A process for production of mono- and di-acylglycerides

Abstract

The present invention relates to a process for producing mono-acylglycerides (MAG) and / or di-acylglycerides (DAG) comprising steps of: a. providing a mixture comprising free fatty acids (FFA) and / or fatty acid alkyl esters, glycerol and lipase; b. reacting the mixture of a) to produce a mixture of MAG and DAG wherein the lipase is CalB from Candida antarctica which was found to produce MAG and DAG but almost no triacylglycerides using FFA and glycerol.

Description

[0001] A PROCESS FOR PRODUCTION OF MONO- AND DI-ACYLGLYCERIDES

[0002] FIELD OF INVENTION

[0003] The present invention relates to a process for producing monoglycerides (MAG) and diacylglycerides (DAG) using an enzyme. More particularly, the present invention relates to a process for synthesizing monoglycerides and diacylglycerides by reacting a fatty acid or a fatty acid ester with glycerol in the presence of a lipase, wherein, as the lipase, a monoglyceride lipase and / or a diglyceride lipase is used to produce monoglycerides and diacylglycerides containing little or no triacylglyceride (TAG) and consisting substantially of a monoglyceride and a diglyceride.

[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] Today, MAG and DAG are commonly produced together as a mixture through thermal glycerolysis. Glycerolysis is a broad term, used for several reactions involving glycerol. Typically, glycerolysis is a reaction between TAG and glycerol to form lower triglycerides (MAG and DAG). Thermal glycerolysis runs at temperatures of about 200°C, and often with a catalyst such as sodium hydroxide. High temperature processes, especially in presence of harsh chemical catalysts, result in byproducts that must be removed along with unconverted reactants, and this downstream processing often involves washing, bleaching and deodorization with associated yield losses and expenses. Glycerolysis can also alternatively be conducted by use of enzymes as catalysts. Typically, an immobilized lipase catalyzes conversion by glycerolysis of TAG and glycerol to mixtures of MAG and DAG. However, usually a significant amount of the product will consist of TAG.

[0007] Apart from glycerolysis, esterification of FFA onto glycerol can be achieved without the use of catalysts at temperatures exceeding 180°C, and the temperature and reaction time can be reduced by employing various catalysts. However, acid catalyzed esterification using glycerol is problematic industrially, especially because of corrosion of the equipment, darkening of the product and side reactions reducing yields and requiring downstream processing.

[0008] Enzymatic esterification of FFA onto glycerol is an alternative to the chemical or thermal processes and have been described in the prior art. However, such previous disclosures primarily apply immobilized lipases, which are expensive and requires large enzyme dosages. Furthermore, esterification of FFA onto glycerol producing DAG or MAG using immobilized lipase results in significant amounts of TAG in the final product.

[0009] Wang, Lili, et al., (Preparation of diacylglycerol-enriched oil from free fatty acids using lecitase ultra-catalyzed esterification." Journal of the American Oil Chemists' Society 88 (2011): 1557-1565), discloses production of DAG-enriched oil by esterification of FFA with glycerol using Lecitase Ultra. Significant amounts of TAG were formed.

[0010] Pinsirodom, Praphan, et al., (Critical temperature for production of MAG by esterification of different FA with glycerol using Penicillium camembertii lipase. Journal of the American Oil Chemists' Society 81.6 (2004): 543-547), describe use of free Penicillium Camembertii lipase for solvent free DAG formation.

[0011] US 5270188 A discloses esterification of FFA onto glycerol using a lipase derived from Penicillium cyclopium.

[0012] US 2020146307 A discloses esterification of FFA onto glycerol using a commercial lipase, Amano lipase AY.

[0013] US 2021 / 388401 discloses a method of refining a grain oil composition feedstock to provide a grain oil product.

[0014] WO 2023 / 222648 discloses a process for reducing level of free fatty acids in biodiesel / fatty acid alkyl esters.

[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] Therefore, there is a need for alternative approaches. The present invention discloses an industrially and economically viable process by which MAG and DAG are produced using enzymes at low dosage and at ambient operating conditions that can substitute high temperature processes.

[0019] SUMMARY OF INVENTION

[0020] The present invention relates to a process for producing mono-acylglycerides (MAG) and / or di-acylglycerides (DAG) comprising steps of: a) providing a mixture comprising free fatty acids (FFA) and / or fatty acid alkyl esters, glycerol and lipase; b) reacting the mixture of a) to produce a mixture of MAG and DAG; wherein the lipase has at least 80% sequence identity to SEQ ID NO: 1 , and wherein the lipase is applied in granular, liquid or water-soluble form.

[0021] SEQUENCE OVERVIEW

[0022] SEQ ID NO: 1 is a lipase obtained from Candida antarctica.

[0023] DEFINITIONS

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The term "hydrolysis" as used herein, refers to the reaction of water with an ester to produce an acid and an alcohol.

[0029] 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.

[0030] 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.

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

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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).

[0037] 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.

[0038] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0039] 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:

[0040] (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:

[0041] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0042] 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 fatty acid alkyl esters, triglyceride, diglyceride, monoglyceride, free fatty acid or any combination thereof. Any oils and fats of vegetable or animal origin comprising fatty acids may be used as substrate for producing fatty acid alkyl esters in the process of the invention. Also, fatty acid feedstock consisting substantially of fatty acid alkyl esters is suitable as feedstock (biodiesel feedstock) for the present invention.

[0043] 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.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates to a process for producing mono- and diacylglycerides without substantial production of triglycerides. How to avoid significant TAG formation is a key aspect of the present invention. It has been observed that there is an increase in TAG over long reaction time, however, TAG only starts to increase significantly once FFA has been reduced to the practical equilibrium of around 10 wt%. Further, this surprising finding is made possible due to the newly found poor selectivity of the prior art lipase known as CalB regarding TAG formation at the conditions disclosed herein. Particularly, the present invention relates to a process for synthesizing monoglycerides and diacylglycerides by reacting a fatty acid or a fatty acid ester with glycerol in the presence of a lipase, wherein, as the lipase, a monoglyceride lipase and / or a diglyceride lipase is used to produce monoglycerides and diacylglycerides containing little or no triacylglyceride (TAG) and consisting substantially of a monoglyceride and a diglyceride.

[0046] The inventors have found a surprising effect using liquid and free lipase in production of MAG and DAG with little or no formation of TAG. The CalB lipase was previously described in the art as a lipase able to form and hydrolyze TAG, and as being an example of an unspecific lipase capable of catalyzing reaction on all positions of the glycerides. The present invention provides benefits related to low amounts of TAG formation. This allows for optional purification of pure form of MAG and DAG fractions by distillation, while the resulting mixture of MAG and DAG has utility without further fractionation too.

[0047] Thus, the present invention relates to a process for producing mono-acylglycerides (MAG) and / or di-acylglycerides (DAG) comprising the steps of: a) providing a mixture comprising free fatty acids (FFA) and / or fatty acid alkyl esters, glycerol and lipase; b) reacting the mixture of a) to produce a mixture of MAG and DAG; wherein the lipase has at least 80% sequence identity to SEQ ID NO: 1 , and wherein the lipase is applied in granular, liquid or water-soluble form.

[0048] In one embodiment of the present invention, the free fatty acids are present in an oil having free fatty acid (FFA) concentration above 90 wt%, preferably above 95 wt%, and most preferably above 98 wt%.

[0049] In one embodiment of the present invention, the fatty acid alkyl esters are present in an oil having fatty acid alkyl ester concentration above 90 wt%, preferably above 95 wt%, and most preferably above 98 wt%.

[0050] In one embodiment of the present invention, fatty acid alkyl esters are selected from fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE).

[0051] In one embodiment of the present invention, MAG and DAG are produced in reaction step b) by esterification and / or transesterification of FFA and / or fatty acid alkyl ester with glycerol.

[0052] In one embodiment of the present invention, more than 50%, preferably more than 70% and most preferably more than 90% of FFA and / or fatty acid alkyl esters is converted to MAG and / or DAG.

[0053] In one embodiment of the present invention, ratio between synthesized MAG and DAG after step b) is about 0.5:9 - 9:0.5, preferably 2:8-8:2, and most preferably 2:8-6:4.

[0054] In one embodiment of the present invention, the amount of MAG exceeds 30 wt% and preferably 40 wt%, while the remainder comprises DAG, TAG and FFA.

[0055] The amount of glycerol present or added is important, and the preferable dosage will depend on the use of optional drying. Dryness of the glycerol used is also an important point and is described below. Increased amounts of dry initial glycerol dosage will lead to lower final FFA concentrations because the hygroscopic nature of glycerol will act as a drying agent itself. It absorbs the water formed during reaction and reduces the chemical potential of the water, thus lowering FFA at equilibrium. Conversely, if drying is conducted during reaction, then a reduced amount of glycerol is preferable, as water evaporates more easily from the system when present in higher concentrations in a smaller glycerol phase than when highly diluted in a large amount of glycerol. The expert in the art would design a production plant, with typical practical limitations in mind such as available existing equipment and decide the best means of drying. For example, one might choose to use a large glycerol phase, while running reaction without drying during reaction, collecting the reaction product MAG / DAG phase, and then thoroughly drying the large glycerol phase before next reaction runs. Otherwise, a smaller glycerol phase could be employed, with continuous drying during reaction of the whole mixture by employing a reactor under vacuum, or for example by drying the glycerol heavy phase, separated and dried continuously in an external separator / flash loop before reentering the reaction vessel. Such exemplified configurations are not meant to be limiting, but rather meant to teach the currently known preferable implementation options. When employing alkyl esters such as FAME or FAEE as feedstock, the alcohol byproduct of reaction will behave in a manner similar to water as explained above, however leading to residual alkyl esters rather than FFA after reaction. The benefit of employing those is the ease of byproduct alcohol removal relative to water when esterifying FFA.

[0056] In one embodiment of the present invention, glycerol in step a) is present in the feedstock oil, added from an exogenous source, and / or recycled from a down- or upstream process step.

[0057] 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 one embodiment of the invention, the total dosage of all incoming glycerol-containing 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, the amount of glycerol present in step a) is between 0.5 and 200%, preferably 20-180%, more preferably 40-150%, most preferably 1-100% wt / wt of fatty acid feedstock oil.

[0060] In one embodiment, 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 dosage.

[0061] In one embodiment of the invention, initially present glycerol is fully, or almost fully, consumed during reaction, leading to a single phase or a mixture containing little residual heavy phase glycerol after reaction. This is the simplest possible setup, where you add the stoichiometric amount of glycerol required for reaction, along with enzyme, and after reaction, you do not necessarily need to reuse the heavy phase, because it is fully (or near-fully) consumed. It has benefits of simplicity and will allow for use of higher temperatures, because the enzyme is not necessarily recycled. However, in cases where a significant part of the enzyme activity is intact after reaction, especially in cases where low temperature has been used during reaction, it is not the preferred option, because reuse of enzyme is economically beneficial whenever possible.

[0062] In one embodiment of the present invention, the amount of glycerol present in step a) is less than 200%, less than 150%, less than 140%, less than 130%, less than 125%, particularly less than 110% of the amount consumed in reaction step b).

[0063] 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.

[0064] In one embodiment of the present invention, lipase has at least 81%, at least 82%, at least

[0065] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0066] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

[0067] 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 .

[0068] In a preferred embodiment of the present invention, the lipase is a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 1 .

[0069] In an embodiment of the present invention, the lipase comprises or consists of the amino acid sequence shown in SEQ ID NO 1.

[0070] Free fatty acid and / or fatty acid alkyl esters 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 (refined, bleached and deodorized). 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 may be pure triglyceride or a mixture of triglyceride and free fatty acids, commonly seen in waste vegetable oil and animal fats. 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.

[0071] 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 fatty acid alkyl esters, triglyceride, diglyceride, monoglyceride, free fatty acid or any combination thereof. Any oils and fats of vegetable or animal origin comprising fatty acids may be used as substrate for producing fatty acid alkyl esters in the process of the invention. Also, fatty acid feedstock consisting substantially of fatty acid alkyl esters is suitable as feedstock (biodiesel feedstock) for the present invention.

[0072] The fatty acid feedstock may be crude, refined, bleached, deodorized, degummed, or any combination thereof.

[0073] In one embodiment of the present invention, the free fatty acid and / or fatty acid alkyl esters is obtained from fatty acid feedstock oil 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 oil, meadow foam 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.

[0074] In one embodiment of the present invention, the FFA are selected from the group consisting of caprylic acid, capric acid, lauric acid, stearic acid, and oleic acid.

[0075] In one embodiment of the present invention, the FFA are selected from the group consisting of lauric acid, stearic acid, and oleic acid, particularly stearic acid.

[0076] The fatty acid feedstock may be derived from any of the above list, having undergone various significant conversion processes. Most importantly, partial or, preferably, full hydrogenation of double bonds is optionally conducted on the feedstock in cases where fully saturated MAG and DAG products are sought. This allows for obtaining of fully saturated feedstocks from otherwise significantly unsaturated origins. Similarly, partial or full hydrolysis or transesterification of the original feedstock are relevant significant conversion processes, as they allow for TAG reduction while yielding relevant FFA or alkyl ester reactants.

[0077] In one embodiment of the invention, any feedstock comprising fatty acids may be used.

[0078] In another embodiment of the present invention, the feedstock comprises less than 20 wt% TAG.

[0079] In another embodiment of the present invention, the feedstock does not comprise substantial amounts of TAG, such as less than 5 wt%, preferably less than 2 wt% and most preferably less than 1 wt%.

[0080] In another embodiment of the present invention, substantially pure FFA is utilized as feedstock.

[0081] In one embodiment of the present invention, lipase of choice exhibits low relative activity in formation of- and preferably also hydrolysis of TAG relative to formation- and preferably also to hydrolysis of DAG.

[0082] Preferably, the lipase of choice will form and / or hydrolyse 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.

[0083] Preferably, the lipase applied, e.g., shown in SEQ ID NO: 1 , should have sufficient activity to esterify the FFA present onto glycerol to form DAG and MAG.

[0084] In one embodiment of the present invention, the FFA is selected from the group consisting of caproic acid, caprylic acid, lauric acid, stearic acid, and oleic acid. Preferably lauric acid, stearic acid, and oleic acid, and most preferably stearic acid.

[0085] In an embodiment of the present invention, the process is performed in the absence of short chain alcohol, such as e.g., ethanol and / or methanol.

[0086] In an embodiment of the present 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.

[0087] In cases where high amounts of TAG are present in the feedstock, a prior hydrolysis or transesterification reaction step may be used to bring TAG concentrations to acceptable levels. Acceptable levels will depend on the desired product composition. In most cases the desired composition will be substantially free of TAG. Prior arts describe how such a prior hydrolysis or transesterification may be conducted. Non-exhaustive examples are: hydrolysis by subjecting the oil to water at high-temperatures and pressures, hydrolysis in presence of TAG-active esterase, methylate- or ethylate catalyzed transesterification with short chain alcohols, and esterase- catalyzed transesterification.

[0088] In one embodiment of the present invention, the feedstock has undergone a partial or full hydrogenation.

[0089] In another embodiment of the present invention, the resulting product or fractions thereof are further fully or partially hydrogenated, leading to lower degrees of unsaturation or full saturation of the fatty acids of the MAG and DAG product(s).

[0090] In one embodiment of the process of the invention, the final composition mixture after reaction of step b) comprises less than 20 wt%, such as 10 wt%, such as less than 5 wt%, such as less than 2 wt% and such as less than 0.5 wt% of triacylglycerides (TAG).

[0091] In an embodiment of the present invention, the amount of triglyceride in the feedstock is substantially unchanged after treatment with lipase.

[0092] 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..

[0093] In an embodiment of the present invention, the lipase is preferably employed as a liquid product, a dry powder, or similar formulations that allow the enzyme to dissolve and act as unbound molecules. Most preferably, the lipase is employed in liquid form.

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

[0095] In an embodiment of the present invention, pH during step b) is in the range of 2.0-8.0, preferably 3.0-8.0.

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

[0097] 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.

[0098] In an embodiment of the present 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.

[0099] In an embodiment of the present invention, the process is performed in a batch, semi- continuous or continuous mode.

[0100] In an embodiment of the present invention, the process further comprises contacting the fatty acid feedstock oil with an enzyme selected from a group consisting of chlorophyllase, and phospholipase, and combinations thereof.

[0101] In an embodiment of the present invention, the reaction mixture is dried before and / or during and / or after step b).

[0102] In an embodiment of the present invention, the process further comprises separating the mixture of step b) into light and heavy phase. In an embodiment of the present invention, the heavy phase and / or the light phase is partially or fully recycled into step b).

[0103] In an embodiment of the present invention, the light phase comprises increased amounts of MAG and / or DAG and reduced amounts of FFA and / or fatty acid alkyl ester.

[0104] In an embodiment of the present invention, the heavy phase comprises water, lipase, glycerol. the heavy phase is dried.

[0105] In an embodiment of the present invention, the heavy phase is partially or fully recycled into step b).

[0106] In an embodiment of the present invention, the light phase is partially or fully recycled into step b).

[0107] In an embodiment of the present invention, the light phase is optionally subjected to purification or fractionation.

[0108] In an embodiment of the present invention, the purification or fractionation comprises distillation, short path distillation, fractional distillation, winterization, cold fractionation, or a combination thereof.

[0109] In an embodiment of the present invention, the different fractions each comprising increased concentrations of FFA and / or MAG and / or DAG are obtained.

[0110] 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.

[0111] In another embodiment of the present invention, the process is performed in optionally countercurrent, optionally compartmentalized, reactor(s). This 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.

[0112] In one embodiment of the invention, the reaction mixture is separated into a light and heavy phase. The light product phase is subjected to further downstream processing, while the heavy phase, comprising glycerol and most of the residual enzyme activity, may be reused.

[0113] In one embodiment of the invention, one or more separation steps run continuously during reaction, with part or all of the light phase being recycled into the reaction step, and / or with part or all of the heavy phase being recycled into the reaction step. Drying off water or alcohol from the reaction mixture will be an important part of most implementations of the invention because they are byproducts of the reaction, which will negatively affect the achievable conversion to MAG and DAG.

[0114] In one embodiment of the invention, the reaction mixture and / or one or more incoming streams are dried.

[0115] 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. Such drying may be continuous or stepwise.

[0116] In one embodiment of the invention, the heavy glycerol phase, isolated by a separation step, is subjected to a drying step prior to re-entering the reaction step. Such a drying step may run continuously during reaction, or in steps. An example is a batch configuration, where enzymecontaining glycerol is first collected, and then prepared for the next reaction by drying. Another example is a continuous setup, where reaction runs continuously along with a separation step yielding product light phase and heavy phase, with the heavy phase being dried, reducing water concentrations during reaction.

[0117] In one embodiment of the invention, drying is affected by use of vacuum distillation, sparging with an inert gas such as N2, use of membrane technologies, addition of water absorbents and subsequent filtration, or similar common industrial drying techniques.

[0118] In one embodiment, the light phase is dried after separation.

[0119] In one embodiment of the invention, drying is done at mild conditions, where the enzyme activity is not substantially degraded. Preferably, more than 50% of the enzyme activity is kept intact after drying. This can be achieved by low temperature drying at correspondingly deeper vacuum. Different esterases will have different thermal stabilities, with SEQ ID NO:1 having good thermal stability in glycerol in liquid form up to about 60°C, above which degradation depending on time will start. A chemical engineer will be able to identify correct temperatures and pressures along with drying equipment designs depending on the choice of esterase and its stability profile.

[0120] In one aspect of the invention, alkyl esters of short chain alcohols are transesterified onto glycerol rather than FFA being esterified. The difference is that preferred alkyl esters, being methyl- or ethyl-esters, will release the short chain alcohol, methanol or ethanol, in transesterification rather than water in FFA esterification, and this brings a benefit in drying efficiency at the expense of other costs. Therefore, throughout this text, and especially when describing drying and water, the same generally applies to short chain alcohols for the use cases involving alkyl esters. Drying thus refers to removal of volatile molecules in general, including water and short chain alcohols where applicable. Similarly, FFA, the most preferred reactant, and alkyl esters, can be viewed as the same. Meaning descriptions of residual FFA, conversion of FFA, and the like also applies to select cases involving alkyl esters and transesterification rather than FFA and esterification. The reaction mechanism of esterification and transesterification is largely the same, with the main difference being alcohol or water as reactants.

[0121] Throughout this text, heavy phase generally refers to the polar water / glycerol / alcohol phase generally containing most enzyme activity, and light phase refers to the substantially nonpolar oil / FFA / alkyl-ester / glyceride phase.

[0122] In another embodiment of the present invention, substantially pure FFA fractions or species are used as feedstock, for example high-melting C16:0-C18:0 fractions from FFA fractionations, or even substantially pure fractions of FFA, such as stearic acid (C18:0). This is the most desirable choice of feedstock because it allows for control of the exact MAG and DAG product composition and properties. The expert in the art will know how to arrive at such feedstocks, and the pathway is insignificant for the application of the invention.

[0123] In one embodiment of the invention, the reaction product light phase composition comprises 0-50 wt% FFA, 10-90 wt% MAG, 20-90 wt% DAG, and 0-20 wt% TAG. Most preferably, the reaction product composition comprises 0-10 wt% FFA, 25-60 wt% MAG, 40-70 wt% DAG and 0-5 wt% TAG. Most preferably, TAG is below 2 wt% or even substantially non-existent, while MAG and DAG can be present at any ratio, because those components can to a large extent be isolated from one another and blended to yield target compositions. For example, products largely comprising 40 wt% MAG and 60 wt% DAG are commercially available and used in food emulsifiers, while >90 wt% MAG is similarly available and can be used to make oil blends with controlled MAG levels. Higher concentration DAG products are theoretically producible by removal of MAG, as described in the introduction, by largely avoiding TAG formation, but as this is not a possibility today, this is one point where the invention brings a significant benefit.

[0124] In one embodiment of the invention, the reaction product light phase composition is subjected to further separation steps after the initial isolation of the reaction product light phase from the heavy phase.

[0125] In one embodiment of the invention, the reaction product light phase is separated by use of distillation-based processes, such as flashing, distillation, fractional distillation and / or short path distillation. Short path distillation is most preferably used, as it is mild and efficient, although the capital expenditure per productivity is typically high.

[0126] In one embodiment of the invention, a reaction product light phase comprising 25-45 wt% MAG and 40-65 wt% DAG is short-path distilled to yield fractions of higher concentrations of each component.

[0127] In a further embodiment of the invention, the fraction of DAG exceeds 80 wt% and preferably 90 wt% DAG concentration. This is possible due to the low concentration of TAG resulting from use of largely TAG-inactive esterase.

[0128] In one embodiment of the invention, residual FFA in the product light phase is removed e.g. by chemical neutralization or by deodorization. Preferably, FFA is removed prior to optional further isolation of MAG and DAG.

[0129] In one embodiment of the invention, FFA and MAG are isolated from DAG in a combined fraction, which is optionally recycled to the reaction step for further conversion into DAG.

[0130] In one embodiment of the present invention, there is no drying during reaction, and the equilibrium FFA level will be determined by the initial FFA concentration, and thereby the releasable amount of water, as well as the glycerol dosage and initial dryness of the system. This may be a feasible implementation of the invention, e.g. if FFA and MAG are subsequently distilled off and recycled, with DAG collected as main product.

[0131] In one embodiment of the invention, the reaction product is subjected to washing. This is because the reaction product, containing high amounts of emulsifier MAG, may cause emulsification with the glycerol phase, especially at low temperatures of <60°C. Therefore, it may be difficult to fully separate the phases by simple gravitational methods such as centrifugation, thereby leaving unacceptable amounts of glycerol in the product. In such cases a washing step may be required. Such a step is preferably conducted using water or glycerol and may require high temperatures at which the emulsion breaks, such as temperatures above 100°C.

[0132] The invention is further described in the following numbered paragraphs.

[0133] Paragraph 1. A process for producing mono-acylglycerides (MAG) and / or diacylglycerides (DAG) comprising steps of: a) providing a mixture comprising free fatty acids (FFA) and / or fatty acid alkyl esters, glycerol and lipase; b) reacting the mixture of a) to produce a mixture of MAG and DAG; wherein the lipase has at least 80% sequence identity to SEQ ID NO: 1 , and wherein the lipase is applied in granular, liquid or water-soluble form.

[0134] Paragraph 2. The process according to paragraph 1 , wherein the free fatty acids are present in a fatty acid feedstock oil having a free fatty acid (FFA) concentration above 90 wt%, preferably above 95 wt%, and most preferably above 98 wt%.

[0135] Paragraph 3. The process according to paragraph 1 , wherein the fatty acid alkyl esters are present in a fatty acid feedstock oil having a fatty acid alkyl ester concentration above 90 wt%, preferably above 95 wt%, and most preferably above 98 wt%.

[0136] Paragraph 4. The process according to paragraph 3, wherein the fatty acid alkyl esters are selected from fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE).

[0137] Paragraph 5. The process according to any of the preceding paragraphs, wherein MAG and DAG are produced in reaction step b) by esterification and / or transesterification of FFA and / or fatty acid alkyl ester with glycerol.

[0138] Paragraph 6. The process according to any of the preceding paragraphs, wherein the final composition mixture after reaction of step b) comprises less than 20 wt%, such as 10 wt%, such as less than 5 wt%, such as less than 2 wt% and such as less than 0.5 wt% of triacylglycerides (TAG).

[0139] Paragraph 7. The process according to any of the preceding paragraphs, wherein more than 50%, preferably more than 70% and most preferably more than 90% of FFA and / or fatty acid alkyl esters is converted to MAG and / or DAG.

[0140] Paragraph 8. The process according to paragraph 5, wherein the synthesized MAG and DAG after reaction step b) is about 0.5:9 - 9:0.5, preferably 2:8-8:2, and most preferably 2:8-6:4.

[0141] Paragraph 9. The process according to paragraph 1 , wherein the amount of glycerol present in step a) is between 0.5 and 200%, preferably 20-180%, more preferably 40-150%, most preferably 1-100% wt / wt of fatty acid feedstock oil.

[0142] Paragraph 10. The process according to any of the preceding paragraphs, where 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, obtained by reuse and accumulation of excess.

[0143] Paragraph 11 . The process according to any of the preceding claims, wherein the amount of glycerol present in step a) is less than 200%, less than 150%, less than 140%, less than 130%, less than 125%, particularly less than 110% of the amount consumed in reaction step b).

[0144] Paragraph 12. The process according to any of the preceding paragraphs, wherein the glycerol in step a) is present in the feedstock oil, added from an exogenous source, and / or recycled from a down- and / or upstream process step.

[0145] Paragraph 13. The process according to paragraph 1 , wherein the lipase has 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.

[0146] Paragraph 14. The process according to paragraph 13, wherein the lipase comprises or consists of amino acid as shown in SEQ ID NO: 1 .

[0147] Paragraph 15. The process according to paragraph 1 , wherein the fatty acid feedstock oil 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 oil, meadow foam 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. Paragraph 16. The process according to paragraph 1 , wherein the fatty acid feedstock comprises of less than 20 wt% TAG.

[0148] Paragraph 17. The process according to paragraph 1 , wherein the fatty acid feedstock does not comprise substantial amounts of TAG, such as less than 5 wt%, preferably less than 2 wt% and most preferably less than 1 wt%.

[0149] Paragraph 18. The process according to any of the preceding paragraphs, wherein substantially pure FFA is utilized as feedstock.

[0150] Paragraph 19: 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.

[0151] Paragraph 20. 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, 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.

[0152] Paragraph 21. The process according to paragraph 1 , wherein pH of step b) is optionally adjusted during and / or prior to contacting.

[0153] Paragraph 22. The process according to paragraph 23, wherein the pH during step b) is in the range of 2.0-8.0, preferably 3.0-8.0.

[0154] Paragraph 23. The process according to paragraphs 23-24, wherein the pH is preferably adjusted using citric acid, phosphoric acid, sodium hydroxide and / or potassium hydroxide.

[0155] Paragraph 24. The process according to paragraph 1 , wherein process is performed in less than 48 hours, preferably less than 24 hours and most preferably less than 12 hours.

[0156] Paragraph 25. The process according to any of the preceding paragraphs, wherein the process is performed in a batch, semi-continuous or continuous mode.

[0157] Paragraph 26. The process according to any of the preceding paragraphs, further comprises contacting the fatty acid feedstock oil with an enzyme selected from a group consisting of chlorophyllase, and phospholipase, and combinations thereof.

[0158] Paragraph 27. The process according to paragraph 1 , wherein the reaction mixture is dried before and / or during and / or after step b).

[0159] Paragraph 28. The process according to paragraph 1 , further comprising separating the mixture of step b) into light and heavy phase.

[0160] Paragraph 29. The process according to paragraph 30, wherein the heavy phase and / or the light phase is partially or fully recycled into step b). Paragraph 30. The process according to paragraph 29, wherein the light phase comprises increased amounts of MAG and / or DAG and reduced amounts of FFA and / or fatty acid alkyl ester.

[0161] Paragraph 31. The process according to paragraph 29, wherein the heavy phase comprises water, lipase, glycerol.

[0162] Paragraph 32. The process according to paragraph 29, wherein the heavy phase is dried.

[0163] Paragraph 33. The process according to paragraph 29, 31 or 32, wherein the heavy phase is partially or fully recycled into step b).

[0164] Paragraph 34. The process according to paragraph 29, wherein the light phase is partially or fully recycled into step b).

[0165] Paragraph 35. The process according to paragraph 29, wherein the light phase is optionally subjected to purification or fractionation.

[0166] Paragraph 36. The process according to paragraph 35, wherein purification or fractionation comprises distillation, short path distillation, fractional distillation, winterization, cold fractionation, or a combination thereof.

[0167] Paragraph 37. The process according to paragraph 36, wherein different fractions each comprising increased concentrations of FFA and / or MAG and / or DAG are obtained.

[0168] Paragraph 38. The process according to paragraph 1 , wherein the FFA are selected from the group consisting of caprylic acid, capric acid, lauric acid, stearic acid, and oleic acid.

[0169] Paragraph 39. The process according to paragraph 37, wherein the FFA are selected from the group consisting of lauric acid, stearic acid, and oleic acid, particularly stearic acid.

[0170] Paragraph 40. 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.

[0171] Paragraph 41. 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.

[0172] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0173] EXAMPLES

[0174] Example 1 : Esterification of FFA onto glycerol using lipase of SEQ ID NO: 1

[0175] 20g of dry technical grade glycerol was mixed with 5, 10 or 20 g technical grade oleic acid. 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.

[0176] 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.

[0177] Table 1 : MAG, DAG, and TAG produced at different ratios of glycerol to FFA

[0178] From Table 1 , it is observed that at high amounts of g ycerol relative to oleic acid, FFA decreases and TAG increases. The relative ratio of MAG to DAG is, counter to stoichiometric considerations, increasing as the amount of glycerol relative to oleic acid decreases.

[0179] Example 2: Esterification of FFA onto glycerol with different lipase dosages (SEQ ID NO: 1)

[0180] 20g of dry technical grade glycerol was mixed with 20 g technical grade oleic acid. 0.1 or 0.5 % (wt / wt of oleic acid) 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 70°C in 100 mL closed square bottles for 24 hours at 250 rpm shaking stirring and the resulting product light phase was measured.

[0181] 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.

[0182] Table 2: MAG and DAG production at different lipase dosages From Table 2, it is observed that 0.5 % enzyme solution, corresponding to 42.5 mg enzyme protein / kg oleic acid feedstock is required for acceptable conversion of FFA to MAG and DAG within 24 hours of reaction. TAG was not measurable in most cases, and below our threshold of confidence in the 24h sample with 0.5% enzyme solution.

[0183] Example 3: Esterification of FFA onto glycerol at different FFA / glycerol levels using the lipase of SEQ ID NO: 1

[0184] 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.

[0185] 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.

[0186] Table 3: Achievable equilibria at various ratios of FFA / glycerol

[0187] From table 3, results show achievable equilibria at various ratios of FFA to glycerol. Relative to the experiments above, these data show resulting compositions at much higher amounts of FFA to glycerol. Generally, as initial FFA increases relative to glycerol, the achievable conversion (without water removal during reaction) is reduced. This is the basis for the described and recommended use of high amounts of glycerol when practicing the invention without use of water removal during reaction. Generally, very low amounts of TAG were observed in the HPLC chromatogram, making quantization impossible.

[0188] Example 4: Esterification of FFA onto glycerol under vacuum using the lipase of SEQ ID NO: 1 Technical grade glycerol was mixed with technical grade oleic acid in ratios of 20 / 20, 15 / 25 and 25 / 15. 0.1 or 0.5 % (wt / wt of feedstock 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 square bottles at 250 rpm shaking stirring for 96 hours and the resulting product light phase was measured. Reactions ran under continuous vacuum of around 5 mbara.

[0189] 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.

[0190] Table 4: Glycerol / Oleic acid ratios

[0191] Results from Table 4 show, that after 20h reaction time, all reactions containing 0.5 % enzyme had desirable product profiles considering conversion of FFA to MAG / DAG, the distribution between MAG and DAG, and the limited concentrations of TAG. FFA can be removed by various means after reaction as described above and as is well known in industry, and does not constitute a significant problem, as it may be recycled for eventual full net conversion in later reaction batches.

[0192] Even though vacuum was applied to the reaction flasks as a means for drying during the full extent of reaction, the conversion of FFA was slow and incomplete. In other relevant industries, where enzymes are applied together with drying, such as in enzymatic biodiesel production, it is known and possible to reduce FFA levels much more efficiently than in this example. We believe this trial conclusively shows the feasibility of drying for obtaining good FFA conversions, but that overall drying efficiency was insufficient due to the simple laboratory setup available. Industrially, one would preferably employ specialized equipment with much better efficiency in water removal, meaning the equilibrium FFA level could be brought down much more quickly than observed here. One significant point is the increase in TAG over long reaction time. TAG only starts to rise significantly once FFA has been reduced to the practical equilibrium of around 10 wt% below which extensive drying is required. The inventors have found that the fact that high conversions to MAG and DAG are achievable with little to no TAG formation. This is only possible due to the newly found poor selectivity of SEQ ID NO: 1 regarding TAG formation at the conditions reported here.

[0193] TAG rises to around 20 wt% with long reaction time and high enzyme dosage. Industrially, when applying efficient means of drying, such long reaction times can be avoided, because conversion of FFA to MAG and DAG is much more efficient than conversion to TAG. So, with sufficient chemical driving force to low FFA levels through low water concentrations, FFA conversion can likely be kept high while TAG formation is constantly low.

[0194] Example 5: Esterification of FFA to MAG / DAG using the lipase of SEQ ID NO: 1

[0195] Technical grade glycerol was mixed with technical grade oleic acid in ratios as indicated in the results table. Liquid enzyme solution of SEQ ID NO: 1 was added in an amount as indicated in the results table 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 the results table.

[0196] 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 we measured were: Oleic acid 0.79, 0.80 and 0.81 , Mono-olein: 1 .0, 1 .0 and 1 .0, Diolein: 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 we choose to report the normalized relative peak areas of FFA, MAG, DAG and TAG without accounting for diluting compounds, which are removable in industrial post-treatment.

[0197] Table 5: Production of MAG, DAG, and TAG using preferred parameters

[0198] From table 5, it is seen that obtaining high conversion of FFAto MAG and DAG with low formation of TAG is possible and proven by the experiments above. When employing the invention, the expert in the art would be able to reduce FFA to acceptable levels (such as around 2.5-3 wt% in some product specifications, e.g. European commission E-number 471 specification specifying an acidity value of 6, which roughly translates to 3 wt% FFA based on oleic acid. For example by preferred means of short path distillation, enabling efficient separation of FFA from glyceride components without materially affecting the MAG / DAG composition, and while operating at relatively low temperatures.

[0199] With increased temperature comes improved water evaporation and reduction of FFA. However, TAG increases significantly. With further optimization, the data suggests that FFA within specification levels of around 2.5-3 wt% can be achieved while at least reaching TAG concentrations below 10 wt%. Generally, the examples show reaction results after 24h. Example 4 showed that TAG formation significantly increases at end of reaction, so shortening reaction time at the expense of elevated FFA levels is preferable in cases where a low TAG product is desired and comes at the cost of employing well known methods of FFA removal, such as stripping or short path distillation.

[0200] Example 6: Thermostability of liquid (free) lipase of SEQ ID NO:1 in presence of glycerol 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] Table 6: Stability of lipase in presence of methanol-glycerol

[0205] Results in table 6 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 6 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.

[0206] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

Claims:

1. A process for producing mono-acylglycerides (MAG) and / or di-acylglycerides (DAG) comprising steps of: a) providing a mixture comprising free fatty acids (FFA) and / or fatty acid alkyl esters, glycerol and lipase; b) reacting the mixture of a) to produce a mixture of MAG and DAG; wherein the lipase has at least 80% sequence identity to SEQ ID NO: 1 , and wherein the lipase is applied in granular, liquid or water-soluble form.

2. The process according to claim 1 , wherein the free fatty acids are present in an oil having a free fatty acid (FFA) concentration above 90 wt%, preferably above 95 wt%, and most preferably above 98 wt%.

3. The process according to claim 1 , wherein the fatty acid alkyl esters are present in an oil having a fatty acid alkyl ester concentration above 90 wt%, preferably above 95 wt%, and most preferably above 98 wt%.

4. The process according to claim 3, wherein the fatty acid alkyl esters are selected from fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE).

5. The process according to any of the preceding claims, wherein MAG and DAG are produced in reaction step b) by esterification and / or transesterification of FFA and / or fatty acid alkyl ester with glycerol.

6. The process according to any of the preceding claims, wherein the final composition mixture after reaction of step b) comprises less than 20 wt%, such as 10 wt%, such as less than 5 wt%, such as less than 2 wt% and such as less than 0.5 wt% of triacylglycerides (TAG).

7. The process according to any of the preceding claims, wherein more than 50%, preferably more than 70% and most preferably more than 90% of FFA and / or fatty acid alkyl esters are converted to MAG and / or DAG.

8. The process according to any of the preceding claims, wherein synthesized MAG and DAG after reaction step b) is about 0.5:9 - 9:0.5, preferably 2:8-8:2, and most preferably 2:8-6:4.

9. The process according to any of the preceding claims, wherein the glycerol in step a) is present in the feedstock oil, added from an exogenous source, and / or recycled from a down- and / or upstream process step.

10. The process according to any of the preceding claims, wherein the amount of glycerol present in step a) is between 0.5 - 200 %, preferably 20-180%, more preferably 40-150%, most preferably 1-100% wt / wt of fatty acid feedstock oil.

11. The process according to any of the preceding claims, wherein the amount of glycerol present in step a) is less than 200%, less than 150%, less than 140%, less than 130%, less than 125%, most particularly less than 110% of the amount consumed in reaction step b).

12. 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.

13. The process according to claim 1 , wherein the reaction mixture is dried before and / or during and / or after step b).

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

15. The process according to any of claims 11 to 14, wherein the heavy phase and / or the light phase is partially or fully recycled into step b).

16. 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.

17. 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.