Enzymatic process for increasing 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 a stearic acid (C18: 0) residue and a palmitic acid (C16: 0) residue and O represents an oleic acid (C18: 1) residue, said method comprising: a) providing a reaction environment comprising: i) an sn-1, 3-specific lipase immobilized on a support, ii) a vegetable oil wherein, based on total C6-C24 fatty acid residues, the lipase is immobilized on the support; the vegetable oil comprises at least 45% oleic acid fatty acid residues, and wherein the vegetable oil has an oleic acid content in the sn-2 position of at least 75% by weight of the total sn-2 fatty acid residues of the vegetable oil, iii) an aliphatic alcohol ester of stearic acid, palmitic acid or a mixture thereof, optionally in combination with stearic acid and / or palmitic acid, and iv) water; wherein the weight ratio of the aliphatic alcohol ester and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4: 1; and wherein the reaction environment has a water activity in the range of 0.1 to 0.6; b) heating the reaction environment to a temperature of 30-60 DEG C for transesterification to obtain a mixture comprising a triglyceride phase, fatty acid esters and optionally free fatty acids; and c) separating the fatty acid ester and the free fatty acid from the mixture obtained in step (b) to obtain the triglyceride composition. The triglyceride phase can be used as a cocoa butter substitute for its component in chocolate or chocolate-like products.

Description

Technical field of the invention

[0001] The present invention relates to a method for increasing the SOS triglyceride content of vegetable oils and a triglyceride composition obtainable by the method. The triglyceride composition having a high content of SOS is used as a cocoa butter substitute or a component thereof in the manufacture of chocolate or chocolate-like products Background of the Invention

[0002] Triglycerides contain three fatty acid residues bound to a glycerol backbone. Their structure can be described using the "sn" notation, which stands for specific stereo numbering. In a Fischer projection of a natural L-glycerol derivative, the secondary hydroxyl group is shown to the left of C-2; the carbon atom above it then becomes C-1 and the one below becomes C-3. The prefix "sn" is placed before the stem name of the compound.

[0003]

[0004] Fischer projection of natural L-glycerol derivatives

[0005] The physical properties of triglycerides depend on the nature of the fatty acid residues and their position on the glycerol backbone. It would therefore be desirable to provide methods for altering the type and position of fatty acid residues in vegetable oils.

[0006] Triglyceride compositions with high SOS triglyceride content (wherein S represents stearic acid (C18:0) residues and palmitic acid (C16:0) residues, and O represents oleic acid (C18:1) residues) are particularly commercially valuable products. SOS triglycerides are present in high levels in cocoa butter and contribute to the unique physical properties of chocolate. Therefore, triglyceride compositions with high SOS triglyceride content can be used as cocoa butter substitutes or components thereof.

[0007] 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, which can be fractionated to provide shea stearin, which has a higher level of SOS triglycerides and is therefore useful in the production of cocoa butter substitutes. The availability of shea stearin depends on the supply of shea nuts.

[0008] An enzymatic interesterification process for producing a triglyceride composition having a high content of SOS triglycerides is known. This process provides a source of SOS triglycerides that is independent of the availability of shea butter.

[0009] US4268527A discloses a method for producing a cocoa butter substitute by transesterification of fats and oils of aliphatic alcohol esters of stearic acid and palmitic acid as a source of stearic acid residues and palmitic acid residues using sn-1,3-specific lipase. The process uses fats and oils having a high oleic acid content at the sn-2 position, but most of which already contain relatively high levels of stearic acid residues and palmitic acid residues at the sn-1 and sn-3 positions, such as fractionated palm oil and salfat. The water content of the reaction mixture is not more than 0.18% by weight. To obtain a satisfactory cocoa butter substitute, fatty acids and aliphatic alcohol esters thereof are distilled out, and the resulting composition is subjected to fractionation.

[0010] EP2251428B1 discloses a method for preparing a composition having a high concentration of SOS (50-80% by weight) from trioleic acid (e.g. high oleic sunflower oil), stearic acid and a sn-1,3-specific enzyme from Rhizopus oryzae. The temperature used is preferably between 65-80°C, and the transesterified triglycerides are fractionated by solvent fractionation.

[0011] WO 2010 / 053244A1 discloses a cocoa butter substitute prepared by enzymatic transesterification of various fats and oils with fatty acids or fatty acid esters using sn-1,3-specific enzymes, distillation of the reactants and fractionation of the separated products. The molar ratio of fat or oil to fatty acid or fatty acid ester is in the range of 1:2 to 1:6, the temperature for the reaction is between 40 and 50°C, and the water content of the oil is less than 0.02%.

[0012] KR101344491B1 discloses a method for producing an oil and fat composition for cocoa butter replacer (CBE) or cocoa butter improver (CBI), the method comprising a first step of preparing a mixed oil by blending fatty acids or fatty acid esters with oil or fat; a second step of transesterifying the mixed oil at a temperature of 20°C lower to 20°C higher than the melting point of the mixed oil (usually 40-70°C) using a sn-1,3-specific enzyme; and a third step of removing side reactants by molecular distillation and fractionation after the transesterification reaction.

[0013] EP2508078A1 discloses a method for preparing a fat having a high content of POS triglycerides (wherein P represents palmitic acid and S represents stearic acid) by enzymatic transesterification of vegetable fats or vegetable oils with fatty acids or fatty acid derivatives, distillation to remove the fatty acids or fatty acid derivatives and subsequent fractionation. The preferred vegetable fat or vegetable oil is palm oil or a fraction thereof.

[0014] GB2205850B discloses a low-water enzymatic transesterification method, which comprises subjecting a reaction liquid 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 of 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 liquid during the reaction.

[0015] EP245076A2 discloses a method for preparing edible fat suitable for use in candy, which method rearranges unsaturated glyceride oil and fat with high oleic acid content by using lipase in the presence of saturated fatty acids or their esters ("acidolysis reactants"). Preferably, 1 to 5 moles of acidolysis reactants are used per mole of oil, and more preferably 3 to 5 moles are used per mole. In the method, the weight ratio of acidolysis reactants to oil is 1:1 (Example 1), 1:2.5 (Example 3) and 2.4:2.5 (Example 4).

[0016] Therefore, known enzymatic processes for producing triglyceride compositions containing high SOS triglyceride content generally involve fractionation to obtain suitable triglyceride compositions (dry fractionation or solvent fractionation). Solvent fractionation can produce high-quality products, but the cost of setting up this method is very high, and the use of solvents may cause safety and customer acceptance issues. Dry fractionation is a more economical and feasible option, but is not so efficient in the separation of SOS triglycerides. This leads to the need to recycle the product, which increases the cost of the method. More recycling also means the formation of more byproducts, so the total yield also decreases. More than one fractionation step may be required to achieve the desired quality. Therefore, both types of fractionation are complex and expensive process steps that affect the method economy.

[0017] Therefore, there is a need for a more efficient enzymatic interesterification process that produces a triglyceride composition suitable for use as a cocoa butter substitute or a component thereof, has improved process economy and fewer process steps, and does not require fractionation of the triglyceride composition produced by the enzymatic interesterification. It is also desirable to provide such an enzymatic interesterification process that can utilize high oleic oil as a starting material and that therefore does not require the use of vegetable oils that already contain high levels of stearic acid and / or palmitic acid, such as shea stearin or palm oil. SUMMARY OF THE INVENTION

[0018] The inventors surprisingly found that by using specific reactants and conditions in an enzymatic interesterification process, a triglyceride composition containing high levels of SOS triglycerides can be provided and can be used as a cocoa butter substitute or a component thereof after simple separation of fatty acid esters and free fatty acids without the need for fractionation of the triglyceride phase.

[0019] Therefore, the present invention provides a method for increasing the SOS triglyceride content of vegetable oil, wherein S represents stearic acid (C18:0) residues and palmitic acid (C16:0) residues and O represents oleic acid (C18:1) residues, the method comprising:

[0020] a) providing a reaction environment, the reaction environment comprising:

[0021] i) a sn-1,3-specific lipase immobilized on a support,

[0022] ii) a vegetable oil, wherein the vegetable oil comprises at least 45% oleic fatty acid residues, based on total C6-C24 fatty acid residues, and wherein the vegetable oil has an oleic acid content at the sn-2 position of at least 75% by weight of the total sn-2 fatty acid residues of the vegetable oil,

[0023] iii) aliphatic alcohol esters of stearic acid, palmitic acid or mixtures thereof, optionally mixed with stearic acid and / or palmitic acid, and

[0024] iv) water;

[0025] wherein the weight ratio of the aliphatic alcohol ester and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1; and

[0026] wherein the water activity of the reaction environment is in the range of 0.1 to 0.6;

[0027] 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; and

[0028] c) separating fatty acid esters and free fatty acids, if present, from said mixture obtained in step (b) to obtain said triglyceride composition.

[0029] Method disclosed herein can produce a triglyceride phase having an SOS content of at least 65% by weight of the triglyceride phase. In addition, the production of unwanted SSO triglycerides can be minimized so that the triglyceride phase of the mixture obtained in step (b) has a weight ratio of at least 80:1 SOS triglycerides to SSO triglycerides. The production of unwanted SSS triglycerides can also be minimized so that the triglyceride phase of the mixture has an SSS triglyceride content of ≤4% by weight of the triglyceride phase. These amounts and ratios are calculated by the weight of the triglyceride phase of the mixture. The triglyceride composition is suitable for use as a cocoa butter substitute or a component thereof.

[0030] After the steps of the above method, any one of the three optional steps d1), d2) and d3) can be carried out, wherein the fatty acid esters and optionally free fatty acids from step c) are returned to the reaction environment of step a) in order to be used as substrates for transesterification:

[0031] d1) recycling the fatty acid esters separated in step (c) and the free fatty acids, if present, to the reaction environment;

[0032] d2) if the fatty alcohol ester (iii) comprises an fatty alcohol ester of stearic acid, optionally mixed with stearic acid, hydrogenating the fatty acid esters and, if present, the free fatty acids separated in step (c) and recycling the hydrogenated fatty acid esters and the free fatty acids to the reaction environment;

[0033] d3) separating the fatty acid esters and, if present, the free fatty acids separated in step (c) into:

[0034] a first fraction comprising stearate and / or palmitate and optionally comprising stearic acid and / or palmitic acid; and

[0035] a second fraction comprising oleate and optionally oleic acid; and

[0036] The first fraction is recycled to the reaction environment.

[0037] Wherein the aliphatic alcohol ester (iii) comprises an aliphatic alcohol ester of stearic acid optionally mixed with stearic acid, the process may further comprise a further step (e3) of hydrogenating the second fraction to provide stearic acid ester and optionally stearic acid, and recycling the hydrogenated second fraction to the reaction environment.

[0038] The vegetable oil (ii) may be selected from high oleic rapeseed oil / rapeseed oil, olive oil, high oleic soybean oil, high oleic sunflower oil, high oleic safflower oil, shea butter, 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 fractions or combinations thereof.

[0039] The method may further comprise using the triglyceride composition obtained in step (c) as a cocoa butter substitute or a component thereof in the manufacture of chocolate or chocolate-like products.

[0040] The present invention also provides a triglyceride composition obtainable by the method.

[0041] The present invention also provides a chocolate or chocolate-like product containing the triglyceride composition.

[0042] The present invention also provides a chocolate or chocolate-like product containing CBE.

[0043] definition

[0044] As used herein, the term "vegetable" should be understood as originating from a plant or a unicellular organism. Thus, if all fatty acids used to obtain the triglyceride or oil are of plant or unicellular organism origin, the vegetable oil or vegetable triglyceride should still be understood as a vegetable oil or vegetable triglyceride.

[0045] As used herein, the term "oil" refers to glyceride fats and oils containing fatty acid acyl groups, and does not imply any particular melting point. The terms "fat" and "oil" are used synonymously herein.

[0046] As used herein, the term "oil derived from..." includes any processed oil, that is, an oil that has been processed. For example, this term includes any fraction of an oil, that is, an oil that has been fractionated.

[0047] As used herein, the term "fatty acid" includes free fatty acids and fatty acid residues in triglycerides.

[0048] The nomenclature CX is used to indicate that the fatty acid contains X carbon atoms, for example a C16-fatty acid has 16 carbon atoms and a C18-fatty acid has 18 carbon atoms.

[0049] The nomenclature CX:Y is used to indicate 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.

[0050] As used herein, the abbreviations "SOS" and "SSO" include triglycerides (TAGs) in which S represents a saturated fatty acid ester (FAE) palmitate (C16:0) or stearate (C18:0), and O represents an unsaturated oleate (C18:1). Thus, "SOS" represents a monounsaturated triglyceride having an O at the sn-2 position and an S (stearic acid residue or palmitic acid residue) at each of the sn-1 and sn-3 positions. "SSO" represents an asymmetric monounsaturated triglyceride having an O at the sn-1 or sn-3 position, an S at the sn-2 position, and an S at the sn-1 or sn-3 position.

[0051] Cocoa butter substitutes are edible fats well known in the art, generally composed of one or more vegetable fats, having a composition and properties similar to cocoa butter, and can coexist with cocoa butter without significantly affecting the characteristics of the chocolate in which they are used.

[0052] As used herein, if nothing else is stated, "%" or "percentage" relates to percentage by weight, ie weight % (wt.%) or weight % (wt.-%).

[0053] As used herein, the expression "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. In equilibrium, the activity of water is equal to the relative humidity. A simple and commonly used method for adjusting the water activity is to pre-equilibrate with a saturated salt solution via the gas phase in a sealed 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 Fouwé, WM Guffens, SM Van Cauwenbergh, PP Tobback, Lipase-catalyzed ester exchange reactions in organic media with controlled humidity, Biotechnol. Bioeng. 30 (1987) pp. 258-266). Another method is the spraying of the substrate with dry or humid air or nitrogen for water removal or supply (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 AEV Petersson, P. Adlercreutz, B. Mattiasson, A wateractivity control system for enzymatic reactions in organic media, Biotechnol. Bioeng. 97 (2007) 235–241).

[0054] As used herein, the expression "reaction environment" is an environment in which enzymatic transesterification occurs, comprising the sn-1,3-specific lipase immobilized on a support, water, reactants, i.e., vegetable oil and aliphatic alcohol esters of stearic acid, palmitic acid or a mixture thereof, optionally mixed with stearic acid and / or palmitic acid.

[0055] As used herein, the expression "feed" is defined as the sum of the input by weight of the substrate for the sn-1,3-specific lipase, i.e. the sum of the weight of the vegetable oil (ii) and the fatty alcohol esters of stearic acid, palmitic acid or mixtures thereof (iii) optionally mixed with stearic acid and / or palmitic acid.

[0056] As used herein, the expression "substrate ratio" is the weight ratio of the aliphatic alcohol ester of stearic acid, palmitic acid or a mixture thereof (iii) optionally mixed with stearic acid and / or palmitic acid to the vegetable oil (ii). BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 depicts a flow chart of three different embodiments of the method of the present invention.

[0058] Figure 1A A method is described for recycling fatty acid esters (FAE) and free fatty acids (FFA) separated from the triglyceride phase after an enzymatic transesterification reaction to the reaction environment.

[0059] Figure 1B A process is described in which fatty acid esters (FAE) and free fatty acids (FFA) separated from the triglyceride phase after an enzymatic transesterification reaction are hydrogenated and then recycled to the reaction environment.

[0060] Figure 1C A method is described for separating fatty acid esters (FAE) and free fatty acids (FFA) separated from a triglyceride phase after an enzymatic transesterification reaction into a first fraction comprising stearate and / or palmitate and optionally comprising stearic acid and / or palmitic acid and a second fraction comprising oleate and optionally comprising oleic acid. The first fraction is recycled to the reaction environment and the second fraction is also recycled to the reaction environment after hydrogenation. DETAILED DESCRIPTION OF THE INVENTION

[0061] In describing the following embodiments, the present invention contemplates all possible combinations and permutations of the below described embodiments and the above disclosed aspects.

[0062] The present invention relates to a method for increasing the SOS triglyceride content of vegetable oil, wherein S represents stearic acid (C18:0) residues and palmitic acid (C16:0) residues, and O represents oleic acid (C18:1) residues, the method comprising:

[0063] a) providing a reaction environment, the reaction environment comprising:

[0064] i) a sn-1,3-specific lipase immobilized on a support,

[0065] ii) a vegetable oil, wherein the vegetable oil comprises at least 45% oleic fatty acid residues, based on total C6-C24 fatty acid residues, and wherein the vegetable oil has an oleic acid content at the sn-2 position of at least 75% by weight of the total sn-2 fatty acid residues of the vegetable oil,

[0066] iii) aliphatic alcohol esters of stearic acid, palmitic acid or mixtures thereof, optionally mixed with stearic acid and / or palmitic acid, and

[0067] iv) water;

[0068] wherein the weight ratio of the aliphatic alcohol ester and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1; and

[0069] wherein the water activity of the reaction environment is in the range of 0.1 to 0.6;

[0070] 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; and

[0071] c) separating the fatty acid esters and free fatty acids from the mixture obtained in step (b) to obtain the triglyceride composition.

[0072] The triglyceride phase of the mixture obtained in step (b) can have a 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% by weight of the triglyceride phase. The SSS content of the triglyceride phase can be ≤4% by weight of the triglyceride phase, preferably ≤3% by weight of the triglyceride phase, more preferably ≤2% by weight of the triglyceride phase, and most preferably ≤1.5% by weight of the triglyceride phase. Therefore, the composition can be used as a cocoa butter substitute or a component thereof without further fractionation of the triglyceride phase.

[0073] The SOS triglyceride, SSO triglyceride, and SSS triglyceride content of the triglyceride phase can be determined using non-aqueous reversed phase HPLC. Suitable methods are 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, pp. 1-11.

[0074] The reaction environment for enzymatic transesterification includes the sn-1,3-specific lipase fixed on a support. This enzyme affects transesterification on the sn-1 and sn-3 sites of vegetable oil, thereby replacing the fatty acid residues at these positions in the vegetable oil with stearic acid residues and palmitic acid residues from the aliphatic alcohol esters of stearic acid or palmitic acid and the optional free fatty acids. In one or more embodiments, sn-1,3-specific lipase (i) is a microbial lipase, such as a bacterial lipase or a fungal lipase. Preferably, sn-1,3-specific lipase is derived from fungal species, particularly Rhizopus oryzae, Thermomyces lanuginosus and Rhizomucormiehei species. The lipase derived from these species is found to be particularly suitable for the transesterification method of the present invention in terms of specificity, reaction rate and robustness.

[0075] The immobilization of enzymes on supports is well known in the art, and immobilized sn-1,3-specific lipases are commercially available from various suppliers. It has been found that the immobilization of sn-1,3-specific lipases on support materials can improve the performance of the enzyme, thereby providing higher levels of SOS triglycerides. Various support materials are known, such as various polymers and silica. In an embodiment of the present invention, the support material on which the sn-1,3-specific lipase is immobilized is hydrophobic. A particularly useful enzyme is immobilized Lipase DF "Amano" IM from Rhizopus oryzae (available from AmanoEnzyme).

[0076] 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 oil or fat can be determined by gas chromatography analysis of methyl ester derivatives prepared by transesterification. The technology of gas-liquid chromatography (GLC), also known as gas chromatography (GC), is a form of distribution chromatography, in which the mobile phase is a gas and the stationary phase is a liquid. The sample volatilizes during injection, and a balance is formed between the gas phase and the liquid phase, and the liquid phase is fixed to the inner wall of the column. When different components are contained in the sample, they diffuse into the liquid phase to different degrees according to their respective equilibrium constants, and therefore move down the column at different rates. This leads to different retention times, thereby leading to physical separation. The separated components appear from the end of the column, showing peaks of concentration, ideally in a Gaussian distribution. These peaks are detected by a flame ionization detector (FID, Flame Ionization Detector), which converts the concentration of the components in the gas phase into electrical signals, which are amplified and transmitted to a continuous recorder, so that the separation process can be monitored and quantified. A suitable method is IUPAC method 2.304.

[0077] The vegetable oil (ii) used as a reactant for transesterification should contain at least 45% oleic acid (C18:1) fatty acid residues based on the total C6-C24 fatty acid residues of the vegetable oil provided in the reaction environment. Preferably, the vegetable oil contains at least 50% oleic acid (C18:1) fatty acid residues, more preferably at least 60% oleic acid (C18:1) fatty acid residues, and most preferably at least 70% oleic acid (C18:1) fatty acid residues. A beneficial feature of the present invention is that vegetable oils that also have 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 substitutes and components thereof.

[0078] Since the method of the present invention involves the use of a sn-1,3-specific lipase, in order to provide a composition with a high level of SOS, it is required that the vegetable oil already has a high level of oleic acid residues at the sn-2 position. Therefore, in the reaction environment, the vegetable oil should have an oleic acid content of at least 75% by weight of the total sn-2 fatty acid residues of the provided vegetable oil at the sn-2 position. Preferably, in the reaction environment, the vegetable oil has an oleic acid content of at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% by weight of the total sn-2 fatty acid residues of the provided vegetable oil at the sn-2 position.

[0079] The oleic acid content of vegetable oils at the sn-2 position can be determined by cleaving the fatty acids at the sn-1 and sn-3 positions using pancreatic lipase, isolating the resulting sn-2 monoacylglycerols (MAGs) using TLC or NPLC, and finally analyzing 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", 7th edition, Standard Methods for the Analysis of Oils, Fats and Derivatives, Blackwell, Oxford, 1992.

[0080] In one or more embodiments, the vegetable oil (ii) is selected from high oleic rapeseed oil / rapeseed oil, olive oil, high oleic soybean oil, high oleic sunflower oil, high oleic safflower oil, shea butter or rapeseed oil and / or its fractions or combinations. Preferably, the vegetable oil is selected from high oleic sunflower oil, high oleic safflower oil and / or its fractions or combinations. These oils are particularly suitable because they have high oleic acid content and relatively low cost.

[0081] The reaction environment further comprises the source of stearic acid residue and / or palmitic acid residue, and it is the aliphatic alcohol ester of stearic acid, palmitic acid or its mixture optionally mixed with stearic acid and / or palmitic acid.The use of stearic acid and palmitic acid aliphatic alcohol ester is advantageous, because these esters have a lower fusing point than the corresponding lipid acid.Low temperature can be used like this, thereby improves the stability of enzyme, avoids undesirable crystallization in the process of processing simultaneously.Preferably, the stearic acid or palmitic acid aliphatic alcohol ester is the alkyl ester of stearic acid or palmitic acid ester or its mixture, more preferably the C ester of stearic acid or palmitic acid or its mixture. 1 -C 4 Alkyl esters. Even more preferably, the ester is selected from the group consisting of methyl stearate, ethyl stearate, methyl palmitate, ethyl palmitate and mixtures thereof. Most preferably, the ester is selected from the group consisting of methyl stearate, ethyl stearate and mixtures thereof.

[0082] Stearate is preferred because the SOS triglyceride that wherein S represents stearic acid residue can not be obtained in a large number from widely used vegetable oils such as palm oil. Stearic acid or palmitic aliphatic alcohol ester can be used in combination with stearic acid and / or palmitic acid as free fatty acids. However, preferably, these free fatty acids are not used.

[0083] The reaction environment also contains water. The presence of water is necessary to provide sufficient enzyme activity to obtain a high level of SOS triglycerides. However, when the level of water is too high, it has been found that diacylglycerides and SSO triglycerides are formed in large quantities in the triglyceride phase obtained in step (b) of the method. It has been found that in order to provide a high level of SOS triglycerides and a low level of diacylglycerides and SSO triglycerides, the water activity in step a) should be in the range of 0.1-0.6, preferably 0.2-0.4.

[0084] The ratio of aliphatic alcohol esters and optional stearic acid and / or palmitic acid (iii) to vegetable oil (ii) used in the method of the present invention is particularly high. It has been found that a high substrate ratio of at least 4:1 results in more combination of stearic acid and palmitic acid in the sn-1 and sn-3 positions of triglycerides, thereby producing more SOS triglycerides. Surprisingly, when a high substrate ratio is combined with other reaction conditions of the method of the present invention, the output of SSO triglycerides and SSS triglycerides is not increased to an unacceptably high level. Therefore, the triglyceride composition produced by the method of the present invention can be used as a cocoa butter substitute or a component thereof, without the need for a further fractionation step to separate SOS triglycerides from SSO triglycerides 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. The present invention also contemplates even higher substrate ratios, such as at least 8:1 or at least 9:1. At very high substrate ratios, the efficiency of the method may be reduced due to the need to remove a large amount of fatty acid esters and free fatty acids in step (c). Thus, the substrate ratio is typically at most 15:1, or at most 12:1, or at most 10:1.

[0085] The substrate ratio used in the ad hoc method can change according to the essence of vegetable oil (ii).For example, when vegetable oil has a particularly high oleic acid content at the sn-2 position, even if the substrate ratio used is close to the lower limit of the specified range, a high level of SOS triglyceride can be achieved by transesterification.In this case, due to the reason of method efficiency, the use of the substrate ratio close to the lower limit of the specified range may be advantageous.Therefore, in one embodiment, the vegetable oil has an oleic acid content of at least 90% or at least 95% by weight of the total sn-2 fatty acid residues of vegetable oil at the sn-2 position, and the weight ratio of aliphatic alcohol esters and optional stearic acid and / or palmitic acid (iii) to vegetable oil (ii) is in the range of 4:1 to 7:1.On the contrary, if vegetable oil has a lower oleic acid content at the sn-2 position, a higher substrate ratio can be used to maximize the level of SOS triglyceride in the reaction product. Thus, in another embodiment, the vegetable oil has an oleic acid content in the sn-2 position in the range of 75% to 90%, or in the range of 75% to 85%, based on the weight of the total sn-2 fatty acid residues of the vegetable oil, and the weight ratio of the aliphatic alcohol esters and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 7: 1. Thus, the substrate ratio may vary depending on the triglyceride content of the vegetable oil used and the desired product composition.

[0086] In all embodiments, the temperature of the reaction environment is heated to a range of 30-60° C. Preferably the temperature is in the range of 35-45° C. The temperature of the reaction environment is selected so that the temperature is high enough to provide good enzyme activity to produce high levels of SOS triglycerides, while the temperature is low enough to minimize the production of undesirable byproducts such as SSO triglycerides and SSS triglycerides. The use of lower reaction temperatures promotes enzyme stability, which can be achieved by the use of aliphatic alcohol esters, which generally have lower melting points.

[0087] The process of the present invention can be carried out as a batch process, a fed-batch process or a continuous process.

[0088] In a batch process, components (i)-(iv) are mixed in a reactor for a certain reaction time until the desired yield of product is achieved and then the enzyme is filtered off.

[0089] In a fed-batch process, the substrate is not added to the process reactor all at once, but a little at a time.

[0090] In continuous treatment, described enzyme is fixed in packed bed reactor, described substrate is pumped through reactor, and product stream is discharged from reactor.In continuous treatment, the flow rate of the charging by reactor (that is, the sum of the weight ratio of the aliphatic alcohol ester (iii) of vegetable oil (ii) and stearic acid, palmitic acid or its mixture optionally mixed with stearic acid and / or palmitic acid) is preferably in the scope of 0.5 to 14, more preferably 2 to 10, most preferably 4 to 8 g charging / g enzyme / h.It has been found that continuous treatment helps the simpler recovery of fatty acid ester and lipid acid, and reduces the migration of acyl group in triglyceride.In a preferred embodiment of the present invention, fatty acid ester and free fatty acids that stay in reaction mixture after transesterification are optionally processed and recycled to described reaction environment.

[0091] In one embodiment, the method further comprises a step (d1) comprising recycling the fatty acid esters separated in step (c) and the free fatty acids, if present, 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. Figure 1A Schematically described in .

[0092] In an optional embodiment, wherein the aliphatic alcohol ester (iii) comprises an aliphatic alcohol ester of stearic acid optionally mixed with stearic acid, the method further comprises a step (d2), which comprises hydrogenating the fatty acid ester separated in step (c) and the free fatty acids, if present, and recycling the hydrogenated fatty acid esters and free fatty acids to the reaction environment. In this embodiment, the free oleic acid and oleic acid esters in the components separated in step (c) can be converted into stearic acid and its esters to further supplement the aliphatic alcohol ester of stearic acid and optional stearic acid in the reaction environment. This process is Figure 1B Schematically described in .

[0093] In a further alternative embodiment, the process further comprises a step (d3), which comprises separating the fatty acid esters and, if present, free fatty acids separated in step (c) into a first fraction comprising stearates and / or palmitates and optionally stearic acid and / or palmitic acid, and a second fraction comprising oleates and optionally oleic acid, and recycling the first fraction to the reaction environment. Optionally, in this embodiment, the second fraction may be hydrogenated to provide stearates and optionally stearic acid, and recycled to the reaction environment. This embodiment is Figure 1C Schematically described in .

[0094] In one embodiment, the separated fatty acid esters and free fatty acids, i.e. the fatty acid esters and optional 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) (before or after hydrogenation), are bleached before recycling to the reaction environment. The bleaching removes impurities, ensuring better enzymatic life and therefore more effective transesterification.

[0095] As mentioned above, one advantage of the process of the present invention is that the triglyceride composition produced has a sufficiently high level of SOS triglycerides, and a sufficiently low level of SSO triglycerides and SSS triglycerides, to be used as a cocoa butter substitute or a component thereof without the need for a further fractionation step. Therefore, in a preferred embodiment, the triglyceride composition obtained in step (c) is not subjected to a fractionation step to provide a fractionated triglyceride composition having a further increased content of SOS triglycerides.

[0096] The method may further comprise using the triglyceride composition obtained in step (c) as a cocoa butter substitute or a component 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 fractionation step to provide a fractionated triglyceride composition having a further increased content of SOS triglycerides, and the triglyceride composition is used as a cocoa butter substitute or a component thereof in the manufacture of chocolate or chocolate-like products.

[0097] The present invention also provides a triglyceride composition that can be obtained by the method of the present invention. The triglyceride composition can be used as a component of a cocoa butter substitute, typically in an amount of 10-70% by weight. Therefore, the present invention also provides a cocoa butter substitute, which contains 10-70% by weight of the triglyceride composition. The present invention also provides a chocolate or chocolate-like product containing the triglyceride composition. Example

[0098] In embodiments, one may want to determine the amount of single positional isomers such as SOS, SSO, and SSS in a triglyceride phase, using non-aqueous reversed phase HPLC (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, pp. 1-11). The method is also used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides, and triacylglycerides.

[0099] Example 1

[0100] Enzymatic transesterification was performed in a continuous processing unit to produce SOS from a feed containing high oleic safflower oil (HOSFO) and methyl stearate (Me-St). A column filled with 15 g of fixed LipaseDF "Amano" IM (Amano Enzyme) from Rhizopus oryzae was used, which is described in the table below along with the reaction conditions. The flow was controlled by a gear pump, pumping the oil from the tank through the enzyme column. The temperature was controlled and set by a heater, and a water bath was heated at the substrate tank, connection, and enzymatic column locations.

[0101] S = C18:0 and C16:0, O = C18:1

[0102] As can be seen from these results, the use of a high substrate ratio, together with the further 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 relatively low, as in Experiments 6 and 7, an SOS content exceeding 65% is not achieved. In addition, in Experiments 1-5, the SSO content of the triglyceride phase remains sufficiently low, so that the SOS / SSO ratio is acceptable. In contrast, when the temperature and water activity are high, as in Experiment 8, SSO triglycerides are produced in higher amounts, resulting in a low SOS / SSO ratio.

Claims

1. A method for increasing the SOS triglyceride content of a vegetable oil, wherein S represents stearic acid (C18:0) residues and palmitic acid (C16:0) residues, and O represents oleic acid (C18:1) residues, the method comprising: a) providing a reaction environment, the reaction environment comprising: i) a sn-1,3-specific lipase immobilized on a support, ii) a vegetable oil, wherein the vegetable oil comprises at least 45% oleic fatty acid residues, based on total C6-C24 fatty acid residues, and wherein the vegetable oil has an oleic acid content at the sn-2 position of at least 75% by weight of the total sn-2 fatty acid residues of the vegetable oil, iii) aliphatic alcohol esters of stearic acid, palmitic acid or mixtures thereof, optionally mixed with stearic acid and / or palmitic acid, and iv) water; wherein the weight ratio of the aliphatic alcohol ester and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 4:1; and wherein 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; and c) separating the fatty acid esters and free fatty acids from the mixture obtained in step (b) to obtain the triglyceride composition.

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

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

4. A process according to any preceding claim, 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. The process according to any preceding claim, wherein the triglyceride phase of the mixture obtained in step (b) has a SSO content of ≤ 1.2% by weight of the triglyceride phase.

6. A method according to any preceding claim, wherein the water activity of the reaction environment is in the range of 0.2 to 0.

4.

7. The method according to any preceding claim, 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, and more preferably at least 70% by weight of oleic acid (C18:1) fatty acid residues, based on total C6-C24 fatty acid residues.

8. A method according to any preceding claim, wherein the vegetable oil has an oleic acid content at the sn-2 position of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% by weight of the total sn-2 fatty acid residues of the vegetable oil.

9. A method according to any preceding claim, wherein the vegetable oil is selected from high oleic rapeseed oil / canola oil, olive oil, high oleic soybean oil, high oleic sunflower oil, high oleic safflower oil, shea butter, or rapeseed oil, and / or fractions or combinations thereof.

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

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

13. A process according to any preceding claim, wherein the reaction environment is heated to a temperature of 35-45°C.

14. A method according to any preceding claim, wherein the weight ratio of the fatty alcohol ester and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is at least 5:1, such as at least 6:1 or at least 7:

1.

15. A method according to any preceding claim, wherein the vegetable oil has an oleic acid content in the sn-2 position of at least 90%, preferably at least 95%, by weight of the total sn-2 fatty acid residues of the vegetable oil, and the weight ratio of the fatty alcohol esters and optionally stearic acid and / or palmitic acid (iii) to the vegetable oil (ii) is in the range of 4:1 to 7:

1.

16. The method according to any preceding claim, wherein the aliphatic alcohol ester of stearic acid or palmitic acid is an alkyl ester of stearic acid or palmitic acid or a mixture thereof, preferably a C1-C4 alkyl ester of stearic acid or palmitic acid or a mixture thereof, more preferably selected from the group consisting of methyl stearate, ethyl stearate, methyl palmitate, ethyl palmitate and mixtures thereof, most preferably selected from methyl stearate and ethyl stearate.

17. A process according to any preceding claim, wherein the transesterification is carried out as a batch process, as a fed batch process or as a continuous process.

18. The method according to any preceding claim, further comprising: d1) recycling the fatty acid esters separated in step (c) and the free fatty acids, if present, to the reaction environment.

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

20. The method according to any one of claims 1 to 18, further comprising: d3) separating the fatty acid esters and, if present, free fatty acids separated in step (c) into: a first fraction comprising stearate and / or palmitate and optionally comprising stearic acid and / or palmitic acid; and a second fraction comprising oleate and optionally oleic acid; and The first fraction is recycled to the reaction environment.

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

22. The process according to any one of claims 18 to 21, wherein any of the following is bleached before being recycled to the reaction environment: the fatty acid esters and optionally the 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) before or after hydrogenation.

23. A method according to any preceding claim, further comprising using the triglyceride composition obtained in step (c) as a cocoa butter substitute or a component thereof in the manufacture of chocolate or chocolate-like products.

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

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

26. A chocolate or chocolate-like product comprising the triglyceride composition according to claim 24.

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