Process for degumming a vegetable oil

Abstract

A process for degumming a vegetable oil comprising the steps of: a) providing a vegetable oil containing a quantity of phospholipids, b) contacting the oil with one or more phospholipases in presence of water, c) separating the reaction mixture of step b) into a light phase and a heavy phase, and d) producing a degummed vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1, wherein more than 75% or 85% or 90% of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is hydrolyzed, and more than 25% or 35% or 50% of phosphatidic acid (PA) is hydrolyzed and wherein the process is carried out at a temperature of 70°C or above.

Description

[0001] PROCESS FOR DEGUMMING A VEGETABLE OIL

[0002] REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] Field of the Invention

[0005] The present invention relates to a process for degumming vegetable oil. The present invention relates to the field of oil processing, and more specifically to an enzymatic process for degumming vegetable oils. The invention pertains to the use of phospholipases for the selective hydrolysis of phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidic acid (PA) in vegetable oils under elevated temperature conditions, thereby improving oil quality.

[0006] BACKGROUND OF THE INVENTION

[0007] Vegetable oils, such as oils from soybean, sunflower and rapeseed, must be refined to remove the impurities in order for them to be suitable for direct human consumption. Some of the impurities, such as seed fragments and meal fines, are oil insoluble and thus can be readily removed by filtration. Others, including free fatty acids, hydrocarbons, ketones, tocopherols, glycolipids, phytosterols, phospholipids, proteins, pigments, and resins, are soluble or form stable colloidal suspensions in the oil. Most of these have an unfavourable effect on the flavour, odour, appearance, and / or shelf life of the oil, and therefore have to be removed from the oils by chemical or physical refining processes.

[0008] In particular, phospholipids pose many problems for the storage and processing of the crude oil and can be removed from oil by processes such as water or wet degumming, acid degumming, caustic refining and enzymatic degumming or refining.

[0009] Various processes are known for enzymatic degumming or enzymatic refining of oils using enzymes such as for example enzymes having phospholipase A1 or A2 activity, phospholipase C activity or phosphatidyl inositol phospholipase C activity.

[0010] Several types of phospholipases are known which differ in their specificity according to the position of the bond hydrolyzed in the phospholipid molecule. Phospholipase A1 (PLA1) removes the sn1-position fatty acid to produce free fatty acid and 2-acyl-1-lysophospholipid. Phospholipase A2 (PLA2) removes the sn2-position fatty acid to produce free fatty acid and 1- acyl-2-lysophospholipid. The term phospholipase B (PLB) is used for phospholipases having both A1 and A2 activity. Phospholipase C (PLC) removes the phosphate moiety to produce 1 ,2 diacylglycerol and phosphate ester. Phospholipase D (PLD) produces 1 ,2-diacylglycero- phosphate and base group Before consumption vegetable oils are degummed to provide refined storage stable vegetable oils of neutral taste and light color. The degumming process comprises removing the phospholipid components (the gum) from the triglyceride rich oil fraction. The most commonly used processes in the industry are water degumming, chemical / caustic refining and physical refining including acid assisted degumming and / or enzyme assisted degumming. Due to the emulsifying properties of the phospholipid components, the degumming procedure has resulted in a loss of oil; i.e. of triglycerides.

[0011] Enzymatic degumming reduces the oils loss due to an efficient hydrolysis of the phospholipids which decrease the emulsifying properties. For a review on enzymatic degumming see Dijkstra 2010 Eur. J. Lipid Sci. Technol. 112, 1178. The use of Phospholipase A and / or phospholipase C in degumming is for example described in Clausen 2001 Eur J Lipid Sci Techno 103 333-340.

[0012] W02007 / 059927 describes a thermostable Bacillus PLC for degumming.

[0013] WO2012 / 062817 describes a fungal PLC with specificity towards all four phospholipids.

[0014] WO 2018 / 171552 describes a process for refining of vegetable oil in which phospholipids present in the vegetable oil are hydrolysed and the oil is subsequently subject to chemical refining.

[0015] US 2019 / 284503 describes a method of reducing the phospholipid content in an oil or fat composition and polypeptides having Pl-specific phospholipase C activity as well as polypeptides having PC, PE-specific phospholipase C activity and combinations thereof.

[0016] US 2013 / 011887 describes phospholipase enzymes, polynucleotides encoding the enzymes, methods of making and using these polynucleotides and polypeptides. In alternative embodiments, the invention provides phosphatidylinositol- specific phospholipase C (PI-PLC) enzymes, nucleic acids encoding them, antibodies that bind specifically to them, and methods for making and using them.

[0017] WO 2023 / 135541 describes mutated phospholipase C enzyme and its application in oil degumming.

[0018] There is a need for further enzymes having phospholipase C activity and suitable for application in enzymatic degumming of edible oils.

[0019] Summary of the Invention

[0020] The present invention relates to a process for degumming a vegetable oil, comprising the steps of: a) providing a vegetable oil containing a quantity of phospholipids, b) contacting the oil with one or more phospholipases in presence of water, c) separating the reaction mixture of step b) into a light phase and a heavy phase, and d) producing a degummed vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1 ; wherein more than 75% or 85% or 90% of phosphatidylcholine (PC) and phosphatidylethanolamine(PE) is hydrolyzed; wherein more than 25% or 35% or 50% of phosphatidic acid (PA) is hydrolyzed; and wherein the process is carried out at a temperature of 70°C or above.

[0021] A key feature of the invention is the use of a phospholipase enzyme having at least 75% sequence identity to SEQ ID NO: 1 , which ensures high specificity and efficiency in hydrolyzing phospholipids. The process achieves hydrolysis of more than 75%, 85%, or 90% of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and more than 25%, 35%, or 50% of phosphatidic acid (PA). The reaction is carried out at a temperature of 70°C or above, which promotes optimal enzyme activity and facilitates effective phase separation.

[0022] This invention offers significant advantages over conventional degumming methods by enabling more complete removal of phospholipids, reducing oil loss, and improving the physical and chemical properties of the final oil product.

[0023] SEQUENCE OVERVIEW

[0024] SEQ ID NO: 1 is a phospholipase obtained from Bacillus macauensis.

[0025] SEQ ID NO: 2 is a phospholipase obtained from Pseudomonas sp.

[0026] SEQ ID NO: 3 is a phospholipase obtained from Bacillus thuringiensis.

[0027] Definitions

[0028] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] 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.

[0030] 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.

[0031] Reference to “about” a value or parameter herein includes embodiments that are directed to that value or parameter perse. 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.

[0032] 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.

[0033] 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.

[0034] Alkali: In the present context “alkali” refers interchangeably to a base that is soluble in water and forms hydroxide ions, such as NaOH, KOH, sodium carbonate, Ca(OH)2, and Mg(OH)2and to the solution of a base in water.

[0035] Bleaching: The term “bleaching” refers to the process for removing color producing substances and for further purifying the fat or oil. Normally, bleaching is accomplished after the oil has been refined.

[0036] Chemical refining: In the present application, the term “chemical refining” is used synonymously with “alkali refining” and “alkaline refining”; the term also covering “caustic refining” and ’’caustic neutralization”.

[0037] Crude oil: The term “crude oil” refers to (also called a non-degummed oil) a pressed or extracted oil or a mixture thereof from, e.g. vegetable sources, including but not limited to acai oil, almond oil, babassu oil, blackcurrent seed oil, borage seed oil, canola oil, cashew oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil, flax seed oil, grape seed oil, hazelnut oil, hempseed oil, jatropha oil, jojoba oil, linseed oil, macadamia nut oil, mango kernel oil, meadowfoam oil, mustard oil, neat's foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, pecan oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil, tsubaki oil, walnut oil, varieties of "natural" oils having altered fatty acid compositions via Genetically Modified Organisms (GMO) or traditional "breading" such as high oleic, low linolenic, or low saturated oils ( high oleic canola oil, low linolenic soybean oil or high stearic sunflower oils). Degummed oil: The term "degummed oil" refers to an oil obtained after removal of nonhydratable phospholipids, hydratable phospholipids, and lecithins (known collectively as "gums") from the oil to produce a degummed oil or fat product that can be used for food production and / or non-food applications, e.g. biodiesel.

[0038] Gum: In the context of the present invention “gum”, “gums” or “gum fraction” refers to a fraction enriched in phosphatides, which is separated from the bulk of vegetable oil during a degumming process. “Gums” consist mainly of phosphatides but also contain entrained oil, contain nitrogen and sugar and meal particles.

[0039] Lysophospholipase: A “lysophospholipase” (EC 3.1.1.5) is an enzyme that can hydrolyze 2-lysophospholids to release fatty acid.

[0040] Phospholipase: The term "phospholipase" refers to an enzyme that hydrolyses phospholipids into fatty acids (saturated or unsaturated), lysophospholipids, diacylgycerols, choline phosphate and phophatidates, depending on the site of hydrolysis. Phospholipases are further classified into types A, B, C and D.

[0041] Phospholipase activity: In the context of the present invention, the term “phospholipase activity” refers to the catalysis of the hydrolysis of a glycerophospholipid or glycerol-based phospholipid.

[0042] Phospholipase A activity: In the context of the present invention the term “phospholipase A activity” comprises enzymes having phospholipase A1 and / or phospholipase A2 activity (A1 or A2, EC 3.1.1.32 or EC 3.1.1.4), i.e., hydrolytic activity towards one or both carboxylic ester bonds in phospholipids such as lecithin. A phospholipase having both A1 and A2 activity is also referred to as a phospholipase B.

[0043] Phospholipase Al and A2 catalyze the deacylation of one fatty acid group in the snl and sn2 positions, respectively. Hence, phospholipase Al (also sometimes referred to herein as PLA1) hydrolyzes the l-acyl group of a phospholipid, hydrolyzing the bond between the fatty acid and the glycerin residue at the one position. Phospholipase A2 (also sometimes referred to herein as PLA2) catalyzes hydrolysis of the 2-acyl group.

[0044] The term “Phospholipase B” cleaves both SN-1 and SN-2 acyl chains.

[0045] The term “Phospholipase C” cleaves before the phosphate, releasing diacylglycerol and a phosphate-containing head group.

[0046] The term “Phospholipase D” cleaves after the phosphate, releasing phosphatidic acid and an alcohol.

[0047] Phospholipase C activity: The term “phospholipase C activity” or “PLC activity” relates to an enzymatic activity that removes the phosphate ester moiety from a phospholipid to produce a 1 ,2 diacylglycerol (see Figure 1). Most PLC enzymes belong to the family of hydrolases and phosphodiesterases and are generally classified as EC 3.1.4.3, E.C. 3.1.4.11 or EC 4.6.1.13. Phospholipase C activity may be determined according to the procedure described in the materials and method section.

[0048] Phospholipase C specificity: The term “phospholipase C specificity” relate to a polypeptide having phospholipase C activity where the activity is specified towards one or more phospholipids, with the four most important once being phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidyl inositol (PI). Phospholipase C specificity may be determined by31P-NMR as described above in relation to the term “phospholipase activity”.

[0049] PC- and PE-specific phospholipase C: The terms “PC- and PE-specific phospholipase C” and “phospholipase C having specificity for phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE)” and “polypeptide having activity towards phosphatidylcholine (PC) and phosphatidylethanolamine (PE)” are used interchangeably. They relate to a polypeptide having activity towards phosphatidylcholine (PC), phosphatidylethanolamine (PE). In addition to the PC and PE specificity it may also have some activity towards phosphatidic acid (PA) and phosphatidyl inositol (PI).

[0050] Pl-Specific Phospholipase C: The terms “Pl-specific phospholipase C”, “Phosphatidylinositol phospholipase C” and “polypeptide having activity towards phosphatidylinositol (PI)” are used interchangeably. They relate to a polypeptide having activity towards phosphatidyl inositol (PI), meaning that its activity towards phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) is low compared to the PI activity. Plspecific phospholipase C enzymes can either belong to the family of hydrolases and phosphodiesterases classified as EC 3.1 .4.11 or to the family of lyases classified as EC 4.6.1 .13.

[0051] PC-, PE-, PA- and Pl-Specific Phospholipase C: The terms “PC-, PE-, PA,- and Plspecific phospholipase C”, and “polypeptide having activity towards phosphatidylcholine (PC), phosphatidylethanoamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI)” are used interchangeably. They relate to a polypeptide having activity towards phosphatidylcholine (PC), phosphatidylethanoamine (PE), phosphatidic acid (PA), and phosphatidyl inositol (PI).

[0052] Sequence identity:

[0053] OPTION 1 :

[0054] Sequence identity: For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0055] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0056] OPTION 2:

[0057] 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 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The percent sequence identity is calculated as follows:

[0058] (Identical Residues x 100) / (Length of the Shortest Sequence in the Alignment)

[0059] The sequence identity between two polynucleotide sequences can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The percent sequence identity is calculated as follows:

[0060] (Identical Deoxyribonucleotides x 100) / (Length of the Shortest Sequence in the Alignment) OPTION 3:

[0061] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using 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 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The percent identity is calculated as follows:

[0062] (Identical Residues x 100) / (Length of the Alignment)

[0063] The sequence identity between two polynucleotide sequences can be determined using the same Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The percent sequence identity is calculated as follows: (Identical Deoxyribonucleotides x 100) / (Length of the Alignment)

[0064] Water degumming: The term “water degumming” refers to a process which involves treating crude oil with an amount of water to hydrate phospholipids present in the oil and make them separable by centrifugation.

[0065] The term "degummed oil" refers to an oil obtained after partial- or full removal of phospholipids. Such as removal of non-hydratable phospholipids, hydratable phospholipids, and lecithin’s (known collectively as "gums") from the oil to produce a degummed oil or fat product that can be used for food production and / or non-food applications, e.g. biodiesel. “Water degummed oil” for example relates to an oil, which has only been partially degummed by water washing, which leads to substantial reduction in the concentration of hydratable phospholipids only.

[0066] The term “phosphatides” relates mainly to intact phospholipids, but it might refer to partially hydrolyzed phospholipids as lyso-phospholipids, and glycerol-phosphoesters.

[0067] Detailed Description of the Invention

[0068] The invention is directed to a process for producing degummed vegetable oil using a phospholipase that is thermostable at temperature of 70°C or above.

[0069] In a first aspect, the invention relates to a process for degumming a vegetable oil, comprising the steps of: a) providing a vegetable oil containing a quantity of phospholipids, b) contacting the oil with one or more phospholipases in presence of water, c) separating the reaction mixture of step b) into a light phase and a heavy phase, and d) producing a degummed vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1 , wherein more than 75% or 85% or 90% of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is hydrolyzed, and more than 25% or 35% or 50% of phosphatidic acid (PA) is hydrolyzed and wherein the process is carried out at a temperature of 70°C or above.

[0070] In an embodiment of the present invention, the phospholipase comprises a phospholipase C, and / or phosphatidylinositol-specific phospholipase C.

[0071] In an embodiment of the present invention, the phospholipase is selected from a group consisting of phospholipase C having specificity for Phosphatidylinositol (PI), phospholipase C having specificity for phosphatidyl choline (PC) and Phosphatidyl ethanolamine (PE) and phospholipase C having specificity for Phosphatidyl choline (PC), Phosphatidyl ethanolamine (PE) Phosphatidic acid (PA) and Phosphatidylinositol (PI).

[0072] In an embodiment of the present invention, the phospholipase C hydrolyses phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and / or phosphatidic acid. In an embodiment of the present invention, the phospholipase has at least at least about

[0073] 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent,

[0074] 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent,

[0075] 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 1.

[0076] The phospholipase of the present invention cleaves phospholipids (phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl inositol (PI)) just before the phosphate group into diglyceride or phosphate ester. The diglyceride stays in the oil phase (improving oil yield) and the phosphorous-containing moieties separates into the aqueous phase where they are removed as components of the heavy phase during separation e.g. by centrifugation.

[0077] The phospholipases of the invention can be used in the industrial application of enzymatic degumming of triglyceride oils as described, e.g., in EP 513 709.

[0078] In an embodiment of the present invention, the oil is selected from crude oil, water degummed oil, caustic refined oil, acid degummed oil and combination thereof.

[0079] In an embodiment of the present invention, the oil is crude oil or water degummed oil.

[0080] The water-degumming of crude oil or fat may be achieved by thoroughly mixing hot water and warm oil or fat having a temperature of between 50°C to 90°C for 30 to 60 minutes. This process serves to partially remove the hydratable phospholipids. Also, an acid treatment may be performed before the enzymatic degumming, where the acid used is selected from the group consisting of phosphoric acid, acetic acid, citric acid, tartaric acid, succinic acid, and mixtures thereof, in particular a treatment using citric acid or phosphoric acid are preferred. The acid treatment is preferably followed by a neutralization step to adjust the pH between about 4.0 to 7.0, more preferably from 4.5 to 6.5, preferably using sodium hydroxide (NaOH) or potassium hydroxide (KOH). The acid treatment serves to chelate metals bound to the phospholipids hereby making a more hydratable form. Preferably, the phospholipase as described herein are added after water degumming or acid treatment of the oil. It is also possible to perform the degumming step using the phospholipases as described herein on a crude oil or fat, i.e. an oil or fat not previously water degummed or acid treated.

[0081] In an embodiment of the present invention, the phospholipase is provided in the form of an aqueous solution, granular, liquid, powder and / or immobilized on a carrier or carrier particles.

[0082] In an embodiment of the present invention, the vegetable oil comprises canola oil, corn oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, sesame oil, soybean oil, or sunflower seed oil.

[0083] In an embodiment of the present invention, the oil comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl inositol (PI) and or phosphatidic acid (PA). In another embodiment of the present invention, the phospholipases may be added into a physical refining process applying citric acid or phosphoric acid and sodium hydroxide to facilitate hydratability of insoluble phospholipids and ensure an environment suitable for the enzyme with preferably less than 0.15% citric acid or phosphoric acid.

[0084] The phospholipase of the invention may be incorporated into either water degumming or a chemical or physical oil refining process.

[0085] In an embodiment of the present invention, the phospholipase is incorporated into a water degumming process, caustic refining process or acid degumming process.

[0086] In an embodiment of the present invention, the process further comprises adding an acid, chelating agent, alkali, or combinations thereof to vegetable oil prior to step a).

[0087] In an embodiment of the present invention, the alkali is selected from sodium hydroxide, potassium hydroxide, sodium silicate, sodium carbonate, calcium carbonate, sodium bicarbonate, ammonia, or sodium citrate, and combinations thereof.

[0088] In an embodiment of the present invention, the acid is selected from phosphoric acid, acetic acid, citric acid, tartaric acid, and succinic acid, or a combination thereof.

[0089] In another aspect of the present invention, the process for reducing the content of phospholipids in an oil, the process comprising a) contacting said oil with phospholipase, under conditions sufficient for the phospholipase to react with the phospholipids to create diglyceride and phosphate ester, and; b) separating the phosphate ester from the oil.

[0090] In one embodiment of the present invention, the oil provided for treatment with the phospholipase thereof contains a quantity of phospholipids.

[0091] Phospholipids are commonly measured in oil as "phosphorous content" in parts per million.

[0092] Table 1 sets forth the typical amounts of phospholipids present in the major oilseed crops, and the distribution of the various functional groups as a percentage of the phospholipids present in the oil.

[0093] Table 1 : Typical levels and phospholipid distributions for common oilseeds

[0094] The processes of the invention can be used to achieve a more complete degumming of high phosphorous oils, e.g., an oil with more than 10 ppm of phosphorous, preferably more than 20 ppm, 30ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 80 ppm, 90 ppm even more preferred the oil contains more than 100 ppm phosphorous. Preferably, the oil for treatment in a process of the present invention comprises phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl inositol (PI).

[0095] In one embodiment of the present invention, the oil contains more than 10 ppm phosphorous originating from phosphatidyl inositol (PI), more preferably it contains more than 20 ppm, 30 ppm, 40 ppm phosphorus originating from PI.

[0096] In one embodiment of the present invention, the oil contains more than 10 ppm phosphorous originating from phosphatidylcholine (PC), more preferably it contains more than 20 ppm, 30 ppm, 40 ppm phosphorous originating from PC.

[0097] In one embodiment of the present invention, the oil contains more than 10 ppm phosphorous originating from phosphatidylethanolamine (PE), more preferably it contains more than 20 ppm, 30 ppm, 40 ppm phosphorous originating from PE.

[0098] In one embodiment of the present invention, the oil contains more than 10 ppm phosphorous originating from phosphatidic acid (PA), more preferably it contains more than 20 ppm, 30 ppm, 40 ppm phosphorous originating from PA.

[0099] In one embodiment of the present invention, the process of enzymatic degumming under conditions of low water, e.g., in the range of between about 0.1 % to 20 % water or 0.5% to 10% water.

[0100] In an embodiment of the present invention, the quantity of water is at least about 1.5 percent by weight of the total mixture.

[0101] In one embodiment of the present invention, the separation of a heavy phase from the oil phase during centrifugation. The improved separation of these phases can result in more efficient removal of phospholipids from the oil, including both hydratable and nonhydratable phospholipids.

[0102] In an embodiment of the present invention, the process further comprises adding phospholipase C to gums from water degummed oil to enable de-oiling of gums.

[0103] In an embodiment of the present invention, one or more phospholipases are added to gums.

[0104] In an embodiment of the present invention, the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent,

[0105] 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent,

[0106] 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent,

[0107] 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 1.

[0108] In an embodiment of the present invention, the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent,

[0109] 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent,

[0110] 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent,

[0111] 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 2.

[0112] In an embodiment of the present invention, the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent,

[0113] 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent,

[0114] 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent,

[0115] 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 3.

[0116] In an embodiment of the present invention, the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent,

[0117] 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent,

[0118] 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent,

[0119] 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 2 and 3.

[0120] In an embodiment of the present invention, the phospholipase C comprises or consists of amino acid as shown in SEQ ID NO: 1 .

[0121] In an embodiment of the present invention, the phospholipase C comprises or consists of amino acid as shown in SEQ ID NO: 2.

[0122] In an embodiment of the present invention, the phospholipase C comprises or consists of amino acid as shown in SEQ ID NO: 3.

[0123] In one aspect of the present invention, the separation of gum fraction that contains less entrained neutral oil (triglycerides), thereby improving the overall yield of oil during the process.

[0124] In one aspect of the present invention, phospholipase of the invention is used to treat oils to reduce gum mass and increase neutral oil gain through reduced oil entrapment. In one aspect, phospholipases of the invention e.g., a polypeptide having PLC activity, are used for diacylglycerol (DAG) production and to contribute to the oil phase.

[0125] The gum phase (heavy phase) may be treated further with a phospholipase of the present invention to increase hydrolysis of phospholipids in the gum fraction from water degumming to release entrapped triglyceride oil. This is particular useful when the degumming process has not already applied phospholipases.

[0126] The phospholipase treatment can be conducted by dispersing an aqueous solution of the phospholipase, preferably as droplets with an average diameter below 10 microM. The amount of water is preferably 0.5-5% by weight in relation to the oil. An emulsifier may optionally be added. Mechanical agitation may be applied to maintain the emulsion. Agitation may be done with a high shear mixer with a tip speed above 1400 cm / s.

[0127] In an embodiment, the oil is contacted with 0.5-200 mg enzyme protein (EP) / Kg oil of said phospholipase; such as with 0.5-100 mg enzyme protein (EP) / Kg oil of said phospholipase, with 0.5-25 mg enzyme protein (EP) / Kg oil of said phospholipase, with 0.5-15 mg enzyme protein (EP) / Kg oil of said phospholipase, with 0.5-10 mg enzyme protein (EP) / Kg oil of said phospholipase, with 0.5-5 mg enzyme protein (EP) / Kg oil of said phospholipase, with 1-200 mg enzyme protein (EP) / Kg oil of said phospholipase, with 1-100 mg enzyme protein (EP) / Kg oil of said phospholipase, with 1-25 mg enzyme protein (EP) / Kg oil of said phospholipase, with 1-15 mg enzyme protein (EP) / Kg oil of said phospholipase, with 1-10 mg enzyme protein (EP) / Kg oil of said phospholipase, with 1-5 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-200 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-100 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-50 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-25 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-15 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-10 mg enzyme protein (EP) / Kg oil of said phospholipase, with 2-7 mg enzyme protein (EP) / Kg oil of said phospholipase, or with 2-5 mg enzyme protein (EP) / Kg oil of said phospholipase.

[0128] In an embodiment of the present invention, the process is performed in a batch, semi- continuous or continuous mode.

[0129] In an embodiment of the present invention, the phospholipase treatment may be conducted batch wise, e.g., in a tank with stirring, or it may be continuous, e.g., a series of stirred tank reactors. The phospholipase treatment may be followed by separation of an aqueous phase and an oil phase. The separation may be performed by conventional means, e.g., centrifugation. When phospholipase is used the aqueous phase will contain phospholipase, and the phospholipase may be re-used to improve the process economy.

[0130] In an embodiment, the total phosphorous content of the oil is reduced to preferably below 200 ppm, below 100 ppm, below 50 ppm, below 40 ppm, 30 ppm, 20 ppm, 15 ppm, more preferably below 10 ppm, below 9 ppm, below 8 ppm, below 7 ppm, below 6 ppm, most preferably below 5 ppm.

[0131] In an embodiment of the present invention, step b) of the process further comprises contacting the oil with an enzyme selected from a group consisting of cellulases, endoglucanases, cellobiohydrolases, xylanase, mannanases, hemicellulases, pectinases, proteases, phytases, chlorophyllases, and combination thereof.

[0132] In an embodiment of the present invention, step b) of the process further comprises contacting the oil with an enzyme selected from a group consisting of phospholipase A1 , phospholipase A2, lyso-phospholipase, and combination thereof. In an embodiment, the process further comprising refining the degummed vegetable oil.

[0133] In an embodiment, the process is carried out at a temperature of 70°C or above, preferably 75°C or above, more preferably 80°C or above, even more preferably 85°C or 90°C or above.

[0134] In an embodiment, the phospholipase has a maximum of its phospholipase activity at a pH in the range of 4.0 to 9.0, preferably in the range of 4.5 to 8.0.

[0135] In an embodiment, the phospholipase (activity) is thermotolerant or thermostable.

[0136] In an embodiment, the phospholipase retains a phospholipase activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to about 85°C, between about 60°C to about 95°C.

[0137] In another embodiment of the present invention, use of a phospholipase in degumming a vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1. In embodiment of the present invention, the phospholipase retains a phospholipase activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to 85°C, between about 60°C to about 95°C.

[0138] In embodiment of the present invention, the phospholipase has a maximum of its phospholipase activity at a pH in the range of 4.0 to 9.0, preferably in the range of 4.5 to 8.0.

[0139] The invention is further disclosed in the following numbered paragraphs.

[0140] Paragraph 1 . A process for degumming a vegetable oil, comprising the steps of: a) providing a vegetable oil containing a quantity of phospholipids; b) contacting the oil with one or more phospholipases in presence of water; c) separating the reaction mixture of step b) into a light phase and a heavy phase; and d) producing a degummed vegetable oil; wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1 , wherein more than 75% or 85% or 90% of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is hydrolyzed, more than 25% or 35% or 50% of phosphatidic acid (PA) is hydrolyzed and wherein the process is carried out at a temperature of 70°C or above.

[0141] Paragraph 2. The process according to paragraph 1 , wherein the phospholipase comprises a phospholipase C, and / or a phosphatidylinositol-specific phospholipase C.

[0142] Paragraph 3. The process according to paragraph 1 , wherein the phospholipase is selected from a group consisting of phospholipase C having specificity for Phosphatidylinositol (PI), phospholipase C having specificity for phosphatidyl choline (PC) and Phosphatidyl ethanolamine (PE) and phospholipase C having specificity for Phosphatidyl choline (PC), Phosphatidyl ethanolamine (PE) Phosphatidic acid (PA) and Phosphatidylinositol (PI).

[0143] Paragraph 4. The process according to paragraph 1 , wherein the phospholipase C hydrolyses phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and / or phosphatidic acid. Paragraph 5. The process according to any of the preceding paragraphs, wherein the vegetable oil is selected from crude oil, water-degummed oil, caustic refined oil, acid degummed oil and combination thereof.

[0144] Paragraph 6. The process according to paragraph 5, wherein the vegetable oil is crude oil or water-degummed oil.

[0145] Paragraph 7. The process according to any one of the paragraphs 1 to 6, further comprisising adding an acid, chelating agent, alkali, or combinations thereof to the vegetable oil prior to step a).

[0146] Paragraph 8. The process according to paragraph 7, wherein the alkali is selected from sodium hydroxide, potassium hydroxide, sodium silicate, sodium carbonate, calcium carbonate, sodium bicarbonate, ammonia, or sodium citrate, and combinations thereof.

[0147] Paragraph 9. The process according to paragraph 7, wherein the acid is selected from phosphoric acid, acetic acid, citric acid, tartaric acid, and succinic acid, or a combination thereof.

[0148] Paragraph 10. The process according to any of the preceding paragraphs, wherein step b) further comprises contacting the oil with an enzyme selected from a group consisting of cellulases, endoglucanases, cellobiohydrolases, xylanase, mannanases, hemicellulases, pectinases, proteases, phytases, chlorophyllases, and combination thereof.

[0149] Paragraph 11 . The process according to any of the preceding paragraphs, wherein step b) further comprises contacting the oil with an enzyme selected from a group consisting of phospholipase A1 , phospholipase A2, lyso-phospholipase, phospholipase C and combination thereof.

[0150] Paragraph 12. The process according to preceding paragraphs, further comprising refining the degummed vegetable oil.

[0151] Paragraph 13. The process according to preceding paragraphs, wherein the vegetable oil comprises of canola oil, corn oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, sesame oil, soybean oil, or sunflower seed oil.

[0152] Paragraph 14. The process according to any of the preceding paragraphs, wherein the process is carried out at a temperature of 70°C or above, preferrably 75°C or above, more preferably 80°C or above or even more preferably 85°C or 90°C or above.

[0153] Paragraph 15. The process according to any of the preceding paragraphs, wherein the phospholipase is provided in the form of an aqueous solution granular, liquid, powder and / or immobilized on a carrier or carrier particles.

[0154] Paragraph 16. The process according to any of the preceding paragraphs, wherein the phospholipase has a maximum of its phospholipase activity at a pH in the range of 4.0 to 9.0, preferably in the range of 4.5 to 8.0.

[0155] Paragraph 17. The process according to any of the preceding paragraphs, wherein the phospholipase (activity) is thermotolerant or thermostable. Paragraph 18. The process according to any of the preceding paragraphs, wherein the phospholipase retains a phospholipase activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to about 85°C, between about 60°C to about 95°C.

[0156] Paragraph 19. The process according to paragraph 1 , wherein the phospholipase has at least about 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent,

[0157] 99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 1 .

[0158] Paragraph 20. The process according to any of the preceding paragraphs, wherein the oil comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl inositol (PI) and or phosphatidic acid (PA).

[0159] Paragraph 21. The process according to any of the preceding paragraphs, wherein the process further comprises adding phospholipase C to gums from water degummed oil to enable de-oiling of gums.

[0160] Paragraph 22. The process according to paragraph 21 , wherein the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is

[0161] 100 percent sequence identity to SEQ ID NO: 1 .

[0162] Paragraph 23. The process according to paragraph 21 , wherein the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is

[0163] 100 percent sequence identity to SEQ ID NO: 2.

[0164] Paragraph 24. The process according to paragraph 21 , wherein the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is

[0165] 100 percent sequence identity to SEQ ID NO: 3. Paragraph 25. The process according to paragraph 21 , wherein the phospholipase C has at least about 70 percent, at least about 71 percent, at least about 72 percent, at least about 73 percent, at least about 74 percent, at least about 75 percent, at least about 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or more, or is

[0166] 100 percent sequence identity to SEQ ID NO: 2 and 3.

[0167] Paragraph 26. The process according to any of the preceding paragraphs, wherein said quantity of water is at least about 1.5 percent by weight of the total mixture.

[0168] Paragraph 27. The process according to any of the preceding paragraphs, wherein the process further comprises recycling the heavy phase of step c) into step a).

[0169] Paragraph 28. The process according to any of the preceding paragraphs, wherein the phospholipase C comprises or consists of amino acid as shown in SEQ ID NO: 1 .

[0170] Paragraph 29. The process according to any of the preceding paragraphs, wherein the phospholipase C comprises or consists of amino acid as shown in SEQ ID NO: 2.

[0171] Paragraph 30. The process according to any of the preceding paragraphs, wherein the phospholipase C comprises or consists of amino acid as shown in SEQ ID NO: 3.

[0172] Paragraph 31. The process according to any of the preceding paragraphs, wherein the phospholipase is dosed in the range of 0.5 - 200 mg enzyme protein (EP) / kg of oil, such as 0.5- 100 mg enzyme protein (EP) / kg of oil, 0.5-50 mg enzyme protein (EP) / kg of oil, such as 0.5-10 mg enzyme protein (EP) / kg of oil.

[0173] Paragraph 32. The process according to any of the preceding paragraphs, wherein the process is performed in a batch, semi-continuous or continuous mode.

[0174] Paragraph 33. Use of a phospholipase in degumming a vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1 .

[0175] Paragraph 34. Use of phospholipase according to paragraph 33, wherein the phospholipase retains a phospholipase activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to about 85°C, between about 60°C to about 95°C.

[0176] Paragraph 35. Use of phospholipase according to paragraph 33 or 34, wherein the phospholipase has a maximum of its phospholipase activity at a pH in the range of 4.0 to 9.0, preferably in the range of 4.5 to 8.0.

[0177] The invention is further illustrated by the following examples.

[0178] Examples:

[0179] Materials and Methods Phospholipase C assay: Phospholipase C activity assay: Reaction mixtures comprising 10 microL of a 100 mM p-nitrophenyl phosphoryl choline (p-NPPC) solution in 100 mM Borax-HCI buffer, pH 7.5 and 90 microL of the enzyme solution are mixed in a microtiter plate well at ambient temperature. The microtiter plate was then placed in a microtiter plate reader and the released p-nitrophenol was quantified by measurement of absorbance at 410 nm. Measurements are recorded during 30 min at 1 minute intervals. Calibration curves in the range 0.01-1 microL / ml p-nitrophenol are prepared by diluting a 10 micromol / ml p-nitrophenol stock solution from Sigma in Borax-HCI buffer. One unit will liberate 1 .0 micromol / minute of p-NPPC at ambient temperature.

[0180] Example 1 : Degumming vegetable oils

[0181] Substrate: Five crude oils with various quality were used as shown in Table 2.

[0182] Table 2. Crude oils composition

[0183] Performance of the phospholipases were tested in a reaction assay that mimics industrial scale conditions. Laboratory scale enzymatic water degumming was performed at various temperatures (55-65-75-80-85°C) and 3% total water content. Oils were preheated to selected temperature and 40-100g portions were transferred into reaction tubes. In tests samples pH was adjusted by addition of NaOH or citric acid, depending on experimental conditions. Enzymes and water were added accordingly, and samples were sonicated for 5 min at selected temperature to ensure sufficient distribution and mixing of enzymes and water into oil phase. In next step, oil samples were placed in heating cabinet and incubated under gentle rotation, 20rpm, at selected temperature for a selected time. The enzymatic reaction was stopped after predefined time by heating oil samples to 99°C for 10min. In control samples only water was added. Sample of reaction mixture was taken for phospholipids analysis.

[0184] Gums and oil phase were separated by centrifugation at 600g and 85°C for 6 min. An upper light oil phase was transferred to fresh tubes and kept for analysis. A lower heavy phase of gums was kept for analysis.

[0185] Diglycerides (DG, %) and Free Fatty Acids (FFA, %) were analyzed in light oil phase. DG were analyzed by Dionex Ultimate3000 HPLC system with Corona detector, column: HypersilGold Silica 3 pm 150 x 4.6 mm, according to AOCS Official Method Cd 11 d-96. FFA were analyzed by NaOH titration according to the AOCS Ca 5a-40 official method. Phospholipids were analyzed in reaction mixture by31P NMR, using the following procedure: To the oil sample was added 0.5 ml internal standard (IS) solution, followed by 0.5 ml CDCI3and 0.5 ml Cs-EDTA buffer. The sample was shaken for 5min, and then centrifuged (tabletop centrifuge, 5 min) to get phase separation. The lower phase was transferred to a NMR- tube. P-NMR was performed with 128 scans and a delay time of 5 s. All signals were integrated. Assignments (approx, ppm): 1.5 (PA), -0.1 (PE), -0.6(PI), -0.8 (PC). The concentration of each species was calculated as “ppm P”, i.e., mg elemental P per kg oil sample. Hence, ppm P=l / l(IS)*n(IS)’M(P) / m(oil). The internal standard solution was 2 mg / mL triphenyl phosphate in methanol. The Cs-EDTA buffer was prepared as follows: EDTA (17.55 g) was dispersed in water (approx. 20 ml_). The pH was adjusted to 7.5 using 50% w / w CsOH. This gave a clear solution. Water was added up to 100 mL to give a concentration of 0.6 M EDTA.

[0186] Yield loss was determined by gums volume (g) and dry matter content (% w / w) in gums heavy phase. The relative oil loss content was calculated using the following equation: Yield loss= (Gums x Dry Matter Z100) x (100 / Oil) Where

[0187] Oil - initial volume of crude oil before degumming (g) Gums - Measured wet gum volume (g)

[0188] Dry Matter - The dry matter content in the gums (%)

[0189] Yield loss - The yield loss is an estimate of the oil loss volume (%)

[0190] Example 2: Effect of temperature on Enzymatic Water Degumming

[0191] Enzymatic (EWD) and water degumming (WD) of crude soybean oil having composition as shown in table 2 was performed at 55, 65 and 75°C, pH 6.3, reaction time 2h, and total water of 3%, according to the method described in Example 1 . The effect of phospholipase was shown as a difference between enzymatic water degumming and reference water degumming at selected temperature conditions. Yield loss reduction and diglycerides increase were key parameters measured in this experiment. The experiment shows significant improvement of oil yield at all selected temperatures (Table 3) this demonstrates that the phospholipase of SEQ ID NO: 1 (dose 330 ppm) and phospholipase of SEQ ID NO: 2 (17 ppm), work at broad range of temperatures relevant in water degumming.

[0192] Table 3. The effect of phospholipase of yield loss reduction and DAG increase

[0193] Example 3: Effect of reaction time on Enzymatic Water Degumming

[0194] Enzymatic (EWD) and water degumming (WD) of crude soybean oil as shown in table 2 was performed at 75°C, pH 6.3, and reaction time: 0.5, 1 and 2h, total water 3%, according to the method described in Example 1.

[0195] The effect of phospholipase was shown as a difference between enzymatic water degumming and reference water degumming at selected experimental conditions. Yield loss reduction, phospholipids hydrolysis and diglycerides increase were key parameters measured in this experiment. The experiment shows significant improvement of oil yield after all reaction times, and almost complete hydrolysis of phospholipids PE, PC and PI within 30min (Table 4), this demonstrates that phospholipase of SEQ ID NO:1 (dose 330 ppm) and phospholipase of SEQ ID NO: 2 (17 ppm), work at broad range of residence times relevant in water degumming. In addition, PA hydrolysis was observed, with complete hydrolysis after 2h of reaction at 75°C.

[0196] Table 4. The effect of incubation

[0197] Example 4: Enzymatic water degumming at various pH, temperature and reaction time conditions Second soybean oil as shown in table 2 with higher concentration of phospholipids was treated in enzymatic water degumming at two reaction temperatures: 75°C and 80°C, several pH conditions: pH 4, 5, 6, 7, 8 (adjusted by citric acid or sodium hydroxide), and reaction times of 0.5, 1 and 2h. The degumming was performed at total water concentration of 3% and according to the method described in the Example 1 . pH adjustment was used to simulate broad range of pH of water extracts common in rapeseeds and soybean oils and as well for known improvement of heavy phase separation at close to neutral pH. Desing of experiments (DOE) plan was used to map as many as possible conditions and included a comparison of two significantly different enzymes dosages of two enzymes used: the phospholipase of SEQ ID NO:1 (dose 330 ppm) and phospholipase of SEQ ID NO: 2 (17 ppm or 50 ppm).

[0198] The effect of phospholipase was shown as a difference between enzymatic water degumming and reference water degumming at selected experimental conditions. Phospholipids hydrolysis and diglycerides increase were key parameters measured in this experiment. The experiment shows significant effect of phospholipase at broad range of pH. The highest hydrolysis of phospholipids, including PA, was observed at pH 6 and 75°C (Table 5).

[0199] Table 5. Effect of the thermostable PLC SEQ ID NO:1 in combination with PLC-PI of SEQ ID NO: 2 on phospholipids hydrolysis under selected experimental conditions.

[0200] ★Combination of two enzymes SEQ ID NO: 1 + SEQ ID NO: 2 was tested Example 5: Enzymatic water degumming of crude rapeseed oil

[0201] A crude rapeseed oil as shown in table 2 with phospholipids concentration similar to soybean oil used in examples 2 and 3 was treated with four various dosages of thermostable phospholipase C SEQ ID NO:1. Enzymatic water degumming and water degumming were performed at optimal conditions identified in previous experiments, i.e. temperature 75°C, and pH 5.8 (pH close to 6), reaction time 30min or 2h, total water 3%, as described in the Example 1. The tested enzymes dosages were: 75, 100, 150 and 200ppm of the thermostable PLC SEQ ID NO: 1.

[0202] The effect of phospholipases shows a difference between enzymatic water degumming and reference water degumming at selected experimental conditions. Yield loss reduction, phospholipids hydrolysis and diglycerides increase were the key parameters measured in this experiment. In general, addition of the thermostable PLC SEQ ID NO:1 alone provides significant increase in DAG and yield loss reduction at all selected dosages and reaction times (Table 6). The highest yield loss reduction was observed after treatment at 100-200 ppm dose of the thermostable PLC SEQ ID NO: 1 , and 2 h reaction time, while for DAG % increase and phospholipids hydrolysis 75 ppm dose of the thermostable PLC SEQ ID NO:1 and 30min reaction time are sufficient. PE and PC were completely hydrolyzed within 30min and at low dose of the thermostable PLC SEQ ID NO: 1 , while PA hydrolysis was in range of 21-29% with highest hydrolysis at 200 ppm dose of the thermostable PLC SEQ ID NO:1 (Table 7).

[0203] Table 6. Effect of enzyme dose and reaction time on enzymatic water degumming

[0204] Table 7. Phospholipids hydrolysis under specifies experimental conditions and show as difference between crude oil and remaining phospholipids.

[0205] Example 6: A comparison of Water Degumming and Enzymatic Water Degumming on high phosphorus crude soybean oil.

[0206] Reference water degumming at temperature 80°C, and pH 6.2, reaction time 30min, total water 3%, defined here as industry typical, was compared to enzymatic water degumming assisted by the thermostable PLC SEQ ID NO: 1 or a combination of PLC-PI SEQ ID NO: 2 and PLC-PE / PC SEQ ID NO: 3, at reaction time 30min or 120min, temperature 55°C or 75°C, total water 3%. Three dosages of the thermostable PLC SEQ ID NO: 1 or the combination of PLC-PI SEQ ID NO: 2 and PLC-PE / PC SEQ ID NO: 3, were used, based on previous experiments, 75 ppm, 150ppm and 200ppm. Crude soybean oil with high level of phospholipids 1117ppm (Table 2) was selected for this experiment, and 100ppm of NaOH was added for separation.

[0207] The effect of phospholipase was compared to water degumming alone and yield loss was the key parameter measured in this experiment. In general, addition of the thermostable PLC SEQ ID NO: 1 alone provides significant improvement in yield (Table 8), which is on pair with standard industrial EWDG, but in reduced reaction time which eliminates a need for additional enzyme reactor and higher temperature, which reduces need for heat exchanges as crude oil from extraction plant can be directly treated in EWDG plant (Table 8, Raw 3,13,14, and 15 versus Raw 11 and 12). The new thermostable PLC SEQ ID NO: 1 offers an opportunity for existing and new EWDG plants to choose between broader reaction conditions, for higher productivity and / or higher oil yields, without compromising on oil quality

[0208] Table 8. Experimental conditions of WDG, EWDG, yield loss and yield loss reduction.

[0209] Example 7: Comparison of Water Degumming and Enzymatic Water Degumming on crude pressed canola oil

[0210] A crude rapeseed oil as shown in table 2 with phospholipids phosphorus concentration of 687ppm was treated with 2 various dosages of thermostable phospholipase C of SEQ ID NO:

[0211] 1. Enzymatic water degumming and water degumming were performed at. temperature 75°C or 90°C, and reaction time of 30vmins, total water 3%, as described in Example 1. The tested enzymes dosages were: 0, 150 and 250 ppm of the thermostable PLC SEQ ID NO: 1.

[0212] The effect of phospholipase shows a difference between enzymatic water degumming and reference water degumming at selected experimental conditions. Yield loss reduction, phospholipids hydrolysis and diglycerides increase were the key parameters measured in this experiment. In general, addition of the thermostable PLC SEQ ID NO: 1 provides significant increase in DAG and yield loss reduction at all selected dosages and reaction temperatures (Table 9). The highest yield loss reduction was observed after treatment at 250 ppm dose of the thermostable PLC SEQ ID NO: 1 , and 30min reaction time, at 75°C, while at 90°C slightly reduced performance has been observed. This result further demonstrates a broad application range of the thermostable PLC SEQ ID NO: 1 .

[0213] Table 9. Effect of enzyme dose and reaction temperature on enzymatic water degumming

[0214] Example 8: Comparison of Water Degumming and Enzymatic Water Degumming on crude soybean oil with lower P

[0215] A crude soybean oil as shown in table 2 with phospholipids phosphorus concentration of 627 ppm was treated with 2 various dosages of thermostable phospholipase C SEQ ID NO: 1. Enzymatic water degumming and water degumming were performed at. temperature 85°C, and reaction time of 2h, total water 3%, as described in Example 1. The tested enzymes dosages were: 0, 92 and 245 ppm of the thermostable PLC SEQ ID NO: 1.

[0216] The effect of phospholipase shows a difference between enzymatic water degumming and reference water degumming at selected experimental conditions. Yield loss reduction, phospholipids hydrolysis and diglycerides increase were the key parameters measured in this experiment. In general, the addition of the thermostable PLC SEQ ID NO: 1 provides significant increase in DAG and yield loss reduction at all selected dosages and reaction conditions (Table 10).

[0217] Table 10. Effect of enzyme and reaction conditions on enzymatic water degumming

[0218] Example 9: Enzymatic Water Degumming of crude soybean oil at 80°C

[0219] Additional one more crude soybean oil with 878 ppm phospholipids phosphorus was treated with thermostable phospholipase C of SEQ ID NO: 1 under industrial relevant conditions, at 80°C and various reaction times. The effect of enzyme was compared to water degumming alone. Yield loss reduction, diglycerides increase, and phospholipids hydrolysis were the key parameters measured in this experiment. In general, addition of the thermostable PLC SEQ ID NO: 1 provides significant improvement in yield, up to 1.9% reduced yield losses (Table 11), both in short (30min) and longer (2 hours) reaction time which offers an opportunity for existing and new EWDG plants to choose between broader reaction conditions, for higher productivity and / or higher oil yields. In addition, PA, PC and PE were significantly hydrolyzed (Table 12) contributing to reduced emulsification of neutral oil in gums and reduced oil yield losses.

[0220] Table 11 . Effect of enzyme dose and reaction time on enzymatic water degumming

[0221] Table 12. Phospholipids hydrolysis under specifies experimental conditions shown as a difference between crude oil and remaining phospholipids

Claims

CLAIMS:

1. A process for degumming a vegetable oil, comprising the steps of: a) providing a vegetable oil containing a quantity of phospholipids, b) contacting the oil with one or more phospholipases in presence of water, c) separating the reaction mixture of step b) into a light phase and a heavy phase, and d) producing a degummed vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1 ; wherein more than 75% or 85% or 90% of phosphatidylcholine (PC) and phosphatidylethanolamine(PE) is hydrolyzed; wherein more than 25% or 35% or 50% of phosphatidic acid (PA) is hydrolyzed; and wherein the process is carried out at a temperature of 70°C or above.

2. The process according to claim 1 , wherein the phospholipase is selected from a group consisting of phospholipase C having specificity for Phosphatidylinositol (PI), phospholipase C having specificity for phosphatidyl choline (PC) and Phosphatidyl ethanolamine (PE) and phospholipase C having specificity for Phosphatidyl choline (PC), Phosphatidyl ethanolamine (PE) Phosphatidic acid (PA) and Phosphatidylinositol (PI).

3. The process according to claim 1 , wherein the phospholipase C hydrolyses phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and / or phosphatidic acid (PA).

4. The process according to any of the preceding claims, wherein the oil is selected from crude oil or water degummed oil.

5. The process according to any of the claims 1 to 4, wherein the process further comprises adding an acid, chelating agent, alkali, or combinations thereof to vegetable oil prior to step a).

6. The process according to any of the preceding claims, wherein step b) further comprises contacting the oil with an enzyme selected from a group consisting of cellulases, endoglucanases, cellobiohydrolases, xylanase, mannanases, hemicellulases, pectinases, proteases, phytases, chlorophyllases, and combination thereof.

7. The process according to any of the preceding claims, wherein step b) further comprises contacting the oil with an enzyme selected from a group consisting of phospholipase A1 , phospholipase A2, lyso-phospholipase, and combination thereof.

8. The process according to any of the preceding claims, further comprising refining the degummed vegetable oil.

9. The process according to any of the preceding claims, wherein the vegetable oil comprises canola oil, corn oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, sesame oil, soybean oil, or sunflower seed oil.

10. The process according to any of the preceding claims, wherein the temperature is above 75°C, preferably above 80°C, in particular 85°C or above or even 90°C or above.

11. The process according to any of the preceding claims, wherein the phospholipase has at least about 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent,83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent,91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent,99 percent, or more, or is 100 percent sequence identity to SEQ ID NO: 1 .

12. The process according to any of the preceding claims, wherein the process further comprises adding phospholipase C to gums from water degummed oil to enable de-oiling of gums.

13. The process according to any of the preceding claims wherein said quantity of water is at least about 1 .5 percent by weight of the total mixture.

14. The process according to any of the preceding claims, wherein the process further comprises recycling the heavy phase of step c) into step a).

15. Use of phospholipase in degumming a vegetable oil, wherein the phospholipase has at least 75 percent sequence identity to SEQ ID NO: 1 .

Images