Studies in the Solvent Interesterification of Acidolysed Cottonseed Oil

The directed rearrangement reaction of an acidolysed cottonseed oil (acidolysed with palmitic acid) in solvent has been studied to find out the mode of distribution of the acyl radicals in the triglyceride moiety and also the changes in glyceride pattern and configuration of the oil after such reaction using pancreatic lipase hydrolysis. The glyceride compositions as calculated by the application of 1,3-random 2-random distribution hypothesis, of M. H. Coleman and W. C. Fulton of acidolysed cottonseed oil before and after directed interesterification reaction point out remarkable changes in the pattern of glycerides namely a notable increase of trisaturated glycerides with the diminution in the content of triunsaturated glycerides. Solid fat indices as determined by measuring the dilatation of fat, content of trisaturated glycerides and moderate slip points of the order of 37°C denote the suitability of using the modified fat thus produced as a good margarine base fat as the said fat has been shown to have a fairly long and extended plastic range as evidenced by low slopes in dilatometric charts compared to other conventional plastic fats.     

Concentration of docosahexaenoic acid in glyceride by hydrolysis of fish oil withcandida cylindracea lipase

In an attempt to concentrate the content of DHA (docosahexaenoic acid) in a glyceride mixture containing triglyceride, diglyceride and monoglyceride, fish oil was hydrolyzed with six kinds of microbial lipase. After the hydrolysis, free fatty acid was removed and fatty acid components of the glyceride mixtures were analyzed. When the hydrolysis withCandida cylindracea lipase was 70% complete, the DHA content in the glyceride mixture was three times more than that in the original fish oil. The EPA (eicosapentaenoic acid) content became almost 70% of the original fish oil. Hydrolysis with other lipases did not result in an increase in the DHA content in the glyceride mixtures. Hydrolysis of DHA-rich tuna oil (DHA content is about 25%) withCandida cylindracea lipase resulted in 53% DHA in the glyceride mixture. The EPA content, however, remained close to that of the original tuna oil. In this report, the acyl chain specificity of lipases is evaluated in terms of hydrolysis resistant value (HRV). HRV is the ratio between the DHA contents in the glyceride mixture of hydrolyzed oil and original oil. HRV clearly indicates differences in hydrolysis between DHA and other fatty acids (e.g., saturated and monoenoic acids).     

Studies in the Glyceride Composition of Partially Hydrogenated Rearranged Glycerides of Cottonseed Oil

The directed rearrangement reaction in solvents of partially hydrogenated cottonseed oil was investigated with special reference to the influence of polarity of solvents and amount of trisaturated glycerides formed. The results as obtained by selective enzymatic hydrolysis, gas liquid chromatography and infrared spectrophotometry of the whole fat triglycerides and of the corresponding monoglycerides of cottonseed oil and partially hydrogenated cottonseed oil, before and after directed interesterification, indicate a general trend of the increase of the saturated fatty acyl radicals in the 2-position of the glyceride moiety with the corresponding decrease of the unsaturated acids. The considerable decrease in the concentration of cis unsaturated acid in the 2-position of the triglycerides of partially hydrogenated cottonseed oil has been observed after the directed rearrangement reaction with the simultaneous enrichment of trans unsaturated acid. It was also observed that cottonseed oil does not show any plasticity, whereas after directed interesterification it shows remarkable plasticity. The plasticity of partially hydrogenated cottonseed oil is further diminished after directed rearrangement reaction.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

Determination of the glyceride structure of fats

A method has been described for the quantitative determination of the following six glyceride types in fats: SSS, SSU, SUS, SUU, USU, and UUU. The method involved a quantitative oxidation of the unsaturated acids in the whole fat to the corresponding dicarboxylic acids. The oxidized fat was separated on a liquid-liquid partition column into two fractions, the first containing glycerides having no dicarboxylic acid or one dicarboxylic acid and the second containing glycerides with two or three dicarboxylic acids. Analysis of these fractions by gas chromatography coupled with lipase hydrolysis allowed the calculation of the proportions of the above six glyceride types. The oxidation, fractionation, lipase hydrolysis, gas chromatographic analysis, and the over-all method were checked on natural fats and mixtures of synthetic glycerides. The final glyceride composition appeared to be reliable to within plus or minus 2 unit per cent. Analyses are given for five natural fats. The compositions found agree very well with those calculated by a distribution theory recently proposed by Vander Wal. Contribution from the National Research Council of Canada, Prairie Regional laboratory, Saskatoon, Saskatchwan. Issued as N.R.C. No. 6161.     

Limit of the solid fat content modification of butter

Three ways have been undertaken to modify solid fat content of butter oil: (i) interesterification, (ii) adjunction of high-melting glycerides and (iii) joint effect of adjunction of high-melting glycerides and interesterification. A solvent-free interesterification, carried out with 1,3-specific lipase fromMucor miehei, resulted in an increase of the solid fat content (SFC) by about 114% after 48 h of interesterification. The changes in triglyceride composition induced by this method were followed by quantitative determination of triglycerides of different equivalent carbon number (ECN) and different theoretical carbon number. The major changes in the triglyceride composition occurred mainly in the concentration of three groups of triglycerides with the same ECN (ECN=38). Adding high-melting glycerides trimyristin (MMM) and tripalmitin (PPP) led to an increase of the SFC measured at 20°C as these proportions increased in the mixture. The joint effect of the addition of MMM or PPP and interesterification was quite significant, mainly for triglycerides that included myristic and palmitic acids. As far as the increase of SFC is concerned, the effect of interesterification decreases when both substrate amounts increase.     

Interesterification of edible oils

Types of interesterification discussed are (a) interchange between a fat and free fatty acids, in which the most important reaction is the introduction of acids of low mol wt into a fat with higher fatty acids; (b) interchange between a fat and an alcohol, e.g., with glycerol, in order to produce emulsifiers like monoglycerides; (c) rearrangement of fatty acid radicals in triglycerides, the so-called transesterification which in recent years has taken on the same importance as hydrogenation or fractionation. In natural fats, the fatty acid radicals are not usually randomly distributed but become so by rearrangement; the distinctive physical properties of natural fats and oils can be changed within limits by this transesterification. Well-known examples are cocoa butter, palm oil, and lard. More important is the transesterification of a mixture of different fats and oils; e.g., the combination of hydrogenation and interesterification allows the production of a solid fat with high linoleic acid content. The composition of glycerides after random interesterification can be calculated by formulas. Distinct from random is such directed interesterification. This is done by working at low temperatures that glycerides with higher melting point crystallize from the reaction mixture. Directed interesterification can be combined with fractionation, for instance, to get a higher yield of liquid fraction from palm oil than is obtained by fractionation alone. The transesterification process can be performed in a batch or continuously. A small amount of metallic sodium or sodium ethylate is used as catalyst, which is destroyed by water or acid and removed after the reaction.     

Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids

Lipase hydrolysis was evaluated as a means of selectively enriching long-chain ω3 fatty acids in fish oil. Several lipases were screened for their ability to enrich total ω-3 acids or selectively enrich either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). The effect of enzyme concentration, degree of hydrolysis, and fatty acid composition of the feed oil was studied. Because the materials that were enriched in long-chain ω3 acids were either partial glycerides or free fatty acids, enzymatic reesterification of these materials to triglycerides by lipase catalysis was also investigated. Hydrolysis of fish oil by eitherCandida rugosa orGeotrichum candidum lipases resulted in an increase in the content of total ω3 acids from about 30% in the feed oil to 45% in the partial glycerides. The lipase fromC. rugosa was effective in selectively enriching either DHA or EPA, resulting in a change of either the DHA/EPA ratio or the EPA/DHA ratio from approximately 1:1 to 5:1. Nonselective reesterification of free fatty acids or partial glycerides that contained ω3 fatty acids could be achieved at high efficiency (approximately 95% triglycerides in the product) by using immobilizedRhizomucor miehei lipase with continuous removal of water.     

Studies in Acidolysis

Acidolysis of a natural triglyceride oil with free fatty acids of different chain length has been studied to find out the degree of interchange of fatty acids, the mode of distribution of the free acids incorporated in the triglyceride molecules and the glyceride composition of the oil after acidolysis. The results as determined by gas-liquid chromatography and pancreatic lipase hydrolysis indicate that the degree of interchange of fatty acids depends on the chain length of the free acids, and the incorporation of the free acids in the glyceride molecules occurs in a purely random manner. Glyceride compositions of the oils are also randomly rearranged after acidolysis reaction.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

On the Mechanism of the Thermal Polymerization of Linseed Oil

The monomeric and polymeric glycerides present in thermally polymerized linseed oils can be separated quantitatively by molecular distillation. Analysis of the fatty acids of monomeric and polymeric glycerides indicate the occurence of intra- and intermolecular condensations respectively. Whereas in the former reaction, leading to the formation of bicyclic, dimeric fatty acid groups within the orginal glycerides, no increase in the molecular weight of the oil takes place, in the latter one, characterized by an increase in the molecular weight, fatty acids of different glyceride molecules are involved. It is shown, however, that the overall increase in molecular weight is due to the dimeric glycerides which are formed by interesterification reactions between glyceride monomers containing dimeric fatty acid groups, the latter resulting from intramolecular condensation.     

Fatty acid composition and glyceride structure of piglet body fat from different sampling sites

Pigs were slaughtered at 16 weeks of age, and fat samples were obtained from outer and inner shoulder, outer and inner loin, and kidney. Fatty acid composition and glyceride type of distribution were determined. Glyceride structure was determined by pancreatic lipase hydrolysis. There were highly significant differences in fatty acid composition between sites. Fatty acids containing less than 18 carbon atoms were preferentially incorporated in the internal positions of the glycerides. The content of saturated fatty acids and fatty acids containing less than 18 carbon atoms at the 2-position of shoulder and loin glycerides was higher than in kidney glycerides. Differences in glyceride types were noted between sites.     

Selective hydrolysis of polyunsaturated fatty acid-containing oil withGeotrichum candidum lipase

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can be concentrated in glycerides by hydrolyzing tuna oil withGeotrichum candidum lipase, the main components in the resulting oil being triglycerides. The reaction mechanism of this selective hydrolysis was investigated. Although the lipase acted well on the esters of oleic, linoleic, and α-linolenic acids, it did not affect the esters of γ-linolenic acid, arachidonic acid, EPA, and DHA as much. The action of PUFA-glycerides was mono-> di- > triglycerides. Furthermore, the condensation of PUFA-partial glycerides and PUFA occurred even in the presence of a large amount of water, and the partial glycerides converted to the triglycerides by transacylation. These results suggested that the PUFA-rich triglycerides were accumulated in the glyceride fraction by the following mechanism: The PUFA-partial glycerides generated by the hydrolysis were converted to PUFA-triglycerides by condensation and transacylation reactions. As the PUFA-triglycerides formed were the poor substrates of lipase, they were accumulated in the reaction mixture.     

Continuous production of structured lipid containing γ-linolenic and caprylic acids by immobilized Rhizopus delemar lipase

Production of a structured lipid containing γ-linolenic acid (GLA) achieved by the continuous acidolysis of borage oil with caprylic acid (CA) using 1,3-specific Rhizopus delemar lipase as a catalyst. The lipase immobilized on a ceramic carrier was activated by feeding the borage oil/CA (1:2, w/w) mixture saturated with water into a column packed with the enzyme. However, the generation of partial glycerides (20%) in the reaction mixture showed that hydrolysis occurred concomitantly with acidolysis. The concomitant hydrolysis was completely repressed by feeding the oil/CA substrate mixture without adding additional water. When the substrate mixture was fed at 30°C and a flow rate of 4.5 mL/h into a column packed with 8 g of the carrier with immobilized lipase, the content of CA incorporated in glycerides was 50 to 55 mol%. The acidolysis activity scarcely changed even though the substrate mixture was continuously fed for 60 d; then it gradually decreased. The CA content in glycerides was decreased to 73% of the initial value after 100 d, but returned to the initial level when the flow rate was reduced to 3.1 mL/h. Molecular distillation was employed to separate the transesterified oil from the reaction mixture. No glycerides were detected in the distillate, and the transesterified oil was recovered as the residue (acid value, 2.6). Regiospecific analysis of the transesterified oil showed that only fatty acids at the 1- and 3-positions of borage oil were exchanged for CA. It was additionally found by high-performance liquid chromatography analysis that all the triglycerides contained one or two CA, and that the triglyceride with two GLA and one CA was also present, because the lipase acted on GLA very weakly.     

Production of triglycerides enriched in long-chain n-3 polyunsaturated fatty acids from fish oil

Processes that combine enzymic and physical techniques have been studied for concentrating and separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil.Candida rugosa lipase was used in hydrolysis reactions to concentrate these acids in the glyceride fraction. By controlling the degree of hydrolysis, two products have been obtained, one enriched in total n-3(∼50%), the other enriched in DHA and depleted in EPA (DHA∼40%, EPA∼7%). The glyceride fraction from these reactions was recovered by evaporation and converted back to triglycerides by partial enzymic hydrolysis, followed by enzymic esterification. Both reactions were carried out withRhizomucor miehei lipase. DHA-depleted free fatty acids from aC. rugosa hydrolysis were fractionated to increase the EPA level (∼30%) and re-esterified to triglycerides by reaction with glycerol andR. miehei.     

Effect of enzymatic interesterification on the melting point of tallow-rapeseed oil (LEAR) mixture

To reduce the melting point of a tallow-rapeseed oil mixture, the triglyceride composition of the mixture was altered by enzymatic interesterification in a solvent-free system. The interesterification and hydrolysis were followed by melting point profiles and by free fatty acid determinations. The degree of hydrolysis was linearly related to the initial water content of the reaction mixture. The rate of the interesterification reaction was influenced by the amount of enzyme but not much by temperature between 50 and 70°C. The melting point reduction achieved by interesterification depended on the mass fractions of the substrates: the lower the mass fraction of tallow, the larger the reduction of the melting point.     

Fractionation procedures for obtaining cocoa butter-like fat from enzymatically interesterified palm olein

Solvent-free lipase-catalyzed incorporation of stearic acid in palm olein by the 1,3-regiospecific Novo Lipase Lipozyme IM20 resulted in the formation of a complex mixture of fatty acid glycerides and free fatty acids. The stearoyl incorporation in palm olein gave rise to the formation of 39.3% of the desired cocoa butter-like triglycerides in the fatty acid glyceride portion, namely distearoyl-oleoyl-glycerol (SOS), palmitoyl-oleoyl-stearoyl-glycerol (POS) and dipalmitoyl-oleoyl-glycerol (POP). A combination of fractionation steps involving initially the removal of free fatty acids (FFA) from the product mixture by steam distillation under vacuum, followed by fractional crystallization of the fatty acid-free glycerides in hexane and/or acetone, gave a fat, whose triglyceride composition and melting profile were comparable to that of cocoa butter as adduced by reversed-phase high performance liquid chromatography (HPLC) and differential scanning calorimetry (DSC). The yield of the cocoa butter-like fat was approximately 25% of the weight of the original palm olein.     

Oat (Avena sativa) caryopses as a natural lipase bioreactor

The caryopses of oats, when moistened and immersed in oils, constitute a natural lipase bioreactor. Hydrolysis was monitored by titration of the free fatty acids in the oil phase and by thin-layer chromatography. The optimum amount of additional water was about 20% of the weight of the caryopses, and the optimum temperature was about 40°C. The reaction was accelerated by gentle agitation, by reducing the viscosity of the oil phase by the addition of nonpolar solvents, and by increasing the amount of lipase on the caryopses. The reaction was inhibited by the accumulation of glycerol in the interior of the caryopses and free fatty acids in the oil phase. The lipase hydrolzyed all three positions of glycerol and there was little accumulation of mono- or diglyceride in the lipid phase. The time necessary to obtain 90% hydrolysis varied for a few days to several weeks. Greater degrees of hydrolysis could be obtained by replacing the caryopses when they became inhibited or by diluting the oil phase with hexane. The glycerol that was released could be recovered by extracting the caryopses with water. The moist oat bioreactor also was capable of catalyzing transesterification and interesterification reactions. Journal Paper No. 13447 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project No. 2799.     

The effect of dietary fat on the glyceride structure of rat carcass fat

Carcass fats were obtained from weanling rats fed a complete diet for 8 weeks, which consisted of 2% cottonseed oil and 10% of the following fats: (1) corn oil; (2) the fatty acids of corn oil; (3) triricinolein; (4) ricinoleic acid; (5) the hydrogenated fatty acids of castor oil ; and (6) commercial hydrogenated shortening. The fats were subjected to both pancreatic lipase and nonspecific hydrolysis ; the resulting acids converted into methyl esters by conventional methods, and subjected to gas Chromatographie analysis. From these data, the positional distri-bution of the component fatty acids, glyceride types, and isomeric forms were calculated. The results indicated a preferential placement of un-saturated acids in the 2- position of the carcass triglycerides and that the carcass fat composition in terms of unsaturated (U) and saturated (S) fatty acid composition is not greatly influenced by the S and U compositions of the dietary fat. It was found that hydroxy acids or their tri-esters are metabolized much the same as are normal triglycerides and exert no particular in-fluence upon the fat structure of the rat. Some type of relationship between the dietary U and the U₃ in the carcass fat appears to be present. The glycerides of the carcass fats examined here are essentially a random mixture of the major glyceride types, but the isomeric forms (SUS, S SU, USU and UUS) are a definite non-random mixture. Carried out at the Food Res. Div., Armour & Co., and at The Burnsides Research Laboratory under research grant No. EF 225 from the National Institutes of Health, U. S. Public Health Service, and Deparmtent of Health, Education, and Welfare.     

Solvent-free enzymatic glycerolysis of marine oils

Marine triglyceride oils (cod liver oil and oils from blubber of harp seal and minke whale) were reacted with glycerol using lipase as a catalyst at low temperature. A solvent-free batch system with magnetic stirring was used. Solidification of the reaction mixture occurred, and a mixture of mono-, di-, and triglycerides was obtained in all cases. The recovered glyceride mixtures were solid at room temperature. The yield of monoglyceride (MG) and the fatty acid profile of the MG fractions were dependent on oil and the type of lipase used as a catalyst. Of the commercially-available lipases investigated, lipase AK fromPseudomonas sp. synthesized the highest yield of MG (42–53%) at 5°C. These MG fractions were low in saturated fatty acids (4–11%) and high in long-chain monounsaturated fatty acids (52–69%). The concentration of n-3 polyunsaturated fatty acids was 12–20%.