Antimicrobial Activity of Positively Charged Oligopeptides with Theoretical High α-Helix Content against Cutibacterium acnes.

Cutibacterium acnes is abundant and commonly exists as a superficial bacteria on human skin. Recently, the resistance of C. acnes to antimicrobial agents has become a serious concern, necessitating the development of alternative pharmaceutical products with antimicrobial activity against C. acnes. To address this need, we evaluated the antimicrobial activity of CKR-13-a mutant oligopeptide of FK-13 with increased net charge and theoretical α-helical content-against C. acnes in modified Gifu Anaerobic Medium broth by determining the minimum inhibitory concentration (MIC). CKR-13 exerted greater antimicrobial activity against C. acnes than FK-13 in the broth at pH 7.0. The antimicrobial activity of CKR-13 with RXM against C. albicans was pH-dependent. The ionization of CKR-13 and pH-dependent growth delay of C. albicans was suggested to be associated with the increase in CKR-13 antimicrobial activity.

Synthesis and Photophysical Characterization of Fluorescent Naphtho[2,3-d]thiazole-4,9-Diones and Their Antimicrobial Activity against Staphylococcus Strains.

The chemical reaction of 2-(methylsulfinyl)naphtho[2,3-d]thiazole-4,9-dione (3) using different amines, including benzylamine (4a), morpholine (4b), thiomorpholine (4c), piperidine (4d), and 4-methylpiperazine (4e), produced corresponding new tricyclic naphtho[2,3-d]thiazole-4,9-dione compounds (5a-e) in moderate-to-good yields. The photophysical properties and antimicrobial activities of these compounds (5a-e) were then characterized. Owing to the extended π-conjugated system of naphtho[2,3-d]thiazole-4,9-dione skeleton and substituent effect, 5a-e showed fluorescence both in solution and in the solid state. The introduction of nitrogen-containing heterocycles at position 2 of the thiazole ring on naphtho[2,3-d]thiazole-4,9-dione led to large bathochromic shifts in solution, and 5b-e exhibited orange-red fluorescence with emission maxima of over 600 nm in highly polar solvents. Staphylococcus aureus (S. aureus) is a highly pathogenic bacterium, and infection with its antimicrobial-resistant pathogen methicillin-resistant S. aureus (MRSA) results in serious clinical problems. In this study, we also investigated the antimicrobial activities of 5a-e against S. aureus, MRSA, and S. epidermidis. Compounds 5c with thiomorpholine group and 5e with 4-methylpiperazine group showed potent antimicrobial activity against these bacteria. These results will lead to the development of new fluorescent dyes with antimicrobial activity in the future.

Antimicrobial Activity of 2-(Piperazin-1-yl)naphtho[2,3-d]thiazole-4,9-dione against Staphylococcus Strains.

There is an urgent need to discover and develop novel antibacterial agents. Accordingly, we synthesised 2-(piperazin-1-yl)naphtho[2,3-d]thiazole-4,9-dione (PNT), which exhibits antimicrobial activity. The aim of this study was to characterise PNT as an effective antimicrobial agent. Fluorescence microscopy was used to measure PNT's uptake into microbial cells (strains of Staphylococcus epidermidis, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA)), transmission electron microscopy (TEM) was used to investigate the influence of PNT on the configuration of microbial cells, and a DNA gyrase supercoiling assay was used to investigate whether PNT inhibits DNA gyrase. PNT was taken up by more than 50% of microbial cells within 30 min. Using TEM, hollowed-out bacterial cytoplasms were observed in the specimen treated with PNT, although there was no disintegration of the bacterial membrane. In the DNA gyrase supercoiling assay, a dose-dependent reduction in fluorescence intensity was observed as the concentration of PNT increased. This suggests that PNT is taken up by microbial cells, resulting in cell disruption, and it reveals that one of the mechanisms underlying the antimicrobial activity of PNT is the inhibition of DNA gyrase.

Production of a selective antibacterial fatty acid against Staphylococcus aureus by Bifidobacterium strains.

Aims: C16 monounsaturated fatty acid (C16:1) show antibacterial activity against Staphylococcus aureus, a pathogen associated with various diseases such as atopic dermatitis and bacteremia, while the compound does not exhibit antibacterial activity against Staphylococcus epidermidis, an epidermal commensal that inhibits the growth of S. aureus. In this study, we aimed to find bifidobacterial strains with the ability to produce C16:1 and to find a practical manner to utilize C16:1-producing strains in industry. Methods: Various Bifidobacterium strains were screened for their content of C16:1. The chemical identity of C16:1 produced by a selected strain was analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Medium components that affect the C16:1 content of the selected strain were investigated. Antibacterial activity against staphylococci was compared between the authentic C16:1 isomers and total fatty acids (TFA) extracted from the selected strain. Results: B. adolescentis 12451, B. adolescentis 12-111, B. boum JCM 1211, and Bifidobacterium sp. JCM 7042 showed high C16:1 content among the tested strains. TFA extracted from Bifidobacterium sp. JCM 7042 contained C16:1 at 2.3% as the fatty acid constituent (2.4 mg/L of broth). Through GC-MS and LC-MS analyses, the C16:1 synthesized by Bifidobacterium sp. JCM 7042 was identified as 7-cis-hexadecenoic acid (7-cis-C16:1). The authentic 7-cis-C16:1 showed strong and selective antibacterial activity against S. aureus, similar to 6-cis-C16:1, with a minimum inhibitory concentration (MIC) of < 10 µg/mL. Components that increase C16:1 productivity were not found in the MRS and TOS media; however, Tween 80 was shown to considerably reduce the C16:1 ratio in TFA. Antibacterial activity against S. aureus was observed when the TFA extracted from Bifidobacterium sp. JCM 7042 contained high level of 7-cis-C16:1 (6.1% in TFA) but not when it contained low level of 7-cis-C16:1 (0.1% in TFA). Conclusion: The fatty acid, 7-cis-C16:1, which can selectively inhibit the S. aureus growth, is accumulated in TFA of several bifidobacteria. The TFA extracted from cultured cells of Bifidobacterium sp. JCM 7042 demonstrated antibacterial activity. From a practical viewpoint, our findings are important for developing an efficient method to produce novel skin care cosmetics, functional dairy foods, and other commodities.

Targeted Delivery of Miconazole Employing LL37 Fragment Mutant Peptide CKR12-Poly (Lactic-Co-Glycolic) Acid Polymeric Micelles.

We previously reported that conjugates of antimicrobial peptide fragment analogues and poly (lactic-co-glycolic) acid (PLGA) enhance antimicrobial activity and that the conjugated micelle structure is an effective tool for antimicrobial drug delivery. In recent years, the delivery of antimicrobial peptides to targets for antimicrobial activity has attracted attention. In this study, we targeted Candida albicans, a causative organism of catheter-related bloodstream infections, which is refractory to antimicrobial agents and is currently a problem in medical practice. We evaluated the antifungal activity of CKR12 (a mutant fragment of the human cathelicidin peptide, LL-37)-PLGA-miconazole (MCZ) micelles using nanotechnology with MCZ delivery. The prepared CKR12-PLGA-MCZ micelles were characterised by measuring dynamic light scattering, zeta potential, dilution stability, and drug release. CKR12-PLGA-MCZ micelles showed higher antifungal activity than CKR12-PLGA micelles and MCZ solution. Furthermore, scanning and transmission electron microscopy suggested that CKR12-PLGA-MCZ micelles disrupted both cell wall and cell membrane of C. albicans. Our results revealed a synergistic effect of antifungal activity using a combination of antimicrobial peptide fragment analogues and MCZ, and that MCZ is a promising tool for the delivery to target microorganisms.

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus, Escherichia coli, and Candida albicans.

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.

Dietary cis-9, trans-11-conjugated linoleic acid reduces amyloid ß-protein accumulation and upregulates anti-inflammatory cytokines in an Alzheimer's disease mouse model.

Conjugated linoleic acid (CLA) is an isomer of linoleic acid (LA). The predominant dietary CLA is cis-9, trans-11-CLA (c-9, t-11-CLA), which constitutes up to ~ 90% of total CLA and is thought to be responsible for the positive health benefits associated with CLA. However, the effects of c-9, t-11-CLA on Alzheimer's disease (AD) remain to be elucidated. In this study, we investigated the effect of dietary intake of c-9, t-11-CLA on the pathogenesis of an AD mouse model. We found that c-9, t-11-CLA diet-fed AD model mice significantly exhibited (1) a decrease in amyloid-ß protein (Aß) levels in the hippocampus, (2) an increase in the number of microglia, and (3) an increase in the number of astrocytes expressing the anti-inflammatory cytokines, interleukin-10 and 19 (IL-10, IL-19), with no change in the total number of astrocytes. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatographic analysis revealed that the levels of lysophosphatidylcholine (LPC) containing c-9, t-11-CLA (CLA-LPC) and free c-9, t-11-CLA were significantly increased in the brain of c-9, t-11-CLA diet-fed mice. Thus, dietary c-9, t-11-CLA entered the brain and appeared to exhibit beneficial effects on AD, including a decrease in Aß levels and suppression of inflammation.

Health Beneficial Food Emulsifier Produced from Fishery Byproducts.

The bioavailability of DHA-bound phospholipids, especially the DHA-bound lysophospholipid (DHA-LPL) could be considered the most effective DHA chemical forms for DHA accretion in the brain. Such a DHA-LPL should also have very high emulsifying stability performance based on its analogy with conventional soy LPL. Therefore, in this study, we describe two fishery byproducts, rich in DHA-bound phospholipids, to derive DHA-LPL via sn-1 positional specific lipase partial hydrolysis of the phospholipids. Through this reaction, the DHA composition increased to 43.8 % from 29.1 % in the salmon head phospholipid-derived DHA-LPL, and to 84.0 % from 47.4 % in the squid meal phospholipid-derived DHA-LPL. In fact, these obtained DHA-LPLs exhibited far higher emulsifying stability than the conventional food emulsifiers in the market. For example, the prepared high-purity squid meal phospholipid-derived LPL sustained an emulsion form for a week even under 80°C. Thus, food emulsifiers produced from fishery byproducts are considered to exhibit very high values of both in a sense of outstandingly high health benefits and sustaining emulsions even under very high temperatures.

Effect of Dietary Oil Rich in Docosahexaenoic Acid-Bound Lysophosphatidylcholine Prepared from Fishery By-Products on Lipid and Fatty Acid Composition in Rat Liver and Brain.

The possibility of improving brain function coupled with its preferential uptake in the brain has garnered attention for docosahexaenoic acid-bound lysophosphatidylcholine (DHA-LPC). However, studies focusing on the health benefits of dietary DHA-LPC are lacking. We prepared a dietary oil rich in DHA-LPC (DHA-LPC rich oil) via enzymatic modification of phospholipids (PL) extracted from squid (Todarodes pacificus) meal and purification of active carbon, ion exchange resin, and silica gel. We then examined the effects of dietary DHA-LPC rich oil on male Wistar rats by evaluating serum and liver lipid profiles, fatty acid (FA) metabolizing enzyme activity, and the FA composition of serum and brain. The rats were fed a basal diet containing either soybean oil alone (7%) or soybean oil (4.5%) with DHA-LPC rich oil (2.5%) for 28 days, and then evaluated. The rats fed the diet containing DHA-LPC rich oil showed reduced triacylglycerol concentration due, in part, to the enhancement of carnitine palmitoyltransferase 2 and acyl-CoA oxidase activities and suppression of acetyl-CoA carboxylase and glucose-6-phosphate dehydrogenase activities in the liver. Moreover, the dietary DHA-LPC rich oil moderately increased DHA in the FA composition of the rat hippocampus, which may be due to elevated DHA composition in serum LPC. These results suggest that DHA-LPC rich oil has hypolipidemic effect and moderate increase in hippocampal DHA amount in normal rats.

Tissue-dependent effects of cis-9,trans-11- and trans-10,cis-12-CLA isomers on glucose and lipid metabolism in adult male mice.

Mixtures of the two major conjugated linoleic acid (CLA) isomers trans-10,cis-12-CLA and cis-9,trans-11-CLA are used as over the counter supplements for weight loss. Because of the reported adverse effects of CLA on insulin sensitivity in some mouse studies, we sought to compare the impact of dietary t10c12-CLA and c9t11-CLA on liver, adipose tissue, and systemic metabolism of adult lean mice. We fed 8 week-old C57Bl/6J male mice with low fat diets (10.5% Kcal from fat) containing 0.8% t10c12-CLA or c9t11-CLA for 9 or 38 days. Diets containing c9t11-CLA had minimal impact on the endpoints studied. However, 7 days after starting the t10c12-CLA diet, we observed a dramatic reduction in fat mass measured by NMR spectroscopy, which interestingly rebounded by 38 days. This rebound was apparently due to a massive accumulation of lipids in the liver, because adipose tissue depots were visually undetectable. Hepatic steatosis and the disappearance of adipose tissue after t10c12-CLA feeding was associated with elevated plasma insulin levels and insulin resistance, compared to mice fed a control diet or c9t11-CLA diet. Unexpectedly, despite being insulin resistant, mice fed t10c12-CLA had normal levels of blood glucose, without signs of impaired glucose clearance. Hepatic gene expression and fatty acid composition suggested enhanced hepatic de novo lipogenesis without an increase in expression of gluconeogenic genes. These data indicate that dietary t10c12-CLA may alter hepatic glucose and lipid metabolism indirectly, in response to the loss of adipose tissue in mice fed a low fat diet.

Effect of Dietary Partial Hydrolysate of Phospholipids, Rich in Docosahexaenoic Acid-Bound Lysophospholipids, on Lipid and Fatty Acid Composition in Rat Serum and Liver.

Lysophospholipids have been recognized as potent biologically active lipid mediators. However, attention has not been paid to the health benefits of dietary partial hydrolysate of phospholipids (PH-PL), which is rich in docosahexaenoic acid (DHA)-bound lysophospholipids. In this study, the effects of PH-PL on serum and liver lipid profiles of rats upon administration of PH-PL are demonstrated in comparison to those of fish oil (FO), which comprises eicosapentaenoic acid (EPA) and DHA-bound triglyceride (TG). PH-PL containing EPA and DHA was prepared via enzymatic modification of squid (Todarodes pacificus) meal that is rich in phospholipids. Male Wistar rats were fed a basal diet containing soybean oil alone (7%), FO, and PH-PL. The FO and PH-PL diets had similar EPA and DHA contents. After the rats had been fed their respective diets for 28 d, their serum and liver lipid contents, fecal lipid excretion, and hepatic gene expression level were measured. The results demonstrated that compared with the soybean oil diet alone, the PH-PL diet decreased serum and liver TG contents partially because of the enhancement of liver acyl-CoA oxidase activity and suppression of liver fatty acid synthase activity. In addition, compared with the soybean oil diet, the PH-PL group exhibited lower serum cholesterol content at least in part because of the reduction of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase mRNA expression level. We found that dietary administration of EPA and DHA containing PH-PL has a hypolipidemic effect that may help prevent the development lifestyle-related diseases.

Production of a Health-Beneficial Food Emulsifier by Enzymatic Partial Hydrolysis of Phospholipids Obtained from the Head of Autumn Chum Salmon.

Phospholipids and their partial hydrolysates, namely lysophospholipids (LPLs), have been widely used in food, pharmaceutical, and cosmetic products as highly efficient emulsifiers. This study was conducted to produce docosahexaenoic acid (DHA)-esterified LPLs by enzymatic modification of phospholipids obtained from the head of autumn chum salmon (Oncorhynchus keta). The emulsifying properties of the obtained LPLs were also evaluated. Two different types of substrates of salmon head phospholipids were prepared via silica gel and cold acetone precipitation. Enzymatic partial hydrolysis was carried out using immobilized phospholipase A1 (PLA1) and Lipozyme RM IM. Results showed that the increase in DHA in the LPLs was much higher in the silica-separated phospholipids than in the acetone-precipitated phospholipids. When silica-separated phospholipids were used as the substrate, the DHA content of the LPLs increased from 23.1% to 40.6% and 42.6% after 8 h of partial hydrolysis with Lipozyme RM IM and immobilized PLA1, respectively. The yield of the LPLs was comparatively higher in the Lipozyme RM IM than in the immobilized PLA1 hydrolysis reaction. The critical micelle concentration values of the LPLs and purified lysophosphatidylcholine (LPC) were 100 mg/L and 5 mg/L, respectively. The surface tension values of the LPLs and LPC were reduced to 30.0 mN/m and 30.5 mN/m, respectively. The hydrophilic-lipophilic balance of the LPLs and LPC were 6.0 and 9.4, respectively. Based on the emulsifying properties observed, we conclude that LPLs derived from the phospholipids of salmon head lipids could be used as a health-beneficial emulsifier in the food industry.

Promotion of the lipase-catalyzed hydrolysis of conjugated linoleic acid l-menthyl ester by addition of an organic solvent.

Conjugated linoleic acid l-menthyl ester was hydrolyzed in water by the lipase from Candida rugosa with the addition of an organic solvent. The degree of hydrolysis (yield) greatly improved when a tertiary alcohol, such as t-butyl alcohol, was added. However, the addition of a less polar solvent, such as hexane, decreased the degree of hydrolysis, and some water-miscible solvents, such as acetone, caused inactivation of the lipase. With the addition of t-butyl alcohol, the reaction mixture formed a one- or two-phase system, and the mixing ratio of substrates and t-butyl alcohol determined the number of phases. Although the degree of hydrolysis at 10 d was higher in the one-phase system, the initial reaction rate was generally lower. Meanwhile, the reaction was much faster in the two-phase system while maintaining a moderate degree of hydrolysis.

Direct GC analysis of the fatty acid compositions of conjugated linoleic acid and its L-menthyl esters.

Commercially available conjugated linoleic acid (CLA) is a mixture of two main isomers. Fractionation of the two isomers was performed by a lipase-catalyzed esterification of CLA with L-menthol. In this study, a GC analytical method was developed to simultaneously determine the degree of esterification and fatty acid (FA) compositions of CLA in the free fatty acid (FFA) and ester forms without separation of the FFA and the ester. The methylation of the oil phase of the reaction mixture was performed using trimethylsilyldiazomethane in a mixture of toluene/methanol. Only FFA was quantitatively methylated, whereas the other compounds were little changed. A GC analysis using a polar column was performed to simultaneously determine the degree of esterification and the fatty acid compositions.

One-pot enzymatic synthesis of docosahexaenoic acid-rich triacylglycerols at the sn-1(3) position using by-product from selective hydrolysis of tuna oil.

Docosahexaenoic acid (DHA)-rich oil has been industrially produced by selective hydrolysis of tuna oil with a lipase that acts weakly on DHA. The free fatty acids (FFAs) generated in this process as by-products contain a high DHA concentration (46wt%) but are treated as industrial waste. This study attempted to reuse these by-product FFAs using a one-pot process, and succeeded in producing triacylglycerols (TAGs) through the esterification of the by-product FFAs with glycerol using immobilized Rhizomucor miehei lipase. Regiospecific analysis of the resulting TAGs showed that the content of DHA at the sn-1(3) position (51.7mol%) was higher than the content of DHA at the sn-2 position (17.3mol%). The DHA distribution in TAGs synthesized in this study was similar to the DHA distribution in TAGs from seal oil.

Control of the regiospecificity of Candida antarctica lipase by polarity.

Candida antarctica lipase has generally been known to be nonregiospecific. This report, however, showed that its regiospecificity was linearly correlated to the index of polarity of the reaction mixture, which was calculated based on the dielectric constant of the components. Thus, it was strongly indicated that the regiospecificity depended on the polarity of the environment; the higher the polarity, the stricter the regiospecificity. The highest 1(3)-regiospecificity was obtained in the transesterification of oil with ethanol among other alcohols investigated. Although methanol, which is more polar than ethanol, was expected to give highest regiospecificity, it was fatal to the lipase. The transesterification of oil with ethanol (1:10, wt/wt) at 30 degrees C for three hours efficiently accumulated 2-monoacylglycerols without significant fatty acid (FA) specificity. Thus, it was successfully applied to regiospecifically analyze FA composition of triacylglycerols containing saturated and unsaturated FAs of C4-C24.

Linoleic acid-menthyl ester reduces the secretion of apolipoprotein B100 in HepG2 cells.

The effect of linoleic acid-menthyl ester (LAME) on lipid metabolism were assessed in HepG2 cells. It is well known that high level of apolipoprotein (apo) B100 in the serum is risk for atherosclerosis. Although linoleic acid (LA) treatment and LA plus L-mentol treatment increased apo B100 secretion, LAME treatment significantly decreased apo B100 secretion in HepG2 cells compared with control medium. The hypolipidemic effect of LAME was attributable to the suppression of triglyceride synthesis in HepG2 cells. It is also known that the risk of coronary heart disease is negatively related to the concentration of serum apo A-1. In the present study, LAME treatment increased apo A-1 secretion as compared with LA treatment in HepG2 cells. These results suggest that mentyl-esterification of fatty acids may be beneficial in anti-atherogenic dietary therapy.

Isomer-specific anti-obese and hypolipidemic properties of conjugated linoleic acid in obese OLETF rats.

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.

Effects of oral glucose intake on gastric myoelectrical activity and gastric emptying.

To investigate the effect of oral glucose intake on gastric motility, we measured gastric myoelectrical activity and gastric emptying on two test conditions: 1) glucose intake and 2) water intake in the same 10 healthy male volunteers (20 to 29 years old). Gastric motility was evaluated with cutaneous-recorded electrogastrography (EGG) for 30 min both on fasting and after glucose or water intake, while gastric emptying was measured using acetaminophen-absorption method. There were no significant changes in EGG dominant frequency after water intake, but the frequency increased significantly after glucose intake. A postprandial dip (i.e., a transient decrease in frequency immediately after the food intake) was observed in 3 subjects after water intake and in 8 subjects following glucose intake. The EGG power ratio was significantly larger after glucose than water intake, with delayed gastric emptying in the former case. These results suggest that glucose is one of the components responsible for postprandial gastric motility.

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols.

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1:2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40 degrees C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7-36.5 degrees C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5 degrees C; JAOCS 80, 341-346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.