Antibacterial Activity of a Linolenic Acid Stigmasterol Ester Produced by Lipase-Mediated Transesterification

IntroductionCholesterol is essential for various physiological functions.It is a key component of skin lipids, playing a crucial role in maintaining the skin barrier.It helps prevent moisture loss and blocks penetration of external substances [1,2].Additionally, cholesterol can be used as a valuable biomaterial in cosmetics and foods, serving as an emulsifier or oil additive [3,4].However, excessive accumulation of cholesterol in the body can lead to health issues such as hypercholesterolemia [5].Phytosterol, a plant-derived sterol with a chemical structure similar to cholesterol, is a beneficial alternative as it does not pose the same clinical risks [6].Found in plant oils and seeds, phytosterols are effective in lowering serum cholesterol levels, which is why they are added to various food products such as spreadable fats, yogurts, and milk [7,8].Moreover, phytosterols possess additional advantages including anticancer, anti-inflammatory, antifungal, and antibacterial properties [9,10].However, phytosterols have relatively high melting points and low solubility in oils, limiting their industrial applications [11].For example, phytosterols have melting points of at least 135C and a solubility of 1.24 g/100 ml in soybean oil [12].To address this, some studies have focused on chemical modifications to enhance oil solubility of phytosterols for broader use [13].Among these modifications, forming phytosterol fatty acid esters has been proven to be particularly effective in improving solubility in fats and oils [14].Esterification with unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid has significantly enhanced solubility of phytosterols [15].Chemical synthesis of phytosterol esters requires complex separation and purification processes due to byproducts and toxic chemical catalysts [16].As a result, enzyme-catalyzed synthesis has gained increasing attention due to its advantages, including milder and more environmentally friendly reaction conditions, higher selectivity, and fewer by-products than chemical synthesis [17].In enzyme-catalyzed reactions, esterification using free fatty acids produces water as a by-product, which can lead to hydrolysis and limit the equilibrium shift necessary for ester formation [18].Meanwhile, when transesterification is performed, the reaction yield can be increased because water molecules are not generated [19].That is, when plant oil is used as an acyl donor for a reaction, the target ester compound can be synthesized with a high efficiency through a transesterification reaction [20].Recent efforts have used enzyme catalysts to synthesize phytosterol esters, addressing drawbacks of chemical methods [21].However, studies on their detailed synthesis and characterization are limited, with studies on properties beyond cholesterol-lowering effects being scarce [12].In this study, a linolenic acid stigmasterol ester (LASE) was synthesized using -linolenic acid-rich linseed oil to improve both oil and emulsion solubility of stigmasterol, one of the most common plant sterols in human diet [22].Furthermore, antibacterial activities of LASE against four food spoilage bacteria, Bacillus coagulans, Bacillus subtilis, Alcaligenes faecalis, and Pseudomonas fluorescens [23][24][25], were evaluated.Phytosterols are naturally found in lipid-rich plant foods and oils.These compounds exhibit various pharmacological effects, including anti-inflammatory, antimicrobial, antioxidant, and cholesterollowering properties.However, their industrial application has been limited due to their high melting points and poor solubility in both water and oil.Some unsaturated fatty acids can enhance phytosterols' oil solubility while exhibiting antimicrobial activities against various bacterial strains.In this study, we synthesized a linolenic acid stigmasterol ester (LASE) to improve oil and emulsion solubility of stigmasterol, a phytosterol known for its beneficial physiological effects in humans.LASE was synthesized with Candida antarctica lipase A through a transesterification reaction using stigmasterol and -linolenic acid-rich linseed oil as substrates.The reaction was conducted at 50C in isooctane with 20 mM of both stigmasterol and linseed oil.Following synthesis, LASE was purified using preparative liquid chromatography.The purified LASE demonstrated a 20-fold improvement in solubility in tricaprylin (TCN) compared to stigmasterol.Additionally, its antibacterial activities against specific food spoilage bacteria were confirmed using a TCN-based emulsion system.