Enzymatic methods for increasing the SOS triglyceride content of vegetable oils.

Abstract

The present invention relates to a method for increasing the SOS triglyceride content of a vegetable oil (wherein S represents stearic acid (C18:0) and palmitic acid (C16:0) residues and O represents oleic acid (C18:1) residues), comprising: a) a method for increasing the SOS triglyceride content of a vegetable oil, comprising: i) an sn-1,3-specific lipase immobilized on a support; ii) a vegetable oil containing at least 45% oleic acid fatty acid residues, based on the total C6-C24 fatty acid residues, and having an oleic acid content at the sn-2 position of at least 75% by weight, relative to the total sn-2 fatty acid residues of the vegetable oil; and iii) a fatty alcohol ester of stearic acid, palmitic acid, optionally mixed with stearic acid and / or palmitic acid. The method comprises the steps of: a) providing a reaction environment comprising a vegetable oil (ii) and a fatty alcohol ester, or a mixture thereof; and iv) water, wherein the weight ratio of the fatty alcohol ester and optional stearic and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1, and the water activity of the reaction environment is in the range of 0.1 to 0.6; b) heating the reaction environment to a temperature of 30 to 60°C to perform interesterification, thereby obtaining a mixture comprising a triglyceride phase, fatty acid esters, and optionally free fatty acids; and c) separating the fatty acid esters and free fatty acids from the mixture obtained in step (b) to obtain a triglyceride composition. The triglyceride phase can be used as a cocoa butter equivalent or component thereof in chocolate or chocolate-like products.

Description

[Technical Field]

[0001] The present invention relates to a method for increasing the SOS triglyceride content of vegetable oils and to triglyceride compositions obtained thereby. Triglyceride compositions having a high SOS content are useful as cocoa butter equivalents or components thereof for use in the production of chocolate and chocolate-like products. [Background technology]

[0002] Triglycerides contain three fatty acid residues attached to a glycerol backbone. Their structures can be described using the "sn" notation, which stands for stereospecific numbering. In the Fischer projection of natural L-glycerol derivatives, the secondary hydroxyl group is shown to the left of C-2, the carbon atom above this is C-1, and the carbon atom below it is C-3. The prefix "sn" is placed before the systematic name of the compound. [ka]

[0003] The physical properties of triglycerides depend on the nature of the fatty acid residues and their position on the glycerol backbone, and therefore it may be desirable to provide a method for varying the type and position of fatty acid residues in vegetable oils.

[0004] Triglyceride compositions with a high SOS triglyceride content (S represents stearic acid (C18:0) and palmitic acid (C16:0) residues, and O represents oleic acid (C18:1) residues) are particularly commercially valuable products. SOS triglycerides are present at high levels in cocoa butter and contribute to the characteristic physical properties of chocolate. Therefore, triglyceride compositions with a high SOS triglyceride content can be useful as cocoa butter equivalents or components thereof.

[0005] Shea butter is used commercially as a source of SOS triglycerides, particularly when the saturated fatty acid residue S is a stearic acid residue. Shea butter is obtained from the shea tree, Butyrospermum parkii, and can be fractionated to provide shea stearin, which has higher levels of SOS triglycerides and is therefore useful in the production of cocoa butter equivalents. The availability of shea stearin depends on the supply of shea nuts.

[0006] Enzymatic interesterification processes are known for producing triglyceride compositions having a high content of SOS triglycerides. Such processes provide a source of SOS triglycerides that is not dependent on the availability of shea nuts.

[0007] U.S. Patent No. 4,268,527(A) discloses a method for producing cocoa butter substitutes by interesterification of fats and oils using an sn-1,3-specific lipase and fatty alcohol esters of stearic and palmitic acid as a source of stearic and palmitic acid residues. This method uses fats and oils, such as fractionated palm oil and sal fat, which have a high oleic acid content at the sn-2 position, but most of which already contain relatively high levels of stearic and palmitic acid residues at the sn-1 and sn-3 positions. The water content of the reaction mixture is 0.18% by weight or less. To obtain a satisfactory cocoa butter substitute, fatty acids and their fatty alcohol esters are distilled, and the resulting composition is subjected to fractionation.

[0008] EP 2251428 B1 discloses a method for producing a composition with a high concentration of SOS (50-80% by weight) from triolein, e.g., high oleic sunflower oil, stearic acid, and sn-1,3-specific enzyme from Rhizopus oryzae. The temperature used is preferably 65-80°C, and the transesterified triglycerides are fractionated by solvent fractionation.

[0009] WO 2010 / 053244 A1 discloses cocoa butter equivalents produced by enzymatic transesterification of a wide variety of fats and oils with fatty acids or fatty acid esters using sn-1,3-specific enzymes, distillation of the reaction mixture, and fractionation to separate the products. The molar ratio of fat or oil to fatty acid or fatty acid ester ranges from 1:2 to 1:6, the temperature used in the reaction is 40-50°C, and the moisture content of the oil is less than 0.02%.

[0010] Korean Patent No. 101344491 (B1) discloses a method for producing an oil and fat composition for use in a cocoa butter equivalent (CBE) or a cocoa butter improver (CBI), which comprises the following steps: a first step of mixing a fatty acid or a fatty acid ester with an oil or fat to prepare a mixed oil; a second step of transesterifying the mixed oil using an sn-1,3-specific enzyme at a temperature 20°C lower to 20°C higher than the melting point of the mixed oil (typically 40-70°C); and a third step of removing by-products after the transesterification reaction by molecular distillation and fractionation.

[0011] EP 2508078 A1 discloses a method for preparing fats with a high content of POS triglycerides (P stands for palmitic acid and S stands for stearic acid) by enzymatic interesterification of vegetable fats or oils with fatty acids or fatty acid derivatives, distillation to remove the fatty acids or fatty acid derivatives, and subsequent fractionation. A preferred vegetable fat or oil is palm oil or a fraction thereof.

[0012] British Patent Specification No. 2205850(B) discloses a method for enzymatic interesterification with low water content, which comprises subjecting a reaction solution containing (a) a fat or oil and (b) a member selected from the group consisting of (i) another fat or oil, (ii) a fatty acid ester with a lower alcohol, and (iii) a fatty acid to the action of a lipase in the form of an immobilized enzyme catalyst, and adjusting the water concentration in the reaction solution during the reaction.

[0013] EP 245076 A2 discloses a method for preparing edible fats suitable for use in confectionery by rearrangement of unsaturated glyceride oils with a high oleic acid content using a lipase enzyme in the presence of saturated fatty acids or their esters ("oxidative hydrolysis reactants"). Preferably, 1 to 5 moles of oxidative hydrolysis reactant are used per mole of oil, more preferably 3 to 5 moles of oxidative hydrolysis reactant per mole of oil. In exemplified methods, the weight ratio of oxidative hydrolysis reactant to oil is 1:1 (Example 1), 1:2.5 (Example 3), and 2.4:2.5 (Example 4).

[0014] Therefore, known enzymatic methods for producing triglyceride compositions containing high SOS triglyceride content generally involve fractionation (dry fractionation or solvent fractionation) to achieve the appropriate composition of triglycerides. While solvent fractionation can produce a quality product, the cost of establishing the process is high, and the use of solvents can raise safety and customer acceptance issues. Dry fractionation is a more economically viable alternative, but is less efficient at separating SOS triglycerides. This requires product recycling, increasing process costs. More recycling also means the formation of more by-products, thereby reducing overall yield. More than one fractionation step may be required to achieve the desired quality. Therefore, both types of fractionation are complex and costly process steps, affecting process economics. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Patent No. 4,268,527(A) [Patent Document 2] European Patent No. 2251428(B1) [Patent Document 3] International Publication No. 2010 / 053244(A1) Pamphlet [Patent Document 4] Korean Patent No. 101344491(B1) [Patent Document 5] European Patent Application Publication No. 2508078(A1) [Patent Document 6] British Patent No. 2205850(B) [Patent Document 7] European Patent Application Publication No. 245076(A2) Summary of the Invention [Problem to be solved by the invention]

[0016] Therefore, there is a need for a more efficient enzymatic interesterification process that results in a triglyceride composition suitable for use as a cocoa butter equivalent or component thereof, with improved process economics, fewer process steps, and without the need to fractionate the triglyceride composition resulting from the enzymatic interesterification. It is also desirable to provide such an enzymatic interesterification process that can utilize high oleic oils as a starting material, thereby eliminating the need to use vegetable oils that already contain high levels of stearic and / or palmitic acid, such as shea stearin or palm oil. [Means for solving the problem]

[0017] The inventors have surprisingly found that by using certain reactants and conditions in an enzymatic interesterification process, it is possible to provide a triglyceride composition containing high levels of SOS triglycerides, which can be used as a cocoa butter equivalent or component thereof, without the need to fractionate the triglyceride phase, simply after separation of the fatty acid esters and free fatty acids.

[0018] The present invention therefore provides a method for increasing the SOS triglyceride content of a vegetable oil, where S represents stearic acid (C18:0) and palmitic acid (C16:0) residues and O represents oleic acid (C18:1) residues, comprising: a) i) an sn-1,3-specific lipase immobilized on a support; and ii) a vegetable oil containing at least 45% oleic acid fatty acid residues, based on the total C6-C24 fatty acid residues, and having an oleic acid content at the sn-2 position of at least 75% by weight relative to the total sn-2 fatty acid residues of the vegetable oil; iii) fatty alcohol esters of stearic acid, palmitic acid, or mixtures thereof, optionally mixed with stearic acid and / or palmitic acid; iv) Water and wherein the weight ratio of the fatty alcohol ester and optional stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1, and the water activity of the reaction environment is in the range of 0.1 to 0.6; b) heating the reaction environment to a temperature of 30-60°C to carry out transesterification, thereby obtaining a mixture comprising a triglyceride phase, fatty acid esters, and optionally free fatty acids; c) separating fatty acid esters and, if present, free fatty acids from the mixture obtained in step (b) to obtain a triglyceride composition; The present invention provides a method comprising:

[0019] The methods disclosed herein can produce a triglyceride phase having an SOS content of at least 65% by weight of the triglyceride phase. Furthermore, the undesired production of SSO triglycerides can be minimized, such that the triglyceride phase of the mixture obtained in step (b) has a weight ratio of SOS triglycerides to SSO triglycerides of at least 80:1. The undesired production of SSS triglycerides can also be minimized, such that the triglyceride phase of the mixture has an SSS triglyceride content of no more than 4% by weight of the triglyceride phase. These amounts and ratios are calculated by weight based on the triglyceride phase of the mixture. The triglyceride composition is suitable for use as a cocoa butter equivalent or component thereof.

[0020] The above process steps can be followed by any of the following three alternative steps d1), d2) and d3), in which the fatty acid esters and any free fatty acids from step c) are returned to the reaction environment of step a) for use as substrates for transesterification: d1) recycling the fatty acid esters separated in step (c) and, if present, the free fatty acids to the reaction environment; d2) if the fatty alcohol esters (iii) comprise fatty alcohol esters of stearic acid, optionally mixed with stearic acid, hydrogenating the fatty acid esters separated in step (c) and, if present, the free fatty acids, and recycling the hydrogenated fatty acid esters and the free fatty acids to the reaction environment, d3) combining the fatty acid esters separated in step (c) and, if present, the free fatty acids, a first fraction comprising stearic acid and / or palmitic acid esters, and optionally stearic acid and / or palmitic acid; a second fraction comprising oleic acid esters and optionally oleic acid; and recycling the first fraction to the reaction environment.

[0021] When the fatty alcohol ester (iii) comprises a fatty alcohol ester of stearic acid, optionally mixed with stearic acid, the process may also comprise a further step (e3) of hydrogenating the second fraction to provide the stearic acid ester and optionally stearic acid, and recycling the hydrogenated second fraction to the reaction environment.

[0022] The vegetable oil (ii) may be selected from high oleic rapeseed / canola oil, olive oil, high oleic soybean oil, high oleic sunflower oil, high oleic safflower oil, shea oil, or rapeseed oil, and / or fractions or combinations thereof. Preferably, the vegetable oil (ii) is selected from high oleic sunflower oil, high oleic safflower oil, and / or combinations or fractions thereof.

[0023] The method may further comprise using the triglyceride composition obtained in step (c) as a cocoa butter equivalent or ingredient thereof in the manufacture of chocolate or chocolate-like products.

[0024] The present invention also provides a triglyceride composition obtainable by the method.

[0025] The present invention also provides a chocolate or chocolate-like product comprising the triglyceride composition.

[0026] The present invention also provides a chocolate or chocolate-like product comprising a CBE.

[0027] definition As used herein, the term "vegetable" is to be understood as being derived from a plant or a single-cell organism. Thus, a vegetable oil or vegetable triglyceride is understood as a vegetable oil or vegetable triglyceride even if all fatty acids used to obtain said triglyceride or oil are derived from a plant or a single-cell organism.

[0028] As used herein, the term "oil" refers to a glyceride fat containing fatty acyl groups and does not denote a particular melting point. The term "fat" is used synonymously with "oil" herein.

[0029] As used herein, "oil derived therefrom" encompasses any processed oil, i.e., oil that has undergone processing. For example, the term includes any fraction of an oil, i.e., oil that has undergone fractionation.

[0030] As used herein, the term "fatty acid" includes free fatty acids and fatty acid residues in triglycerides.

[0031] Using the nomenclature, CX means that the fatty acid contains X carbon atoms, for example, a C16 fatty acid has 16 carbon atoms, a C18 fatty acid has 18 carbon atoms, and so on.

[0032] Using the nomenclature, CX:Y means that the fatty acid contains X carbon atoms and Y double bonds, for example, a C16:0 fatty acid has 16 carbon atoms and 0 double bonds, while a C18:1 fatty acid has 18 carbon atoms and 1 double bond.

[0033] As used herein, the abbreviations "SOS" and "SSO" encompass triglycerides (TAGs), where S denotes the saturated fatty acid esters (FAEs) palmitic acid (C16:0) or stearic acid (C18:0), and O denotes the unsaturated oleic acid (C18:1). Thus, "SOS" refers to a monounsaturated triglyceride with one O at the sn-2 position and one S (either a stearic or palmitic acid residue) at each of the sn-1 and sn-3 positions. "SSO" refers to an asymmetric monounsaturated triglyceride with one O at either the sn-1 or sn-3 position, one S at the sn-2 position, and one S at either the sn-1 or sn-3 position.

[0034] Cocoa butter equivalents are known in the art as edible fats and are typically composed of one or more vegetable fats, have composition and properties similar to cocoa butter, are compatible with cocoa butter and do not significantly affect the behavior of the chocolate in which they are used.

[0035] As used herein, "%" or "percentage" refers to weight percent, i.e., weight % (wt.%) or weight % (wt.-%), unless otherwise specified.

[0036] As used herein, the term "water activity" is defined as the partial vapor pressure of water in oil divided by the vapor pressure of pure water at the same temperature. At equilibrium, water activity is equal to the relative humidity. One simple and commonly used method for adjusting water activity is pre-equilibration with a saturated salt solution through the vapor phase in a closed container (RH Valivety, PJ Halling, AR Macrae, Reaction rate with suspended lipase catalyst shows similar dependence on water activity in different organic solvents, Biochim. Biophys. Acta (BBA) / Protein Struct. Mol. (1992) and HL Goderis, G. Ampe, MP Feyten, BL Fouwe, WM Guffens, SM Van Cauwenbergh, PP Tobback, Lipase-catalyzed ester exchange reactions in organic media with controlled humidity, Biotechnol. Bioeng. 30 (1987) 258-266). Another method is to inject the substrate with dry or humid air or nitrogen gas to remove or replenish water (K. Won, Sun Bok Lee, Computer-aided control of water activity for lipase-catalyzed esterification in solvent-free systems, Biotechnol. Prog. 17 (2001) 258-264 and A. E. V. Petersson, P. Adlercreutz, B. Mattiasson, A water activity control system for enzymatic reactions in organic media, Biotechnol. Bioeng. 97 (2007) 235-241).

[0037] As used herein, the expression "reaction environment" refers to the environment in which the enzymatic transesterification occurs and includes the sn-1,3-specific lipase immobilized on a support, water, reactants, i.e., vegetable oil, and stearic acid, a fatty alcohol ester of palmitic acid, or a mixture thereof, optionally mixed with stearic acid and / or palmitic acid.

[0038] As used herein, the term "feed" is defined as the sum by weight of the incoming substrates for the sn-1,3-specific lipase, i.e., vegetable oil (ii) and stearic acid, and / or fatty alcohol esters of palmitic acid, or mixtures thereof, optionally mixed with palmitic acid (iii).

[0039] As used herein, the expression "substrate ratio" refers to the weight ratio of stearic acid, a fatty alcohol ester of palmitic acid, or a mixture thereof (iii), optionally mixed with stearic acid and / or palmitic acid, to vegetable oil (ii). [Brief explanation of the drawings]

[0040]

[0013] FIG. 1 shows a process diagram of three different embodiments of the method of the present invention. [Figure 1A] It is shown how fatty acid esters (FAEs) and free fatty acids (FFAs) separated from the triglyceride phase after enzymatic interesterification are recycled to the reaction environment. [Figure 1B] We show how fatty acid esters (FAEs) and free fatty acids (FFAs) separated from the triglyceride phase after enzymatic interesterification are hydrogenated before being recycled to the reaction environment. [Figure 1C]The method is shown in which the fatty acid esters (FAEs) and free fatty acids (FFAs) separated from the triglyceride phase after the enzymatic interesterification are separated into a first fraction containing stearic and / or palmitic acid esters and optionally stearic and / or palmitic acid, and a second fraction containing oleic acid esters and optionally oleic acid, where the first fraction is recycled to the reaction environment and the second fraction is hydrogenated and also recycled to the reaction environment. DETAILED DESCRIPTION OF THE INVENTION

[0041] In describing the following embodiments, the present invention contemplates all possible combinations and permutations of the aspects disclosed above and the embodiments described below.

[0042] The present invention provides a method for increasing the SOS triglyceride content of vegetable oils, where S represents stearic acid (C18:0) and palmitic acid (C16:0) residues and O represents oleic acid (C18:1) residues, comprising: a) i) an sn-1,3-specific lipase immobilized on a support; and ii) a vegetable oil containing at least 45% oleic acid fatty acid residues, based on the total C6-C24 fatty acid residues, and having an oleic acid content at the sn-2 position of at least 75% by weight relative to the total sn-2 fatty acid residues of the vegetable oil; iii) fatty alcohol esters of stearic acid, palmitic acid, or mixtures thereof, optionally mixed with stearic acid and / or palmitic acid; iv) Water and wherein the weight ratio of the fatty alcohol ester and optional stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1, and the water activity of the reaction environment is in the range of 0.1 to 0.6; b) heating the reaction environment to a temperature of 30-60°C to carry out transesterification, thereby obtaining a mixture comprising a triglyceride phase, fatty acid esters, and optionally free fatty acids; c) separating the fatty acid esters and free fatty acids from the mixture obtained in step (b) to obtain a triglyceride composition; The present invention relates to a method, comprising:

[0043] The triglyceride phase of the mixture obtained in step (b) can have an SOS triglyceride content of at least 65% by weight of the triglyceride phase and a weight ratio of SOS triglycerides to SSO triglycerides of at least 80:1. The SSO content of the triglyceride phase can be 1.2% or less by weight of the triglyceride phase. The SSS content of the triglyceride phase can be 4% or less by weight of the triglyceride phase, preferably 3% or less by weight of the triglyceride phase, more preferably 2% or less by weight of the triglyceride phase, and most preferably 1.5% or less by weight of the triglyceride phase. Thus, the composition can be used as a cocoa butter equivalent or a component thereof without the need for further fractionation of the triglyceride phase.

[0044] The SOS triglyceride, SSO triglyceride, and SSS triglyceride content of the triglyceride phase can be determined using a non-aqueous reversed-phase HPLC method. A suitable method is described in "Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy," Causevic, A. et al., Journal of Chromatography A, Vol. 1652, 2021, pages 1-11.

[0045] The reaction environment for enzymatic interesterification includes an sn-1,3-specific lipase immobilized on a support. This enzyme transesterifies the vegetable oil at the sn-1 and sn-3 positions, replacing the fatty acid residues at these positions with fatty alcohol esters of stearic or palmitic acid and stearic and palmitic acid residues from any free fatty acids. In one or more embodiments, the sn-1,3-specific lipase (i) is a microbial lipase, such as a bacterial or fungal lipase. Preferably, the sn-1,3-specific lipase is derived from a fungal species, particularly Rhizopus oryzae, Thermomyces lanuginosus, or Rhizomucor miehei. Lipases derived from these species have been found to be particularly suitable for the interesterification process of the present invention due to their specificity, reaction rate, and robustness.

[0046] Immobilization of enzymes on supports is well known in the art, and immobilized sn-1,3-specific lipases are commercially available from various sources. It has been found that immobilization of 1,3-specific lipases on support materials improves enzyme performance and thus provides higher levels of SOS triglycerides. Various support materials are known, including various polymers and silica. In embodiments of the present invention, the support material on which the sn-1,3-specific lipase is immobilized is hydrophobic. One particularly useful enzyme is immobilized lipase DF "Amano" IM from Rhizopus oryzae (available from Amano Enzyme Co., Ltd.).

[0047] The vegetable oil (ii) used in the method of the present invention should have a fatty acid composition containing a relatively high level of oleic acid. The fatty acid composition of an oil or fat can be determined by gas chromatographic analysis of the methyl ester derivatives prepared by transesterification. Gas-liquid chromatography (GLC), also known as gas chromatography (GC), is a form of partition chromatography in which the mobile phase is gas and the stationary phase is liquid. The sample is volatilized during injection, and equilibrium is formed between the gas and liquid phases, resulting in immobilization on the inner wall of the column. If the sample contains different components, they will diffuse to different degrees into the liquid phase according to their individual equilibrium constants and move down the column at different rates. This results in different retention times and thus physical separation. The separated components emerge from the end of the column exhibiting concentration peaks, ideally in a Gaussian distribution. These peaks are detected by a flame ionization detector (FID), which converts the concentrations of the components in the gas phase into electrical signals that are amplified and passed to a continuous recording device, allowing the progress of separation to be monitored and quantified. A suitable method is IUPAC method 2.304.

[0048] The vegetable oil (ii) used as a transesterification reactant should contain at least 45% oleic (C18:1) fatty acid residues, based on the total C6-C24 fatty acid residues of the vegetable oil provided to the reaction environment. Preferably, the vegetable oil contains at least 50% oleic (C18:1) fatty acid residues, more preferably at least 60% oleic (C18:1) fatty acid residues, and most preferably at least 70% oleic (C18:1) fatty acid residues. One advantageous feature of the present invention is that vegetable oils with high levels of oleic acid not only at the sn-2 position but also at the sn-1 and sn-3 positions can be used to provide cocoa butter equivalents and their components.

[0049] Because the method of the present invention involves the use of sn-1,3-specific lipases, providing a composition with a high level of SOS requires that the vegetable oil already have a high level of oleic acid residues at the sn-2 position. Therefore, the vegetable oil should have an oleic acid content at the sn-2 position of at least 75% by weight, based on the total sn-2 fatty acid residues of the vegetable oil provided to the reaction environment. Preferably, the vegetable oil has an oleic acid content at the sn-2 position of at least 80% by weight, more preferably at least 85% by weight, even more preferably at least 90% by weight, and most preferably at least 95% by weight, based on the total sn-2 fatty acid residues of the vegetable oil provided to the reaction environment.

[0050] The oleic acid content at the sn-2 position of vegetable oils can be determined by a method involving cleavage of fatty acids at the sn-1 and sn-3 positions using the enzyme pancreatic lipase, followed by separation of the resulting sn-2 monoacylglycerols (MAGs) using TLC or NPLC, and finally analysis of the fatty acid methyl esters by gas chromatography. A suitable method is IUPAC official method 2.210: "Determination of fatty acids in the 2-position in the triglycerides of oils and fats," seventh ed., Standard Methods for the Analysis of Oils, Fats and Derivatives, Blackwell, Oxford, 1992.

[0051] In one or more embodiments, the vegetable oil (ii) is selected from high oleic rapeseed / canola oil, olive oil, high oleic soybean oil, high oleic sunflower oil, high oleic safflower oil, shea oil, or rapeseed oil, and / or fractions or combinations thereof. Preferably, the vegetable oil (ii) is selected from high oleic sunflower oil, high oleic safflower oil, and / or combinations or fractions thereof. These oils are particularly suitable due to their high oleic acid content and relatively low cost.

[0052] The reaction environment further includes a source of stearic acid and / or palmitic acid residues, which may be stearic acid, a fatty alcohol ester of palmitic acid, or a mixture thereof, optionally mixed with stearic acid and / or palmitic acid. The use of fatty alcohol esters of stearic acid and palmitic acid is advantageous because these esters have lower melting points than the corresponding fatty acids. This allows for lower temperatures to be used, improving enzyme stability and avoiding undesirable crystallization during processing. Preferably, the fatty alcohol ester of stearic acid or palmitic acid is an alkyl ester of stearic acid or palmitic acid, or a mixture thereof, more preferably a C1-C4 alkyl ester of stearic acid or palmitic acid, or a mixture thereof. Even more preferably, the ester is selected from the group consisting of methyl stearate, ethyl stearate, methyl palmitate, ethyl palmitate, and a mixture thereof. Most preferably, the ester is selected from the group consisting of methyl stearate, ethyl stearate, and a mixture thereof.

[0053] Stearic acid esters are preferred because SOS triglycerides, where S represents a stearic acid residue, are not available in large quantities from widely available vegetable oils such as palm oil. Fatty alcohol esters of stearic or palmitic acid can be used in admixture with stearic and / or palmitic acid as free fatty acids. However, preferably, these free fatty acids are not used.

[0054] The reaction environment also contains water. To obtain high levels of SOS triglycerides, the presence of water is necessary to provide sufficient enzyme activity. However, it has been found that if the water level is too high, more diacylglycerides and SSO triglycerides are formed in the triglyceride phase obtained in step (b) of the process. It has been found that to provide high levels of SOS triglycerides and low levels of diacylglycerides and SSO triglycerides, the water activity in step (a) should be in the range of 0.1 to 0.6, preferably in the range of 0.2 to 0.4.

[0055] The method of the present invention utilizes a particularly high ratio of fatty alcohol ester and optional stearic and / or palmitic acid (iii) to vegetable oil (ii). It has been found that a high substrate ratio of at least 4:1 results in greater incorporation of stearic and palmitic acids at the sn-1 and sn-3 positions of the triglycerides, thereby resulting in the production of more SOS triglycerides. Surprisingly, when combined with the other reaction conditions of the method of the present invention, a high substrate ratio does not increase the production of SSO and SSS triglycerides to unacceptably high levels. Thus, the triglyceride composition produced by the method of the present invention can be used as a cocoa butter equivalent or component thereof without the need for an additional fractionation step to separate SOS triglycerides from SSO and SSS triglycerides. Preferably, the substrate ratio is at least 5:1, more preferably at least 6:1, and even more preferably at least 7:1. Even higher substrate ratios, such as at least 8:1 or at least 9:1, are also contemplated within the present invention. At very high substrate ratios, the need to remove large amounts of fatty acid esters and free fatty acids in step (c) can reduce the efficiency of the process. Thus, the substrate ratio is typically at most 15:1, or at most 12:1, or at most 10:1.

[0056] The substrate ratio used in a particular method can vary depending on the nature of the vegetable oil (ii). For example, if the vegetable oil has a particularly high sn-2 oleic acid content, high levels of SOS triglycerides can be achieved by transesterification even when using a substrate ratio near the lower end of the specified range. In such cases, using a substrate ratio near the lower end of the specified range can be advantageous for process efficiency reasons. Thus, in one embodiment, the vegetable oil has an oleic acid content at the sn-2 position of at least 90% or at least 95% by weight, based on the total sn-2 fatty acid residues of the vegetable oil, and the weight ratio of the fatty alcohol ester and optional stearic and / or palmitic acid (iii) to the vegetable oil (ii) is in the range of 4:1 to 7:1. Conversely, if the vegetable oil has a lower sn-2 oleic acid content, a higher substrate ratio can be used to maximize the level of SOS triglycerides in the reaction product. Thus, in another embodiment, the vegetable oil has an oleic acid content at the sn-2 position in the range of 75% to 90% by weight or in the range of 75% to 85% by weight, based on the total sn-2 fatty acid residues of the vegetable oil, and the weight ratio of the fatty alcohol ester and optional stearic acid and / or palmitic acid (iii) to vegetable oil (ii) is at least 7: 1. The substrate ratio can therefore vary depending on the triglyceride content of the vegetable oil used and the desired product composition.

[0057] In all embodiments, the temperature to which the reaction environment is heated is in the range of 30-60° C. Preferably, the temperature is in the range of 35-45° C. The temperature of the reaction environment is selected to be high enough to provide good enzyme activity and thus high levels of SOS triglycerides, while being low enough to minimize the production of undesirable by-products such as SSO and SSS triglycerides. The use of low reaction temperatures is made possible by the use of fatty alcohol esters, which promote enzyme stability and generally have low melting points.

[0058] The method of the present invention can be carried out as a batch process, a fed-batch process, or a continuous process.

[0059] In a batch process, components (i)-(iv) are mixed in a single reactor for a specific reaction time until a desired yield of product is produced, and the enzyme is filtered off.

[0060] In a fed-batch process, the substrate is added to a batch reactor in small amounts at a time, rather than all at once.

[0061] In a continuous process, the enzyme is immobilized in a packed-bed reactor, the substrate is pumped through the reactor, and a product stream is removed from the reactor. In a continuous process, the flow rate of the feed (i.e., the combined weight of the vegetable oil (ii) and the fatty alcohol ester of stearic acid, palmitic acid, or a mixture thereof (iii), optionally mixed with stearic acid and / or palmitic acid) through the reactor is preferably in the range of 0.5 to 14, more preferably 2 to 10, and most preferably 4 to 8 g feed / g enzyme / h. Continuous processes have been found to be advantageous for easier recycling of fatty acid esters and fatty acids and for reduced migration of acyl groups within triglycerides. In an advantageous embodiment of the present invention, the fatty acid esters and free fatty acids remaining in the reaction mixture after transesterification are optionally treated and recycled to the reaction environment.

[0062] In one embodiment, the method further comprises step (d1), which comprises recycling the fatty acid esters separated in step (c) and, if present, free fatty acids, to the reaction environment. In this way, a more efficient reaction can be achieved, and the substrate ratio can be increased without the cost of providing additional reactants. Such a method is shown schematically in Figure 1A.

[0063] In an alternative embodiment in which the fatty alcohol ester (iii) comprises a fatty alcohol ester of stearic acid, optionally mixed with stearic acid, the method further comprises step (d2) comprising hydrogenating the fatty acid ester and, if present, the free fatty acid separated in step (c) and recycling the hydrogenated fatty acid ester and free fatty acid to the reaction environment. In this embodiment, the free oleic acid and oleic acid esters among the components separated in step (c) can be converted to stearic acid and its esters to further replenish the fatty alcohol ester of stearic acid and any stearic acid in the reaction environment. This embodiment is shown schematically in FIG. 1B.

[0064] In a further alternative embodiment, the method further comprises step (d3) comprising separating the fatty acid esters and, if present, free fatty acids separated in step (c) into a first fraction comprising stearic acid and / or palmitic acid esters, and optionally stearic acid and / or palmitic acid, and a second fraction comprising oleic acid esters and optionally oleic acid, and recycling the first fraction to the reaction environment. Optionally, in this embodiment, the second fraction can be hydrogenated to provide stearic acid esters and optionally stearic acid, which can be recycled to the reaction environment. This embodiment is shown schematically in FIG. 1C.

[0065] In an embodiment, the separated fatty acid esters and free fatty acids, i.e. the fatty acid esters and optionally free fatty acids separated in step (c), or the hydrogenated fatty acid esters and free fatty acids provided in step (d2), or the first fraction provided in step (d3), or the second fraction provided in step (d3) (either before or after hydrogenation), are bleached before being recycled to the reaction environment. Bleaching removes impurities and ensures good enzymatic life and thus efficient transesterification.

[0066] As noted above, one advantage of the process of the present invention is that the triglyceride composition produced has sufficiently high levels of SOS triglycerides and sufficiently low levels of SSO and SSS triglycerides to be used as a cocoa butter equivalent or component thereof without the need for a further fractionation step. Thus, in a preferred embodiment, the triglyceride composition obtained in step (c) is not subjected to a further fractionation step to provide a fractionated triglyceride composition having an increased content of SOS triglycerides.

[0067] The method may further comprise using the triglyceride composition obtained in step (c) as a cocoa butter equivalent or an ingredient thereof in the manufacture of chocolate or chocolate-like products. In a particularly preferred embodiment, the triglyceride composition obtained in step (c) is not subjected to a further fractionation step to provide a fractionated triglyceride composition having an increased content of SOS triglycerides, and the triglyceride composition is used as a cocoa butter equivalent or an ingredient thereof in the manufacture of chocolate or chocolate-like products.

[0068] The present invention also provides a triglyceride composition obtainable by the method of the present invention. The triglyceride composition can be used as a component of a cocoa butter equivalent, typically in an amount of 10 to 70% by weight. Accordingly, the present invention also provides a cocoa butter equivalent comprising 10 to 70% by weight of the triglyceride composition. The present invention also provides a chocolate or chocolate-like product comprising the triglyceride composition. [Example]

[0069] In embodiments where the amounts of individual positional isomers, such as SOS, SSO, and SSS, in the triglyceride phase can be determined, a non-aqueous reversed-phase HPLC method was used (Causevic, A. et al. "Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy," Journal of Chromatography A, Vol. 1652, 2021, pages 1-11). The method was also used to separate and quantify various free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides, and triacylglycerides. [Example]

[0070] Enzymatic interesterification was carried out in a continuous process setup to produce SOS from a feed containing high oleic safflower oil (HOSFO) and methyl stearate (Me-St). A column packed with 15 g of immobilized lipase DF "Amano" IM from Rhizopus oryzae (Amano Enzyme Co., Ltd.) was used, with the reaction conditions listed in the table below. A gear pump controlled the flow rate, delivering oil from the tank to the enzyme column. A heater controlled and set the temperature, heating the substrate tank, connections, and the water bath in which the enzyme column was located. [Table 1]

[0071] These results demonstrate that a high substrate ratio, combined with the additional process conditions of the present invention, can produce a high-quality product containing a high SOS content and a high SOS / SSO ratio. In contrast, when the substrate ratio is low, as in Experiments 6 and 7, an SOS content of more than 65% is not achieved. Furthermore, in Experiments 1-5, the SSO content of the triglyceride phase remains sufficiently low, resulting in an acceptable SOS / SSO ratio. In contrast, when the temperature and water activity are high, as in Experiment 8, SSO triglycerides are produced in greater amounts, resulting in a low SOS / SSO ratio.

Claims

1. 1. A method for increasing the SOS triglyceride content of a vegetable oil, wherein S represents stearic acid (C18:0) and palmitic acid (C16:0) residues and O represents oleic acid (C18:1) residues, comprising: a) i) a sn-1,3-specific lipase immobilized on a support; and ii) a vegetable oil containing at least 45% oleic acid fatty acid residues, based on the total C6-C24 fatty acid residues, and having an oleic acid content at the sn-2 position of at least 75% by weight relative to the total sn-2 fatty acid residues of said vegetable oil; iii) fatty alcohol esters of stearic acid, palmitic acid, or mixtures thereof, optionally mixed with stearic acid and / or palmitic acid; iv) Water and wherein the weight ratio of the fatty alcohol ester and optional stearic and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1, and the water activity of the reaction environment is in the range of 0.1 to 0.6; b) heating the reaction environment to a temperature of 30-60°C to carry out transesterification, thereby obtaining a mixture comprising a triglyceride phase, fatty acid esters, and optionally free fatty acids; c) separating the fatty acid esters and free fatty acids from the mixture obtained in step (b) to obtain a triglyceride composition; The method comprising:

2. 10. The method of claim 1, wherein the triglyceride composition obtained in step (c) is not subjected to a fractionation step, further providing a fractionated triglyceride composition having an increased content of SOS triglycerides.

3. 3. The method of claim 1 or 2, wherein the triglyceride phase of the mixture obtained in step (b) has an SOS triglyceride content of at least 65% by weight of said triglyceride phase.

4. 4. The method of any one of claims 1 to 3, wherein the triglyceride phase of the mixture obtained in step (b) has a weight ratio of SOS triglycerides to SSO triglycerides of at least 80:

1.

5. 5. The method of any of claims 1 to 4, wherein the triglyceride phase of the mixture obtained in step (b) has an SSO content of not more than 1.2% by weight of said triglyceride phase.

6. The method according to any one of claims 1 to 5, wherein the water activity of the reaction environment is in the range of 0.2 to 0.

4.

7. 7. The method according to any one of claims 1 to 6, wherein the vegetable oil comprises at least 50% by weight of oleic acid (C18:1) fatty acid residues, preferably at least 60% by weight of oleic acid (C18:1) fatty acid residues, more preferably at least 70% by weight of oleic acid (C18:1) fatty acid residues, based on total C6-C24 fatty acid residues.

8. 8. The method according to any one of claims 1 to 7, wherein the vegetable oil has an oleic acid content at the sn-2 position of at least 80% by weight, preferably at least 85% by weight, more preferably at least 90% by weight, and most preferably at least 95% by weight, based on the total sn-2 fatty acid residues of said vegetable oil.

9. 9. The method of any one of claims 1 to 8, wherein the vegetable oil is selected from high oleic rapeseed / canola oil, olive oil, high oleic soybean oil, high oleic sunflower oil, high oleic safflower oil, shea oil, or rapeseed oil, and / or fractions or combinations thereof.

10. 10. The method of any one of claims 1 to 9, wherein the vegetable oil is selected from high oleic sunflower oil, high oleic safflower oil, and / or fractions or combinations thereof.

11. The method according to any one of claims 1 to 10, wherein the sn-1,3-specific lipase is a microbial lipase such as a bacterial lipase or a fungal lipase.

12. 12. The method of claim 11, wherein the sn-1,3-specific lipase is derived from a fungal species selected from Rhizopus oryzae, Thermomyces lanuginosus, and Rhizomucor miehei.

13. The process according to any one of claims 1 to 12, wherein the reaction environment is heated to a temperature of from 35 to 45°C.

14. 14. The method of any one of claims 1 to 13, wherein the weight ratio of fatty alcohol ester and optional stearic acid and / or palmitic acid (iii) to vegetable oil (ii) is at least 5:1, such as at least 6:1 or at least 7:

1.

15. 15. The method according to any one of claims 1 to 14, wherein the vegetable oil has an oleic acid content in the sn-2 position of at least 90% by weight, preferably at least 95% by weight, based on the total sn-2 fatty acid residues of the vegetable oil, and the weight ratio of the fatty alcohol ester and optional stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is in the range of 4:1 to 7:

1.

16. The fatty alcohol ester of stearic acid or palmitic acid is an alkyl ester of stearic acid or palmitic acid or a mixture thereof, and preferably is a C ester of stearic acid or palmitic acid. １ -C ４ 16. The method according to any one of claims 1 to 15, wherein the hydroxybenzoate is an alkyl ester or a mixture thereof, more preferably selected from the group consisting of methyl stearate, ethyl stearate, methyl palmitate, ethyl palmitate, and mixtures thereof, and most preferably selected from methyl stearate and ethyl stearate.

17. 17. The method of any of claims 1 to 16, wherein the transesterification is carried out as a batch process, as a fed-batch process, or as a continuous process.

18. d1) recycling the fatty acid esters separated in step (c) and, if present, the free fatty acids to the reaction environment. The method of any one of claims 1 to 17, further comprising:

19. 19. The method of any one of claims 1 to 18, wherein the fatty alcohol ester (iii) comprises a fatty alcohol ester of stearic acid, optionally mixed with stearic acid, d2) hydrogenating the fatty acid esters separated in step (c) and, if present, the free fatty acids, and recycling the hydrogenated fatty acid esters and the free fatty acids to the reaction environment. The method further comprises:

20. d3) reacting the fatty acid esters separated in step (c) and, if present, the free fatty acids with a first fraction comprising stearic acid and / or palmitic acid esters, and optionally stearic acid and / or palmitic acid; a second fraction comprising oleic acid esters and optionally oleic acid; and recycling said first fraction to the reaction environment. The method of any one of claims 1 to 18, further comprising:

21. 21. The method of claim 20, wherein the fatty alcohol ester (iii) comprises a fatty alcohol ester of stearic acid, optionally mixed with stearic acid. e3) hydrogenating the second fraction to provide stearic acid esters and optionally stearic acid and recycling the hydrogenated second fraction to the reaction environment. The method further comprises:

22. the fatty acid esters and optionally free fatty acids separated in step (c), the hydrogenated fatty acid esters and free fatty acids provided in step (d2); the first fraction provided in step (d3), or the second fraction provided in step (d3), either before or after hydrogenation The process according to any of claims 18 to 21, wherein any of the above is bleached before being recycled to the reaction environment.

23. 23. The method of any of claims 1 to 22, further comprising using the triglyceride composition obtained in step (c) as a cocoa butter equivalent or ingredient thereof in the manufacture of chocolate or chocolate-like products.

24. A triglyceride composition obtainable by the method according to any one of claims 1 to 22.

25. 25. A cocoa butter equivalent comprising 10 to 70% by weight of the triglyceride composition of claim 24.

26. 25. A chocolate or chocolate-like product comprising the triglyceride composition of claim 24.

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