Production of linolenic acid in yeast cells expressing an omega-3 desaturase from tung (<Emphasis Type="Italic">Aleurites fordii</Emphasis>)

Tung oil is an industrial drying oil containing ca. 90% PUFA. We previously reported on enzymes required for the synthesis of linoleic (6% of FA) and eleostearic (80%) acids and here describe the cloning and functional analysis of an omega-3 FA desaturase (FAD3) required for the synthesis of linolenic acid (1%). The tung FAD3 cDNA was identified by screening a tung seed cDNA library using the polymerase chain reaction and degenerate primers encoding conserved regions of the FAD3 enzyme family. Expression of this cDNA in yeast cells, cultured in the presence of linoleic acid, resulted in the synthesis and accumulation of linolenic acid, which accounted for up to 18% w/w of total cellular FA. Tung FAD3 activity was significantly affected by cultivation temperature, with the greatest amount of linolenic acid accumulating in yeast cells grown at 15°C. The amount of linolenic acid synthesized in yeast cells by tung FAD3 is ca. 10-fold higher than that observed by expression of a rapeseed (Brassica napus) FAD3 in yeast, suggesting that tung FAD3 might be useful for biotechnological production of omega-3 FA in transgenic organisms.     

Thermal deactivation and inhibition of D‐Amino acid oxidase in permeabilized cells of the yeast Trigonopsis variabilis

The inhibition of D ‐amino acid oxidase contained in permeabilized cells of the yeast Trigonopsis variabilis by α‐keto acids (pyruvic acid, phenylpyruvic acid and 4‐methylthio‐2‐oxobutanoic acid), products of the transformation of the corresponding D ‐amino acids, was studied. In all cases, inhibition was of the mixed type and significant differences with respect to the inhibition shown by the enzyme from other sources such as pig kidney or the yeast Rhodotorula gracilis were observed. A study was also made of the thermal deactivation of the enzyme contained in permeabilized cells of T variabilis in the temperature range 30–50 °C in sodium phosphate and Tris hydroxylmethyl aminomethane + CaCl₂ buffers. A deactivation mechanism with two steps in series is proposed to account for the variation in activity with time. The results suggest that the enzyme shows greater stability in phosphate buffer, with half‐lives between 7.6 days at 30 °C and 8.6 h at 50 °C. Copyright © 2004 Society of Chemical Industry     

An Unprecedented Occurrence of Δ5,9- and Δ9,15-Dienoic Fatty Acids in Ovaries of the Archaeogastropod Limpet Cellana toreuma

A detailed structural diversity of dienoic fatty acids (FA), including non‐methylene‐interrupted dienoic FA, of triacylglycerols and polar lipids in ovaries of Cellana toreuma was clarified for the first time by using capillary gas chromatography–mass spectrometry of their 3‐pyridylcarbinol esters and argentation thin‐layer chromatography. Interestingly, in addition to 5,9‐octadecadienoic (18:2Δ5,9), 5,9‐eicosadienoic (20:2Δ5,9), 5,9‐heneicosadienoic (21:2Δ5,9), 5,9‐docosadienoic (22:2Δ5,9), 5,9‐tricosadienoic (23:2Δ5,9), and 5,9‐tetracosadienoic (24:2Δ5,9) acids, previously identified in ovaries of C. grata, rare FA 5,9‐hexadecadienoic (16:2Δ5,9), 5,9‐nonadecadienoic (19:2Δ5,9), and 21‐methyl‐5,9‐docosadienoic (iso 23:2Δ5,9) were newly recognized in ovaries of C. toreuma. Detectable amounts of four Δ9,15‐dienoic FA were present in the ovary lipids. The FA identified were one novel 9,15‐heneicosadienoic (21:2Δ9,15) acid and known 9,15‐docosadienoic (22:2Δ9,15), 9,15‐tricosadienoic (23:2Δ9,15), and 9,15‐tetracosadienoic (24:2Δ9,15) acids. The findings help to explain the broad evidence of the structural diversity in marine gastropods and suggest biomarkers to evaluate marine food web relations.     

Cloning of Flax Oleic Fatty Acid Desaturase and Its Expression in Yeast

Fatty acid desaturases dehydrogenate acyl chains, which results in the formation of a double bond. Using PCR on flax genomic DNA, we cloned a putative Δ12 fatty acid desaturase (Fad2) gene encoding a 378 amino acid protein. Heterologous expression of this protein in yeast as an N-terminal fusion to GFP showed its localization within endoplasmic reticulum. Analysis of membrane lipids revealed the production of dienoic fatty acids, decreased levels of FAD2 substrates and an increased concentration of longer fatty acids. Higher peroxidation of lipids in FAD2-containing strains is not reflected by any visible phenotype in YPD medium. However, FAD2-containing strains with deleted superoxide dismutase genes exhibited significant growth reductions under oxidative stress.     

Characterization of expression of a plant oleate desaturase in yeast

We have expressed the oleate desaturase gene of Arabidopsis thaliana in Saccharomyces cerevisiae by means of a galactose-inducible promoter. Transfer of yeast cells to galactose-containing media and growth at 30°C resulted in the production of active oleate desaturase, as shown by the appearance of the diunsaturated octadecadienoic acid (18:2) and hexadecadienoic acid (16:2). The highest level of these two fatty acids was 46.2%. In induced cultures, 18:2 appeared before 16:2, and we show that both of these fatty acids were present first on phosphatidylcholine and then appeared in other phospholipids and a neutral lipid fraction. We raised antibodies against the C-terminal 100 amino acids of oleate desaturase and demonstrated that this protein was in the microsomal fraction of induced cells.     

Roasted Sunflower Kernel Paste (Tahini) Stability: Storage Conditions and Particle Size Influence

Sunflower tahini faces quality problems due to the tendency of oil to exudate, causing accelerated rancidity and low marketability. In this study, the colloidal and oxidative stability of different trimodal particle size distributions of sunflower tahini (cumulative volume percentages of small, middle and large class populations of 9.61–16.67, 43.03–55.03 and 47.36–28.3) was evaluated during storage at three different temperatures. Decreasing sample particle size decreased the sunflower tahini colloidal stability. The coarsest prototype, being the most stable tahini in terms of oil separation, was included in the oxidative stability storage test together with a commercial tahini. The peroxide values of the studied sunflower tahini samples increased significantly (p < 0.05) with storage time, irrespective of storage temperature, while the p‐anisidine values showed an irregular pattern. Considering 30‐meq O₂/kg as a PV limit, the commercial tahini showed good oxidative stability as it was under this when stored 3 months at 4 °C, 2 months at room temperature and 1 month at 40 °C. The coarsest tahini sample was under the limit when stored 2 months at 4 °C, 1 month at room temperature and 2 weeks at 40 °C. Particle size was shown to be an essential parameter for controlling sunflower tahini's overall stability.     

Sphingolipidomics of Thermotolerant Yeasts

Mass spectrometry‐based shotgun lipidomics was applied to the analysis of sphingolipids of 11 yeast strains belonging to four genera, that is Cryptococcus, Saccharomyces, Schizosaccharomyces, and Wickerhamomyces. The analysis yielded comprehensive results on both qualitative and quantitative representation of complex sphingolipids of three classes—phosphoinositol ceramide (PtdInsCer), mannosyl inositol phosphoceramide (MInsPCer), and mannosyl diinositol phosphoceramide (M(InsP)₂Cer). In total, nearly 150 molecular species of complex sphingolipids were identified. Individual strains were cultured at five different temperatures, that is 5, 10, 20, 30, and 40 °C (Wickerhamomyces genus only up to 30 °C), and the change in the culture temperature was found to affect the representation of both the sphingolipid classes and the length of the long‐chain bases (LCB). Individual classes of sphingolipids differing in polar heads differed in the temperature response. The relative content of PtdInsCer increased with increasing temperature, whereas that of M(InsP)₂Cer decreased. Molecular species having C18‐LCB were associated with low cultivation temperature, and a higher proportion of C20‐LCB molecular species was produced at higher temperatures regardless of the type of polar head. On the other hand, the influence of temperature on the representation of very long‐chain fatty acids (VLCFA) was less noticeable, the effect of the taxonomic affiliation of the strains being more pronounced than the cultivation temperature. For example, lignoceric and 2‐hydrocylo‐lignoceric acids were characteristic of the genera Cryptococcus and Schizosaccharomyces, and of Saccharomyces genus cultivated at high temperatures.     

Effects of rainbow trout freshness on n‐3 polyunsaturated fatty acids in fish offal

The effects of rainbow trout cold storage on the quality of offal left after fish processing to fillets with skin were determined. The intact farmed rainbow trout were kept at 2 °C in ice for 0, 4, 7, and 14 days of storage. The offal was, immediately after processing, frozen at ?20 °C and analysed after a month‐long frozen storage; fillets (non‐frozen) were analysed as well. Non‐protein nitrogen, volatile bases, trimethylamine, lipid oxidation (peroxide value, anisidine value, UV‐VIS spectra, and fluorescence) and fatty acid composition were determined. The offal consists in 15% of protein and in about 20% of chloroform/methanol‐extractable lipids, with n‐3 polyunsaturated fatty acids (n‐3 PUFA) accounting for 20.37 ± 1.25% of the fatty acids. The fish storage duration was found to exert a significant (p = 0.05) effect on the changes in lipids and nitrogen compounds. No losses of long‐chain n‐3 PUFA in the offal were detected during the 2 wk of storage in ice plus 1 month at ?20 °C. The rainbow trout offal is a valuable – rich and stable – source of n‐3 PUFA.     

Evaluation of antioxidant capacity of sesamol and free radical scavengers at different heating temperatures

Sesamol is a natural antioxidant found in sesame oil from roasted sesame seeds. Activation energy and antioxidant capacity of sesamol were determined and compared with other free radical scavengers (FRSs) including tert‐butylated‐hydroxyquinone (TBHQ), butylated‐hydroxyanisol (BHA), or α‐tocopherol in a lard model system treated with different heating temperature. Each FRS was added in lard and heated at 90, 120, 150, and 180°C for 48, 24, 8, or 2 h, respectively and antioxidant capacity was evaluated by conjugated dienoic acid (CDA) value, conjugated diene hydroperoxides, p‐anisidine value (p‐AV), and a modified 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) method. Apparent activation energy of sesamol was determined as 12.98 kcal/mol in a lard model system. Antioxidant capacity of sesamol was as good as that of TBHQ and was higher than those of BHA and α‐tocopherol at 90, 120, and 150°C based on CDA, conjugated diene hydroperoxides, and p‐AV assays. The results of a modified DPPH method showed that each FRS showed different distribution of radical scavenging compounds from oxidized lipids (RSOLs) during oxidation. Sesamol may replace synthetic FRSs like TBHQ and BHA in processed foods treated with high temperature. Practical application: Processed foods are frequently treated with high temperature during oven‐drying, roasting, baking, and deep‐fat frying. This study showed that sesamol, one of natural antioxidants, had stronger antioxidant capacities than other synthetic FRSs at the temperature ranges from 90 to 180°C. The results of this study can be applied in food industries producing deep‐fat fried foods including snacks, chips, and French fries to extend the shelf‐life of final foods with high temperature treatment.     

Comparison of supercritical CO2 and hexane extraction of lipids from sorghum distillers grains

Total yields and compositions of sorghum dried distillers grains with solubles (DDGS) lipids obtained by supercritical CO₂ (SC‐CO₂) extraction were compared with those obtained by recirculated solvent extraction (RSE) with hexane. The total yield of lipids obtained by SC‐CO₂ extraction at 27.5 MPa and 70 °C was 150 g lipids/kg DDGS, while the yield obtained by RSE with hexane at 69 °C was only 85 g lipids/kg DDGS. The contents of four high‐value compounds, i.e., policosanols, phytosterols, free fatty acids (FFA) and tocols, in the lipids obtained by SC‐CO₂ extraction were 31.2, 15.6, 155.3 and 0.50 mg/g at 27.5 MPa and 70 °C, compared to 26.6, 9.6, 57.3 and 0.03 mg/g for RSE with hexane at 69 °C. The profiles of phytosterols and FFA in the sorghum DDGS lipids were relatively independent of the extraction methods and operating conditions.     

Synthesis and polymerization of the bromoacrylated plant oil triglycerides to rigid,flame‐retardant polymers

Simultaneous addition of bromine and acrylate to the double bonds of fatty acids in triglycerides was achieved. In the first part of the study, methyl oleate was bromoacrylated in the presence of acrylic acid and N‐bromosuccinimide as a model compound for the application of the reaction to the triglycerides. Next, soybean oil and high oleic sunflower oil were bromoacrylated by using the same procedure. The products were characterized by GC, IR, ¹H‐NMR, ¹³C‐NMR, and mass spectrometry. The bromoacrylation yields for soybean oil and sunflower oil were 75 and 55%, respectively. A rigid thermoset polymer was prepared from the radical copolymerization of bromoacrylated soybean oil with styrene. The bromoacrylated sunflower oil–styrene copolymer showed semirigid properties. The crosslinked network structure of the copolymers was examined by their swelling behavior in different solvents. Glass‐transition temperatures were also determined and soybean oil–based polymer and sunflower oil–based polymer showed a glass transition at 55–65 and 20–30°C, respectively. The storage moduli of the soybean‐based and sunflower‐based polymers at room temperature were approximately 1.0 × 10¹⁰ and 1.1 × 10⁸ Pa, respectively. Photopolymerization was also carried out by using 2,2‐dimethoxy‐2‐phenyl‐acetophenone as initiator. The response of the cured polymers to the thermal energy produced by a small flame was also tested by the ignition respond index method according to ASTM D 3713‐78 and was found to be 5 B at 2.00 mm. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91:2700–2710, 2004     

Synthesis of fatty acid esters from acid oils using lipase B from Candida antarctica

Esterification of corn and sunflower acid oils with straight‐ and branched‐chain alcohols were conducted using lipase B from Candida antarctica (Novozym 435) in n‐hexane. Sunflower acid oil consisted of 55.6% free fatty acids and 24.7% triacylglycerols, while the free fatty acids and triacylglycerols contents of corn acid oil were 75.3% and 8.6%, respectively. After 1.5 h of methanolysis of sunflower acid oil, the highest fatty acid methyl ester content (63.6%) was obtained at 40 °C and the total fatty acid/methanol molar ratio was 1/1, using 15% enzyme based on acid oil weight. The conversion of both acid oils with straight‐ and branched‐chain alcohols was not significantly affected by the chain length of the alcohols. However, the lowest fatty acid methyl ester content (50%) was obtained in the reaction of corn acid oil with methanol. Sunflower acid oil was converted to fatty acid esters using primer alcohols such as n‐propanol, i‐ and n‐butanol, n‐amylalcohols, n‐octanol, and a mixture of amylalcohol isomers, resulting in a fatty acid ester content of about 70% at 40 °C.     

Modelling lipase‐catalysed transesterification of fats containing n‐3 fatty acids monitored by their solid fat content

Transesterification of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA), catalysed by a commercial immobilised thermostable lipase from Thermomyces lanuginosa, was carried out batch‐wise. Experiments were performed, following central composite rotatable designs (CCRDs) as a function of reaction time, temperature and media formulation. Mixtures of palm stearin, palm kernel oil and a commercial concentrate of triacylglycerols rich in n‐3 PUFA (“EPAX 2050TG” in CCRD‐1 and “EPAX 4510TG” in CCRD‐2) were used. The time‐course of transesterification was indirectly followed by the solid fat content (SFC) values of the blend at 10 °C, 20 °C, 30 °C and 35 °C. A decrease in all SFC values of the blends at 10 °C, 20 °C, 30 °C and 35°C was observed upon transesterification. The SFC_{10 °C} and SFC_{20 °C} of transesterified blends varied between 18 and 48 and SFC_{35 °C} between 6 and 24. These values fulfil the technological requirements for the production of margarines. Under our conditions, lipid oxidation may be neglected. However, the accumulation up to 8.3% free fatty acids in reaction media is a problem to overcome. The development of response surface models, describing both the final SFC value and the SFC decrease, will allow predicting results for novel proportions of fats and oils and/or a novel combination time‐temperature.     

Effect of Different Drying Procedures on the Nutritional Value of the Mantle and Tentacles of <Emphasis Type="Italic">Sepia officinalis</Emphasis>

The aim of this work was to investigate the influence of two different drying methods, traditionally by sun and controlled temperatures of 45, 50, 60 and 70 °C and relative humidity of 20 and 30% on the chemical composition of Sepia officinalis. When compared to fresh samples, lipid content increased under both drying methods whereas protein levels significantly increased only under controlled conditions. Drying procedures increased the percentage of saturated fatty acids mainly in sun‐dried samples and at high temperature (70 °C). A significant decrease in the amount of polyunsaturated fatty acids was detected in sun‐dried samples. No significant differences were found between the levels of polyunsaturated fatty acids of fresh cuttlefish and those under controlled conditions. The highest value of malondialdehyde in mantles was observed in sun‐dried samples, while in tentacles the highest level was seen at 70 °C. The highest level of advanced oxidation protein products was noted in traditionally sun‐dried samples. The results obtained showed that 45–50 °C and 20–30% humidity conserved the value of proteins and lipids more than traditional drying. Results of this study may be suitable for developing a reliable industrial index.     

Effect of vacuum‐packaging on the changes of Russian sturgeon muscle lipids during frozen storage

The changes in fatty acid composition, non‐polar (triglycerols) and polar lipids (phospholipids), total free fatty acids and total cholesterol of Russian sturgeon (Acipenser gueldenstaedtii) were studied during 360 days of storage at ?18°C. It was established that total neutral lipids and phospholipids content decreased and total free fatty acids concentration increased significantly during the frozen storage. Lower non‐polar and polar lipids content and higher free fatty acids concentration of vacuum‐packaged samples in comparison with air‐packaged samples were found. The changes in total cholesterol concentration and phospholipid classes of frozen stored sturgeon were not influenced by the frozen storage period and the type of packaging. It was established that the sturgeon polar lipids consisted mainly of phosphatidylcholine – 54.98 ± 0.85%, phosphatidylethanolamine – 28.42 ± 0.61%, and phosphatidylserine – 8.64 ± 0.45%. The increase of the total free fatty acids concentration was associated with the free n ? 3 PUFA accumulation as a result of hydrolysis of non‐polar and polar lipids. During the frozen storage DHA percentage of non‐polar lipids and phospholipids decreased approximately 3 and 1.75%, respectively. After 360 days of storage at ?18°C the n ? 3/n ? 6 PUFA ratio of total lipids decreased 4.9%.     

Anti-mutagenic Properties of Mono- and Dienoic Acid Biohydrogenation Products from Beef Fat

Unsaturated fatty acid biohydrogenation products from beef fat and pure fatty acids were subjected to the Ames Salmonella mutagenicity testing, including monounsaturated fatty acids [MUFA: oleic acid, vaccenic acid, elaidic acid; beef fatty acid fractions rich in trans (t)11/t13‐t14‐18:1 (t11,13,14‐Frac), t10‐18:1 (t10‐Frac)] and dienoic fatty acids [linoleic acid, conjugated linoleic isomers cis (c)9,t11‐18:2 and t10,c12‐18:2, and a mixed beef dienoic fatty acid fraction high in c9,t13‐/t8,c12/t11c15‐18:2 (MD)]. Significantly higher anti‐mutagenic effects of oleic acid, vaccenic acid, t11, 13, 14‐Frac, and t10‐Frac against daunomycin were observed at 2.5 mg. All dienoic acids except MD significantly reduced daunomycin mutagenicity at ≥0.25 mg. Anti‐mutagenicity of oleic and vaccenic acids against 2‐aminoanthracene was found at 2.5 and 0.25 mg, respectively. All dienoic acids significantly reduced 2‐aminoanthracene mutagenicity at ≥0.25 mg. Findings of this study show that unsaturated fatty acids, including trans‐fatty acids commonly found in beef, can act as strong anti‐mutagens.     

In situ development of bio‐based polyurethane‐blend‐epoxy hybrid materials and their nanocomposites with modified graphene oxide via non‐isocyanate route

We developed a series of sunflower oil‐based non‐isocyanate polyurethane (NIPU)‐blend‐epoxy hybrid materials (HNIPUs) and their nanocomposites with amine‐functionalized graphene oxide (AF‐GO). Firstly, carbonated sunflower oil (CSFO) containing five‐membered cyclocarbonate groups was synthesized by the reaction of epoxidized sunflower oil with carbon dioxide (CO₂) at a pressure of 50 bar and temperature of 110 °C. Then, a series of HNIPUs were synthesized using a mixture of CSFO and a commercially available epoxy resin in various amounts (10, 20 and 30 wt% with respect to CSFO) using isophorone diamine as the curing agent. The HNIPU with 30 wt% epoxy showed the best mechanical properties. Finally, nanocomposites of 30 wt% HNIPU‐based composition were prepared with various amounts of AF‐GO (0.3, 0.6 and 1.0 wt%) and were characterized using Fourier transform infrared and ¹H NMR spectroscopies, X‐ray diffraction and scanning electron microscopy. These results emphasize the potentiality of this environmentally friendly approach for preparing renewable HNIPU and nanocomposite materials of high performances. © 2018 Society of Chemical Industry     

Microbial mediated dimerization of fattyacids of sunflower oil: An effective role of lipase and biosurfactant

This study emphasizes microbial mediated transformation of sunflower oil to an adhesive product and characterization in detail. Marine bacterial isolates Bacillus (MTCC 5514), when grown in mineral medium, releases both hydrolytic enzymes and surface‐active components during the log phase of growth. When this species was grown in the presence of sunflower oil at an optimized concentration of 5% (w/v) under room temperature, enzymatic hydrolysis of oil proceeds with the release of fatty acids and glycerol. Further, on increasing the incubation period, the presence of surface‐active components, lipase and glycerol, influence the dimerization of the fatty acids, which further, transformed to a polymerized product sunflower oil‐based adhesive product with adhesive nature. Liquid chromatography‐mass spectrometry (electrospray ionization) analysis further authenticates the presence of dimeracids. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40555.     

Foam processing and cellular structure of polypropylene/clay nanocomposites

Polypropylene (PP)/clay nanocomposites (PPCNs) were autoclave‐foamed in a batch process. Foaming was performed using supercritical CO₂ at 10 MPa, within the temperature range from 130.6°C to 143.4°C, i.e., below the melting temperature of either PPCNs or maleic anhydride‐modified PP (PP‐MA) matrix without clay. The foamed PP‐MA and PPCN2 (prepared at 130.6°C and containing 2 wt% clay) show closed cell structures with pentagonal and/or hexagonal faces, while foams of PPCN4 and PPCN7.5 (prepared at 143.4°C, 4 and 7.5 wt% clay) had spherical cells. Scanning electron microscopy confirmed that foamed PPCNs had high cell density of 10⁷–10⁸ cells/mL, cell sizes in the range of 30–120 μm, cell wall thicknesses of 5–15 μm, and low densities of 0.05–0.3 g/mL. Interestingly, transmission electron microscopic observations of the PPCNs' cell structure showed biaxial flow‐induced alignment of clay particles along the cell boundary. In this paper, the correlation between foam structure and rheological properties of the PPCNs is also discussed.