Enzymatic modification of triacylglycerols of high eicosapentaenoic and docosahexaenoic acids content to produce structured lipids

Immobilized lipase, IM60, from Rhizomucor miehei was used as a biocatalyst for the incorporation of capric acid (C10:0) into fish oil originally containing 40.9 mol% eicosapentaenoic (20:5n-3) and 33.0 mol% docosahexaenoic (22:6n-3) acid. Acidolysis was performed with and without organic solvent. Pancreatic lipase-catalyzed sn-2 positional analysis was performed after enzymatic modification. Tocopherol analysis was performed before and after enzymatic modification. Products were analyzed by gas-liquid chromatography. After a 24-h incubation in hexane, there was an average of 43.0±1.6 mol% incorporation of C10:0 into fish oil, while 20:5 and 22:6 decreased to 27.8±2.2 and 23.5±1.3 mol%, respectively. The solvent-free reaction produced an average of 31.8±8.5 mol% C10:0 incorporation, while 20:5 and 22:6 decreased to 33.2±3.3 and 28.3±3.9 mol%, respectively. The effect of incubation time, substrate molar ratio, enzyme load, and added water were also studied. In general, as the enzyme load, molar ratio, and incubation time increased, mol% C10:0 incorporation also increased. The optimal mol% C10:0 incorporation was 41.2% at 48 h for the reaction in hexane and 46.4% at 72 h for the solvent-free reaction. The highest C10:0 incorporation (65.4 mol%) occurred at a molar ratio of 1:8 (fish oil triacylglycerols/capric acid) in hexane. For the solvent-free reaction, the optimal mol% C10:0 incorporation (56.1 mol%) occurred at a molar ratio of 1:6. An enzyme load of 10% gave the highest mol% C10:0 incorporation (41.4 mol%) in hexane; the highest incorporation (38.3 mol%) for the solvent-free reaction occurred at 15% enzyme load. Mol% incorporation of C10:0 declined with increasing amounts of added water. The optimal mol% C10:0 incorporation occurred at 1% added water (47.9 mol%) for the reaction in hexane, and at zero added water for the solvent-free reaction (21.8 mol%). Fish oil containing capric acid was successfully produced and may be nutritionally more beneficial than unmodified oil.     

Preparation of polyunsaturated phospholipids by lipase-catalyzed transesterification

Transesterifications were investigated to determine a means for preparing polyunsaturated phospholipids simply from soy phospholipid, sardine oil, and two kinds of microbial lipases originating fromCandida cylindracea andRhizopus delemar. The optimum reaction conditions forCandida cylindracea lipase were: 4 g of sardine oil, 10 mL of water, 0.7 g of lipase, 10 mL of hexane, 48 hr of reaction time at 37°C for 3 g of soy phospholipid, for which the transesterification ratio reached approximately 45%. Recovery of phospholipid was low, because hydrolysis also occurred under these reaction conditions. However, hydrolysis could be suppressed by using glycerine instead of water, and the recovery of phospholipid increased to 47%, although the transesterification ratio was reduced to 32%. Rhizopus delemar lipase has 1,3-specificity for triglycerides, and the transesterification ratio was approximately 37% in the 1-position of phospholipid. The resulting phospholipid was rich in polyunsaturated fatty acids and linoleic acid, while the total percentage of polyunsaturated fatty acids incorporated was 18.4%. Therefore, polyunsaturated phospholipids can be prepared easily by transesterification of soy phospholipid with fish oil by means of commercial lipases.     

Lipase-catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids

Structured lipids were synthesized by acidolysis of perilla oil and caprylic acid using two lipases, Lipozyme RM IM from Rhizomucor miehei and Lipozyme TL IM from Thermomyces lanuginosa. Effects of molar ratio, reaction time, reaction temperature, enzyme load, and solvent content on acidolysis reactions were studied. The solvent content ranged from 0.0 (solvent-free) to 85.3%. The results showed that the incorporation increased in parallel with solvent content to 49.0% with Lipozyme RM IM and to 63.8% with Lipozyme TL IM. After 24 h incubation in n-hexane, caprylic acids were incorporated to 48.5 mol% with Lipozyme RM IM and to 51.4 mol% with Lipozyme TL IM, respectively, whereas linolenic acid content was reduced from 61.4 to 31.5 mol% with Lipozyme RM IM and to 28.4 mol% with Lipozyme TL IM, respectively. Lipozyme TL IM showed a higher acyl migration rate than Lipozyme RM IM when acidolysis was performed in the reaction system containing n-hexane as a solvent, whereas the difference in acyl migration between the two lipases in the solvent-free system was negligible.     

Lipase-catalyzed acidolysis of tristearin with oleic or caprylic acids to produce structured lipids

Two different structured lipids (SL) were synthesized by transesterifying tristearin with caprylic acid (C8∶0) or oleic acid (C18∶1). The objective was to synthesize SL containing stearic acid (C18∶0) at the sn-2 position as possible nutritional and low-calorie fats. The reaction was catalyzed by IM60 lipase from Rhizomucor miehei in the presence of n-hexane. The effects of reaction parameters affecting the incorporation of caprylic acid into tristearin were compared with those for incorporating oleic acid into tristearin. For all parameters studied, oleic acid incorporation was higher than caprylic acid. The range of conditions favorable for synthesizing high yields of C8∶0-containing SL was narrower than for oleic acid. An incubation time of 12–24 h and an enzyme content of 5% (w/w total substrates) favored C8∶0 incorporation. The mole percentage of incorporated C18∶1 did not increase further at enzyme additions greater than 10%. C18∶1 incorporation decreased with the addition of more than 10% water (w/w total substrates) to the tristearin-oleic acid reaction mixture. Increasing the mole ratio of fatty acid (FA) to triacylglycerol increased oleic acid incorporation. The highest C8∶0 incorporation was obtained at a 1∶6 mole ratio of tristearin to FA. Positional analysis confirmed that C18∶0 remained at the sn-2 position of the synthesized SL. The melting profiles of tristearin-caprylic acid and tristearin-oleic acid SL displayed peaks between −20 to 30°C and −20 to 40°C, respectively. Their solid fat contents (∼25%) at 25°C suggest possible use in spreads or for inclusion with other fats in specialized blends.     

Characterization of enzymatically synthesized structured lipids containing eicosapentaenoic, docosahexaenoic, and caprylic acids

Structured lipids (SL) containing n-3 polyunsaturated (eicosapentaenoic or docosahexaenoic) and mediumchain (caprylic) fatty acids were synthesized in gram quantities and characterized. Tricaprylin was mixed with n-3-rich polyunsaturated fatty acids in a 1:2 molar ratio and transesterified by incubating at 55°C in hexane with SP 435 lipase (10% by wt of total substrates) in a 125-mL Erlenmeyer flask as the bioreactor. After several batches of reaction, the products were pooled and hexane was evaporated. Short-path distillation was used for purification of synthesized SL. The distillation conditions were 1.1 Torr and 170°C at a feed flow rate of 3 mL/min. Up to 240 g of SL was isolated and deacidified by alkaline extraction or ethanol-water solvents. The fatty acid profile, free fatty acid value, saponification number, iodine value, peroxide value, thiobarbituric acid, and conjugated diene contents were determined. Oxidation stability, with α-tocopherol as antioxidant, and the oxidative stability index were also determined.     

代谢工程改造酵母生产多不饱和脂肪酸的研究进展

多不饱和脂肪酸因其在食品和医药领域的广泛作用而得到人们极大的关注,当前利用微生物发酵生产多 不饱和脂肪酸具有诸多优点,由于酵母生产迅速且生物量较高,利用酵母生产多不饱和脂肪酸已成为人们关注 的热点。本文综述了代谢工程改造酵母生产多不饱和脂肪酸的研究进展,以常规酵母-酿酒酵母和非常规酵母- 解脂耶氏酵母为例,介绍了酵母菌中多不饱和脂肪酸的代谢途径、酵母产油脂的生化机制、代谢工程改造酵母 产多不饱和脂肪酸以及不饱和脂肪酸积累对酵母耐受性的影响。以后研究工作的重点是进一步加强对酵母生产 多不饱和脂肪酸的机理研究,并以此为来指导代谢工程改造酵母生产多不饱和脂肪酸。     

Enzymatic enrichment of C20 cis-5 polyunsaturated fatty acids fromBiota orientalis seed oil

Enrichment ofcis-5 polyunsaturated fatty acids [20:3(5c,11c,14c), 4.3% and 20:4(5c,11c,14c,17c), 11.3%] fromBiota orientalis seed oil was carried out by lipase-catalyzed selective esterification and hydrolysis reactions. Lipases fromRhizomucor miehei (Lipozyme),Candida cylindracea and porcine pancreas were used. Lipozyme-catalyzed esterification ofBiota fatty acids withn-butanol inn-hexane allowed 20:3 and 20:4 (as fatty acids) to be enriched to a maximum level of 52.9%, and in the presence ofC. cylindracea lipase 61.5% enrichment was achieved. Esterification with pancreatic lipase was poor with low levels of enrichment of 20:3 and 20:4 (22%). A multigram scale esterification of the free fatty acids fromBiota seed oil by repeated treatment of the isolated fatty acid fraction withn-butanol inn-hexane in the presence ofC. cylindracea lipase furnished an enrichment yield of 72.5% of a mixture of 20:3 and 20:4 fatty acids. Urea fractionation of the free fatty acids ofBiota oil gave an initial enriched fraction of 20:3 (9.5%) and 20:4 (25.2%) which, upon treatment withC. cylindracea lipase inn-butanol andn-hexane, gave an enriched fraction of 85.3% of 20:3 and 20:4 fatty acids. Partial hydrolysis of the triglycerides ofBiota oil byC. cylindracea lipase in potassium phosphate buffer at 25°C resulted in a 2.8-fold enrichment ofcis-5 polyunsaturated fatty acids (40.8% of 20:3 and 20:4) as contained in the unhydrolyzed acylglycerol fractions.     

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (V_max app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s⁻¹·mg protein⁻¹), with a K_m value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg²⁺, Ca²⁺, Cu²⁺ and Cd²⁺, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.     

Kinetics of lipase‐catalysed interesterification of triolein and caprylic acid to produce structured lipids

The influence of the molar ratio caprylic acid/triolein, enzyme concentration and water content on the kinetics of the interesterification reaction of triolein (TO) and caprylic acid (CA) were studied. The enzyme used was the 1,3‐specific Rhizomucor miehei lipase. Data modelling was based on a simple scheme in which the acid was only incorporated in positions 1 and 3 of the glyceride backbone. In addition, it was assumed that positions 1 and 3 of the triglycerides were equivalent and that the events at position 1 did not depend on the nature of the fatty acid in position 3 and vice versa. Monoglycerides and diglycerides were not detected during the experiments. This was attributed to the low water content of the immobilised enzyme particles. The value of the equilibrium constant, K, for the exchange of caprylic and oleic acids was 2.7, which indicated that the incorporation of caprylic acid into triglycerides was favoured compared with the incorporation of oleic acid. Simple first order kinetics could describe the interesterification reaction. Using this model and the calculated equilibrium constant, the apparent kinetic constants were calculated. The model fitted all the experimental data except for the CA/TO molar ratios larger than 6. Moreover, the interesterification reaction rate had a maximum value at CA/TO molar ratios of 4–6 mol mol^?1. Copyright © 2003 Society of Chemical Industry     

Effect of lipids and surfactants on extracellular lipase production by Yarrowia lipolytica

The production of extracellular lipase in submerged cultures of Yarrowia lipolytica CECT 1240 has been investigated. Several compounds have been added to the culture medium, in order to assess their efficiency as inducers of lipase production. First, the effect of triglycerides (olive oil, sunflower oil, tributyrin) and fatty acids (oleic acid) has been studied. The highest activity level was obtained with sunflower oil (58 U cm^?3), followed by olive oil (49 U cm^?3). The cultures with tributyrin and oleic acid attained similar activities (33 U cm^?3). Then, several surfactants (Tween 80, Triton X‐100, gum arabic, polyethylene glycol 200) were added to the cultures with sunflower oil, in an attempt to increase the levels of extracellular lipase activity. The obtained activities were slightly lower than those achieved without surfactants. The assay of a wide range of surfactant concentrations in the case of PEG‐200 (with which the highest activity levels had been attained) did not improve the results. This strain secreted lipase concentrations two‐fold higher and showed significantly different behaviour towards the presence of surfactants in the culture medium, compared with other wild‐type Yarrowia lipolytica strains. Copyright © 2003 Society of Chemical Industry     

Alteration of Fatty Acid Profile in Fragile X Syndrome

Fragile X Syndrome (FXS) is the most prevalent monogenic cause of Autism Spectrum Disorders (ASDs). Despite a common genetic etiology, the affected individuals display heterogenous metabolic abnormalities including hypocholesterolemia. Although changes in the metabolism of fatty acids (FAs) have been reported in various neuropsychiatric disorders, it has not been explored in humans with FXS. In this study, we investigated the FA profiles of two different groups: (1) an Argentinian group, including FXS individuals and age- and sex-matched controls, and (2) a French-Canadian group, including FXS individuals and their age- and sex-matched controls. Since phospholipid FAs are an indicator of medium-term diet and endogenous metabolism, we quantified the FA profile in plasma phospholipids using gas chromatography. Our results showed significantly lower levels in various plasma FAs including saturated, monosaturated, ω-6 polyunsaturated, and ω-3 polyunsaturated FAs in FXS individuals compared to the controls. A decrease in the EPA/ALA (eicosapentaenoic acid/alpha linoleic acid) ratio and an increase in the DPA/EPA (docosapentaenoic acid/eicosapentaenoic acid) ratio suggest an alteration associated with desaturase and elongase activity, respectively. We conclude that FXS individuals present an abnormal profile of FAs, specifically FAs belonging to the ω-3 family, that might open new avenues of treatment to improve core symptoms of the disorder.     

Effects of some leaf-emitted volatile compounds on aphid population increase

A role of some volatile compounds produced by plant tissues may be as defensive molecules against various pests, including arthropods. Volatile six-carbon compounds derived in plant tissue from polyunsaturated fatty acids via lipoxygenase/hydroperoxide lyase reduced tobacco aphid fecundity at certain concentrations when added to headspace vapor to which aphids were exposed. Both C₆ aldehydes and alcohols were effective, with the alcohols having greater activity. (Z)-3-Hexenyl acetate at levels in the headspace similar to those of the alcohols and aldehydes did not reduce aphid fecundity. A 6-hr exposure period to the C₆ aldehydes and alcohols was needed for maximum effect on the aphids feeding on tobacco leaves. Analysis of the direct versus indirect effects of these compounds indicates that the volatile aldehydes had both direct effects on aphid fecundity and indirect effects due to induced changes in the leaves upon which the aphids were feeding, while only indirect effects were observed for the alcohols. Tomato leaves have the capacity to produce volatile compounds at levels that impact aphid population increase, with the volatiles produced from crushed leaves having a much larger effect. The C₆ aldehydes and alcohols may be components of the fecundity reduction seen with tomato volatiles; however, volatile terpenes showed no effect. These results can be of significance for the genetic alteration of plants for improved aphid resistance.The investigation reported in this paper (No. 93-3-1) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director.     

Characterization and oxidative stability of enzymatically produced fish and canola oil-based structured lipids

Two-kilogram quantities of structured lipids (SL) of menhaden fish and canola oils containing caprylic acids (8∶0) were produced in a laboratory-scale packed-bed bioreactor by acidolysis catalyzed by an immobilized lipase, Lipozyme IM, from Rhizomucor miehei. SL were characterized and their oxidative stabilities investigated. The SL contained 29.5% 8∶0 for fish oil and 40.15 for canola oil. Polyunsaturated fatty acids (PUFA) of fish oil remained unchanged after the modification while PUFA of canola oil were reduced from 29.6 to 21.2%. Monoenes, especially 18∶1n−9, were completely replaced by 8∶0 in fish oil and reduced from 61.9 to 34.7% in canola oil. Downstream processing of enzymatically produced SL led to loss in natural total tocopherol contents of the fish and canola oils. The effects of antioxidants such as α-tocopherol (TOC), tert-butylhydroxyquinone (TBHQ), and combinations thereof on the oxidative stability of SL were investigated. SL were analyzed for oxidative stability index, peroxide value, conjugated diene content, free fatty acid content, iodine value, saponification number, and thiobarbituric acid value. Iodine value of unmodified fish oil (154.71) was reduced to 144.10 and that of canola oil (114.49) to 97.27 after modification. The SN increased from 183.72 to 242.63 for fish oil and from 172.50 to 227.90 for canola oil. TBHQ exhibited better antioxidant effects than TOC. A combination of TBHQ/TOC also proved to be an effective antioxidant for SL. We suggest the addition of antioxidants to enzymatically produced and purified SL.     

Assessment of lipase- and chemically catalyzed lipid modification strategies for the production of structured lipids

The purpose of the present study was to devise a two-step reaction to produce partial glycerides, which would subsequently be used as substrates in both lipase-catalyzed and chemically catalyzed esterification reactions with caprylic acid. The yields and kinetics of these two-step reactions were compared to established lipase-catalyzed acidolysis and transesterification as well as to chemical transesterification reactions. Acyl migration did not occur during the hydrolysis or short-path distillation steps in the preparation of free fatty acid-free partial glycerides for esterification reactions. No significant differences in final yields (59.9% to 82.8% w/w of total triacylglycerols) of new structured lipids were detected among lipase-catalyzed (24 h) and chemically catalyzed (5 h) reactions; however, the yield of new structured triacylglycerols (TAG) after 2 h was lower for acidolysis than for the other lipase-catalyzed reactions (P≤0.05). Since no differences in final yields were detected among the reactions, chemical esterification using hydrolyzed oil could represent the best synthetic option, since it offers the advantage of positional distribution control associated with lipase-catalyzed reactions as well as rapid reaction times associated with chemically catalyzed reactions. Attempts to evaluate the positional distribution of caprylic acid using allyl magnesium bromide (Grignard reagent) were hampered by production of unknown species, which prevented accurate determination of the concentration of some key fatty acids.     

Hydrolysis of acylglycerols and phospholipids of milled rice surface lipids during storage

The relative contribution of acylglycerols and phospholipids to the lipid hydrolysis in milled rice was investigated during storage at 37°C and 70% RH for 50 d. The MAG, DAG, and lysophospholipid contents of surface lipid were determined by reversed-phase HPLC. The MAG and DAG content of milled rice increased during storage from 0.06 to 0.18% (w/w milled rice), with the MAG content increasing more than that of the DAG. Lysophosphatidylcholine increased throughout the study from 0.013 to 0.034% (w/w), whereas lysophosphatidylinositol and lysophosphatidylethanolamine contents initially increased from 0.002 to 0.003% and from 0.005 to 0.009% (w/w), respectively, and then stabilized until day 50. The relative percentage of TAG and phospholipids hydrolyzed in milled rice increased rapidly during the first 3 d of storage from 12.3 to 37.6% and 25.0 to 62.5% (w/w), respectively, and steadily increased to 53.1 and 73.8% (w/w) on day 50. A higher percentage (62.5%) of phospholipids was hydrolyzed relative to TAG (37.6%) after 3 d and probably contributed significantly to milled rice lipid hydrolysis during early storage. However, TAG concentration (0.57%, w/w) was much higher relative to phospholipids (0.08%, w/w) in surface lipids, and therefore TAG hydrolysis was the major contributor to FFA development and the quality of stored milled rice.     

Analysis of regiospecific distribution of FA of TAG using the lipase-catalyzed ester-exchange

A very simple and versatile GC method has been developed that can be utilized for quick analysis, in many samples, of the FA compositions at the sn-2- and sn-1,3-positions of TAG. By using the lipase-catalyzed, sn-1,3-regioselective esterexchange reaction of TAG with ethyl acetate, followed by direct injection of the crude reaction mixture into the GC apparatus without any pretreatment, the FA located at the sn-2-position could accurately be analyzed as a TAG derivative in which the sn-1,3-positions were substituted by an acetate residue. Furthermore, the FA located at the sn-1,3-positions could simultaneously be analyzed as the corresponding ethyl ester derivatives using this method. The reliability of the analytical method was compared with conventional methods by analyzing the TAG consisting of caprylic acid (C) at the sn-2-position and oleic acid (O) at the sn-1,3-positions, giving comparable analytical results. The present method was applied to the analysis of the structured lipids CCD and CCE, consisting of TAG as a major component in which C and the highly unsaturated FA, DHA (D) or EPA (E), were specifically bound at the sn-2- and the sn-1,3-positions, respectively.     

Enzymatic modification of triolein: Incorporation of caproic and butyric acids to produce reduced-calorie structured lipids

Lipase-catalyzed acidolysis of triolein with caproic and butyric acids was performed to produce reduced-calorie structured lipids (SL). The SL were obtained by incubating a 1:4:4 mole ratio of triolein, caproic acid, and butyric acid, respectively, with 10% of lipase (w/w of total substrates) in 1.5 mL hexane at 55°C for 24 h. Of nine commercially avaialble lipases screened, IM60, which contains the lipase from Rhizomucor miehei, was the most effective and produced 13 mol% unreacted triolein, 49% disubstituted, and 38% monosubstituted triacylglycerols that contained short-chain fatty acids. The products were analyzed by reverse-phase high performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters studied included time course, temperature, enzyme load, and substrate mole ratio. The yields obtained demonstrate that a structured lipid with long-chain and short-chain fatty acids can be synthesized by using IM60 lipase in organic medium.     

Comparison between extraction of lipids and fatty acids from microalgal biomass

Seven solvent mixtures have been used to extract the lipid fraction of lyophilized biomass ofIsochrysis galbana. Six of them were composed of biocompatible solvents. Each method was carried out under relaxed operating conditions (i.e., one hour at room temperature) with extraction in a nitrogen atmosphere to prevent autooxidation and degradation of polyunsaturated fatty acids (PUFAs). Apart from the well-established Bligh and Dyer method [Can. J. Biochem. Physiol. 37:911 (1959)] (Cl₃CH/MeOH/H₂O, 1∶2∶0.8, vol/vol/vol), which rendered the highest yield of lipids (93.8%), ethanol (96%) and hexane/ethanol (96%), 1∶2.5 vol/vol produced the best results (84.4 and 79.6%, respectively). To obtain free fatty acids, KOH was added to the solvent mixtures used to extract the total lipids, except for Cl₃CH/MeOH/H₂O, and direct saponification was carried out at 60°C for 1 h or at room temperature for 8 h. The highest yields obtained by direct saponicification were 81% with hexane/ethanol (96%), 1∶2.5, vol/vol and 79.8% with ethanol (96%). Partial yields of the mainn-3 PUFAs found inI. galbana, stearidonic acid (SA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were calculated for both extraction methods. For lipid extraction with ethanol (96%), yields of 91, 82 and 83% were obtained for SA, EPA and DHA, respectively. When direct saponification was used, hexane/ethanol (96%; 1∶2.5, vol/vol) produced the best yields of (91, 79 and 69% for SA, EPA and DHA, respectively).     

Extrusion deactivation of rice bran enzymes by pH modification

Rice bran is considered in Mexico as “waste”, useful only for feeds. As considerable amounts of oil are available in rice bran, it might be worthwhile to stabilize it and extract the edible oil before using it for feedstuffs. Precisely these oils are responsible for rice bran rapid deterioration, particularly in climatic conditions such as those prevalent in Mexico's tropical areas (high humidity and high temperature). This paper deals with the study of the effect of pH during extrusion of fresh rice bran in order to inactivate lipid‐breaking enzymes. Hydrochloric acid or calcium hydroxide, Ca(OH)₂, were added at 0, 1, 5, 10% (dry basis), and moisture content of the bran samples was varied (20, 30, 40%, dry basis) in a 3² factorial design to corroborate its effect at acid and alkaline pH range. Free fatty acids (FFA) increase was the control variable. Extruded samples were stored at room temperature (between 20 and 28 °C) using a non‐extruded sample as control to assess the shelf life effects. Results indicate that in acid‐extruded samples, the increase in FFA concentration after 98 days was much less than in the unmodified‐pH or alkaline samples. The lowest FFA increase after 3 months of storage time was <10 mg FFA/g rice bran using extrusion with no water or chemicals added or using extrusion adding HCl, irrespective of the moisture content of rice bran.