无溶剂体系中脂肪酶改造猪油制备功能性脂的研究

我国有丰富廉价的动植物油资源,但这一资源并未得到有效的利用,为了充分利用这些资源,开展了无溶剂系统脂肪酶催化猪油与辛酸酸解制备功能性脂的研究工作。脂肪酶筛选实验表明,在所选用的五种脂肪酶中,来自T .languginosa的固定化脂肪酶LiopzymeTLIM的催化效果最好。以LipozymeTLIM为催化剂,进一步研究了酶量、底物比率、反应时间、反应温度和水分添加量对猪油中辛酸插入率的影响。反应产物通过高效液相色谱法(HPLC)进行分析。研究结果表明,当脂肪酶量为20% (底物重量百分比)时,猪油中辛酸插入率最高。反应时间研究表明,当反应时间达到24h时,辛酸的插入率最高,达到38.77mol%。当猪油与辛酸的比率为1∶2 (摩尔比)时,辛酸的插入率最高,达到30 95mol%。在4 5～6 0℃范围之内,反应温度对辛酸插入率没有明显的影响,温度高于6 0℃时,辛酸插入率降低。水分添加量为2 5 %时,辛酸插入率最高,高达35 76mol%。     

<Emphasis Type="Italic">In Vitro</Emphasis> effect of C<Subscript>2</Subscript>–C<Subscript>18</Subscript> fatty acids on salmonellas

The susceptibility of Salmonella spp. to 15 fatty acids was determined in vitro in cultures grown on glucose. Antimicrobial activity was expressed as IC50 (a concentration at which only 50% of the initial glucose in cultures was utilized). Caprylic acid was the only acid inhibiting glucose utilization. In cultures of S. enteritidis, S. infantis and S. typhimurium, IC50 of caprylic acid ranged from 0.75 to 1.17 mg/mL. A moderate adaptation effect was observed as these values increased 1.5-1.8 times when bacteria were subcultured 10 times in media containing a low concentration of caprylic acid (1/3 IC50). No effect of calcium ions added in excess on antimicrobial activity of caprylic acid was observed. Incubation of salmonellas with caprylic acid (1 mg/mL; 30 min) at pH 5.2-5.3 led to a reduction in the concentration of viable cells below the detection limit; 2-6% of Salmonella cells survived at pH 6.3-6.6.     

Caprylic acid precipitation method for impurity reduction: An alternative to conventional chromatography for monoclonal antibody purification

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes. Biotechnol. Bioeng. 2012; 109: 2589–2598. © 2012 Wiley Periodicals, Inc.     

Parameters affecting incorporation and by-product formation during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent-free system

By-product formation is a serious problem in the lipase-catalyzed acyl exchange of phospholipids (PL). By-products are formed due to parallel hydrolysis reactions and acyl migration in the reaction system. A clear elucidation of these side reactions is important for practical operation in order to minimize by-products during reaction. In the present study we examined the lipozyme RM IM-catalyzed acidolysis for the production of structured phospholipids between phosphatidylcholine (PC) and caprylic acid in the solvent-free system. A five-factor response surface design was used to evaluate the influence of major factors and their relationships on a number of responses reflecting the turnover of main reactions as well as side reactions. The five factors, including enzyme dosage, reaction time, reaction temperature, substrate ratio (mol/mol caprylic acid/PC) and water addition, were varied at three levels with two star points. All parameters besides water addition had an effect on the incorporation of caprylic acid into PC and lysophosphatidylcholine (LPC). Reaction time and enzyme dosage showed increased effect on incorporation into PC, while substrate ratio and reaction temperature showed opposite effect. The PC content decreased with increase of all parameters except for substrate ratio. Optimal conditions are recommended as enzyme dosage 40%, reaction temperature 55 °C, water addition 1%, reaction time 70 h, and substrate ratio 6 mol/mol caprylic acid/PC. Under these conditions an incorporation of 46% with PC accounting for 53% of the PL fraction can be obtained. Regiospecific analysis of the product revealed that the caprylic acid was mainly incorporated into the sn-1 position accounting for 80% of the fatty acids incorporated.     

Susceptibility of<Emphasis Type="Italic">Escherichia coli</Emphasis> to C<Subscript>2</Subscript>-C<Subscript>18</Subscript> fatty acids

The antimicrobial activity of C2-C18 fatty acids was determined in vitro in cultures of two strains of Escherichia coli grown on glucose. Antimicrobial activity was expressed as IC50 (a concentration at which only 50% of the initial glucose in the cultures was utilized). Utilization of glucose was inhibited by caprylic acid (IC50 0.30-0.85 g/L) and capric acid (IC50 1.25-2.03 g/L). Neither short-chain fatty acids (C2-C6) nor fatty acids with longer chain (C12-C18) influenced substrate utilization. Caproic acid, however, decreased cell yield in cultures of E. coli in a dose-dependent manner. No inhibition of glucose utilization was produced with unsaturated fatty acids, oleic and linoleic. Calcium ions added in excess reversed the antimicrobial effect of capric acid, but not that of caprylic acid. Antimicrobial activity of caprylic and capric acid decreased when the bacteria were grown in the presence of straw particles, or repeatedly subcultured in a medium containing these compounds at low concentrations. Counts of viable bacteria determined by plating decreased after incubation with caprylic and capric acid (30 min; 1 g/L) at pH 5.2 from > 10(9) to approximately 10(2)/mL. A reduction of a mere 0.94-1.96 log10 CFU was observed at pH 6.5-6.6. It can be concluded that caprylic acid, and to a lesser extent also capric acid, has a significant antimicrobial activity toward E. coli. Effects of other fatty acids were not significant or absent.     

Partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins

Caprylic acid (octanoic acid), has been used for over 50 years as a stabilizer of human albumin during pasteurization. In addition caprylic acid is of great interest, by providing the advantage of purifying mammalian immunoglobulins and clearing viruses infectivity in a single step. Exploiting these two properties, we sequentially used the caprylic acid precipitation and the pasteurization to purify horse hyperimmune globulins used in the manufacturing of Sérocytol. To evaluate the effectiveness of the process for the removal/inactivation of viruses, spiking studies were carried out for each dedicated step. Bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and minute virus of mice (MVM) were used for the virological validation. Our data show that the treatment with caprylic acid 5% (v/v) can effectively be used as well to purify or to ensure viral safety of immunoglobulins. Caprylic acid precipitation was very efficient in removing and/or inactivating enveloped viruses (PRV, BVDV) and moderately efficient against non-enveloped viruses (MVM, ECMV). However the combination with the pasteurization ensured an efficient protection against both enveloped and non-enveloped viruses. So that viruses surviving to the caprylic acid precipitation will be neutralized by pasteurization. Significant log reduction were achieved > or =9 log(10) for enveloped viruses and 4 log(10) for non-enveloped viruses, providing the evidence of a margin of viral safety achieved by our manufacturing process. Its a simple and non-expensive manufacturing process of immunoglobulins easily validated that we have adapted to a large production scale with a programmable operating system.     

Enzymatic synthesis of tricaprylin in a solvent-free system: lipase regiospecificity as controlled by glycerol adsorption on silica gel

Enzymatic synthesis of tricaprylin from caprylic acid (octanoic acid) and glycerol was carried out under solvent-free conditions. Kinetics of mono-, di- and tricaprylin synthesis and caprylic acid esterification in standard conditions (free glycerol) were slightly different from those obtained in the case of silica gel adsorbed glycerol. The critical role of the silica gel which behaves as a "reservoir" is proposed and it was shown that the use of silica gel facilitates reactions and improves the conversion yields at high glycerol concentrations.     

Inclusion/exclusion of fatty acids in amylose complexes as a function of the fatty acid chain length

Structural models are proposed for amylose-fatty acid complexes depending on the respective chain lengths of their constituents. The three studied fatty acids induce the Vh amylose crystalline type. However, in contrast to lauric and palmitic acids, caprylic acid is not present in crystals. On the basis of the relative amounts of amylose and fatty acid determined in complexes and previous results of molecular modelling, inclusion of lauric and palmitic acids inside the amylose helices is proposed; the acyl chains are included in crystalline areas and the car☐ylic groups in amorphous areas. The absence of caprylic acid in crystals could be due to the solubility of this compound in the crystallization medium.     

Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis

Acyl modification of the sn-2 position in phospholipids (PLs) was conducted by acidolysis reaction using immobilized phospholipase A(2) (PLA(2)) as the catalyst. In the first stage we screened different carriers for their ability to immobilize PLA(2). Several carriers were able to fix the enzyme and maintain catalytic activity; however, the final choice of carrier for the continued work was a non-ionic weakly polar macroreticular resin. Response surface methodology was applied to evaluate the influence of substrate ratio, reaction temperature, and water addition during acidolysis reaction between caprylic acid and soybean phosphatidylcholine (PC). Reaction temperature and water addition had significant effect on acidolysis reaction, however no effect was observed for substrate ratio (mol caprylic acid/mol PC) in range tested. In general an inverse relationship between incorporation of caprylic acid and PC recovery was observed. Highest incorporation obtained during acidolysis reactions was 36%. Such incorporation could be obtained under reaction temperature, 45 degrees C; substrate ratio, 9mol/mol caprylic acid/PC; water addition of 2%; 30wt.% immobilized enzyme; and reaction time, 48h. The yield under these conditions was however only 29%. Lysophosphatidylcholine (LPC) was the major by-product formed during the reaction. Incorporation of acyl donor into LPC was very low (<4%), which indicates that acyl migration is only a minor problem for PLA(2) catalyzed synthesis reaction. Conjugated linoleic acid and docosahexaenoic acid were also tested as acyl donors, and were able to be incorporated into PC with 30 and 20%, respectively.     

Preparation of immunoglobulins G,A,M (IgGAM) for therapeutic use. Conditions for enrichment in IgA or in IgM]

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...     

Influence of pH on antimicrobial activity of organic acids against rabbit enteropathogenic strain of<Emphasis Type="Italic">Escherichia coli</Emphasis>

Susceptibility of the rabbit enteropathogenic strain Escherichia coli C6 (O128 serogroup) to C6-C14 fatty acids, oleic, citric, lactic and fumaric acid at 5 mg/mL was determined by the plating technique in the near-neutral pH region (pH approximately 6.5), and in a weakly acid and acid environment (pH 5.4 +/- 0.1 and 2.2-2.5, respectively). In the near-neutral pH region caproic and caprylic acid reduced the concentration of viable cells by 3 and 6 orders, respectively. At lower pH the bactericidal effect of caproic acid remained similar, but caprylic acid decreased the concentration of viable cells to < 100/mL. The bactericidal activity of capric acid was low at pH 6.5 but increased at pH 5.3. High environmental acidity was intrinsically bactericidal and at very low pH the effects of fatty acids were thus less pronounced. Citric acid reduced the counts of viable cells to 1/10. Antimicrobial activity of other acids examined was marginal or absent. Medium-chain fatty acids, caprylic and, to a lesser extent, also caproic and capric acid were better antimicrobials than other organic acids examined; the antimicrobial activity of fatty acids toward the C6 strain was pH-dependent. Beneficial effects of citric, lactic and fumaric acid reported by animal nutritionists are thus probably related to factors other than their direct antimicrobial action.     

Chinese whip scorpion using 2-ketones in defense secretion (Arachnida: Uropygi)

Summary North American as well as East Asian whip scorpions of the generaMastigoproctus andTypopeltis are known to use acetic acid and caprylic acid as a defense mechanism. In the secretion from defense glands of a ChineseTypopeltis species, 2-ketones (2-heptanone, 2-octanone, and 2-nonanone) were identified as constituents for the first time. While acetic acid is also present as a secretory product, no caprylic acid has been found.Abbreviations CI chemical ionization - EI electron impact - eV electron Volt - GCMS gas chromatography-mass spectrometry - m/z mass/charge ratio (Dalton)     

Caprylic Triglyceride as a Novel Therapeutic Approach to Effectively Improve the Performance and Attenuate the Symptoms Due to the Motor Neuron Loss in ALS Disease

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and finally death. ALS patients suffer from asthenia and their progressive weakness negatively impacts quality of life, limiting their daily activities. They have impaired energy balance linked to lower activity of mitochondrial electron transport chain enzymes in ALS spinal cord, suggesting that improving mitochondrial function may present a therapeutic approach for ALS. When fed a ketogenic diet, the G93A ALS mouse shows a significant increase in serum ketones as well as a significantly slower progression of weakness and lower mortality rate. In this study, we treated SOD1-G93A mice with caprylic triglyceride, a medium chain triglyceride that is metabolized into ketone bodies and can serve as an alternate energy substrate for neuronal metabolism. Treatment with caprylic triglyceride attenuated progression of weakness and protected spinal cord motor neuron loss in SOD1-G93A transgenic animals, significantly improving their performance even though there was no significant benefit regarding the survival of the ALS transgenic animals. We found that caprylic triglyceride significantly promoted the mitochondrial oxygen consumption rate in vivo. Our results demonstrated that caprylic triglyceride alleviates ALS-type motor impairment through restoration of energy metabolism in SOD1-G93A ALS mice, especially during the overt stage of the disease. These data indicate the feasibility of using caprylic acid as an easily administered treatment with a high impact on the quality of life of ALS patients.     

羊抗人IgG的纯化及其在抗—HCV检测中的应用

单独或联合应用辛酸沉淀、饱和硫酸铵沉淀、阴离子交换等方法对羊抗人IgG进行纯化,对纯化前后抗体的纯度和免疫学活性进行比较,并与辣根过氧化物酶连接,作为二抗用于抗－HCV的ELISA检测。结果表明,不同方法纯化的抗体其纯度和免疫学活性具有一定程度的差别,其中经辛酸＋饱和硫酸铵沉淀纯化的抗体为最佳,凝胶扫描纯度为98．05％,比活性近1800,为纯化前的6．8倍。用痞根过氧化物酶标记后,作为酶标二抗检测HCV阴性和阳性标准血清各40份,阴性符合率为97．5％,阳性符合率为95％,可用于抗－HCV的ELISA检测。     

Demonstration of glycoprotein antigens associated with stomach tumors

After precipitation of the glycoproteins from gastric tumor tissues by caprylic acid, immunochemical methods were applied to the research of specific gastric tumor antigens; this precipitation process by caprylic acid was less aggressive than the use of urea or proteases. The purification of a fraction associated with tumor tissue has been carried out by affinity chromatography with a specific antiserum immobilized on Sepharose. The obtained fraction contains 4 proteic antigens. One of them is common with all the gastric extracts. Neither CEA, nor alpha-foetoprotein has been detected in this fraction. The corresponding antiserum seems to show a tumor specificity, whereas tissue specificity has to be demonstrated.     

Prevalence and antimicrobial resistance of Campylobacter in US dairy cattle

AIMS: To obtain an overview of the prevalence and antimicrobial resistance of Campylobacter in faeces of US dairy cows in 2002. METHODS AND RESULTS: Faeces from 1435 cows, representing 96 dairy operations in 21 US states, were collected for the culture of Campylobacter. A total of 735 Campylobacter strains were isolated (51.2% positive samples) with 94 operations positive (97.9%) for Campylobacter. From this collection, 532 isolates (473 Campylobacter jejuni and 59 Campylobacter coli) were randomly selected for susceptibility testing to eight antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. The C. jejuni isolates exhibited resistance to tetracycline (47.4%), nalidixic acid (4.0%) and ciprofloxacin (2.5%), while the C. coli strains exhibited some resistance to all antimicrobials except chloramphenicol and ciprofloxacin. Only 3.6% of the C. jejuni isolates were resistant to two or more antimicrobials but 20.3% of the C. coli strains were multiresistant. CONCLUSIONS: On most operations, at least one cow was positive for Campylobacter and more than half of the cows sampled were shedding Campylobacter. The C. coli isolates had significantly higher levels of resistance to macrolides and to tetracycline compared with the C. jejuni strains, but were susceptible to ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a high prevalence of Campylobacter on US dairy operations; however, US dairy cattle have not been recognized as a major source of human infection compared with poultry. Campylobacter coli appears to develop antimicrobial resistance more readily than C. jejuni from the same environment.     

Changes in free fatty acids of awamori during aging

The fatty acid components of awamori during aging were as follows. The total amount of volatile acids calculated as acetic acid ranged from 20 to 140 mg/l, the main acid was acetic acid, and the proportion of acetic acid to total acids ranged from 35 to 80 per cent. The main acids other than acetic acid were propionic acid and i-butyic acid. Differences were observed in fatty acid constituents between awamori and other alcoholic beverages.Certain components tended to increase during maturation in kame (porous earth-enware pots): acetic acid, i-butyric acid, i-valeric acid, valeric acid, capric acid, lauric acid, myristic acid and total fatty acids. Others, however, showed no distinct changes: propionic acid, butyric acid, caproic acid, caprylic acid, palmitic acid, stearic acid, oleic acid and linoleic acid.During maturation in non-porous containers (stainless-steel or glass-linked tanks), on the other hand, caprylic acid, capric acid, lauric acid and myristic acid components tended to increase, while no distinct changes however were shown by acetic acid, propionic acid, i-butyric, butyric acid, i-valeric acid, valeric acid, caproic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and total fatty acids.     

Protein precipitation by caprylic acid: Equilibrium composition data

The precipitation equilibria of hen egg-white lysozyme and bovine serum albumin from aqueous solutions by caprylic acid were studied. A thermodynamic equilibrium was obtained, and the compositions of both the precipitate and the aqueous phases were measured to establish phase diagrams for both systems. The precipitate was not pure protein, but also contained large amounts of water and caprylic acid. At constant initial pH and ionic strength, the composition of both phases depended only on the overall composition of the system. This suggests that protein precipitation by fatty acids should be pictured as liquid-liquid rather than solid-liquid equilibria. The precipitated proteins retained high biological activity. (c) 1996 John Wiley & Sons, Inc.     

Lipase-catalyzed acyl exchange of soybean phosphatidylcholine in n-Hexane: a critical evaluation of both acyl incorporation and product recovery

Lipase-catalyzed acidolysis was examined for the production of structured phospholipids in a hexane system. In a practical operation of the reaction system, the formation of lyso-phospholipids from hydrolysis is often a serious problem, as demonstrated from previous studies. A clear elucidation of the issue and optimization of the system are essential for the practical applications in reality. The effects of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio were optimized in terms of the acyl incorporation, which led to the products, and lyso-phospholipids formed by hydrolysis, which led to the low yields. The biocatalyst used was the commercial immobilized lipase Lipozyme TL IM and substrates used were phosphatidylcholine (PC) from soybean and caprylic acid. A response surface design was used to evaluate the influence of selected parameters and their relationships on the incorporation of caprylic acid and the corresponding recovery of PC. Incorporation of fatty acids increased with increasing enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio. Enzyme dosage had the most significant effect on the incorporation, followed by reaction time, reaction temperature, solvent amount, and substrate ratio. However the parameters had also a negative influence on the PC recovery. Solvent amount had the most negative effect on recovery, followed by enzyme dosage, temperature, and reaction time. Individually substrate ratio had no significant effect on the PC recovery. Interactions were observed between different parameters. On the basis of the models, the reaction was optimized for the maximum incorporation and maximum PC recovery. With all of the considerations, the optimal conditions are recommended as enzyme dosage 29%, reaction time 50 h, temperature 54 degrees C, substrate ratio 15 mol/mol caprylic acid/PC, and 5 mL of hexane per 3 g substrate. No additional water is necessary. Under these conditions, an incorporation of caprylic acid up to 46% and recovery of PC up to 60% can be obtained from the prediction. The prediction was confirmed from the verification experiments.     

Integrated enzymatic production of specific structured lipid and phytosterol ester compositions

Mixtures of specific structured lipids and phytosterol esters, valuable food components, were synthesized by an enzymatic one-pot process in organic-solvent-free medium starting from a mixture of phytosterol, caprylic acid and sunflower oil. Nine biocatalysts, seven commercially available lipases and two air-dried solid state (SSF) fermentation preparations of Aspergillus oryzae NRRL 6270 (AoSSF) and Aspergillus sojae NRRL 6271 (AsSSF), were screened for lipase activity in the transesterification reactions of sunflower oil with caprylic acid and for sterol esterase activity in the direct esterification of phytosterols with free fatty acids. The best process variant using a sequence of sterol esterase (AoSSF)-catalyzed esterification reaction of the free fatty acids and phytosterols, followed by water removal in vacuum and lipase-catalyzed transesterification with immobilized lipase from Rhizomucor miehei (Lipozyme) resulted in 92.1% conversion to phytosterol esters and 44.1% conversion to triacylglycerols containing two caprylic esters.