Process for free fatty acid reduction

Abstract

The present invention relates to reparation of oil through esterification of FFA in especially crude oils, by converting the FFA back into edible MAG and DAG, without substantial production of TAG and furthermore avoiding interesterification of TAG.

Description

[0001] PROCESS FOR FREE FATTY ACID REDUCTION

[0002] FIELD OF INVENTION

[0003] The present invention relates to a process for enzymatic reduction of free fatty acid (FFA) in oils and fats using a lipase.

[0004] BACKGROUND OF THE INVENTION

[0005] Oils and fats consist of complex mixtures of triacylglycerols (TAGs), diacylglycerols (DAGs), monoacylglycerols (MAGs), free fatty acids (FFA) and other minor components. MAG and to a lesser extent DAG are emulsifiers and are used in various commercial foods for that purpose. Examples are stabilizers in baked goods, chips, dressings, margarines and so on. DAG is additionally sold as a healthy oil, with 1 ,3-DAG being most favored, the claimed benefit being less associated body weight gain by human consumption.

[0006] Most crude oils and fats or derivates thereof will comprise a significant amount of FFA. Relevant examples are crude soybean, typically comprising 0.5-3 wt% FFA, and crude palm oil, typically comprising 2-10 wt% FFA. In vegetable oil refining, those two oils essentially describe the main two refining processes being chemical or physical refining.

[0007] Chemical refining involves alkaline neutralization and subsequent separation of FFA as precipitated soaps, forming a soapstock byproduct. 1 wt% FFA becomes 1 .5-2 wt% soap stock, because valuable, and often edible, TAG-oil is emulsified into the soaps and lost as a non-food byproduct. At increasing FFA levels in the feedstock crude oil, the yield loss of TAG-oil will increase accordingly. Chemical refining is thus typically used for refining of crude oils with relatively low residual FFA concentrations.

[0008] Physical refining involves a distillation / stripping (deodorization) step, where FFA is removed as fatty acid distillate (FAD) of relatively high purity and value. The deodorization step is expensive to procure, however, but contrary to chemical refining, it results in an insignificant loss of TAG-oil.

[0009] In both processes, FFA is lost as a technical grade and often non-edible byproduct, and in the case of chemical refining, edible TAG-oil is lost in the FFA-removal process. The literature, and especially American Oil Chemists Society (AOCS) presentations at the annual conference, have been discussing re-esterification of FFA into TAG to address the issues above. TAG formation from the FFA can easily be done, using either high-temperature esterification or enzymes. But high-temperature esterification is not a viable option, because the temperature will cause unacceptable degradation and losses that exceed the economical benefit of reducing FFA, especially for unsaturated oils. Further, enzymatic TAG-formation is not a viable option either, due to the process of interesterification, which cannot easily be avoided when employing a TAG- active enzyme for this purpose. Interesterification is the net reaction affecting randomization of especially TAG-molecules in the oil. For example, interesterification between tri-oleic (OOO) with tri-palmitic (PPP) would result in a number of new species such as POO, OPO, PPO, and so on. Randomization of the TAG species does not change the edibility of the oil, but is still unacceptable, because the properties of the oil will change significantly, with melting behavior being most important. This means a producer of palm oil may no longer be producing a palm oil with the expectable palm oil properties, making product sales difficult or even impossible.

[0010] Steen Balchen et al., 2017, (Plant Scale Enzymatic FFA-remediation of Rice Bran Oil, AOCS annual Meetings an Industry Showcases, Abstracts Page 4) describes relatively high- dosage immobilized lipase. Immobilized lipase is known in industry to result in an unacceptably high degree of interesterification and will affect formation of TAG.

[0011] Pinsirodom, Praphan, et al., (Critical temperature for production of MAG by esterification of different FA with glycerol using Penicillium camemberti lipase. Journal of the American Oil Chemists' Society 81.6 (2004): 543-547), describe use of free Penicillium Camemberti lipase for solvent free DAG formation.

[0012] US 5270188 A discloses esterification of FFA onto glycerol using a lipase derived from Penicillium cyclopium.

[0013] US 2020146307 A discloses esterification of FFA onto glycerol using a commercial lipase, Amano lipase AY.

[0014] US 5,219,744 discloses a method of synthesizing triglycerides from partial glycerides and fatty acids using a lipase from Penicillium cyclopium.

[0015] Kim, Y. H. et al., (Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and Rosetta Design. Enzyme and Microbial Technology 47 (2010) 1-5, describes CalB mutants showed highest residual activity in the range from 40°C to 60°C compared to that of wildtype CalB at 55°C.

[0016] Saikia. et.al., (Amino-functionalised mesoporous silica microspheres for immobilisation of Candida antarctica lipase B - application towards greener production of 2,5-furandicarboxylic

[0017] Acid, Journal of IET Nanobiotechnol., 2020, Vol. 14 Iss. 8, pp. 732-738) describes CalB was immobilised on mesoporous silica microspheres, which exhibited maximum esterification efficiency. However, the lowered yield was achieved due to the inactivation of CaLB after prolonged incubation at a high temperature at 50°C.

[0018] US 2021 / 388401 discloses a method of refining a grain oil composition feedstock to provide a grain oil product.

[0019] WO 2023 / 222648 discloses a process for reducing level of free fatty acids in biodiesel / fatty acid alkyl esters.

[0020] Therefore, there is a need in the art for a process capable of transforming FFA in a feedstock oil to edible and valuable MAG and DAG, which largely stay in the final edible oil, and at the same time largely avoid randomization of the TAG species present in the oil. SUMMARY OF INVENTION

[0021] The present invention relates to an enzymatic process for reducing FFA (Free fatty acid) content in an oil feedstock, without significantly affecting the TAG species present in the oil, by avoiding randomization / interesterification of TAG.

[0022] An aspect of the present invention therefore relates to a process for reducing FFA (Free fatty acid) content in an oil feedstock comprising the steps of: a) providing an oil feedstock comprising FFA; b) reacting the oil feedstock of a) by esterification of FFA with glycerol in the presence of a lipase to convert FFA into MAG and DAG; wherein the lipase is applied in granular, liquid or water-soluble form, and wherein the lipase is selected from lipases which are inactive on TAG, or substantially inactive on TAG.

[0023] SEQUENCE OVERVIEW

[0024] SEQ ID NO: 1 is a lipase obtained from Candida antarctica.

[0025] SEQ ID NO: 2 is a lipase from Penicillium camemberti.

[0026] DEFINITIONS

[0027] 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.

[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 to which this invention belongs.

[0029] 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.

[0030] 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.

[0031] The term "hydrolysis" as used herein, refers to the reaction of water with an ester to produce an acid and an alcohol.

[0032] The term “glycerolysis” as used herein, refers to a reaction for combining glycerol and TAG to form MAG and DAG. It also includes the reaction between glycerol and DAG to form two molecules of MAG.

[0033] 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. 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”.

[0034] 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 each component (MAG and DAG especially), and depending on target product composition, it can be useful to stop reaction at almost any point in time during reaction.

[0035] The terms "alkyl" or "alkyl group" is to be construed according to its broadest meaning, to describe a univalent aliphatic compound comprising hydrocarbons.

[0036] 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); 1 ,3-benzylideneglycerol (or 1 ,3- O-benzylideneglycerol); and glycerol 2- phosphate (or 2-phosphoglycerol) among others. If the substitution is on a carbon atom, rather than on the oxygen of the hydroxyl group than the compound may be considered as a derivative of glycerol (e.g., 1 ,2,3-nonadecanetriol for C16H33CHOH-CHOH-CH2OH, which may be also considered as 1-C-hexadecyl glycerol). The term "glycerol" as used herein is intended to encompass glycerol derivatives including glycerol.

[0037] The terms “oil” and “fat” are used interchangeably and relate to oils and fats comprising fatty acids or derivatives thereof such as MAG, DAG and TAG. Oils are generally liquid at room temperature, while fats are not, however chemically they are largely similar.

[0038] The terms mono-, di- and tri-glycerol / glycerides, mono-, di- and tri- acylglycerol / acylglycerides, MG / DG / TG and MAG / DAG / TAG are used herein interchangeably, and all refer to fatty acid based glycerides.

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

[0041] 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:

[0042] (Identical Residues x 100) / (Length of Alignment - T otal Number of Gaps in Alignment)

[0043] 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:

[0044] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0045] 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), monoglyceride (MAG)), phospholipids or lyso-phospholipids. Triglycerides are formed by combining glycerol with three fatty acid molecules. The hydroxyl (HO-) group of glycerol and the carboxyl (-COOH) group of the fatty acid 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.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention relates to reparation of oil through esterification of FFA in especially crude oils, converting the FFA back into edible MAG and DAG. This yields edible oil from all or part of the FFA, which would otherwise become non-edible byproducts of the refining process, while also avoiding or reducing the TAG-losses associated with FFA removal in refining. This is achieved, while additionally avoiding interesterification by employing an enzyme with little or no activity on the TAG molecules of the oil. Further, the invention relates to use of cheap, preferentially liquid, formulation of lipase rather than expensive immobilized lipases requiring high numbers of reuses for economic viability.

[0048] None of the referenced prior art describe reduction of FFA concentrations in oils, in the presence of added glycerol, and where the main goal is converting FFA to MAG and / or DAG, as a means to avoid the associated refining losses of food oil, by use of cheap and nonimmobilized, largely TAG-inactive, lipase, and while avoiding significant interesterification.

[0049] The inventor has found a surprising benefit of employing liquid lipase in esterification of FFA with glycerol, producing primarily MAG and DAG with low TAG formation, while avoiding interesterification.

[0050] The main aspect of the present invention therefore relates to a process for reducing FFA (Free fatty acid) content in an oil feedstock comprising steps of: a) providing an oil feedstock comprising FFA; b) reacting the oil feedstock of a) by esterification of FFA with glycerol in the presence of a lipase to convert FFA into MAG and DAG; wherein the lipase is applied in granular, liquid or water-soluble form, and wherein the lipase is selected from lipases which are inactive on TAG, or substantially inactive on TAG.

[0051] In an embodiment of the invention, the invention is utilized as a means for reduction of FFA to achieve higher refining yields.

[0052] The resulting relatively high amounts of DAG in the product oil of the invention comes at the cost of increased costs associated with avoiding formation of glycidyl esters in later refining steps. Formation of glycidyl esters, a product of thermal decomposition of DAG e.g., in deodorization (up to 260 °C), and mitigation thereof, is a well-established and solved problem in the industry. The expert in the art will be able to arrive at an edible oil product by correct operation and / or retrofitting of the downstream refining operations when necessary. Therefore, the invention brings a net increase in edible oil yields with associated great benefit to food availability among others.

[0053] In an embodiment of the invention, the glycerol present in step b) is 0.5-200% (wt / wt of oil feedstock), preferably 0.5-50% (wt / wt of oil feedstock), more preferably 1-25% (wt / wt of oil feedstock), such as 2-15% (wt / wt of oil feedstock).

[0054] The relevant dosage depends on the process setup. More glycerol leads to higher conversion however, higher glycerol dosage occupies reactor space and thus limits productivity per reactor volume. For virgin oils comprising less than 10 wt% FFA, a dosage of glycerol around 5 wt% will be sufficient to achieve great effect, however, for feedstocks comprising higher concentrations of FFA, there will be a requirement for more glycerol and / or higher drying efficacy.

[0055] Note the difference in description of present glycerol and added glycerol. Present glycerol describes the amount of glycerol present initially when starting a batch reaction, or continuously, when at steady state. Added glycerol is the addition of new glycerol on top of the present amount of glycerol in the system, which may be zero.

[0056] As production plant seldom achieve perfect steady state operation, especially in a series of batch operations, there may be fluctuations in added glycerol amounts, and the following embodiments may be viewed as a whole, describing various scenarios in operation from a fresh startup to perfect steady state operation between batches or in employment of a continuous CSTR system.

[0057] In an embodiment of the invention, net added glycerol corresponds to the stoichiometrically required amount to allow reaction and the amount of glycerol lost by inefficient separation, while an excess of glycerol optionally exists in the reactor and is recycled. This is because the amount of glycerol present during reaction is preferably primarily recycled glycerol from previous reactions. Most of the range of present glycerol described above will be far exceeding the stoichiometrically required amount for reaction. Therefore, it is beneficial to reuse unconverted glycerol and only add whatever amount is required to arrive at the desired glycerol dosage. In one embodiment of the present invention, the amount of glycerol added corresponds to the stoichiometrically required amount or above. This requirement will depend on the desired product. Conversion of 100 g oleic acid will require around 33 % glycerol (wt / wt of oleic acid feedstock) to result in 100% MAG stoichiometrically, and 22% will stoichiometrically result in 50 / 50 MAG / DAG. However, chemical equilibrium through reaction conditions, reaction time, and enzyme dosage will dictate the required stoichiometry depending on target product profile.

[0058] In a further embodiment of the invention, the total dosage of all incoming glycerolcontaining streams results in a stoichiometric excess of glycerol, relative to the chosen resulting product composition, and the residual glycerol is used to allow for enzyme reuse through reuse of the glycerol heavy phase.

[0059] In a further embodiment, glycerol is added in an amount of between 0.5%-200% preferably 20%-180%, more preferably 40-150% most preferably 1-100% wt / wt of fatty acid feedstock oil.

[0060] The expert in the art will know, that due to production plant variations, net added glycerol will fluctuate between batches or during continuous operation. It must at times correspond to a large part or the full steady state desired amount of glycerol present, for example after a maintenance shutdown. But generally, the invention is preferably practiced in a manner requiring only stoichiometric amounts of glycerol as add-up between reactions, with a large accumulated and reused amount of glycerol constantly available or present in the system.

[0061] In one embodiment of the present invention, net added glycerol corresponds to the stoichiometrically required amount to allow reaction and the amount of glycerol lost by inefficient separation, while an excess of glycerol optionally exists in the reactor and is recycled.

[0062] The invention may also be practiced without reuse of residual heavy phase, and the added amount of glycerol would thus correspond to the present amount. This is beneficial and possible, especially when employing high temperatures in exchange for high reaction rate. But high temperatures will degrade the enzyme, allowing for little to no enzyme activity recycling, and as a result, there is no inherent utility in accumulating glycerol. Full or near-full consumption of glycerol brings simplicity at the cost of increased enzyme consumption, which may be prohibitive economically.

[0063] In one embodiment of the present invention, glycerol in step b) is present in the feedstock oil, added from an exogenous source, and / or recycled from a down- or upstream process step.

[0064] The relevant substrate in accordance with the present invention are a broad variety of vegetable oils and fats; rapeseed and soybean oils are most commonly used, though other crops such as mustard, sunflower, canola, coconut, hemp, palm oil and even algae show promise. The substrate can be of crude quality or further processed (especially degummed and / or bleached). Also, animal fats including tallow, lard, poultry, marine oil as well as waste vegetable and animal fats and oil, commonly known as yellow and brown grease can be used. The suitable fats and oils are mixtures of glycerides and free fatty acids, commonly seen in waste vegetable oil and animal fats as well as crude oils. The substrate may also be obtained from vegetable oil deodorizer distillates. The type of fatty acids in the substrate comprises those naturally occurring as glycerides in vegetable and animal fats and oils. These include oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, and lauric acid to name a few. Minor constituents in crude vegetable oils are typically phospholipids, free fatty acids and partial glycerides i.e., mono- and diglycerides.

[0065] The term “fatty acid feedstock” or “oils and / or fats” or “vegetable oil feedstock” is defined herein as a substrate comprising fatty acid derivatives. The substrate may comprise triglyceride, diglyceride, monoglyceride, free fatty acid or any combination thereof.

[0066] The fatty acid feedstock may be oil 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, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, shea oil, tall oil, 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 combinations thereof.

[0067] In an embodiment of the invention, the lipase is selected from lipases belonging to EC 3.1.1. In another embodiment the lipase is selected from lipases belonging to EC 3.1 .1 .3.

[0068] The lipase of choice importantly does not exhibit substantial activity in esterification of DAG to TAG, hydrolysis of TAG to DAG and FFA, and in interesterification of TAG.

[0069] In an embodiment of the invention, the lipase is selected from the group consisting of: i) a lipase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 ; or ii) a lipase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0070] In an embodiment of the present invention, the lipase has at least 80% sequence identity to SEQ ID NO:1.

[0071] In another embodiment, the lipase has at least at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1 .

[0072] In a preferred embodiment of the invention, the lipase is a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 1.

[0073] In a preferred embodiment of the invention, the lipase comprises, consists essentially of, or consists of SEQ ID NO: 1 .

[0074] In an embodiment of the invention, the lipase comprises or consists of the amino acid sequence shown in SEQ ID NO 1.

[0075] In an embodiment of the invention, the lipase has at least 80% sequence identity to SEQ ID NO: 2.

[0076] In another embodiment of the invention, the lipase has at least at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0077] In a preferred embodiment of the invention, the lipase comprises, consists essentially of, or consists of SEQ ID NO: 2.

[0078] In an embodiment of the invention, the lipase comprises or consists of the amino acid sequence shown in SEQ ID NO 2.

[0079] In an embodiment of the invention, less than 10%, preferably less than 5%, more preferably less than 2% and most preferably less than 1%, such as less than 0.5% of FFA in step b) is converted into triacylglycerides (TAG).

[0080] In an embodiment of the invention, the feedstock comprises TAG levels exceeding 10 wt%, and preferably those exceeding 90 wt%.

[0081] Most relevant feedstocks will be virgin oils, with crude palm oil typically comprising up to 12 wt% FFA, roughly, and soybean up to 4 wt%.

[0082] In an embodiment of the invention, the FFA content in the oil feedstock is less than 15 wt%, less than 12 wt%, less than 10%, less than 8 wt%, such as less than 5 wt%, and the TAG content in the oil feedstock is more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, more than 50 wt%, more than 60 wt%, more than 70 wt%, more than 80 wt%, such as more than 90 wt%.

[0083] However, other relevant feedstocks are used cooking oil (UCO), brown grease, and the like. Such waste quality feedstocks typically comprise FFA concentration exceeding 10 wt%, such as 50 wt%, and therefore TAG concentrations between 5 and 90 wt%. Such feedstocks are also relevant, even though they are inedible, because reduction of FFA has utility outside the scope of virgin food oils, and the invention is similarly relevant and applicable on those. For example, this is very relevant for preparation of feedstocks in production of sustainable aviation fuel or other derivatives of hydrotreated vegetable oils (HVO), where hydrogen gas is used for full reduction of carboxylic acids to aliphatic hydrocarbons. In the hydrotreatment process, a heterogeneous catalyst is typically employed under high temperatures and pressures (>300 °C and >20 barg), where the acidity of FFA, while being mild at ambient conditions, becomes critically difficult to handle from a corrosion perspective. As such, FFA concentrations in the feedstocks must be below 20 wt% in many HVO plants, and levels below 2.5 wt% are even common in older retrofitted crude oil refineries employing the original steel alloys rather than modern HVO-customized alloys. This means such waste oils are today largely only applicable when blended into large amounts of high-quality edible oils, with significant negative effects on food prices and availability. Employment of the known means of FFA reduction, described by the prior arts mentioned in the introduction, brings yield losses or are too expensive or impractical (for example, the P. camemberti enzyme will not work well at temperatures exceeding 50°C and is to the knowledge of the authors not commercially available at scale). For this purpose, the present invention brings down cost significantly by preferably employing a known, available, and thermostable enzyme in cheap and liquid form, preferably SEQ ID NO:1 , without issues associated with irreversible clogging of immobilized and therefore more expensive formulations.

[0084] In an embodiment of the invention the FFA content in the oil feedstock is more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, such as about 50 wt%, and the TAG content in the oil feedstock is more than 5 wt%, more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, more than 50 wt%, more than 60 wt%, more than 70 wt%, more than 80 wt%, such as more than 90 wt%.

[0085] Particularly, for seed oils the FFA content in the oil feedstock may be in the range from 0.1-100 wt%, 0.25-90 wt%, 0.5-70 wt%, 0.5-50 wt%, 0.5-40 wt%, 0.5-30 wt%, 0.5-25 wt%, 0.5- 20 wt%, 0.5-15 wt%, 0.5-12.5 wt%, such as 1-10 wt%.

[0086] Particularly, for waste oils the FFA content in the feedstock oil may be in the range from 5-100 wt%, 20-95 wt%, 25-85 wt%, such as 30-80 wt %.

[0087] In one embodiment of the invention, more than 25 %, preferably more than 50 %, and most preferably more than 75 % of the FFA in the feedstock is converted into MAG and DAG. The MAG and DAG composition after esterification is, however, not as important, since the main purpose is FFA reduction because the reduction in FFA will lead to higher yields of refined oil by reduction of soapstock formation in chemical refining.

[0088] Preferably, the lipase of choice will form and / or hydrolyze DAG at a 10 times higher rate than TAG. Such hydrolysis rate can be measured by rate of FFA formation in reaction between 1g water and 1g DAG or TAG, along with 0.1 mg esterase protein at 30 °C. Esterification rate can be measured as rate formation of DAG or TAG by reaction between 0.5g MAG or DAG, 0.5g FFA with 1 mg esterase at 30 °C under vacuum. The expert in the art will be able to identify other relevant measures of relative rate of formation and hydrolysis of DAG and TAG.

[0089] In an embodiment of the invention, the degree of interesterification measured as degree of randomization of the triglycerides is below 20%, preferably 10%, most preferably below 5% relative to a fully randomized feedstock.

[0090] In an embodiment of the present invention, lipase is dosed in the range of 0.1 - 50000 mg enzyme protein (EP) / kg of oil, preferably in the range of 0.1-200 mg enzyme protein (EP) / kg of oil, 5-100 mg enzyme protein (EP) / kg of oil, such as 10-50 mg enzyme protein (EP) / kg of oil.

[0091] In an embodiment of the invention, the pH of step b) is optionally adjusted during and / or prior to reacting / esterification.

[0092] In an embodiment of the invention, the pH during step b) is in the range of 2.5-8.0, 3.0- 7.5, such as 4.0-7.0.

[0093] In an embodiment of the invention, the pH is preferably adjusted using citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide.

[0094] In an embodiment of the present invention, the process is performed at temperatures in the range of 5°C-100°C, 10°C-95°C, 10°C-90°C, 10°C-85°C, 25°C-85°C, 40°C-85°C, 50°C-85°C, 55°C-85°C, 60°C-85°C, 65°C-85°C, 70°C-85°C, 75°C-85°C, 60°C-80°C, 65°C-80°C, 70°C-80°C, 75°C-80°C such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C or 85°C.

[0095] In an embodiment of the invention, the process is performed in less than 48 hours, preferably less than 24 hours and most preferably less than 12 hours. The reaction time will primarily depend on the desired degree of conversion.

[0096] In an embodiment of the invention, the process is performed in a batch, semi-continuous or continuous mode.

[0097] In another embodiment of the present invention, the process runs in several sequential reaction steps such as 2-10 reactors in series, preferably 2-5 reactors in series. Most preferably a single reactor will be sufficient, but this will depend on the target product and choice of implementation of the invention. For example, in large continuous plants, requiring a high degree of conversion, several CSTR reactors in series is typically the optimal choice of setup. For smaller setups, producing a small amount of relatively valuable product, a single batch reactor is often sufficient. A process engineer will be able to evaluate and design a proper reactor setup capable of achieving target conversions most efficiently.

[0098] In another embodiment of the present invention, the process is performed in optionally countercurrent, optionally compartmentalized, reactor(s). Countercurrent operation has the benefit of transferring water, through absorption into the glycerol phase, in the opposite direction of the light phase product, yielding a net effect similar to that of drying by reducing water levels at the final stages of the conversion. While compartmentalized CSTR reactors, as an example, will approach a plug flow, which a process engineer will recognize as the optimal design, while being more costly.

[0099] In an embodiment of the invention, the process further comprises a reaction with one or more additional enzyme(s) that do not exhibit a detrimental activity to the target of the invention. An undesirable choice of second enzyme would be a TAG-active lipase, which would result in TAG interesterification and / or conversion and quality reduction through unnecessarily elevated concentrations of MAG and DAG through glycerolysis (TAG+glycerol --> MAG + DAG). A desirable choice, depending on feedstock, would be a phospholipase, chlorophyllase and so on, each bringing further benefits without exhibiting substantial activity in interesterification, formation or hydrolysis of TAG. The expert in the art will be able to identify relevant combinations of enzyme activities.

[0100] In an embodiment of the invention, one or more enzymes are applied in combination with the main TAG-inactive lipase, either in a combined single reaction step or in sequential reaction steps with one, some or all of the enzymes active in each.

[0101] In an embodiment of the present invention, one or more additional enzymes are applied only in combination with the TAG-inactive lipase.

[0102] In an embodiment of the present invention, one or more additional enzymes are phospholipases and / or chlorophyllases.

[0103] In an embodiment of the invention, the feedstock oil is degummed, before, during or after the process.

[0104] In one embodiment of the invention, the reaction mixture and / or one or more incoming streams are dried. The reaction mixture may be dried before and / or during and / or after step b). Drying after step b) has utility within the process of the invention, especially when part of the reaction mixture or phases thereof are recycled, but may also have utility for later processing steps such as prior to a bleaching step following the process of the invention. If the invention is practiced without employing recycling, and if chemical neutralization directly follows the process of the invention (with reduced soapstock byproduct production as a result of the process of the invention), then drying after reaction will have no utility. A beneficial and preferred way of practicing the invention is to allow for reduced soapstock byproduct formation by employing the invention prior to a chemical refining process in an edible oil refinery. Example 1 shows such a relevant reduction of FFA, starting from 1 .5 wt% FFA in crude soybean oil, and bringing the concentration down to 0.6 wt% in 4 hours of reaction time, and further down to 0.2 wt% in 24 hours. The reaction is conducted under vacuum to continuously remove water. This drives FFA at equilibrium towards zero. In practice, an existing refinery will often not have 24 hours of reaction time available due to the plant throughput and associated required reactor sizes. As such, accepting only 4 hours of reaction (and drying time), by employing very efficient drying by known industrial means to quickly reduce FFA levels will be preferable in most cases. The dryness prior to and during reaction is therefore important. For example, if all incoming streams are completely dried prior to reaction, and the process of the invention is employed without further drying, the formation of water through the esterification reaction will determine the final FFA concentration achievable through chemical equilibrium. And to further reduce the FFA level, drying will be necessary during reaction, to remove a sufficient part of the formed water. If the user of the invention requires full conversion of FFA, drying during reaction is required. However, if the user of the invention intends for a fast reaction and accepts only 50% reduction in FFA, the initial dryness of the system may be sufficient without further drying. It is thus clear that a preferable process would include the possibility of drying of incoming streams and / or during reaction, and employ the drying equipment when appropriate, depending on the initial FFA concentration of the feedstock and desired degree of conversion. Another important point in terms of dryness is the impact of the amount of glycerol on the FFA level. Glycerol is known to absorb and lower the chemical potential of water, effectively acting as a drying agent. Therefore, with increased amounts of initial glycerol, lower FFA concentrations are achievable, but at the cost of increased glycerol procurement and recovery costs. From this description it is also clear that higher initial FFA concentrations in the feedstock increases the necessity of dryness when low FFA concentrations are desired.

[0105] In one embodiment of the invention, the reaction mixture is dried during the reaction. Such drying may be affected by directly applying vacuum to the reaction mixture, or by a drying system such as a flash drying column.

[0106] In an embodiment of the invention, further comprising separating the mixture of step b) into light and heavy phase.

[0107] In an embodiment of the invention, the process is performed in the absence of short chain alcohol, such as e.g., ethanol and / or methanol.

[0108] In an embodiment of the invention, the process is performed without addition of solvent, wherein the solvent is an insert solvent, e.g., an aliphatic or an aromatic solvent, such as hexane, heptane, toluene, xylene, or benzene. In an embodiment of the invention, the light phase comprises increased amounts of MAG and / or DAG and decreased FFA.

[0109] In an embodiment of the invention, the heavy phase comprises water, lipase, glycerol.

[0110] In an embodiment of the invention, the heavy phase is dried.

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

[0112] In an embodiment of the invention, the light phase is partially or fully recycled into step b).

[0113] In an embodiment of the invention, the light phase is optionally subjected to purification.

[0114] In an embodiment of the invention, the reaction mixture is initially seeded by product from prior reaction or relevant material with elevated concentrations of emulsifiers, which may improve the initial emulsification rate. This is not required, but relevant because good emulsification increases reaction rate, and initial emulsification of heavy glycerol (density 1 .25 g / mL) into light oil (density around 0.9 g / mL) can delay reaction onset.

[0115] The process according to the invention may be applied in combination with further process steps, e.g., in one embodiment the process comprises a refining step, and / or degumming step.

[0116] The invention is further described in the following numbered paragraphs.

[0117] Paragraph 1 : A process for reducing FFA (Free fatty acid) content in an oil feedstock comprising steps of: a) Providing an oil feedstock comprising FFA; b) Reacting the oil feedstock of a) by esterification of FFA with glycerol in the presence of a lipase to convert FFA into MAG and DAG; wherein the lipase is applied in granular, liquid or water-soluble form, and wherein the lipase is selected from lipases which are inactive on TAG, or substantially inactive on TAG.

[0118] Paragraph 2: The process according to paragraph 1 , wherein the glycerol present in step b) is 0.5-200% (wt / wt of oil feedstock), preferably 0.5-50% (wt / wt of oil feedstock), more preferably 1-25% (wt / wt of oil feedstock), such as 2-15% (wt / wt of oil feedstock).

[0119] Paragraph 3: The process according to paragraph 1 , wherein the glycerol is added in an amount of between 0.5%-200% preferably 20%-180%, more preferably 40-150% most preferably 1-100% wt / wt of fatty acid feedstock oil.

[0120] Paragraph 4: The process according to paragraph 1 , wherein the lipase is selected from the group consisting of: i) a lipase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 ; or ii) a lipase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0121] Paragraph 5: The process according to any of the preceding c paragraphs, wherein less than 10%, preferably less than 5%, more preferably less than 2% and most preferably less than 1%, such as less than 0.5% of FFA in step b) is converted into triacylglycerides (TAG).

[0122] Paragraph 6: The process according to any of the preceding paragraphs, wherein the FFA content in the oil feedstock is less than 15 wt%, less than 12 wt%, less than 10%, less than 8 wt%, such as less than 5 wt%, and the TAG content in the oil feedstock is more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, more than 50 wt%, more than 60 wt%, more than 70 wt%, more than 80 wt%, such as more than 90 wt%.

[0123] Paragraph 7: The process according to any of the preceding paragraphs, wherein the FFA content in the oil feedstock is more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, such as about 50 wt%, and the TAG content in the oil feedstock is more than 5 wt%, more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, more than 50 wt%, more than 60 wt%, more than 70 wt%, more than 80 wt%, such as more than 90 wt%.

[0124] Paragraph 8: The process according to any of the preceding paragraphs, wherein the FFA content in the oil feedstock is in the range from 0.1-100 wt%, 0.25-90 wt%, 0.5-70 wt%, 0.5- 50 wt%, 0.5-40 wt%, 0.5-30 wt%, 0.5-25 wt%, 0.5-20 wt%, 0.5-15 wt%, 0.5-12.5 wt%, such as 1- 10 wt%.

[0125] Paragraph 9: The process according to any of the preceding paragraphs, wherein feedstock oil comprises waste oils having an FFA content in the range from 5-100 wt%, 20-95 wt%, 25-85 wt%, such as 30-80 wt %.

[0126] Paragraph 10: The process according to any of the preceding paragraphs, wherein more than 25%, particularly more than 50%, most particularly more than 75% of the FFA in the oil feedstock is converted to MAG and DAG.

[0127] Paragraph 11 : The process according to any of the preceding paragraphs, wherein the degree of interesterification measure as degree of randomization of the triglycerides will be below 20%, 10%, preferably below 5% relative to a fully randomized feedstock.

[0128] Paragraph 12: The process according to paragraph 1 , wherein the process is performed at temperatures in the range of 5°C-100°C, 10°C-95°C, 10°C-90°C, 10°C-85°C, 25°C-85°C, 40°C-85°C, 50°C-85°C, 55°C-85°C, 60°C-85°C, 65°C-85°C, 70°C-85°C, 75°C-85°C, 60°C-80°C, 65°C-80°C, 70°C-80°C, 75°C-80°C such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C or 85°C. Paragraph 13: The process according to paragraph 1 , 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 10-50 mg enzyme protein (EP) / kg of oil.

[0129] Paragraph 14: The process according to paragraph 1 , wherein pH in step b) is optionally adjusted during and / or prior to reacting.

[0130] Paragraph 15: The process according to paragraph 14, wherein the pH in step b) is in the range of 2.5-8.0, 3.0-7.5, such as 4.0-7.0.

[0131] Paragraph 16: The process according to paragraphs 14 and 15, wherein the pH is preferably adjusted using citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide.

[0132] Paragraph 17: The process according to any of the preceding paragraphs, wherein the process is performed in less than 48 hours, preferably less than 24 hours and most preferably less than 12 hours.

[0133] Paragraph 18: The process according to any of the preceding paragraphs, wherein the process is performed in a batch, semi-continuous or continuous mode.

[0134] Paragraph 19: The process according to any of the preceding paragraphs, further comprises contacting the oil feedstock with chlorophyllase and / or phospholipase.

[0135] Paragraph 20: The process according to any of the preceding paragraphs, wherein the feedstock oil is degummed, before, during or after the process of paragraph 1.

[0136] Paragraph 21 : The process according to paragraph 1 , wherein the reaction mixture is dried before and / or during and / or after step b).

[0137] Paragraph 22: The process according to paragraph 1 , further comprising separating the mixture of step b) into a light and a heavy phase.

[0138] Paragraph 23. The process according to any of the preceding paragraphs, wherein the process is performed in the absence of short chain alcohol, such as e.g., ethanol and / or methanol.

[0139] Paragraph 24. The process according to any of the preceding paragraphs, wherein the process is performed without addition of solvent, wherein the solvent is an insert solvent, e.g., an aliphatic or an aromatic solvent, such as hexane, heptane, toluene, xylene, or benzene.

[0140] Paragraph 25: The process according to paragraph 22, wherein the light phase comprises increased amounts of MAG and / or DAG and decreased FFA.

[0141] Paragraph 26: The process according to paragraph 22, wherein the heavy phase comprises water, lipase, glycerol.

[0142] Paragraph 27: The process according to paragraph 22, where the heavy phase is dried.

[0143] Paragraph 28: The process according to any of paragraphs 22-27, wherein the heavy phase is partially or fully recycled into step b).

[0144] Paragraph 29: The process according to paragraph 22, wherein the light phase is partially or fully recycled into step b). Paragraph 30: The process according to paragraph 22, wherein the light phase is optionally subjected to purification.

[0145] Paragraph 31 : The process according to paragraph 1 , wherein the oil feedstock 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.

[0146] Paragraph 32: The process according to any of the preceding paragraphs, wherein the glycerol present in step b) is present in the feedstock oil, added from an exogenous source, or recycled from a downstream process step.

[0147] Paragraph 33: The process according to any of the preceding paragraphs, wherein net added glycerol corresponds to the stoichiometrically required amount to allow reaction, and the amount of glycerol lost by inefficient separation, while an excess of glycerol optionally exists in the reactor and is recycled.

[0148] Paragraph 34: The process according to any of the preceding paragraphs, comprising a refining step, and or degumming step.

[0149] The invention is further illustrated in the following examples.

[0150] Examples

[0151] Example 1 : FFA reduction in crude soybean oil at 55 °C using the lipase of SEQ ID NO:1.

[0152] 30g of crude soybean oil was mixed with 0% (0g) or 5% (1 .5 g) (wt / wt of oil) technical grade glycerol. 0.1 or 0.5 % (wt / wt of oil) liquid enzyme solution of SEQ ID NO: 1 was added. The solution contained around 10 mg / mL active enzyme protein. The mixture was incubated at 55°C in 100 mL closed square bottles for 24 hours at 250 rpm shaking stirring in a shaking incubator oven. The reaction bottles were placed in a custom-made metal box with rubber gasket and lid allowing for vacuum to be applied. Vacuum was turned on and applied continuously at <5 mbara during the extent of reaction. The resulting product light phase was measured.

[0153] Analyses: wt% FFA by titration (AOCS 5a-40 Free Fatty Acid in Crude and Refined Fats and Oils) and the diglycerides were measured by HPLC (AOCS method Cd11d-96, using heptane rather than hexane).

[0154] Table 1 : FFA reduction

[0155] The results in table 1 show reduction of FFA from 1 .5 wt% in crude soybean oil by app ying SEQ ID NO:1 while running reaction under vacuum. Without presence of glycerol, there was insignificant change in the concentration of FFA. When glycerol was added, however, there was a significant drop in the concentration of FFA. With the low enzyme dosage of 0.1%, 33% conversion of FFA was achieved within 4 hours of reaction, and 66% after 24 hours. With the higher enzyme dosage of 0.5%, >85% conversion of FFA was achieved. DAG does not immediately rise proportionally with the reduction in FFA. This was likely because MAG was initially formed, and then becomes DAG towards the end of reaction. In the final datapoint, the sum of DAG and FFA exceeds the sum in the crude oil. This indicates that MAG in the crude oil was also converted to DAG, yielding lower MAG in the resulting mixture than was in the crude oil, while an amount of TAG may have been converted too.

[0156] Example 2: Esterification of FFA onto glycerol without use of vacuum with high amounts of glycerol dosed using the lipase of SEQ ID NO: 1.

[0157] 20g of dry technical grade glycerol was mixed with 5, 10 or 20 g technical grade oleic acid (FFA). 0.1 % (wt / wt of oleic acid) liquid enzyme solution of SEQ ID NO: 1 was added. The solution contained around 45 mg / mL active enzyme protein. The mixture was incubated at 60°C in 100 mL closed square bottles for 24 hours at 250 rpm shaking stirring and the resulting product light phase was measured.

[0158] Analyses: wt% FFA by titration (AOCS 5a-40 Free Fatty Acid in Crude and Refined Fats and Oils) and the mono-, di- and triglycerides were measured by a customized HPLC method. The customized HPLC method was not corrected by standard curve or response factor, so the reported MAG and DAG concentrations are normalized peak areas while subtracting wt% FFA from the normalization basis.

[0159] Table 2: Effect of high dose of glycerol

[0160] First note that the feedstock in this case is oleic acid, and therefore 100% FFA, which makes this an example of the worst possible case in terms of required conversion. With higher amounts of glycerol relative to FFA, achievable FFA concentrations at equilibrium drops. This example was thus meant to show that with largely dry initial incoming streams, a significant degree of conversion of FFA is achievable, when employing a high dosage of glycerol (100% up to 400% here) as shown in table 2. The following example, Example 3, shows lower relative dosages of glycerol. TAG formation is surprisingly low and may be kept even lower by reducing the reaction time.

[0161] Example 3: Esterification of FFA onto glycerol without vacuum at around stoichiometrically relevant glycerol dosages using the lipase of SEQ ID NO: 1.

[0162] Technical grade glycerol was mixed with technical grade oleic acid in the ratios indicated in the table below. 0.1 % (wt / wt of 50g total reaction mixture) liquid enzyme solution of SEQ ID NO: 1 was added. The solution contained around 45 mg / mL active enzyme protein. The mixture was incubated at 55°C in 100 mL closed square bottles for 48 hours at 250 rpm shaking stirring and the resulting product light phase was measured.

[0163] Table 3: glycerol:oleic acid ratios

[0164] Analyses: wt% FFA by titration (AOCS 5a-40 Free Fatty Acid in Crude and Refined Fats and Oils) and the mono-, di- and triglycerides were measured by a customized HPLC method. The customized HPLC method was not corrected by standard curve or response factor, so the reported MAG, DAG and TAG concentrations are normalized peak areas while subtracting wt% FFA from the normalization basis.

[0165] Table 4: Equilibria at various ratios of FFA to glycerol

[0166] The results show achievable equilibria at various ratios of FFA to glycerol. Again, starting from near-pure FFA feedstock. Relative to the experiment above, Example 2, table 4 shows resulting compositions at higher amounts of FFA relative to glycerol. Generally, as initial FFA increases relative to glycerol, the achievable conversion (without water removal during reaction) is reduced. This was the basis for the described and recommended use of high amounts of glycerol when practicing the invention without use of water removal during reaction due to the net drying effect of presence of glycerol. Generally, very low amounts of TAG were observed in the HPLC chromatogram, making quantization impossible. Note that 3 mol FFA / glycerol was stoichiometric in terms of formation of TAG, while lower ratios represent a stoichiometric excess of glycerol.

[0167] Example 4: Esterification of FFA onto glycerol using the lipase of SEQ ID NO: 1 .

[0168] Technical grade glycerol was mixed with technical grade oleic acid in ratios as indicated in the table 5. Liquid enzyme solution of SEQ ID NO: 1 was added in an amount shown in table 5 with units of % (wt / wt of oil). The solution contained around 10 mg / mL active enzyme protein. The mixture was incubated in a heated 4x6 1.5 mL well plate mixed by vibration with 4000 rpm set point and cross-formed-magnet in each well for 24 hours using a Tumblemixer (type VP 710C5-7A by VP Scientific) with custom built airtight vacuum hood. The resulting product light phase was measured. Reactions ran under continuous vacuum of around 100-200 mbara (house vacuum) at temperatures as indicated in table 5. Analyses: wt% FFA by titration (AOCS 5a-40 Free Fatty Acid in Crude and Refined Fats and Oils) and the mono-, di- and triglycerides were measured by a customized GC method. Note that other experiments were measured using HPLC. This GC method yields better quality measurements. The customized GC method was not corrected by standard curve, so the reported FFA, MAG, DAG and TAG concentrations are normalized peak areas. The response factors that were measured were: Oleic acid 0.79, 0.80 and 0.81 , Mono-olein: 1.0, 1.0 and 1.0, Di-olein: 1.1 , 0.99 and 0.90, Tri-Olein: 1.19, 0.97 and 1.06. As an example, for FFA (oleic acid), a relative peak area of 10 % is thus closer to 8 wt%. Response factors of mono-, di-, and triglycerides of olein are around 1 and correction from relative peak area to wt% will be largely the same. However, it is not possible to convert the reported numbers below directly into true wt% using these response factors, as they do not account for non-fatty-material, especially dissolved glycerol, which dilutes the results in reality. Therefore, normalized relative peak areas of FFA, MAG, DAG and TAG without accounting for diluting compounds, which are removable in industrial post-treatment were reported. Table 5: Conversion of FFA to MAG and DAG with low formation of TAG Obtaining high conversion of FFA to MAG and DAG with low formation of TAG under vacuum is possible and proven as shown in table 5. Combined prior examples, this scales to feedstocks comprising lower (and relevant) FFA concentrations.

[0169] With increased temperature comes improved water evaporation and reduction of FFA. However, TAG increases significantly if reaction runs for too long, which is why the final, and most important, datapoint above shows the 16h timepoint of a 1 glycerol: 1 FFA molar stoichiometric reaction, while generally the examples show reaction results after 24h.

[0170] Example 5: Esterification of FFA in crude palm oil showing near-zero interesterification using the lipase of SEQ ID NO: 1 .

[0171] 100g of crude palm oil was added to a round bottle and heated to 70°C. Then technical grade glycerol was added in amounts as shown in table 6. Liquid enzyme solution of SEQ ID NO: 1 was added in amounts as shown in table 6 with units of % (wt / wt of oil). The solution contained around 10 mg / mL active enzyme protein. The mixture was high-shear mixed at 24000 rpm for 30 seconds. Then the flasks were attached to vacuum and ran at 0-20 mbara and 70 °C with 350 rpm magnetic stirring.

[0172] Analyses: wt% FFA by titration (AOCS 5a-40 Free Fatty Acid in Crude and Refined Fats and Oils) and the mono-, di- and triglycerides were measured by GO using a customized method. Table 6: Esterification of FFA in crude palm oil

[0173] This trial shows FFA reduction in crude palm oil using SEQ ID NO:1 at relatively high dosages of 1 and 2 % (wt / wt of oil). In all cases, FFA was reduced and a largely corresponding amount of MAG and DAG were formed. Comparing reaction 1-8 with reaction 9, presence of significant amounts of glycerol was required for near-full conversion. The thorough initial mixing using high shear mixing, high enzyme dosage, as well as deep vacuum and use of 350 rpm magnetic stirring yields a very efficient reaction and >90% FFA conversion in just 3 hours in reaction 9. In that reaction, conversion of TAG was below 1 %, when comparing FFA conversion to the MAG / DAG formation, and the degree of interesterification was insignificant, judging from the most notable POP, POO and PPP. T riglyceride species are typically denoted by three-letter abbreviations with letter P = palmitic (C16:0), S = stearic (C18:0), O = oleic (C18:1), M = myristic (C14:0), L = Linoleic (C18:2), and so on. PPP for example denotes a triglyceride with three palmitic acids bound to the glycerol molecule. POP would have oleic acid in the middle position. Degree of interesterification was thus a combined measure of the change in distribution of triglyceride species from the natural distribution determined by the oil origin, towards the theoretically fully randomized, and unnatural, distribution achievable statistically and thermodynamically. The expert in the art will recognize the insignificant changes in the results of this experiment as a measure of little to no interesterification. This example proves that interesterification can be kept low and FFA conversion high, enabling production of higher amounts of edible oil rather than inedible technical grade byproducts as shown in table 6.

[0174] Example 6: Thermostability of liquid (free) lipase of SEQ ID NO:1 in presence of glycerol

[0175] A mixture of crude palm oil (4.4 wt% FFA) and oleic acid was made comprising a combined FFA concentration of 7.8 wt%. 30g of the oil blend was preheated to 35 °C and then mixed with 0 or 3 g of glycerol, 1.17 mg of SEQ ID NO:1 as 0.85 wt% active enzyme protein aqueous solution, and 0, 0.5 or 1 % (wt / wt oil) of water. 50 ppm of sodium hydroxide was added as 1 M solution. Finally, 1 or 2 % (wt / wt oil) of methanol was added upon reaction initiation and reaction ran at 35 °C and at 250 rpm in a shaking incubator oven in square 100 mL bottles.

[0176] Analyses: wt% FFA by titration (AOCS 5a-40 Free Fatty Acid in Crude and Refined Fats and Oils). FAME by 1 H NMR, measured as relative peak area of terminal CH3 and fatty acid methyl ester bond peak.

[0177] This example employs free lipase of SEQ ID NO:1 in reduction of FFA but results in FFA esterification with methanol rather than with glycerol. It is an example of the significant limitations in using SEQ ID NO:1 at conditions not involving glycerol. In this example, glycerol is present, but does not participate in the reaction, because methanol competes for the fatty acids because thermodynamically, methanol preferentially esterifies instead of glycerol. Therefore, this example depicts the significant and previously unknown improvement brought by presence of glycerol itself to the stability and efficacy of SEQ ID NO:1 in esterification reactions because its effect as a reactant was removed.

[0178] Methanol itself has known to be very destabilizing to lipases, lowering the thermostability significantly. Thermomyces lanuginosus lipase are often the choice of enzyme experiences a drop in thermostability from around 60-75 °C down to 35-45°C at industrially relevant dosages of methanol, as an example. Using lipase of SEQ ID NO:1 at 35°C was performed because of the known negative impact of methanol on the thermostability of the enzyme, but as the results show, presence of glycerol markedly increases the stability of the enzyme at these difficult conditions of methanol presence.

[0179] Table 7: Stability of lipase in presence of methanol-glycerol

[0180] Results in table 7 show a significant improvement in rate of reaction and final FFA concentration when glycerol was added. FFA concentration at equilibrium (assumed to be at 24 h) was mainly determined by the ratio of methanol and water. Glycerol does provide some degree of water inactivation, as known in the art, thereby impacting the achievable equilibrium. However, this effect of glycerol cannot explain the significant difference in achieved FFA concentrations with and without the presence of glycerol, only improved enzyme stability can. Therefore, results in table 7 prove that glycerol significantly improves the stability of the enzyme. In the context of this invention this example serves to show that inventors have found a previously unknown effect of glycerol, namely that it allows for employment of lipase at temperatures exceedingly previously known acceptable temperatures of the lipase in free form.

Claims

Claims:

1. A process for reducing FFA (Free fatty acid) content in an oil feedstock comprising steps of: a) Providing an oil feedstock comprising FFA; b) Reacting the oil feedstock of a) by esterification of FFA with glycerol in the presence of a lipase to convert FFA into MAG and DAG; wherein the lipase is applied in granular, liquid or water-soluble form, and wherein the lipase is selected from lipases which are inactive on TAG, or substantially inactive on TAG.

2. The process according to claim 1 , wherein the glycerol present in step b) is 0.5-200% (wt / wt of oil feedstock), preferably 0.5-50% (wt / wt of oil feedstock), more preferably 1-25% (wt / wt of oil feedstock), such as 2-15% (wt / wt of oil feedstock).

3. The process according to claim 1 , wherein the glycerol is added in an amount of between 0.5%-200% preferably 20%-180%, more preferably 40-150% most preferably 1-100% wt / wt of fatty acid feedstock oil.

4. The process according to claim 1 , wherein the lipase is selected from the group consisting of: i) a lipase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 ; or ii) a lipase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

5. The process according to any of the preceding claims, wherein less than 10%, preferably less than 5%, more preferably less than 2% and most preferably less than 1%, such as less than 0.5% of FFA in step b) is converted into triacylglycerides (TAG).

6. The process according to any of the preceding claims, wherein the FFA content in the oil feedstock is less than 15 wt%, less than 12 wt%, less than 10%, less than 8 wt%, such as less than 5 wt%, and the TAG content in the oil feedstock is more than 10 wt%, more than 20 wt%,more than 30 wt%, more than 40 wt%, more than 50 wt%, more than 60 wt%, more than 70 wt%, more than 80 wt%, such as more than 90 wt%.

7. The process according to any of the preceding claims, wherein the FFA content in the oil feedstock is more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, such as about 50 wt%, and the TAG content in the oil feedstock is more than 5 wt%, more than 10 wt%, more than 20 wt%, more than 30 wt%, more than 40 wt%, more than 50 wt%, more than 60 wt%, more than 70 wt%, more than 80 wt%, such as more than 90 wt%.

8. The process according to any of the preceding claims, wherein the FFA content in the oil feedstock is in the range from 0.1-100 wt%, 0.25-90 wt%, 0.5-70 wt%, 0.5-50 wt%, 0.5-40 wt%, 0.5-30 wt%, 0.5-25 wt%, 0.5-20 wt%, 0.5-15 wt%, 0.5-12.5 wt%, such as 1-10 wt%.

9. The process according to any of the preceding claims, wherein feedstock oil comprises waste oils having an FFA content in the range from 5-100 wt%, 20-95 wt%, 25-85 wt%, such as 30-80 wt %.

10. The process according to any of the preceding claims, wherein more than 25%, particularly more than 50%, most particularly more than 75% of the FFA in the oil feedstock is converted to MAG and DAG.

11. The process according to any of the preceding claims, wherein the degree of interesterification measure as degree of randomization of the triglycerides will be below 20%, 10%, preferably below 5% relative to a fully randomized feedstock.

12. The process according to claim 1 , wherein the process is performed at temperatures in the range of 5°C-100°C, 10°C-95°C, 10°C-90°C, 10°C-85°C, 25°C-85°C, 40°C-85°C, 50°C- 85°C, 55°C-85°C, 60°C-85°C, 65°C-85°C, 70°C-85°C, 75°C-85°C, 60°C-80°C, 65°C-80°C, 70°C-80°C, 75°C-80°C such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C or 85°C.

13. The process according to claim 1 , 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 10-50 mg enzyme protein (EP) / kg of oil.

14. The process according to any of the preceding claims, wherein the process is performed in less than 48 hours, preferably less than 24 hours and most preferably less than 12 hours.

15. The process according to claim 1 , further comprising separating the mixture of step b) into a light and a heavy phase.

16. The process according to any of the preceding claims, wherein the process is performed in the absence of short chain alcohol, such as e.g., ethanol and / or methanol.