A selective hydrolysis method for diglycerides in oils / fats using Candida Antarctica lipase B

Abstract

1. A method for reducing and / or removing the diglyceride content of an oil without substantial transesterification of the triglycerides, comprising the steps of providing an oil or fat and hydrolyzing the mono- 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 enzymatically removing and / or reducing diglycerides from oils. [Background technology]

[0002] Oils and fats consist of complex mixtures of triacylglycerols (TAGs), diacylglycerols (DAGs), monoacylglycerols (MAGs), free fatty acids, and other minor components. Crystallization of these mixtures depends on the properties of the TAGs (such as the fatty acid combination, their chain length, and their degree of unsaturation) and the interactions 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 physical properties vary from crystallization rate, polymorphic changes, melting point, crystal size, and crystal habit (Siew, 2001).

[0003] In some oils extracted from oilseeds, the effect of DAG is less pronounced as it is often present in small amounts, but palm oil, rice bran, shea butter, and olive oil, for example, usually have large amounts of DAG after extraction, and their presence would impair the quality of these oils.

[0004] The presence of these diglycerides in the main product (=triglycerides) is undesirable because the diglycerides have a detrimental effect on the properties of the triglyceride product. Diglycerides are also reactive during parts of the refining process, especially during deodorization, and tend to form food safety regulated by-products under certain conditions, such as glycidyl esters.

[0005] Several methods to address this problem have been disclosed in the literature, which focus on the removal of diglycerides from mixtures with triglycerides, the enzymatic conversion of which is carried out by using enzymes specific for the hydrolysis of diglycerides to glycerol and free fatty acids.

[0006] For example, JP 62 / 51997 A discloses a method for improving fats, in which a mixture containing at least 70 weight percent triglycerides and 2 weight percent or more diglycerides is contacted with an enzyme specific for partial glycerides in the presence of a small amount of water.

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

[0008] A similar method is disclosed in JP 62 / 287 A, whereby the hydrolysis of monoglycerides and / or diglycerides is carried out using a lipase produced by Penicillium cyclopium ATCC 34613.

[0009] Thus, the prior art teaches methods to reduce or remove the diglyceride content in palm oil and other edible oils by enzymatic reactions. These processes rely on the hydrolysis of diglycerides using specific diglyceride-hydrolyzing lipases to form free fatty acids and glycerol. The free fatty acids can then be removed by different processes such as vacuum distillation, saponification, or fractional distillation.

[0010] The use of certain diglyceride hydrolases results in the formation of free fatty acids, which often must be removed from the oil. However, in current market conditions where fatty acid distillates are relatively valuable for various reasons, combining the improvement in TAG oil quality with the cost of additional FFA production (often a distillate) can result in a combined economic improvement. A further benefit of reducing MAG and DAG can be expected, namely a significant improvement in the quality of the distillate compared to standard quality, since hydrolysis of MAG and DAG increases the FFA concentration in the distillate. This means that such distillates will require less extensive processing and will save energy. To achieve such economic benefits, a sufficiently inexpensive lipase is required, and to the inventors' knowledge, no such lipase is currently available.

[0011] The present invention provides a solution to overcome the problem of high diglyceride content in palm oil and other vegetable oils and derivatives throughout the supply chain by using lipases that are sufficiently easy to produce and available to manufacturers today at a cost low enough to allow economically viable production of oils with the low diglyceride benefits, particularly the health benefits, mentioned above. Summary of the Invention [Means for solving the problem]

[0012] 1. A method for reducing and / or removing the diglyceride content of an oil without substantial transesterification of the triglycerides, comprising the steps of providing an oil or fat and hydrolyzing the mono- 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 the enzymatic removal and / or reduction of diglycerides from oils that allows for profitable and competitive large-scale processes.

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

[0015] These and other objects and advantages of the present invention will become apparent from the following description. In the following detailed description, preferred embodiments of the present invention are described with reference to the accompanying drawings. These embodiments do not represent the full scope of the present invention. Rather, the present invention may be used in other embodiments. Therefore, reference should be made to the claims herein to interpret the scope of the present invention.

[0016] definition 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 methods and materials disclosed herein, as such may vary to some extent. 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, since the scope of the present invention will be defined only by the appended claims and equivalents thereof.

[0017] In describing and claiming the present invention, the following terminology will be used.

[0018] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a step" includes reference to one or more of such steps.

[0019] As used herein, "substantial," when used in reference to an amount or quantity of a material, or a particular property thereof, refers to an amount sufficient to provide the effect that the material or property is intended to provide. The exact degree of tolerance may depend on the particular context. Similarly, "substantially free," etc., refers to the absence of a specified element or agent in a process. In particular, an element specified as "substantially free" is either completely absent from the process or present in an amount small enough that it does not adversely affect the process.

[0020] As used herein, a reference "about" a value or parameter includes embodiments related to the value or parameter itself. For example, a reference "about X" includes the embodiment "X." When used in conjunction 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.

[0021] Similarly, reference to a gene or polypeptide "derived from" another gene or polypeptide X includes that gene or polypeptide X.

[0022] It will be understood that the embodiments described herein include "consisting of" and / or "consisting essentially of" embodiments. As used herein, unless the context requires otherwise by clear language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify that stated features are present, but do not exclude that further features are present or added to various embodiments.

[0023] Concentrations, amounts, and other numerical data may be expressed in range format herein. It will 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 limiting the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. For example, a weight range of about 1 percent to about 20 percent should be interpreted not only to include 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, etc., and subranges such as 5 percent to 15 percent, 10 percent to 20 percent, etc.

[0024] 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.

[0025] The term "esterification" as used herein refers to the reaction to combine an organic acid, such as a fatty acid, with any alcohol or with a polyol, such as glycerol.

[0026] The term "hydrolysis," as used herein, refers to the reaction of water with an ester to produce an acid and an alcohol.

[0027] The term "transesterification," as used herein, refers to the reaction of a first ester with a second ester to produce intermixture between the acyl and alcohol moieties.

[0028] The terms "alkyl" or "alkyl group" are to be interpreted in their broadest sense to refer to monovalent aliphatic compounds, including hydrocarbons.

[0029] The terms "glycerol derivative" and "glyceride" are used interchangeably herein to refer to esters, ethers, and other derivatives of glycerol in which at least one of the hydrogen atoms of any of the hydroxyl groups attached to the C1, C2, or C3 carbons has been replaced. Examples of glycerol derivatives are tristearoylglycerol (or tri-O-stearoylglycerol or glycerol tristearate or glyceryl tristearate); 1,3-benzylideneglycerol (or 1,3-O-benzylideneglycerol); and, among others, glycerol 2-phosphate (or 2-phosphoglycerol). When 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., 1,2,3-nonadecanetriol, which has the formula CHCHOH-CHOH-CHOH, and can also be considered 1-C-hexadecylglycerol). The term "glycerol" as used herein is intended to encompass glycerol derivatives, including glycerol.

[0030] The terms mono-, di-, and tri-glycerols / glycerides, mono-, di-, and tri-acylglycerols / acylglycerides, MG / DG / TG, and MAG / DAG / TAG are used interchangeably herein and all refer to fatty acid-based glycerides.

[0031] Lipase: The terms "lipase," "lipase enzyme," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to enzymes in class EC 3.1.1 as defined by enzyme nomenclature, which 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).

[0032] The terms "parent" or "parent lipase" refer to a lipase that is altered to produce an enzyme variant. A parent lipase can be a naturally occurring (wild-type) polypeptide, but can also be a variant and / or fragment thereof.

[0033] The relatedness between two amino acid sequences is expressed by a parameter known as "sequence identity."

[0034] For purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably 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), version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a 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, calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0035] Suitable substrates for the present invention include a wide variety of vegetable oils and vegetable fats, with rapeseed oil and soybean oil being the most commonly used, but other crops such as mustard oil, sunflower oil, canola oil, coconut oil, hemp oil, palm oil, and even algae also being promising. The substrates can be crude or further processed (refined, bleached, and deodorized). Animal fats, including tallow, lard, poultry oil, and fish oil, as well as discarded vegetable and animal fats and oils (commonly known as yellow and brown greases), can also be used. Suitable fats and oils can be pure triglycerides or mixtures of triglycerides and free fatty acids commonly found in discarded vegetable and animal oils. The substrate can also be obtained from deodorized distillates of vegetable oils. The types of fatty acids in the substrate include those that occur naturally as glycerides in vegetable and animal fats and oils. These include oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, and lauric acid, to name a few. The minor components in crude vegetable oils are typically phospholipids, free fatty acids, and partial glycerides, i.e., monoglycerides and diglycerides.

[0036] The terms "fatty acid feedstock," or "oil and / or fat," or "vegetable oil feedstock" are defined herein as a substrate containing fatty acid derivatives. The substrate may include fatty acid alkyl esters, triglycerides, diglycerides, monoglycerides, free fatty acids, or any combination thereof. Any oils and fats of plant or animal origin that contain fatty acids can be used as a substrate for producing fatty acid alkyl esters in the process of the present invention. Fatty acid feedstocks that consist essentially of fatty acid alkyl esters are also suitable as feedstocks (biodiesel feedstocks) for the present invention.

[0037] The fatty acid source can be an oil selected from the group consisting of microbial oil, algal 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, distilled 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 derived from halophytes, shepherd's purse oil, camelina oil, jojoba oil, coriander seed oil, meadowfoam oil, sea mallow oil, or any combination thereof.

[0038] The fatty acid source can be a fat selected from the group consisting of animal fats, including tallow from porcine, beef and ovine, lard, chicken fat, fish oil, or any combination thereof.

[0039] The fatty acid feedstock may be crude, refined, bleached, deodorized, degummed, or any combination thereof.

[0040] The term free fatty acid (FFA) refers to a carboxylic acid with a long carbon chain. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, ranging 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 combining glycerol with three fatty acid molecules. The hydroxyl (HO-) group of glycerol and the carboxyl (-COOH) group of the fatty acid combine to form an ester. A glycerol molecule has three hydroxyl (HO-) groups. Each fatty acid has a carboxyl (-COOH) group. Diglycerides are formed by combining glycerol with two fatty acid molecules. Monoglycerides are formed by combining glycerol with one fatty acid molecule. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to a method for reducing and / or eliminating the diglyceride content of oils without substantial transesterification of the triglycerides.

[0042] In one aspect, the present invention relates to a method for reducing and / or removing diglycerides without substantial transesterification of triglycerides in an oil, the method comprising the steps of providing an oil or fat and hydrolyzing mono- and diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO:1.

[0043] In one aspect, the lipolytic enzyme or lipase applied in the method of the present invention is selected from lipases, phospholipases, cutinases, acyltransferases, or mixtures of one or more of lipases, phospholipases, cutinases and acyltransferases. The lipolytic enzyme or lipase is selected from enzymes of EC 3.1.1, EC 3.1.4 and EC 2.3.

[0044] Suitable lipolytic enzymes include polypeptides having lipase activity, such as Candida antarctica lipase A (CALA) disclosed in WO 88 / 02775, C. antarctica lipase B (CALB) disclosed in WO 88 / 02775 and set forth in SEQ ID NO: 1 of WO 2008065060, Thermomyces lanuginosus (formerly Humicola lanuginosus) lipase disclosed in EP 258068, Thermomyces lanuginosus (formerly Humicola lanuginosus) lipase disclosed in WO 2000 / 60063 or WO 1995 / 22615, and the like. lanuginosus variants, in particular the lipase shown at positions 1 to 269 of SEQ ID NO: 2 in WO 95 / 22615, Hyphozyma sp. lipase (WO 98 / 018912), and Rhizomucor miehei miehei lipase (SEQ ID NO: 5 in WO 2004 / 099400), P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. glumae, P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas spp. lipases from P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012); Bacillus lipases, e.g., B. subtilis (Dartois et al.(1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus or G. stearothermophilus (JP 64 / 744992 A) or B. pumilus (WO 91 / 16422 A). The following organisms were identified: Fusarium oxysporum, Absidia reflexa, Absidia corymbefera, Rhizomucor miehei, Rhizopus delemar (oryzae), Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum, Aspergillus oryzae, Penicillium camembertii, Aspergillus foetidus, and Thermomyces lanuginosus. Also preferred are lipases derived from any of the Lactobacillus sp. lanuginosus, for example, lipases selected from any of SEQ ID NOS: 1 to 15 in WO 2004 / 099400.

[0045] Lipase activity: In the context of the present invention, lipase activity can be determined as Lipase Units (LU) using tributyrate as substrate. This method is based on the enzymatic hydrolysis of tributyrin, during which alkali consumption to maintain a constant pH is recorded as a function of time. [ka]

[0046] One lipase unit (LU) can be defined as the amount of enzyme that liberates 1 micromole of titratable butyric acid per minute under standard conditions (i.e., at 30°C, pH 7.0, 0.1% (w / v) gum arabic as an emulsifier, and 0.16 M tributyrin as a substrate).

[0047] 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, 30°C; the lipase hydrolyzes the ester bond, releasing pNP, which is yellow in colour and can be detected at 405 nm. [ka]

[0048] The term "selective," as used herein, means that in an edible oil environment, the lipase preferentially utilizes diglycerides (DAGs) over triacylglycerides (TAGs) as substrates. Thus, diglycerides can be removed and / or reduced from edible oils while the amount of triglycerides in the oil remains unchanged (or substantially unchanged). The amount of monoglycerides in the oil is also substantially hydrolyzed during processing, particularly if sufficient water is available.

[0049] In one embodiment of the invention, the lipase is a polypeptide having at least 80% sequence identity to SEQ ID NO:1.

[0050] In another embodiment, the lipase is a polypeptide having at least 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.

[0051] In a preferred embodiment of the invention, the lipase is a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:1.

[0052] In one embodiment of the invention, the lipase comprises or consists of the amino acid sequence set forth in SEQ ID NO:1.

[0053] In one embodiment of the invention, the oil is derived from one or more of algal oil, canola oil, coconut oil, castor oil, palm oil, copra oil, corn oil, distilled corn oil, cottonseed oil, flax 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 oil, tall oil, oils derived from halophytes, and / or animal fats including tallow from pigs, beef and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soda cake fatty acid material, yellow grease, used cooking oil, palm oil mill waste oil, and brown grease, or any combination thereof.

[0054] In one embodiment of the present invention, the process is carried out at a temperature in the range of 10 to 100°C, preferably 20 to 90°C.

[0055] In one embodiment 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 acyl groups of the DAGs in the oil are converted to free fatty acids.

[0056] In one embodiment of the present invention, the total amount of lipase added during hydrolysis is in the range of 0.1 to 50,000 mg enzyme protein (EP) / kg oil, preferably 0.1 to 200 mg enzyme protein (EP) / kg oil when a liquid enzyme preparation is used, and preferably 500 to 50,000 mg enzyme protein (EP) / kg oil when an immobilized enzyme preparation is used.

[0057] In one embodiment of the present invention, the lipase is preferably used as a liquid product, an immobilized product, or a dry powder.

[0058] In one embodiment of the present invention, the total reaction time of the process is at least 15 minutes.

[0059] In one embodiment of the present invention, the total reaction time of the process is up to 48 hours.

[0060] In one embodiment of the present invention, the amount of water added during hydrolysis is between 0.01 and 100% (w / w) of the oil.

[0061] In one embodiment of the present invention, the pH is optionally adjusted during or before the hydrolysis to optimize the reaction.

[0062] In one embodiment of the present invention, the pH during hydrolysis is between 3.0 and 7.0.

[0063] In one embodiment of the present invention, the pH is adjusted using citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide.

[0064] In one embodiment of the present invention, the method is carried out in a batch, semi-continuous, or continuous mode.

[0065] In another embodiment of the invention, the process is carried out in multiple successive reaction steps, for example in 2 to 10 successive reactors, preferably in 2 to 5 successive reactors.

[0066] In another embodiment of the invention, the process is carried out in a countercurrent reactor, optionally in a compartment reactor.

[0067] In another embodiment of the invention, the lipase is used in an immobilized formulation, for example using a column or bed.

[0068] In another embodiment of the invention, the method further comprises adding one or more additional lipases and / or phospholipases during hydrolysis.

[0069] In one embodiment of the present invention, the amount of triglycerides in the oil remains unchanged (or substantially unchanged) after treatment with lipase.

[0070] In another embodiment of the present invention, the feedstock oil or fat is a feedstock for a degumming process. Optionally, the feedstock is treated according to the present invention before degumming. Optionally, the feedstock is treated according to the present invention after degumming. Optionally, the feedstock is treated according to the present invention in combination with degumming. Such degumming can be, for example, but not limited to, water degumming, acid degumming, or enzymatic degumming as performed by existing mills.

[0071] In another embodiment of the present invention, the raw oil or fat is previously refined and / or bleached, and the present invention is utilized as a pre-treatment prior to deodorization to improve the quality of the deodorized product by reducing the production of undesirable by-products such as 3MCPD and glycidol esters during deodorization.

[0072] In another embodiment of the present invention, the feed oil or fat is intended for fractionation and / or winterization, and the use of the present invention is used, for example, to improve the yield of the desired fraction.

[0073] In another embodiment, the process of diglyceride hydrolysis is carried out sequentially or simultaneously with the degumming process.

[0074] In another embodiment, the diglyceride hydrolysis process is carried out in the extracted oil "miscella" mixture before evaporation. This mixture, a mixture of oil and organic solvent, leaves the primary oil extraction step behind. The miscella mixture is then separated into a product crude oil and organic solvent for reuse. This mixture primarily contains oil and organic solvent. Preferably, the solvent is acetone, hexane, or heptane. Most preferably, the organic solvent is hexane. Therefore, the method disclosed herein can be used on the miscella mixture by adding enzymes and water, followed by a separation step, after which the treated oil and hexane mixture can be subjected to the conventional evaporation process. This results in a crude oil with improved quality by reducing DAGs and forming FFAs even before entering the refinery, which is particularly advantageous for mills that wish to provide an improved crude product to external refineries.

[0075] The invention is further described in the following sections.

[0076] Item 1. A method for reducing and / or removing the diglyceride content of an oil without substantial transesterification of triglycerides, comprising: a. providing an oil or fat; b. hydrolyzing diglycerides in the oil with water in the presence of a lipase having at least 80% sequence identity to SEQ ID NO:1; A method comprising:

[0077] Item 2. The method of item 1, further comprising separating the light and heavy phases after hydrolysis.

[0078] Item 3. The method of item 2, wherein the light phase comprises an oil having reduced diglycerides and increased FFAs.

[0079] Item 4. The method of item 2, wherein the heavy phase comprises water, lipase, and glycerol.

[0080] Item 5. The method according to any one of items 2 to 4, wherein the heavy phase is recycled, partially or completely, to the hydrolysis step.

[0081] Item 6. The method of any one of items 2 to 4, wherein the free fatty acids are separated from the oil present in the light phase.

[0082] Item 7. The method of item 1, wherein less than 10%, preferably less than 5%, more preferably less than 2%, and most preferably less than 0.5% of the triglycerides present in the oil are hydrolyzed.

[0083] Item 8. The method of item 1, wherein the diglyceride concentration is reduced by at least 30%, more preferably at least 40%, and most preferably at least 50%.

[0084] Item 9. The method of item 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.

[0085] Item 10. The method of any one of items 1 to 9, wherein the oil is derived from one or more of, for example, algal oil, canola oil, palm oil, castor oil, copra oil, corn oil, distilled corn oil, cottonseed oil, flax 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 oil, tall oil, oils derived from halophytes, and / or animal fats including tallow from pigs, beef and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soda cake fatty acid material, yellow grease, used cooking oil, palm oil mill waste oil and brown grease or any combination thereof.

[0086] Item 11. The method according to any one of items 1 to 10, carried out at a temperature in the range of 10 to 100°C, preferably 20 to 90°C.

[0087] Item 12. The method according to any one of items 1 to 11, wherein the lipase is added in an amount ranging from 0.1 to 50,000 mg enzyme protein (EP) / kg oil.

[0088] Item 13. The method according to any one of items 1 to 12, wherein the lipase is in the form of a liquid product, an immobilized product, or a dry powder.

[0089] Item 14. The method of any one of items 1 to 13, wherein the total reaction time of the method is at least 15 minutes.

[0090] Item 15. The method of any one of items 1 to 14, wherein the total reaction time of the method is up to 48 hours.

[0091] Item 16. The method according to any one of items 1 to 15, wherein the amount of water added is between 0.01 and 100% (w / w) of the oil.

[0092] Item 17. The method of any one of items 1 to 16, wherein the pH is optionally adjusted during or before the hydrolysis to optimize the reaction.

[0093] Item 18. The method according to item 14, wherein the pH during hydrolysis is preferably between 3.0 and 7.0.

[0094] Item 19. The method of items 14 or 15, wherein the pH is adjusted preferably using citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide.

[0095] Item 20. The method of any one of items 1 to 19, carried out in a batch, semi-continuous, or continuous mode.

[0096] Item 21. The method of any one of items 1 to 20, further comprising adding one or more additional lipases and / or phospholipases during hydrolysis.

[0097] Item 22. The method of any one of items 1 to 21, further comprising the presence of an organic solvent during the reaction.

[0098] Item 23. The method of item 22, wherein the organic solvent is acetone, hexane, or heptane. [Example]

[0099] SEQ ID NO: 1 of the present invention is shown as SEQ ID NO: 1 in WO2008065060.

[0100] SEQ ID NO: 2 of the present invention is shown as SEQ ID NO: 2 in WO2011067349.

[0101] Example 1: Surprising effects of SEQ ID NO: 1 Heat palm stearin until fully melted. Weigh out required amount, add required weight of water and incubate the mixture at 60°C. Add required dose of lipase of SEQ ID NO: 1. Allow to react with mixing at 60°C. After sampling and centrifugation at 2000g for 5 minutes, take the light phase and analyze the oil for wt% FFA by titration (AOCS5a-40, Free Fatty Acids in Crude and Refined Fats and Oils).

[0102] [Table 1]

[0103] [Table 2]

[0104] FFAs increase very rapidly initially, suggesting that mono- and diglycerides are converted rapidly initially. Then, over the course of 20 hours of reaction, a significant amount of triglycerides is converted. When hydrolyzing palm stearin, one would not expect 22 wt% FFAs to result from the hydrolysis of mono- and diglycerides alone. Therefore, triglycerides must have been converted, especially in Tests 1 and 2 above, where glycerol was not initially present. The lower conversion rate in the presence of glycerol is due to the equilibrium between hydrolysis and the re-esterification of fatty acids with glycerol.

[0105] These results demonstrate the general recognition in the industry that SEQ ID NO: 1 has a significant effect on triglycerides, which is why SEQ ID NO: 1 is not considered a viable enzyme for the present application. SEQ ID NO: 1 is commonly used, for example, as an enzymatic catalyst for the production of triglycerides by esterification of FFAs with glycerol in the opposite reaction direction to the same hydrolysis reaction.

[0106] The inventors have found that ordinary use of SEQ ID NO: 1 would be expected to have significant activity toward triglycerides, especially when sufficiently high temperatures are combined with high amounts of water, long reaction times, and relatively high amounts of enzyme. It is therefore surprising that SEQ ID NO: 1 can be reacted with diglycerides with an unfathomably low activity toward triglycerides.

[0107] Example 2: Hydrolysis of diglycerides Crude palm oil (CPO) is heated to 70°C until fully melted. The required oil is weighed into a 250 mL Schott square bottle. The required weight of water is added and the mixture is incubated at 50°C or 60°C with stirring. The required dose of lipase of SEQ ID NO: 1 is added. The reaction is carried out in a water bath at 60°C with a stirrer at 350 rpm. Samples are taken into test tubes after 4 and 24 hours. After sampling and centrifugation at 2000g for 5 minutes, the light phase is collected and the oil is analyzed for %FFA by titration and for the content of mono- and diglycerides by GC and TG profile by GC (AOCS5a-40, Free Fatty Acids in Crude and Refined Fats and Oils, and AOCS Ce5-86, Triglycerides by Gas Chromatography).

[0108] [Table 3]

[0109] [Table 4]

[0110] As shown in Table 4, T4, which used a higher water dosage of 2% at 50°C and 3.4 mg lipase / kg oil, achieved the lowest DG content, reducing it from 6.9 wt% in CPO to 2.4 wt% after 24 hours of reaction. For DG hydrolysis from 4 to 24 hours, 1% water appears to be too low for the system, where conversion stalled after 4 hours (for both FFA and DG), and presumably the free water available at that stage (fully utilized after 4 hours of reaction) was too low to promote further conversion. T2, which used a 10-fold higher enzyme dosage of 34 mg lipase / kg oil, was able to promote a more rapid DG reduction (from 6.9 wt% to 3.2 wt% after 4 hours). However, with the low amount of available free water, condensation occurred, increasing DG to 4.5 wt% after 20 hours of reaction. Comparing T1 and T3 (60 °C vs. 50 °C), the difference in FFA is almost the same, but it appears that lower DG values ​​can be achieved at 60 °C (4.7 wt% vs. 5.3 wt%). Referring to Table 5, the TG profile remained unchanged after enzymatic hydrolysis and showed no signs of interesterification, further substantiating the claim of little to no activity towards triglycerides.

[0111] Furthermore, it can be concluded that SEQ ID NO: 1 is effective in the specific hydrolysis of DG at a low dose of lipase without interacting with TG in the oil. In addition, it shows a significant and pronounced reduction in DG after about 4 hours.

[0112] [Table 5]

[0113] Example 3: Hydrolysis of DG using SEQ ID NO:2 Crude palm oil (CPO) is heated to melt thoroughly. The required amount is weighed out, 5% (wt / wt) water is added and the mixture is incubated at 75°C. The required dose of lipase of SEQ ID NO: 2 is added. React at 75°C with mixing. After sampling, heat to 99°C for 10 minutes and centrifuge at 2000g for 5 minutes, the light phase is collected and the oil is analyzed for %FFA by titration (AOCS5a-40, Free Fatty Acids in Crude and Refined Fats and Oils) and for mono- and diglycerides by a customized HPLC method.

[0114] [Table 6]

[0115] [Table 7]

[0116] Table 7 shows that SEQ ID NO: 2 significantly and markedly reduces DG.

[0117] Example 4: SEQ ID NO: 1 in combination with PLC (Quara Boost). Laboratory-scale enzymatic water degumming was carried out at 55°C and a total water content of 3 wt%. Two samples of crude soybean oil of varying qualities were used in this experiment (Table 8). The oil was preheated to 55°C, and 30 g portions were transferred to glass tubes. Enzyme and water were added accordingly, and the samples were sonicated at 50°C for 5 minutes to ensure adequate distribution of the enzyme and water and mixing into the oil phase. In the next step, the oil samples were placed in a warming cabinet and incubated at 55°C with gentle rotation for selected times. Sequential processing was investigated. In the first step, phospholipase C was added to the oil samples, and after 2 hours, SEQ ID NO: 1 was added to selected samples. After that, both PLC and SEQ ID NO: 1 were simultaneously present in the reaction mixture. After 24 hours, the enzymatic reaction was stopped by heating the oil samples to 95°C for 10 minutes. In control samples, phospholipase C alone was added for 24 hours, or SEQ ID NO: 1 alone was added for 22 hours. The gum and oil phases were separated by centrifugation at 600 g and 85°C for 6 minutes. The upper oil phase was transferred to a new tube and retained for analysis. Diglycerides (DG, wt%) and free fatty acids (FFA, wt%) were analyzed in the oil phase. DG was analyzed according to AOCS official method Cd11d-96 on a Dionex Ultimate 3000 HPLC system equipped with a Corona detector, using a HypersilGold Silica 3 μm, 150 x 4.6 mm column. FFA was analyzed by NaOH titration according to AOCS official method Ca5a-40. Phospholipids were analyzed in crude oil samples. 31 The product was analyzed by P NMR.

[0118] [Table 8]

[0119] Table 8 shows that combining SEQ ID NO:1 with a PLC-type phospholipase results in oils with reduced DG levels compared to conventional PLC degumming without the lipase of SEQ ID NO:1. PLC-type phospholipases convert phospholipids to diglycerides, liberating the phosphate side groups. Such phospholipid conversion is a well-known type of enzymatic degumming and provides increased yields over traditional non-enzymatic degumming methods, such as water / acid degumming. As a result of PLC catalysis, diglyceride levels can sometimes increase to problematic levels, and therefore it is desirable to combine PLC with an enzyme active on diglycerides, such as SEQ ID NO:1.

[0120] Example 5: SEQ ID NO: 1 as a liquid and immobilized formulation 50 g of the same CPO as in Example 3 above was used. 4% (wt / wt) 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 100 mL blue-capped square bottle in a shaking incubator with stirring at 250 rpm. After sampling, heating to 99°C for 10 minutes and centrifugation at 2000g for 5 minutes was used to collect the light phase, and the oil was analyzed for % FFA by titration (AOCS5a-40, Free Fatty Acids in Crude and Refined Fats and Oils) and for monoglycerides and diglycerides by a customized HPLC method.

[0121] [Table 9]

[0122] Comparing the results, the liquid formulations provide faster reaction rates than the immobilized formulations. The lipases of SEQ ID NOs: 1 and 2 can be used both as liquid, dry or immobilized formulations.

[0123] Example 6: DG hydrolysis in the presence of solvent 60 g of CPO containing 4.4 wt% FFA, 0.6 wt% MG, and 4.9 wt% DG was used. 2% or 5% (wt / wt) water was added to this oil along with 15 g, 30 g, or 60 g of hexane. The mixture was preheated to 50°C. 0.25% (wt / wt of CPO) of a formulation of SEQ ID NO: 1 with 0.95 wt% active enzyme protein was added to the mixture. The reaction was carried out in a 250 mL blue-capped square bottle in a water bath with magnetic stirring at 500 rpm. After sampling, the mixture was heated to 99°C for 10 minutes. The hexane was evaporated from the sample under reduced pressure overnight. The samples are then centrifuged at 2000 g for 5 minutes and the light phase is then analysed for % FFA by titration (AOCS5a-40, Free Fatty Acids in Crude and Refined Fats and Oils) and for mono- and diglycerides by a customised HPLC method.

[0124] [Table 10]

[0125] DAG hydrolysis using a hexane / oil mixture carried out directly from the extraction step before removing the hexane and isolating the oil. The results above show that 50% hexane is preferred over 25% and 100% because it can hydrolyze the most DAG.

[0126] While we have shown and described what are presently considered to be the preferred embodiments of the present invention, it would 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. 1. A method for reducing and / or removing the diglyceride content of an oil without substantial transesterification of the triglycerides, comprising: a. providing an oil or fat; 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; A method comprising:

2. 10. The method of claim 1, further comprising the step of separating the light and heavy phases after hydrolysis.

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

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

5. 5. The method according to any one of claims 2 to 4, wherein the heavy phase is partially or completely recycled to the hydrolysis step.

6. 5. The method according to any one of claims 2 to 4, wherein free fatty acids are separated from the oil present in the light phase.

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

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

9. 2. 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. 10. The method of any one of claims 1 to 9, wherein the oil is derived from one or more of, for example, algal oil, canola oil, palm oil, castor oil, copra oil, corn oil, distilled corn oil, cottonseed oil, flax 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 oil, tall oil, oils derived from halophytes, and / or animal fats including tallow from pigs, beef and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soda cake fatty acid material, yellow grease, used cooking oil, palm oil mill waste oil and brown grease or any combination thereof.

11. A method according to any one of claims 1 to 10, wherein the amount of water added is between 0.01 and 100% (w / w) of the oil.

12. 12. The method according to any one of claims 1 to 11, wherein the pH is optionally adjusted during or before the hydrolysis to optimize the reaction.

13. 13. The method of any one of claims 1 to 12, further comprising adding one or more additional lipases and / or phospholipases during hydrolysis.

14. The process of any one of claims 1 to 13, further comprising the presence of an organic solvent during the reaction.

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

Images