Enzymatic modification of canola/palm oil mixtures: Effects on the fluidity of the mixture

The bioreactor system to interesterify edible oils and fats at an ultra-micro aqueous phase of 100 ppm and less was investigated. The adsorption of lecithin, together with lipase onto a carrier, was effective for conducting the interesterifying reaction efficiently for oils and fats in micro aqueous phase. To improve the handling properties of palm oil at rather low temperature, palm oil was blended with canola or soybean oil, and then these blended oils were modified by enzymatic selective interesterification in a solvent-free, ultra-micro aqueous bioreactor system with an immobilized lipase that had 1,3-positional specificity. The effects of enzymatic interesterification were confirmed by triglyceride determination, by solid fat content profiles and by cloud point profiles, which were also compared to products of chemical interesterification. The improvement in the fluidity of blended oils with canola oil by the enzymatic reaction was bigger than with soybean oil, and chemical interesterification had no effects on the fluidity of blended oils.     

Lipase-catalyzed alcoholysis of triglycerides for short-chain monoglyceride production

Lipase from Pseudomonas fluorescens efficiently catalyzed the alcoholysis of various TG in dry alcohols. For TG with short-chain FA, more MG were accumulated. The yields of MG were affected by the alcohols used. The maximum yields of MG were as follows: 85% for monoacetin in n-butanol, 96% for monobutyrin in ethanol or n-butanol, 50% for monocaprylin in n-butanol, 48% for monolaurin in isopropanol, and 45% for monopalmitin in isopropanol. The MG produced were judged to be 2-MG by TLC analysis. The presence of organic cosolvent affected the reaction rate of the lipase-catalyzed alcoholysis of TG. For the alcoholysis of various TG in ethanol and cosolvent (1∶1, vol/vol), the rates had the following orders: (i) for tributyrin, hexane > toluene > acetone > ethyl acetate > chloroform > acetonitrile > pyridine; (ii) for tricaprylin, hexane > acetone > toluene > acetonitrile > ethyl acetate > pyridine > chloroform; and (iii) for trialurin, hexane > acetonitrile=acetone > ethyl acetate > pyridine=chloroform > toluene.     

Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sn-diacylglycerols

Regioisomerically pure 1,3-sn-diacylglycerols are conveniently prepared in high yields (>80%) and in large quantities by enzymatic esterification of glycerol in the presence of various 1,3-selective lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) and a variety of different acyl donors like free fatty acids, fatty acid alkyl esters and vinyl esters. All reactions are carried out in aprotic organic solvents of low water content, namelyn-hexane, diethyl ether or tBuOMe. The creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction media by the adsorption of glycerol onto a solid support prior to use was essential for the success of these transformations. The effects of reaction conditions and the regioselectivities of the lipases on the product yields are described in detail.     

Continual conversion of free fatty acid in rice bran oil to triacylglycerol by immobilized lipase

Rice bran oil containing 30–50% free fatty acid was continually converted to an oil containing more than 75% of triacylglycerol (TG) by means of immobilized lipase. The reaction was carried out at 60°C for 24 h with dehydration and reactant mixing by dry nitrogen flow under a positive nitrogen atmosphere. Enzymatic TG synthesis with evaporation by heating was not suitable because of the increasing peroxide value of the oil. Part of this article was presented at the annual meeting of the Japan Oil Chemists' Society at Sendai, Japan, October, 16, 1990.     

Hydrolysis of fats and oils with moist oat caryopses

To improve the economic feasibility of hydrolyzing fats and oils with moist oat caryopses, various factors affecting the efficiency of the process were studied. Caryopses produced with an impact-type dehuller exhibited greater lipase activity than those produced by a wringer-type dehuller. Abrasion of oat caryopses against each other in a fluidized bed released particles rich in lipase. Such lipase concentrates could be added to moist caryopsis reactors to speed fat hydrolysis. Beef tallow, lard, soybean oil and crambe oil were hydrolyzed more efficiently than corn oil, castor oil and milk fat. The poor hydrolysis of castor oil was attributed to the formation of esters with the hydroxy group of ricinoleic acid, and the hydrolysis of castor oil was increased by dilution of the substrate with hexane. Diglycerides inhibited the hydrolysis and accounted for the slower hydrolysis of corn oil. Hydrolysis of milk fat by moist oat caryopses resulted in preferential hydrolysis of C₆ to C₁₀ acids. Erucic acid was released from crambe oil at significantly slower rates than the other acyl groups. High conversions of fats and oils to free fatty acids could be attained by (i) exposing the fats and oils to two to three lots of moist caryopses, (ii) the use of special oat varieties with elevated lipase content, (iii) the addition of oat lipase concentrates to moist caryopsis reactors, and (iv) dilution of the substrate with hexane. Estimates of the cost of producing free fatty acids with these processes indicated that the first three should be profitable. Growth ofClostridium sporogenes spores could not be demonstrated in caryopsis reactors. During the incubation of moist oat caryopses immersed in oil, the free fatty acid content of the internal caryopsis lipid increased only slightly, but there were changes in its fatty acid composition.     

Fractionation and enrichment of CLA isomers by selective esterification with Candida rugosa lipase

A commercial product of CLA contains almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA. We attempted to enrich the two isomers by a two-step selective esterification using Candida rugosa lipase that acted on c9,t11-CLA more strongly than on t10,c12-CLA. An FFA mixture containing CLA isomers was esterified with an equimolar amount of lauryl alcohol in a mixture of 20% water and the lipase. When the esterification of total FA reached 50%, two isomers were fractionated in a good yield: t10,c12-CLA was enriched in FFA, and c9,t11-CLA was recovered in lauryl esters. The FFA were esterified again to enrich t10,c12-CLA. At 27.3% esterification of total FA, the t10,c12-CLA content in FFA increased to 64.8 wt% with 89.3% recovery: The ratio of the content of t10,c12-CLA to that of two isomers was 95.9%. Lauryl esters obtained by the single esterification were employed for enrichment of c9,t11-CLA. After the esters were hydrolyzed, the resulting FFA were esterified again with lauryl alcohol. At 62.0% esterification of total FA, the c9,t11-CLA content in lauryl esters increased to 73.3 wt% with 79.4% recovery: The ratio of the content of c9,t11-CLA to that of two isomers was 95.6%. In a 600-g-scale purification, molecular distillation was effective in separating the reaction mixture into lauryl alcohol, FFA, and lauryl ester fractions.     

Lipase PS-catalyzed transesterification of citronellyl butyrate and geranyl caproate: Effect of reaction parameters

Pseudomonas sp. lipase PS was immobilized by adsorption and tested for its ability to catalyze the synthesis of citronellyl butyrate and geranyl caproate by transesterification in n-hexane. The reaction parameters investigated were: enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse. Yields as high as 96 and 99% were obtained for citronellyl butyrate and geranyl caproate, respectively, with 300 units (approx. 15% w/w of reactants) of lipase PS. Increasing amounts of terpene alcohol inhibited lipase activity, while excess acyl donor (triacylglycerol) concentration enhanced ester production. Optimal yields were obtained at temperatures from 30–50°C after 24-h incubation time. Yields of 90 and 99% were obtained for citronellyl and geranyl esters, respectively, with 2% added water. Solvents with log P values ≥ 2.5 showed the highest conversion yields. pH 7 and 6–8 seemed to be ideal for citronellyl butyrate and geraniol caproate, respectively. The lipase remained active after reusing 12 times.     

Production of cocoa butter-like fat from interesterification of vegetable oils

Cocoa butter-like fat was prepared from completely hydrogenated cottonseed and olive oils by enzymatic interesterification. The optimum reaction time to produce the major-component of cocoa butter, 1(3)-palmitoyl-3(1)-stearoyl-2-monoolein (POS), was 4 hr. The cocoa butter-like fat was isolated from the reaction mixture by two filtration steps. The yield of cocoa butter-like fat was 19%, based on the weight of the original oils. Chromatographic analysis of the product by reversephase high-performance liquid chromatography (HPLC) has shown it contains triglyceride components similar to those of cocoa butter, but that it has slightly more diglycerides. The melting point of this product, as measured by a differential scanning calorimeter, is 39°C, which compares well to the 36°C melting point of natural cocoa butter. Presented in part at the AOCS meeting in Cincinnati, Ohio, in 1989.     

Oil and fat hydrolysis with lipase fromAspergillus sp.

Hydrolysis of olive oil, soybean oil, mink fat, lard, palm oil, coconut oil, and a hydrogenated, hardened oil with lipase from anAspergillus sp. has been studied. The lipase had high specific activity (60,000 U/g) and did not show any positional specificity. The lipase proved to be a more effective catalyst than Lipolase fromA. oryzae, with an optimal activity at 37°C and pH 6.5–7.0. It was activated by Ca²⁺ but inactivated by organic solvents such as isopropanol and propanone. All substrates examined could be hydrolyzed to corresponding fatty acids with this enzyme at concentrations of 5–30 U/meq with yields of 90–99% in 2–24 h. The degree of hydrolysis was almost logarithmically linear with reaction time and occurred in two stages. The lipase was stable and could be repeatedly recycled for hydrolysis.     

Isolation of erucic acid from rapeseed oil by lipase-catalyzed hydrolysis

Three lipases were compared for their ability to hydrolyze high erucic acid rapeseed oil, with the objective of concentrating the erucic acid in a single glyceride fraction. Lipase fromPseudomonas cepacia released all fatty acids rapidly and did not result in selective distribution of erucic acid.Geotrichum candidum lipase released C20 and C22 fatty acids extremely slowly, resulting in their accumulation in the di- and triglyceride fractions. Less than 2% of the total erucic acid was found in the free fatty acid (FFA) fraction. Lipase fromCandida rugosa released erucic acid more slowly than C20 and C18 fatty acids at 35°C but only resulted in a limited accumulation of the erucic acid in the di- and triglyceride fractions. However, when hydrolysis catalyzed byC. rugosa lipase was carried out below 20°C, the reaction mixture solidified and was composed solely of FFAs and diglycerides. The diglyceride fraction contained approximately 95% erucic acid while about 20% of the total erucic acid was found in the FFA fraction. It is concluded that hydrolysis at low temperature withC. rugosa lipase results in a higher purity of erucic acid in the glyceride fraction than can be obtained withG. candidum lipase, but with considerable loss of erucic acid to the FFA fraction.