Glyceride Fraction Research Articles

Concentration of docosahexaenoic acid from tuna oil via a two lipase-catalyzed reaction

Docosahexaenoic acid (DHA) was concentrated successfully in the glyceride fractions from tuna oil via a two-step enzyme reaction involving hydrolysis and ethanolysis. In the first step, Candida rugosa lipase-catalyzed hydrolysis was carried out to concentrate DHA in the glyceride fractions. The DHA content in the glyceride fraction after hydrolysis increased from 30% in the initial tuna oil to 46%. In the second step, Lipozyme RM IM-catalyzed ethanolysis was conducted with the reaction mixture from the first step to further concentrate DHA in the glyceride fraction. In this step, the reaction mixture obtained from the first step was employed directly in Lipozyme RM IM-catalyzed ethanolysis without additional steps needed to remove free fatty acid. Finally, DHA was concentrated from an initial content of 30% in the tuna oil to 68.4% in the glyceride fractions via a novel two-step enzyme reaction strategy.

Pencucian Crude Palm Oil (CPO) untuk Mitigasi Penurunan 3-MCPDE dan GE pada Pemurnian Minyak Sawit

Crude palm oil (CPO) purification aims to remove or degrade unwanted components such as free fatty acids (FFA), phospholipids, impurities, color pigments, metals, and odors. The CPO purification generally consists of degumming, bleaching, and deodorization process. During the stages, some undesirable compounds are formed, including 3-monochloropropane-1,2-diol ester (3-MCPDE) and glycidyl ester (GE). The main precursors responsible for formation of the two compounds are chlorides and acyl glycerol fractions, especially diacylglycerol (DAG) and this occurs high temperatures in the deodorization process. Therefore, it is necessary to reduce their precursors in order to supress the contaminants. To cope with the problem, a washing process of CPO was proposed as an extra process before a purification stage. The washing process was carried out in two ways, namely wet washing by using water, and dry washing by using bleaching earth (BE) by 2 types of commercial BE (natural and acid activated). Washing processes using wet and dry washing (natural and activated BE) showed comparable effectiveness in reducing 3-MCPDE and GE levels in refined palm oil. As a result, the process successfully reduced 3-MCPDE content to comply with a standard Commission Regulation (EU) 2020/1332, but GE content did not conform to standard.

Preparation of glycerides enriched with n-3 long-chain polyunsaturated fatty acids by Thermomyces lanuginosus lipase-catalyzed hydrolysis of tuna oil

n-3 Long-chain polyunsaturated fatty acids (LC-PUFAs) in glyceride form have attracted much attention due to their nutritional value and high bioavailability. In this work, Thermomyces lanuginosus lipase (TL 100 L) was used to selectively hydrolyze tuna oil for preparing glycerides enriched with n-3 LC-PUFAs. Effects of reaction variables on the hydrolysis reaction were optimized by response surface methodology, and the optimal conditions were as follows: reaction temperature 54 °C, enzyme load 3%, reaction time 6 h, mass ratio of water to tuna oil 1.5:1. Under these conditions, the resulting degree of hydrolysis was 55.01%, and the total content of eicosapentaenoic acid and docosahexaenoic acid in the glyceride fraction was found to be 46.39%. The final product was mainly composed of diacylglycerols (53.93%) and triacylglycerols (34.58%) after removal of the free fatty acids. This paper provides a simple and suitable method for industrial production of glycerides enriched with n-3 LC-PUFAs.

Lipases from different yeast strains: Production and application for n-3 fatty acid enrichment of tuna eyeball oil

Enrichment of n-3 polyunsaturated fatty acids (PUFA) in the glyceride fraction of skipjack tuna eyeballs oil (STEO) was investigated by using six extracellular and cell-bound lipases from three different yeast strains. Among all lipases tested, Candida rugosa extracellular lipase (Cr-ECL) showed promising enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in STEO. Firstly, various parameters including types of solvent, solvent to oil ratio, water to oil ratio, hydrolysis time, temperature, and lipase concentration were studied to select the major independent parameters. Finally, response surface methodology (RSM) was used to optimize the reaction conditions. Optimal condition included 450 U of Cr-ECL at water to oil ratio of 1:8 for 11.28 h, which resulted in 1.9-fold increase in DHA concentration in STEO. EPA was increased by 2.1-fold, when 191.35 U of Cr-ECL, water to oil ratio of 1:6 for 5.14 h was used. The Cr-ECL catalyzed hydrolysis was non-stereoselective, but had acyl chain specificity, and recognition of the whole triacyl-glycerol molecule as shown by 1H NMR and 13C NMR. Therefore, the established hydrolysis protocol for DHA and EPA enrichment was successful in converting fish oil from tuna waste to useful and healthy fish oil enriched with n-3 fatty acids. These findings pave the way for the future valorization of industrial marine waste oil.

Preparation of γ‐linolenic acid rich triacylglycerol from borage oil via a two‐step lipase‐catalyzed reaction

Abstractγ‐Linolenic acid (GLA) rich triacylglycerol (TAG) was successfully synthesized from glyceride, instead of glycerol, and fatty acid (FA) via Lipozyme TL IM‐catalyzed esterification as a novel strategy. In the first step, GLA was enriched into glyceride fraction from borage oil by Candida rugosa lipase‐catalyzed hydrolysis. The glyceride was separated from the reaction mixture by molecular distillation. GLA was enriched from 20.64% in borage oil to 45.94% in the glyceride fraction under optimum conditions. In the second step, the Lipozyme TL IM‐catalyzed synthesis of TAG was carried out with the glyceride, and the FA obtained by saponification of a portion of the glyceride. The optimum conditions were the temperature of 50°C, the enzyme loading of 10%, and the vacuum level of 20 mmHg, respectively. The maximum TAG content of approximately 92% was achieved after 12 h under the optimum conditions.

LIPASE-CATALYZED TRANSESTERIFICATION OF MEDIUM-LONG-MEDIUM STRUCTURED LIPID (MLM-SL) USING PALM OLEIN AND PALM KERNEL OIL IN BATCH AND CONTINUOUS SYSTEMS

Lipase-catalyzed transesterification between refined bleached deodorized palm olein (RBDO) and palm kernel oil (RBDPKO) has been investigated to produce medium-long-medium structured lipid (MLM-SL). The synthesis was catalyzed by a specific sn-1,3 commercial lipase (Lipozyme TL IM) in batch and continuous systems. Progress of the transesterification of this study was monitored as triacylglycerol (TAG) with equivalent carbon number (ECN) 40, presumably that of 1,3-dilauryl-2-oleoyl-sn-glycerol (LaOLa). The results showed that lipase-catalyzed transesterification using RBDO and RBDPKO could potentially be used as the main substrate for MLM-SL synthesis, both for batch and continuous systems. In batch system, transesterification of RBDO and RBDPKO at the ratio of 1:2 at 50oC yielded in the highest concentration of ECN 40 (LaLaO/LaOLa, 7.34%) but also a higher total concentration of partial acylglycerol fractions (Di- and Monoacylglycerols; DAGs and MAGs). Thus, this condition also obtained transesterified lipid rich in EC N40 with a lower slip melting point as compared to other substrate ratios. In a continuous system, transesterification at RDBO:RBDPKO of 1:1 at 50oC and 15 min of residence time were selected as the optimum conditions, resulting in 5.39% EC N40 with a minimum concentration of DAGs and MAGs.

1H NMR to analyse the lipid profile in the glyceride fraction of different categories of Iberian dry-cured hams

The extraordinary organoleptic qualities of Iberian ham derive from the singular producing pig breed and from the traditional rearing conditions, both of which define its lipid content and composition. In this work 1H NMR spectroscopy is used for the first time to analyse the lipid profile of Iberian hams as determinant of quality. Quantification of fatty acids is readily obtained from the spectra, with the monounsaturated fatty acids standing out, especially in the higher quality hams. Unprecedently, triacylglyceride hydrolysis products formed during the curing process can also be directly detected and quantified. Furthermore, chemometric analysis of the NMR data allows to classify Iberian hams according to the pig’s crossbreed and feeding regime. Principal component analysis shows fatty acid unsaturation and triacylglyceride hydrolysis as discriminating variables. 1H NMR spectroscopy has thus revealed as a convenient and powerful tool for the lipid analysis and classification of Iberian hams and for detection of fraud.

Efficient Synthesis of Stearidonic Acid Enriched Triacylglycerol from Ahiflower Seed Oil via a Two-Step Enzyme Reaction.

Stearidonic acid (SDA) is a plant-based n-3 polyunsaturated fatty acid with multiple biological activities. The enrichment of SDA and synthesis of triacylglycerol (TAG) were carried out consecutively via two lipase-catalyzed reactions, hydrolysis, and esterification. First, SDA was enriched into a glyceride fraction from ahiflower seed oil by Candida rugosa lipase-catalyzed hydrolysis. Under the optimum conditions of 35°C, 0.1% lipase powder of Lipase OF, and 50% buffer solution (based on the weight of total substrate), SDA was enriched from 21.6 to 40.7 wt% in glyceride fraction. SDA-enriched TAG was then synthesized from the SDA-enriched glyceride and the SDA-enriched fatty acid via esterification using an in-house immobilized lipase as a biocatalyst. The SDA-enriched fatty acid was obtained from part of the SDA-enriched glyceride by saponification and the in-house immobilized lipase was prepared from Eversa® Transform 2.0 using Lewatit VP OC 1600 as a carrier. The optimum reaction conditions for the synthesis of TAG were a temperature of 50°C, an enzyme loading of 10%, and a vacuum of 10 mmHg. A maximum conversion to TAG of ca. 94% was achieved after 12 h under the optimum conditions.

Enzymatic enrichment of n-3 polyunsaturated fatty acid glycerides by selective hydrolysis

Most natural oils are low in n-3 polyunsaturated fatty acids (n-3 PUFAs) content, which limits their application in health products. In this study, n-3 PUFAs in glyceride form were selectively enriched by lipase-mediated hydrolysis of n-3 PUFA-containing oils. First, commercial lipases were screened, and the lipase AY “Amano” 400SD from Candida cylindracea was the best choice in producing n-3 PUFA glycerides from tuna oil. Subsequently, the hydrolysis conditions were optimized. Under the optimal conditions, the highest n-3 PUFA content in the glyceride fraction was found to be 57.7% after enzymatic hydrolysis. Addition of Ca2+ to the system significantly shortened the reaction time from 10 to 4 h. When algal oil was used as substrate, total PUFA contents in the glyceride fraction were 89.9%. This study provides an efficient enzymatic process to produce n-3 PUFA-enriched glyceride concentrates and demonstrates that AY “Amano” 400SD can effectively discriminate against n-3 PUFAs during hydrolysis.

Direct flow injection profiling of acyl glycerols from food products using isopropanol as solvent.

This work proposes a novel method for the direct flow injection profiling of acylglycerols in edible oils and fats without preliminary extraction and consequent reconstitution in the injection solvent. The work exploits the outstanding performance of high-resolution mass spectrometry to target unique elemental compositions even in the most complex matrices. The performance of isopropanol as the unique solvent for both the solubilization and analysis of acylglycerols was investigated in comparison with other classical methods involving preliminary extractions, sample recovery, and analysis. The calibrations of two triglyceride standards (triolein and trilinolenin) were successfully performed in presence and absence of oil matrix. As final application, the effects on the acylglycerol fraction of a heat treatment on three different fat samples (extra virgin olive oil, lard oil, and fat from dry cured ham-speck) were monitored. The proposed method is therefore suitable for a rapid evaluation of acylglycerol fractions in food lipid samples.

Highly Efficient and Enzyme-Recoverable Method for Enzymatic Concentrating Omega-3 Fatty Acids Generated by Hydrolysis of Fish Oil in a Substrate-Constituted Three-Liquid-Phase System.

A novel three-liquid-phase system which contained fish oil as the nonpolar phase was developed for the lipase-based hydrolysis of fish oil and subsequent enrichment of the omega-3 polyunsaturated fatty acids (n-3 PUFA) in the glyceride fraction of the fish oil. In comparison with the traditional oil/water system, the enrichment factor of n-3 PUFA in this system was increased by 363.4% as a result of a higher dispersity, higher selectivity of the lipase for the other fatty acids except for n-3PUFA, and relief of product inhibition. The content of n-3 PUFA in the glyceride fraction could be concentrated to 67.97% by repeated hydrolysis after removing the free fatty acids. Furthermore, the lipase could be reused for at least eight rounds. This method would be an ideal approach for enriching n-3 PUFA because it is cost-effective, low in toxicity, and easily scaled up.

Enzymatic ethanolysis subjected to Schizochytrium biomass: Sequential processing for DHA enrichment and biodiesel production

To valorize oleaginous Schizochytrium sp. biomass, a novel sequential process was addressed and developed to simultaneously produce high value DHA-enriched glycerides and low value bulky biodiesels. In this process, Schizochytrium sp. biomass was first treated by alkaline protease; then the resulting biomass was directly subjected to enzymatic ethanolysis catalyzed by liquid enzymes from C. antarctica (CALA), T. lanuginosus (TL), R. miehei (RM) and A. oryzae (PLA). For all four liquid lipases, the alkaline protease treatment on Schizochytrium sp. biomass significantly enhanced total lipid recovery, biodiesel yield and yield of DHA-enriched glycerides. Under optimized conditions, CALA and TL achieved the highest biodiesel yields 76.28–76.66 wt% based on microalgae TFAs while PLA had the lowest (55.25 wt%). Moreover, CALA attained the highest DHA (77.93 wt%) and LC PUFAs (93.08 wt%) contents in glyceride fraction, suggesting a superior performance to concentrate DHA and LC PUFAs in glycerides. 13C NMR analysis illustrated that non-regiospecificity and unfavorability towards LC PUFAs of CALA made it possible to transesterify saturated fatty acids located in all 3 positions of microalgal oil; thus LC PUFAs were highly enriched in glycerides. Overall, the sequential process using enzymes and ethanol as green media was successfully established for the co-production of high-value microalgal bioproducts rich in LC PUFAs and sustainable biodiesels in a comprehensive manner.

Lipase-catalyzed selective enrichment of omega-3 polyunsaturated fatty acids in acylglycerols of cod liver and linseed oils: Modeling the binding affinity of lipases and fatty acids

Present study employed molecular modeling method to elucidate the binding affinity of lipases with fatty acids of different chain lengths; and investigated the effects of lipases positional and fatty acids specificity on omega-3 polyunsaturated fatty acids (ω-3 PUFAs) enrichment in cod liver and linseed oils. Among the lipases studied, molecular modeling showed the active sites of Candida rugosa lipase (CRL) had a low C-Docker interactive energy for saturated (SFA) and monounsaturated (MUFA) fatty acids which predicted CRL to have highest preferences to selectively hydrolyze resulting in efficient enrichment of ω-3 PUFAs. Verification experiments showed the SFA and MUFA in the acylglycerol fraction includes monoacylglcyerols (MAG), diacyglycerols (DAG), and triacylglycerols (TAG) of CRL-hydrolyzed cod liver oil decreased from the initial 25.21 to 16.88% and 45.25 to 32.17%, respectively. In addition, CRL-hydrolyzed cod liver oil demonstrated 88.36% of ω-3 PUFAs enrichment. The regio-distribution of fatty acids in CRL-hydrolyzed cod liver oil were not significantly different than that of cod liver oil indicating the ω-3 PUFAs enrichment was due to fatty acids selectivity and not positional selectivity of CRL.

Combination of modern plant breeding and enzyme technology to obtain highly enriched erucic acid from Crambe oil

Fatty acids from vegetable oils are useful building blocks for industrial materials. The purpose of this work was to prepare erucic acid with high purity from a vegetable oil. High purity erucic acid is used for the production of erucamide with applications in plastics manufacturing. A newly developed transgenic Crambe line produces seed oil with 68 % erucic acid compared to 53 % in the wild type oil. Further enrichment of erucic acid from Crambe (wild type and transgenic) oil was achieved by selective enzymatic hydrolysis. Using Candida rugosa lipase as catalyst, other fatty acids were preferentially hydrolysed from the triacylglycerols and erucic acid was enriched in the acylglycerol fraction. The highest content of erucic acid achieved in that fraction was 95 %. The combination of modern plant breeding and enzyme technology is a promising approach for preparation of fatty acids of high purity.

GC/MS Analysis of Fatty Acids in Italian Dry Fermented Sausages

The present investigation reports a study about the evolution, during ripening, of the fatty acid profile and the fatty acid composition in acylglycerols of three different fermented sausages industrially produced in the Calabria region (Southern Italy). Statistical analysis (ANOVA) was applied to the results obtained for the profiles to check all the differences between samples. The study comprised also an evaluation of the lipid oxidation level. All kind of sausages showed a free fatty acids profile in which the monounsaturated fatty acids were predominant, followed by saturated and polyunsaturated fatty acids. In acylglycerols, a low content in linoleic acid (approximately 2% of total methyl esters) was displayed, while that found in the free fatty acids profile was higher (approximately 17% of total FFAs). In addition, the generation of aldehydes through secondary lipid oxidation was clearly confirmed after long ripening period by classical qualitative colorimetric method supported by 1H NMR spectroscopy. The differences in fatty acids profiles observed in the free fatty acids and the acylglycerol fractions were certainly due to the high selectivity of lypase activity during the ripening.

Enzymatic synthesis of 1,3-dicaproyglycerol by esterification of glycerol with capric acid in an organic solvent system

Abstract In this work, the esterification of glycerol with capric acid catalyzed by an immobilized form of a 1,3-positionally selective lipase ( Rhizomucor miehei ) showed to be effective for the synthesis of 1,3-dicaprin in n -heptane as the reaction medium. The effects of the reaction parameters were studied using an experimental factorial design of three factors and three levels with two central points. The selected experimental variables were amount of glycerol adsorbed on silica gel ( G ), biocatalyst load ( E ) and reaction temperature ( T ), and the response variables were total conversion of capric acid, acylglycerol fractions, selectivity and yield of dicaprin, and acyl migration reaction. The range of each parameter was selected as follows: G = 50–250 mg, E = 20–40 mg and T = 40–60 °C. At optimum conditions 73% capric acid conversion was achieved, with 76% dicaprin selectivity, and selectivity to the specific 1,3-dicaprin of 70% of total products. An adequate selection of the reaction conditions is necessary not only to maximize the conversion of capric acid, but also to minimize the acyl migration reaction and the generation of undesired products. Evidence of kinetically controlled enzymatic acyl migration from sn-3/sn-1 to sn-2 is presented.

Concentration of Docosahexaenoic Acid (DHA) by Selective Alcoholysis Catalyzed by Lipases

AbstractThe aim of this work was to obtain acylglycerols from tuna oil (23 % weight DHA) rich in docosahexaenoic acid (DHA) by selective ethanolysis, catalyzed by lipases. First, seven immobilized lipases were tested and the best DHA concentration and recovery in the acylglycerol fraction were attained with Lipozyme TL IM® from Thermomyces lanuginosus, Lipozyme RM IM from Rhizomucor miehei, and lipase from Thermomyces lanuginosus immobilized on Immobead 150. As it is the cheapest, Lipozyme TL IM® was selected to optimize the reaction conditions. The influence of temperature, reaction time, and ethanol/oil and lipase/oil ratios were studied. Under the optimized conditions (35 °C, ethanol/oil molar ratio 2.3, lipase/oil ratio 5 % weight and 48 h) and for a 56 % conversion, acylglycerols were obtained with a 45 % DHA concentration and 90 % recovery. In these optimized conditions the reaction was scaled up to 766 g of tuna oil and carried out in a batch stirred tank reactor, with the lipase contained in a cartridge filter attached to the stirring rod. The results were similar to those obtained on the smaller scale. The DHA‐enriched acylglycerols were separated from the ethyl esters by evaporation of the latter in a short‐path vacuum distiller, where the influence of distillation temperature was studied. At 170 °C DHA‐rich acylglycerols (44 % DHA) were recovered in the residue with 94.5 % purity and 72 % recovery.

Repeated hydrolysis process is effective for enrichment of omega 3 polyunsaturated fatty acids in salmon oil by Candida rugosa lipase

Enrichment of omega 3 polyunsaturated fatty acids (PUFA) in the glyceride fraction of salmon oil was performed by Candida rugosa lipase (CRL)-catalysed hydrolysis. Total omega 3 PUFA content in the product was 38.71% (mol.%), more than double of the initial level. The hydrolysis reaction was scaled up to 1l in a stirred tank reactor without any decrease in the selectivity of the reaction. Moreover, non-steroselectivity and acyl chain specificity of CRL-catalysed hydrolysis, as well as CRL’s recognition of the whole triacylglycerol molecule, were proved. The product was purified by short path distillation, which was also shown to contribute to the recovery of omega 3 PUFA in residue. After the removal of free fatty acids, the final product was subjected to a second round of hydrolysis to concentrate omega 3 PUFA further to 50.58%. Recoveries of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) decreased slightly after the second round of hydrolysis, while oleic acid (OA) content was not affected.

Enzymatic enrichment of omega‐3 polyunsaturated fatty acids in Nile perch (Lates niloticus) viscera oil

AbstractOil was extracted from fatty material obtained from Nile perch viscera using the protease Protex 30L. Enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the glyceride fraction was carried out by hydrolysis of extracted oils with lipases from Candida rugosa, Thermomyces lanuginosus and Pseudomonas cepacia. The unusual fatty acid distribution of the oil influenced the apparent lipase specificity to a large extent. In the unhydrolysed oil, only 16% of EPA was in sn‐2 position while 51% of palmitic acid was located in this position of the triacylglycerol (TAG) molecules. Non‐regioselective lipase from C. rugosa was the most effective in combined enrichment of both EPA and DHA. This was partly because it was able to hydrolyse off palmitic acid from the sn‐2 position, which 1‐, 3‐specific lipases were unable to do. Hydrolysis with C. rugosa lipase enriched EPA from 3 to 6 mol% and DHA from 9 to 23 mol%, with recoveries of 42 and 55%, respectively. The 1‐, 3‐specific lipase from T. lanuginosus was ineffective in enriching EPA, but gave best DHA enrichment, 38 mol% with a recovery of 39%. DHA was rather equally distributed in sn‐1, ‐2 and ‐3 positions of TAG. The results show that both the fatty acid specificity and regiospecificity of the lipase as well as the fatty acid distribution of the oil should be considered when choosing the strategy for fatty acid enrichment.

13C NMR spectroscopy for determining the acylglycerol positional composition of lampante olive oils. Chemical shift assignments and their dependence on sample concentration

Two Lampante olive oils with free acidity expressed as oleic acid of 25.9 and 8.4 g per 100 g were used to carry out the 13C NMR study for the assignment of all resonances of free fatty acid fraction and of the acylglycerol fraction comprising 1(3)-mono- (1(3)-MAG), 1,3-di- (1,3-DAG), 1,2-di- (1,2-DAG) and triacylglycerols (TAG). The glycerol carbon resonances were found to be resolved according to the esterification degree of the glycerol molecule but no data were available on the length and the unsaturation pattern of the acyl chains. It was the spectral region where the C-1 carboxy carbons resonate that enabled the determination of free fatty acids and of different acylglycerol species. Moreover, within each acylglycerol class, the acyl chain distribution between the 1,3- and 2- glycerol positions was also calculated where the saturated, vaccenate, oleate and linoleate chains were detected as baseline resolved resonances in 1(3)-MAG, 1,3-DAG, 1,2-DAG and TAG. The variability of chemical shifts to sample concentration changes was also evidenced in all the frequency ranges being studied where the methylene C-2 and C-3 carbons underwent the most dramatic changes in their resonance patterns which prevented any resonance assignment.