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Crude olive pomace oil (OPO) is a by-product of olive oil extraction. In this study, low-calorie structured triacylglycerols (TAGs) were produced by acidolysis of crude OPO with medium-chain fatty acids (caprylic, C8:0; capric, C10:0) or interesterification with their ethyl ester forms (C8EE, C10EE). These new TAGs present long-chain fatty acids (L) at position sn-2 and medium-chain fatty acids (M) at positions sn-1,3 (MLM). Crude OPO exhibited a high acidity (12.05-28.75% free fatty acids), and high contents of chlorophylls and oxidation products. Reactions were carried out continuously in a packed-bed bioreactor for 70 h, using sn-1,3 regioselective commercial immobilized lipases (Thermomyces lanuginosus lipase, Lipozyme TL IM; and Rhizomucor miehei lipase, Lipozyme RM IM), in solvent-free media at 40 °C. Lipozyme RM IM presented a higher affinity for C10:0 and C10EE. Lipozyme TL IM preferred C10:0 over C8:0 but C8EE over C10EE. Both biocatalysts showed a high activity and operational stability and were not affected by OPO acidity. The New TAG yields ranged 30-60 and the specific productivity ranged 0.96-1.87 g NewTAG/h.g biocatalyst. Lipozyme RM IM cost is more than seven-fold the Lipozyme TL IM cost. Therefore, using Lipozyme TL IM and crude acidic OPO in a continuous bioreactor will contribute to process sustainability for structured lipid production by lowering the cost of the biocatalyst and avoiding oil refining.

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Our study offers a novel sn-1,3 specific lipase MAJ1 from marine member Janibacter sp. strain HTCC2649 for preparing long-medium-long (LML) type structured triacylglycerols (TAGs). Firstly, the resin ECR1030 was selected as a suitable support for the immobilization of lipase MAJ1. An efficient synthesis of LML-type structured TAGs by the immobilized lipase MAJ1-catalyzed interesterification of methyl palmitate and tricaprylin was studied in a solvent-free system. The reaction conditions, including substrate molar ratio, temperature and enzyme loading, were optimized. Under the optimum conditions (immobilized lipase MAJ1 of 45 U/g, substrate molar ratio of 4:1, temperature of 35 °C, reaction time of 24 h), the structured TAGs with double long chains (DLCST) were obtained in a yield of 44.3 mol%. Secondly, multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) was employed to quantify each TAG positional isomer in DLCST. The content of 1,3-dipalmitoyl-2-capryloyl-sn-glycerol in DLCST was 97.6% determined by the MDMS-SL technology.

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Enzymatic interesterification (EIE) is one of the emerging technologies in the specialty fats industry. EIE has several advantages over the conventional chemical interesterification method, such that the process has higher flexibility and efficiency, is environmentally friendly and the immobilized enzyme can be recycled besides of the lower requirement for substrate's acid value. The physical properties and nutritional qualities of the fats and oils are modified after EIE, depending on the change in the position of fatty acids on the triacylglycerol (TAG) molecules. Evaluation of the interesterification reaction are important and useful in terms of its technological applications. This paper summarizes the conventional methods and the advancement for evaluating EIE processes, e.g., determination of the change in slip melting points, solid fat contents, TAG with equivalent carbon numbers, and sn-2 fatty acid compositions of the end product. Nonetheless, these methods are not comprehensive because during the EIE process, acyl migration occurs. A novel and convenient evaluation model which is based on the fatty acid distribution on the glycerol-backbone is proposed as a perspective. This model can be employed to monitor the interesterification degree and acyl migration during a regiospecific EIE process, which serves as a reaction rule that can be employed to control and optimize the EIE process, thereby producing structured TAG with desired properties.

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The present study aims to evaluate the suitability of structured triacylglycerol (ST) consisting of medium chain triacylglycerol (MCT) as low calorie and instant energy source in combination with essential fatty acids for formulations in different food products to add value to existing products. The study investigates the effect of presence of ST on the physicochemical and sensory properties of various food products such as mayonnaise enriched with ST synthesized from medium chain fatty acid (MCFA) and rice bran oil, cookies enriched with ST synthesized from MCFA and oleic acid rich moringa oil, energy bar with ST synthesized from MCFA and omega 3 enriched fish oil, as well as yogurt drink with ST synthesized from MCFA and omega 6 enriched flaxseed oil. The obtained results established the suitability of incorporation of ST in food products and comparison with standard market products revealed the validity of the products to sustain the demand with value addition. The oxidative stability studies of all the products in the presence of natural antioxidants from moringa leaves and pomegranate peel using the peroxide value test and rancimat analysis demonstrated similar stability to the standard marketed product. Application of plant wastes and residues in the form of leaves or peels as the likely sources for isolating bioactive compounds demonstrates potential of the sustainable approach also giving benefit of increasing the stability of the product. The study also points towards possible replacement option for synthetic carcinogenic antioxidants with natural antioxidants obtained from moringa leaves and pomegranate peel. The present work clearly demonstrates the effective use of structured triacylglycerols synthesized using green methodologies for creating unique health enriched food products with all worthy and green components.

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The regiodistribution of fatty acids (FAs) in triacylglycerols (TAGs) is highly correlated to the digestion and absorption of dietary fats. During in vitro gastric digestion, medium-long-medium/short chain triacylglycerols (MLM) showed the fastest digestibility among 105 TAG molecular species, for both raw and homogenised milk, resulting from the higher activity of gastric lipase towards medium-chain fatty acids (MCFAs) than towards long-chain fatty acids (LCFAs). After 60 min of in vitro gastric digestion, the percentage of digestion of each MLM was 16.6-31.2% and 12.3-18.6% respectively in raw sheep and cow milk, and it reached 33.4-45.5% and 23.0-38.9% respectively, after homogenisation. Sheep milk TAGs were digested faster than cow milk TAGs in raw milk (16.1% and 12.4%, respectively) as well as after homogenisation (24.1% and 20.3%, respectively). This is due to the higher percentages of MCFAs esterified on the sn-1/3 positions of sheep milk TAGs (48.3-67.3% and 11.9-51.5% respectively for sheep and cow milk prior to and after homogenisation). Homogenisation favoured TAG lipolysis due to the increased fat globule surface area; however, it did not affect lipase regiospecificity towards TAG molecular species.

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Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterized by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or ß) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study, we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (vs. outer) positions by more than 20-fold (i.e., from ∼3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.

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This article reviews the application of enzymes in lipid modification. Lipases are the most established biocatalysts used for the synthesis of structured triacylglycerols, fats, and margarine and for the release of flavoring fatty acids for food applications. In addition, the various enzymes, such as P450 monooxygenases, hydratases, lipoxygenases, and certain lyases, used for oxyfunctionalization and the phospholipases used for degumming are covered. Basic aspects of enzyme catalysis and the modern tools used for their discovery and improvement by protein engineering provide insight into how suitable biocatalysts can be identified and optimized for an application. In addition to isolated enzymes, whole-cell engineered microorganisms are also used for lipid modification. Thus, the polyunsaturated fatty acid EPA (eicosapentaenoic acid) can be produced in a yeast using sugar as a renewable resource.

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The positional distribution pattern of fatty acids (FAs) in the triacylglycerols (TAGs) affects intestinal absorption of these FAs. The aim of this study was to compare lymphatic absorption of pinolenic acid (PLA) present in structured pinolenic TAG (SPT) where PLA was evenly distributed on the glycerol backbone, with absorption of pine nut oil (PNO) where PLA was predominantly positioned at the sn-3 position. SPT was prepared via the nonspecific lipase-catalyzed esterification of glycerol with free FA obtained from PNO. Lymphatic absorption of PLA from PNO and from SPT was compared in a rat model of lymphatic cannulation. Significantly (P < 0.05) greater amounts of PLA were detected in lymph collected for 8 h from an emulsion containing SPT (28.5 ± 0.7% dose) than from an emulsion containing PNO (26.2 ± 0.6% dose), thereby indicating that PLA present in SPT has a greater capacity for lymphatic absorption than PLA from PNO.

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Human milk fat-style structured triacylglycerols were produced from microalgal oil in a continuous microfluidic reactor packed with immobilized lipase for the first time. A remarkably high conversion efficiency was demonstrated in the microreactor with reaction time being reduced by 8 times, Michaelis constant decreased 10 times, the lipase reuse times increased 2.25-fold compared to those in a batch reactor. In addition, the content of palmitic acid at sn-2 position (89.0%) and polyunsaturated fatty acids at sn-1, 3 positions (81.3%) are slightly improved compared to the product in a batch reactor. The increase of melting points (1.7°C) and decrease of crystallizing point (3°C) implied higher quality product was produced using the microfluidic technology. The main cost can be reduced from $212.3 to $14.6 per batch with the microreactor. Overall, the microfluidic bioconversion technology is promising for modified functional lipids production allowing for cost-effective approach to produce high-value microalgal coproducts.

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This study describes the identification of regioisomers and enantiomers of triacylglycerols of C20 polyunsaturated fatty acids (PUFAs) in the alga Trachydiscus minutus cultivated at different temperatures using reversed- and chiral-phase liquid chromatography-mass spectrometry. The use of the two different phases contributes to ready identification, both qualitative and semiquantitative, of regioisomers and enantiomers of triacylglycerols containing eicosapentaenoic and arachidonic in the molecule. The ratio of regioisomers and enantiomers of triacylglycerols (TAG) depends on the temperature of cultivation; with lowering temperature the proportion of the achiral TAG increases and the enantiomer ratio diverges from 1:1.

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Novel structured triacylglycerols (STAGs) enriched with unsaturated fatty acids (UFAs) and low palmitic acid (PA) content were firstly synthesized from Schizochytrium sp. oil and oleic acid (OA) via solvent-free acidolysis catalyzed by Lipozyme RM IM. The results indicated that, the PA content decreased from 24.49% to 6.95%, while the UFAs content increased from 70.20% to 90.9% at the sn-1,3 positions in the STAGs under the optimal condition (i.e., lipase load of 7%, molar ratio of microalgae oil TAGs to OA of 1:3, and temperature of 65 °C). The lipase Lipozyme RM IM could be reused 16 times without significant loss of activity. The improved plastic and storage ranges of STAGs are useful for infant formula formulations, by which a possible method is blending of this product and 1,3-dioleoyl-2-palmitoylglycerol enriched fats and minor lipids based on the corresponding chemical compositions of human milk fat.

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Structured triacylglycerols (TAGs) were isolated from nine cultivated strains of microalgae belonging to different taxonomic groups, i.e. Audouinella eugena, Balbiania investiens, Myrmecia bisecta, Nannochloropsis limnetica, Palmodictyon varium, Phaeodactylum tricornutum, Pseudochantransia sp., Thorea ramosissima, and Trachydiscus minutus. They were separated and isolated by means of NARP-LC/MS-APCI and chiral LC and the positional isomers and enantiomers of TAGs with two polyunsaturated, i.e. arachidonic (A) and eicosapentaenoic (E) acids and one saturated, i.e. palmitic acid (P) were identified. Algae that produce eicosapentaenoic acid were found to biosynthesize more asymmetrical TAGs, i.e. PPE or PEE, whereas algae which produced arachidonic acid give rise to symmetrical TAGs, i.e. PAP or APA, irrespective of their taxonomical classification. Nitrogen and phosphorus starvation consistently reversed the ratio of asymmetrical and symmetrical TAGs.

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Structured triacylglycerols, containing medium chain fatty acids, were produced by acidolysis of virgin olive oil with caprylic or capric acid, at a molar ratio of olive oil:fatty acid of 1:2, at 45°C for 24h, in solvent-free media or in n-hexane, catalysed by Thermomyces lanuginosa (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) immobilised lipases. Incorporations were always greater for capric than for caprylic acid. For both acids, higher incorporations were always attained in solvent-free media: the highest caprylic acid incorporations were obtained with Novozym 435 (25.5mol%) and Lipozyme RM IM (25.7mol%), while similar capric acid incorporations were obtained with all biocatalysts (27.1-30.4mol%). After 10 repeated uses of Lipozyme RM IM, the same incorporation level of capric acid was obtained at the end of each 23h batch. However, with caprylic acid, a first-order deactivation was observed (half-life time, t1/2=299h). During acidolysis with both acids, Novozym 435 (t1/2=217h, for caprylic, t1/2=225h, for capric acid) and Lipozyme TL IM (t1/2=50.4h, for caprylic; t1/2=47.2h, for capric acid) presented first-order deactivation. All biocatalysts presented 1,3-regioselectivity.