Method for selectively hydrolyzing diglyceride in oil / fat by using candida antarctica lipase B

Abstract

Disclosed is a method of reducing and / or removing diglyceride content in an oil without substantial transesterification of triglycerides, the method comprising the steps of: providing an oil or fat; and hydrolyzing the monoglycerides and diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention relates to a method for the enzymatic removal and / or reduction of diglycerides from oil. Background Art

[0002] Oils and fats consist of complex mixtures of triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acids and other minor components. The crystallization of these mixtures depends on the characteristics of the TAGs (combination of fatty acids, their chain length, their degree of unsaturation, etc.) and the interaction of these TAGs with each other. Regarding the presence of DAGs, previous studies have shown that they have a significant effect on the physical properties of oils and fats. These effects vary in crystallization rate, polymorphic changes, melting point, crystal size and habit (Siew, 2001).

[0003] In some oils extracted from oilseeds, the effect of DAG is less pronounced because DAG is usually present only in small amounts. However, in, for example, palm oil, rice bran oil, shea butter and olive oil, large amounts of DAG are typically present after extraction, and the quality of these oils is affected if DAG is present.

[0004] The presence of these diglycerides in the main product (= triglycerides) is disadvantageous, since the diglycerides can have an adverse effect on the product properties of the triglycerides. The diglycerides are also reactive during parts of the refining process, especially during deodorization, and under certain conditions (e.g. glycidyl esters) tend to form by-products that are subject to food safety regulation.

[0005] Many methods for solving this problem are disclosed in the literature. These methods mainly focus on removing diglycerides from a mixture containing triglycerides, wherein the enzymatic conversion of diglycerides is carried out by using enzymes specifically for hydrolyzing diglycerides into glycerol and free fatty acids.

[0006] JP 62 / 51997, for example, discloses a method for improving fats, wherein a mixture containing at least 70 weight percent triglycerides and not less than 2 weight percent diglycerides is contacted with an enzyme particularly for partial glycerides in the presence of a small amount of water.

[0007] A similar process is disclosed in JP 62 / 61590. The hydrolysis of the partial glycerides is followed by an esterification process using a 1,3-specific enzyme.

[0008] A similar process is disclosed in JP 62 / 287, wherein a lipase produced by Penicillium cyclopium ATCC 34613 is used for the hydrolysis of mono- and / or diglycerides.

[0009] Therefore, the prior art teaches methods for reducing or removing the diglyceride content in palm oil and other edible oils by enzymatic reactions. These methods rely on the hydrolysis of diglycerides with specific diglyceride hydrolyzing lipases during the formation of free fatty acids and glycerol. The free fatty acids can then be removed by different methods such as vacuum distillation, saponification or fractionation.

[0010] Use specific diacylglycerol hydrolase to cause the formation of free fatty acids.Usually these free fatty acids must be removed from oil.But under current market conditions, due to various reasons, fatty acid distillates are relatively valuable, and the combination of improving TAG oil quality by producing FFA (normally distillate) in addition can bring economical combination improvement.The other advantage of reducing MAG and DAG is that the quality of distillate can even be expected to significantly improve with respect to standard quality, because the hydrolysis of MAG and DAG causes the FFA concentration in the distillate to increase.This means that such distillate will need less extensive processing, thereby saves energy consumption.In order to bring such economic benefit, need enough cheap lipase, and as far as we know, there is no such lipase at present.

[0011] The present invention provides a solution to overcome the high diglyceride content of palm oil and other vegetable oils and their derivatives throughout the supply chain using lipase. Lipase production is simple enough to enable today's producers to use lipase at a sufficiently low cost to enable economically viable production of low diglyceride oils with the above-mentioned benefits, especially health benefits. Summary of the invention

[0012] A method for reducing and / or removing the diglyceride content in an oil without substantially transesterifying triglycerides, the method comprising the steps of providing an oil or fat; and hydrolyzing the monoglycerides and diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO: 1.

[0013] A general object of the present invention is to provide a method for enzymatically removing and / or reducing diglycerides from oils which enables a large scale process that is both advantageous and competitive.

[0014] This object is achieved by the features of each of the independent claims. Advantageous further embodiments are defined in the dependent claims.

[0015] These and other objects and advantages of the present invention will be apparent from the following description. In the following detailed description, preferred embodiments of the present invention will be described with reference to the accompanying drawings. These embodiments do not represent the full scope of the present invention. On the contrary, the present invention can be used for other embodiments. Therefore, the breadth of the present invention should be explained with reference to the claims herein.

[0016] definition

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

[0018] In describing and claiming the present invention, the following terminology will be used.

[0019] 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.

[0020] As used herein, "substantially free," when used to refer to the amount or quantity of a material or a particular characteristic thereof, refers to an amount sufficient to provide the effect that the material or characteristic is intended to provide. In some cases, the exact degree of deviation allowed may depend on the particular circumstances. Similarly, "substantially free" and the like refer to the absence of a determined element or agent in a process. In particular, an element determined to be "substantially free" is either completely absent from the process or is included only in a sufficiently small amount so as to have no deleterious effect on the process.

[0021] Reference herein to "about" a value or parameter includes reference to embodiments of the value or parameter itself. For example, a description referring to "about X" includes the embodiment "X". When used in conjunction with a measured value, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value, and may include a range of plus or minus two standard deviations around the stated value.

[0022] Likewise, reference to a gene or polypeptide "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" and / or "consisting essentially of" embodiments. As used herein, unless the context requires otherwise due to express language or necessary meaning, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. specifying the presence of stated features but not excluding the presence or addition of other features in various embodiments.

[0024] Concentration, amount and other numerical data can be presented in the form of ranges herein. It should be understood that such range forms are used only for convenience and brevity, and should be flexibly interpreted as not only including the values ​​clearly listed as range limits, but also including all individual values ​​or sub-ranges contained within the range, as if each value and sub-range were clearly listed. For example, a weight range of about 1% to about 20% should be interpreted as not only including the clearly listed concentration limits of 1% to about 20%, but also including individual concentrations such as 2%, 3%, 4%, and sub-ranges such as 5% to 15%, 10% to 20%.

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

[0026] As used herein, the term "esterification" is a reaction used to combine an organic acid (such as a fatty acid) with any alcohol or polyol (such as glycerol).

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

[0028] As used herein, the term "transesterification" refers to the reaction of a first ester with a second ester, inducing mixing between an acyl moiety and an alcohol moiety.

[0029] The term "alkyl" or "alkyl group" should be interpreted according to its broadest sense to describe a monovalent aliphatic compound containing hydrocarbons.

[0030] The terms "glycerol derivatives" and "glycerides" are used interchangeably herein to describe esters, ethers, and other derivatives of glycerol in which at least one hydrogen of any hydroxyl group attached to a C1, C2, or C3 carbon is substituted. Examples of glycerol derivatives are: tristearoylglycerol (or tri-stearoylglycerol or tristearate, or glyceryl tristearate); 1,3-benzylpropylene glycol (or 1,3-O-benzylpropylene glycol); and glycerol-2-phosphate (or 2-phosphoglycerol), etc. If the substitution is on a carbon atom, rather than on the oxygen of the hydroxyl group, the compound can be considered a derivative of glycerol (e.g., for 1,2,3-nonadecantriol (C16H33CHOH-CHOH-CH2OH), it can also be considered 1-C-hexadecylglycerol). As used herein, the term "glycerol" is intended to encompass glycerol derivatives that include glycerol.

[0031] The terms monoglycerol, diglycerol, and triglyceride, monoacylglycerol, diacylglycerol, and triacylglycerol, MG / DG / TG, and MAG / DAG / TAG are used interchangeably herein and all refer to fatty acid-based glycerides.

[0032] Lipase: The terms "lipase" or "lipase enzyme", "lipolytic enzyme", "lipid esterase", "lipolytic polypeptide" and "lipolytic protein" refer to an enzyme in the EC 3.1.1 class as defined in the enzyme nomenclature. It may have lipase activity (triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74), sterol esterase activity (EC 3.1.1.13) and / or wax ester hydrolase activity (EC 3.1.1.50).

[0033] The term "parent" or "parent lipase" means a lipase that is altered to produce an enzyme variant. The parent lipase may be a naturally occurring (wild-type) polypeptide, but may also be a variant and / or fragment thereof.

[0034] The relatedness between two amino acid sequences is described by the parameter "sequence identity".

[0035] For the 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. [Molecular Biology] 48: 443-453), as implemented by the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277, preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows:

[0036] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0037] Suitable substrates according to the present invention are a wide variety of vegetable oils and fats; rapeseed oil and soybean oil are the most commonly used, but other crops such as mustard, sunflower, canola, coconut, hemp, palm oil and even algae also show application prospects. The substrate can be of crude quality or further processed (refined, bleached and deodorized). Animal fats can also be used, including animal fats, lard, poultry oils, aquatic animal oils and waste animal and vegetable fats, which are commonly referred to as yellow greases and brown greases. Suitable greases can be pure triglycerides or mixtures of triglycerides and free fatty acids, which are commonly found in waste vegetable oils and animal fats. The substrate can also be obtained from a vegetable oil deodorizer distillate. The types of fatty acids in the substrate include those fatty acids that naturally occur as glycerides in plant and animal fats. These fatty acids include, to name a few: oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid and lauric acid. The minor components in crude vegetable oils are typically phospholipids, free fatty acids and partial glycerides, i.e. monoglycerides and diglycerides.

[0038] The term "fatty acid feedstock" or "oil and / or fat" or "vegetable oil feedstock" is defined herein as a substrate comprising fatty acid derivatives. The substrate may comprise fatty acid alkyl esters, triglycerides, diglycerides, monoglycerides, free fatty acids or any combination thereof. Oils and fats of any plant or animal origin comprising fatty acids may be used as substrates for producing fatty acid alkyl esters in the method of the present invention. In addition, a fatty acid feedstock consisting essentially of fatty acid alkyl esters is suitable as a feedstock (biodiesel feedstock) for use in the present invention.

[0039] The fatty acid feedstock can be an oil selected from the group consisting of microbial oil, algae oil, canola oil, coconut oil, castor oil, coconut oil (coconut kernel oil), corn oil, cottonseed oil, linseed 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 seed oil, seashore mallow oil, or any combination thereof.

[0040] The fatty acid source may be a fat selected from the group consisting of animal fat, including tallow from pigs, cattle and sheep, lard, chicken fat, fish oil or any combination thereof.

[0041] The fatty acid feedstock may be crude, refined, bleached, deodorized, degummed, or any combination thereof.

[0042] The term free fatty acid (FFA) is a carboxylic acid with a long carbon chain. Most naturally occurring fatty acids have unbranched chains with an even number of carbon atoms from 4 to 24. Free fatty acids are usually derived from fats (triglycerides (TAG), diglycerides (DAG), monoglycerides (MAG)), phospholipids or lysophospholipids. Triglycerides are formed by glycerol combined with three fatty acid molecules. The hydroxyl groups (HO-) of glycerol and the carboxyl groups (-COOH) of fatty acids combine to form esters. A glycerol molecule has three hydroxyl groups (HO-). Each fatty acid has a carboxyl group (-COOH). A diglyceride is formed by glycerol combined with two fatty acid molecules. A monoglyceride is formed by glycerol combined with one fatty acid molecule. DETAILED DESCRIPTION

[0043] The present invention relates to a method for reducing and / or removing the diglyceride content of oil without substantially transesterifying the triglycerides.

[0044] In one aspect, the present invention relates to a method for reducing and / or removing diglycerides in oil without substantially transesterifying triglycerides, the method comprising the steps of providing oil or fat, and hydrolyzing monoglycerides and diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO: 1.

[0045] In one aspect, the lipolytic enzyme or lipase used in the method of the invention is selected from lipase, phospholipase, cutinase, acyltransferase, or a mixture of one or more of lipase, phospholipase, cutinase and acyltransferase. The lipolytic enzyme or lipase is selected from the enzymes in EC 3.1.1, EC 3.1.4 and EC 2.3.

[0046] Suitable lipolytic enzymes may be polypeptides having lipase activity, for example, selected from Candida antarctica lipase A (CALA) as disclosed in WO 88 / 02775; Candida antarctica lipase B (CALB) as disclosed in WO 88 / 02775 and shown in SEQ ID NO: 1 of WO 2008065060; Thermomyces lanuginosus (formerly Humicola lanuginosus) lipase disclosed in EP 258 068; Thermomyces lanuginosus variants disclosed in WO 2000 / 60063 or WO 1995 / 22615 (in particular, shown in SEQ ID NO: 2 of WO 95 / 22615); 2, 1-269 of position 1-269 of NO:2), yeast-like Hyphozyma species lipase (WO 98 / 018912), and Rhizomucor miehei lipase (SEQ ID NO: 5 in WO 2004 / 099400); from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218 272), Pseudomonas cepacia (EP 331 376), Pseudomonas glumae, Pseudomonas stutzeri (GB 1,372,034), Pseudomonas fluorescens, Pseudomonas sp. strain SD 705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); Bacillus lipases, for example from Bacillus subtilis (B. subtilis) (Dartois et al. (1993), Biochemica et Biophysica Acta [Biochemistry and Biophysics], 1131, 253-360), Bacillus stearothermophilus or G. stearothermophilus (JP 64 / 744992) or Bacillus pumilus (WO 91 / 16422).Also preferred are lipases from any one of the following organisms: Fusarium oxysporum, Absidia reflexa, Absidia corymbefera, Rhizomucor miehei, Rhizopus delemar (rice), Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum, Aspergillus oryzae, Penicilium camembertii, Aspergillus foetidus and Thermomyces lanuginosus, such as a lipase selected from any one of SEQ ID NOs: 1 to 15 in WO 2004 / 099400.

[0047] Lipase activity:

[0048] In the context of the present invention, lipase activity can be determined as lipase units (LU) using tributyrate as substrate. The method is based on the hydrolysis of tributyrin by the enzyme and the consumption of alkalinity to maintain a constant pH during the hydrolysis is registered as a function of time.

[0049]

[0050] One lipase unit (LU) can be defined as the amount of enzyme that releases 1 micromole titratable butyrate per minute under standard conditions (ie at 30°C; pH 7.0; with 0.1% (w / v) gum arabic as emulsifier and 0.16 M tributyrin as substrate).

[0051] Alternatively, lipolytic activity can be determined as long chain lipase units (LCLU) using the substrate pNP-palmitate (C:16), when incubated at pH 8.0 at 30°C, the lipase hydrolyzes the ester bond and releases pNP, which is yellow and can be detected at 405 nm.

[0052]

[0053] As used herein, the term "selective" means that in the context of an edible oil, the lipase preferentially utilizes diglycerides (DAG) rather than acyl triglycerides (TAG) as substrates. Thus, diglycerides can be removed and / or reduced from an edible oil while maintaining the amount of triglycerides in the oil unchanged (or substantially unchanged). During the treatment process, monoglycerides in the oil can also be hydrolyzed in large quantities, especially in the presence of sufficient water.

[0054] In an embodiment of the present invention, the lipase is a polypeptide having at least 80% sequence identity to SEQ ID NO:1.

[0055] In another embodiment, the lipase is a polypeptide having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1.

[0056] In a preferred embodiment of the present invention, the lipase is a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:1.

[0057] In an embodiment of the present invention, the lipase comprises or consists of the amino acid sequence shown in SEQ ID NO 1.

[0058] In embodiments of the invention, the oil is derived from one or more of: algae oil; canola oil; coconut oil; castor oil; coconut oil; coconut kernel oil; corn oil, distillers corn oil; cottonseed oil; linseed oil; fish oil; grape seed oil, hemp oil; jatropha oil; jojoba oil; mustard oil; canola oil; palm oil; palm stearin; palm olein; palm kernel oil; peanut oil; rapeseed oil; rice bran oil; safflower oil; soybean oil; sunflower oil; shea butter; tall oil; oil from halophytes; and / or animal fats, including animal fats from pigs, cattle and sheep, lard, chicken fat, fish oil; palm oil free fatty acid distillate; soybean oil free fatty acid distillate; soap stock fatty acid material; yellow grease; used cooking oil; palm oil mill waste liquor; and brown grease or any combination thereof.

[0059] In an embodiment of the present invention, the method is carried out at a temperature in the range of 10°C-100°C, preferably 20°C-90°C.

[0060] In embodiments of the invention, the hydrolysis comprises reacting free fatty acids and / or fatty acids in the oil with water in the presence of a lipase until at least 30% (w / w), such as more than 50% (w / w) or such as at least 70% (w / w), of the fatty acid acyl groups of the DAGs in the oil have been converted to free fatty acids.

[0061] In an embodiment of the present invention, the total amount of the lipase added during the hydrolysis process is in the range of 0.1-50000 mg enzyme protein (EP) / kg oil. When a liquid enzyme preparation is used, preferably 0.1-200 mg enzyme protein (EP) / kg oil, when an immobilized enzyme preparation is used, preferably 500-50000 mg enzyme protein (EP) / kg oil.

[0062] In the embodiments of the present invention, the lipase is preferably used in the form of a liquid product, an immobilized product or a dry powder.

[0063] In an embodiment of the present invention, the total reaction time of the method is at least 15 minutes.

[0064] In an embodiment of the present invention, the total reaction time of the method is up to 48 hours.

[0065] In an embodiment of the present invention, the amount of water added during the hydrolysis process is between 0.01% (w / w) and 100% (w / w) of the oil.

[0066] In embodiments of the present invention, the pH is optionally adjusted during or prior to hydrolysis to optimize the reaction.

[0067] In an embodiment of the present invention, the pH during the hydrolysis process is between 3.0 and 7.0.

[0068] In an embodiment of the present invention, citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide are used to adjust the pH.

[0069] In an embodiment of the present invention, the method is carried out in a batch mode, a semi-continuous mode or a continuous mode.

[0070] In another embodiment of the present invention, the process is run in multiple sequential reaction steps (eg, 2-10 reactors in series, preferably 2-5 reactors in series).

[0071] In another embodiment of the present invention, the process is carried out in a countercurrent, optionally partitioned reactor.

[0072] In another embodiment of the invention, the lipase is used in an immobilized preparation using, for example, a column or a bed.

[0073] In another embodiment of the present invention, the method further comprises adding one or more additional lipases and / or phospholipases during the hydrolysis process.

[0074] In embodiments of the invention, the amount of triglycerides in the oil is unchanged (or substantially unchanged) after treatment with lipase.

[0075] In another embodiment of the present invention, the raw material oil or fat is the raw material of the degumming process. Optionally, the raw material is treated according to the present invention before degumming. Optionally, the raw material is treated according to the present invention after degumming. Optionally, the raw material is treated in combination with degumming according to the present invention. Such degumming can be, for example, water degumming, acid degumming, enzyme degumming performed by existing factories, but is not limited to those.

[0076] In another embodiment of the present invention, the raw oil or fat is previously refined and / or bleached, and the present invention uses it as a pretreatment before deodorization to improve the quality of the deodorized product by reducing the generation of unwanted by-products (such as 3MCPD and glycidyl esters) during deodorization.

[0077] In another embodiment of the present invention, the raw oil or fat is intended for fractionation and / or winterization, and the present invention uses this to improve, for example, the yield of the desired fraction.

[0078] In another embodiment, the diglyceride hydrolysis process and the degumming process are performed sequentially or simultaneously.

[0079] In another embodiment, before evaporation, the hydrolysis method of diglyceride is carried out in the "mixed oil" mixture of extracted oil. This mixture is a mixture of oil and organic solvent obtained after the main oil extraction step. Then the mixed oil mixture is separated into product crude oil and organic solvent for reuse. This mixture is mainly composed of oil and organic solvent. Preferably, the solvent is acetone, hexane or heptane. Most preferably, the organic solvent is hexane. Therefore, the method disclosed in this document can be used in the mixed oil mixture by adding enzyme and water, and then carrying out the separation step, after which the mixture of treated oil and hexane can be subjected to a common evaporation method. This produces a crude oil with improved quality by reducing DAG and forming FFA even before entering the refinery, and is particularly advantageous for a pressing plant that wishes to provide a crude product with improved quality to an external refinery.

[0080] The present invention is further described in the following paragraphs.

[0081] Paragraph 1. A method for reducing and / or removing the diglyceride content of an oil without substantially transesterifying triglycerides, the method comprising the steps of:

[0082] a. provide oil or fat; and

[0083] b. hydrolyzing the diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO: 1.

[0084] Paragraph 2. The method according to paragraph 1 further comprises the following steps: separating the light phase and the heavy phase after hydrolysis.

[0085] Paragraph 3. The method of paragraph 2, wherein the light phase comprises the oil having reduced diglycerides and increased FFAs.

[0086] Paragraph 4. The method according to paragraph 2, wherein the heavy phase comprises water, lipase, and glycerol.

[0087] Paragraph 5. The process according to paragraphs 2-4, wherein the heavy phase is partially or completely recycled to the hydrolysis step.

[0088] Paragraph 6. The method according to paragraphs 2-4, wherein free fatty acids are separated from the oil present in the light phase.

[0089] Paragraph 7. The method according to paragraph 1, wherein the hydrolysis rate of the triglycerides present in the oil is less than 10%, preferably less than 5%, more preferably less than 2%, and most preferably less than 0.5%.

[0090] Paragraph 8. The method of Paragraph 1, wherein the concentration of diglycerides is reduced by at least 30%, more preferably at least 40%, and most preferably at least 50%.

[0091] Paragraph 9. A method according to paragraph 1, wherein the lipase is a polypeptide having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1.

[0092] Paragraph 10. A method according to any of the preceding paragraphs, wherein the oil is derived, for example, from one or more of: algae oil; canola oil; coconut oil; castor oil; coconut kernel oil; corn oil; distiller's grains corn oil; cottonseed oil; linseed oil; fish oil; grapeseed oil; hemp oil; jatropha oil; jojoba oil; mustard oil; canola oil; palm oil; palm stearin; palm olein; palm kernel oil; peanut oil; rapeseed oil; rice bran oil; safflower oil; soybean oil; sunflower oil; shea butter; tall oil; oil from halophytes; and / or animal fats, including animal fats from pigs, cattle and sheep, lard, chicken fat, fish oil; palm oil free fatty acid distillate; soybean oil free fatty acid distillate; soap stock fatty acid material; yellow grease; used cooking oil; palm oil mill waste liquor; and brown grease or any combination thereof.

[0093] Paragraph 11. The process according to any one of the preceding paragraphs, wherein the process is carried out at a temperature in the range of 10°C to 100°C, preferably 20°C to 90°C.

[0094] Paragraph 12. The method according to any of the preceding paragraphs, wherein the dosage of the lipase ranges from 0.1-50000 mg enzyme protein (EP) / kg oil.

[0095] Paragraph 13. The method according to any of the preceding paragraphs, wherein the lipase is a liquid product, an immobilized product or a dry powder.

[0096] Paragraph 14. The method according to any of the preceding paragraphs, wherein the total reaction time of the method is at least 15 minutes.

[0097] Paragraph 15. A method according to any of the preceding paragraphs, wherein the total reaction time of the method is up to 48 hours.

[0098] Paragraph 16. The method of any of the preceding paragraphs, wherein the amount of water added is between 0.01% (w / w) and 100% (w / w) of the oil.

[0099] Paragraph 17. The method according to any of the preceding paragraphs, wherein the pH is optionally adjusted during or prior to hydrolysis to optimize the reaction.

[0100] Paragraph 18. The method according to paragraph 14, wherein the pH during hydrolysis is preferably between 3.0-7.0.

[0101] Paragraph 19. The method according to paragraphs 14-15, wherein the pH is preferably adjusted using citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide.

[0102] Paragraph 20. The method according to any of the preceding paragraphs, wherein the method is carried out in a batch mode, a semi-continuous mode or a continuous mode.

[0103] Paragraph 21. The method according to any of the preceding paragraphs, further comprising adding one or more additional lipases and / or phospholipases during the hydrolysis process.

[0104] Paragraph 22. The method according to any of the preceding paragraphs, further comprising the presence of an organic solvent during the reaction.

[0105] Paragraph 23. The method according to paragraph 22, wherein the organic solvent is acetone, hexane or heptane.

[0106] Examples

[0107] The SEQ ID NO: 1 of the present invention is shown as SEQ ID NO: 1 of WO 2008065060.

[0108] SEQ ID NO: 2 of the present invention is shown as SEQ ID NO: 2 of WO 2011067349.

[0109] Example 1: Surprising effect of SEQ ID NO:1.

[0110] Palm stearin was completely melted by heating. The required amount was weighed, the required weight of water was added, and the mixture was incubated to 60°C. The required dose of lipase of SEQ ID NO: 1 was added. The reaction occurred while mixing at 60°C. After sampling and centrifugation at 2000g for 5 min, the light phase was taken and analyzed by titration for wt% FFA (AOCS 5a-40 free fatty acids in crude and refined fats).

[0111] Table 1: Experimental setup

[0112]

[0113] Table 2: Results

[0114]

[0115] The initial increase in FFA was very rapid, indicating that the mono- and di-glycerides were converted very quickly initially. Then over the course of the >20 hour reaction, a significant amount of triglycerides were converted. When hydrolyzing palm stearin, one would not expect that the hydrolysis of mono- and di-glycerides would produce 22 wt% FFA. Therefore, the triglycerides must have been converted, especially in the above tests 1 and 2, where glycerol was not initially present. The lower conversion in the presence of glycerol is attributed to the balance between the hydrolysis and esterification of fatty acids into glycerol.

[0116] These results are intended to show the common view in the industry that SEQ ID NO: 1 does have a significant effect on triglycerides, which is why SEQ ID NO: 1 has not yet been considered a viable enzyme for this application. For example, SEQ ID NO: 1 is commonly used as an enzyme catalyst to produce triglycerides by esterification of FFA and glycerol, which is the opposite direction of the same hydrolysis reaction.

[0117] The inventors found that the use of SEQ ID NO: 1 would generally be expected to have significant activity on triglycerides, especially when combined with sufficiently high temperatures and high water dosages, long reaction times and relatively high enzyme dosages. Therefore, it is surprising that SEQ ID NO: 1 can react with diglycerides, but has very little activity on triglycerides.

[0118] Example 2: Hydrolysis of diglycerides

[0119] Crude palm oil (CPO) was completely melted by heating it to 70°C. The desired oil was weighed into a 250mL square Schott bottle. The desired weight of water was added and the mixture was incubated to 50°C or 60°C with stirring. The desired dose of SEQ ID NO:1 lipase was added. The mixture was reacted at 350rpm in a water bath with a stirrer at 60°C. Samples were taken in test tubes after 4h and 24h. After sampling and centrifugation at 2000g for 5min, the light phase was taken and the %FFA in the oil was analyzed by titration, the monoglyceride and diglyceride content was analyzed by GC, and the TG curve was analyzed by GC (AOCS 5a-40 free fatty acids and AOCS Ce 5-86 triglycerides in crude and refined oils and fats were analyzed by gas chromatography).

[0120] Table 3: Experimental setup

[0121]

[0122]

[0123] Table 4: Data list (wt% FFA and wt% DG)

[0124]

[0125] As shown in Table 4, T4 with a higher water content of 2% and 3.4 mg lipase / kg oil at 50°C produced the lowest DG content: after 24 h of reaction, the content in CPO decreased from 6.9 wt% to 2.4 wt%. DG hydrolysis from 4 to 24 h, 1% water seems too little for the system with stagnant conversion after 4 h (both FFA and DG), perhaps the free water available at this stage (fully utilized after 4 hr of reaction) is too little to stimulate further conversion. The 10-fold increase in enzyme dosage of T2 (34 mg lipase / kg oil) can promote a faster reduction of DG from 6.9 wt% to 3.2 wt% in 4 hr. However, due to less free water available, DG increased to 4.5 wt% after another 20 hr of reaction, resulting in condensation. Comparing T1 and T3 (60°C vs. 50°C), the difference in FFA is almost the same, but 60°C seems to be able to achieve lower DG values ​​(4.7 wt% vs. 5.3 wt%). Referring to Table 5, the TG profile remained unchanged after enzymatic hydrolysis, showing no signs of transesterification and further confirming the claim that there was little activity on triglycerides.

[0126] In addition, it can be concluded that SEQ ID NO: 1 specifically hydrolyzes DG at low lipase dosage without interacting with TG in the oil. In addition, a significant and important effect on the reduction of DG was shown within about 4 hours.

[0127] Table 5: Interesterification (% of each identifiable oil component)

[0128]

[0129]

[0130] Example 3: Hydrolysis of DG using SEQ ID NO:2.

[0131] Crude palm oil (CPO) was completely melted by heating. The required amount was weighed, 5% (wt / wt) water was added, and the mixture was incubated to 75°C. The desired dose of SEQ ID NO:2 lipase was added. The reaction occurred while mixing at 75°C. After sampling, it was heated to 99°C for 10 minutes and centrifuged at 2000g for 5min, the light phase was taken and the % FFA (AOCS 5a-40 free fatty acids in crude and refined oils) in the oil was analyzed by titration, and monoglycerides and diglycerides were analyzed by a customized HPLC method.

[0132] Table 6: Experimental setup

[0133] Test 1 Temperature / ℃ 75 Crude palm oil (g) 30 Types of enzymes SEQ ID NO:2 mg enzyme protein / kg oil 8.5 %(wt / wt)H2O 5

[0134] Table 7: Results

[0135] % FFA (by AOCS Ca 5a-40 method, based on palmitic acid), DG and TG are expressed as normalized relative HPLC peak areas.

[0136]

[0137] As can be seen from Table 7, SEQ ID NO: 2 has a significant and important effect on the reduction of DG.

[0138] Example 4: Combination of SEQ ID NO: 1 and PLC (Quara Boost).

[0139] Laboratory-scale enzymatic water degumming was performed at 55°C and 3wt% total water content. Two different masses of crude soybean oil samples were used in this experiment (Table 8). The oil was preheated to 55°C and then 30g portions were transferred to glass tubes. Enzymes and water were added accordingly and the samples were sonicated at 50°C for 5min to ensure that the enzymes and water were fully distributed and mixed into the oil phase. In the next step, the oil samples were placed in a heating cabinet and incubated at 55°C for a selected time with gentle rotation. Check the sequential processing. In the first step, phospholipase C was added to the oil sample, and after 2h, SEQ ID NO: 1 was added to the selected sample, after which PLC and SEQ ID NO: 1 were simultaneously present in the reaction mixture. After 24h, the enzymatic reaction was stopped by heating the oil sample to 95°C for 10min. In the control sample, only phospholipase C was added for 24h or only SEQ ID NO: 1 was added for 22h. The gum and oil phases were separated by centrifugation at 600g and 85°C for 6min. The upper oil phase was transferred to a new tube and saved for analysis. Diacylceride (DG, wt%) and free fatty acid (FFA, wt%) were analyzed in the oil phase. DG was analyzed by Dionex Ultimate 3000 HPLC system (column: Hypersil Gold Silica 3 μm 150 x 4.6 mm) equipped with Corona detector according to AOCS official method Cd11d-96. FFA was analyzed by NaOH titration according to AOCS Ca 5a-40 official method. 31 Phospholipids in crude oil samples were analyzed by P NMR.

[0140] Table 8: Results of enzymatic degumming of soybean oil by PLC and SEQ ID NO: 1 at 55°C and 3% water, 24h reaction time

[0141]

[0142]

[0143] *NA – not analyzed

[0144] As can be seen from Table 8, compared with the normal PLC degumming without the lipase of SEQ ID NO:1, the combination of SEQ ID NO:1 and PLC-type phospholipidase produces an oil with reduced DG level.PLC-type phospholipidase converts phospholipids into diglycerides and releases the phosphate side groups.Such phospholipid conversion is a well-known enzymatic degumming type, and its productivity increases compared with traditional non-enzymatic degumming methods (e.g., water / acid degumming).Due to the PLC catalytic reaction, the diglyceride level can be increased to disturbing levels in some cases, so a combination of PLC and diglyceride active enzymes (like SEQ ID NO:1) is needed.

[0145] Example 5: SEQ ID NO: 1 as liquid and immobilized formulations

[0146] 50 g of the same CPO as in Example 3 above was used. 4% (wt / wt) of water was added to the oil and the mixture was preheated to 50°C. Lipase was added to the mixture and the reaction was carried out in a shaking incubator at 250 rpm using a 100 mL square blue cap bottle. After sampling, heating to 99°C for 10 minutes and centrifuging at 2000 g for 5 min, the light phase was taken and the % FFA (AOCS 5a-40 free fatty acids in crude and refined oils) in the oil was analyzed by titration, and monoglycerides and diglycerides were analyzed by a customized HPLC method.

[0147] Table 9: Comparison results between liquid and immobilized SEQ ID NO: 1

[0148]

[0149]

[0150] Comparing the results, the liquid formulation provided a higher reaction rate than the immobilized formulation.The lipases of SEQ ID NOs: 1 and 2 can be used as both liquid, dry or immobilized formulations.

[0151] Example 6: Hydrolysis of DG in the presence of solvent

[0152] Use 60g of CPO containing 4.4wt%FFA, 0.6wt%MG, 4.9wt%DG. 2% or 5% (wt / wt) water is added to the oil together with 15, 30 or 60g of hexane. The mixture is preheated to 50°C. 0.25% (wt / wt CPO) preparation of SEQ ID NO:1 containing 0.95wt% active enzyme protein is added to the mixture, and a 250mL square blue cap bottle is used to react in a water bath under 500rpm magnetic stirring. After sampling, it is heated to 99°C for 10 minutes. The hexane in the sample is evaporated overnight under vacuum. The sample is then centrifuged at 2000g for 5min, and then the %FFA (AOCS 5a-40 free fatty acids in crude oil and refined oil) in the oil is analyzed by titration, and monoglyceride and diglyceride are analyzed by a customized HPLC method.

[0153] Table 10: Reaction results of SEQ ID NO: 1 in the presence of hexane

[0154]

[0155]

[0156] DG hydrolysis was performed with the hexane / oil mixture directly from the extraction step before removing the hexane and separating the oil. The above results indicate that 50% hexane is superior to 25% hexane and 100% hexane because it can hydrolyze DAG to the greatest extent.

[0157] While there has been shown and described what are presently considered to be the preferred embodiments of the invention, it will be obvious to those skilled in the art that various changes and modifications can be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A method for reducing and / or removing the diglyceride content in an oil without substantially transesterifying triglycerides, the method comprising the steps of: a. Provide oil or fat; as well as b. hydrolyzing the diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO:

1.

2. The method according to claim 1, further comprising the following steps: After the hydrolysis the light and heavy phases were separated.

3. The method of claim 2, wherein the light phase comprises the oil having reduced diglycerides and increased FFAs.

4. The method according to claim 2, wherein the heavy phase comprises water, lipase, and glycerol.

5. The process according to claims 2-4, wherein the heavy phase is partially or completely recycled to the hydrolysis step.

6. A process according to claims 2-4, wherein free fatty acids are separated from the oil present in the light phase.

7. The method of claim 1, wherein the hydrolysis rate of the triglycerides present in the oil is less than 10%, preferably less than 5%, more preferably less than 2%, and most preferably less than 0.5%.

8. The method of claim 1, wherein the concentration of diglycerides is reduced by at least 30%, more preferably at least 40%, and most preferably at least 50%.

9. The method of claim 1, wherein the lipase is a polypeptide having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

1.

10. The method of any one of the preceding claims, wherein the oil is derived, for example, from one or more of algal oil; canola oil; coconut oil; castor oil; coconut kernel oil; corn oil; distiller's corn oil; cottonseed oil; linseed oil; fish oil; grapeseed oil; hemp oil; jatropha oil; jojoba oil; mustard oil; canola oil; palm oil; palm stearin; palm olein; palm kernel oil; peanut oil; rapeseed oil; rice bran oil; safflower oil; soybean oil; sunflower oil; shea butter; tall oil; oils from halophytes; and / or animal fats, including animal fats from pigs, cattle and sheep, lard, chicken fat, fish oil; palm oil free fatty acid distillate; soybean oil free fatty acid distillate; soap stock fatty acid material; yellow grease; used cooking oil; palm oil mill waste liquor; and brown grease or any combination thereof.

11. A method according to any one of the preceding claims, wherein the amount of water added is between 0.01% (w / w) and 100% (w / w) of the oil.

12. A process according to any one of the preceding claims, wherein the pH is optionally adjusted during or prior to the hydrolysis to optimize the reaction.

13. The method according to any one of the preceding claims, further comprising adding one or more additional lipases and / or phospholipases during the hydrolysis process.

14. The method according to any one of the preceding claims, further comprising the presence of an organic solvent during the reaction.

15. The method according to claim 14, wherein the organic solvent is acetone, hexane or heptane.

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