A process for improved oil yield in refining

Abstract

The present invention relates to a process for reducing diacylglycerides and / or phospholipids content in oil or fat without substantial interesterification and / or hydrolysis of triglycerides comprising the steps of: a. contacting an oil or fat with a lipase and a phospholipase in presence of water; and b. Hydrolyzing the diacylglycerides and / or phospholipids present in the oil; wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

Description

[0001] A PROCESS FOR IMPROVED OIL YIELD IN REFINING REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0003] FIELD OF INVENTION

[0004] The present invention relates to a process for reducing diacylglycerides and / or phospholipids content in oil or fat. without, substantial interesterification and / or hydrolysis of triglycerides.

[0005] BACKGROUND OF THE INVENTION

[0006] Vegetable oils, such as oils from palm, 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, giycolipids, phytosterols, phospholipids, proteins, pigments, and resins, are soluble or form stable colloidal suspensions in the oil. Most of these have an unfavorable effect on the flavor, odor, appearance, and / or shelf life of the oil, and therefore have to be removed from the oils by chemical or physical refining processes.

[0007] 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.

[0008] 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.

[0009] Several types of phospholipases are known to 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 comprisesremoving the phospholipid components (the gum) from the triglyceride rich oil fraction. The most 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. Enzymatic degumming both reduces the emulsifying properties of the phospholipids, so less oil is lost, and it recovers free fatty acids and / or diacylglycerols (DAG) from the phospholipid molecules themselves, resulting in overall improved yield. Diacylglycerols (DAG) are both present in the original oil in various quantities, depending on oil type, and are formed when using a PLC type phoshpolipase and the quality of the oil is reduced in proportion to the concentration of DAG present therein. The presence of diglycerides in the main product (= triglycerides (TAG)) is unfavorable as the diglycerides have an adverse effect on the product properties. Diglycerides are also reactive during parts of the refining process, especially during deodorization, and tend to form food-safety-regulated byproducts at certain conditions, e.g. glycidyl esters (GE) and, according to some literature, monochloropropanediol (esters) (MCPD(E)). It would therefore be desirable to enable a process for reducing DAG content and phosphorous content before later refining processing steps.

[0010] Despite recent advances in oil degumming and refining, there is a need for providing improved process for degumming and refining of vegetable oil in which the oil loss is minimized. This invention discloses a new process for simultaneous conversion of phospholipids and DAG using different enzyme classes, with the surprising effect of improved phospholipids conversion and oil yield recovery when, in combination, utilizing a phospholipase A and a lipase that is active in hydrolysis of DAG, but substantially inactive on TAG.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention relates to a process for reducing diacylglycerides and / or phospholipids content in oil or fat without substantial interesterification and / or hydrolysis of triglycerides.

[0013] An aspect of the present invention therefore relates to a process for reducing diacylglycerides and / or phospholipids content in oil or fat without, substantial interesterification and / or hydrolysis of triglycerides comprising the steps of: a) contacting an oil or fat with a lipase and a phospholipase in presence of water; and b) hydrolyzing the diacylglycerides and / or phospholipids present in the oil; wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.In a second aspect, the invention relates to a composition comprising a lipase and a phospholipase, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A In a third aspect, the invention provides a use of a lipase and a phospholipase for increasing oil yield, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0014] In a fourth aspect, the invention provides a use of a lipase and a phospholipase for hydrolysis of phospholipids, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0015] In a fifth aspect, the invention provides a use of a lipase and a phospholipase for reducing phospholipid content in an oil, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0016] SEQUENCE OVERVIEW SEQ ID NO: 1 is a lipase obtained from Candida antarctica

[0017] SEQ ID NO: 2 is a lipase obtained from Penicillium camemberti

[0018] SEQ ID NO: 3 is a lipase obtained from Aspergillus oryzae

[0019] SEQ ID NO: 4 is a phospholipase obtained from Talaromyces leycettanus

[0020] SEQ ID NO: 5 is a phospholipase obtained from Thermomyces lanuginosus

[0021] DEFINITIONS

[0022] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise

[0023] Unless defined otherwise or clearly indicated by context, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] The term "lipid" refers to phospholipids and their derivatives, triglycerides and derivatives, sterols, stands, cholesterol, sphingolipids, ceramides, fatty acids, fatty alcohols, glycolipids, proteolipids, lipopolysaccharides, ether- lipids, polar and non- polar lipids and derivatives thereof.

[0025] The term "esterification" as used herein, refers to a reaction for combining an organic acid such as a fatty acid with any alcohol or polyol such as a glycerol.The term "hydrolysis" as used herein, refers to the reaction of water with an ester to produce an acid and an alcohol.

[0026] The term “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)2 and to the solution of a base in water.

[0027] The term “bleaching” refers to the process for removing especially color and for further purifying the fat or oil. Normally, bleaching is accomplished after the oil has been refined by addition of a bleaching powder, typically bleaching clay or earth or active carbon, which adsorbs color, metal ions, and various organic compounds depending on choice of bleaching powder substance.

[0028] The term “chemical refining” is used synonymously with “alkali refining” and “alkaline refining”; the term also covering “caustic refining” and “caustic neutralization”. “Chemical refining” is sometimes also referred to as “super degumming” as it involves addition of base thereby removing both FFA as soap, and phospholipids by ionization thereby achieving good water solubility and removal of phospholipids with the soap byproduct phase.

[0029] The term "interesterification" as used herein, refers to the reaction of a first ester with a second ester leading to a mix up between the acyl and the alcohol moieties. Typically, interesterification refers to reaction between two TAGs comprising different fatty acids and interchanging of the fatty acids between the two original TAGs forming two new TAGs of different FA composition and position.

[0030] The term “crude oil” (also called a non-degummed oil) refers to a pressed or extracted oil or a mixture thereof.

[0031] The term “transesterification” as used herein, refers to the reaction of an ester with an alcohol, with change of alcohol as a result. This can be reaction between TAG and methanol or ethanol, forming DAG and fatty acid methyl or ethyl ester (FAME or FAEE), generally termed “alkyl esters”.

[0032] The term “reaction mixture” refers to the reaction mixture at any stage of reaction progression. This is because, and as evident from the cited literature, there is often an optimum in concentrations of DAG, and depending on target product composition, it can be useful to stop reaction at almost any point in time during reaction.

[0033] The terms "alkyl" or "alkyl group" is to be construed according to its broadest meaning, to describe a univalent aliphatic compound comprising hydrocarbons.

[0034] The term Free fatty acid (FFA) is a carboxylic acid with a long carbon chain. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 24. Free fatty acids are usually derived from fats (triglycerides (TAG), diglycerides (DAG)), phospholipids or lyso-phospholipids. Triglycerides are formed bycombining glycerol with three fatty acid molecules. The hydroxyl (HO-) group of glycerol and the carboxyl (-COOH) group of the fatty acid join to form an ester. The glycerol molecule has three hydroxyl (HO-) groups. Each fatty acid has a carboxyl group (-COOH). Diglycerides are formed by combining glycerol with two fatty acid molecules. Monoglycerides are formed by combining glycerol with one fatty acid molecule.

[0035] The terms "glycerol derivatives" and "glycerides" are interchangeably used herein to describe esters, ethers and other derivatives of glycerol in which at least one of the hydrogens, of any of the hydroxyl group attached to the C1, C2 or C3 carbons, is substituted. Examples of glycerol derivatives are: tristearoylglycerol (or tri-Ostearoyl glycerol or glycerol tristearate, or glyceryl tristearate);l,3-benzylideneglycerol (or 1,3- 0- benzylideneglycerol); and glycerol 2-phosphate (or 2-phosphoglycerol) among others. If the substitution is on a carbon atom, rather than on the oxygen of the hydroxyl group than the compound may be considered as a derivative of glycerol (e.g., 1,2,3-nonadecanetriol for C16H33CHOH-CHOH-CH2OH, which may be also considered as 1-C-hexadecyl glycerol). The term "glycerol" as used herein is intended to encompass only glycerol.

[0036] The terms mono, di- and tri-glycerol / glycerides, di- and tri- acylglycerol / acylglycerides, MG / DG / TG and MAG / DAG / TAG are used herein interchangeably and all refer to fatty acid based glycerides.

[0037] The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipid esterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to an enzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may have lipase activity (triacylglycerol lipase, EC3.1.1.3).

[0038] The terms “Lysophospholipase” relates to a “lysophospholipase” (EC 3.1.1.5) is an enzyme that can hydrolyze 2-lysophospholids to release fatty acid.

[0039] The term “parent” or “parent lipase” means a lipase to which an alteration is made to produce the enzyme variants. The parent lipase may be a naturally occurring (wild-type) polypeptide but may also be a variant and / or fragment thereof.

[0040] 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] The term "phospholipase A" refers to enzymes that catalyse the hydrolysis of the ester bond of the fatty acid components of phospholipids. There are two different types of phospholipase A activity that can be distinguished. Phospholipase Al, as defined in enzyme entry EC 3.1.1.32, and phospholipase A2, as defined in enzyme entry EC 3.1.1.4, catalyse the deacylation of one fatty acyl group in the snl and sn2 positions, respectively, from a diacylglycerophospholipid to produce lysophospholipid.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.

[0042] The term “Phospholipase B” cleaves both SN-1 and SN-2 acyl chains.

[0043] The term “Phospholipase C” cleaves before the phosphate, releasing diacylglycerol and a phosphate-containing head group.

[0044] The term “Phospholipase D” cleaves after the phosphate, releasing phosphatidic acid and an alcohol.

[0045] The term Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0046] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” 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. 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:

[0047] (Identical Residues x 100) / (Length of Alignment -Total Number of Gaps in Alignment) 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: (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0048] The term “oils and / or fats” is defined herein as a substrate comprising fatty acid derivatives. The substrate may comprise fatty acid alkyl esters, triglyceride, diglyceride, monoglyceride, free fatty acid or any combination thereof. Any oils and fats of vegetableor animal origin comprising fatty acids may be used as substrate in the process of the invention.

[0049] The fatty acid feedstock is preferably crude, but may be any combination of crude, refined, bleached, deodorized, degummed oils, with the only limitation being the loss of benefits relating to the invention proportional to dilution of phospholipids and DAG concentrations in the combined oil.

[0050] 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.

[0051] The term “phosphatides” relates mainly to intact phospholipids, but it might refer to partially hydrolyzed phospholipids as lyso-phospholipids, and glycerol¬ phosphoesters.

[0052] 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

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention relates to a process for reducing diacylglycerides and / or phospholipids content in oil or fat without substantial interesterification and / or hydrolysis of triglycerides. The inventors observed a significant and surprising oil yield increase when applying the combination of a phospholipase and a lipase inactive on TAG as compared to performing degumming without the presence of the TAG-inactive lipase.

[0055] The present invention provides combined application of a lipase and a phospholipase, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides, and the phospholipase is selected from phospholipase A, and where the combined action of the lipase and the phospholipase A, results in improved conversion of phospholipids and increased oil yield compared to only applying a phospholipase.

[0056] The present invention therefore relates to a process for reducing diacylglycerides and / or phospholipids content in oil or fat without substantial interesterification and / or hydrolysis of triglycerides comprising the steps of: a) contacting an oil or fat. with a lipase and a phospholipase in presence of water; and b) hydrolyzing the diacylglycerides and / or phospholipids present in the oil; wherein the lipase is selected from lipases substantiallylacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0057] In the present invention, interesterification between two TAG molecules is most relevant. Interesterification thus especially relates to randomization of specific types of TAG, such as in reaction between one holding 3 palmitic acids (PPP) and another holding 3 oleic acids (OOO), which through interesterification would lead to a distribution of PPP, PPO, POO, POP, and 000. In relation to oil refining, interesterification would be detrimental, as the properties of a substantially interesterified oil is often significantly different from the original oil, meaning an interesterified palm oil cannot be sold as ordinary palm oil. Interesterification can be defined by a degree of interesterification, which can be measured by various means of various accuracies, with one rough method being measurement of the delta in solid fat content at a defined temperature between original oil and fully interesterified oil, and another more precise method being full quantification of all TAG species and statistical analysis of the degree of randomization.

[0058] It is known that none of the exemplified lipases exhibit substantial TAG activity and ability to interesterify. Interesterification and hydrolysis, in the context of lipases, are similar reactions, because both follow the Ping-Pong Bi-Bi mechanism, wherein a first ester molecule delivers a fatty acid to be covalently bound to the active site of the enzyme while liberating a first alcohol, after which a second alcohol (or water) picks up the fatty acid to form a new ester (or free fatty acid). This means that lack of hydrolysis activity on TAG molecules directly translates to lack of interesterification activity.

[0059] The invention relates to improved yields of oil in a degumming process obtainable by combining a lipase capable of hydrolyzing DAG in combination with a phospholipase. This effect is obtainable by applying enzymes each exhibiting at least one of those effects. The invention is preferably practiced in a single batch or CSTR reactor where all enzymes are applied simultaneously. However, it is possible, and might be found to be optimal, to apply one or more of the enzymes in sequence. This is especially the case when one or more enzyme have different optimal operating points regarding temperature and pH. For example, in a continuous system using enzymes X and Y, it may be necessary to run a first reactor at mild temperature and at a certain pH optimal for enzyme X to run, with or without enzyme Y, followed by transfer to a second reactor optionally with pH and / or temperature change in the piping line or later directly in the second reactor, where enzyme Y is either already-present from the first reactor, applied directly into the piping line before reaching the second reactor, or added directly into the second reactor.

[0060] The effects observed according to the invention, e.g., increased oil yield and increased reduction in phospholipids, are present when comparing to only applying a phospholipase.In an embodiment of the invention, oil yield is increased when applying the phospholipase A in combination with the lipase, as compared to applying the phospholipase alone.

[0061] In an embodiment of the invention, the phospholipids conversion is increased when applying the phospholipase A in combination with the lipase, as compared to applying the phospholipase alone

[0062] The lipase and the phospholipase may perform hydrolysis simultaneously or sequentially.

[0063] In an embodiment of the claimed invention, the phospholipase and the lipase are added / applied simultaneously or sequentially.

[0064] Especially for sequential reactions, no separation steps are envisioned between reactions, e.g., for sequential reactions reaction conditions such as pH and temperature may be adjusted without performing any separation steps.

[0065] In one embodiment at least one phospholipase and at least one lipase are applied in the process of the invention.

[0066] In another embodiment, at least one phospholipase and at least two lipases or at least one lipase and at least two phospholipases are applied.

[0067] In another embodiment, two or more phospholipase(s) and two or more lipase(s) are applied.

[0068] In one embodiment, thus DAG hydrolysis may be performed in a first step and then move the oil to a new reactor for phospholipid hydrolysis, but without separating heavy phase from oil.

[0069] In another embodiment, sequential reactions may be performed by first enzymatic degumming followed by a second step of DAG hydrolysis, but without intermediate separation.

[0070] It may also be necessary to do a first non-enzymatic treatment of the oil prior to enzymatic reaction steps.

[0071] In an embodiment of the invention, the process optionally has a separation step wherein the heavy phase comprising the lipase and phospholipase are separated from oil between reaction steps. However, this separation step is applied after both DAG hydrolysis and phospholipid hydrolysis has been performed.

[0072] It may also be necessary to apply the same enzyme several times for increased efficacy.

[0073] There is also a conceivable setup with a counter-current operation, e.g. where heavy phase is separated from oil following a second reactor, and where that, heavy phase flows back to a first reactor. This would lead to re-usage of the enzyme, and conceivably higher conversion of phospholipids and DAG with further improved yield as a result.In an embodiment of the invention, the process comprises of separating of light oil phase and heavy water phase after reaction step b.

[0074] In an embodiment of the invention, the light phase comprises the oil with reduced diacylglyceride and / or phospholipid content.

[0075] In an embodiment of the invention, the recovery of light phase product, is increased compared to using the phospholipase alone

[0076] In an embodiment of the invention, the recovery of light phase product is at least 0.1, 0.2, 0.3, 0.4, or even 0.5 % increased based on total recovered oil mass compared to using the phospholipases alone.

[0077] In an embodiment of the invention, water added is between 0.01% -100% (w / w) of oil, such as between 0.5%-50%, 0.5% -25%, 0.5%-15%, 0.5%-10%, 0.5%-8%, 0.5%-7%, 1%-6%, or 1%-5% (wt / wt) of oil.

[0078] In an embodiment of the invention, water added is less than 10% wt / wt, such as below 9%, below 8%, below 7%, below 6%, below 5% wt / wt of the oil or fat.

[0079] In an embodiment of the invention, the oil or fat is e.g. derived from one or more of microbial oil, algae oil, canola oil, coconut oil, castor oil, copra oil, corn oil, distiller’s corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard 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, oil from halophytes, pennycress oil, camelina oil, coriander seed oil, meadowfoam oil, seashore mallow oil, and / or animal fat.

[0080] The most suitable oil or fat is one comprising substantial amounts of phospholipids and DAG, leading to yield improvements with reduced DAG concentrations in the resulting oil. The oils may be, such as crude, hexane extracted soyabean oil, crude corn oil or similar kind. Any other oil, especially those with low phospholipids concentrations, may still be treated in the process of the invention, but with relatively reduced benefits relating to yield In an embodiment of the invention, said process has 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.

[0081] In an embodiment of the invention, the degree of interesterification is less than 10%, preferably less than 5 %, such as preferably less than 4%, 3%, or even 2 % of the triglycerides present in the oil.

[0082] In an embodiment of the invention, the diglyceride conversion is at least 20%, such as at least 25%, 30%, preferably at least 40%, and most preferably at least 50%.

[0083] In an embodiment of the invention, the two or more phospholipases may comprise phospholipase B, phospholipase A1, phospholipase A2, phospholipase C, lysophospholipase or phospholipase D type phospholipases.

[0084] :0In an embodiment of the invention, the phospholipases comprise phospholipase A and phospholipase C, particularly phospholipase A1 and phospholipase C.

[0085] In an embodiment of the invention, the process is performed in a batch, semi-continuous or continuous mode.

[0086] In an embodiment of the invention, the process is performed in any number of sequential and / or parallel reactors, wherein the reaction can be partial or full in each / any.

[0087] In an embodiment of the invention, said one or more lipase(s) is any lipase that is substantially TAG-inactive while being DAG-active, with TAG-activity relating to both hydrolysis and interesterification as per other embodiments. That is, such lipase is substantially without TAG-activity at dosages resulting in hydrolysis of DAG to the required extent as per previous embodiments. This embodiment is to make it clear that the invention is possible to practice only at lipase dosages that are not extreme. It is evident to the expert in the field of use of enzymes in oils and fats, that, most enzymes can be used for almost any theoretically possible reaction with successful results, if the dosage of enzyme is extreme. For example, a phospholipase will often have a tiny activity in hydrolysis of TAG, but as dosages are typically optimized for sufficient phospholipid hydrolysis, and thus very low at economically optimal dosages, the TAG-activity is insignificant, and a phospholipase can truly be said to be without activity on TAG when applied optimally. The same is true for substantially TAG-inactive lipases - they are only inactive on TAG if used at dosages that are optimized for the purpose of being DAG-active and TAG-inactive. The dosage ranges of another embodiment of the invention will satisfy this requirement. To achieve substantially TAG-inactive enzyme in the context of this invention, the user of the invention should choose an enzyme that exhibits at least 10 times higher initial rate of reaction in hydrolysis of DAG relative to TAG, preferably 20 times, such as 30, 40, 50 times.

[0088] This can, as a simple example, be measured by mixing 100g of 50 wt% DAG, 50wt% TAG with 100g of water, and reacting at 50 °C for 30 minutes. If, following reaction, the concentration of DAG is 40 wt%, while TAG is still 49wt%, then the rate of reaction of DAG is 10 wt% / 30 minutes, relative to 1wt / 30 minutes for TAG, and therefore the relative initial rate of hydrolysis reaction would satisfy the requirement of a factor 10.

[0089] Then, when applying the enzyme, the dosage of enzyme must, be chosen to account for this relative rate of conversion of DAG relative to TAG and the chosen reaction time. It is evident to the expert in the field, that this will depend on the choice of enzyme, as different enzymes exhibit a wide range of inherent efficacy. It will also depend on operating conditions such as temperature and pH, and whatever other conditions might affect the efficacy of the enzyme. As such, to properly practice the invention, the dosage of TAG-inactive enzyme must be low enough that it satisfies the other embodimentsrelating to the required conversion of DAG, maximum conversion of TAG, and minimum yield of oil.

[0090] In an embodiment of the invention, said lipase having at least about 70 percent, has 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 100 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0091] In an embodiment of the invention, said lipase comprises or consists of amino acid as shown in SEQ ID NO: 1.

[0092] In an embodiment of the invention, said lipase comprises or consists of amino acid as shown in SEQ ID NO: 2

[0093] In an embodiment of the invention, the process is performed at temperatures in the range of 10°C -100°C, preferably 20°C -90°C, such as 30°C -90°C, 40°C -90°C, 45°C - 90°C, 50°C -90°C, 55°C -90°C, 55°C -85°C, 55°C -80°C.

[0094] In an embodiment of the invention, pH in step a) and / or step b) of the process is optionally adjusted during and / or prior to reacting.

[0095] In an embodiment of the invention, pH in step a) and / or step b) of the process is in the range of 2.5-9.0, 3.0-8.5, such as 4.0-8.0.

[0096] In an embodiment of the invention, pH is preferably adjusted using citric acid, acetic acid, lactic acid, phosphoric acid, formic acid, sodium hydroxide and / or potassium hydroxide.

[0097] In an embodiment of the invention, prior to or during the hydrolysis step b) the oil or fat is acid degummed or water degummed or both, especially the sequence of water degumming followed by acid degumming and finally enzymatic degumming, as described in the embodiments of the invention, is relevant when very low phosphorus content is required.

[0098] In an embodiment of the invention, said lipase is dosed in the range of 0.1-50000 mg enzyme protein (EP) / kg of oil, such as 0.1-200 mg enzyme protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 5-50 mg enzyme protein (EP) / kg of oil. In an embodiment of the invention, said phospholipase is dosed in the range of 0.1 - 50000 mg enzyme protein (EP) / kg of oil, such as 0.1-200 mg enzyme protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 5-50 mg enzyme protein (EP) / kg of oil.

[0099] In an embodiment of the invention, said lipase and the phospholipase are dosed in the ratio of 0.1 - 50000 mg enzyme protein (EP) / kg of oil, such as 0.1-200 mg enzyme

[0100] :2protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 5-50 mg enzyme protein (EP) / kg of oil.

[0101] At the very high dosage ranges of the embodiments above, the dosage especially relates to use of one or more immobilized enzymes, which are typically applied at extreme enzyme dosages that are then reused several times. In such a case, which is possible but not preferred, it is very important to optimize and especially reduce reaction time to reduce the risk of TAG-activity becoming detrimental to the targets of applying the invention. The most preferable application of the enzymes are as liquid products in single-use low dosages, which allows for easy operation and generally low cost Generally, the efficacy of a liquid, or any type of formulation of enzyme that becomes free (non-immobilized) in the application, is markedly higher than that of an immobilized enzyme when considering the activity per enzyme protein on a mass basis.

[0102] In an embodiment of the invention, said phospholipase A having 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 100 percent sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0103] In an embodiment of the invention, said phospholipase A comprises or consists of amino acid as shown in SEQ ID NO: 3.

[0104] In an embodiment of the invention, said phospholipase A comprises or consists of amino acid as shown in SEQ ID NO: 4.

[0105] In an embodiment of the invention, the process further comprises contacting the oil or fat with is a chlorophyllases or other enzymes active on chlorophyll, and combination thereof.

[0106] In an embodiment of the invention, the total phosphorous content of the oil is reduced to preferably below 300 ppm, 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.

[0107] In an embodiment of the invention the process further comprises refining the oil. In an embodiment of the invention, the oil is subjected to chemical refining or physical refining.

[0108] In an embodiment of the invention, the refining comprises degumming.

[0109] In an embodiment of the invention, the lipase is provided in the form of an aqueous solution, granular, liquid, powder and / or immobilized on a carrier or carrier particles.In an embodiment of the invention, the phospholipase is provided in the form of an aqueous solution, granular, liquid, powder and / or immobilized on a carrier or carrier particles.

[0110] In an embodiment of the invention, the heavy phase is partially or fully recycled into step b).

[0111] In an embodiment of the invention, the light phase is partially or fully recycled into step b).

[0112] In an embodiment of the invention, the light phase is optionally subjected to purification or fractionation.

[0113] In an embodiment of the invention, the total reaction time of the process is at least 15 minutes, preferably at least 30 minutes, such as 45, 60, 75, 90, 120, 160, 200, 240 minutes.

[0114] In an embodiment of the invention, the total reaction time of the process is up to 48 hours, preferably up to 12 hours, such as up to 10, 8, 6, 4 hours.

[0115] In an embodiment of the invention, process further optionally comprises of addition of alcohol in step b).

[0116] In an embodiment of the invention, said alcohol is short chain alcohol such as methanol, ethanol, propanol, butanol or mixtures thereof

[0117] In an embodiment of the invention, the process is performed in presence of solvent. In an embodiment of the invention, the solvent is an organic and inert solvent, e.g., an aliphatic or an aromatic solvent, such as hexane, heptane, toluene, methyloxolane, xylene, or benzene.

[0118] In an embodiment of the invention, said the process is used after or within the solvent extraction process.

[0119] In an embodiment of the invention, purification or fractionation comprises deodorization, stripping, distillation, short path distillation, fractional distillation, winterization, dry- and / or solvent fractionation, or a combination thereof.

[0120] In an embodiment of the invention, said lipase is selected from the group consisting of: Aspergillus lipase; Aspergillus niger lipase; Thermomyces lanuginosa lipase; Candida Antarctica lipase A; Candida Antarctica lipase B; Candida cylindracae lipase; Candida deformans lipase; Candida lipolytica lipase; Candida parapsilosis lipase; Mucor miehei, Chromobacterium, Candida rugosa lipase; Corynebacterium acnes lipase; Humicola lanuginosa, Cryptococcus spp. S-2 lipase; Fusarium culmorum lipase; Fusarium heterosporum lipase; Fusarium oxysporum lipase; Mucorjavanicus lipase; Rhizomucor miehei lipase; Rhizomucor delemar lipase; Burkholderia (Pseudomonas) cepacia lipase; Pseudomonas sp, ATCC 21808, Pseudomonas camembertii lipase; Pseudomonas fluorescens lipase; Rhizopus lipase; Rhizopus arrhizus lipase; Staphylococcus aureus

[0121] :4lipase; Geotrichium candidum lipase; Hyphozyma sp. lipase; Klebsiella oxytoca lipase; and wildtype orthologs and homologs thereof; and variants thereof.

[0122] In another embodiment of the invention, a composition comprising a lipase and a phospholipase, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0123] In an embodiment of the invention, the composition further comprises additional enzyme, wherein the additional enzyme is a chlorophyllases or other enzymes active on chlorophyll, and combination thereof.

[0124] In another embodiment of the invention, use of a lipase and a phospholipase in a process for improving oil yield, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0125] In another embodiment of the invention, use of a lipase and a phospholipase in a process for hydrolysis of phospholipids, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0126] In another embodiment of the invention, use of a lipase and a phospholipase in a process to reduce the phospholipid content in an oil, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0127] The invention is further described in the following numbered paragraphs.

[0128] Paragraph 1. A process for reducing diacylglycerides and / or phospholipids content in oil or fat without substantial interesterification and / or hydrolysis of triglycerides comprising the steps of:

[0129] a. contacting an oil or fat with a lipase and a phospholipase in presence of water; and

[0130] b. hydrolyzing the diacylglycerides and / or phospholipids present in the oil; wherein the lipase is selected from lipases substantially lacking activity towards triacylgiycerides and the phospholipase is selected from phospholipase A.

[0131] Paragraph 2. The process according to paragraph 1, comprising separation of light oil phase and heavy water phase after reaction step b.Paragraph 3. The process according to paragraph 2, wherein the light phase comprises the oil with reduced diacylglyceride and / or phospholipid content.

[0132] Paragraph 4. The process according to paragraph 3, wherein the recovery of light phase product is increased compared to using the phospholipase alone.

[0133] Paragraph 5. The process according to paragraph 4, wherein the recovery of light phase product is at least 0.1, 0.2, 0.3, 0.4, or even 0.5 % increased based on total recovered oil mass compared to using the phospholipases aione.

[0134] Paragraph 6. The process according to any of the preceding paragraphs, wherein water added is less than 10% wt / wt, such as below 9%, below 8%, below 7%, below 6%, below 5% wt / wt. of the oil or fat

[0135] Paragraph 7. The process according to paragraph 6, wherein water added is between 0.01% -100% (w / w) of oil, such as between 0.5%-50%, 0.5% -25%, 0.5%-15%, 0.5%-10%, 0.5%-8%, 0.5%-7%, 1%-6%, or 1%-5% (wt / wt) of oil.

[0136] Paragraph 8. The process according to any of the preceding paragraphs, wherein the oil or fat is e.g. derived from one or more of microbial oil, algae oil, canola oil, coconut oil, castor oil, copra oil, corn oil, distiller’s corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard 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, oil from halophytes, pennycress oil, camelina oil, coriander seed oil, meadowfoam oil, seashore mallow oil, and / or animal fat, including tallow from pigs, beef and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soy oil free fatty acid distillate, soap stock fatty acid material, yellow grease, used cooking oil, palm oil mill effluent and brown grease or any combination thereof.

[0137] Paragraph 9 The process according to paragraph 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.

[0138] Paragraph 10. The process according to paragraph 1, wherein the degree of interesterification is less than 10%, preferably less than 5 %, such as preferably less than 4%, 3%, or even 2 % of the triglycerides present in the oil.

[0139] !6Paragraph 11. The process according to paragraph 1, wherein the diglyceride conversion is at least 20%, such as at least 25%, 30%, preferably at least 40%, and most preferably at least 50%.

[0140] Paragraph 12. The process according to paragraph 1, wherein oil yield is increased when applying the phospholipase in combination with the lipase as compared to applying the phospholipase alone.

[0141] Paragraph 13. The process according to paragraph 1, wherein the phospholipids conversion is increased when applying the phospholipase in combination with the lipase as compared to applying the phospholipase alone.

[0142] Paragraph 14. The process according to any of the preceding paragraphs, wherein the phospholipase and the lipase are added / applied simultaneously or sequentially.

[0143] Paragraph 15. The process according to any of the preceding paragraphs, wherein at least one phospholipase and at least one lipase are applied.

[0144] Paragraph 16. The process according to any of the preceding paragraphs, wherein at least one phospholipase and at least two lipases or wherein at least one lipase and at least two phospholipases are applied.

[0145] Paragraph 17. The process according to any of the preceding paragraphs, two or more phospholipase(s) and two or more lipase(s) are applied.

[0146] Paragraph 18. The process according to any of the paragraphs 16-17, wherein the two or more phospholipases may comprise phospholipase B, phospholipase A1, phospholipase A2, phospholipase C, lysophospholipase or phospholipase D type phospholipases.

[0147] Paragraph 19. The process according to paragraphs 18, wherein the phospholipases comprise phospholipase A and phospholipase C.

[0148] Paragraph 20. The process according to any of the preceding paragraphs, wherein the process is performed in a batch, semi-continuous or continuous mode.Paragraph 21. The process according to any of the preceding paragraphs, wherein the process is performed in any number of sequential and / or parallel reactors, wherein the reaction can be partial or full in each.

[0149] Paragraph 22. The process according to any of the preceding paragraphs, wherein the lipase having at least about 70 percent, has 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 100 percent sequence identity to SEQ ID NO: 1.

[0150] Paragraph 23. The process according to any of the preceding paragraphs, wherein the lipase having at least about 70 percent, has 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 100 percent sequence identity to SEQ ID NO: 2.

[0151] Paragraph 24. The process according to any of the preceding paragraphs, wherein the lipase having at least about 70 percent, has 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 100 percent sequence identity to SEQ ID NO: 3.

[0152] Paragraph 25. The process according to any of the preceding paragraphs, wherein the lipase comprises or consists of amino acid as shown in SEQ ID NO: 1.

[0153] Paragraph 26. The process according to any of the preceding paragraphs, wherein the lipase comprises or consists of amino acid as shown in SEQ ID NO: 2.

[0154] :8Paragraph 27. The process according to any of the preceding paragraphs, wherein the lipase comprises or consists of amino acid as shown in SEQ ID NO: 3.

[0155] Paragraph 28. The process according to any of the preceding paragraphs, wherein the process is performed at temperatures in the range of 10°C -100°C, preferably 20°C -90°C, such as 30°C -90°C, 40°C -90°C, 45°C -90°C, 50°C -90°C, 55°C -90°C, 55°C -85°C, 55°C -80°C.

[0156] Paragraph 29. The process according to any of the preceding paragraphs, wherein pH in step a) and / or step b) is optionally adjusted during and / or prior to reacting.

[0157] Paragraph 30. The process according to any of the preceding paragraphs, wherein the pH in step a) and / or step b) is in the range of 2.5-9.0, 3.0-8.5, such as 4.0-8.0.

[0158] Paragraph 31. The process according to any of the preceding paragraphs, wherein the pH is preferably adjusted using citric acid, acetic acid, lactic acid, phosphoric acid, formic acid, sodium hydroxide and / or potassium hydroxide.

[0159] Paragraph 32. The process according to any of the preceding paragraphs, wherein the lipase is dosed in the range of 0.1-50000 mg enzyme protein (EP) / kg of oil, such as 0.1- 200 mg enzyme protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 5-50 mg enzyme protein (EP) / kg of oil.

[0160] Paragraph 33. The process according to any of the preceding paragraphs, wherein the phospholipase is dosed in the range of 0.1 - 50000 mg enzyme protein (EP) / kg of oil, such as 0.1-200 mg enzyme protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 5-50 mg enzyme protein (EP) / kg of oil.

[0161] Paragraph 34. The process according to any of the preceding paragraphs, wherein lipase and the phospholipase are dosed in the ratio of 0.1 - 50000 mg enzyme protein (EP) / kg of oil, such as 0.1-200 mg enzyme protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 5-50 mg enzyme protein (EP) / kg of oil.

[0162] Paragraph 35. The process according to any of the preceding paragraphs, wherein the phospholipase A having 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, 82percent, 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: 4.

[0163] Paragraph 36. The process according to any of the preceding paragraphs, wherein the phospholipase A having 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 100 percent sequence identity to SEQ ID NO: 5.

[0164] Paragraph 37. The process according to any of the preceding paragraphs, wherein the phospholipase A comprises or consists of amino acid as shown in SEQ ID NO: 4.

[0165] Paragraph 38. The process according to any of the preceding paragraphs, wherein the phospholipase A comprises or consists of amino acid as shown in SEQ ID NO: 5

[0166] Paragraph 39. The process according to any of the preceding paragraphs, further comprises contacting the oil or fat with is a chlorophyllases or other enzymes active on chlorophyll, and combination thereof.

[0167] Paragraph 40. The process according to any of the preceding paragraphs, wherein the total phosphorous content of the oil is reduced to preferably below 300 ppm, 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.

[0168] Paragraph 41. The process according to any of the preceding paragraphs, further comprising refining the oil.

[0169] Paragraph 42. The process according to paragraph 41, wherein the oil is subjected to chemical refining or physical refiningParagraph 43. The process according to paragraph 41-42, wherein refining comprises degumming.

[0170] Paragraph 44. The process according to any of the preceding paragraphs, wherein the lipase is provided in the form of an aqueous solution, granular, liquid, powder and / or immobilized on a carrier or carrier particles.

[0171] Paragraph 45. 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.

[0172] Paragraph 46. The process according to any of the preceding paragraphs, wherein the heavy phase is partially or fully recycled into step b).

[0173] Paragraph 47. The process according to any of the preceding paragraphs, wherein the light phase is partially or fully recycled into step b).

[0174] Paragraph 48. The process according to any of the preceding paragraphs, wherein the light phase is optionally subjected to purification or fractionation.

[0175] Paragraph 49. The process according to any of the preceding paragraphs, wherein the total reaction time of the process is at least. 15 minutes, preferably at least. 30 minutes, such as 45, 60, 75, 90, 120, 160, 200, 240 minutes.

[0176] Paragraph 50. The process according to any of the preceding paragraphs, wherein the total reaction time of the process is up to 48 hours, preferably up to 12 hours, such as up to 10, 8, 6, 4 hours.

[0177] Paragraph 51. The process according to paragraph 1, further optionally comprises of addition of alcohol in step b).

[0178] Paragraph 52. The process according to paragraph 51, wherein said alcohol is short chain alcohol such as methanol, ethanol, propanol, butanol or mixtures thereof.

[0179] Paragraph 53. The process according to any of the preceding paragraphs, wherein the process is performed in presence of solvent.Paragraph 54. The process according to paragraph 53, wherein the solvent is an organic or inert solvent, e.g., an aliphatic or an aromatic solvent, such as hexane, heptane, toluene, methyloxolane, xylene, or benzene.

[0180] Paragraph 55 The process according to paragraph 53, wherein said the process is used after or within the solvent extraction process.

[0181] Paragraph 56. The process according to paragraph 48, wherein purification or fractionation comprises distillation, short, path distillation, fractional distillation, winterization, dry- and / or solvent fractionation, ora combination thereof.

[0182] Paragraph 57. The process according to anyone of the preceding paragraphs, wherein said lipase is selected from the group consisting of: Aspergillus lipase; Aspergillus niger lipase; Thermomyces lanuginosa lipase; Candida Antarctica lipase A; Candida Antarctica lipase B; Candida cylindracae lipase; Candida deformans lipase; Candida lipolytica lipase; Candida parapsilosis lipase; Mucor miehei, Chromobacterium; Candida rugosa lipase; Corynebacterium acnes lipase; Humicola lanuginosa, Cryptococcus spp. S-2 lipase; Fusarium culmorum lipase; Fusarium heterosporum lipase; Fusarium oxysporum lipase; Mucorjavanicus lipase; Rhizomucor miehei lipase; Rhizomucor delemar lipase; Burkholderia (Pseudomonas) cepacia lipase; Pseudomonas sp, ATCC 21808, Pseudomonas camembertii lipase; Pseudomonas fluorescens lipase; Rhizopus lipase; Rhizopus arrhizus lipase; Staphylococcus aureus lipase; Geotrichium candidum lipase; Hyphozyma sp. lipase; Klebsiella oxytoca lipase; and wildtype orthologs and homologs thereof; and variants thereof.

[0183] Paragraph 58. The process according to any of the preceding claims, wherein prior to or during the hydrolysis step b) the oil or fat is acid degummed or water degummed.

[0184] Paragraph 59. A composition comprising a lipase and a phospholipase, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

[0185] Paragraph 60. The composition of paragraph 59 further comprises additional enzyme, wherein the additional enzyme is a chlorophyllases or other enzymes active on chlorophyll, and combination thereof.Paragraph 61. Use of a lipase and a phospholipase for increasing oil yield, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A or composition according to paragraphs 59-60.

[0186] Paragraph 70. Use of a lipase and a phospholipase for hydrolysis of phospholipids, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A or composition according to paragraphs 59-60

[0187] Paragraph 71. Use of a lipase and a phospholipase for reducing phospholipid content in an oil, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A or composition according to paragraphs 59-60.

[0188] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0189] EXAMPLES

[0190] Example 1: General method of degumming vegetable oils

[0191] A. Laboratory scale water degumming and enzymatic water degumming was performed at 55°C and 3% total water content. Oils were preheated to selected temperature and 40g portions were transferred into reaction tubes. In tests samples pH was adjusted by addition of NaOH or citric acid, depending on experimental conditions. Enzymes (lipase and phospholipase) 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 pre-defined time by heating oil samples to 99°C for 10min. In control samples only water was added (water degumming).

[0192] B. Laboratory scale deep degumming and enzymatic deep degumming was performed at 55°C and 3% total water content. Oils were preheated to 70°C and 40g portions were transferred into reaction tubes. In next step, 650 ppm of citric acid was added to all samples, and samples were sonicated for 5 min at 70°C toensure sufficient distribution and mixing of acid into oil phase. The chelation step was performed by placing oil samples in a heating cabinet at 70°C and incubated under gentle rotation, 20rpm, for 25min. After chelation step, pH of tests samples was adjusted by addition of NaOH. Enzymes and water were added accordingly, and samples were sonicated for 5 min at 55°C 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 55°C for a 4hours. The enzymatic reaction was stopped after pre-defined time by heating oil samples to 99°C for 10min. In control samples only chelation step was included, followed by pH adjustment and water addition (acid degumming).

[0193] Sample of reaction mixture was taken for phospholipids analysis. 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. 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 µm 150 x 4.6 mm, according to AOCS Official Method Cd 11d-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 CDCl3and 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 (I S)*n(IS)’M (P) / m(oil). The internal standard solution is 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.

[0194] Total phosphorus of degummed oil (light phase) was measured by ICP according to AOCS Official Method 20-99 Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy, and is shown as P (ppm).

[0195] 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 / 100) x (100 / Oil)Where

[0196] Oil - initial volume of crude oil before degumming (g)

[0197] Gums - Measured wet gum volume (g)

[0198] Dry Matter - The dry matter content in the gums (%)

[0199] Yield loss - The yield loss is an estimate of the oil loss volume (%)

[0200] Substrate: Three crude oils with various quality were used (Table 1).

[0201] Table 1. Crude oils composition

[0202] Table 1 data as shown in image with proper table structure

[0203]

[0204] Example 2: Synergistic effect of phospholipase A1 and partial glycerides lipase in deep degumming of rapeseed oil

[0205] Enzymatic (EDD) and acid deep degumming (ADD) of crude rapeseed oil (Table 1) was performed at 55°C, pH 6.3, reaction time 4h, and total water of 3%, according to the method described in Example 1. Two phospholipase A1 enzymes were used: SEQ ID NO: 4 (dose 50 ppm) & SEQ ID NO:.4 (50 ppm), alone and in combination with partial glycerides lipase SEQ ID NO: 1 (0.2%)

[0206] The effect of enzymes is evaluated as a difference between enzymatic deep degumming and reference acid degumming at selected pH conditions. Yield loss reduction, and diglycerides decrease and final phosphorus in the degummed oil were key parameters measured in this experiment, along with phospholipids hydrolysis. The experiment shows significant improvement of oil yield in presence of phospholipases A1 and additional yield improvement when PLA1 was combined with lipase (Table 2). This demonstrates that the enzymes SEQ ID NO: 1 & SEQ ID NO: 4 or SEQ ID NO: 5, have synergistic effect in degumming of vegetable oils.

[0207] Table 2. The effect of enzymes on yield loss reduction and DAG reduction in vegetable oil deep degumming assisted by enzymes.

[0208] Treatment conditions Results

[0209] Acid (ADD) or

[0210] pH in Yield Phospholipids Enzymatic Deep Degumming DAG (%) FFA (%)

[0211] reaction loss (%) hydrolysis (%) (EDD)

[0212]

[0213] ADD 4.5 4.29 0.4 1.5 0 EDD: SEQ ID NO:4 (50ppm) 4 3.28 0.4 2.2 71

[0214] EDD: SEQ ID NO: 4 (50ppm)

[0215] 4 3.26 0.23 2.9 76

[0216] + SEQ ID NO: 1 (0.2%)

[0217] EDD: SEQ ID NO:5 (50ppm) 5 4.05 0.4 2.1 56

[0218] EDD: SEQ ID NO: 5 (50ppm)

[0219] 5 3.78 0.35 2.2 61

[0220] + SEQ ID NO: 1 (0.2%)

[0221]

[0222] Example 3: Dose response of partial glycerides lipase in deep degumming of rapeseed oil

[0223] Enzymatic (EDD) and acid deep degumming (ADD) of crude pressed rapeseed oil (Table 1) was performed at 55°C, pH 4, reaction time 4h, and total water of 3%, according to the method described in Example 1. The phospholipase A1 enzymes SEQ ID NO: 4 (dose 50 ppm) was tested in combination with partial glycerides lipase SEQ ID NO: 1 (dose 0.05% or 0.1%)

[0224] The effect of enzymes is evaluated as a difference between enzymatic deep degumming and reference acid degumming at selected pH conditions. Yield loss reduction, and final phosphorus in the degummed oil were key parameters measured in this experiment, along with phospholipids hydrolysis. The experiment shows significant improvement of oil yield in presence of phospholipases A1 combined with lipase SEQ ID NO: 1 and additional improvement with increased does of lipase SEQ ID NO: 1 (Table 3). This demonstrates that the enzymes SEQ ID NO: 1 & SEQ ID NO: 4 have synergistic effect in degumming of vegetable oils, especially of crude pressed rapeseed oil.

[0225] Table 3. Increased yield in presence of PLA1 SEQ ID NO: 4 and partial glyceride lipase in enzymatic deep degumming of crude pressed rapeseed oil.

[0226] Treatment conditions Results

[0227] Acid (ADD) or

[0228] pH in Yield loss Phospholipids Enzymatic Deep Degumming

[0229] reaction (%) hydrolysis (%) (EDD)

[0230] ADD 4 2.5 0

[0231] EDD: SEQ ID NO: 4 (50ppm)

[0232] 4 1.8 76

[0233] + SEQ ID NO: 1 (0.05%)

[0234] EDD: SEQ ID NO: 4 (50ppm)

[0235] 4 1.6 84

[0236] + SEQ ID NO: 1 (0.1%)

[0237]

[0238] Example 4: Enzymatic water degumming in presence of PLA1 and partial glyceride lipase

[0239] Enzymatic (EWD) and water degumming (WD) of crude rapeseed oils (Table 1) was performed at 55°C, reaction time 4h, and total water of 3%, according to the method described in Example 1. The phospholipase A1 enzymes SEQ ID NO: 4 or SEQ ID NO: 5 (dose 50 or 100 ppm) was tested in combination with partial glycerides lipase SEQ ID NO: 1 (dose 0.2%)

[0240] The effect of enzymes is evaluated as a difference between enzymatic water degumming and reference water degumming at selected reaction conditions. Yield loss reduction and DAG decrease were key parameters measured in this experiment. The experiment shows significant improvement of oil yield in presence of phospholipases A1 SEQ ID NO: 4 or SEQ ID NO: 5 combined with lipase SEQ ID NO: 1 (Table 4). This demonstrates that the enzymes SEQ ID NO: 1 & SEQ ID NO: 4 or SEQ ID NO: 5 have synergistic effect in enzymatic water degumming of vegetable oils, and addition of partial glyceride lipase on its own does not impact phospholipids hydrolysis or even increases yield losses.

[0241] Table 4. Water and enzymatic water degumming of crude rapeseed oils in presence of phospholipase A1 enzymes SEQ ID NO: 4 or SEQ ID NO: 5 in combination with partial glycerides lipase SEQ ID NO: 1.

[0242] Treatment conditions Results

[0243] pH in Yield P

[0244] Water (WD) or DAG reacti loss (PP Enzymatic Water Degumming (EWD) (%)

[0245] on (%) m)

[0246] WD 4.7 5 79 0.6 EWD: SEQ ID NO: 1 (0.2%) 4.7 5.9 - 0.28 EWD: SEQ ID NO: 5 (50ppm)

[0247] 4.7 4.4 49 0.49 + SEQ ID NO: 1 (0.2%)

[0248] EWD: SEQ ID NO: 5 (100ppm)

[0249] 4.7 4.0 - 0.54 + SEQ ID NO: 1 (0.2%)

[0250] EWD: SEQ ID NO: 4 (50ppm)

[0251] 4.7 4.13 68 0.36 + SEQ ID NO: 1 (0.2%)

[0252] EWD: SEQ ID NO: 5 (50ppm)

[0253] 4.7 4.3 - 0.38 + SEQ ID NO: 1 (0.2%)

[0254]

[0255] Example 5: Synergistic effect, of phospholipase A1 and partial glycerides lipase in degumming of water degummed soy bean oil

[0256] Acid and enzymatic degumming of water degummed soybean oil was assessed by total phosphorus which was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). Citric acid (50% solution) was added at the ratios indicated in Table 5 relative to the oil. After addition of acid to oil (30g), the tubes were sonicated for 5 min at 70°C. The chelation step was performed by placing oil samples in a heating cabinet at 70°C and incubated under gentle rotation, 40rpm, for 15min. Enzymes, water and ethanol were added as indicated in Table 5, and samples were sonicated for 5min at 60°C. in the next step, oil samples were placed in a heating cabinet and incubated under gentle rotation, 40rpm, at 60°C for a 4hours. The enzymatic reaction was stopped by incubation at 90°C in a water bath for a minimum of 15min, and the tubes were then centrifuged at 600xg for 6 minutes.

[0257] The phosphorous in the oils after degumming treatment was shown in Table 5. Neither enzymatic treatment alone provided a benefit compared to acid degumming (650 ppm citric acid). However, the combination of SEQ ID NO: 4 (60ppm) + SEQ ID NO: 1 (100ppm) provided a benefit that was better than acid degumming indicating a synergistic effect of combining the two enzymes in degumming.

[0258] Table 5. Acid and enzymatic degumming of water degummed soybean oil in presence of phospholipase Al enzyme SEQ ID NO: 4 in combination with partial glycerides lipase SEQ ID NO: 1

[0259] P (ppm) Treatment Mean Std Err

[0260] 650 ppm citric acid 41.4 0.45 SEQ ID NO: 1 (100ppm) + 150 ppm citric acid 53.4 0.64 SEQ ID NO: 1 (100ppm) + 0.6% EtOH + 150 59.9 0.05 ppm citric acid

[0261] SEQ ID NO: 4 (60ppm) + SEQ ID NO: 1 30.4 10.3 (100ppm) + 0.6% EtOH + 150 ppm citric acid

[0262] SEQ ID NO: 4 (60ppm) + 150 ppm citric acid 47.6 2.31

[0263]

Claims

CLAIMS1. A process for reducing diacylglycerides and / or phospholipids content in oil or fat without substantial interesterification and / or hydrolysis of triglycerides comprising the steps of:a. contacting an oil or fat with a lipase and a phospholipase in presence of water; andb. hydrolyzing the diacylglycerides and / or phospholipids present in the oil; wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

2. The process according to claim 1, comprising separation of light oil phase and heavy water phase after reaction step b.

3. The process according to claim 2, wherein the light phase comprises the oil with reduced diacylglyceride and / or phospholipid content.

4. The process according to claim 3, wherein the recovery of light phase product is increased compared to using the phospholipase alone.

5. The process according to claim 4, wherein the recovery of light phase product is at least 0.1, 0.2, 0.3, 0.4, or even 0.5 % increased based on total recovered oil mass compared to using the phospholipases alone.

6. The process according to any of the preceding claims, wherein water added is less than 10% wt / wt, such as below 9%, below 8%, below 7%, below 6%, or below 5% wt / wt of the oil or fat.

7. The process according to claim 6, wherein water added is between 0.01% -100% (w / w) of oil, such as between 0.5%-50%, 0.5% -25%, 0.5%-15%, 0.5%-10%, 0.5%- 8%, 0.5%-7%, 1%-6%, or 1%-5% (wt / wt) of oil.

8. The process according to claim 1, wherein less than 10%, preferably less than 5 %, more preferably less than 2 % and most preferably less than 05% of the triglycerides present in the oil are hydrolyzed.

9. The process according to claim 1, wherein the degree of interesterification is less than 10%, preferably less than 5 %, such as preferably less than 4%, 3%, or even 2 % of the triglycerides present in the oil.

10. The process according to claim 1, wherein the diglyceride conversion is at least 20%, such as at least 25%, 30%, preferably at least 40%, and most preferably at least 50%.

11. The process according to claim 1, wherein oil yield is increased when applying the phospholipase in combination with the lipase as compared to applying the phospholipase alone.

12. The process according to claim 1, wherein the phospholipids conversion is increased when applying the phospholipase in combination with the lipase as compared to applying the phospholipase alone.

13. The process according to any of the preceding claims, wherein the lipase having at least about 70 percent, has 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 100 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.

14. The process according to any of the preceding claims, wherein the phospholipase A having 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 100 percent sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5.

15. A composition comprising a lipase and a phospholipase, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A.

16. Use of a lipase and a phospholipase for increasing oil yield, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A or composition according to claim 15.

17. Use of a lipase and a phospholipase for hydrolysis of phospholipids, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A or composition according to claim 15.

18. Use of a lipase and a phospholipase for reducing phospholipid content in an oil, wherein the lipase is selected from lipases substantially lacking activity towards triacylglycerides and the phospholipase is selected from phospholipase A or composition according to claim 15

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