WO2025052383A1

WO2025052383A1 - Oils enriched with phytosterol esters, preparation, compositions and uses thereof

Info

Publication number: WO2025052383A1
Application number: PCT/IL2024/050893
Authority: WO
Inventors: Sobhi Basheer; Ramez MASRI
Original assignee: Enzymofit Ltd
Current assignee: Enzymofit Ltd
Priority date: 2023-09-05
Filing date: 2024-09-05
Publication date: 2025-03-13
Anticipated expiration: 2026-03-05

Abstract

Disclosed are processes for producing an oil enriched with solubilized phytosterol esters and oils enriched with phytosterol esters produced thereby. Also disclosed is a process for the preparation of an oil/fat of high solid fat content comprising a mixture of solubilized phytosterol esters in oil obtained by enzymatically/chemically transesterified oil with phytosterols, and fully hydrogenated oil at different ratios. Further disclosed are fats enriched with phytosterol fatty acid esters, and stable oleogels comprising oil/s enriched with sterol fatty acid esters and protein(s), exhibiting high plasticity, and food products and supplements, cosmetic and pharmaceutical products comprising these fats and oleogels.

Description

OILS ENRICHED WITH PHYTOSTEROL ESTERS, PREPARATION,

COMPOSITIONS AND USES THEREOF

TECHNOLOGICAL FIELD

[0001 ] Disclosed herein are methods for the preparation of oils and fats enriched with tatty acid esters of phytosterols, compositions comprising them and various uses thereof in food and cosmetic industry and technologies.

TECHNOLOGIAL BACKGROUND

[0002] Phytosterols are naturally occurring plant compounds that structurally belong to the same family of cholesterol. They are commonly found in seeds, vegetable oils, and nuts. The use of phytosterols in oils and fats has gained attention due to their potential health and other functional benefits.

[0003] Well-known documented advantages are associated with the use of phytosterols, such as cholesterol-lowering effects, thus contributing to reduction of the risk of heart disease; cardiovascular health as the cholesterol -lowering effects of phytosterols can be reduce LDL cholesterol levels, thus maintain a healthier lipid profile, important for reducing the risk of atherosclerosis, heart attacks, strokes, and other related body functional disorders; and phytosterols offer natural plant-based alternative to synthetic cholesterol-lowering drugs, they can be incorporated into oils and fats without altering their taste or texture significantly, which makes them suitable for individuals seeking natural ways to manage their cholesterol levels.

[0004] Some known disadvantages of phy tosterols are, for example, Limited solubility in fats and oils, typically 1-3% at 25°C depending on the chemical structure of phytosterols, at ambient temperatures (Hariklia Vaikousi et al., J. Agric. Food Chem., 2007, 55, 1790-1798), limiting their concentrations in functional food products. This issue is knowm to be overcome using esterifi ed phytosterols, which are more soluble in fats and oils. Nonetheless, the esterification process may involve chemical modifications and adversely affect the natural properties of phytosterols. Another disadvantage is the susceptibility of phytosterols to oxidation, especially under certain processing conditions, which can negatively affect the taste, odor, and nutritional quality of oils and fats, and therefore appropriate processing and storage conditions must be provided to ensure the desired quality of the product. Further, there is no sufficient evidence on long-term effects of phytosterols: while their short-term benefits in reducing blood cholesterol levels are well -documented in the literature, the- e is limited research on their long-term effects, alone or in combinations with other medications or health conditions, and their use in oils or fats is subject to regulatory guidelines and limitations, which may vary in different countries.

[0005] As mentioned, the poor solubility of free form of phytosterols oil-based systems, limits their applications in functional foods, cosmeceuticals and pharmaceuticals, therefore, different approaches have been developed for the synthesis of phytosterols with enhanced solubility and stability in hydrophobic media. These methods include esterification with fatty acids, employing lipases or chemical catalysts such as sulfuric acid or p-toluene sulfonic acid, where the reaction can be carried out under various conditions, including solvent-based or solvent-free microaqueous systems. Solvent-free esterification methods have gained popularity' due to their eco-friendly nature and the ease of product recovery. Common fatty acids used for esterification of phytosterols include palmitic acid, stearic acid, and oleic acid, typical ly of vegetable oil sources. Another widely used method for synthesizing phytosterol esters is transesterification, involving random exchange of ester groups between phytosterols and fatty acid esters. Thi s reaction can be catalyzed by chemical catalysts such as sodium methoxide/ethoxide, or by enzymes such as lipases. Transesterification offers advantages such as one-step reaction under milder reaction conditions, making it a versatile method for phytosterol esters synthesis. Enzymatic synthesis methods have gained attention as greener alternatives for synthesizing phytosterol esters. Free or immobilized lipases, particularly those derived from Candida antarctica, Candida rugosa. and Rhizomucor miehei, are commonly employed as biocatalysts. Lipase-catalyzed transesterification offers advantages such as high selectivity, mild reaction conditions, and the avoidance of toxic chemicals. The reaction can be performed in various systems, including solvent-based under none or supercritical conditions, and solvent-free systems. After the desired transesterification reaction conversion is achieved, the produced phytosterol esters can be separated from the reaction mixture, by, for example solvent extraction, distillation, crystallization, or chromatographic techniques, can be applied to obtain high purity phytosterol esters.

[0(106] Interesterification of oils and fats is a process that involves rearranging of the fatty acyl groups within the glycerol backbone of different triglyceride molecules. This reaction results in modifying the physical and chemical properties of interesterified oils and fats. The process typically uses alkali alkoxide, such as sodium methoxide/ethoxide to catalyze random rearrangement of fatty acyl groups bound to glycerol backbone comprising oils and fats triglycerides from different sources. After the interesterification reaction is complete, the product is typically separated from the enzyme and other reaction by-products. This can be achieved through various physical separation methods such as filtration, water-wash, or distillation. This process has gained significant attention in the food industry as an alternative for the hydrogenation process, allowing the synthesis of trarrr-free fats and oils, improvement of melting properties, alteration of texture, and enhancement of nutritional profiles of fats and oils.

[0007] It has been reported that certain types of lipases in their native or immobilized forms possess the ability to catalyze interesterification reactions randomly or with sn-1,3 specificity depending on the source of the enzyme. The reaction conditions include parameters such as temperature, pH, enzyme concentration, and reaction time. Lipases are often activated prior to their use in interesterification processes. Activation methods can include pre-incubation with water, organic solvents, specific additives, immobilizing of the enzyme on an i norgam c/organic carrier. This step helps optimize the enzyme's activity and stability.

[0008] Lipase-catalyzed interesterification offers several advantages over other modification methods of oils and fats, such as chemical interesterifi cation. It is a milder process, operating under milder reaction conditions, and it can be performed with greater selectivity, leading to more precise control over the final fatty acid composition. Additionally, enzymatic interesterification can result in products with improved functional properties, such as altered melting points (Solid Fat Content-SFC), enhanced stability, and modified nutritional characteristics.

[0009] Interesterified oils and fats produced using immobilized lipases find applications in various industries, including food and beverage, cosmetics, and pharmaceuticals. These modified lipids can be used as ingredients in margarines, spreads, confectionery products, bakery goods, and other food products. They can also be incorporated into personal care products, such as creams, lotions, and cosmetics, as well as in drug delivery systems and in plant-based food alternatives for meat, eggs, fish, and dairy products.

SUMMARY OF INVENTION

[0010] Disclosed herein is a process for producing an oil enriched with solubilized phytosterol esters, the process comprising subjecting a reaction substrate comprising a homogenous mixture of oil and free fatty acids optionally supplemented with free monoglycerides, fatty acid short-chain alkyl esters, specifically fatty acid ethyl esters, and phytosterol(s) solubilized in said mixture at an amount of up to saturation while said mixture maintains homogeneity, to enzymatic transesterification/ inter-esterification/esterification reaction/s in said oil, in an aqueous/micro-aqueous reaction having pH of about 4-11 in the presence of a crude or immobilized lipase, and allowing the reach on/s to proceed for a suitable time period, stopping the reach on/s by removing the lipase, water or buffer and by- products, giving as a product a homogenous mixture comprising said oil and at about 1%- 90%w/w phytosterol fatty acid esters solubilized in said oil, thereby obtaining an oil enriched with solubilized phytosterol fatty acid esters.

[0011 ] Specifically disclosed is a process for producing an oil enriched with solubilized phytosterol esters, the process comprising: subjecting a reaction substrate comprising a homogenous mixture of:

- an oil comprising mono-, di-, and triglycerides and free fatty acids, wherein the oil may be optionally supplemented with free monoglycerides;

- fatty acid short-chain alkyl esters, specifically fatty acid ethyl esters; and

- phytosterol(s) solubilized in said mixture at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more ,such as about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s); to enzymatic transesterification and/or interesterification and/or esterification reactions in said oil, wherein said transesterification occurs between said solubilized phytosterols and between said oil glycerides and optional supplemented monoglycerides, dominantly between said oil monoglycerides and optionally supplemented monoglycerides, and optionally between said oil diglycerides and free fatty acids as fatty acyl donor, in an aqueous/micro- aqueous reaction medium containing an aqueous alkaline buffer or water and having pH of about 4-11 and comprising a crude or immobilized lipase, and allowing the reaction/s to proceed for a suitable time period, and stopping the reaction/s by removing the lipase, water or buffer and by-products, giving as a product a homogenous mixture comprising said oil and at about 1%-90%W/W, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w phytosterol fatty acid esters solubilized in said oil, thereby obtaining an oil enriched with solubilized phytosterol fatty acid esters.

[0012] In said process, the interesterification occurs as a side-reaction exchanging fatty acyl groups simultaneously between said oil mono-, di-, and tri-glycerides and optionally supplemented monoglycerides and between fatty acid ethyl esters, wherein exchange of fatty acyl groups occurs between all reactants having ester bonds, and the esterification occurs as a side-reaction between free fatty acids and phytosterols. [0013] In the product of said process, the amounts of the oil monoglycerides and optional supplemented monoglycerides, and optionally of said oil diglycerides in the product are lower than their amounts in the starting reaction substrate, while the amount of triglycerides is not significantly changed.

[0014] Specifically disclosed is a process for producing oil enriched with solubilized phytosterol esters, the process comprising the steps of:

(a) providing an oil-based homogenous reaction substrate comprising an oil which comprises fatty acid alkyl esters, monoglycerides, diglycerides, triglycerides and free fatty acids at suitable amounts, and phytosterol(s) solubilized in said mixture at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more ,such as about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s);

(b) subj ecting said reaction substrate to at least one of enzymatic transesterification/ interesterification/esterification reaction/s in said oil, in an aqueous or microaqueous reaction mixture having a pH of about 4-11 in the presence of a crude lipase or an immobilized lipase whilst stirring, preferably at about 250 rpm for a suitable time period, preferably about 5-12 hours, at a temperature of about 10-90°C, whereby transesterification/esterification reactions occur between said solubilized phytosterols and said glycerides, dominantly monoglycerides and optionally diglycerides and free fatty acids as fatty acyl donor, thereby obtaining a homogenous mixture comprising solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual solubilized non-reacted phytosterols, and as by-product of the transesterification reaction free fatty acids; and

(c) collecting the reaction mixture of step (b) by filtering off the enzyme and removing water to give as a product a homogenous oil mixture comprising at least l%w/w, preferably at least 2%w/w, more preferably at least 3%w/w solubilized phytosterol fatty acid esters at room temperature, mono-, di- and tri-glycerides, residual solubilized non-reacted phytosterol(s), residual fatty acid alkyl esters and free fatty acids, wherein the amounts of said monoglycerides, optionally said diglycerides in the product are lower than their amounts in the said reaction substrate, while the amount of triglycerides is not significantly changed; and

(d) optionally distilling off said free fatty acids and said residual fatty acid alkyl esters, to give a homogenous product comprising about l-90%w/w, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual non- reacted solubilized phytosterol(s).

[0015] In this specific process, water or aqueous alkaline buffer is added to said reaction substrate at from at least 100 ppm and up to about 70% w/w to form a reaction medium having a pH of about 4-11.

[0016] Alao disclosed is a further specific process, for producing an oil enriched with solubilized phytosterol fatty acid esters, the process comprising the steps of:

(a) providing an oil-based homogenous reaction substrate comprising an oil which comprises fatty acid alkyl esters, monoglycerides, diglycerides, triglycerides and free fatty acids at suitable amounts, and phytosterol(s) solubilized in said mixture at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10% w/w, about 15%w/w, about 20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more, such as about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s);

(b) adding to said reaction substrate water or aqueous alkaline buffer at more than 100 ppm up to about 70% w/w to form a reaction medium having a pH of about 4-11;

(c) adding to said reaction medium a lipase which is a crude lipase or a lipase immobilized on an organic or inorganic support to form a reaction mixture;

(d) stirring the said reaction mixture for a period of time of about 5-12 hours, at a temperature of about 10-90°C at about 250 rpm, to allow for at least one of transesterification/interesterification/esterification reactions between solubilized phytosterols and said glycerides and free fatty acids, dominantly said monoglycerides, optionally said diglycerides, as a fatty acyl donor to occur in said oil, thereby obtaining a homogenous mixture comprising phytosterol fatty acid esters, mono-, di- and tri- glycerides, residual phytosterols, and as by-product of the transesterification reaction free fatty acids;

(e) collecting the reaction mixture of step (d) by filtering off the enzyme and removing water to give as a product a homogenous oil mixture comprising at least l%w/w, preferably at least 2%w/w, more preferably at least 3%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides, residual solubilized non- reacted phytosterol(s), residual fatty acid alkyl esters and free fatty acids, wherein the amounts of said monoglycerides, optionally said diglycerides in the product are lower than their amounts in the said reaction substrate, while the amount of triglycerides is not significantly changed; and

(f) optionally distilling off said free fatty acids and said residual fatty acid alkyl esters, to give a homogenous product comprising about l-90%w/w, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual non-reacted solubilized phytosterol(s).

[0017] In any of said disclosed processes the said reaction substrate can be prepared by (i) enzymatic partial transesterification of oil triglycerides with a short-chain alcohol in the presence of a free crude lipase or a lipase immobilized on an inorganic or organic support, or chemically by using a chemical catalyst, optionally a strong alkaline catalyst, preferably sodium methoxide or ethoxide, and removing the enzyme, respectively the chemical catalyst, from the reaction mixture at the end of the reaction by filtration or water wash and optionally adding free monoglycerides to the product obtained; or (ii) by mixing an oil with mono- glycerides and fatty acid alkyl esters, preferably fatty acid ethyl esters, to give a mixture comprising fatty acid alkyl esters, mono-, di- and tri-glycerides.

[0018] In any of said processes, the period of time of conducting the reaction can be a period of time sufficient to achieve reaction equilibrium.

[0019] Any of the said processes can further comprise monitoring the transesterification/ interesterification/esterification reaction/s by drawing samples of the reaction mixture at suitable volumes, such as about 60-100 microliters, at desired time points, which can be pre- determined or random, and determining concentrations of all species contained in the reaction mixture, reactants and the products, including primarily monoglycerides, diglycerides, triglycerides, free fatty acids, phytosterol/s and phytosterol esters, whereby equilibrium stage of the reaction is detected when there is no significant change in the levels of the reactants/products compared to the immediately preceding measurement.

[0020] In any of said process/s the said monoglycerides, optionally said di glycerides, in the presence of fatty acid short-chain alkyl esters lead to solubilization of said phytosterol(s) in the reaction substrate and wherein under the reaction conditions monoglycerides, optionally diglycerides, serve as dominant donor of fatty acyl groups in the transesterification process as compared to triglycerides or free fatty acids. Specifically, the amount of monoglycerides in the final reaction product is significantly reduced compared to the amount of said monoglycerides in said reaction substrate, while the amounts of said di-glycerides, triglycerides and free fatty acids is not significantly changed compared to their amounts in said reaction substrate [0021] In any of said processes, the amount of the monoglycerides in the reaction substrate is about l-50%w/w, the amount of the diglycerides is about 1-50 %w/w, the amount of the triglycerides is about l-90%w/w and the amount of the fatty acid alkyl, specifically ethyl esters is about 2-90%w/w (as determined by GC area ratios), where the reaction substrate may optionally comprise free fatty acids 0-90%w/w.

[0022] In any of said processes, the reaction substrate comprises at least l%w/w, 2%w/w, 3%w/w, 5%w/w, 10% w/w, 15%w/w, 20%w/w, 30%w/w and up to 40%w/w, 50%w/w, 60%w/w, 70%w/w, or more, up to saturation of said oil mixture with phytosterol(s). The reaction product comprises at least l%w/w, 2%w/w, 3%w/w, 5%w/w, 10% w/w, 15%w/w, 20%w/w, 30%w/w and up to 40%w/w, 50%w/w, 60%w/w, 70%w/w, 80%w/w or 90%w/w of phytosterol fatty acid esters solubilized in said oil enriched with phytosterol fatty acid esters. Further, in any of said processes, the reaction substrate can comprise about 10-70% w/w fatty acid alkyl esters, about 10-30% w/w monoglycerides, about 10-30% w/w di glycerides, and about 5-50% w/w said triglycerides, and optionally further comprise free fatty acids.

[0023] In any of said processes, the short-chain alcohol is short-chain Ci-6 alcohol, preferably ethanol. Said short-chain alkyl esters are short-chain Ci-6 alkyl esters, preferably ethyl esters.

[0024] In any of said processes, lipase can be a lipase derived from any of Candida rugosa, Candida cylindracea, Candida antarctica A (CALA), Candida antarctica B (CALB), Pseudomonas fluorescens, Pseudomonas sp., Alcaligenes sp., Burkholderia sp., Burkholderia ubonensis (strain PL266-QLM), Pseudomonas (Burkholderia) cepacia, Rhizomucor miehei, Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Thermomyces lanuginosus, Chromobacterium viscosum, and Pseudomonas stutzeri, in free form or immobilized on an organic or inorganic support. Specifically disclosed lipases are Candida rugosa, preferably Candida rugosa (Enzyme Development Corporation, USA), or Candida rugosa (Lipase AY, Amano Enzymes, Japan), Candida cylindracea (rugosa) (Lipase OF, (Meito Sangyo Japan) or Burkholderia ubonensis, preferably Burkholderia ubonensis strain PL266-QLM (Lipase QLM, Meito Sangyo, Japan).

[0025] In any of said processes, the immobilizing support for the lipase can be a neutral adsorbent, anion- or cation-exchange resin, a hydrophilic organic polymer, such as polyacrylate, polymethyl methacrylate, polymethyl methacrylate or cross-linked phenol formaldehyde condensate, or mild hydrophobic polymer, for example divinylbenzene, or a mixed hydrophobic/hydrophilic polymer, specifically cross linked divinylbenzene-methyl methacrylate polymer, hydrophobic/mild hydrophobic organic polymer such as polyvinyl alcohol, polydivinyl-benzene and polystyrene, or a hydrophobic polymer, such as polypropylene, and any mixture thereof, or said support is an inorganic support, optionally as silica, Celite, diatomaceous earth and perlite.

[0026] In any of said processes, the said oil is a food-grade oil or fat or cosmetic-grade oil or fat, or plant, oleaginous single cell oil, animal or synthetic origin, such as, for example, plant- derived fat or oil, optionally soybean, sunflower, canola, palm, rice bran, sesame, corn, coconut, algal oil, oleaginous single cell oil, cocoa butter or medium chain triglycerides (MCT); or said oil is animal-derived, optionally fish oil, bovine milk fat or anhydrous milk fat; a structured oil, optionally fully hydrogenated plant oil enzymatically or chemically interesterified with a single plant oil or any mixture of at least two thereof.

[0027] In any of said processes, the aqueous alkaline buffer can be a bicarbonate, carbonate, acetate, phosphate, citrate or tris buffer salt or any of their combination. The said water or aqueous alkaline buffer can be added to said reaction substrate at an amount of from about 0.05%, 0.1, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60% and up to 70% by weight of said reaction medium. The pH of the reaction medium can be any of 4, 5, 6, 7, 8, 9, 10 or 11.

[0028] In any of said processes, the transesterification reaction can be carried out at a temperature of about 20, 30, 40, 50, 60, 70, 80 or 90 °C.

[0029] In any of said processes, the said phytosterol can be any one of beta-sitosterol, campesterol, stigmasterol, stanol, and brassicasterol and any mixture of at least two thereof.

[0030] In any of the disclosed processes, the enzyme filtered off after the reaction is stopped can be suitable for recurring use over multiple reaction cycles, where the immobilized lipase retains more than 70% of its original activity after the 10^}h reaction cycle.

[0031] In any of said disclosed processes, the said phytosterol fatty alkyl esters are C2-C24, optionally Cie-Cis saturated or unsaturated fatty acids, which may be, acetic, propanoic, butanoic, caproic, caprylic, capric, myristic, palmitic, oleic, linoleic, linolenic or stearic, eicosapentaenoic (EPA) or docosahexaenoic (DHA) acids.

[0032] The said disclosed processes can be conducted in a stirred batch or continuous reactor or in a packed- or fluidized- bed column reactor and said lipase is an immobilized lipase.

[0033] Further disclosed in a homogenous oil product, comprising about l-90%w/w, about 2- 3%w/w to about 10-50%w/w, or about 5-20%w/w solubilized phytosterol fatty acid esters, prepared by any of the said disclosed processes.

[0034] Yet further, any of the disclosed processes can further comprising the steps of: subjecting a homogenous oil mixture enriched with about 10-30%w/w, preferably about 15-25%w/w, more preferably about 20%w/w solubilized phytosterol fatty acid esters, produced by any of the said processes from a reaction substrate which comprises 10- 30%w/w, respectively 15-25%w/w, respectively 20%w/w phytosterols, to chemically or enzymatically catalyzed interesterification with a fully hydrogenated oil of less than 10 iodine value by incubating said homogenous oil mixture enriched with solubilized phytosterol fatty acid esters and said fully hydrogenated oil or fat at a temperature of about 30-70°C, preferably whilst stirring, preferably at 160 rpm for a suitable period of time, preferably from about 3-14 hours, stopping the reaction upon achieving equilibrium and removing the catalyst, whereby said enriched homogenous oil mixture and said fully hydrogenated oil are interesterified and form a reaction product that is a homogenous oil mixture in which said phytosterol esters, optionally residual phytosterols, are solubilized; and optionally deodorizing and bleaching the said reaction product to give a homogenous fat enriched with at least l%w/w phytosterol fatty acid esters, in which the ratio of saturated fatty acids/unsaturated fatty acids is less than 60%, preferably less than 50%, having a melting point higher than 25°C.

[0035] In this last disclosed process, said fully hydrogenated oil or fat can be plant-derived, optionally soybean, sunflower, canola, palm, rice bran, sesame, com, coconut, algal oil, oleaginous single cell oil, cocoa butter or medium chain triglycerides (MCT), a structured oil, optionally fully hydrogenated plant oil enzymatically or chemically inter-esterified with a single plant oil or any mixture of at least two thereof.

[0036] Further in this last disclosed process, the said lipase can be any one of Lipozyme TL IM, lipases derived from Rhizomucor miehei, Rhizopus niveus, Rhizopus oryzae, Aspergillus niger, Asperguillus oryzae, Penicillium camembertii, Thermomyces lanuginosus, Candida antarctica A (CALA), Candida antarctica B (CALB), Pseudomonas sp., Alcaligenes sp., Burkholderia sp., Burkholderia ubonensis (strain PL266-QLM), Candida rugosa, Pseudomonas (Burkholderia) cepacia, Rhizomucor miehei, Mucor javanicus, Mucor miehei, Chromobacterium viscosum, and Pseudomonas stutzeri, or any combinations of at least two thereof, in their free or immobilized forms. The lipase immobilizing support can be a neutral adsorbent or anion- or cation-exchange resin, a hydrophilic organic polymer, such as polyacrylate, polymethyl methacrylate, polymethyl methacrylate or cross-linked phenol formaldehyde condensate, or mild hydrophobic polymer, for example divinylbenzene, or a mixed hydrophobic/hydrophilic polymer, specifically cross linked divinylbenzene-methyl methacrylate polymer, hydrophobic/mild hydrophobic organic polymer such as polyvinyl alcohol, polydivinyl-benzene and polystyrene, or a hydrophobic polymer, such as polypropylene, and any mixture thereof, or said support is an inorganic support, optionally as silica, Celite, diatomaceous earth and perlite.

[0037] Further in this last disclosed process, when the interesterification is chemically catalyzed, the catalyst can be potassium/sodium methoxide or ethoxide.

[0038] Further disclosed herein is a homogenous fat enriched with phytosterol fatty acid esters, having a melting point higher than 25°C, wherein the ratio between phytosterol fatty acid esters and other constituents of said fat, primarily mono-, di- and triglycerides is at least 1%, and wherein the ratio of saturated fatty acids/unsaturated fatty acids is less than 60%. This product can be used as or in a food product, food ingredient, food supplement, cosmetic product, or pharmaceutical product.

[0039] Thus, the present disclosure also relates to food product, food ingredient, a food supplement, a cosmetic product, or a pharmaceutical product comprising the said solid homogenous fat product, where said phytosterol fatty acid esters, optionally said residual phytosterols, can replace cholesterol/cholesterol fatty acids esters typically comprised in fats and oils of animal origin.

[0040] Yet further, the said disclosed processes of the present disclosure, for producing oils enriched with solubilized phytosterol fatty acid esters, can further comprises the step of (i) subjecting an amount of the obtained homogenous oil mixture comprising more than 3% wt. phytosterol fatty acid esters product to vigorous mixing for a suitable period of time, optionally 1 minute, together with an amount of water and an amount of an aqueous native or enzymatically hydrolyzed protein solution, wherein the relative amounts of said product, said water and said hydrolyzed protein being pre-determined, optionally 40-90% oil mixture and 60-10% aqueous native or hydrolyzed protein solution of -l-50%w/w protein, and (ii) cooling the mixture to a temperature of less than 7°C, optionally 4°C for 1 hour; to give a product being a stable oleogel comprising oil, proteins, water and phytosterol fatty acid esters, phytosterol, and optionally fatty acid alkyl esters.

[0041] Thus, the present disclosure also relates to a stable oleogel exhibiting high plasticity, comprising 40% w/w oil(s) enriched with phytosterol fatty acid esters and intact or hydrolyzed protein(s), wherein the content of phytosterol fatty acid esters is at least about 1%, produced by the method of claim 41. The disclosed oleogel can serve as or in the preparation of food products, food ingredients, food supplements, cosmetic products or pharmaceutical products.

DETAILED DESCRIPTION OF EMBODIMENTS

General description [0042] The processes and methods of the present disclosure are essentially novel biocatalyst- based batch/continuous transesterification/interesterification/esterification processes for producing compositions of phytosterol esters suspended/dissolved/solubilized in homogenous oil media, the compositions containing more than 3% wt. phytosterols suspended/dissolved/solubilized in the oil media.

[0043] Throughout the present disclosure, the following abbreviations are used in singular and plural forms: MGs (monoglycerides): DGs (di glycerides); TGs (triglycerides); FFAs (free fatty acids); PSs (phytosterols); PSEs (phytosterol esters); FAEEs (fatty acid ethyl esters, which at times may be used herein for fatty acid alkyl esters).

[0044] In a first aspect, the present disclosure relates to a process for producing an oil enriched with solubilized phytosterol esters, the process comprising subjecting a reaction substrate comprising a homogenous mixture of: (i) an oil comprising mono-, di-, and triglycerides and free fatty acids, wherein the oil may be optionally supplemented with free monoglycerides, (ii) fatty acid short-chain alkyl esters, specifically fatty acid ethyl esters, and ( ill ) about 1%-90%W/W, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s) solubilized in said mixture, such as at least l%w/w, or 2%w/w, or 3%w/w, or 5%w/w, or 10%w/w or 20%w/w phyto sterol (s) solubilized in said mixture to enzymatic transesterification and/or interesterification and/or esterification reactions in said oil, wherein said transesterification occurs between said solubilized phytosterols and between said oil glycerides and optional supplemented monoglycerides, dominantly between said oil monoglycerides and optionally supplemented monoglycerides, and optionally or at times between said oil diglycerides and free fatty acids as fatty acyl donor, in an aqueous/micro-aqueous reaction medium containing an aqueous alkaline buffer or water and having pH of about 4-11 and comprising a free or immobilized lipase, and allowing the reaction/s to proceed for a suitable time period, stopping the reaction/s by removing the lipase, water or buffer and by-products, giving as a product a homogenous mixture comprising said oil and at least about 1%-90%W/W, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s) solubilized in said mixture, such as at least l%w/w, or 2%w/w, or 3%w/w, or 5%w/w, or 10%w/w or 20%w/w of total oil medium phytosterol fatty acid esters, solubilized in said oil thereby obtaining an oil enriched with solubilized phytosterol fatty acid esters.

[0045] In said process of the first aspect of the disclosure and all embodiments thereof, the interesterification occurs as a side-reaction exchanging fatty acyl groups simultaneously between said oil mono-, di-, and tri-glycerides and optionally supplemented monoglycerides and between fatty acid ethyl esters, wherein exchange of fatty acyl groups occurs between all reactants having ester bonds, and said esterification occurs as a side-reaction between free fatty acids and phytosterols.

[0046] Further in said process of the first aspect of the disclosure, the amounts of the oil monoglycerides and optionally supplemented monoglycerides, optionally of said oil diglycerides in the product are lower than their amounts in the said reaction substrate, while the amount of triglycerides is not significantly changed.

[0047] In one specific embodiment of the process of said first aspect disclosed is a process for producing oil enriched with solubilized phytosterol esters comprising the steps of

(a) providing an oil-based homogenous reaction substrate comprising an oil which comprises fatty acid alkyl esters, monoglycerides, diglycerides, triglycerides and free fatty acids at suitable amounts, and about 1%-90%W/W, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s) solubilized in said mixture, such as at least l%w/w, or 2%w/w, or 3%w/w, or 5%w/w, or 10%w/w or 20%w/w phytosterol(s) solubilized in said oil;

(b) subj ecting said reaction substrate to at least one of enzymatic transesterification/ interesterification/esterification reaction/s in said oil, in an aqueous or microaqueous reaction mixture having a pH of about 4-11 in the presence of a crude lipase or an immobilized lipase whilst stirring, preferably at about 250 rpm for a suitable time period, preferably about 5-12 hours, at a temperature of about 10-90°C, whereby transesterification/esterification reactions occur between said solubilized phytosterols and said glycerides, dominantly monoglycerides and optionally or at times diglycerides and free fatty acids as fatty acyl donor, thereby obtaining a homogenous mixture comprising solubilized phytosterol fatty acid esters, mono-, di- and tri- glycerides and residual solubilized non-reacted phytosterols, and as by-product of the transesterification reaction free fatty acids;

(d) optionally distilling off said free fatty acids and said residual fatty acid alkyl esters, to give a homogenous product comprising at least about l%w/w, at least about 2%w/w, at least about 3%w/w, at least about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 30%w/w, about 40%w/w, about 50%w/w, about 60%w/w, about 70%w/w, about 80%w/w and up to about 90%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual non-reacted solubilized phytosterol(s).

[0048] The said aqueous or microaqueous reaction mixture can comprise from about 100 ppm to about 10%w/w water or aqueous alkaline buffer.

[0049] In another specific embodiment of the process of said first aspect of the disclosure, the present disclosure relates to a process for producing an oil enriched with solubilized phytosterol fatty acid esters, the process comprising the steps of:

(al) providing a homogenous reaction substrate comprising fatty acid short-chain alkyl esters, an oil comprising mono-, di- and tri-glycerides and free fatty acids a suitable amounts, and about 1%-90%W/W, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s) solubilized in said mixture, such as at least l%w/w, or 2%w/w, or 3%w/w, or 5%w/w, or 10%w/w or 20%w/w solubilized phytosterol(s);

(bl) adding to said reaction substrate water or aqueous alkaline buffer at more than 100 ppm up to about 70% w/w to form a reaction medium having a pH of about 4-11;

(cl) adding to said reaction medium a lipase which is a crude lipase or a lipase immobilized on an organic or inorganic support to form a reaction mixture;

(dl) stirring the said reaction mixture for a period of time of about 5-12 hours, at a temperature of about 10-90°C at about 250 rpm, to allow for transesterification/ interesterification/esterification reactions between the solubilized phytosterols and said mono-, di- and triglycerides and said free fatty acids, dominantly monoglycerides, optionally or at times said diglycerides, as a fatty acyl donor, to occur, thereby obtaining a homogenous mixture comprising solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides, residual solubilized phytosterols, and as by-product of the transesterification reaction free fatty acids;

(el) collecting the reaction mixture of step (dl) by filtering off the enzyme and removing water to give as a product a homogenous oil mixture comprising at least l%w/w, preferably at least 2%w/w, more preferably at least 3%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides, residual solubilized non-reacted phytosterol(s), residual fatty acid alkyl esters and free fatty acids, wherein the amounts of said monoglycerides, optionally said diglycerides in the product are lower than their amounts in the said reaction substrate, while the amount of triglycerides is not significantly changed; and (fl) optionally distilling off said free fatty acids and said residual fatty acid alkyl esters, to give a homogenous product comprising at least about l%w/w, at least about 2%w/w, at least about 3%w/w, at least about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 30%w/w, about 40%w/w, about 50%w/w, about 60%w/w, about 70%w/w, about 80%w/w and up to about 90%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual non-reacted solubilized phytosterol(s).

[0050] In all embodiments of the process of said first aspect of the present disclosure, the said reaction substrate may be prepared by a method comprising providing oil triglycerides and subjecting the oil triglycerides to:

(i) partial enzymatic transesterification with a short-chain alkyl alcohol, specifically ethyl alcohol, in the presence of a free crude lipase or a lipase immobilized on an inorganic or organic support, and removing the enzyme from the reaction mixture at the end of the reaction by filtration and optionally adding free monoglycerides to the product;

(ii) chemical partial transesterification using a chemical catalyst, optionally a strong alkaline catalyst, preferably sodium methoxide; and removing the chemical catalyst from the reaction mixture at the end of the reaction by water wash and optionally adding free monoglycerides to the product; or

(iii) mixing an oil with mono-glycerides and fatty acid alkyl esters, preferably fatty acid ethyl esters; to give an oil mixture comprising monoglycerides at an amount of about l-50%w/w, diglycerides at an amount of about l-50%w/w, triglycerides at an amount of about l-90%w/w, and fatty acid alkyl esters, specifically ethyl esters at an amount of about 2-90%w/w (as determined by GC area ratios), where said reaction substrate optionally comprises free fatty acids at an amount of up to 90%w/w; and solubilizing in said oil mixture phytosterol(s) at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more ,such as about 10-50%w/w, more preferably from about 5- 20%w/w phytosterol(s), to give a homogenous reaction substrate.

[0051] In all embodiments of the process of said first aspect of the present disclosure, the weight ratios between said mono-, di- and tri-glycerides in the homogenous reaction substrate can vary, and the ratios between these glycerides and said fatty acid short-chain alkyl esters, specifically fatty acid ethyl esters, and, where present, free fatty acids can also vary. In particular embodiments the amount (in w/w%) of said monoglycerides in said reaction substrate is about 1-50%, for example but not limited to 5-40%w/w, 10-30%w/w and ranges therebetween, the amount of said diglycerides is about 1-50 %, for example but not limited to 5-40%w/w, 10-30%w/w and ranges therebetween, the amount of said triglycerides is about 1-90%, for example, but not limited to 5-50%w/w and ranges therebetween, and the amount of said fatty acid alkyl esters, specifically ethyl esters is about 2-90%, for example but not limited to 10-70%w/w (as determined by GC area ratios) and ranges therebetween, where said reaction substrate optionally comprises free fatty acids at up to 90%w/w. Some representative compositions of the MGs, DGs, TGs, FAEEs (alternatively fatty acid alkyl esters) in the reaction substrate are presented in Tables 3, 13, 14 and 15 below. For example, the reaction substrate prepared in Example 2, namely canola oil transesterified with ethanol, comprised (w/w%) fatty acid ethyl esters (FAEEs, representing fatty acid alkyl esters) (67.4%) of which about 2-4% are free fatty acids (FFAs); (monoglycerides (MGs) (12.5%), diglycerides (DGs) (12.5%) and triglycerides (TGs) (7.6%) as determined by GC analysis. FFAs were a by-product of the hydrolytic reaction catalyzed by the lipase, and were mostly present at a range of about 0.5-10% depending on the initial amount of water in the reaction system. Where FFAs values are not shown separately, they were added to the value for FAEEs; otherwise they are shown separately.

[0052] Generally, as shown in Examples 3, 13, 14 and 15 below, the amounts of the reaction components can be (in w/w% as determined by GC area ratios) FAEEs about 2-90%; MGs about 1-50%, DGs about 1-50%; and TGs aboutl-90%.

[0053] In some embodiments of the process of the first aspect of the present disclosure, the said homogenous reaction substrate (provided in step (a)) can be prepared by enzymatic partial transesterification of oil triglycerides with a short-chain alkyl alcohol in the presence of a crude free lipase or a lipase immobilized on a inorganic or organic support as catalyst, as described, for example, in Example 2 below, or chemically catalyzed by using a chemical catalyst, optionally a strong alkaline catalyst, preferably sodium methoxide or ethoxide, and removing the lipase, respectively the chemical catalyst, from the reaction mixture at the end of the reaction by filtration or water wash. The said short-chain alkyl alcohol can be any Ci-6 alkyl alcohol, preferably ethanol. Alternatively, the said homogenous reaction substrate (provided in step (a)) can be prepared by mixing an oil with mono-glycerides and fatty acid alkyl esters, preferably fatty acid ethyl esters, to give a mixture comprising fatty acid alkyl esters, mono-, di- and tri-glycerides (as shown, for example, in Example 11), or by mixing an oil with fatty acid alkyl esters to give a mixture comprising the oil glycerides and fatty acid alkyl esters (as presented, for example, in Examples 6 and 7, where the fatty acid alkyl ester is ethyl oleate).

[0054] As shown in the following examples, during the transesterification reaction, the MGs proved to be the dominant acyl donor for the phytosterol(s) (PSs) when FAEEs were present. While the concentrations of FFAs, DGs and TGs remained almost unchanged, the levels of MGs and PSs dropped, and the levels of the PSEs increased, thus enriching the reaction substrate with PSEs The addition of fatty acid alkyl esters, for example and specifically fatty acid ethyl esters improved the solubilization of PSs in the reaction medium. Without being bound by theory, the improved solubilization of the PSs under the reaction medium under the reaction conditions, specifically the presence of FAEEs alone or in combination with the presence of monoglycerides, leads to solubilization of the phytosterols (PSs) in the reaction medium, and consequently preference of MGs as acyl donor. Phytosterol esters (PSEs) have greater solubility in the reaction medium as compared to PSs. The produced oil-based compositions enriched with PSEs can thus contain high amounts of PSEs, for example from at least about l%w/w, at least about 2%w/w, at least about 3%w/w, at least about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 30%w/w, about 40%w/w, about 50%w/w, about 60%w/w, about 70%w/w, about 80%w/w and up to about 90%w/w of total oil medium, and specific amounts therebetween. In this connection it is noted that phytosterols are more soluble in monoglycerides than in diglycerides, least soluble in triglycerides.

Without being bound by theory, this may explain applicant’s finding that in the transesterification reaction between oil glycerides in all aspects and embodiments of the present disclosure, monoglycerides are first to react, making them the dominant acyl donor, and at times, diglycerides may also serve as acyl donors, as reflected by the results presented in the following Examples.

[0055] Thus, in all embodiments of said first aspect of the present disclosure, the amount of monoglycerides in the final product following the transesterification reaction is significantly reduced compared to the amount of said monoglycerides in the reaction substrate, while the amounts of said di-glycerides, triglycerides and free fatty acids is not significantly changed compared to their amounts in said reaction substrate. By the term “significantly reduced” when referring in particular to amount of monoglycerides, optionally or at times diglycerides, is meant a reduction of more than 60% on molar basis with the initial amount of phytosterols compared to their amount in the reaction mixture before initiation of the reaction by addition of the biocatalyst into the reaction medium. By “not significantly changed” with respect to, for example, monoglycerides, diglycerides, triglycerides and free fatty acids is meant a change of no more than about 20% on a weight basis, where concentrations/levels of these reactants in the initial reaction mixture are compared to the final reaction product.

[0056] In all aspects and embodiments of the process of the present disclosure, the progress of the transesterification reaction can be monitored at suitable time points. Samples of the reaction mixture can be drawn at suitable volumes at desired time points, which can be pre- determined or random, and concentrations of all species contained in the reaction mixture, reactants and the products, including primarily MGs, DGs, TGs, FFAs, FAEEs, PSs (phytosterols) and PSEs, are determined, for example by Gas chromatography peak area ratios. The transesterification reaction can be stopped at any stage, preferably when equilibrium is reached, i.e. when there is no significant change in the level of the reactants/products compared to the preceding measurement.

[0057] In some embodiments of the first aspect of the present disclosure, while the MGs serve as the dominant acyl donor for the transesterification of the PSs, DGs can also exhibit measurable contribution of acyl moieties in die transesterification reaction, while TGs and FFAs levels in the final reaction mixture remain essentially unchanged. Reference is may also be made to Example 14 and Table 15 below. This example shows that MGs were dominantly the donor of fatty acyl group to phytosterols, rather than TGs under the transesterification reaction conditions described in Table 15, for both starting mixtures, namely (A) partially enzymatically transesterified canola oil (PTO) produced according to Example 2, and (B) an oil mixture prepared by mixing appropriate weight ratios of FAEEs (as ethyl oleate), MGs, DGs (both from commercially available distilled MGs) and TGs (as canola oil).

[0058] In all aspects and embodiments of the process of the present disclosure, the oil used in the reaction substrate is a food grade oil or fat or cosmetic grade oil or fat, or plant, oleaginous single cell, animal or synthetic origin. Specific oils for use in the processes of the present disclosure are, but not limited to, oil or fat is plant-derived, for example soybean, sunflower, canola, palm, rice bran, sesame, corn, coconut, algal oil, cocoa butter or medium chain triglycerides (MCT). Other oils/fats for use in the processes of the present disclosure are, for example, animal-derived, optionally fish oil, bovine milk fat or anhydrous milk fat. The oil used in the processes of the present disclosure can also be a structured oil, for example fully hydrogenated plant oil enzymatically or chemically interesterified with a single plant oil. The oil/fat used in the presently disclosed process can be a single oil or fat, or a mixture of at least two thereof. Mixtures of oil/s and fat/s are also useful in the present processes. [0059] In all embodiments of said first and aspect of the present disclosure, said short-chain alcohol is short-chain Ci-6 alcohol, preferably ethanol.

[0060] In ah embodiments of said first and aspect of the present disclosure, said short-chain alkyl esters are short-chain Ci-6 alkyl esters, preferably ethyl esters.

[0061] In all aspects and embodiments of the process of the present disclosure, the said phytosterol is any one of beta-sitosterol, campesterol, stigmasterol, stanol, and brassicasterol and any mixture of at least two thereof

[0062] In all aspects and embodiments of the process of the present disclosure, the phytosterol fatty acid esters can be, but are not limited to, C2-C24, for example Cie-Cis saturated or unsaturated fatty acids, which may be, acetic, propanoic, butanoic, caproic, caprylic, capric, myristic, palmitic, oleic, linoleic, linolenic or stearic, eicosapentaenoic (EPA) or docosahexaenoic (DHA) acids.

[0063] In all aspects and embodiments of the processes of the present disclosure, the said lipase can be derived from any one of Candida rugosa, Candida cylindracea, Candida antarctica A (CALA), Candida antarctica B (CALB), Pseudomonas fluorescens, Pseudomonas sp., Alcaligenes sp., Burkholderia sp., Burkholderia ubonensis (strain PL266- QLM), Pseudomonas (Burkholderia) cepacia, Rhizomucor miehei, Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Thermomyces lanuginosus, Chromobacterium viscosum, and Pseudomonas stutzeri, in free form or immobilized on an organic or inorganic support.

[0064] Specific lipases useful in the said processes are Candida rugosa, for example Candida rugosa (Enzyme Development Corporation, USA), or Candida rugosa (Lipase AY, Amano Enzymes, Japan), Candida cylindracea (rugosa) (Lipase OF, (Meito Sangyo Japan) or Burkholderia ubonensis, preferably Burkholderia ubonensis strain PL266-QLM (Lipase QLM, Meito Sangyo, Japan).

[0065] The lipases used in the transesterification processes of the present disclosure can be in crude, free form, or they may be immobilized on various solid supports, for example, porous beads or other suitable carriers. The support can be, but is not limited to polymeric support, such as but not limited to a neutral adsorbent, an anion- or cation-exchange resin, hydrophilic organic polymers, such as polyacrylate, polymethyl methacrylate, polymethyl methacrylate or cross-linked phenol formaldehyde condensate, or mild hydrophobic polymers, for example divinylbenzene, or mixed hydrophobic/hydrophilic polymers, specifically cross-linked divinylbenzene-methyl methacrylate polymer, hydrophobic/mild hydrophobic organic polymers such as polyvinyl alcohol, polydivinyl-benzene and polystyrene, or hydrophobic polymers, such as polypropylene, and any mixture thereof, or said support is an inorganic support, optionally as silica, diatomaceous earth, for example Celite^R, and perlite.

[0066] It is noted that the immobilized enzyme preparations used in the processes of the present disclosure exhibit stability over numerous reaction cycles. Once the enzymatic reaction is terminated, the enzyme, specifically immobilized enzyme, is filtered off and is suitable for recurring uses over multiple additional reaction cycles, while the immobilized lipase retains more than 70% of its original activity after the 10^th reaction cycle

[0067] In all embodiments of the processes of said aspect of the present disclosure, the said aqueous alkaline buffer used in the transesterification reaction can be, but is not limited to a bicarbonate, carbonate, acetate, phosphate, citrate or tris, buffer salt or any combination.

[0068] The said water or aqueous alkaline buffer can be added to said reaction substrate at an amount of from about 0.01%, 0.05%, 0.1, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60% and up to 70% by weight of said reaction medium.

[0069] The pH of the reaction medium is adjusted to any of pH 4, 5, 6, 7, 8, 9, 10 or 11.

[0070] The said transesterification reaction can be carried out at a temperature of about 20, 30, 40, 50, 60, 70, 80 or 90°C.

[0071] In all embodiments of the processes of said first aspect of the present disclosure, the said transesterification step of the process can be conducted in a stirred batch or continuous reactor or in a packed- or fluidized-bed reactors and said lipase is an immobilized lipase. Nonetheless, when using a transesterification reaction medium in the absence of fatty acid alkyl esters, such as, for example in Example 2, the reaction is preferably be conducted in stirred-tank reactors, as the solubility of phytosterols in oil is limited to below 3%, and reacting in a stirred tank reactor helps avoiding clogging problems caused by insoluble phytosterols. This approach can be adopted whenever reacting non-homogenous (heterogenous) reaction systems.

[0072] In a further aspect, the present disclosure relates to a homogenous oil product comprising at least 3% w/w of solubilized phytosterol fatty acid esters. In specific embodiments, this homogenous oil product is prepared by the said processes of the present disclosure. Many such homogenous oil products are described in the following Examples, particularly Examples 3, 13, 14 and 15, and are referred to in detail above.

[0073] In a second aspect, the present disclosure relates to a process for producing inter- esterified oil mixtures having a melting point higher than 30°C in which the ratio of saturated/unsaturated fatty acids is lower than 0.5. This process essentially comprises subjecting the homogenous oil mixture comprising solubilized 1-90%, for example but not limited to 10-30%w/w or 15-25%w/w, or 20%w/w phytosterol esters, specifically where prepared by a process of the said first aspect of the current disclosure and its embodiments, to chemically or enzymatically catalyzed interesterification with a fully hydrogenated oil of less than 10 iodine value by incubating said homogenous oil mixture and said fully hydrogenated oil or fat at a temperature of about 30-70°C, preferably whilst stirring, preferably at 160 rpm for a suitable period of time, preferably from about 4-14 hours, whereby said homogenous oil mixture and said fully hydrogenated oil are interesterified and form a reaction product that is a homogenous oil mixture in which said phytosterol esters, optionally residual phytosterols are solubilized. The reaction is stopped upon reaching equilibrium by removing the catalyst, optionally by filtration. Optionally, the product is deodorized and bleached.

[0074] In this aspect of the present disclosure, as mentioned, the starting material can be the homogenous oil enriched with phytosterol esters prepared by the process of said first aspect and its embodiments of the present disclosure. For example, the starting oil transesterified with phytosterol(s) can be the product of the above-described step (c) or step (d) of said embodiment of the first aspect of the current disclosure, or of the above described step (el) or of step (fl) of said other embodiment, or the above described product of step (5) or step (6) of the process of said yet another embodiment of the present disclosure or embodiments thereof. The interesterification reaction mixture between the oil enriched with phytosterol esters and a fully hydrogenated oil can be monitored at suitable time points. Generally, the first oil is comprised of phytosterol esters, MGs, DGs, TGs and optionally unreacted residual phy tosterols and free fatty acid byproduct, while the second oil is comprised of fully hydrogenated plant oil comprised of dominantly palmitic and stearic acids, and optionally other saturated fatty acids of C2-C14 carbon atoms, at different ratios. Samples of the reaction mixture can be drawn at suitable volumes at desired time points, which can be pre- determined or random, and concentrations of all species contained in the reaction mixture, reactants and the products, are determined, for example by Gas chromatography peak area ratios. The interesterification reaction can be stopped at any stage, preferably when equilibrium is reached, i.e. when there is no significant changes in the levels of the reactants/products compared to the Immediately preceding measurement.

[0075] The reaction between the said first oil, namely homogenous oil comprising phytosterol esters, MGs, DGs, TGs and optionally unreacted residual phytosterols and free fatty acid byproduct (which can be prepared by the various processes of the present disclosure as described above) and between the fully hydrogenated oil triglycerides is mainly an interesterification reaction between glycerides of the homogenou s oil enriched with phytosterol esters and between the second, fully hydrogenated oil triglycerides, to form a homogenous oil mixture wherein phytosterol esters are solubilized, and which results in increasing the melting point of the oil mixture as compared to mixture prepared by interesterification of fully hydrogenated oils with a non-partially transesterified/esterified oil (non-processed oil) under the same reaction conditions, as shown in Example 15.

[0076] Example 15, describes an enzymatic interesterification reaction between an oil mixture comprised of 20% phytosterol fatty acid esters prepared by an enzymatic partial transesterification of sunflower oil triglycerides and ethanol and afterwards removal of ethanol and then enzymatically reacting the oil mixture with phytosterols followed by removal of fatty acid ethyl esters and free fatty acids byproduct (as in the process/es described above), with fully hydrogenated sunflower oil. A control experiment was carried out by interesterification of non-processed sunflower oil and fully hydrogenated sunflower oil under similar reaction conditions. The melting points of the resulting interesterified oil mixtures are presented in Table 16. As shown, mixtures of enzymatically interesterified oils containing 20% phytosterol esters exhibit higher melting points as compared to the same oil composition, however without phytosterol esters. The data in Table 16 show that fat mixtures of melting point higher than 30^cC with a low ratio of saturated/unsaturated fatty acids less than 50%, can be prepared by introducing phytosterol esters in the reaction mixture. These modified products according to the present disclosure, make another use of the benefits of the high content of phytosterol esters, which exhibit improved functional properties, such as the higher melting point (SFC) while maintaining lower ratios of saturated/unsaturated fatty acids, as well as enhanced stability, and modified nutritional characteristics.

[0077] Example 15 further shows that enriching liquid oil mixtures with phytosterol esters leads to increasing the solid fat content of the resulting oil, thus decreasing the ratio of unsaturated fatty acids/saturated fatty acids, as compared to the composition of the same oil, however free of phytosterol esters. Thus, the fats produced according to this second aspect of the present disclosure have improved physical properties, as well as improved nutritional and dietary properties compared to animal-derived oils and fats. These properties render them particularly suitable for use in various food technology processes and in the production of non-animal based food products, for example in the artificial meat and other artificial foods such as, for example vegan food industries, and also in the production of food ingredients, food supplements, cosmetic products and pharmaceutical products.

[0078] The said fully hydrogenated oil may be fat is plant-derived, optionally soybean, sunflower, canola, palm, rice bran, sesame, corn, coconut, algal oil, oleaginous single cell oil, cocoa butter or medium chain triglycerides (MCT), a structured oil, optionally fully hydrogenated plant oil enzymatically or chemically inter-esterified with a single plant oil or any mixture of at least two thereof.

[0079] I 'he lipase, where enzymatic catalysis is used, can be a crude free lipase or a lipase immobilized on an organic or inorganic support. Specific lipases are, but not limited to Lipozyme TL IM, lipases derived from Rhizomucor miehei, Rhizopus niveus, Rhizopus oryzae, Aspergillus niger, Asperguillus oryzae, Penicillium camembertii, Thermomyces lanuginosus, Candida antarctica A (C ALA), Candida antarctica B (CALB), Pseudomonas sp., Alcaligenes sp., Burkholderia sp., Burkholderia ubonensis (strain PL266-QLM), Candida rugosa, Pseudomonas (Burkholderia) cepacia, Rhizomucor miehei, Mucor javanicus, Mucor miehei, Chromobacterium viscosum, and Pseudomonas stutzeri, or any combinations of at least two thereof. Specific supports can be, but are not limited to a neutral adsorbent, an anion- or cation-exchange resin, a hydrophilic organic polymer, such as polyacrylate, polymethyl methacrylate, polymethyl methacrylate or cross-linked phenol formaldehyde condensate, or mild hydrophobic polymer, for example divinylbenzene, or a mixed hydrophobic/hydrophilic polymer, specifically cross linked divinylbenzene-methyl methacrylate polymer, hydrophobic/mild hydrophobic organic polymer such as polyvinyl alcohol, polydivinyl-benzene and polystyrene, or a hydrophobic polymer, such as polypropylene, and any mixture thereof, or said support is an inorganic support, optionally as silica, Celite, diatomaceous earth and perlite.

[0080] Where chemical catalysis is used, the catalyst can be, but is not limited to potassium or sodium methoxide, or potassium or sodium ethoxide/

[0081] The present disclosure thus provides a solid homogenous fat enriched with phytosterol fatty acid esters, having a melting point higher than 25°C, wherein the ratio between phytosterol esters and other constituents of said fat, primarily mono-, di- and triglycerides is at least 1%, and wherein the ratio of saturated fatty acids/unsaturated fatty acids is less than 60%, preferably less than 55%, more preferably less than 50%. This low ratio contributes to dietary products comprising the same, moreover, it comprises phytosterol(s) that replace the cholesterol comprised in animal-derived fats and oils.

[0082] The present disclosure thus also relates to a food product, a food supplement, a cosmetic product or a pharmaceutical product comprising the solid homogenous fat according to the present disclosure or prepared by the process of the present disclosure. In such food product, food supplement, cosmetic product, or pharmaceutical product the phytosterol fatty acid esters, optionally said residual phytosterols, replace cholesterol/cholesterol fatty acids esters typically comprised in fats and oils of animal origin. The high melting point fats and oil disclosed herein may find specific use, for example in sausages production, as they may prevent leakage of fat to the enveloping layer of the product.

[0083] In a third aspect, the present disclosure relates to the production of edible (food grade) and/or cosmetically/pharmaceutically acceptable oleogels comprising oils comprising solubilized phytosterol esters prepared according to the process/es of the present disclosure, and either an intact or hydrolyzed protein, or an emulsifier such as methyl cellulose aqueous solution. Oleogels, in which the continuous liquid phase is an oil, exhibit high stability and high elasticity. These properties make them useful in the food, pharmaceutical and cosmetic industries, and can replace solid fats.

[0084] Thus, the present disclosure relates to a process for producing an oleogel, the process comprising mixing together a. partially transesterified oil enriched with solubilized phytosterol esters prepared by the process/es of the present disclosure, and an aqueous solution of a native or enzymatically hydrolyzed protein, preferably at about 1-70% w/w , wherein the relative amounts of said partially transesterified oil containing solubilized phytosterol esters, and said intact or hydrolyzed protein are pre-determined, optionally 40- 90% oil mixture and 5-70% aqueous protein or protein hydrolysate solution, for example of 1-70% w/w; and cooling the mixture to a temperature of less than 7°C, optionally 4°C for a suitable period of time, for example 1-24 hour; to give a product being a stable homogenous oleogel comprising oil, proteins, water and phytosterol fatty acid esters, and optionally fatty acid alkyl esters.

[0085] Example 16 shows the production of oleogels using an oil partially transesterified produced in accordance with the present disclosure, and protein solutions. As shown in Example 16 and Table 17, the oleogels prepared using an oil partially transesterified with phytosterols as the fat component, give stable homogenous oleogels characterized with higher plasticity when mixed with protein isolate solutions of 10% wt. compared to using only oil triglycerides with the same protein isolate solution.

[0(186] Example 17 and Table 18 show that the plasticity of oleogels produced by using the oil canola partially transesterified with phytosterols (30%) mixed with protein isolate solution (10%) pretreated enzymatically with different proteases was higher when mixed with hydrolyzed protein isolate solutions of 10% wt. compared to using only oil triglycerides with the same protein isolate solution.

[0087] Thus, in a yet further fourth aspect, the present disclosure relates to a stable homogenous oleogel exhibiting high plasticity, comprising about 40% w/w oil(s) comprising about 1-70% solubilized phytosterol esters, and about 10-80%w/w intact protein or hydrolyzed protein(s) of 1-50% w/w protein or protein hydrolysate. In some embodiments, the oil(s) partially trans-esterified with phytosterol(s) is/are prepared by any of the processes of the present disclosure.

[0088] Also envisaged are food products, food supplements and additives, cosmetic products and pharmaceutical products comprising the oleogel according to the present disclosure. In these products comprising the oleogel according to the present disclosure phytosterol/phytosterol fatty acid esters serve as substitutes for cholesterol/cholesterol fatty acids esters typically comprised in fats and oils of animal origin. The disclosed oleogels may be used as an egg yolk substitute in many vegan food and bakery products, and also in low- cholesterol products.

Definitions

[0089] The terms “fatty acid alkyl esters” and “fatty acid ethyl esters” may be used herein interchangingly, and the abbreviations “FAEE” or “FAEEs” defined above may be used to designate both.

[0090] The term “lipase" as used herein generally refers to a naturally occurring lipase enzyme obtained from a natural source by industrial fermentation process/es. The term “lipase" is also referred to herein as “the enzyme” or the “biocatalyst” which may be used interchangingly.

[0091] Further, the term “lipase” as used herein encompasses both “crude” or “free” or “nonimmobilized" lipase, and a lipase immobilized on a support as described herein, which may be referred to as “immobilized lipase” . Both crude and immobilized lipase may be referred to herein as “lipase preparation” .

[0092] Further, the term “lipase” as used herein encompasses both “crude” or “free” or “nonimmobilized" lipase, and a lipase immobilized on a support as described herein, which may be referred to as “immobilized lipase” . Both crude and immobilized lipase may be referred to herein as “lipase preparation”. It is to be understood that a “commercially available lipase” encompasses lipases derived from different strains of microorganisms, suspended in excipients, such as, for example, lactose or cyclodextrins, to produce the solid form of a lipase preparation, or a lipase dissolved in water optionally containing a polyol, such as glycerol, ethylene or propylene glycol, or salt such as sodium chloride to produce a liquid lipase preparation.

[0093] The term “support” is to be taken to mean a solid matrix or polymer or polymeric resin on which the biocatalyst is immobilized by either physical bonding or chemical bonding. The terms “support”, “matrix”, “supporting polymer” “polymeric resin” may be used herein interchangingly. Admixed' hydrophobic/ hydrophilic polymer recited herein is also referred to herein as ''mild' hydrophobic.

[9094] The term “microaqueous' ' as used herein, particularly in connection with water content of the reaction systems described herein, is to be taken to mean a weight content of water of from about lOOppp to 20% w/w of oil, and where an immobilized lipase is employed may also refer to or include water confined in the immobilized lipase preparation.

[0095] The term “homogenous oil mixture (or homogenous reaction substrate) saturated with solubilized phytosterol(s)” as used herein is to be taken to mean an oil mixture (or reaction substrate) comprising any given amount of phytosterol(s) while homogeneity of the oil mixture (or reaction substrate) is maintained. Such amounts can be, but are not limited to, from about l%w/w, 2%w/w 3%w/w, 5%w/w, 10%w/w, 15%w/w, 20%w/w, 25% w/w, 30%w/w and up to 5()%w/w or more.

[9096] Unless otherwise indicated, the term percent (%) is to be taken to mean weight by weight percent (%w/w or %wt.).

[0097] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[9098] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicated number and a second indicated number and "ranging/ranges from" a first indicated number "to" a second indicated number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. It should be noted that where various embodiments are described by using a given range, the range is given as such merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.

[0099] Disclosed and described, it is to be understood that this disclosure is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present disclosure will be limited only by the appended claims and equivalents thereof.

[00199] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present disclosure. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the disclosure, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the disclosure.

DESCRIPTION OF NON-LIMITING EXAMPLES

Example 1:

Screening of lipases of transesterification activity to p roduce phytosterol fatty acid est e rs A mixture of canola oil (3g) containing phytosterols (0.3 g) was shaken at 60°C. The reaction was initiated by the addition of 150mg commercial lipase preparation, and then the reaction mixture containing partly solubilized phytosterols, was shaken at 160rpm and 60°C for 24 hours. Samples of 60 microliters were withdrawn periodically for GC analysis for determining the concentrations (as peak area ratios) of free fatty acids (FFAs), monoglycerides (MGs), phytosterols (PSs), diglycerides (DGs), phytosterol esters (PSEs), and triglycerides (TGs). Tables I and 2 show the concentrations (GC peak area ratios) of the reaction components after 6 and 24 hours of reaction.

Table 1: The concentrations (GC peak area ratios) of the reaction components after 6 hours of reaction using different types of lipases.

Reaction conditions: A mixture of canola oil (3g) and phytosterols (0.3g) were mixed in screw-cap vial at 60°C and shaken at 160rpm. The reaction was initiated by the addition of 150mg of a commercial lipase preparation into the oil mixture and shaking and heating were resumed for 24 hours.

Table 2: The concentrations (GC peak area ratios) of the reaction components after 24 hours of reaction.

Reaction conditions: Same as in Table 1.

The results presented in Tables 1 and 2 show that lipases derived from Candida rugosa, Candida antarctica A , Pseudomonas sp., and Burkholderia ubonensis are preferred enzymes for transesterification of oil triglycerides to produce mixtures of phytosterol fatty acid esters, MGs, DGs and residual TGs. Specifically preferred were Candida rugosa (Enzyme Development Corporation (EDC), USA), Candida antarctica A (Novocor AD L, Novozymes, Denmark), Pseudotnonas sp., (Lipase PS and Lipase PF, Amano Enzymes, Japan) and Burkholderia ubonensis (Lipase QLM, Meito Sangyo, Japan). Since the solubility of phytosterol in oil is limited to 2-3%, the reaction medium is heterogeneous when containing more than 2% phytosterols, and therefore such reactions can normally be conducted batchwise.

Example 2

Preparation of partially transesterified mixture of plant ails and ethanol in the presence of a lipase

A mixture of canola oil (100g) containing 10% wt. ethanol and 0.1% sodium bicarbonate solution of 0.1 M, was incubated at 60°C. TransZyme ET (7g) -• Thermomyces lanuginosus lipase immobilized on microporous acrylic polymer beads, was added into the mixture, and the reaction mixture was mixed at 160 rpm, and 60^°C for 24 hours. Samples of 60 microliters were withdrawn periodically for GC analysis for determining the concentrations of fatty acid ethyl esters (FAEEs), FFAs, MGs, DGs and TGs. The reaction mixture after 6 hours of reaction w^ras filtered off for the removal of the biocatalyst to obtain a partially transesterified canola oil having the composition of: 67.4% FAEEs, 12.5% MGs, 12.5% DGs, and 7.6% TGs, and nonreacted ethanol. Residual ethanol and added water were flash evaporated, and the remaining reaction mixture was used as a medium for solubilization of phytosterols at concentrations of higher than 2% in oil, and as a potent fatty acyl group donor for transesterification reactions between oil glycerides and phytosterols to produce phytosterol esters and other related components.

Example 3

Screening of lipases of transesterification activity to produce phytosterol fatty acid esters using partially transesterified plant oils with ethanol as a reaction medium

A mixture of partially transesterified canola oil obtained in Example 2 (3g) containing phytosterols (0.3g), was shaken at 6()°C till complete solubilization of phytosterols and produce a homogenous reaction medium. The reaction w^ras initiated by the addition of a commercial free lipase preparation and then mixing at 160 rpm and 60°C for 24 hours. Samples of 60 microliters were withdrawn periodically for GC analysis for determining the concentrations of the reaction components. Tables 3 and 4 show the concentrations (GC peak area ratios) of the reaction components after 6 and 24 hours of reaction.

Table 3: The concentrations (GC peak area ratios) of the reaction components after 6 hours of reaction ,

Reaction conditions: A partially transesterified canola oil with ethanol of composition as described in Example 2 (3g) and phytosterols (0.3g) was shaken at 60°C till complete solubilization of phytosterols in the oil. The reaction was initiated by the addition of 150 mg commercial lipase preparation, and then the reaction mixture was shaken at 160rpm and 60°C for 24hours. FFAs, a by-product of the hydrolytic reaction catalyzed by the lipase, were mostly present at a range of about 0.5-10% depending on the initial amount of water in the reaction system. Where FFAs values are not shown separately, they were added to the value for FAEEs.

'Table 4: The concentra tions (GC peak area ratios) of the reaction components after 24 hours of reaction. Reaction conditions: See Table 3.

The results presented in Tables 3 and 4 are similar with the results obtained in Example 1 where lipases derived from Candida rugosa (EDC, USA), Candida antarctica A , (Nonozytnes, Denmark). Pseudomonas sp. (Lipase PS or Lipase PF - Amano Enzymes, Japan), Candida cylindracea (Meito Sangyo, Japan), and Burkholderia ubonensis (Meito Sangyo, Japan) are the most active enzymes for transesterification of phytosterols with a mixture of fatty acid ethyl esters and oil glycerides to produce a mixture of phytosterol fatty acid esters. The results show surprisingly that MGs are the predominantly most, favored fatty acyl donor among all other reaction medium components. The concentrations of FAEEs, DGs and TGs remained approximately unchanged before and after the transesterification reaction, while the levels of MGs dropped, and levels of PSEs increased.

Example 4 - Comparative

Transesterification of plant oils and phytosterols using immobilized lipases

A mixture of canola oil (2.7g) containing 10%wt. phytosterols (0.3g) was shaken at 60°C. The heterogeneous reaction was initiated by the addition of 220mg of a lipase of different origin immobilized on microporous methyl methacrylate/divinylbenzene polymer beads, and then the reaction mixture containing partly solubilized phytosterols, was shaken at 160rpm and 60°C for 24hours. The reaction was carried out without and with the addition of 0.5% wt./wt of sodium bicarbonate solution of 0. 1 M. Samples of 60microliters were withdrawn periodically for GC analysis for determining the concentrations (as peak area ratio) for FFAs, MGs, PSs, DGs, PSEs. and TGs. Table 5 shows the concentrations of the reaction components after 6 hours of reaction.

Table 5: The concentrations (GC peak area ratios) of the reaction components after 6 hours of reaction.

Reaction conditions: A mixture of canola oil (2.7g) and 10% wt. phytosterols (0.3g) incubated at 60°C was mixed at 160rpm for 6hours. The reaction was initiated by the addition of 220mg of different origin immobilized on microporous methyl methacrylate (MMA)/diviny Ibenzene

(DVB) polymer beads. Reactions were performed without and with the addition of 0.5% wt/wt. of sodium bicarbonate solution of 0.1 M into the reaction medium

The results presented in Table 5 show that lipases derived from Candida sps. (Candida antarctica A (NovoCor ADL, Novozymes, Denmark) and Candida rugosa, EDC, USA), and from Burkholderia ubonensis (Lipase QLM Meito Japan), immobilized on polymeric resin beads, were also able to catalyze the transesterification of phytosterols with oil triglycerides. The results show also that the transesterification activity of immobilized Burkholderia ubonensis lipase, unlike the other lipases, is intolerant to the addition of water in the reaction medium while lipases derived from Candida sps. immobilized on the same beads, favored the addition of micro-amount of water as sodium bicarbonate aqueous solution into the reaction medium. Example 5

Transesterification of partially transesterified oils and phytosterols using immobilized lipases A mixture of partially transesterified canola oil (2.7g) with a composition as described in Example 2, containing 10%wt. fully solubilized phytosterols (0.3g) was incubated at 60°C. The reaction was initiated by the addition of 220m g of a lipase of different origin immobilized on microporous methyl methacrylate/divinylbenzene polymer beads. The reaction mixture was shaken at 160rpm and 60°C for 24hours. The reaction was carried out without and with the addition of 0.5% wt./wt. of sodium bicarbonate aqueous solution of 0.1 M. Samples of 60 microliters were withdrawn periodically for GC analysis for determining the concentrations (as peak area ratio) for fatty acids ethyl esters (FAEEs), monoglycerides (MGs), phytosterols (PSs), diglycerides (DGs), phytosterol esters (PSEs), and triglycerides (TGs). Table 6 shows the concentrations (GC peak area ratios) of the reaction components after 6 hours of reaction.

Table 6: The concentrations (GC peak area ratios) of the reaction components after 6 hours of reaction.

Reaction conditions: A mixture of partially transesterified canola oil (2.7 g) and 10% wl. solubilized phytosterols (0.3g) (as described in Example 2 - different weight ratios were used) incubated at 60°C, was mixed at 160rpm for 6 hours. The reaction was initiated by the addition of 220mg of lipases of different origin immobilized on microporous methyl methacrylate/diviny [benzene polymer beads. Reactions were performed without and with the addition of 0.5% wt./wt. of sodium bicarbonate aqueous solution of 0.1 M.

The results presented in Table 6 were similar to the results obtained in Example 4 where lipases derived from Candida sps. immobilized on a polymeric resin favor the addition of water into the reaction medium while lipase derived from Burkholderia ubonensis is intolerant to the presence of micro-amounts of water in the reaction medium.

Example 6

Transesterification of canola oil and ethyl oleate mixture at different weight ratios with phytosterols to produce phytosterol esters using free lipases

A mixture of canola oil and ethyl oleate at different weight ratios (3g) containing phytosterols (0.3g), was shaken at 60°C till complete solubilization. The reaction was initiated by the addition of a commercial lipase preparation derived either from Burkholderia vtboneiisis (strain PL266-QLM, Lipase QLM, Mello Sangyo, Japan) or from Candida rugosa (EDC, USA), and then mixing at 160 rpm and 60°C for 24 hours. Samples of 60 microliters were withdrawn periodically for GC analysis for determining the concentrations of the reaction components. Table 7 shows the concentrations (GC peak area ratios) of the reaction components after 0, 3, 6 and 24 hours of reaction.

Table 7: The concentrations (GC peak area ratios) of the reaction components at 0, 6 and 24 hours of reaction.

Reaction conditions: A mixture of canola oil and ethyl oleate (3g) at different weight ratios and phytosterols (0.3g) was shaken at 60°C until complete solubilization. The reaction was initiated by the addition of 150mg commercial lipase derived either from Burkholderia ubonensis or from Candida rugosa, and then the reaction mixture was shaken at 160rpm and 60°C for 24hours.

The results presented in Table 7 show that the different lipases utilized preferably TGs as a fatty acyl donor, where the concentration of triglycerides is reduced with time till reaching equilibrium concentration after 24 hours of reaction. The results in Table 7 show that the concentrations of FAEEs remained approximately with no significant changes indicating that the lipase does not favor FAEEs as fatty acyl group donor in the transesterification reaction.

Example 7

Transesterification of canola oil and ethyl oleate mixture at different weight ratios with phytosterols to produce phytosterol esters using immobilized lipases

A mixture of canola oil and ethyl oleate at different weight ratios (3g) and phytosterols (0.3g), was shaken at 60°C till complete solubilization of phytosterols. The reaction was initiated by the addition of a lipase derived either from Burkholderia ubonensis (strain PL266-QLM, Lipase QLM, Meito Sangyo, Japan) or from Candida rugosa (EDC, USA), both immobilized on microporous acrylic/divinylbenzene polymer beads, and then mixing at 160 rpm and 60°C for 24 hours. Samples of 60 microliters were withdrawn periodically for GC analysis for determining the concentrations of the reaction components. Table 8 shows the concentrations (GC peak area ratios) of the reaction components at 0, 3, 6 and 24 hours of reaction.

Table 8: The concentrations (GC peak area ratios) of the reaction components after 0, 3, 6 and 24 hours of reaction.

Reaction conditions: A mixture of canola oil and ethyl oleate (3g) at different weight ratios and phytosterols (0.3g) was shaken at 60°C till complete solubilization. The reaction was initiated by the addition of 300mg lipase derived either from BurkJiolderla ubonensis or from Candida rugosa, both immobilized on microporous acrylic/divinylbenzene polymer beads, and then the reaction mixture was shaken at 160rpm and 60°C for 24hours.

Similar results to those obtained in Example 6 when the same lipases, however immobilized on a polymeric resin were used for the transesterification of oil mixtures comprised of different ratios of FAEEs and oil, as a source of fatty acyl groups. The results show that triglycerides are the primary source of fatty acyl group in the transesterification reaction while FAEEs are not favored as a fatty acid source by any enzyme. Example 8 - Comparative

Transesterification of a mixture of canola oil and MGs at different weight ratios with phytosterols to produce phytosterol esters using immobilized lipases

The following experiments were conducted to determine the tatty acyl group donor to the phytosterols for producing the phytosterol esters. A mixture of canola oil and a commercial distilled monoglycerides (predominantly MGs) was transesterified with phytosterols in the presence of lipases derived either from Burkholderia ubonensis (QLM, Meito Sangyo, Japan) or from Candida rugosa (EDC, USA), both immobilized on microporous acrylic/divinyl benzene polymer beads. Table 9 shows the concentrations (Peak area ratios %) of the different reaction components at different periods of time.

Table 9: The concentrations (GC peak area ratios) of the reaction components after 0, 3, 6 and 24 hours of reaction.

Reaction conditions: A mixture of canola oil and distilled MGs (3g) at different weight ratios and phytosterols (0.3g) was shaken at 60°C till complete solubilization. The reaction was initiated by the addition of 300mg lipase derived either from Burkholderia ubonensis or from Candida rugosa, both immobilized on microporous acrylic/divinylbenzene polymer beads, and then the reaction mixture was shaken at 160rpm and 60°C for 24hours.

The results presented in Table 9 show explicitly that TGs are better fatty acyl donors than MGs for phytosterols to produce sterol fatty acid esters under the described reaction conditions. Example 9 ~ Comparative

Transesterification of a mixture of canola oil, MGs and FFAs at different weight ratios with phy toste rols to produce phytosterol esters using free lipases

The following experiments were conducted to determine the fatty acyl group donor to the phytosterols for producing the phytosterol esters. A mixture of canola oil and distilled MGs containing a fixed concentration of free fatty acids was transesterified/esterified with solubilized phytosterols in the presence of free lipases derived either from Burkholderia ubonensis or from Candida rugosa. Table 10 shows the concentrations (Peak area ratios %) of the different reaction components at different reaction times.

Table 19: T he concentrations (GC peak area ratios) of the reaction components after 0, 3, 6 and 24 hours of reaction.

Reaction conditions: A mixture of canola oil, FFAs and MGs (3g) at different weight ratios, 0.5% wt. of oil sodium bicarbonate solution of 0.5M. and phytosterols (0.3g) was shaken at 60°C till complete solubilization. The reaction was initiated by the addition of 150mg commercial lipase derived either from Burkholderia ubonensis or from Candida rugosa, and then the reaction mixture was shaken at 160rpm and 60°C for 24hours.

The results presented in Table 10 show explicitly that 'TGs are better fatty acyl donors than MGs as well as than FFAs for phytosterols to produce sterol fatty acid esters under the described reaction conditions where free lipases were used. Example 10- Comparative

Transesterification of a mixture of canola oil, MGs and FFAs at differen t weight ratios with phytosterols to produce phytosterol esters using immobilized lipases

The following experiments were conducted to determine the fatty acyl group donor to the phytosterols for producing the phytosterol esters. A mixture of canola oil and distilled MGs containing a fixed concentration of free fatty acids was transesterified/esterified with phytosterols in the presence of lipases derived either from Burkholderia ubonensis or front Candida rugosa, both immobilized on microporous acrylic/divinylbenzene polymer beads.

Table 9 shows the concentrations (peak area ratios %) of the different reaction components at different periods of time.

Table 11 : The concentrations (GC peak area ratios) of' the reaction components after 0, 3, 6 and 24 hours of reaction

Reaction conditions: A mixture of canola oil, FFAs and MGs (3g) at different weight ratios, and phytosterols (0.3g) was shaken at 60°C till complete solubilization. The reaction was initiated by the addition of 300mg lipase derived either from Burkholderia vtbonensis or from

Candida riisosa, both immobilized on microporous acrylic/divinylbenzene polymer beads, and then the reaction mixture was shaken at 160rpm and 60^cC for 24 hours.

The results presented in Table 11 show explicitly that TGs are better fatty acyl donors than MGs as well as than FFAs for phytosterols to produce sterol fatty acid esters under the described reaction conditions when immobilized lipases were used.

Example 11

Transesterification of partially transesterified oils (PTO) with ethanol mixed with MGs at different weight ratios, and phytosterols using immobilized lipases A mixture of partially transesterified canola oil with ethanol with product composition as described in Example 2. and distilled MGs at different ratios (2.7g). containing fully solubilized phytosterols (0.3g), was prepared. The reaction was initiated by the addition of 300mg of a lipase of different origin immobilized on microporous methyl methacrylate/divinylbenzene polymer beads. The reaction mixture was shaken at 160rpm and 60°C for 24hours. The reaction was carried out with the addition of 0.5% wt./wt. of sodium bicarbonate solution of 0. 1 M. Samples of 60microliters were withdrawn periodically for GC analysis for determining the concentrations (as peak area ratio) for FAEEs, MGs, PSs, DGs, PSEs, and TGs. Table 12 shows the concentrations (GC peak area ratios) of the reaction components at different time intervals.

Table 12: The concentrations (GC peak area ratios) of the reaction components at different time intervals.

Reaction conditions: A mixture of partially transesterified canola oil (PTO) with ethanol (product composition of Example 2), and MGs at different weight ratios (2.7g) and solubilized phytosterols (0.3g) incubated at 60°C, was mixed at 160rpm for 24hours. The reaction was initiated by the addition of 300mg lipase derived either from Burkholderia ubonensis or from Candida rugosa, both immobilized on microporous acrylic/divinylbenzene polymer beads, and then the reaction mixture was shaken at 160rpm and 60°C for 24hours. Reactions were performed with the addition of 0.5% wt./wt. of sodium bicarbonate aqueous solution of 0.1 M

The results presented in Table 12 show explicitly that in the presence of low concentrations of

TGs, MGs would act as fatty acyl donors for phytosterols to produce sterol fatty acid esters under the described reaction conditions. The results also show that MGs serve as better fatty acyl donor than FAEEs to produce phytosterol esters under the above-described conditions. Example 12

Transesterification of partially transesterified oils with ethanol, and phytosterols using imniobiiized lipases

A mixture of partially transesterified canola oil (2.7g) with ethanol containing reduced concentration of FAEEs (partially removed by distillation to reach a composition of 27.5% FAEEs, 32.2% MGs, 23.5% DGs, and 16.8% TGs), containing 10%wt. fully solubilized phytosterols (0.3g), was prepared. The reaction was initiated by the addition of 220mg of a lipase of different origin immobilized on microporous methyl methacrylate/divinylbenzene polymer beads. The reaction mixture was shaken at 160rpm and 60°C for 24hours. The reaction was carried out without and with the addition of 0.5% wt./wt. of sodium bicarbonate solution of 0.1 M. Samples of 60microliters were withdrawn periodically for GC analysis for determining the concentrations (as peak area ratio) for FAEEs, MGs, PSs, DGs, PSEs, and TGs. Table 13 shows the concentrations (GC peak area ratios) of the reaction components at different time intervals. The values for GC peak area ratios for FAEEs represent the sum of peaks for FAEEs and FFAs where FFAs values are typically in the range of 3-9% when the concentration of water (as sodium bicarbonate solution of 0.1M) was below 0.5%.

Table 13: The concentrations (GC peak area ratios) of the reaction components at different time intervals.

Reaction conditions: A mixture of partially transesterified canola oil with ethanol containing reduced amount of FAEEs (2.7g), and 10% wt. solubilized phytosterols (0.3g) incubated at 60°C, was mixed at 160rpm for 24hours. The reaction was initiated by the addition of 220mg of different origin immobilized on microporous methyl methacrylate/divinylbenzene polymer beads. Reactions were performed without and with the addition of 0.5% wf. of sodium bicarbonate solution of 0. 1 M.

The results presented in Table 13 show that when TGs concentration is low in the reaction medium, MGs would serve as a fatty acyl donor for phytosterols to produce phytosterol esters under the described reaction conditions. The results show also that MGs are a better fatty acyl donor than FAEEs under the above-described reaction conditions. The increase in the values for FAEEs in all experiments where water was added, is attributed to the increase of FFAs concentrations as by product of hydrolysi s side reaction, which were summed up to the GC peak areas of FAEEs.

Example 13

Transesterification of partially transesterified oils with ethanol, under different concentrations of phytosterols using immobilized lipases

A mixture of partially transesterified canola oil (2.7g) with ethanol with the composition as described in Example 2, was mixed with different concentrations of phytosterols. The reaction was initiated by the addition of 220mg of a lipase of different origin immobilized on microporous methyl methacrylate/divinylbenzene polymer beads. The reaction mixture was shaken at 160rpm and 60°C for 24hours. The reaction was carried out wdth the addition of 0.5% wt./wt, of sodium bicarbonate solution of 0. 1 M. Samples of bOmicroliters were -withdrawn periodically for GC analysis for determining the concentrations (as peak area ratio) for FAEEs, MGs, PSs, DGs, PSEs, and TGs. As a control experiment, canola oil was used containing phytosterols (20%wt.) under the same reaction conditions. Table 14 shows the concentrations (GC peak area ratios) of the reaction components at different time intervals. The values for GC peak area ratios for FAEEs represent, the sum of peaks for FAEEs and FFAs where FFAs values are typically in the range of 3-9% when the concentration of water (as sodium bicarbonate solution of 0.1M) below' 0.5%. The values for GC peak area ratios for FAEEs represent the sum of peaks for FAEEs and FFAs where FFAs values are typically in the range of 3-9% when the concentration of water (as sodium bicarbonate solution of 0.1M) below' 0.5%.

Table 14: The concentrations (GC peak area ratios) of the reaction components at different time internals.

Reaction conditions: A mixture of partially transesterified canola oil with ethanol, with the composition as described in example 2 (3g), and 0.5% wt./wt. of sodium bicarbonate solution of 0. 1 M, was mixed with different concentrations of phytosterols. The reaction was initiated by the addition of 220mg of different origin immobilized on microporous methyl- methacrylate/divinylbenzene polymer beads. The reaction mixture was mixed at 160rpm and incubated at 60°C for 24hours. As a control experiment, canola oil was used instead of PSs (20%wt.) under the same reaction conditions.

The results presented in Table 14 show explicitly that in the presence of FAEEs in the reaction medium, MGs become better fatty acyl group donor for phytosterols as compared to TGs to produce phytosterol esters under the above-described reaction conditions.

Example 14

Transesterification of partially enzymatically transesterified oil with ethanol/or oil mixture prepared by mixing appropriate weight ratios of FAEEs (as ethyl oleate), MGs (as distilled monoglycerides), DGs (as a part of the commercially available distilled MGs) and Canola oil triglycerides, with phytosterols using immobilized lipases

The following is the composition of two oil mediums A and B which have been transesterified with 10% wt./wt. phytosterols, separately, under the same reaction conditions as described in Example 13. .A. Partially enzymatically transesterified canola oil (PTO) with ethanol with the following composition as produced according to Example 2:

B. An oil mixture prepared by mixing appropriate weight ratios of FAEEs (as ethyl oleate), MGs, DGs (both from commercially available distilled MGs) and TGs (as canola oil).

Table 15 shows tire concentration profile (GC peak area ratios) of the reaction components for each reaction mixture with 10% phytosterols, separately at different time intervals. Both reactions were catalyzed using immobilized lipases QLM and Candida rugosa.

Table 15: The concentrations (GC peak area ratios) of the reaction components at different time intervals.

Reaction conditions: A mixture of enzymatically partially transesterified canola oil (3 g) with ethanol with the composition as described above (Composition A) or a synthetic mixture of FAEEs, MGs, DGs and TGs (3g of Composition B), were transesterified, separately, each with phytosterols (0.3g) in the presence of 0.5% wt./wt. of sodium bicarbonate solution of 0.1 M. The reaction was initiated by the addition of 220mg of different lipase origin immobilized on raicroporous methyl methacrylate/divinylbenzene polymer beads. The reaction mixture was mixed at 160 rpm and incubated at 60°C for 24hours.

The results presented in Table 15 show clearly that MGs in both reaction mixtures were dominantly the donor of fatty acyl group to phytosterols, rather than TGs or FAEEs under the transesterification reaction conditions described in Table 15.

Example 15

Preparation of solid-state oils of low ratios of saturated/unsaturated fatty acids via enzymatic interesterification of plant oils mixtures

This Example describes an enzymatic interesterification reaction between different weight ratios of sunflower oil triglycerides which has been enzymatically transesterified with 20% phytosterol till achieving equilibrium, with fully hydrogenated sunflower oil. A control experiment was carried out by interesterification of the same weight ratios between sunflower oil triglycerides (without phy tosterol esters), and fully hydrogenated sunflower oil under similar reaction conditions. The melting points of the resulting interesterified oil mixtures and the ratio between saturated and unsaturated fatty acids in each mixture are presented in Table 16.

Table 16: The physical state at different temperatures for enzymatically interesterified different weight ratios of sunflower oil transesterified with 20% w/w with phytosterol till achieving equilibrium, with fully hydrogenated sunflower oil. A control experiment was carried out by interesterification of the same weight ratios between sunflower oil and fully hydrogenated sunflower oil (without supplemented phytosterols trader similar reaction conditions. Reaction conditions: A mixture of oil containing different ratios between the two types of oils, and Lipozyme TL IM (10% Wt.), was incubated and shaken at IbOrpm and 60°C for 12 hours. The ratio of saturated fatty acids/unsaturated fatty acids for each oil mixture is also given in the below Table. Table has been provided below.]

(* ): Ratio of saturated fatty acids/unsaturated fatty acid in the oil mixture as determined by GC analysis.

*SFO - Sunflower oil, FH-SFO - Fully Hydrogenated Sunflower Oil

**PSEs/Oil - Transesterified Sunflower Oil containing 20%wt. phytosterol esters.

The results presented in Table 16 show clearly that mixtures of enzymatically interested fled oils with 20% w/w phytosterols until achieving equilibrium to produce phytosterol esters, with fully hydrogenated sunflower oil exhibit higher melting points as compared to the same oil composition however without phytosterol esters. The data in Table 16 show that fat mixtures of melting point higher than 30°C with a ratio of saturated/un saturated fatty acids less than 50%, can be prepared by introducing phytosterol esters in the reaction mixture. Other compositions of oil mixtures containing phytosterol esters interesterifled with fully hydrogenated plant oils of melting points in the range of 20-80°C can be prepared in accordance with the disclosed process.

Example 16

Production of oleogels using partially transesterified oil with phytosterols and protein solutions

Sunflower oil has been enzymatically transesterified with phytosterols to produce a mixture of phytosterol esters (approx. 30%wt.), MGs, DGs and TGs with residual free phytosterols (below 5%wt.) and free fatty acids (0-10%) as a byproduct of the lipase-catalyzed hydrolysis side reaction. Different weight ratios of this oil mixture mixed with 10% wt. pea protein isolate (90% purity) in distilled water, were prepared. The oil-protein mixtures were vigorously mixed at 15000 rpm for 2 mins and then kept at 4°C for 24 hours. Table 17 shows the stability and texture of the oleogels produced by using the partially transesterified canola oil with phytosterols mixed either with 10% wt. protein isolate solution or with 2% wt. methylcellulose solution as a comparative mixture. Control experiments were conducted to prepare oleogels, using canola oil in combination with either protein or methyl cellulose aqueous solutions prepared under the same conditions.

Table 17: The stability of oleogels produced by using the partially transesterified sunflower oil with 20% w/w phytosterols, with 10% wt. aqueous solution of pea protein isolate (90%), or with 2% wt. methylcellulose aqueous solution. Control experiments were conducted to prepare oleogels however using canola oil in combination with either 10% protein isolate solution or 2% methyl cellulose solution under the same preparation conditions.

The results presented in Table 17 show that partially transesterified oil with 20% phytosterols produce stable homogenous oleogels characterized with higher plasticity when mixed with 10% w/w protein isolate aqueous solutions compared to using only oil triglycerides with the same protein isolate solution under the same preparation conditions.

The oleogels produced can be used in food, cosmetics and pharmaceutical applications of various semi-gel, gel or solid-state properties at different temperatures is demonstrated in the following experiments.

Example 17

Production of oleogels using partially transesterified oil with phytosterols and enzymatically hydrolyzed protein isolate solutions

Oleogels comprising partially transesterified sunflower oil with phytosterols, with the composition as described in Example 16, and an aqueous solution of enzymatically hydrolyzed protein isolates were prepared. Proteins used in these experiments were enzymatically pretreated either with protein glutaminase or with an endo/exo-protease, such as Alcalase, separately. Table 18 shows the stability and texture of oleogels produced by using the partially transesterified canola oil with phytosterols (20%) mixed with protein isolate solution (10% w/w) pretreated enzymatically with different proteases.

Table 18: The stability of oleogels produced by using the partially transesterified sunflower oil with phytosterols 20% wt. and aqueous solutions of enzymatically pretreated 10% w/w protein isolate solutions.

The results presented in Table 18 show that partially transesterified oil with phytosterols produce stable homogenous oleogels characterized with higher plasticity when mixed with hydrolyzed protein isolate solutions of 10% wt. compared to using only oil triglycerides with the same protein isolate solution producing unstable oleogels as presented in Table 17.

Claims

1 . A process for producing an oil enriched with solubilized phytosterol esters, the process comprising subjecting a reaction substrate comprising a homogenous mixture of: an oil comprising mono-, di-, and triglycerides and free fatty acids, wherein the oil may be optionally supplemented with free monoglycerides; fatty acid short-chain alkyl esters, specifically fatty acid ethyl esters, ami phytosterol(s) solubilized in said mixture at an amount up to saturation while said reaction substrate maintains homogeneity; to enzymatic transesterification and/or interesterification and/or esterification reactions in said oil, wherein said transesterification occurs between said solubilized phytosterols and between said oil glycerides and optional supplemented monoglycerides, dominantly between said oil monoglycerides and optionally supplemented monoglycerides, and optionally between said oil diglycerides and free fatty acids as fatty acyl donor, in an aqueous/micro- aqueous reaction medium containing an aqueous alkaline buffer or water and having pH of about 4-11 and comprising a crude or immobilized lipase, and allowing the reaction/s to proceed for a suitable time period, stopping the reaction/s by removing the lipase, water or buffer and by-products, giving as a product a homogenous mixture comprising said oil and at about 1%-90%W/W, preferably from about 2-3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w of total oil medium phytosterol fatty acid esters solubilized in said oil, thereby obtaining an oil enriched with solubilized phytosterol fatty acid esters.

2. The process according to claim 1, wherein said interesterification occurs as a side- reaction exchanging fatty acyl groups simultaneously between said oil mono-, di-, and tri- glycerides and optionally supplemented monoglycerides and between fatty acid ethyl esters, wherein exchange of fatty acyl groups occurs between all reactants having ester bonds.

3. The process according to claim 1 or 2, wherein said esterification occurs as a side- reaction between free fatty acids and phytosterols.

4. The process according to any one of claims 1-3, wherein the amounts of said oil monoglycerides and optionally supplemented monoglycerides, optionally of said oil diglycerides in the product are lower than their amounts in the said reaction substrate, while the amount of triglycerides is not significantly changed.

5. A process for producing oil enriched with solubilized phytosterol esters, the process comprising the steps of:

(a) providing an oil-based homogenous reaction substrate comprising an oil which comprises fatty acid alkyl esters, monoglycerides, diglycerides, triglycerides and free fatty acids at suitable amounts, and phytosterol(s) solubilized in said mixture at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more such as about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s);

(b) subj ecting said reaction substrate to at least one of enzymatic transesterification/ interesterification/esterification reaction/s in said oil, in an aqueous or microaqueous reaction mixture having a pH of about 4-11 in the presence of a crude lipase or an immobilized lipase whilst stirring, preferably at about 250 rpm for a suitable time period, preferably about 5-12 hours, at a temperature of about 10-90°C, whereby transesterification/esterification reactions occur between said solubilized phytosterols and said glycerides, dominantly monoglycerides and optionally diglycerides and free fatty acids as fatty acyl donor, thereby obtaining a homogenous mixture comprising solubilized phytosterol fatty acid esters, mono-, di- and tri- glycerides and residual solubilized non-reacted phytosterols, and as by-product of the transesterification reaction free fatty acids; and

(d) optionally distilling off said free fatty acids and said residual fatty acid alkyl esters, to give a homogenous product comprising about l-90%w/w, preferably from about 2- 3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual non-reacted solubilized phytosterol(s).

6. A process according to any one of claims 1-5, wherein water or aqueous alkaline buffer is added to said reaction substrate at from at least 100 ppm and up to about 70% w/w to form a reaction medium having a pH of about 4-11.

7. A process for producing an oil enriched with solubilized phytosterol fatty acid esters, the process comprising the steps of:

(a) providing an oil-based homogenous reaction substrate comprising an oil which comprises fatty acid alkyl esters, monoglycerides, diglycerides, triglycerides and free fatty acids at suitable amounts, and phytosterol(s) solubilized in said mixture at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more, such as about 10-50%w/w, more preferably from about 5-20%w/w phytosterol(s);

(d) stirring the said reaction mixture for a period of time of about 5-12 hours, at a temperature of about 10-90°C at about 250 rpm, to allow for at least one of transesterification/interesterification/esterification reactions between solubilized phytosterols and said glycerides and free fatty acids, dominantly said monoglycerides, optionally said diglycerides, as a fatty acyl donor to occur in said oil, thereby obtaining a homogenous mixture comprising phytosterol fatty acid esters, mono-, di- and tri-glycerides, residual phytosterols, and as by-product of the transesterification reaction free fatty acids;

(e) collecting the reaction mixture of step (d) by filtering off the enzyme and removing water to give as a product a homogenous oil mixture comprising at least l%w/w, preferably at least 2%w/w, more preferably at least 3%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides, residual solubilized non-reacted phytosterol(s), residual fatty acid alkyl esters and free fatty acids, wherein the amounts of said monoglycerides, optionally said diglycerides in the product are lower than their amounts in the said reaction substrate, while the amount of triglycerides is not significantly changed; and

(f) optionally distilling off said free fatty acids and said residual fatty acid alkyl esters, to give a homogenous product comprising about l-90%w/w, preferably from about 2- 3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w solubilized phytosterol fatty acid esters, mono-, di- and tri-glycerides and residual non-reacted solubilized phytosterol(s).

8. The process of any one of claims 1-7, wherein said homogenous reaction substrate is prepared by

(i) enzymatic partial transesterification of oil triglycerides with a short-chain alcohol in the presence of a free crude lipase or a lipase immobilized on an inorganic or organic support, or chemically by using a chemical catalyst, optionally a strong alkaline catalyst, preferably sodium methoxide or ethoxide, and removing the enzyme, respectively the chemical catalyst, from the reaction mixture at the end of the reaction by filtration or water wash and optionally adding free monoglycerides to the product obtained; or

(ii) by mixing an oil with mono-glycerides and fatty acid alkyl esters, preferably fatty acid ethyl esters, to give a mixture comprising fatty acid alkyl esters, mono-, di- and tri- glycerides.

9. The process according to any one of claims 1-8, wherein said period of time of conducting the reaction is a period of time sufficient to achieve reaction equilibrium.

10. The process according to any one of claims 1-9, further comprising monitoring the transesterification/interesterification/esterification reaction/s by drawing samples of the reaction mixture at suitable volumes, such as about 60-100 microliters, at desired time points, which can be pre-determined or random, and determining concentrations of all species contained in the reaction mixture, reactants and the products, including primarily monoglycerides, diglycerides, triglycerides, free fatty acids, phytosterol/s and phytosterol esters, whereby equilibrium stage of the reaction is detected when there is no significant change in the levels of the reactants/products compared to the immediately preceding measurement.

11. The process according to any one of the preceding claims, wherein said monoglycerides, optionally said diglycerides, in the presence of fatty acid short-chain alkyl esters lead to solubilization of said phytosterol(s) in the reaction substrate and wherein under the reaction conditions monoglycerides, optionally diglycerides, serve as dominant donor of fatty acyl groups in the transesterification process as compared to triglycerides or free fatty acids.

12. The process according to any one of the preceding claims, wherein the amount of monoglycerides in the final reaction product is significantly reduced compared to the amount of said monoglycerides in said reaction substrate, while the amounts of said di-glycerides, triglycerides and free fatty acids is not significantly changed compared to their amounts in said reaction substrate.

13. The process according to any one of the preceding claims wherein the amount of said monoglycerides in said reaction substrate is about l-50%w/w, the amount of said diglycerides is about 1-50 %w/w, the amount of said triglycerides is about l-90%w/w and the amount of said fatty acid ethyl esters is about 2-90%w/w (as determined by GC area ratios), said reaction substrate optionally comprising free fatty acids 0-90%w/w.

14. The process according to any one of the preceding claims, wherein said homogenous reaction substrate comprises at least phytosterol(s) solubilized in said mixture at an amount up to saturation, such as from about l%w/w, about 2-3%w/w, about 5%w/w, about 10%w/w, about 15%w/w, about 20%w/w, about 5-20%w/w, about 25%w/w, about 30%w/w, about 35%w/w and up to about 50%w/w or more such as about 10-50%w/w phytosterol(s).

15. The process according to any one of the preceding claims, wherein the reaction product comprises at least l%w/w, 2%w/w, 3%w/w, 5%w/w, 10% w/w, 15%w/w, 20%w/w, 30%w/w and up to 40%w/w, 50%w/w, 60%w/w, 70%w/w, 80%w/w or 90%w/w of phytosterol fatty acid esters solubilized in said oil enriched with phytosterol fatty acid esters.

16. The process according to any one of the preceding claims, wherein the said reaction substrate comprises about 10-70% w/w fatty acid alkyl esters, about 10-30% w/w monoglycerides, about 10-30% w/w diglycerides, and about 5-50% w/w said triglycerides, optionally further comprising free fatty acids.

17. The process according to any one of the preceding claims, wherein said short-chain alcohol is short-chain Ci-6 alcohol, preferably ethanol.

18. The process according claim 17, wherein said short-chain alkyl esters are short-chain Ci-6 alkyl esters, preferably ethyl esters.

19. The process according to any one of the preceding claims, wherein said lipase is derived from any one of Candida rugosa, Candida cylindracea, Candida antarctica A (CALA), Candida antarctica B (CALB), Pseudomonas fluorescens, Pseudomonas sp., Alcaligenes sp., Burkholderia sp., Burkholderia ubonensis (strain PL266-QLM), Pseudomonas ( Burkholderia ) cepacia, Rhizomucor miehei, Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Thermomyces lanuginosus, Chromobacterium viscosum, and Pseudomonas stutzeri, in free form or immobilized on an organic or inorganic support.

20. The process of any one of the preceding claims, wherein said lipase is Candida rugosa, preferably Candida rugosa (Enzyme Development Corporation, USA), or Candida rugosa (Lipase AY, Amano Enzymes, Japan), Candida cylindracea (rugosa) (Lipase OF, (Mei to Sangyo Japan) or Burkholderia ubonensis, preferably Burkholderia ubonensis strain PL266-QLM (Lipase QLM, Meito Sangyo, Japan).

21. The process according to any one of the preceding claims, wherein said support is a neutral adsorbent, anion- or cation-exchange resin, a hydrophilic organic polymer, such as polyacrylate, polymethyl methacrylate, polymethyl methacrylate or cross-linked phenol formaldehyde condensate, or mild hydrophobic polymer, for example divinylbenzene, or a mixed hydrophobic/hydrophilic polymer, specifically cross linked divinylbenzene-methyl methacrylate polymer, hydrophobic/mild hydrophobic organic polymer such as polyvinyl alcohol, polydivinyl-benzene and polystyrene, or a hydrophobic polymer, such as polypropylene, and any mixture thereof, or said support is an inorganic support, optionally as silica, Celite, diatomaceous earth and perlite.

22. The process according to any one of the preceding claims wherein said oil is a food- grade oil or fat or cosmetic-grade oil or fat, or plant, oleaginous single cell oil, animal or synthetic origin.

23. The process according to claim 22, wherein said oil or fat is plant-derived, optionally soybean, sunflower, canola, palm, rice bran, sesame, corn, coconut, algal oil, oleaginous single cell oil, cocoa butter or medium chain triglycerides (MCT); or said oil is animal- derived, optionally fish oil, bovine milk fat or anhydrous milk fat; a structured oil, optionally fully hydrogenated plant oil enzymatically or chemically interesterified with a single plant oil or any mixture of at least two thereof.

24. The process according to any one of the preceding claims, wherein said aqueous alkaline buffer is a bicarbonate, carbonate, acetate, phosphate, citrate or tris buffer salt or any of their combination.

25. The process according to any one of the preceding claims, wherein said water or aqueous alkaline buffer is added to said reaction substrate at an amount of from about 0.05%, 0.1, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60% and up to 70% by weight of said reaction medium.

26. The process according to any one of the preceding claims, wherein the pH of the reaction medium is 4, 5, 6, 7, 8, 9, 10 or 11.

27. The process according to any one of the preceding claims, wherein the transesterification reaction is carried out at a temperature of about 20, 30, 40, 50, 60, 70, 80 or 90 °C.

28. A process according to any one of the preceding claims, wherein said phytosterol is any one of beta-sitosterol, campesterol, stigmasterol, stanol, and brassicasterol and any mixture of at least two thereof.

29. The process according to any one of the preceding claims, wherein the said filtered off enzyme is suitable for recurring use over multiple reaction cycles, where the immobilized lipase retains more than 70% of its original activity after the 10^th reaction cycle.

30. The process according to any one of the preceding claims, wherein said phytosterol fatty alkyl esters are C₂-C₂₄, optionally C₁₆-C₁₈ saturated or unsaturated fatty acids, which may be, acetic, propanoic, butanoic, caproic, caprylic, capric, myristic, palmitic, oleic, linoleic, linolenic or stearic, eicosapentaenoic (EPA) or docosahexaenoic (DHA) acids.

31. The process according to any one of the preceding claims, wherein the said transesterification is conducted in a stirred batch or continuous reactor or in a packed- or fluidized- bed column reactor and said lipase is an immobilized lipase.

32. A homogenous oil product comprising about l-90%w/w, preferably from about 2- 3%w/w to about 10-50%w/w, more preferably from about 5-20%w/w solubilized phytosterol fatty acid esters, prepared by the process of any one of claims 1-31 .

33. A process according to any one of claims 1-27, further comprising the steps of:

(1) subjecting a homogenous oil mixture enriched with about 10-30%w/w, preferably about 15-25%w/w, more preferably about 20%w/w solubilized phytosterol fatty acid esters, produced by the process of any one of claims 1-26 from a reaction substrate which comprises 10-30%w/w, respectively 15-25%w/w, respectively 20%w/w phytosterols, to chemically or enzymatically catalyzed interesterification with a fully hydrogenated oil of less than 10 iodine value by incubating said homogenous oil mixture enriched with solubilized phytosterol fatty acid esters and said fully hydrogenated oil or fat at a temperature of about 30-70°C, preferably whilst stirring, preferably at 160 rpm for a suitable period of time, preferably from about 3-14 hours, stopping the reaction upon achieving equilibrium and removing the catalyst, whereby said enriched homogenous oil mixture and said fully hydrogenated oil are interesterified and form a reaction product that is a homogenous oil mixture in which said phytosterol esters, optionally residual phytosterols, are solubilized; and optionally

(2) deodorizing and bleaching the said reaction product to give a homogenous fat enriched with at least l%w/w phytosterol fatty acid esters, in which the ratio of saturated fatty acids/unsaturated fatty acids is less than 60%, preferably less than 50%, having a melting point higher than 25°C.

34. The process according to claim 33, wherein said fully hydrogenated oil or fat is plant- derived, optionally soybean, sunflower, canola, palm, rice bran, sesame, com, coconut, algal oil, oleaginous single cell oil, cocoa butter or medium chain triglycerides (MCT), a structured oil, optionally fully hydrogenated plant oil enzymatically or chemically inter-esterified with a single plant oil or any mixture of at least two thereof.

35. The process according to claim 33 or 34, wherein said lipase is any one of Lipozyme TL IM, lipases derived from Rhizomucor miehei, Rhizopus niveus, Rhizopus oryzae, Aspergillus niger, Asperguillus oryzae, Penicillium camembertii, Thermomyces lanuginosus, Candida antarctica A (CALA), Candida antarctica B (CALB), Pseudomonas sp., Alcaligenes sp., Burkholderia sp., Burkholderia ubonensis (strain PL266-QLM), Candida rugosa, Pseudomonas (Burkholderia) cepacia, Rhizomucor miehei, Mucor javanicus, Mucor miehei, Chromobacterium viscosum, and Pseudomonas stutzeri, or any combinations of at least two thereof, in their free or immobilized forms.

36. The process according to claim 34, wherein said support is a neutral adsorbent or anion- or cation-exchange resin, a hydrophilic organic polymer, such as polyacrylate, polymethyl methacrylate, polymethyl methacrylate or cross-linked phenol formaldehyde condensate, or mild hydrophobic polymer, for example divinylbenzene, or a mixed hydrophobic/hydrophilic polymer, specifically cross linked divinylbenzene-methyl methacrylate polymer, hydrophobic/mild hydrophobic organic polymer such as polyvinyl alcohol, polydivinyl-benzene and polystyrene, or a hydrophobic polymer, such as polypropylene, and any mixture thereof, or said support is an inorganic support, optionally as silica, Celite, diatomaceous earth and perlite.

37. The process according to claim 33 or claim 34, wherein said interesterification is catalyzed chemically by potassium/sodium methoxide or ethoxide.

38. A homogenous fat enriched with phytosterol fatty acid esters, having a melting point higher than 25°C, wherein the ratio between phytosterol fatty acid esters and other constituents of said fat, primarily mono-, di- and triglycerides is at least 1%, and wherein the ratio of saturated fatty acids/unsaturated fatty acids is less than 60%.

39. A food product, a food supplement, a cosmetic product or a pharmaceutical product comprising the homogenous fat of claim 38.

40. A food product, a food ingredient, a food supplement, a cosmetic product, or a pharmaceutical product comprising the solid homogenous fat of claim 38, or produced by the process of any one of claims 32 to 37, where said phytosterol fatty acid esters, optionally said residual phytosterols, replace cholesterol/cholesterol fatty acids esters typically comprised in fats and oils of animal origin.

41. The process of any one of claims 1 to 31, further comprising the step of:

(i) subjecting an amount of the homogenous oil mixture enriched with phytosterol fatty acid esters product obtained by the process of any one of claims 1-31 to vigorous mixing for a suitable period of time, optionally 1 minute, together with an amount of water and an amount of an aqueous native or enzymatically hydrolyzed protein solution, wherein the relative amounts of said product, said water and said hydrolyzed protein being pre- determined, optionally 40-90% oil mixture and 60-10% aqueous native or hydrolyzed protein solution of -l-50%w/w protein, and

(ii) cooling the mixture to a temperature of less than 7°C, optionally 4°C for 1 hour; to give a product being a stable oleogel comprising oil, proteins, water and phytosterol fatty acid esters, phytosterol, and optionally fatty acid alkyl esters.

42. A stable oleogel exhibiting high plasticity, comprising 40% w/w oil(s) enriched with sterol fatty acid esters and intact or hydrolyzed protein(s), wherein the content of phytosterol fatty acid esters is at least about 1%, produced by the method of claim 41.

43. A food product, a food supplement, a cosmetic product or a pharmaceutical product comprising the oleogel of claim 42.