer solution of pH 6.0, water content of 100% (w/w, with respect to the oil mass), enzyme loading of 120 U/g (U/w, with respect to oil mass) and reaction temperature of 30 °C. During hydrolysis, the immobilized SMG1-F278N exhibited good reusability and TAG purity of over 94% was maintained after being used for six cycles. Subsequently, purification of TAG was accomplished by molecular distillation at low temperature (150 °C) and highly pure (99.85%) TAG with 88.73% n-3 PUFA was obtained. The final glyceride mixtures with low acid, peroxide and anisidine value were promising products for medical and dietetic purposes. Compared with the conventional one-step synthesis of n-3 PUFA-enriched TAG by enzymatic esterification or glycerolysis or the two-step method by combined transesterification and ethanolysis, this improved process allows for higher purity of n-3 PUFA-enriched TAG and significant reduction in reaction time.     

Hypolipidemic Effects of Phospholipids (PL) Containing n-3 Polyunsaturated Fatty Acids (PUFA) Are Not Dependent on Esterification of n-3 PUFA to PL

Phospholipids (PL) containing n‐3 polyunsaturated fatty acids (PUFA) have beneficial effects of maintaining and promoting health compared with triacylglycerols (TAG) containing n‐3 PUFA or general PL. This study evaluated the effects of dietary PL containing n‐3 PUFA and elucidated the effects of the glycerophosphate structure and n‐3 PUFA on fatty acid (FA) metabolism in rats. Rats were fed a basal diet containing soybean oil alone, TAG containing n‐3 PUFA (1.8 %), soybean PL (2.7 %), PL containing n‐3 PUFA (2.7 %), or TAG containing n‐3 PUFA (1.8 %) + soybean PL (2.7 %). The present n‐3 PUFA‐supplemented diets had similar FA compositions, and the PL diets had similar PL compositions. TAG containing n‐3 PUFA reduced serum TAG contents, but did not affect serum cholesterol contents compared with soybean oil alone. PL diets containing n‐3 PUFA and the combination of TAG containing n‐3 PUFA and soybean PL resulted in decreased serum and liver TAG contents compared with the diet containing soybean oil alone, reflecting enhanced liver FA β‐oxidation. The results of this study show that TAG containing n‐3 PUFA with added soybean PL affects serum and liver TAG and cholesterol contents to a similar degree as PL containing n‐3 PUFA. TAG containing n‐3 PUFA and soybean PL are widely used as functional food ingredients and pharmaceutical constituents and are inexpensive compared with PL containing n‐3 PUFA. Therefore, the combination of TAG containing n‐3 PUFA and soybean PL has potential as a useful and inexpensive component of functional foods.     

n-3 Polyunsaturated Fatty Acids Improve Inflammation via Inhibiting Sphingosine Kinase 1 in a Rat Model of Parenteral Nutrition and CLP-Induced Sepsis

The sphingosine kinase 1 (SphK1)/sphingosine‐1‐phosphate (S1P) pathway plays a key role in inflammation. Parenteral nutrition containing n‐3 polyunsaturated fatty acids (n‐3 PUFA) may regulate inflammatory reactions. The aim of this study is to determine whether n‐3 PUFA may improve inflammatory responses by neutralizing SphK1 signaling. Rat models of parenteral nutrition, cecal ligation and puncture (CLP)‐induced sepsis were generated. Male Sprague–Dawley rats were operated for CLP on day 2 after venous catheterization. The rats were randomized to receive normal saline (NS; n = 20), parenteral nutrition (PN; n = 20), or PN + fish oil (FO; n = 20) for 5 days. The daily intake of fish oil (1.25–2.82 g EPA and 1.44–3.09 g DHA per 100 ml) in the FO group was approximately 1.8 g/kg body weight/day. Rats in the control group (n = 10) were subjected to sham operation and received a chow diet. Spleen tissues were collected for SphK1 and S1P receptor expression analysis. Our data showed that n‐3 PUFA ameliorated the survival rate. SphK1 expression and its enzymatic activity were significantly upregulated in sepsis rats. Furthermore, mRNA and protein levels of S1PR3, but not S1PR1, were also facilitated after CLP. However, PN + FO dramatically decreased SphK1 mRNA level and its enzymatic activity. S1PR3 expression was also attenuated by FO addition. In conclusion, the anti‐inflammatory effect of n‐3 PUFA may be linked to the inhibition of the SphK1/S1P pathway in a rat model of parenteral nutrition and CLP‐induced sepsis.     

Dietary Unsaturated Fatty Acids Modulate Maternal Dyslipidemia‐Induced DNA Methylation and Histone Acetylation in Placenta and Fetal Liver in Rats

The present study assessed the role of dietary unsaturated fatty acids in maternal dyslipidemia‐induced DNA methylation and histone acetylation in placenta and fetal liver and accumulation of lipids in the fetal liver. Weanling female Wistar rats were fed control and experimental diets for 2 months, mated, and continued on their diets during pregnancy. At gestation days of 18–20, rats were euthanized to isolate placenta and fetal liver. DNA methylation, DNA methyl transferase‐1 (DNMT1) activity, acetylation of histones (H2A and H2B), and histone acyl transferase (HAT) activity were evaluated in placenta and fetal liver. Fetal liver lipid accumulation and activation of peroxisome proliferator‐activated receptor‐α (PPAR‐α) were assessed. Maternal dyslipidemia caused significant epigenetic changes in placenta and fetal liver. In the placenta, (1) global DNA methylation increased by 37% and DNMT1 activity by 86%, (2) acetylated H2A and H2B levels decreased by 46% and 24% respectively, and (3) HAT activity decreased by 39%. In fetal liver, (1) global DNA methylation increased by 52% and DNMT1 activity by 78%, (2) acetylated H2A and H2B levels decreased by 28% and 26% respectively, and (3) HAT activity decreased by 37%. Maternal dyslipidemia caused a 4.75‐fold increase in fetal liver triacylglycerol accumulation with a 78% decrease in DNA‐binding ability of PPAR‐α. Incorporation of dietary unsaturated fatty acids in the maternal high‐fat diet significantly (p < 0.05) modulated dyslipidemia‐induced effects in placenta and fetal liver. Eicosapentaenoic acid (EPA, 20:5n‐3) + docosahexaenoic acid (DHA, 22:6n‐3) exhibited a profound effect followed by alpha‐linolenic acid (ALA, 18:3n‐3) than linoleic acid (LNA, 18:2n‐6) in modulating the epigenetic parameters in placenta and fetal liver.     

Optimization of Lipase-Catalyzed Interesterification of Flaxseed Oil and Tricaprylin Using Response Surface Methodology

The biosynthesis of structured lipids (SL) in organic solvent media was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC), using Novozym 435. The bioconversion yield (BY, %) of medium-long-medium type SL, including C-caprylic and Ln-linolenic acids (CLnC), La-linoleic acid (CLaC) and O-oleic acid (COC), was monitored. Response surface methodology was used to obtain significant models for the responses, on the basis of a five level, five variable central composite rotatable design. In the experimental preliminary trials significant reaction parameters, including reaction temperature (T _r), TC/FO molar ratio (M _r), enzyme concentration (E _c), reaction time (R _t) and initial water activity (a _w), were considered for optimization. Significant models for CLnC, CLaC and COC were determined after regression analysis with backward elimination. The optimal conditions, generated for a maximum CLnC, CLaC and COC, were found to be 54.50–56.25 °C for T _r, 6.23–6.25 mol/mol for M _r, 2.68–3.13 % for E _c, 36.58–37.50 h for R _t and 0.15–0.33 for a _w. Under these optimum conditions, the BY of CLnC, CLaC and COC was predicted to be 32.48–36.67, 3.26–3.38 and 5.79–6.16 %, respectively.     

Fish Oil Decreases C-Reactive Protein/Albumin Ratio Improving Nutritional Prognosis and Plasma Fatty Acid Profile in Colorectal Cancer Patients

Previous studies have shown that n‐3 polyunsaturated fatty acids n‐3 (n‐3 PUFA) have several anticancer effects, especially attributed to their ability to modulate a variety of genomic and immune responses. In this context, this randomized, prospective, controlled clinical trial was conducted in order to check whether supplementation of 2 g/day of fish oil for 9 weeks alters the production of inflammatory markers, the plasma fatty acid profile and the nutritional status in patients with colorectal cancer (CRC). Eleven adults with CRC in chemotherapy were randomized into two groups: (a) supplemented (SG) daily with 2 g/day of encapsulated fish oil [providing 600 mg/day of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)] for 9 weeks (n = 6), and (b) control (CG) (n = 5). All outcomes were evaluated on the day before the first chemotherapy session and 9 weeks later. Plasma TNF‐α, IL‐1β, IL‐10 and IL‐17A, the pro/anti‐inflammatory balance (ratio TNF‐α/IL‐10 and IL‐1β/IL10) and serum albumin, showed no significant changes between times and study groups (p > 0.05). C‐reactive protein (CRP) and the CRP/albumin ratio showed opposite behavior in groups, significantly reducing their values in SG (p < 0.05). Plasma proportions of EPA and DHA increased 1.8 and 1.4 times, respectively, while the ARA reduced approximately 0.6 times with the supplementation (9 weeks vs baseline, p < 0.05). Patients from SG gained 1.2 kg (median) while the CG lost ?0.5 kg (median) during the 9 weeks of chemotherapy (p = 0.72). These results demonstrate that 2 g/day of fish oil for 9 weeks of chemotherapy improves CRP values, CRP/albumin status, plasma fatty acid profile and potentially prevents weight loss during treatment.     

Optimization of Two-Step Biodiesel Production from Beef Tallow with Microwave Heating

Animal fats are by-products from slaughterhouses that may be utilized as renewable energy source. This study was about biodiesel production from high free fatty acid beef tallow waste using two-step process with microwave heating. Sulfuric acid and NaOH were used as catalysts with methanol for the first esterification and second transesterification step, respectively. Catalyst loadings were between 0.25% and 2.5%, with applied microwave power of 340?W, operation time of 10–50?min, and oil-to-methanol molar ratio between 1:3 and 1:15. These process parameters were optimized using the design of experiments. The yields and properties of the biodiesel were assessed. The results indicated that the two-step process were successful in converting the beef tallow to biodiesel. Statistical analysis of the results showed that significant contributions were from the linear and quadratic terms of these three variables. The optimum conditions for esterification and transesterification were reported. Validity of the predicted models was confirmed by the experimental verification.     

Dietary High‐Oleic Acid Soybean Oil Dose Dependently Attenuates Egg Yolk Content of n‐3 Polyunsaturated Fatty Acids in Laying Hens Fed Supplemental Flaxseed Oil

Chickens can hepatically synthesize eicosapentaenoic acid (20:5 n‐3) and docosahexaenoic acid (22:6 n‐3) from α‐linolenic acid (ALA; 18:3 n‐3); however, the process is inefficient and competitively inhibited by dietary linoleic acid (LNA; 18:2 n‐6). In the present study, the influence of dietary high‐oleic acid (OLA; 18:1 n‐9) soybean oil (HOSO) on egg and tissue deposition of ALA and n‐3 polyunsaturated fatty acids (PUFA) synthesized from dietary ALA was investigated in laying hens fed a reduced‐LNA base diet supplemented with high‐ALA flaxseed oil (FLAX). We hypothesized that reducing the dietary level of LNA would promote greater hepatic conversion of ALA to very long‐chain (VLC; >20C) n‐3 PUFA, while supplemental dietary HOSO would simultaneously further enrich eggs with OLA without influencing egg n‐3 PUFA contents. Nine 51‐week‐old hens each were fed 0, 10, 20, or 40 g HOSO/kg diet for 12 weeks. Within each group, supplemental dietary FLAX was increased every 3 weeks from 0 to 10 to 20 to 40 g/kg diet. Compared to controls, dietary FLAX maximally enriched the total n‐3 and VLC n‐3 PUFA contents in egg yolk by 9.4‐fold and 2.2‐fold, respectively, while feeding hens 40 g HOSO/kg diet maximally attenuated the yolk deposition of ALA, VLC n‐3 PUFA, and total n‐3 PUFA by 37, 15, and 32%, respectively. These results suggest that dietary OLA is not neutral with regard to the overall process by which dietary ALA is absorbed, metabolized, and deposited into egg yolk, either intact or in the form of longer‐chain/more unsaturated n‐3 PUFA derivatives.     

Effect of phospholipid n‐3 polyunsaturated fatty acids on rat lipid metabolism

It has been demonstrated that the amount and type of dietary fat are factors involved in the risk of arteriosclerosis and coronary or cerebral artery disease through lipid metabolism. In this study, we investigated the effects of phospholipids (PLs) containing n‐3PUFAs on lipid metabolism in rats. PLs containing n‐3PUFAs were prepared from squid (Todarodes pacificus) mantle muscle. Groups of male Wistar rats were fed AIN93G diet containing soybean oil (SO, 7%), fish oil (1.2%) + SO (5.8%), soybean PLs (1.8%) + SO (5.2%), or PLs containing n‐3PUFAs (1.8%) + SO (5.2%). The following indicators were assayed as indexes of lipid metabolism: TAG and cholesterol in serum and liver, fecal cholesterol, bile‐acid excretion, and liver mRNA expression levels of genes encoding proteins involved in cholesterol homeostasis. Serum and liver TAG contents decreased significantly in the group fed PLs containing n‐3PUFAs as compared to other groups, accompanied by a significant decline in the expression level of sterol regulatory element binding protein‐1c. The decrease in cholesterol content in the group fed PLs containing n‐3PUFAs was due to the increase in fecal cholesterol excretion and the increase of mRNA expression levels of ATP‐binding cassette (ABC) G5 and ABCG8 in liver. Practical applications : PLs containing n‐3PUFAs decreased serum and liver TAG contents compared with that induced by soybean PLs. Further, PLs containing n‐3PUFAs can induce a reduction in serum and liver cholesterol concentrations as well as the triglyceride‐reducing effect of conventional n‐3PUFAs containing TAG. In other words, dietary n‐3PUFAs contained in PLs can prevent life‐style diseases such as hyperlipidemia, arteriosclerosis and coronary, or cerebral artery disease more effectively than TAG containing n‐3PUFAs. Therefore, it is expected that the risk of lifestyle diseases would be decreased if PL containing n‐3PUFAs can be supplied routinely. In this study, PLs containing n‐3PUFAs were prepared from squid mantle muscle. On an industrial scale, such PLs can be produced from various unused resources and waste materials of fisheries. We conclude that highly functional foods could be developed based on the findings of this study, and would be available for health promotion worldwide.     

Dietary Fatty Acids Differentially Regulate Secretion of Adiponectin and Interleukin‐6 in Primary Canine Adipose Tissue Culture

The aim of this study was to determine the effect of n3 polyunsaturated fatty acids (PUFA) on canine adipose tissue secretion of adiponectin, interleukin‐6 (IL6), and tumor necrosis factor‐α (TNFα). Subcutaneous and omental visceral adipose tissue samples were collected from 16 healthy intact female dogs. Concentrations of adiponectin were measured in mature adipocyte cultures, and concentrations of IL6 and TNFα were measured in undifferentiated stromovascular cell (SVC) cultures following treatment with eicosapentaenic acid (EPA, 20:5n‐3), arachidonic acid (ARA, 20:4n‐6), or palmitic acid (PAM, 16:0) at 25, 50, or 100 μM. Secretion of adiponectin from mature adipocytes was higher (p < 0.001) following EPA treatment at 50 μM compared to control in subcutaneous tissue, and higher following EPA treatment compared to PAM treatment at 25 μM in both subcutaneous (p < 0.001) and visceral tissues (p = 0.010). Secretion of IL6 from SVC derived from subcutaneous tissue was lower following EPA treatment and higher following PAM treatment compared to control both at 50 μM (p = 0.001 and p = 0.041, respectively) and 100 μM (p = 0.013 and p < 0.001, respectively). These findings of stimulation of adiponectin secretion and inhibition of IL6 secretion by EPA, and stimulation of IL6 secretion by PAM, are consistent with findings of increased circulating concentrations of adiponectin and decreased circulating concentration of IL6 in dogs supplemented with dietary fish oil, and show that the effect of fish oil on circulating concentrations of adiponectin and IL6 is, at least partially, the result of local effects of EPA and PAM on adipose tissue.     

Effect of Dietary Replacement of Soybean Oil with Different Sources of Gamma‐Linolenic Acid on Fatty Acid Composition of Nile Tilapia

Gamma‐linolenic acid (GLA) plays an important role in the prevention and/or treatment of certain diseases. In this work, we investigate the incorporation of GLA from supplemented feed diets with borage oil (BO) and evening primrose oil (EPO) as substitutes for soybean oil (SO) into the composition of tilapia fillet lipids. High contents of PUFA and n‐6 fatty acids were quantified in fish fillet after 30 days of treatment with SO, BO, and EPO. Feed diets containing BO and EPO were efficient in the incorporation of GLA into fish. Compared to the initial day of the experiment, the increase of GLA was significant (from 6.43 to 13.99 and 15.12 mg g^?1, in lipids of fish treated for 30 days with BO and EPO, respectively). The increase of GLA was also observed in fish which were fed with SO diet (6.43–11.43 mg g^?1). Principal component analysis (PCA) allowed the separation of the treatments and discriminated BO and EPO in a group of fish that received the GLA supplemented diet. In addition to GLA, n‐3 fatty acids were important in the characterization of SO diet and affected the separation of BO and EPO from SO in the PCA score plot.     

Cell Proliferation and Long Chain Polyunsaturated Fatty Acid Metabolism in a Cell Line From Southern Bluefin Tuna (Thunnus maccoyii)

Southern bluefin tuna (SBT, Thunnus maccoyii) aquaculture is a highly valuable industry, but research on these fish is hampered by strict catch quotas and the limited success of captive breeding. To address these limitations, we have developed a SBT cell line (SBT-E1) and here we report on fatty acid metabolism in this cell line. The SBT-E1 cells proliferated well in standard Leibovitz’s L-15 cell culture medium containing fetal bovine serum (FBS) as the source of fatty acids. Decreasing the FBS concentration decreased the cell proliferation. Addition of the C₁₈ polyunsaturated fatty acids (PUFA) α-linolenic acid (ALA, 18:3n-3) or linoleic acid (LNA, 18:2n-6) to the cell culture medium had little effect on the proliferation of the cells, whereas addition of the long-chain PUFA (LC-PUFA) arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) significantly reduced the proliferation of the cells, especially at higher concentrations and especially for DHA. Addition of vitamin E to the culture medium overcame this effect, suggesting that it was due to oxidative stress. The fatty acid profiles of the total lipid from the cells reflected those of the respective culture media with little evidence for desaturation or elongation of any of the fatty acids. The only exceptions were EPA and ARA, which showed substantial elongation to 22:5n-3 and 22:4n-6, respectively, and DHA, which was significantly enriched in the cells compared with the culture medium. The results are discussed in light of the dietary PUFA requirements of SBT in the wild and in aquaculture.     

17β-Estradiol Increases Liver and Serum Docosahexaenoic Acid in Mice Fed Varying Levels of α-Linolenic Acid

Docosahexaenoic acid (DHA) is considered to be important for cardiac and brain function, and 17β-estradiol (E2) appears to increase the conversion of α-linolenic acid (ALA) into DHA. However, the effect of varying ALA intake on the positive effect of E2 on DHA synthesis is not known. Therefore, the objective of this study was to investigate the effects of E2 supplementation on tissue and serum fatty acids in mice fed a low-ALA corn oil-based diet (CO, providing 0.6 % fatty acids as ALA) or a high ALA flaxseed meal-based diet (FS, providing 11.2 % ALA). Ovariectomized mice were implanted with a slow-release E2 pellet at 3 weeks of age and half the mice had the pellet removed at 7 weeks of age. Mice were then randomized onto either the CO or FS diet. After 4 weeks, the DHA concentration was measured in serum, liver and brain. A significant main effect of E2 was found for liver and serum DHA, corresponding to 25 and 15 % higher DHA in livers of CO and FS rats, respectively, and 19 and 13 % in serum of CO and FS rats, respectively, compared to unsupplemented mice. There was no effect of E2 on brain DHA. E2 results in higher DHA in serum and liver, at both levels of dietary ALA investigated presently, suggesting that higher ALA intake may result in higher DHA in individuals with higher E2 status.     

Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega‐3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl‐CoA substrates in the presence of malonyl‐CoA. To distinguish between enzymes that desaturate CoA‐ and phosphatidylcholine‐linked fatty acid substrates, we developed a novel in vitro method. We show that a delta‐12 desaturase from Phytophthora sojae, an omega‐3 desaturase from Phytophthora infestans and a delta‐4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine‐linked acyl substrates with comparatively low use of acyl‐CoA substrates. To further validate our method, a delta‐9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl‐CoA as substrate, but could not use phosphatidylcholine‐linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.