Interesterification and hydrolysis catalyzed by fatty acid‐modified lipases

Native lipases often exhibit poor interesterification activity. We previously developed a fatty acid modification method to improve the activity of lipases. In this study, we applied this fatty acid modification method to several lipases and evaluated their interesterification and hydrolytic activities. The resulting interesterification activity was strongly dependent on the modifying fatty acid used. Of the saturated fatty acids tested, stearic acid modification substantially improved the interesterification activity of three lipases. Hydrolytic activity was affected slightly by the modifying fatty acid used. Substrate specificity of the modified lipase with triglycerides was also investigated and it was found that fatty acid modification changed the substrate specificity of some lipases.     

Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System

Enzymatic interesterification of palm stearin with coconut oil was conducted by applying a dual lipase system in comparison with individual lipase-catalyzed reactions. The results indicated that a synergistic effect occurred for many lipase combinations, but largely depending on the lipase species mixed and their ratios. The combination of Lipozyme TL IM and RM IM was found to generate a positive synergistic action at all test mixing ratios. Only equivalent amount mixtures of Lipozyme TL IM with Novozym 435 or Lipozyme RM IM with Novozym 435 produced a significant synergistic effect as well as the enhanced degree of interesterification. The interesterification catalyzed by Lipozyme TL IM mixed with thermally inactivated immobilized lipase preparations indicated that the carrier property may play an important role in affecting the interaction of two mixed lipases and the subsequent reactions. A dual enzyme system, consisting of immobilized lipases and a non-immobilized one (Lipase AK), in most cases apparently endows the free lipase with a considerably enhanced activity. 70% Lipase AK mixed with 30% immobilized lipase (Lipozyme TL IM, RM IM and Novozym 435) can achieve an increase in activity greater than 100% over the theoretical value when the reaction proceeds for 2 h. The co-immobilization action of the carrier of the immobilized lipases towards the free lipase was proposed as being one of the reasons leading to the synergistic effect and this has been experimentally verified by a reaction catalyzed by a Lipase AK-inactivated preparation. No apparently synergistic effect of the combinations of Lipozyme TL IM and RM IM was observed when the dual enzyme systems applied to the continuous reaction performed in a packed bed reactor. In brief, this work demonstrated the possibility of increasing the reaction rate or enhancing the degree of conversion by employing a dual lipase system as a biocatalyst.     

Deodorization of lipase‐interesterified butterfat and rapeseed oil blends in a pilot deodorizer

A mixture of butterfat and rapeseed oil (7 : 3, wt/wt) was interesterified using immobilized lipase from Thermomyces lanuginosus at 50 °C. The interesterified mixture was then deodorized at five temperatures (60–180 °C) and three samples were withdrawn at 1, 2, and 3 h. The operation was monitored by free fatty acid (FFA) content, peroxide value (PV), volatiles, and the sensory evaluation of the samples with respect to flavor and odor (most importantly the butter flavor and odor and the off‐flavor and odor from butyric acid). ANOVA partial least squares regression analysis showed that deodorization time, and especially deodorization temperature, significantly affected the sensory properties and levels of volatiles, FFA and peroxides in the samples. The best compromise between removing undesirable off‐flavors while maintaining the desirable butter flavor seemed to be obtained by using a deodorization temperature of 120 °C for 2 h. Response surface methodology analysis showed a significant effect of deodorization temperature and, to a lesser extent, deodorization time. The butter flavor and odor had an optimum at a deodorization temperature of approximately 100–120 °C for 1–2 h. These conditions are therefore recommended in order to remove the off‐flavor and odor, while maintaining as much as possible of the attractive butter flavor and odor.     

Increasing short-chain fatty acid yield during lipase hydrolysis of a butterfat fraction with periodic aqueous extraction

Factors affecting the release of short-chain fatty acids during hydrolysis of a butterfat fraction with a 1,3-positional and short-chain-specific Penicillium roqueforti lipase were investigated. When a short-chain triglyceride fraction was used as substrate, as opposed to whole butterfat, the ratio of desirable flavor short-chain free fatty acids (FFA) to undesirable medium-chain FFA in the FFA fraction increased from 0.75 to 1.80. However, with both substrates, FFA accumulation eventually led to lipase inhibition and limited the total amount of triglyceride hydrolysis. This inhibition phenomenon was principally due to product inhibition. Periodically extracting the FFA with a buffer solution minimized this inhibition phenomenon, thereby significantly increasing lipase activity and the degree of triglyceride hydrolysis. Thus, on-line extraction of FFA in lipase reactors has the potential of greatly increasing system productivity.     

Increasing short-chain fatty acid yield during lipase hydrolysis of a butterfat fraction with periodic aqueous extraction

Factors affecting the release of short-chain fatty acids during hydrolysis of a butterfat fraction with a 1,3-positional and short-chain-specific Penicillium roqueforti lipase were investigated. When a short-chain triglyceride fraction was used as substrate, as opposed to whole butterfat, the ratio of desirable flavor short-chain free fatty acids (FFA) to undesirable medium-chain FFA in the FFA fraction increased from 0.75 to 1.80. However, with both substrates, FFA accumulation eventually led to lipase inhibition and limited the total amount of triglyceride hydrolysis. This inhibition phenomenon was principally due to product inhibition. Periodically extracting the FFA with a buffer solution minimized this inhibition phenomenon, thereby significantly increasing lipase activity and the degree of triglyceride hydrolysis. Thus, on-line extraction of FFA in lipase reactors has the potential of greatly increasing system productivity.     

Polyunsaturated fatty glyceride syntheses by microbial lipases

The degree of glyceride syntheses by lipase TOYO (Chromobacterium viscosum) and lipase OF (Candida cylindracea) using individual free fatty acids C_18∶1, C_18∶2, C_18∶3, C_18∶4, C_20∶4, C_20∶5 and C_22∶6 were compared. Lipase TOYO incorporated each of the fatty acids into glycerol at levels of greater than 89%. Lipase OF incorporated most of the fatty acids at levels above 70% (docosahexaenoic acid incorporation was 63%). It was concluded that these two lipases are feasible for producing glycerides from unsaturated fatty acids.     

Selection of surfactant-modified lipases for interesterification of triglyceride and fatty acid

Interesterification of tripalmitin and stearic acid inn-hexane was investigated with surfactant-modified lipases. Various kinds of lipases and surfactants were screened for high interesterification activity of the modified lipases. The modified-lipase hydrophile-lipophile balance value and fatty acid group of the surfactants. The modified lipase obtained fromRhizopus japonicus with sorbitan monostearate as surfactant had the highest activity in then-hexane system. The interesterification activity of the selected modified lipase in molten substrates at 75°C without solvents was the same as that in then-hexane system at 40°C.     

The effect of immobilization on the hydrolytic/interesterification activity of lipase from Rhizopus niveus

Lipase from Rhizopus niveus was immobilized by physical adsorption on Celite 545 and glass beads. The results showed that the highest immobilization efficiency and specific hydrolytic activity of 96% and 9.2 meq/mg protein/min, respectively, were obtained with Celite as the carrier. However, the specific hydrolytic activity of lipase adsorbed on glass beads by acetone precipitation was similar to that obtained by the Celite carrier, although the protein loading capacity was relatively low. The results showed that lipase immobilized on glass beads exhibited similar activity profiles with respect to reaction time, different enzyme concentrations, and water content, using trimyristin and tripalmitin as substrates, to those obtained with the free enzyme. In contrast, the immobilized lipase on Celite exhibited a considerably lower hydrolytic activity. However, the results also showed that the lipase activities of the free enzyme and the immobilized Celite enzyme were similar when the more hydrophilic triolein was used as the substrate. The interesterification of a mixture of tripalmitin and trimyristin or triolein was carried out using both the free and immobilized enzymes. The results indicated that the hydrolytic activity of lipase was similar in both cases for the first 24 h, after which it decreased dramatically. These findings suggest that at this late stage an equilibrium between the hydrolytic and interesterification reactions was reached.     

微乳液法合成棒状纳米Ni

在25℃时,作出SDS/正丁醇/正庚烷/水体系中SDS和正丁醇质量比为1∶1时的拟三元相图,用电导率曲线讨论了该体系由W/O型→双连续型→棒状液晶→层状液晶→双连续型→O/W型的微观结构转变。并在70℃时,以棒状液晶结构的微乳液为反应模板,用水合肼为还原剂还原NiCl2,合成出棒状纳米Ni。TEM研究表明,棒的直径处于20~30 nm,长度为150~250 nm,长径比为7~10。合成结果表明,通过控制反应模板的结构,可以有效地合成特定形状的纳米粒子。     

Triglyceride interesterification by lipases. 1. Cocoa butter equivalents from a fraction of palm oil

Twelve commercially available triacylglycerol lipase preparations were screened for their suitability as catalysts in the interesterification of palm oil mid fraction and ethyl stearate to form a cocoa butter equivalent. Five fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipase preparations was tested with sunflower seed oil and was independent of their interesterification activity. The operational stability of three of the preparations most suited for production of cocoa butter equivalents was examined. The amount of a commercial lipase preparation loaded onto a support was surveyed for optimum short-term catalytic activity. The influence of solvent concentration on the reaction rate and the purity of the product was examined at two temperatures. The optimum solvent concentration at 40°C was 1–1.5 grams of solvent/gram of substrate; at 60°C, the rate of interesterification diminished and the purity of the product decreased with increasing amounts of solvent. Four of the commercial lipase preparations found to be suitable interesterification catalysts were immobilized on five supports and their ability to catalyze the interesterification of a triglyceride and palmitic acid or ethyl palmitate was measured. The choice of support and substrate form (esterified or free fatty acid) greatly affected the catalytic activity. Some preparations were more affected by the choice of support, others by the form of the substrate. No preparation yielded maximum activity on all supports, and no support was found which produced an immobilized enzyme preparation of high activity with every commercial lipase preparation. Caution is advised in transferring observations about the suitability of a support from tests on one commerical enzyme preparation to others; individual testing is required.     

Modification of butterfat by selective hydrolysis and interesterification by lipase: Process and product characterization

Butterfat was chemically modified via combined hydrolysis and interesterification, catalyzed by a commercial lipase immobilized onto a bundle of hydrophobic hollow fibers. The main goal of this research effort was to engineer butterfat with improved nutritional properties by taking advantage of the sn-1,3 specificity and fatty acid specificity of a lipase in hydrolysis and ester interchange reactions, and concomitantly decrease its level of long-chain saturated fatty acid residues (viz., lauric, myristic, and palmitic acids) and change its melting properties. All reactions were carried out at 40°C in a solvent-free system under controlled water activity, and their extent was monitored via chromatographic assays for free fatty acids, esterified fatty acid moieties, and triacylglycerols; the thermal behavior of the modified butterfat was also assessed via calorimetry. Lipase-modified butterfat possesses a wider melting temperature range than regular butterfat. The total saturated triacylglycerols decreased by 2.2%, whereas triacylglycerols with 28–46 acyl carbons (which contained two or three lauric, myristic, or palmitic acid moieties) decreased by 13%. The total monoene triacylglycerols increased by 5.4%, whereas polyene triacylglycerols decreased by 2.9%. The triacylglycerols of interesterified butterfat had ca. 10.9% less lauric, 10.7% less myristic, and 13.6% less palmitic acid residues than those of the original butterfat.     

Interesterification of sesame oil and a fully hydrogenated fat using an immobilized lipase catalyst in both batch and continuous‐flow processes

Lipase‐mediated interesterification of sesame oil and a fully hydrogenated soybean oil was studied at 70 °C in both a batch reactor (BR) and a continuous‐flow packed‐bed reactor (PBR) using four different initial weight ratios of substrates (90 : 10, 80 : 20, 70 : 30 and 60 : 40) with Lipozyme TL IM (Thermomyces lanuginosa) as the biocatalyst. Reaction rates were determined by following the dependence of the profile of the product triacylglycerols (TAG) on the reaction time (BR) or the space time (PBR) via RP‐HPLC‐ELSD. Product TAG identities were confirmed by HPLC‐APCI‐MS. Primary differences between the performances of the two reactors were the maximum level of net hydrolysis (ca. 3 and 10 wt‐% lower acylglycerols at equilibrium for the PBR and BR, respectively), the time or space time required to approach quasi‐equilibrium conditions, and less migration of acyl groups in the PBR trials. For the BR trials, quasi‐equilibrium conditions were approached in 4–6 h, while for the PBR trials short space times (15 min to 2 h) were sufficient to produce effluent compositions similar to equilibrium BR compositions. The predominant TAG families formed by interesterification were LLS, PSO, PSL, SSL, and SSO (L = linoleic; S = stearic; P = palmitic; O = oleic). Oxidative stabilities, melting profiles and solid fat contents were determined for selected reaction products.     

Specificity of papaya lipase in esterification of aliphatic alcohols a comparison with microbial lipases

Straight-chain saturated C4 to C18 alcohols and unsaturated C18 alcohols such as cis-9-octadecenyl (oleyl) cis-6-octadecenyl (petroselinyl), cis-9, cis-12-octadecadienyl (linoleyl), all-cis-9,12,15-octadecatrienyl (α-linolenyl), and all-cis-6,9,12-octadecatrienyl (γ-linolenyl) alcohols, were esterified with caprylic acid using papaya (Carica papaya) latex lipase (CPL) and immobilized lipase from Candida antarctica (Lipase B, Novozym, NOV) and Rhizomucor miehei (Lipozyme, LIP) as biocatalysts. With CPL, highest activity was found for octyl and decyl caprylate syntheses, whereas both NOV and LIP showed a broad chain-length specificity toward the alcohol substrates. CPL strongly discriminated against all C18 alcohols studied, relative to n-hexanol, whereas the microbial lipases accepted the C18 alcohols as substrates nearly as well as n-hexanol. Both petroselinyl and γ-linolenyl alcohol were very well accepted as substrates by NOV as well as LIP, although the corresponding fatty acids, i.e., petroselinic and γ-linolenic acid, are strongly discriminated against by several microbial and plant lipases, including LIP and CPL.     

Influence of Interesterification of a Stearic Acid-Rich Spreadable Fat on Acute Metabolic Risk Factors

Chemical and enzymatic interesterification are used to create spreadable fats. However, a comparison between the two processes in terms of their acute metabolic effects has not yet been investigated. A randomised crossover study in obese (plasma TAG > 1.69 mmol/L, and BMI > 30 (BMI = kg/m²) or waist circumference > 102 cm, n = 11, age = 59.3 ± 1.8 years) and non-obese (plasma triacylglycerol (TAG) < 1.69 mmol/L, and BMI < 30  or waist circumference < 102 cm, n = 10, age = 55.8 ± 2.2 years) men was undertaken to compare the effects of chemical versus enzymatic interesterification on postprandial risk factors for type 2 diabetes (T2D) and cardiovascular disease (CVD). TAG, cholesterol, glucose, insulin and free fatty acid concentrations were measured for 6 h following consumption of 1 g fat/kg body mass of non-interesterified (NIE), chemically interesterified (CIE), enzymatically interesterified (EIE) stearic acid-rich fat spread or no fat, each with 50 g available carbohydrate from white bread. Interesterification did not affect postprandial glucose, insulin, free fatty acids or cholesterol (P > 0.05). Following ingestion of NIE, increases in serum oleic acid were observed, whereas both oleic and stearic acids were increased with CIE and EIE (P < 0.05). While postprandial TAG concentrations in non-obese subjects were not affected by fat treatment (P > 0.05), obese subjects had an 85% increase in TAGs with CIE versus NIE (P < 0.05). The differences in TAG response between non-obese and obese subjects suggest that interesterification may affect healthy individuals differently compared to those already at risk for T2D and/or CVD.     

生物柴油基吸收剂净化甲苯废气

研究了新型微乳液吸收剂对含甲苯废气的吸收性能。以生物柴油、水、Tween-80和正丁醇制备微乳液,最佳制备条件是:m(Tween-80)∶m(正丁醇)=2∶1,HLB值为12.33,温度为室温。吸收实验结果表明,微乳液对甲苯的吸收率高达91.14%。对吸收废液进行再生实验,二次回收的吸收液对甲苯仍然具有较好的吸收效果,吸收率高达83.85%。     

烷基聚葡糖苷表面活性剂形成微乳液的研究

以烷基聚葡糖苷(APG或CiGi)为表面活性剂，C4～C6正构醇为助表面活性剂，采用肉豆蔻酸异丙酯为油相，与水一起得到微乳液体系。研究了在不同烷基链长度的醇作用下，不同烷基碳链长度的烷基聚葡糖苷形成微乳液体系的拟三元相图以及δ—γ相图，并用HLB方程计算了微乳液界面的一些重要参数。发现使用较长碳链的醇以及用较长碳链的APG均有利于单相及中相微乳液的形成，并对此进行了解释，同时确定了单相微乳液效率最高的体系。     

作为微反应器的微乳液体系研究进展

概述了作为“微反应器”的微乳液体系研究,进展其中包括纳米粒子的制备，聚合反应，有机合成反应等；并阐明了微乳液各组分对于化学反应的影响。     

Interesterification kinetics of triglycerides and fatty acids with modified lipase inn-hexane

The kinetics of lipase-catalyzed interesterification of triglycerides and fatty acids in organic media was studied. First, the lipase Saiken 100,Rhizopus japonicus, was modified by surfactant to form an enzyme precipitate in aqueous solution, which was well dispersed in organic solvents. This modified lipase catalyzed the interesterification of tripalmitin and stearic acid. The enzyme has 1,3-positional specificity and does not distinguish between stearic and palmitic acids. The kinetic model developed to describe the interesterification reaction system is based on mass balance of two consecutive second-order reversible reactions. The reaction rate constant, k, was determined by solving the differential rate equations of the reaction system and by expressing the value of k as a function of concentrations of the substrates with time. The model gave satisfactory results. The best value of the specific reaction rate constant k* that fits all experimental data was 1.2 · 10⁻⁵ [L²/(mmol · mg biocatalyst · h)] under the reaction conditions in this study.     

生物柴油制备过程中磷脂及水分含量对脂肪酶的影响

用叔丁醇作为反应介质,利用固定化脂肪酶催化油脂原料甲醇解反应制备生物柴油,消除了甲醇和甘油对酶的负面影响,酶的使用寿命显著延长,进一步系统研究了以叔丁醇作为反应介质,磷脂及水含量对脂肪酶催化特性的影响。当体系中中含水量达2%以上或磷脂含量达0.1%油重时将对固定化脂肪酶LipozymeIM TL 和Novozym 435的催化活性造成明显的不利影响。同时还发现把脂肪酶LipozymeIM TL 和 1%Novozym 435混合起来能大大提高脂肪酶的耐水、耐磷脂能力。     

10%抗蚜威微乳剂的研制

对国内外文献未见报道的10%抗蚜威微乳剂制剂进行了研制,测定了微乳剂理化性状及生物活性。研制的10%抗蚜威ME透明温度范围-5℃￣60℃,乳液稳定性(20倍,1h)和低温稳定性(-5℃±1℃,14d)合格,热贮稳定性(54℃±2℃,14d)分解率小于5%,经时稳定性(2年)合格。对麦长管蚜室内生物测定结果表明,LC50毒力比25%抗蚜威WG高2.1倍。