Process for producing interesterified fat products

Abstract

A method of producing a fat product is disclosed, the method comprising: (a) contacting a triglyceride and a mixture of one or more free fatty acids or free fatty acid esters with an enzyme composition comprising: (i) an additional ingredient, and (ii) two or more lipases; and (b) recovering the fat product.

Description

Technical Field

[0001] This invention relates to a method for preparing transesterified oil or fat products and their uses. Background Technology

[0002] Fats and oils are important components of food products such as confectionery, baked goods, or cooking products.

[0003] Transesterification, by randomizing the acyl groups of fatty acids, allows for the alteration of the physical properties of fats or oils to meet the requirements of various applications. Chemical transesterification typically requires a basic catalyst (such as sodium methoxide). However, in the presence of large amounts of free fatty acids, transesterification cannot be performed or cannot be completed completely. Therefore, additional deacidification steps (such as deodorization, distillation, or neutralization) are required prior to transesterification. Furthermore, chemical transesterification involves several undesirable side reactions, such as the formation of diacyl ketones, significant color formation (requiring post-bleaching and resulting in corresponding yield losses), and the degradation of nutritionally beneficial antioxidants. In terms of product quality, it has been concluded that chemical transesterification is inferior to its competing enzymatic processes because the use of chemical catalysts is hazardous and may generate trans fats.

[0004] Currently, chemical transesterification is primarily used when the use of enzymes is not economically feasible. Such cases involve oil or fat batches that are too small for enzymatic processes to be practical. This is because enzymes are typically used in packed bed columns with significant volumes, leading to substantial oil loss through batch-to-batch contamination when the batch size is sufficiently small. This is the problem addressed by the present invention—an improved enzymatic solution that reduces the required enzyme volume by significantly increasing the specific reaction rate of the immobilized enzyme.

[0005] Transesterification can also be catalyzed by enzymes. The presence of large amounts of free fatty acids may not affect enzyme-catalyzed reactions. However, after transesterification and before refining, these free fatty acids need to be removed through additional deacidification processes such as deodorization, distillation, or neutralization.

[0006] Immobilization of lipases has been known for many years. Immobilized enzyme products can be used for the enzymatic modification of organic compounds, such as in organic synthesis processes, transesterification of vegetable oils, and biodiesel production.

[0007] Enzyme immobilization involves attaching an enzyme protein to a carrier. The enzyme is immobilized on the carrier but retains its function, meaning it is not released or is substantially not released (washed out) into the liquid it comes into contact with. The most common immobilized enzymes are glucose isomerases used for isomerization reactions, and lipases used for, for example, transesterification of vegetable oils and organic synthesis.

[0008] The industrial applications of enzymes are often limited by their high cost and rapid inactivation. To improve their economic viability in industrial processes, enzymes are typically immobilized onto particles. Immobilization facilitates the reuse of enzymes and can positively impact their selectivity and stability. Immobilization research primarily focuses on methods to enhance enzyme transfer to the support and to ensure that enzymes retain their activity after immobilization.

[0009] For use in non-aqueous solutions, lipases can be immobilized on a variety of porous inorganic supports by absorbing the aqueous solution of the lipase into the pore volume of the support, by adsorbing it onto the surface of the support, or by a combination of adsorption and absorption followed by drying to remove water.

[0010] JP 5-292965A discloses an immobilized lipase and its preparation method.

[0011] WO 95 / 22606 (Pedersen et al.) describes an immobilization method based on a granulation process.

[0012] WO 99 / 33964 (Christensen et al.) describes an immobilization method in which an enzyme is applied to a microparticle porous carrier.

[0013] Immobilized enzymes are known to be used in continuous and batch enzymatic reactions in a variety of industrial applications, including wastewater treatment, pharmaceutical production, high-fructose corn syrup production, vegetable oil processing, and chemical synthesis.

[0014] The efficiency of transesterification processes for fats still needs improvement, especially when the feedstock contains high levels of free fatty acids. Furthermore, the quality of transesterified fat products needs to be improved in various aspects. Summary of the Invention

[0015] A method for producing a fat product, the method comprising: (a) contacting a mixture of triglycerides and one or more free fatty acids or free fatty acid esters with an enzyme composition comprising: (i) additional ingredients, and (ii) two or more lipases; and (b) recovering the oil or fat product.

[0016] These and other objects and advantages of the invention will become apparent from the following description. Preferred embodiments of the invention will be described in the following detailed description with reference to the accompanying drawings. These embodiments do not represent the full scope of the invention. Rather, the invention can be used in other embodiments. Therefore, the scope of the invention should be interpreted with reference to the claims herein.

[0017] definition Before disclosing and describing specific embodiments of the invention, it should be understood that the invention is not limited to the specific methods and materials disclosed herein, as they can vary to some extent. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be restrictive, as the scope of the invention will be defined only by the appended claims and their equivalents.

[0018] The following terms will be used in describing and claiming protection for this invention.

[0019] Unless the context clearly specifies otherwise, the singular forms “a” and “the” include plural indicators. Thus, for example, referring to “step” includes referring to one or more such steps.

[0020] As used herein, when referring to the quantity or amount of a material or its particular characteristic, "a large quantity" means an amount sufficient to provide the intended effect of the material or characteristic. In some cases, the exact extent of permissible deviation may depend on the specific context. Similarly, "substantially absent" means the absence of the identified element or reagent in the composition. In particular, elements identified as "substantially absent" are either completely absent from the composition or included only in a sufficiently small amount to not have an adverse effect on the composition.

[0021] This document uses the term "about" to refer to a value or parameter, including embodiments relating to that value or parameter itself. For example, a description referring to "about X" includes embodiment "X". When used in conjunction with a measurement value, "about" encompasses a range that covers at least the uncertainty associated with the method of measuring the particular value, and may include a range of two standard deviations positive or negative around the said value.

[0022] Similarly, a gene or polypeptide that is “derived from” another gene or polypeptide X includes that gene or polypeptide X.

[0023] It should be understood that the embodiments described herein include “consisting of… embodiments” and / or “substantially consisting of… embodiments.” As used herein, the word “comprise” or variations (such as “comprises” or “comprising”) are used in an inclusive sense, specifying the presence of the described features but not excluding the presence or addition of additional features in various embodiments, unless otherwise required by the context due to the language of expression or necessary meaning.

[0024] Concentration, amount, and other numerical data may be presented in range form throughout this document. It should be understood that such range form is used merely for convenience and brevity, and should be flexibly interpreted to include not only the values ​​explicitly stated as range limits, but also all individual values ​​or subranges covered within that range, as if each value and subrange were explicitly stated. For example, a weight range of approximately 1% to approximately 20% should be interpreted to include not only the explicitly stated concentration limit of 1% to approximately 20%, but also individual concentrations such as 2%, 3%, and 4%, and subranges such as 5% to 15%, 10% to 20%, etc.

[0025] Lipids: The term "lipid" refers to phospholipids and their derivatives, triglycerides and their derivatives, sterols, sterols, cholesterol, sphingolipids, ceramides, fatty acids, fatty alcohols, glycolipids, protein lipids, lipopolysaccharides, ether-lipids, polar and nonpolar lipids and their derivatives.

[0026] Esterification As used herein, the term “esterification” refers to a reaction that combines an organic acid (such as a fatty acid) with any alcohol or polyol (such as glycerol).

[0027] hydrolysis As used in this article, the term "hydrolysis" refers to the reaction of water with an ester to produce an acid and an alcohol.

[0028] alcoholysis As used herein, the term "alcohololysis" refers to the reaction of an ester with a monohydric alcohol (such as ethanol or butanol) or a polyhydric alcohol (such as glycerol) to produce an ester with different alkyl groups.

[0029] Acid hydrolysis: As used in this article, the term "acidolysis" refers to the reaction of an ester with an acid, resulting in the exchange of acyl groups.

[0030] Ester exchange: As used herein, the term "ester exchange" refers to the reaction of a primary ester with a secondary ester, resulting in mixing between the acyl and alcohol moieties.

[0031] Transesterification group function: As used herein, the term "transesterification" refers to any of the following reactions: alcoholysis, acidolysis, and transesterification.

[0032] synthesis: As used herein, the term “synthesis” or “synthesis of fatty acids” refers to the covalent bonding of a fatty acid at the sn-2 position of a glycerol ester, preferably by a one-step reaction selected from any of the following reactions: esterification, transesterification, alcoholysis, acidolysis, transesterification.

[0033] The term “alkyl” or “alkyl group” should be interpreted in its broadest sense to describe monovalent aliphatic compounds containing hydrocarbons.

[0034] The terms “glycerol derivative” and “glycerol ester” are used interchangeably herein to describe esters, ethers, and other derivatives of glycerol in which at least one hydrogen atom in any hydroxyl group attached to a C1, C2, or C3 carbon is substituted. Examples of glycerol derivatives are: tristearylglycerol (or tri-stearoylglycerol or tristearate, or glyceryl tristearate); 1,3-benzylallyltriol (or 1,3-O-benzylallyltriol); and 2-phosphoglycerate (or 2-phosphoglycerol), etc. If the substitution is on a carbon atom rather than on the oxygen atom of the hydroxyl group, the compound can be considered a derivative of glycerol (e.g., 1,2,3-nonadecantriol (C16H33CHOH-CHOH-CH2OH), which can also be considered 1-C-hexadecylglycerol).

[0035] Lipase: The terms "esterase," "lipase enzyme," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to enzymes in class EC3.1.1 as defined by enzyme nomenclature. It can possess lipase activity (triacylglycerol lipase, EC3.1.1.3), keratinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13), and / or wax ester hydrolase activity (EC3.1.1.50). For the purposes of this invention, lipase activity (i.e., lipase hydrolytic activity) can be determined by pNP assay using substrates having various chain lengths as described in the "Materials and Methods" section.

[0036] Parental lineage or parental lipase: The term "parent" or "parental lipase" refers to an esterase that has been altered to produce an enzyme variant. Parental esterases can be naturally occurring (wild-type) polypeptides, but can also be variants and / or fragments thereof.

[0037] Sequence identity: The correlation between two amino acid sequences is described by the parameter "sequence identity".

[0038] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) is used. J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) determines sequence identity between two amino acid sequences using algorithms such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000). Trends Genet.This was implemented using the Needle procedure (preferably version 5.0.0 or later) from [Trends in Genetics] 16: 276-277. The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The "Longest Identity" output (obtained using the -nobrief option) marked by Needle was used as the identity percentage and calculated as follows: (Identical residues × 100) / (Alignment length - Total number of vacancies in the alignment) Substrate: Suitable substrates according to the invention are a wide variety of vegetable oils and fats; rapeseed oil and soybean oil are commonly used. Substrates may be oils selected from the group consisting of: microbial oils, algae oils, low-erucic acid rapeseed oil, coconut oil, castor oil, coconut kernel oil, corn oil, cottonseed oil, linseed oil, fish oil, grapeseed oil, jatropha oil, jojoba oil, mustard oil, low-erucic acid rapeseed oil, palm oil, distillers' grains corn oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, and oils from halophytes, pennycress oil, camellia oil, jojoba oil, coriander seed oil, meadowfoam seed oil, seashore mallow oil, or any combination thereof.

[0039] Other crops, such as mustard, sunflower, canola, coconut, palm oil, and even algae and their derivatives (such as palm stearin), can also be used. The substrate can be crude or further processed (refined, bleached, and / or deodorized). Additionally, animal fats can be used, including animal fats, lard, poultry oil, aquatic animal oil, and waste vegetable and animal fats and oils (often referred to as yellow and brown fats). Suitable fats and oils can be pure triglycerides or a mixture of triglycerides and free fatty acids (often found in waste vegetable oils and animal fats). The types of fatty acids in the substrate include those naturally occurring as glycerides in vegetable and animal fats and oils. These fatty acids include (to name only): oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, and lauric acid. Minor components in crude vegetable oils are typically phospholipids, free fatty acids, and some glycerides (i.e., monoglycerides and diglycerides). When used in this article, the phrase “fatty acid residue” refers to a free or esterified fatty acid, such as in triglycerides, diglycerides, monoglycerides, or alkyl esters of fatty acids.

[0040] Preferably, the free fatty acid content of the substrate is less than 0.25%, less than 0.30%, less than 0.35%, less than 0.50%, less than 0.75%, less than 1.0%, less than 5.0%, less than 10.0%, less than 15.0%, less than 20.0%, less than 25.0%, less than 30.0%, less than 40%, or even less than 50.0%. Detailed Implementation

[0041] The present invention relates to a method for producing a fat product, the method comprising: (a) contacting a mixture of triglycerides and one or more free fatty acids or free fatty acid esters with an enzyme composition comprising: (i) additional ingredients, and (ii) two or more lipases; and (b) recovering the fat product.

[0042] The fatty acid groups in triacylglycerols can rearrange within a single oil (lactolation) or by exchanging fatty acid groups with those of other oils, the latter being called transesterification. As with other oil modification processes, the purpose of transesterification is to alter the melting characteristics of fats or fat blends to improve the functional properties of the product. The reaction requires the presence of a catalyst, and a basic catalyst is typically used. Sodium methoxide (also known as sodium methyl methoxide) is widely used, but metallic sodium is also employed.

[0043] The corrosive nature of chemical catalysts makes it crucial to reduce the water content and FFA content of the oil to be processed to the lowest practical value, as the hydrolysis and consumption of chemical catalysts lead to significant oil loss. It is also essential that process operators handle catalysts with extreme care, as exposure to catalysts can cause serious damage.

[0044] Transesterification has become an important tool for using fully hardened oils because it allows oil processors to minimize the trans fatty acid content in oil blends. The process can be applied to the production of fat blends for spreads and baking fats because it helps to tailor the melt properties of the fat to specific requirements. It is also used in the production of cocoa butter substitutes. Typically, two oils or fats containing fatty acids with different degrees of unsaturation (the least unsaturated being usually the fully hardened fat) are blended in carefully selected ratios according to a known scheme to obtain the desired melt profile of the transesterified oil or fat.

[0045] Directed transesterification is sometimes used in industry. In directed transesterification, it involves transesterification and simultaneous cooling to allow the forming saturated triacylglycerols to crystallize and thus shift the equilibrium towards the formation of more saturated triacylglycerols. This invention also applies to directed transesterification.

[0046] Specifically, the random transesterification process of this invention is proposed for use in this invention. Random transesterification aims to randomize the distribution of available fatty acid groups on the glycerol molecule and thereby alter the melting properties of the fat or oil blend. Random transesterification is a process used when two different oils or fats are mixed and transesterified.

[0047] Immobilized lipases have been successfully used to achieve industrial-scale transesterification. This eliminates the need for irritating chemicals and brings all the associated benefits. Lipases that will remain active at temperatures up to 80°C have been developed; however, elevated temperatures will degrade the enzyme and may result in higher FFA and diacylglycerol contents, which need to be removed through refining or may adversely affect product properties. Although the reuse of immobilized enzymes has been shown to be feasible, the cost of immobilized enzymes remains too high for general applicability to enzymatic transesterification at any scale. Smaller batches, in particular, are currently uneconomical because lipases in today's state-of-the-art solutions must be administered in extreme quantities to achieve complete randomization within acceptable timeframes or reactor sizes. Specifically, today's state-of-the-art solutions typically involve multiple sequential packed-bed reactors filled with enzymes, through which oil flows slowly. For a given system, there is an unavoidable retention volume of oil within the enzyme-filled bed, which can only be flushed out by introducing new oil. This effectively determines the minimum amount of oil required for a given batch to be transesterified economically. Despite the industry's long-standing demand for this technology, no enzymatic transesterification scheme has yet been available for processing small batches of oils without unacceptable intermixing between batches. Therefore, this invention relates to an improved immobilized lipase composition with significantly improved efficacy (measured as randomization rate). It is suitable for one or more reduced-size packed columns, or for applications using a significantly reduced dose of enzyme in single batches, where the immobilized lipase remains reusable. The significantly improved efficacy of the enzyme composition of this patent essentially achieves reduced or eliminated intermixing between batches in transesterification, enabling the use of benign enzymatic transesterification for a wider range of oils, fats, and blends currently transesterified, which in turn can include those blends typically produced in low-frequency, small-batch production. This will allow manufacturers to further reduce the use of chemical processes and deliver all the aforementioned related benefits.

[0048] Lipase The enzyme composition comprising two or more enzymes according to the present invention is a lipase, i.e., an enzyme capable of hydrolyzing carboxylic acid ester bonds to release the carboxyl group (EC 3.1.1). Lipases are enzymes classified under enzyme classification number EC 3.1.1- (carboxylic acid ester hydrolases) according to the recommendations of the International Union of Biochemistry and Molecular Biology (IUBMB) (1992). Therefore, lipases generally exhibit hydrolytic activity at the water / lipid interface against carboxylic acid ester bonds in substrates such as monoglycerides, diglycerides and triglycerides, phospholipids, thioesters, cholesterol esters, wax esters, keratin, cork resins, synthetic lipids, or other lipids mentioned in the context of EC 3.1.1. Lipases may have, for example, triacylglycerol lipase activity (EC 3.1.1.3; 1,3- position specific or nonspecific), phospholipase activity (A1 or A2; EC 3.1.1.32 or EC 3.1.1.4), esterase activity (EC 3.1.1.1) or keratinase activity (EC 3.1.1.74).

[0049] Suitable lipases (such as lipases) include those derived from bacteria or fungi. This also includes chemically modified mutants or protein-engineered mutants. Examples include lipases from the genera *Candida*, *C. antarctica* (e.g., lipases A and B described in WO 88 / 02775), and *C. rugosa* (*C. cylindracea*); lipases from the genera *Rhizomucor* and *R. miehei*; lipases from the genera *Hyphozyma* and *Humicola*; lipases from the genera *Thermomyces*, *T. lanuginosus* (*H. lanuginosa* lipases) (as described in EP 258 068 and EP 305 216); and lipases from the genus *Pseudomonas*, such as those from *P. alkaloidea*. Lipases from *Pseudoalcaligenes* (EP 218 272), *Pseudomonas cepacia* (EP 331 376), *Pseudomonas glumae*, *Pseudomonas stutzeri* (GB 1,372,034), *Pseudomonas fluorescens*, *Pseudomonas* species strains SD 705 (WO 95 / 06720 and WO 96 / 27002), and *Pseudomonas wisconsinensis* (WO 96 / 12012); lipases from *Bacillus*, such as those from *Bacillus subtilis* (Dartois et al. (1993), *Biochemica et Biophysica Acta*, 1131, ...). Lipases from *Bacillus stearothermophilus* (JP 64 / 744992) or *Bacillus pumilus* (WO 91 / 16422); lipases / phospholipases from *Fusarium oxysporum*; lipases from *F. heterosporum*; lysophospholipases from *Aspergillus foetidus*; phospholipase A1 from *A. oryzae*; lipases from *A. oryzae*; lipases / ferulinases from *A. niger*; and lipases from *A. tabingensis*.Lipases / ferulinases from *Aspergillus tubingensis*; lipases from *Aspergillus tubingensis*; lysophospholipases from *Aspergillus niger*; and lipases from *F. solani*.

[0050] When interacting with triglycerides as substrates, lipases can be site-specific (i.e., 1,3 specific) or non-specific.

[0051] In addition, many cloned lipases can be useful, including Penicillium camembertii lipase (described in Yamaguchi et al., (1991), Gene[Gene] 103, 61-67), Geotricum candidum lipase (Shimada, Y. et al., (1989), J. Biochem.[Journal of Biochemistry], 106, 383-388) and various Rhizopus lipases, such as R. delemar lipase (Hass, MJ et al., (1991), Gene[Gene] 109, 117-113), Rhizopus niveus lipase (Kugimiya et al., (1992), Biosci. Biotech. Biochem.[Biological Sciences, Biotechnology and Biochemistry] 56, 716-719) and Rhizopus oryzae (R. oryzae) lipase.

[0052] Other types of lipases, such as keratinase, can also be useful, for example, keratinase from Pseudomonas mendocina (WO 88 / 09367), Fusarium solani pisi (WO 90 / 09446), or H. insolens (US 5,827,719).

[0053] The enzyme can be, for example, an enzyme variant produced by recombinant technology. Examples are lipase variants, such as those described in WO 92 / 05249, WO 94 / 01541, EP 407 225, EP 260 105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079 and WO 97 / 07202.

[0054] Examples of commercially available lipases include Lipex™, Lipoprime™, Lipolase™, Lipolase™ Ultra, Lipozyme™, Palatase™, Novozym™ 435, Quara™, and Lipura. TM And Lecitase™ (all available from Novozymes A / S). Other commercially available lipases include Lumafast™ (Mendoza Pseudomonas lipase from Genencor International Inc.); Lipomax™ (Alcaligenes Pseudomonas pseudoalkanoate lipase from DSM / Genencor International Inc.; and Bacillus species lipases from Genencor. Additional lipases are available from other suppliers.

[0055] Enzymes can be added to the immobilization process in liquid form (such as enzyme-containing liquid (aqueous) media).

[0056] In a particular embodiment of the invention, the enzyme-containing liquid medium is a hydrophilic medium. In another particular embodiment, the liquid medium is aqueous. It may contain other organic or biological substances. Thus, it may be a fermentation broth or an enzyme concentrate that can be purified, for example, by ultrafiltration or by protein precipitation, separation, and redissolution in another aqueous medium. It may also be a substantially pure enzyme dissolved in an aqueous medium. In a particular embodiment of the invention, the enzyme-containing aqueous liquid is not subjected to costly water removal steps (such as evaporation) prior to immobilization, nor is any non-aqueous solvent, such as organic solvents like alcohols, such as polyethylene glycol and / or polypropylene glycol, added.

[0057] Other ingredients The enzyme composition contains one or more additional components selected from the group consisting of: polymers, silica, polysaccharides, alumina, controllable porosity glass (CPG), hybrid controllable porosity glass (hybrid CPG), filter aids, or water-soluble polyols.

[0058] Filter aid Filter aids are a class of essentially inert materials commonly used in filtration. The purpose of adding filter aids is to increase flow rate by reducing the compressibility of the filter cake and increasing its permeability. Similar improvements apply to this invention and to general packed beds.

[0059] According to the present invention, the organic filter aid can be a cellulose or lignocellulose material. Preferably, the organic filter aid is substantially insoluble in both water and oil under standard environmental conditions and at temperatures up to meaningful operating temperatures (20°C-100°C). Therefore, the organic filter aid can be an insoluble cellulose derivative.

[0060] In the embodiments, the organic filter aid is a wood product (such as sawdust) or is derived from wood chemistry. Preferably, the organic filter aid is a water-insoluble polysaccharide that may contain β(1→4) glycosidic bonds.

[0061] In a particularly preferred embodiment, the organic filter aid is cellulose (such as Filtracel from J. Rettenmaier & Sohne, Germany).

[0062] Organic filter aids can be mixed with other materials, as long as the mixture maintains the overall performance of the organic filter aid and can be used as a filter aid for oils / fats.

[0063] Organic filter aids can even be functionalized with silica, so that a portion of the siliceous material used in the particles of this invention is provided as an integrated part of the organic filter aid.

[0064] The enzyme composition of the present invention may contain an amount of organic filter aid in the form of 0.00001%-99% w / w, preferably 0.10%-90% w / w.

[0065] Siliceous materials The enzyme compositions of the present invention may contain siliceous materials. The siliceous materials may be amorphous or crystalline, or mixtures thereof, and may be naturally occurring (clay, talc, diatomaceous earth, sand, quartz, etc.) or synthetic (precipitated, gaseous, colloidal, silica gel, etc.) (usually purer).

[0066] Suitable siliceous materials are, for example, commercially available silica (e.g., Sipernat 22S, Sipernat 50, Sipernat 50s from Evonik, Germany), as well as zeolite, diatomaceous earth, and kaolin. In certain embodiments of the invention, the siliceous material is selected from the group consisting of silica, zeolite, and kaolin. The silica content of the siliceous material may be greater than 85% w / w, greater than 90%, greater than 95%, or greater than 98%. The siliceous material may be silica with an average particle size in the range of 1-2500 µm, such as 1-2000 µm, wherein the purity of the silica is greater than 90%. In another embodiment, the siliceous material is silica with an average particle size of 1-2500 µm and a purity greater than 95%.

[0067] The enzyme composition of the present invention may contain 0.00001%-99% w / w, preferably 0.10%-90% w / w, of a silicon material.

[0068] Water-soluble polyols Optionally, the soluble polyols used in this invention are carbohydrates or sugar alcohols, typically having a solubility of at least 0.1 g per 100 ml of water at ambient temperature (e.g., 20°C). The carbohydrates may consist of 1-20 monosaccharide units. This includes monosaccharides and oligosaccharides, such as disaccharides, trisaccharides, maltodextrin, and dextrin.

[0069] Monosaccharides can be hexoses (ketoses or aldoses), such as glucose, mannose, galactose, fructose, and combinations thereof. Disaccharides can include sucrose, maltose, trehalose, isomaltose, cellobiose, melibiose, primrose, rutinose, gentiobiose, and lactose, and combinations thereof. Trisaccharides can be maltotriose, raffinose, or combinations thereof.

[0070] Carbohydrates can be starch hydrolysates produced by hydrolysis (e.g., enzymatic hydrolysis) (e.g., having an average of 2-20 monomeric glucose units), such as dextrins of starch with DE 6-8 or maltodextrins with DE 20-23.

[0071] Sugar alcohols can be monomers, such as sorbitol or arabinol.

[0072] In a particularly preferred embodiment, the polyol is maltodextrin with a DE between 6 and 52. Maltodextrin with a DE higher than 20 is generally referred to as glucose syrup.

[0073] The amount of polyol (carbohydrate or sugar alcohol) used in the particles of the present invention may be higher than 2% by weight, for example, 2% to 50%, 2% to 30%, 5% to 25% or 7% to 25% by weight of the enzyme.

[0074] The invention is further described in the following paragraphs.

[0075] Paragraph 1. A method for producing randomly transesterified oil or fat products, the method comprising: I. Contacting a mixture of triglycerides and one or more free fatty acids or free fatty acid esters with an enzyme composition, the enzyme composition comprising: a. Other components; b. Two or more lipases; and II. Recycle the oil or fat product.

[0076] Paragraph 2. The method according to paragraph 1, wherein the two or more lipases comprise 1%-99% w / w of active enzyme proteins of 1,3-specific lipases from enzyme class EC 3.1.1.3, representing 1% to 99% of the total weight of the enzyme composition.

[0077] Paragraph 3. The method according to paragraph 1, wherein the two or more lipases comprise 1% to 99% w / w of nonspecific lipases from enzyme class EC 3.1.1.3 of the total weight of the enzyme composition.

[0078] Paragraph 4. The method according to paragraph 1, wherein the two or more lipases comprise 1% to 99% w / w of active enzyme proteins of 1,3-specific and non-specific lipases from enzyme class EC 3.1.1.3, representing 1% to 99% of the total weight of the enzyme composition.

[0079] Paragraph 5. According to the method described in paragraph 1, the additional component comprises 1%-99% w / w of an additional component selected from the group consisting of: polymer, silica, polysaccharide, alumina, controllable porosity glass (CPG), hybrid controllable porosity glass (hybrid CPG), filter aid, or water-soluble polyol.

[0080] Paragraph 6. The method according to paragraph 1, wherein the produced oil or fat product is obtained by transesterification, the transesterification having a randomization degree of at least 70%, preferably at least 75%, more preferably 80% to 100%, even more preferably 84% to 100%, and most preferably 90% to 100%.

[0081] Paragraph 7. According to the method described in Paragraph 1, the triglycerides and one or more free fatty acids or mixtures of free fatty acid esters are derived from (i) milk fat, cocoa butter, cocoa butter substitutes, illipe fat, canola oil, milk fat, monocarboxylic acid, phulawa butter, salsa, shea butter, Borneo butter, lard, lanolin, beef tallow, mutton tallow, animal fat, animal fat, low-erucic acid rapeseed oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, jatropha oil, flaxseed oil, mango kernel oil, meadowfoam seed oil, mustard oil, cow's hoof oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, shea butter, soybean oil, sunflower seed oil, tall oil, ailanthus oil, vegetable oil, marine oil convertible to plastic fat, marine oil convertible to solid fat, herring oil (menhaden oil), candlefish oil, cod liver oil, orange char oil, Pacific pile herd oil, sardine oil, whale oil, herring oil, 1,3-dipalmitoyl-2-monooleoglyceride (POP), 1(3)-palmitoyl-3(1)-stearoyl-2-monooleoglyceride (POSt), 1,3-distearate-2-monooleoglyceride (StOSt), triglycerides, diglycerides, monoglycerides, behenic acid triglycerides, tri-oil, tripalmitin, tri-stearin, palm oil, palm stearin, palm kernel oil, palm kernel stearin, medium-chain fatty acid triglycerides; (ii) partially hydrogenated oils of (i); (iii) fully hydrogenated oils of (i); or (iv) fractionated oils of (i).

[0082] Paragraph 8. The method according to any one of the preceding paragraphs, wherein one or more fatty acids comprise a carbon chain of about 4 to about 22 carbons.

[0083] Paragraph 9. The method according to any one of the preceding paragraphs, wherein one or more lipases and other components are packed into one or more columns.

[0084] Paragraph 10. The method according to any one of the preceding paragraphs, wherein one or more lipases and other components are used as freely movable particles in a stirred tank reactor.

[0085] Paragraph 11. The method according to paragraphs 10 and 11, wherein a combination of freely movable enzyme particles with a packed column or stirred tank reactor is used and connected in series and / or parallel.

[0086] Paragraph 12. The method according to paragraphs 10-11, wherein one or more unit operations, such as phase separation, deodorization, and decolorization, are performed before, after, or between processing steps.

[0087] Paragraph 13. The method according to any one of the preceding paragraphs, wherein the method is performed in a batch or continuous mode or a combination thereof.

[0088] Paragraph 14. The method according to any one of the preceding paragraphs, wherein the 1,3-specific lipase is derived from any of the following: Candida antarcticis A (CALA) Mucor mirifica, species of Pseudomonas, Rhizopus spp., Mucor javanica ( Mucor javanicus ), Rhizopus oryzae, Candida antarcticis B (CALB) Aspergillus niger, Penicillium chebula, and species of the genus Alcaligenes ( Alcaligenes sp. Burkholderia species ( Burkholderia sp. ), cottony thermophilic filamentous ... Chromobacterium viscosum ).

[0089] Paragraph 15. The method according to any one of the preceding paragraphs, wherein the nonspecific lipase is derived from any of the following: Candida antarcticis A (CALA) *Rhizopus oryzae*, *Pseudomonas* species, *Rhizopus sylvatica*, *Rhizopus javanica*, *Rhizopus oryzae*, *Candida antarcticis* B (CALB) Aspergillus niger, Penicillium chebula, species of Alcaligenes, species of Burkholderia, thermophilic filamentosa, or Myxobacterium spp.

[0090] Paragraph 16. The method according to any one of the preceding paragraphs, wherein the 1,3-specific lipase has at least 60% amino acid sequence identity with SEQ ID NO:1.

[0091] Paragraph 17. The method according to any one of the preceding paragraphs, wherein the 1,3-specific lipase has at least 70%, at least 80%, and at least 90% amino acid sequence identity with SEQ ID NO:1.

[0092] Paragraph 18. The method according to any one of the preceding paragraphs, wherein the 1,3-specific lipase has an amino acid sequence as shown in SEQ ID NO:1.

[0093] Paragraph 19. The method according to any one of the preceding paragraphs, wherein the nonspecific lipase has at least 60% amino acid sequence identity with SEQ ID NO: 2.

[0094] Paragraph 20. The method according to any one of the preceding paragraphs, wherein the nonspecific lipase has at least 70%, at least 80%, and at least 90% amino acid sequence identity with SEQ ID NO: 2.

[0095] Paragraph 21. The method according to any one of the preceding paragraphs, wherein the nonspecific lipase has an amino acid sequence as shown in SEQ ID NO: 2.

[0096] Paragraph 22. The method according to any one of the preceding paragraphs, wherein the composition is formulated as a powder, slurry, suspension, granules or pellets.

[0097] Paragraph 23. The method according to any one of the preceding paragraphs, wherein the two or more lipases comprise a 1,3-specific lipase having at least 95%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 1, and a non-specific lipase having at least 95%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 2.

[0098] Paragraph 24. The method described in paragraph 1, wherein two or more identical or different lipases are immobilized with one or more additional components, either identical or different.

[0099] Paragraph 25. Use of randomly transesterified oil or fat products produced according to any of the preceding paragraphs in food applications.

[0100] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0101] Example: The chemical is a commercial product of at least reagent grade. The lipase of *Thermophilus spp.* has the amino acid sequence shown in SEQ ID NO: 1. Candida antarctica Lipase A has the amino acid sequence shown in SEQ ID NO: 2.

[0102] Example 1: Randomized Reaction A total of 23.3 mg of active enzyme protein was added to a 100 mL square flask. As indicated in the results table, the liquid solutions of SEQ ID NO:1 and SEQ ID NO:2 were added as a blend, with the protein weight dose used as the indicated percentage. 3 g of polymethacrylate carrier material was added to the liquid enzyme blend. The mixture was freeze-dried at -18°C for 20 h.

[0103] The procedure is as follows: (1) Preheat palm oil extract. (2) Add 20 g of palm oil extract to the freeze-dried immobilized enzyme (as shown in the table) in a 100 mL square blue-cap flask. (3) Place the blue-cap flask in a shaking incubator at 70 °C and 250 RPM. (4) Sample the oil after 2, 4, and 20 h. (5) Inactivate the enzyme at 99 °C for 10 min. (6) Centrifuge in a benchtop centrifuge. PPP% was analyzed by GC method.

[0104] Table 1: PPP% via enzymatic transesterification As shown in Table 1, SEQ ID NO: 2 alone (0% SEQ ID NO: 1, 100% SEQ ID NO: 2) has a negligible effect on PPP formation. SEQ ID NO: 1 alone (100% SEQ ID NO: 1, 0% SEQ ID NO: 2) has a significant effect on PPP formation. Surprisingly, all mixtures of SEQ ID NO: 1 and SEQ ID NO: 2 tested here have a greater effect on PPP formation than SEQ ID NO: 1 alone.

Claims

1. A method for producing randomly transesterified oil or fat products, the method comprising: i. Contacting a mixture of triglycerides and one or more free fatty acids or free fatty acid esters with an enzyme composition, said enzyme composition comprising: a. Other components; b. Two or more lipases; and ii. Recycle the oil or fat product.

2. The method of claim 1, wherein the two or more lipases comprise 1%-99% w / w of active enzyme protein of 1,3-specific lipases from enzyme class EC 3.1.1.3, representing 1%-99% w / w of the total weight of the enzyme composition.

3. The method of claim 1, wherein the two or more lipases comprise 1%-99% w / w of active enzyme protein of nonspecific lipase from enzyme class EC 3.1.1.3, representing 1%-99% w / w of the total weight of the enzyme composition.

4. The method of claim 1, wherein the two or more lipases comprise 1%-99% w / w of active enzyme proteins of 1,3-specific and non-specific lipases from enzyme class EC 3.1.1.3, representing 1%-99% w / w of the total weight of the enzyme composition.

5. The method of claim 1, wherein the additional component comprises 1%-99% w / w of another component selected from the group consisting of: polymers, silica, polysaccharides, alumina, controllable porosity glass (CPG), hybrid controllable porosity glass (hybrid CPG), filter aids, or water-soluble polyols.

6. The method of claim 1, wherein the oil or fat product produced is obtained by transesterification, the transesterification having a randomization degree of at least 70%, preferably at least 75%, more preferably 80% to 100%, even more preferably 84% to 100%, and most preferably 90% to 100%.

7. The method according to any one of the preceding claims, wherein one or more fatty acids comprise a carbon chain of about 4 to about 22 carbons.

8. The method according to any one of the preceding claims, wherein the 1,3-specific lipase has at least 60% amino acid sequence identity with SEQ ID NO:

1.

9. The method according to any one of the preceding claims, wherein the 1,3-specific lipase has at least 70%, at least 80%, and at least 90% amino acid sequence identity with SEQ ID NO:

1.

10. The method according to any one of the preceding claims, wherein the 1,3-specific lipase has the amino acid sequence shown in SEQ ID NO:

1.

11. The method according to any one of the preceding claims, wherein the nonspecific lipase has at least 60% amino acid sequence identity with SEQ ID NO:

2.

12. The method according to any one of the preceding claims, wherein the nonspecific lipase has at least 70%, at least 80%, and at least 90% amino acid sequence identity with SEQ ID NO:

2.

13. The method according to any one of the preceding claims, wherein the nonspecific lipase has an amino acid sequence as shown in SEQ ID NO:

2.

14. The method of claim 1, wherein the two or more lipases are immobilized with the same or different one or more additional components.

15. Use of a randomly transesterified oil or fat product produced according to any one of the preceding claims in a food application.

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