WO2025083001A2

WO2025083001A2 - Method of producing interesterified fat product

Info

Publication number: WO2025083001A2
Application number: PCT/EP2024/079097
Authority: WO
Inventors: Rasmus Boeg Hansen; Jon Martin Persson; Pavle ANDRIC; Kim Borch; Hans Christian Holm
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-10-16
Filing date: 2024-10-16
Publication date: 2025-04-24
Anticipated expiration: 2026-04-16
Also published as: WO2025083001A3

Abstract

A method of producing fat product comprising: (a) contacting a mixture of triglycerides and free fatty acid(s) or free fatty acid esters with an enzyme composition comprising: (i) additional ingredient and (ii) two or more lipase; and (b) recovering the fat product.

Description

METHOD OF PRODUCING INTERESTERIFIED FAT PRODUCT

FIELD OF THE INVENTION

The present invention relates to a method of preparing interesterified oil- or fat product and the use thereof.

BACKGROUND OF THE INVENTION

Fats and oils are important ingredients of food products such as confectionery products, bakery products or culinary products.

Interesterification is performed in order to effect randomization of the fatty acid acyl groups so that the physical properties of the fat or oil can be altered to fulfill the requirements of various applications. Chemical interesterification often requires an alkaline catalyst, such as sodium methanolate. However, in the presence of a high amount of free fatty acids, the interesterification cannot be performed or not completely performed. Therefore, an extra deacidification step such as deodorization, distillation or neutralization is necessary prior to interesterification. Additionally, chemical interesterification involves several unwanted side-reactions such as formation of diacylketones, significant color formation requiring post-bleaching with corresponding yield losses, and degradation of nutritionally beneficial antioxidants. In terms of product quality, chemical interesterification has been concluded to be inferior to the competing enzymatic process as it is hazardous to work with chemical catalysts and can generate trans-fats.

Chemical interesterification is today mainly used for interesterification in cases where the enzyme is not economically viable to use. Such cases are when the oil or fat batch size is too small to feasibly use the enzymatic process. This is because the enzyme is typically employed in a packed bed column with a significant volume of enzyme, so loss of oil through inter-batch contamination becomes significant when batch sizes are sufficiently small. This is the problem that this invention solves - an improved enzymatic solution requiring reduced volume of enzyme by significantly increasing the specific reaction rate of the immobilized enzyme.

The interesterification reaction can also be catalyzed by an enzyme. The presence of a high amount of free fatty acids may not affect the reaction catalyzed by enzyme. However, after interesterification before refining, these need to be removed by an extra deacidification such as deodorization, distillation or neutralization.

Immobilization of lipolytic enzymes has been known for many years. An immobilized enzyme product may be used in enzymatic modification of an organic compound such as in organic synthesis processes, vegetable oil interesterification, biodiesel production etc.

Enzyme immobilization is the attachment of an enzyme protein onto a carrier on which the enzyme is fixed, yet functional, where the enzyme is not or largely not released into the liquid (washed out) to which it is contacted. The most commonly immobilized enzymes are glucose isomerase used for isomerization reactions, and lipase used for, e.g., interesterification of vegetable oils and organic synthesis.

The industrial use of enzymes is often limited by their high cost and rapid inactivation. To improve their economic feasibility in industrial processes, enzymes are generally immobilized onto a particle. Immobilization facilitates re-use of the enzymes and may positively affect the selectivity and stability of the enzyme. Immobilization research has mainly focused upon means to enhance the transfer of enzymes onto the support and means to ensure that the enzymes remain active after being immobilized.

For use in non-aqueous solutions, lipolytic enzymes, such as lipases, can be immobilized on a number of different porous, inorganic carriers by absorption of an aqueous solution of lipase into the pore volume of the carrier, or by adsorption to the surface of the carrier, or by a combination of both adsorption and absorption followed by water removal by drying.

JP 5-292965A discloses an immobilized lipase and a method for preparing it.

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

WO 99/33964 (Christensen et al.) describes an immobilization process wherein the enzyme is applied to a particulate porous carrier.

Immobilized enzymes are known to be used in both continuous and batch enzymatic reactions within a variety of industrial applications, including wastewater treatment, production of pharmaceuticals, high fructose corn syrup production, vegetable oil processing and synthesis of chemicals.

There remains a need to improve the efficiency of processes for interesterification of fats, particularly when the content of free fatty acid is high in the raw material. There is also a need to improve the quality of the interesterified fat product in different aspects.

SUMMARY OF THE INVENTION

A method of producing fat product comprising: (a) contacting a mixture of triglycerides and free fatty acid(s) or free fatty acid esters with an enzyme composition comprising: (i) additional ingredient and (ii) two or more lipase; and (b) recovering the oil or fat product.

These and still other objectives and advantages of the present invention will be apparent from the description which follows. In the detailed description below, preferred embodiments of the invention will be described in reference to the accompanying drawings. These embodiments do not represent the full scope of the invention. Rather the invention may be employed in other embodiments. Reference should therefore be made to the claims herein for interpreting the breadth of the invention. DEFINITIONS

Before particular embodiments of the present invention are disclosed and described, it is to be understood that this invention is not limited to the particular process and materials disclosed herein as such may vary to some degree. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be defined only by the appended claims and equivalents thereof.

In describing and claiming the present invention, the following terminology will be used.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a step" includes reference to one or more of such steps.

As used herein, "substantial" when used in reference to a quantity or amount of a material, or a specific characteristic thereof, refers to an amount that is sufficient to provide an effect that the material or characteristic was intended to provide. The exact degree of deviation allowable may in some cases depend on the specific context. Similarly, "substantially free of" or the like refers to the lack of an identified element or agent in a composition. Particularly, elements that are identified as being "substantially free of' are either completely absent from the composition or are included only in amounts which are small enough so as to have no deleterious effect on the composition.

Reference to “about” a value or parameter herein includes embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes the embodiment “X”. When used in combination with measured values, “about” includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value and can include a range of plus or minus two standard deviations around the stated value.

Likewise, reference to a gene or polypeptide that is “derived from” another gene or polypeptide X, includes the gene or polypeptide X.

It is understood that the embodiments described herein include “consisting” and/or “consisting essentially of” embodiments. As used herein, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments.

Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a weight range of about 1 percent to about 20 percent should be interpreted to include not only the explicitly recited concentration limits of 1 percent to about 20 percent, but also to include individual concentrations such as 2 percent, 3 percent, 4 percent, and sub-ranges such as 5 percent to 15 percent, 10 percent to 20 percent, etc.

Lipid: The term "lipid" refers to phospholipids and their derivatives, triglycerides and derivatives, sterols, stands, cholesterol, sphingolipids, ceramides, fatty acids, fatty alcohols, glycolipids, proteolipids, lipopolysaccharides, ether-lipids, polar and non-polar lipids and derivatives thereof.

Esterification: The term "esterification" as used herein, refers to a reaction for combining an organic acid such as a fatty acid with any alcohol or polyol such as a glycerol.

Hydrolysis: The term "hydrolysis" as used herein, refers to the reaction of water with an ester to produce an acid and an alcohol.

Alcoholysis: The term "alcoholysis" as used herein, refers to the reaction of an ester with a monohydric alcohol, such as ethanol, butanol, or polyhydric alcohol as glycerol, to produce an ester with a different alkyl group.

Acidolysis: The term "acidolysis" as used herein, refers to the reaction of an ester with an acid leading to the exchange of acyl groups.

Interesterification: The term "interesterification" as used herein, refers to the reaction of a first ester with a second ester leading to a mix up between the acyl and the alcohol moieties.

Transesterification: The term "transesterification" as used herein, refers to any of the following reactions: alcoholysis, acidolysis and interesterification.

Synthesis: The term "synthesis" or "synthesis of fatty acids" as used herein, refer to covalently binding a fatty acid at the sn-2 position of a glyceride, preferably by a one- step reaction selected from any one of the following reactions: esterification, interesterification, alcoholysis, acidolysis, transesterification.

The terms "alkyl" or "alkyl group" is to be construed according to its broadest meaning, to describe a univalent aliphatic compound comprising hydrocarbons.

The terms "glycerol derivatives" and "glycerides" are interchangeably used herein to describe esters, ethers and other derivatives of glycerol in which at least one of the hydrogens, of any of the hydroxyl group attached to the Cl, C2 or C3 carbons, is substituted. Examples of glycerol derivatives are: tristearoylglycerol (or tri-Ostearoyl glycerol or glycerol tristearate, or glyceryl tristearate);l,3-benzylideneglycerol (or 1 ,3- O-benzylideneglycerol); and glycerol 2- phosphate (or 2-phosphoglycerol) among others. If the substitution is on a carbon atom, rather than on the oxygen of the hydroxyl group than the compound may be considered as a derivative of glycerol (e.g., 1 ,2,3-nonadecanetriol for C16H33CHOH-CHOH-CH2OH, which may be also considered as 1-C-hexadecyl glycerol).

Lipases: The terms “Esterase”, “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipid esterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to an enzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may have lipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-ester hydrolase activity (EC3.1.1.50). For purposes of the present invention lipase activity (i.e. the hydrolytic activity of the lipase) may be determined with a pNP assay using substrates with various chain length as described in the “Materials & Methods”-section.

Parent or parent Lipases: The term “parent” or “parent Lipases” means an esterase to which an alteration is made to produce the enzyme variants. The parent esterase may be a naturally occurring (wild-type) polypeptide but may also be a variant and/or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Substrates: Suitable substrates in accordance with the present invention are a broad variety of vegetable oils and fats; rapeseed and soybean oils are commonly used. The substrate may be oil selected from the group consisting of: microbial oil, algae oil, canola oil, coconut oil, castor oil, coconut oil (copra oil), corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, distillers’ 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 oil from halophytes, pennycress oil, camelina oil, jojoba oil, coriander seed oil, meadowfoam oil, seashore mallow oil, or any combination thereof.

Other crops such as mustard, sunflower, canola, coconut, hemp, palm oil and even algae and derivates thereof, such as palm stearin, can also be used. The substrate can be of crude quality or further processed (refined, bleached and/or deodorized). Also, animal fats including tallow, lard, poultry, marine oil as well as waste vegetable and animal fats and oil, commonly known as yellow and brown grease can be used. The suitable fats and oils may be pure triglyceride or a mixture of triglyceride and free fatty acids, commonly seen in waste vegetable oil and animal fats. The type of fatty acids in the substrate comprises those naturally occurring as glycerides in vegetable and animal fats and oils. These include oleic acid, linoleic acid, linolenic acid, palmitic acid, steric acid, and lauric acid to name a few. Minor constituents in crude vegetable oils are typically phospholipids, free fatty acids and partial glycerides i.e., mono- and diglycerides. When used herein the phrase "fatty acid residues" refers to fatty acids, either free or esterified as in triglycerides, diglycerides, monoglycerides or fatty acid alkyl esters. Preferably, the free fatty acid content of the substrate is below 0.25%, below 0.30%, below 0.35%, below 0.50%, below 0.75%, below 1.0%, below 5.0%, below 10.0%, below 15.0%, below 20.0%, below 25.0%, below 30.0%, below 40%, or even below 50.0%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of producing fat product comprising: (a) contacting a mixture of triglycerides and free fatty acid(s) or free fatty acid esters with an enzyme composition comprising: (i) additional ingredient and (ii) two or more lipase; and (b) recovering the fat product.

The fatty acid groups in triacylglycerols can be rearranged within a single oil (intraesterification) or by exchange of fatty acid groups with those of other oils, the latter being known as interesterification. As is the case with the other oil modification processes, the purpose of interesterification is to alter the melting properties of the fat or fat blend in order to improve the functional properties of the product. The reaction requires the presence of a catalyst, and alkaline catalysts are generally used. In particular, sodium methylate, also referred to as sodium methoxide, is extensively used, but metallic sodium has also been used.

The aggressive nature of the chemical catalyst makes it essential that the moisture content and the FFA content of the oil to be processed are reduced to the lowest practical values, as hydrolysis and consumption of the chemical catalyst results in significant oil losses. It is also of crucial importance that the catalyst is handled with great care by the process operators, as exposure to it is likely to inflict serious injury.

Interesterification has now become an important tool in the use of fully hardened oils, as this enables an oil processor to minimize the content of trans fatty acids in an oil blend. The process finds application in the production of fat blends for use in spreads and of bakery fats, as it facilitates the tailoring of the melting properties of fats to specific requirements. It is also used in the production of cocoa-butter replacers. Typically two oils or fats comprising fatty acids of different degrees of unsaturation, the most unsaturated often being fully hardened fat, are mixed in carefully chosen ratios according to known recipes, thus yielding the desired melting profile of the interesterified oil or fat.

Sometimes, directed interesterification is used in the industry. In the directed interesterification, it involves interesterification and simultaneous cooling in order to crystallize the saturated triacylglycerols being formed and thus move the equilibrium towards the formation of more saturated triacylglycerols. The present invention is applicable for use also in directed interesterification.

Random interesterification in the present process is particularly proposed for use with this invention. Random interesterification, aims to randomize the distribution of the available fatty acid groups on the glycerol molecule and thereby to alter the melting properties of the fat or oil blend. Random interesterification is the process used when two different oils or fats are mixed and interesterified.

Immobilized lipases have been used successfully to achieve interesterification on an industrial scale. This eliminates the need for harsh chemicals with all the related benefits. Lipases have been developed that will retain activity at temperatures up to 80 °C, but increased temperatures will degrade the enzyme and can result in higher FFA and diacylglycerol contents, which either require removal by refining or can adversely affect the product properties. Although reuse of the immobilized enzyme has been shown to be feasible, the cost of the immobilized enzyme remains too high for general applicability of enzymic interesterification at any scale. Especially smaller batches are not currently economical because the lipase of today’s state-of- the-art solution must be dosed in extreme amounts to effect full randomization in an acceptable amount of time or reactor size. Specifically, today’s state of the art solution typically involves a number of sequential packed bed reactors filled with enzyme, with slow flow of oil through. For a particular system, there is an unavoidable holding volume of oil within the enzyme packed bed, which can only be flushed out by pushing in new oil. This effectively determines a minimum amount of oil for a certain batch of oil to be interesterified economically. Until today, no enzymatic solution has been available for processing of small batches of oil without unacceptable intermixing between batches, even though industry has voiced such a need for years. Therefore, the present invention relates to improved immobilized lipase compositions with significantly improved efficacy measured as rate of randomization. It is applicable for use in either one or more packed columns of reduced size or for application in single batches with much reduced dosage of the enzyme, wherein the immobilized lipase can still be reused. The significantly improved efficacy of the enzyme composition of this patent essentially effects reduced or eliminated intermixing between batches in interesterification enabling use of benign enzymatic interesterification on a broader spectrum of today’s interesterified oils, fats, and blends which can then include those blends that are typically produced infrequently and in small batches. This will allow producers to further reduce usage of the chemical process with all the associated benefits mentioned above.

Lipolytic Enzyme

The enzyme composition comprises two or more enzymes according to the invention is a lipolytic enzyme, i.e. an enzyme which is capable of hydrolyzing carboxylic ester bonds to release carboxylate (EC 3.1.1). The lipolytic enzyme is an enzyme classified under the Enzyme Classification number E.C. 3.1.1.- (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB). Thus, the lipolytic enzyme may exhibit hydrolytic activity, typically at a water/lipid interface, towards carboxylic ester bonds in substrates such as mono-, di- and triglycerides, phospholipids, thioesters, cholesterol esters, wax-esters, cutin, suberin, synthetic esters or other lipids mentioned in the context of E.C. 3.1.1. The lipolytic enzyme may, e.g., have triacylglycerol lipase activity (EC 3.1.1.3; 1 ,3-positionally specific or non-specific), phospholipase activity (A1 or A2; EC 3.1.1.32 or EC 3.1.1.4), esterase activity (EC 3.1.1.1) or cutinase activity (EC 3.1.1.74).

Suitable lipolytic enzymes (e.g. lipases) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples include lipases from Candida, C. Antarctica (e.g. lipases A and B described in WO 88/02775), C. rugosa (C. cylindracea), Rhizomucor, R. miehei, Hyphozyma, Humicola, Thermomyces, T. lanuginosus (H. lanuginosa lipase) as described in EP 258 068 and EP 305 216, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. glumae, P. stutzeri (GB 1 ,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422), lipase/phospholipase from Fusarium oxysporum, lipase from F. heterosporum, lysophospholipase from Aspergillus foetidus, phospholipase A1 from A. oryzae, lipase from A. oryzae, lipase/ferulic acid esterase from A. niger, lipase/ferulic acid esterase from A. tubingensis, lipase from A. tubingensis, lysophospholipase from A. niger and lipase from F. solani.

The lipase may be positionally site specific (i.e., 1 ,3 specific) or non-specific, upon interaction with triglycerides as substrates.

Furthermore, a number of cloned lipases may be useful, including the Penicillium camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the Geotricum candidum lipase (Shimada, Y. et al., (1989), J. Biochem., 106, 383-388), and various Rhizopus lipases such as a R. delemar lipase (Hass, M.J et al., (1991), Gene 109, 117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech. Biochem. 56, 716-719) and a R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be useful, e.g. cutinase from Pseudomonas mendocina (WO 88/09367), Fusarium solani pisi (WO 90/09446) or H. insolens (US 5,827,719).

The enzyme may be an enzyme variant produced, for example, by recombinant techniques. Examples are lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407225, 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.

Examples of commercially available lipases include Lipex™, Lipoprime™, Lipolase™, Lipolase™ Ultra, Lipozyme™, Palatase™, Novozym™ 435, Quara™, Lipura™ and Lecitase™ (all available from Novozymes A/S). Other commercially available lipases include Lumafast™ (Pseudomonas mendocina lipase from Genencor International Inc.); Lipomax™ (Ps. pseudoalcaligenes lipase from DSM/Genencor Int. Inc.; and Bacillus sp. lipase from Genencor enzymes. Further lipases are available from other suppliers.

The enzyme may be added to the immobilization process in liquid form, such as an enzyme containing liquid (aqueous) medium. The enzyme containing liquid medium is, in a particular embodiment of the present invention, a hydrophilic medium. In another particular embodiment, the liquid medium is aqueous. It may contain other organic or biological matter. Thus, it may be a fermentation broth or an enzyme concentrate solution obtainable by purifying a fermentation broth by e.g. ultra filtration or by protein precipitation, separation and re-dissolution in another aqueous medium. It may further be substantially pure enzyme dissolved in an aqueous medium. In a special embodiment of the present invention the enzyme containing aqueous liquid has not been subjected to costly processing steps prior to immobilization to remove water such as evaporation nor has it been subjected to addition of non-aqueous solvents, e.g. organic solvents such as alcohols, e.g. (poly)ethylene glycol and/or (poly) propylene glycol.

Additional ingredient

The enzyme composition comprises one or more additional ingredients selected from a group consisting of polymer, silica, polysaccharides, alumina, a controlled porosity glass (CPG), hybrid-controlled porosity glass (Hybrid CPG), a filter aid or water-soluble polyol.

Filter aid

Filter aids is a group of substantially inert materials that are typically used in filtration. An objective of adding filter aids is to improve the flow rate by decreasing cake compressibility and increasing cake permeability. Similar improvements apply for this invention and for packed beds in general.

An organic filter aid, according to the invention, may be a cellulosic or lignocellulosic material. Preferably, the organic filter aid is substantially insoluble in both water and oil at standard ambient conditions and up to meaningful operating temperatures (20°C - 100°C). Thus, the organic filter aid may be an insoluble cellulose derivative.

In an embodiment, the organic filter aid is a wooden product (such as saw dust), or chemically derived from wood. Preferably, the organic filter aid is a water-insoluble polysaccharide, which may comprise beta(1^4) glycosidic bonds.

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

The organic filter aid may be mixed with other materials, as long as the mixture retains the overall properties of the organic filter aid, and can be used as a filter aid in oils/fats.

The organic filter aid may even be functionalized with silica, so that a part of the siliceous material used in the particles of the invention is supplied as an integrated part of the organic filter aid.

The enzyme composition of the invention may comprise the organic filter aid in an amount of 0.00001-99% w/w, preferably 0.10-90% w/w. Siliceous material

The enzyme composition of the invention may comprise siliceous material. Siliceous material can be amorphous or crystalline or a mixture thereof, and it can be naturally occurring (clay, talc, diatomaceous earth, sand, quartz, etc.) or synthetic (precipitated, fumed, colloidal, silica gels, etc.) that is typically more purified.

Suitable siliceous materials are, for example, commercially available silicas (e.g. Sipernat 22S, Sipernat 50, Sipernat 50s from Evonik, Germany), but also zeolites, diatomaceous earth and kaolins. In a particular embodiment of the present invention the siliceous material is selected from the group consisting of silica, zeolite and kaolin. The siliceous material may have a silica content of greater than 85% w/w, greater than 90%, greater than 95%, or greater than 98%. The siliceous material may be silica with a mean particle size in the range of 1-2500 pm, such as 1-2000 pm, wherein the silica has a purity of more than 90%. In another embodiment, the siliceous material is a silica with a mean particle size of 1-2500 pm and a purity of more than 95%.

The enzyme composition of the invention may comprise the siliceous material in an amount of 0.00001-99% w/w, preferably 0.10-90% w/w.

Water-soluble polyol

The soluble polyol optionally used in the invention is a carbohydrate or a sugar alcohol, typically with a solubility of at least 0.1 g per 100 ml of water at ambient temperature (e.g. 20°C). The carbohydrate may consist of 1-20 monosaccharide units. This includes monosaccharides and oligosaccharides such as disaccharides, trisaccharides, maltodextrin and dextrin.

The monosaccharide may be a hexose, either a ketose or an aldose, such as glucose, mannose, galactose, fructose and combinations thereof. Disaccharides may include sucrose, maltose, trehalose, isomaltose, cellubiose, melibiose, primeverose, rutinose, gentiobiose and lactose and combinations thereof. The trisaccharide may be maltotriose, raffinose or a combination thereof.

The carbohydrate may be a starch hydrolysate produced by hydrolysis, e.g. enzymatic hydrolysis, for example with an average of 2-20 monomer glucose units, such as dextrin with DE 6-8 or maltodextrin with DE 20-23 of starch.

The sugar alcohol may be monomeric, e.g. sorbitol or arabitol.

In a particularly preferred embodiment, the polyol is maltodextrin having a DE between 6 and 52. Maltodextrins with a DE above 20 are often referred to as glucose sirup.

The amount of the polyol (carbohydrate or sugar alcohol) used in the particle of the invention may be above 2% by weight, e.g. 2 to 50%, 2 to 30%, 5 to 25% or 7 to 25% by weight of the enzyme.

The invention is further described in the following paragraphs. Paragraph 1 . A method of producing randomly interesterified oil or fat product comprising

I. contacting a mixture of triglycerides and free fatty acid(s) or free fatty acid esters with an enzyme composition comprising: a. additional ingredient b. two or more lipase; and

II. recovering the oil or fat product.

Paragraph 2. The method according to paragraph 1 , wherein two or more lipase comprises of 1-99 percent w/w of active enzyme protein of a 1 ,3 specific lipase from enzyme class EC 3.1.1.3, of total weight of enzyme composition.

Paragraph 3. The method according to paragraph 1 , wherein two or more lipase comprises of 1-99 percent w/w of active enzyme protein, of unspecific lipase from enzyme class EC 3.1.1.3, of total weight of enzyme composition.

Paragraph 4. The method according to paragraph 1 , wherein two or more lipase comprises of 1-99 percent w/w of active enzyme protein, of a 1 ,3 specific lipase and unspecific lipase from enzyme class EC 3.1.1 .3, of total weight of enzyme composition.

Paragraph 5. The method according to paragraph 1 , wherein additional ingredient comprises of 1-99 percent w/w of additional ingredient selected from a group consisting of a polymer, silica, polysaccharides, alumina, a controlled porosity glass (CPG), hybrid-controlled porosity glass (Hybrid CPG), a filter aid or water soluble polyol.

Paragraph 6. The method according to paragraph 1 , wherein the oil or fat product produced is obtained by interesterification which has a degree of randomization of at least 70 percent, preferably at least 75 percent, more preferably from 80 percent to 100 percent, even more preferably from 84 percent to 100 percent and most preferably from 90 percent to 100 percent.

Paragraph 7. The method according to paragraph 1 , wherein said mixture of triglycerides and free fatty acid(s) or free fatty acid esters is derived from (i) butterfat, cocoa butter, cocoa butter substitutes, illipe fat, kokum butter, milk fat, mowrah fat, phulwara butter, sal fat, shea fat, boriieo tallow, lard, lanolin, beef tallow, mutton tallow, tallow, animal fat, canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil, jatropha oil, linseed oil, mango kernel oil, meadowfoam oil, mustard oil, neat's foot oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, shea butter, soybean oil, sunflower seed oil, tall oil, tsubaki oil, vegetable oils, marine oils which can be converted into plastic fats, marine oils which can be converted into solid fats, menhaden oil, candlefish oil, codliver oil, orange roughly oil, pile herd oil, sardine oil, whale oils, herring oils, 1 ,3- dipalmitoyl-2- monooleine (POP), l(3)-palmitoyl-3(l)-stearoyl- 2-monooleme (POSt), l,3-distearoyl-2-monooleine (StOSt), triglyceride, diglyceride, monoglyceride, behenic acid triglyceride, trioleine, tripalmitine, tristearine, palm olein, palm stearin, palm kernel olein, palm kernel stearin, triglycerides of medium chain fatty acids; (ii) processed partially hydrogenated oils of (i); (iii) processed fully hydrogenated oils of (i); or (iv) fractionated oils of (i).

Paragraph 8. The method according to anyone of the preceding paragraphs, wherein one or more fatty acids comprise carbon chains from about 4 to about 22 carbons long.

Paragraph 9. The method according to anyone of the preceding paragraphs, wherein one or more lipases and additional ingredient are packed in one or more columns.

Paragraph 10. The method according to anyone of the preceding paragraphs, wherein one or more lipases and additional ingredient are employed as freely movable particles in a stirred tank reactor.

Paragraph 11. The method according to paragraphs 10 and 11 , where a combination of freely movable enzyme particles and packed column or stirred tank reactors are used and connected in series and/or in parallel.

Paragraph 12. The method according to paragraphs 10-11 , where one or more unit operations such as phase separation, deodorization, bleaching are performed before, after, or in between processing steps.

Paragraph 13. The method according to anyone of the preceding paragraphs, wherein said method is performed in batch or continuous mode or in a combination thereof.

Paragraph 14. The method according to anyone of the preceding paragraphs, wherein said 1 ,3 specific lipase is derived from any one of Candida antarctica A (CALA), Rhizomucor miehei, Pseudomonas sp., Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Candida antarctica B (CALB), Aspergillus niger, Penicillium camembertii, Alcaligenes sp., Burkholderia sp., Thermomyces lanuginosa or Chromobacterium viscosum.

Paragraph 15. The method according to anyone of the preceding paragraphs, wherein said unspecific lipase is derived from any one of Candida antarctica A (CALA), Rhizomucor miehei, Pseudomonas sp., Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Candida antarctica B (CALB), Aspergillus niger, Penicillium camembertii, Alcaligenes sp., Burkholderia sp., Thermomyces lanuginosa or Chromobacterium viscosum.

Paragraph 16. The method according to anyone of the preceding paragraphs, wherein said 1 ,3 specific lipases has at least 60 percent amino acid sequence identity to SEQ ID NO:1.

Paragraph 17. The method according to anyone of the preceding paragraphs, wherein said 1 ,3 specific lipases has at least 70 percent, at least 80 percent, at least 90 percent amino acid sequence identity to SEQ ID NO:1.

Paragraph 18. The method according to anyone of the preceding paragraphs, wherein said 1 ,3 specific lipases has the amino acid sequence as shown in SEQ ID NO:1.

Paragraph 19. The method according to anyone of the preceding paragraphs, wherein said unspecific lipases has at least 60 percent amino acid sequence identity to SEQ ID NO: 2.

Paragraph 20. The method according to anyone of the preceding paragraphs, wherein said unspecific lipases has at least 70 percent, at least 80 percent, at least 90 percent amino acid sequence identity to SEQ ID NO: 2.

Paragraph 21. The method according to anyone of the preceding paragraphs, wherein said unspecific lipases has the amino acid sequence as shown in SEQ ID NO: 2.

Paragraph 22. The method according to anyone of the preceding paragraphs, wherein the composition is formulated as powder, slurry, suspension, particles or pellet.

Paragraph 23. The method according to anyone of the preceding paragraphs, wherein said two or more lipases comprises a 1 ,3 specific lipase having at least 95 percent, e.g., at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or 100 percent amino acid sequence identity to SEQ ID NO:1 , and an unspecific lipase having at least 95 percent, e.g., at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or 100 percent amino acid sequence identity to SEQ ID NO: 2.

Paragraph 24. The method according to paragraphs 1 , wherein the two or more same or different lipases are immobilized with the same or different additional ingredient(s).

Paragraph 25. Use of a randomly interesterified oil or fat product produced according to any one of the preceding paragraphs in a food application. The present invention is further described by the following example that should not be construed as limiting the scope of the invention.

Examples:

Chemicals were commercial products of at least reagent grade. Thermomyces lanuginosa lipase has the amino acid sequence shown in SEQ ID NO: 1 . Candida antarctica A lipase has the amino acid sequence shown in SEQ ID NO: 2.

Example 1 : Randomization Reaction

A total of 23.3 mg active enzyme protein is added to a 100 mL square bottle. Liquid solutions of SEQ ID NO:1 and SEQ ID NO:2 are added in blends, as indicated in the results table, with protein weight dosage as basis for the percentages indicated. 3 g of polymethacrylate carrier material is added to the liquid enzyme blends. Freeze drying is done at -18C for 20h.

Procedure is as follows: (1) Pre-heat Palm Olein. (2) Add 20 g Palm Olein to the freeze- dried immobilized enzyme (as shown in table) in the squared 100 mL Bluecap flask. (3) Place bluecap flask in a shaking incubator at 70°C, 250 RPM. (4) Sample oil after 2, 4 and 20 h. (5) Inactivate enzymes at 99°C for 10 min. (6) Centrifuge in tabletop centrifuge. Analyze PPP% by GC method.

Table 1 : PPP% by enzymatic interesterification

From Table 1 , it is shown that the effect of SEQ ID NO: 2 alone (0% SEQ ID NO: 1 , 100% SEQ ID NO: 2) on PPP formation is negligible. SEQ ID NO: 1 alone (100% SEQ ID NO: 1 , 0% SEQ ID NO: 2) has a clear effect on PPP formation. Surprisingly, all mixes of SEQ ID NO: 1 and SEQ ID NO: 2 tested here had a greater effect on PPP formation than SEQ ID NO: 1 alone.

Claims

CLAIMS:

1. A method of producing randomly interesterified oil or fat product comprising i. contacting a mixture of triglycerides and free fatty acid(s) or free fatty acid esters with an enzyme composition comprising: a. additional ingredient; b. two or more lipase; and ii. recovering the oil or fat product.

2. The method according to claim 1 , wherein said two or more lipases comprise of 1-99 percent w/w of active enzyme protein of a 1 ,3 specific lipase from enzyme class EC 3.1.1.3, of total weight of enzyme composition.

3. The method according to claim 1 , wherein said two or more lipases comprise of 1-99 percent w/w of active enzyme protein of unspecific lipase from enzyme class EC 3.1 .1 .3, of total weight of enzyme composition.

4. The method according to claim 1 , wherein said two or more lipases comprise of 1-99 percent w/w of active enzyme protein of a 1 ,3 specific lipase and unspecific lipase from enzyme class EC 3.1.1.3, of total weight of enzyme composition.

5. The method according to claim 1 , wherein additional ingredient comprises of 1-99 percent w/w of additional ingredient selected from a group consisting of a polymer, silica, polysaccharides, alumina, a controlled porosity glass (CPG), hybrid-controlled porosity glass (Hybrid CPG), a filter aid or water soluble polyol.

6. The method according to claim 1 , wherein the oil or fat product produced is obtained by interesterification which has a degree of randomization of at least 70 percent, preferably at least 75 percent, more preferably from 80 percent to 100 percent, even more preferably from 84 percent to 100 percent and most preferably from 90 percent to 100 percent.

7. The method according to anyone of the preceding claims, wherein one or more fatty acids comprise carbon chains from about 4 to about 22 carbons long.

8. The method according to anyone of the preceding claims, wherein said 1 ,3 specific lipases has at least 60 percent amino acid sequence identity to SEQ ID NO:1.

9. The method according to anyone of the preceding claims, wherein said 1 ,3 specific lipases has at least 70 percent, at least 80 percent, at least 90 percent amino acid sequence identity to SEQ ID NO:1.

10. The method according to anyone of the preceding claims, wherein said 1,3 specific lipases has the amino acid sequence as shown in SEQ ID NO:1.

11. The method according to anyone of the preceding claims, wherein said unspecific lipases has at least 60 percent amino acid sequence identity to SEQ ID NO: 2.

12. The method according to anyone of the preceding claims, wherein said unspecific lipases has at least 70 percent, at least 80 percent, at least 90 percent amino acid sequence identity to SEQ ID NO: 2.

13. The method according to anyone of the preceding claims, wherein said unspecific lipases has the amino acid sequence as shown in SEQ ID NO: 2.

14. The method according to claim 1, wherein said two or more lipases are immobilized with the same or different additional ingredient(s).

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