Interesterification Research Articles

Nutritional enrichment of vegetable oils with long-chain n-3 fatty acids through enzymatic interesterification with a new vegetable lipase

The aim of the present work was to produce vegetable oils enriched with long-chain n-3 fatty acids of nutraceutical interest, through an enzyme-catalyzed interesterification with a new lipase, from physic nut (Jatropha curcas L.). The Vegetable Lipase Powder (biocatalyst) called VLP, which has never been applied in functional foods, was obtained from the physic nut seed, and efficiently hydrolyzed the 95% of waste fish oil in 24 h. Urea precipitation was used to concentrate polyunsaturated fatty acids (PUFA) and was further interesterified with oils of different sources by means of enzymatic catalysis. After the interesterification reaction, which was also catalyzed by the VLP, the PUFA content in coconut oil increased almost ten-fold from 1.8% to 17.7%. In palm oil, the PUFA content increased two-fold from 10.5% to 21.8%, while in olive oil the level of PUFA increased from 8.6% to 21.3%. The mixture of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (3.7% to 3.9%) was incorporated into the triacylglycerol fraction of each of the coconut, palm and olive oils. Through the hydroesterification (hydrolysis followed by interesterification) all the interesterified vegetable oils tested presented sufficient EPA and DHA levels to satisfy the levels recommended for intake by human adults in one tablespoon.

Production of 1, 3-dioleoyl-2-palmitoyl glycerol as a human milk fat substitute using enzymatic interesterification of natural fats and oils

Human milk fat substitutes were synthesized using non-preprocessed natural fats and oils with lipase. Enzymatic interesterification of tripalmitin and oleic acid was carried out in isooctane. The reaction reached equilibrium after 12 h and the reaction conditions were partially optimized as a molar ratio of tripalmitin to oleic acid of 1:5, an initial water content of 0.01 g/L, and a water removal time of 1 h. After interesterification of tripalmitin and oleic acid, the reaction products contained 55.2% 1,3-dioleoyl-2-palmitoyl glycerol (OPO). The OPO yield was 25.2% when commercial palm oil and camellia oil were used as substrates, and the OPO concentration was increased to 53.3% after fractional crystallization of the reaction product.

Synthesis of Structured Lipids by Enzymatic Interesterification of Milkfat and Soybean Oil in a Basket-Type Stirred Tank Reactor

Lipase from Rhizopus oryzae immobilized on polysiloxane–poly(vinyl alcohol) (SiO2–PVA) was used to study the interesterification reaction of the milkfat with soybean oil in a stirred tank reactor (STR) containing baskets for the immobilized enzyme retention in two different configurations: central or lateral. The progress of the reaction was followed by determining free fatty acids, composition in triacylglycerols (TAGs), and consistency. The central basket was chosen for assessing the biocatalyst operational stability by running 10 consecutive batch assays lasting 6 h each. Non-notable deactivation of the biocatalyst was observed during the total operation time (60 h). The potential of the evaluated system to make the milkfat-based fat more spreadable under cooling temperature and with lower saturated fatty acids content was demonstrated in this study. Both evaluated basket reactor designs have shown potential to be used in interesterification reactions of industrial interest.

Structured lipids enriched with unsaturated fatty acids produced by enzymatic acidolysis of silkworm pupae oil using oleic acid

Desilked silkworm (Bombyx mori L.) pupae oil has been identified as a valuable source of unsaturated fatty acids (UFAs). However, direct consumption is a food safety issue due to the presence of sn‐1,3 triacylglycerols (TAGs), including palmitic acid (PA). A simple, selective and safe conversion to structured triacylglycerols (STAGs) with high UFA content is highly desirable. Novel STAGs with low PA and high α‐linolenic acid (ALA), oleic acid (OA) and linoleic acid (LA) content were synthesized from silkworm pupae oil using a solvent‐free system catalyzed by Lipozyme RM IM. Crystallization and melting profiles were determined using thermograms, and optimal conditions involved an enzyme load of 7%, a silkworm pupae oil TAG to OA molar ratio of 1:5, and a reaction temperature of 60°C. The content of PA and stearic acid decreased from 41.04 to 20.11%, while UFAs increased from 61.12 to 77.88%. The STAGs exhibited lower crystallization (−15.44°C) and melting (−23.47°C) profiles than TAGs (4.64 and −5.58°C, respectively).Practical applicationsThe presented methods using a solvent‐free system enable STAGs to be synthesized using a natural substrate (desilked silkworm pupae oil). A simple, selective and safe conversion to STAGs with high UFA content and low PA content is highly desirable. This STAG is rare in nature and chemical synthesis is cumbersome. This STAG could be applied as a clinical treatment for nutritional enhancement with a beneficial effect on human health because of the high content of the ALA.Novel structure triacylglycerols (STAGs) with low palmitic acid and rich in oleic acid and linoleic acid were synthesized from silkworm pupae oil and oleic acid, through solvent‐free enzymatic interesterification catalyzed by Lipozyme RM IM. Under the optimal conditions, the content of palmitic acid and stearic acid decreased from 41.04 to 20.11% in the sn‐1, 3 positions, and the content of unsaturated fatty acids (including palm stearin, oleic acid, linoleic acid and α‐linolenic acid) increased from 61.12 to 77.88%. The STAGs could be added into milk fat substitutes, which may be useful as nutrition enhancers for infants and may decrease obesity.

An alternative method for production of microalgal biodiesel using novel Bacillus lipase.

In this study, enzymatic interesterification is carried out using encapsulated lipase as biocatalyst with methyl acetate as acyl acceptor in a solvent-free system. Lipase, isolated from a marine bacterial isolate, Bacillus sp.S23 (KF220659.1) was immobilized in sodium alginate beads. This investigation elaborated on the effects of various parameters, namely enzyme loading, temperature, water, molar ratio, reaction time and agitation for interesterification. The study resulted in the following optimal conditions: 1.5 g immobilized lipase, 1:12 molar ratio of oil to methyl acetate, 35 °C, 8 % water, 60 h reaction time, 250 rpm of agitation. With the standardized condition, the maximum conversion efficiency was 95.68 %. The immobilized beads, even after ten cycles of repeated usage showed high stability in the presence of methyl acetate and no loss of lipase activity. The microalgal biodiesel composition was analyzed using gas chromatography. The current study was efficient in using immobilized lipase for the interesterification process, since the method was cost-effective and eco-friendly, no solvent was involved and the enzyme was encapsulated in a natural polymer.

Comparison of Chemical and Enzymatic Interesterification of Fully Hydrogenated Soybean Oil and Walnut Oil to Produce a Fat Base with Adequate Nutritional and Physical Characteristics.

The optimal physical, chemical and nutritional properties of natural lipids depend on the structure and composition of triacylglycerols. However, they are not always mutually compatible. Lipid modification is a good way to give them specific functionalities, increase their oxidative stability, or improve their nutritional value. As such, chemical and enzymatic interesterification may be used to modify them and produce structured lipids. In accordance, the aim of this study is to compare chemical and enzymatic interesterification of binary blends of fully hydrogenated soybean oil and walnut oil, using sodium methoxide or Lipozyme TL IM, respectively, to produce a fat base with adequate nutritional and physical characteristics. Three different mass ratios of fully hydrogenated soybean oil and walnut oil blends (20:80, 40:60 and 60:40) were interesterified and evaluated. Total interesterification was determined by the stabilization of the solid fat content. Chemical reaction of the 20:80 blend was completed in 10 min and of the 40:60 and 60:40 blends in 15 min. Enzymatically interesterified blends were stabilized in 120 min at all of the mass ratios. Complete interesterification significantly reduced the solid fat content of the blends at any composition. Chemical and enzymatically interesterified fully hydrogenated blend of soybean and walnut oil at mass ratio of 40:60 showed the plastic curve of an all-purpose- -type shortening rich in polyunsaturated fatty acids, with a high linolenic acid (C18:3n3) content and with zero trans-fatty acids.

Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin, Ethyl Oleate And Mixture of VCO, Soybean Oil And Fish Oil

Human Milk is naturally the only source of food for infant in their early life. It contains 2-6% lipid which provides about 50% of the total energy needed by the infant. Human milk fat (HMF) mainly as TAG with the specific fatty acid composition, palmitic acid (C16:0) (20-25%), which is primary located at sn-2 of glycerol bonds (70%) and oleic acid (C18:1), located at sn-1,3 (35%). HMF also provide fatty acids such as linoleic acid, linolenic acid, EPA, DHA and lauric acid that are very important for infant. The purposes of this research are to synthesize of 1,3-dioleoyl-2-palmitoilglycerol (OPO) and to determine the best composition of OPO, VCO, soybean oil and fish oil for HMFS production for infant formula. Interesterification of tripalmitin and ethyl oleate using immobilized lipase from Rhizomucor miehei (Lipozym RM IM) were used to synthesize of OPO. Interesterification product of mixed VCO, soybean oil and fish oil that are source of lauric acid, linoleic acid, α-linolenic, EPA and DHA, were formulated in mass ratio (58:20:20:2) and (70:18:10:2) for obtaining HMFS which have fatty acids composition similar or close to HMF. Composition of fatty acids from product were analyzed by GCMS. From this research, were obtained HMFS containing palmitic acid as much as 28.89% where 84.49% of that are located at sn-2 while sn-1,3 position are dominated by oleic acid as much as 55.11% from the total 38.7% and 70:18:10:2 w/w is the best composition of interesterification product, VCO, soybean oil and fish oil to obtain HMFS similar to HMF.

Functional monoesters of jojoba oil can be produced by enzymatic interesterification: Reaction analysis and structural characterization

AbstractLong‐chain monounsaturated fatty acid ethyl esters (LMFAEE) and fatty alcohol acetates (LMFAA) are important functional compounds in industry, and new sources are needed. In this study, jojoba oil was interesterified with ethyl acetate (EA) for the combined production of LMFAEE and LMFAA. Lipozyme RM IM and Novozym 435 were employed as the biocatalyst and their efficiencies were compared. Aliquot removal and subsequent 1H NMR analysis were used to monitor reaction progress and determine the interesterification yield (IY, %). Optimization of this reaction was conducted for both lipases with respect to four experimental parameters: temperature, EA content, immobilized lipase load, and reaction time. The highest IY in the presence of Novozym 435 was 97%, which was achieved after 9 hours of reaction at 37°C with 75 wt% EA and 5.00 wt% lipase load at 300 rpm, whereas the IY was 76% in the case of Lipozyme RM IM. The interesterified product was purified by preparative TLC and characterized by 1H NMR, FT‐IR and GC‐MS, indicating the main components are ethyl cis‐11‐eicosenoate (44%), cis‐11‐eicosenyl acetate (25%) and cis‐13‐docosenyl acetate (22%). Novozym 435 exhibited excellent operational stability over 17 interesterification cycles with ca. 12% IY decrease.Practical applications: Conversion of the rare LMF‐acid/alcohol source from jojoba oil to LMFAEE and LMFAA was realized in a one step interesterification reaction. The obtained jojobyl LMFAEE and LMFAA, in mixture or purified form, can serve as functional ingredients in various upmarket products, such as functional lipids, fragrances, cosmetics, pharmaceuticals and lubricants. Cis‐11‐eicosenyl acetate and cis‐13‐docosenyl acetate are also important insect pheromones in biological study. Additionally, the lipase reuse can help reduce cost significantly, making jojoba oil more economically attractive for industrial application.Jojoba oil was converted to long‐chain monounsaturated fatty acid ethyl ester (LMFAEE) and fatty alcohol acetate (LMFAA) via enzymatic interesterification with ethyl acetate. The resulting jojobyl LMFAEE and LMFAA have great potential for various upmarket applications in both industry and academia (e.g., fragrance, cosmetic, pharmaceutical and insect pheromone).

Continuous enzymatic interesterification of milkfat with soybean oil produces a highly spreadable product rich in polyunsaturated fatty acids

AbstractThe aim of this study was to tune the physical properties of milkfat by enzymatic interesterification with soybean oil in a continuous fluidized bed reactor (FBR) to obtain healthy interesterified fat blends having suitable texture properties for the food industry. The immobilized commercial non‐regioselective Candida antarctica lipase (Novozym®435) and sn1,3‐regioselective Rhizopus oryzae lipase, immobilized in an organic‐inorganic hybrid matrix of polysiloxane‐polyvinyl alcohol, were used as biocatalysts in a FBR. The minimum value of the ascendant flow of the medium for allowing fluidization in the system was 3.13 mL·min−1. The reaction was evaluated in terms of the interesterification yield (IY), consistency values and solid fat content (SFC). The IY values of 10.50 ± 1.64% and of 5.70 ± 1.46% were attained for Novozym®435 and for immobilized R. oryzae lipase, respectively. The consistency of the initial 65:35 milkfat/soybean oil mixture (1000 gf/cm2) decreased to 732.35 ± 75.30 gf/cm2 and to 478.02 ± 71.80 gf/cm2 in interesterified blends catalyzed by Novozym®435 or by R. oryzae lipase, respectively. SFC was considered an inadequate parameter for following the interesterification of this blend formulation because the values were similar for initial and for interesterified blends. Free fatty acid levels of 1.5%, the non‐notable deactivation of Novozym®435 and a half‐life of 190 h for R. oryzae lipase were observed during the operation time.Practical applications: By interesterification of milkfat with soybean oil, a new product presenting higher spreadability than butter and richer in polyunsaturated fatty acids coming from soybean oil, was obtained. The Rhizopus oryzae lipase immobilized in an organic‐inorganic hybrid matrix, used in a FBR for the modification of milkfat, promoted a higher reduction (approximately 52.0%) in the consistency of the interesterified products in relation to the initial reaction mixture than that obtained with Novozym® 435 (approximately 27.0% reduction). Also, while Novozym® 435 is as a non‐specific lipase, the R. oryzae lipase presents sn‐1,3 regioselectivity, which enables the maintenance of the unsaturated fatty acids at position sn‐2 in triacylglycerols with well‐known nutritional benefits. This biocatalyst showed a better performance than the immobilized commercial lipase (Novozym® 435). The implementation of the interesterification in a continuous FBR results in lower shear rate imposed on the biocatalyst.The consistency of the initial milkfat/soybean blend (65:35,% w/w) decreased about 52% or 27% after interesterification in a continuous fluidized bed reactor, catalyzed by the immobilized Rhizopus oryzae lipase or by Novozym 435, respectively.

Enzymatically interesterified fats based on mutton tallow and walnut oil suitable for cosmetic emulsions

Formation of emulsion systems based on interesterified fats was the objective of the study. Enzymatic interesterification was carried out between enzymatic mutton tallow and walnut oil in the proportions 2:3 (w/w) to produce fats not available in nature. At the beginning of the interesterification process, the balance between the interesterification and fat hydrolysis was intentionally disturbed by adding more water to the catalyst (Lipozyme IR MR) of the reaction to produce more of the polar fraction monoacylglycerols [MAGs] and diacylglycerols [DAGs]. To obtain a greater quantity of MAGs and DAGs in the reaction environment via hydrolysis, water was added (11, 13, 14, 16 w-%) to the enzymatic preparation. The obtained fats were used to form emulsions. The emulsions were evaluated with respect to sensory and skin moisturizing properties by 83 respondents. Determination of emulsion stability using temperature and centrifugal tests was carried out. Morphology and the type of emulsions were determined. The respondents described the skin to which the emulsions in testing were applied as smooth, pleasant to touch and adequately moisturized. The work has demonstrated that interesterification of a mutton tallow and walnut oil blend resulted in new fats with very interesting characteristics of triacylglycerols that are not present in the environment. The results of the present work indicate the possibility of application of fats with the largest quantity of MAGs and DAGs as a fat base of emulsions in the cosmetic industries. The hypothesis assumed in this work of producing additional quantities of MAGs and DAGs (in the process of enzymatic interesterification) responsible for the stability of the system was confirmed. It should be pointed out that the emulsions based on interesterified fats exhibited a greater level of moisturization of the skin than the emulsions containing non-interesterified fat. Also, in the respondents' opinion, the emulsion containing fat, which was modified during enzymatic interesterification when 13% of water was added to the enzymatic preparation, exhibited the best sensory profile.

Preparation of Human Milk Fat Substitutes from Lard by Lipase‐Catalyzed Interesterification Based on Triacylglycerol profiles

AbstractHuman milk fat substitutes (HMFSs) with triacylglycerol profiles highly similar to those of human milk fat (HMF) were prepared from lard by physical blending followed by enzymatic interesterification. Based on the fatty acid profiles of HMF, different vegetable and single‐cell oils were selected and added to the lard. Blend ratios were calculated based on established physical blending models. The blended oils were then enzymatically interesterified using a 1,3‐regiospecific lipase, Lipozyme RM IM (RML from Rhizomucor miehei immobilized on Duolite ES562; Novozymes A/S, Bagsværd, Denmark), to approximate HMF triacylglycerol (TAG) profiles, particularly with respect to the distribution of palmitic acid in the sn−2 position. The optimized blending ratios were determined to be: lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. The optimized reaction conditions were determined to be: enzyme load of 11 wt%, temperature of 60 °C, water content of 3.5 wt%, and reaction time of 3 hours. The resulting product was evaluated for total and sn−2 fatty acids, polyunsaturated fatty acids, and TAG composition. A high degree of similarity was obtained, indicating the great potential of the product as a fat alternative for use in infant formulas.

Fatty Acids Profile, Oxidative and Hydrolysis Stability of Virgin Coconut Oil and Palm Stearin Based Human Milk Fat Analog

The objectives of the research are to evaluate fatty acids profile, oxidative and hydrolysis stability of human milk fat (HMF) analog by using virgin coconut oil (VCO) and palm stearin as raw materials. The HMF analog was synthesized through enzymatic interesterification catalyzed by lipase from Rhizomucor miehei. The fatty acid profiles of interesterification products were monitored using gas chromatography. Oxidative stability test was carried out for up to 72 hours at 60oC. The peroxide value was measured during 0, 24, 48 and 72 hours of storage duration. Hydrolysis stability test was held for up to 8 days at room temperature. The free fatty acid content was monitored during 0, 2, 4, 6 and 8 days of storage. The results showed that the resulted HMF analog having high percentage of palmitic acid in the sn-2 position, similar to that of HMF. The palmitic acid content in the sn-2 position was around 39.71%. The MCFAs were esterified in the sn-1 and sn-3 position and the main fatty acid constituent was lauric acid of 39.37%. The obtained HMF analog was stable to oxidative and hydrolysis deterioration as indicated by the peroxide value and free fatty acid content during storage.

Biosynthesis of myristyl serinate by immobilized Candida antarctica lipase in two-phase system

The significance and novelty of this manuscript is that myristyl serinate was firstly synthesized from myristyl alcohol and l -serine using lipase catalyzed esterification in selected solvents. The effects of reaction system, two-phase system composition, temperature, pH, and reaction time on myristyl serinate ester content in the crude reaction mixture were investigated. The results showed that two-phase could significantly increase the enzymatic interesterification conversion and the optimal ratio of two-phase solvent was 80:20 (dichloromethane:phosphate buffer). The optimal conditions for myristyl serinate enzymatic synthesis were substrate molar ratio of 1:1, Novozym 435 load of 5% (w/w), a solvent dosage of 20 ml, 30 °C and reaction time of 36 h. Myristyl serinate content could reach 72.25% in the reaction system. Myristyl serinate was purified through column chromatography and identified by HPLC/IR/LC-MS/NMR. • Myristyl serinate was firstly enzyme-synthesized in selected solvents. • Two-phase could significantly increase the enzymatic esterification conversion. • The optimal ratio of two-phase was 80:20 (dichloromethane:phosphate buffer). • Myristyl serinate content reached 72.25% in the system under optimal conditions. • Myristyl serinate was purified, determinated and identified by HPLC/IR/LC-MS/NMR. Myristyl serinate was firstly enzyme-synthesized from myristyl alcohol and l -serine in selected solvents (single and two-phase solvents). The effects of reaction system, two-phase system composition, temperature, pH, and reaction time on myristyl serinate synthesis were investigated. The results showed that two-phase solvent reaction system could significantly increase the enzymatic conversion ratio and the optimal ratio of two-phase solvent was 80:20 (dichloromethane:phosphate buffer). The optimal conditions for myristyl serinate enzymatic synthesis were substrate molar ratio of 1:1, Novozym 435 load of 5% (w/w), a solvent dosage of 20 ml, 30 °C and reaction time of 36 h. Under these conditions, 72.25% of myristyl alcohol could be converted to myristyl serinate, which was purified through column chromatography and identified by HPLC/IR/LC-MS/NMR.

Enzymatically Modified Fats Based on Mutton Tallow and Rapeseed Oil Suitable for Fatty Emulsions

AbstractThe aim of this work was to develop a new fat by enzymatic interesterification of mutton tallow with rapeseed oil. It was assumed that by inducing hydrolysis of fats by addition of water to the enzymatic preparation (8, 10, 15 wt%) natural emulsifiers would be produced in the reaction environment. Fat blends obtained from the enzymatic reactions were evaluated as a fat base for emulsion systems. It was found that the fat resulting from interesterification in the presence of Lipozyme RM IM (immobilized lipase from Rhizomucor miehei, Novozymes Bagsvaerd, Denmark) with 15 wt% of water possessed the highest content of polar fraction (MAG and DAG), and served as the most suitable blend for emulsification, producing emulsions that exhibited the highest stability.

효소적으로 합성된 대칭형과 비대칭형 Triacylglycerol 혼합물의 In Vitro Digestion에서의 소화율 비교

For developing indigestible lipids, symmetric triacylglycerol (ST) and asymmetric triacylglycerol (AT) were produced by enzymatic interesterification using high oleic sunflower oil, palmitic ethyl ester, and stearic ethyl ester in a shaking water bath. Used enzymes were Lipozyme RMIM for ST and Lipozyme TLIM for AT. To remove ethyl ester from reactants, methanol fractionation (reactant : methanol=1:5, w/v, 25°C) and florisil separation (reactant : florisil=1:8, w/w) were applied. Acetone fractionation (reactant : acetone=1:9, w/v) was implemented to separate triacylglcerol (TAG) species into ST and AT. Fractions I (before fractionation), II (after fractionation, liquid phase) and III (after fractionation, solid phase) were separated from ST, whereas fractions IV (after 1st fractionation, liquid phase) and V (after 2nd fractionation, solid phase) were from AT. From sn-2 fatty acid composition analysis, the sum of palmitic acid (C16:0) and stearic acid (C18:0) was 4.9∼6.5 area% in ST (I, II, III), and 41.9∼43.9 area% in AT (IV, V). In vitro digestion was performed for 0, 15, 30, 60, and 120 minutes at 37°C in a shaking water bath. For the digestion results, hydrolysis of V was only 40% compared to others (I, II, III, IV) at 120 minutes due to its melting point (49°C). However, initially (15 minutes), hydrolysis (%) was as follows: V<III<IV<I, II. This order was similar with that of melting point, and a higher TAG content (two saturated fatty acids) was associated with a higher melting point. On the other hand, fractions III and IV showed similar complete melting points (32.5°C, 31.8°C) and different slip melting points (31.3°C, 19.5°C). Even though IV has a lower TAG content composed of two saturated fatty acids than III, it had a similar melting point.

Amberlite에 고정화된 Lipase 제조 및 효소적 Interesterification을 이용한 반응 특성 연구

본 연구는 미생물로부터 유래된 5종(AH, AK, AY, PS와 R)의 lipase들을 Amberlite XAD 7에 흡착법으로 고정화 시킨 후 각 immobilized lipase들의 특성을 알아보았다. 고정화 전과 후의 단백질 함량 및 각 free lipase들과 immobilized lipase들을 이용한 interesterification 반응물의 지방산과 TAG 조성을 분석하였다. 또한, immobilized lipase에 있어 중요한 요인인 reusability를 확인하였다. Free lipase의 단백질 함량은 2.22-11.41%로 AH가 가장 높았던 반면, immobilized lipase에서는 AH, PS와 AK가 mg protein/g support이 높았다. 한편, 반응 특성을 알아보기 위해 카놀라유, PEE와 StEE를 기질로 하여 batch type interesterification을 진행하였을 때, free lipase의 경우 free lipase R을 제외한 다른 free lipase들은 반응시간이 1시간에서 72시간으로 증가함에 따라 총 포화지방산 함량이 증가하였으며 그 중 free lipase AH가 반응성이 가장 높았다. 또한, RP-HPLC를 통해 free lipase AK 반응물을 분석한 결과, 반응시간이 길어질수록 카놀라유(0시간)에서 볼 수 있었던 57.49 area%의 <TEX>${\bigcirc}{\bigcirc}{\bigcirc}$</TEX>가 6.53 area%로 감소하였다. 이는 각 free lipase들이 PEE와 StEE를 효소적 반응에 이용했기 때문이라고 판단된다. 한편, immobilized lipase AY와 R의 경우 반응시간이 1시간에서 48시간으로 증가하여도 카놀라유(0시간)의 총 포화지방산 함량과 큰 차이가 없었으나 immobilized AK의 경우 48시간에서 38.3 area%의 포화지방산 함량으로 가장 높은 활성을 보였다. 또한, 이를 사용하여 continuous type으로 반응하였을 때 유속이 느릴수록 효소와 기질 사이의 접촉 시간이 길어져 반응물의 총 포화지방산 함량이 증가함을 알 수 있었다. Reusability는 immobilized AK, AH와 PS 모두에서 두 번째 반복 반응을 하였을 때, 첫 번째 반응보다 총 포화지방산이 120-196.5% 증가하였다. 그러나 bounding protein 함량이 가장 높았던 immobilized AK는 support에 흡착되었던 free lipase AK의 탈착이 일어나 2번째 반응 후부터 활성이 감소한 반면, immobilized AH와 PS는 활성이 비교적 유지되었다. Immobilized lipases were prepared by physical adsorption using lipase AK, AY, AH, PS and R on Amberlite<TEX>$^{(R)}$</TEX>XAD<TEX>$^{(R)}$</TEX>7 HP resin. With the immobilized lipases (10%), structured lipid was synthesized by enzymatic interesterification of canola oil, palmitic ethyl ester, and stearic ethyl ester in order to study the reaction characteristics. Among the lipase, the highest protein content was obtained from lipase AH (11.41%) before immobilization, while the highest levels of bound protein was observed from immobilized lipase AK (63.91%). Immobilized lipase AK had the highest interesterification activity (38.3% of total saturated fatty acid). Lipase AK was also used for a continuous reaction in which the slow flow of reactant resulted in increased reaction rate. Reusability of immobilized AK, AH and PS increased at the second reaction (120-196.5%). However, the activity of immobilized AK, which had the highest bound protein content (63.91%) decreased after the third reaction, while the activity of immobilized AH and PS was maintained until the sixth reaction.

Effect of Palm Oil Enzymatic Interesterification on Physicochemical and Structural Properties of Mixed Fat Blends

AbstractThe physicochemical properties of binary and ternary fat systems made of commercial samples of palm oil (PO) blended with anhydrous milk fat (AMF) and/or rapeseed oil (RO) were studied. Physical properties such as solid fat content by pulsed‐Nuclear Magnetic Resonance (p‐NMR), melting profile by differential scanning calorimetry (DSC), and polymorphism of the blends were investigated. Palm oil was then batch enzymatically interesterified for 27 h, using Lipozyme® TL IM as biocatalyst, and further blended with AMF and/or RO in the same way. The objective of the present work was to evaluate the effect of batch enzymatic interesterification (B‐EIE) of palm oil on physical characteristics of the investigated fat blends. For that purpose, iso‐solid diagrams have been constructed from p‐NMR data. It was shown that B‐EIE of palm oil modifies its melting behaviour, but also its polymorphic stability and miscibility with other fats. Under dynamic conditions, after B‐EIE, the non‐ideal behaviour (eutectic) detected at low temperatures in the ternary PO/AMF/RO system disappears in the corresponding EIE‐PO/AMF/RO. After static crystallization followed by a tempering, the hardness of palm oil is increased after B‐EIE, as well as the hardnesses of the blends containing this fat compared to the native one. Polymorphism stability of the binary and ternary fat systems is also modified after B‐EIE compared to the corresponding native systems.

Application of the calorimetric and spectroscopic methods in analytical evaluation of the human milk fat substitutes

The aim of this paper was the analytical evaluation of human milk fat substitutes (HMFS) by the calorimetric and spectroscopic methods. The HMFS were obtained by enzymatic interesterification of blend of lard or milk fat with rapeseed oil and concentrate of fish oil. The enzymatic reactions were carried out at 60, 70, and 80 °C for 2 h. A commercially immobilized 1,3-specific lipase, Lipozyme RM IM, was used as a biocatalyst. Oxidative stability of HMFS was determined using the calorimetric method. The oxidative induction time was obtained from the pressure differential scanning calorimetry curves. Peroxide value (PV) and anisidine value were determined using spectroscopic method. Interesterification caused a decrease in oxidative stability. Samples with lower induction times were characterized by higher PV. There was also a strong relation between total polar compound content and induction time. The induction times obtained for analyzed fats can be used as primary parameters for the assessment of the resistance of tested fats to their oxidative decomposition.

The effect of enzymatic interesterification on the physico-chemical properties and thermo-oxidative stabilities of beef tallow stearin and rapeseed oil blends

Beef tallow stearin (BTS) was blended with rapeseed oil (RSO) and the blends: 40 % BTS + 60 % RSO, 30 % BTS + 70 % RSO, 20 % BTS + 80 % RSO were interesterified using immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (Novozym 435) as the catalysts. The starting blends and the products of interesterifications were quantitatively separated into the triacylglycerol and non-triacylglycerol fractions containing free fatty acids (FFA) and of mono- and di-acylglycerols. It has been found that after interesterification the contents of the FFA and of MAG and DAG increased. The slip melting temperatures and solid fat contents of triacylglycerols separated from interesterified samples decreased if compared with non-esterified blends. The DSC melting profiles of the blends before and after interesterification were substantially different. The total fatty acid compositions of fats before and after interesterifications remained unchanged. Their distributions into sn-2 and sn-1,3 positions were close to random when Novozym 435 was used. When Lipozyme IM was used, the fatty acid composition at sn-2 position remained practically unchanged compared with the starting blend. Using the DSC and PDSC techniques, the oxidative stabilities of the fats before and after interesterification were also characterized. The interesterified blends were less resistant on thermo-oxidative degradation than the non-esterified ones.

Abstract 207: Interesterified Fat Rich in Stearic Acid Increases Liver Neutrophil and M1 Macrophage Infiltration and Induces Nonalcoholic Steatohepatitis

Introduction: interesterified fats, rich in saturated fatty acid, have been used by food industry to replace trans. Palmitic acid is the most common dietary fat with known deleterious effects. On the hand stearic acid which is consumed in a low amount, has neutral effect on plasma lipids. Unlike vegetable oils, interesterified fats contain palmitic or stearic acid in the sn-2 position and the positional compositon of triglicerides may regulate its metabolic fate. Thus, the aim of this study was to evaluate the effect on the liver metabolism of the interesterified fats rich in stearic or palmitic fatty acids. Methods: Weaning LDLr-KO male mice were randomly distributed into five groups fed a high fat diet (40% of energy as fat) containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER) during 16 weeks. Total plasma cholesterol and triglyceride concentrations, liver total cholesterol, triglycerides and weight, neutrophil (neutr/area) and classically activated/inflammatory M1(CD11b) infiltrated macrophage were determined. Histological markers of NAFLD activity were assessed. Results: There were no differences in dietary intakes among all groups. The interesterification process did not alter plasma lipids concentrations (mg/dL±SD): PALM vs PALM INTER (499.2 ± 81.7 vs 526.8 ± 92.6) and STEAR vs STEAR INTER (342.6 ± 89.9 vs 363.9 ± 90.0). However, PALM and PALM INTER presented higher total plasma cholesterol, and STEAR and STEAR INTER showed total cholesterol concentrations similar to POLI (320.7±47.4). Liver weight, cholesterol and TG contents did not differ among the groups. POLI and STEARIC groups do not elicit NASH (score &lt;3), PALM and PALM INTER presented intermediate stage (score ≤4) between STEARIC/POLI and STEARIC INTER. Nevertheless, STEAR INTER developed NASH (score ≥5), and induce higher neutrophil infiltration (5.53 ±4.1) as compared to all other groups. The M1 macrophage classically activated infiltration was also higher in STEAR INTER group than in all groups as demonstrated by imunofluorescence. Conclusion: STEAR INTER brings on NASH lesion, increased neutrophil and M1 macrophage classically activated/inflammatory infiltration in the liver.