Improved enzymatic decomposition of oils and fats producing free fatty acids

Abstract

The present invention relates to an improved enzymatic decomposition process for the production of free fatty acids in which a fatty acid feedstock is contacted with an enzyme composition comprising a lipase and a phospholipase to produce a reaction mixture, the mixture is hydrolyzed for 16 hours, the light phase and the heavy phase of the reaction mixture are separated, and the free fatty acids are recovered from the light phase. Meanwhile, the invention also relates to an enzyme composition for enzymatic decomposition of soap raw materials to produce free fatty acids, and the enzyme composition comprises lipase and phospholipase. The enzyme composition can significantly reduce the average residence time of the oil in any reactor from at least 24 hours to 16 hours and at the same time achieve a desired acid number.

Description

Field of the Invention

[0001] The present invention relates to the enzymatic breakdown or hydrolysis of oils and fats (ie, acidic oils) into free fatty acids in the presence of water. Background Art

[0002] Acidified oil refers to oil obtained by acidifying soap stock (i.e., a byproduct of an oil refinery). Acidified oil is essentially fatty acids, which also contain pigments, non-acidified triglycerides, diglycerides, monoglycerides, and other components. Fatty acids are used in a wide variety of products, such as in soaps and surfactants, lubricants, paints and coatings, candles, and in a variety of other agricultural, industrial, and personal care products.

[0003] The enzymatic digestion of acidic oils has long been known and has significant major advantages over the pressure digestion predominantly used in industry. The enzymatic digestion can be carried out at normal pressure and, depending on the enzyme and the oil or fat, even at room temperature.

[0004] It has also long been known that this method of lipolysis is the mildest method. Technological advances in industrial biotechnology have also provided enzymes that are currently available and suitable for lipolysis. However, mainly due to the high enzyme consumption, the extremely long lipolysis time and the resulting low efficiency of enzymatic lipolysis, enzymatic lipolysis has not become an industrial alternative to large-scale pressure lipolysis that is widely accepted in the industry.

[0005] Enzymatic lipolysis using enzymes (so-called lipases) as biocatalysts acting on water / oil mixtures is known in the art. By this decomposition technique, oils or fats are decomposed into glycerol and free fatty acids, respectively. Glycerol migrates into the aqueous phase, while the organic phase is enriched with more and more free fatty acids, until finally only the free fatty acids remain in the organic phase.

[0006] Current enzymatic decomposition process always experiences very long reaction time, for example, in simple batch system, surpasses 24 hours to realize the desired decomposition degree close to 100%.Yet long reaction time will destroy enzyme activity.In other words, the activity of enzyme has a certain degree of reduction over time, mainly due to protein denaturation.This reduction can be compensated by further adding enzyme, which in turn increases cost.Therefore, need to constantly improve enzymatic decomposition process, to shorten reaction time, keep desired decomposition degree, still economical and efficient simultaneously, so that be widely used in industrial production free fatty acid. Summary of the invention

[0007] The present invention provides a method for producing free fatty acids, which comprises: (a) providing a fatty acid raw material, (b) contacting the fatty acid raw material with an enzyme composition comprising lipase and phospholipase to produce a reaction mixture, (c) hydrolyzing the reaction mixture of step b within 16 hours, (d) separating a light oil phase from a heavy phase of the reaction mixture, (e) recycling the heavy phase, (f) recovering free fatty acids from the light phase obtained in step d, and (g) recycling unreacted non-volatile glycerides to the reactor of step c. The general object of the present invention is to provide an enzymatic oil or fat decomposition method that realizes a profitable and competitive large-scale process.

[0008] In another aspect, the present invention provides an enzyme composition for enzymatically decomposing a fatty acid feedstock to produce free fatty acids, wherein the enzyme composition comprises a lipase and a phospholipase.

[0009] In one embodiment, the lipase is derived from Mucor miehei, Candida cylindracea, Rhizopus arrhizus, Humicola lanuginosa, and / or Pseudomonas species ATCC 21808. In a preferred embodiment, the lipase is derived from Humicola lanuginosa and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.

[0010] In another embodiment, the phospholipase is phospholipase A. In a preferred embodiment, the phospholipase is derived from Talaromyces leycettanus and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6.

[0011] The general object of the present invention is to provide an improved enzymatic oil or fat decomposition process which enables a profitable competitive large-scale process. This object is achieved by the features of each of the independent claims. Advantageous further embodiments are defined in the dependent claims. The features of the invention provide short reaction times, reduced enzyme consumption and higher glycerol content in the by-product stream.

[0012] These and other objects and advantages of the present invention will be apparent from the following description. In the following detailed description, preferred embodiments of the present invention will be described with reference to the accompanying drawings. These embodiments do not represent the full scope of the present invention. On the contrary, the present invention can be used for other embodiments. Therefore, the breadth of the present invention should be explained with reference to the claims herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A schematic diagram showing an example of an industrial process showing an embodiment of the enzymatic decomposition of soap raw materials according to the invention is shown. DETAILED DESCRIPTION

[0014] The present invention relates to a method for producing free fatty acids, which comprises: (a) providing a fatty acid raw material, (b) contacting the fatty acid raw material with an enzyme composition to produce a reaction mixture, (c) hydrolyzing the reaction mixture of step b within 16 hours, (d) separating a light oil phase from a heavy phase of the reaction mixture, (e) recycling the heavy phase, (f) recovering free fatty acids from the light phase obtained in step d, and (g) recycling unreacted non-volatile glycerides to the reactor of step c.

[0015] The present invention relates to a method for producing fatty acids, which comprises adding water or a buffer to a fatty acid raw material and allowing the mixture to react under temperature and pressure conditions. The "fatty acid" in the present invention includes not only fatty acids but also those in which glycerol, monoacylglycerol, diacylglycerol and / or triacylglycerol are present.

[0016] In the present invention, the fatty acid feedstock oils and fats to be hydrolyzed can be vegetable, algae, microbial and animal oils and fats as well as any triglycerides from future sources, such as genetically manipulated fat-producing microorganisms or synthetically produced glycerides. Specific examples of fatty acid feedstocks are selected from the group consisting of moringa oil, acai oil, almond oil, babassu oil, black currant seed oil, borage seed oil, canola oil, cashew oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil, linseed oil, grape seed oil, hazelnut oil, hemp seed oil, jatropha oil, jojoba oil, linseed oil, macadamia oil, mango kernel oil, meadowfoam oil, mustard oil, neatsfoot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil / groundnut oil, pecan oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil, camellia oil, and walnut oil or animal derived oils such as tallow, chicken fat, poultry fat fish oil, or fats obtained as by-products from processing industries.

[0017] Hydrolysis conditions are understood to mean those reaction conditions which lead to at least partial conversion of fatty acid alkyl esters or fatty acid glycerides (mono-, di- and triglycerides), preferably technically or economically relevant conversion into free fatty acids. The person skilled in the art will be familiar with selecting known hydrolysis conditions from the prior art and, if necessary, modifying them based on routine tests in order to adapt them to other boundary conditions for the implementation of the process.

[0018] In a preferred embodiment of the process according to the invention, reaction step c is carried out at a temperature of at least 50° C., preferably at least 60° C. These reaction temperatures represent a favorable compromise between high reaction rates and incipient side reactions due to thermal decomposition of the substances involved.

[0019] In one aspect, the average residence time of the oil in any reactor ranges from 0.5 to 16 hours.

[0020] In a preferred embodiment of the method according to the present invention, when the fatty acid raw material reacts with water or buffer in step b, the ratio of water to the fatty acid raw material is the fatty acid of at least 1mol water / 1mol glycerol combination. When operating the present invention, it is possible but not very credible to be up to and exceed the ratio of 20mol / ml, because the key benefit of the present invention is to have the possibility to produce high concentration glycerol by-product. Shown, in this way, realize the favorable compromise between desired high conversion degree and required reactor volume.

[0021] The terms "light phase" and "heavy phase" refer to the respective densities ("specific gravities") of the two liquid phases obtained from the hydrolysis under phase separation conditions.

[0022] Phase separation conditions are understood to mean all physico-chemical parameters which enable, facilitate or accelerate the formation of the two liquid phases obtained from the process. Important parameters in this context are temperature and the intensity of the gravitational field (e.g., the earth's gravity or higher gravitational effects, such as in centrifugal processes). Thermal separation processes are understood to mean all separation processes based on the establishment of thermodynamic phase equilibrium. In the context of the present invention, this is a distillation or rectification that utilizes the evaporation equilibrium of the substances involved.

[0023] If it is desired to separate in such a way that the separated product also contains a certain proportion of free fatty acids, then a person skilled in the art will be able to design the basic thermal separation process in such a way as to achieve this purpose. Thus, when using distillation, a person skilled in the art will accordingly select the temperature profile, the reflux ratio and the mass flow rates of the light and heavy phase weights in the distillation apparatus. There are also cases of flash distillation, in which the reaction mixture is preheated and pressurized before entering a container of various designs, operating under reduced pressure, resulting in a sudden evaporation of volatile components.

[0024] If the oil retains a significant amount of dissolved water after separation, it is conceivable that a drying step will be required between separation and distillation-based unit operations. An example of such a drying step is a vacuum vessel that allows the oil sufficient residence time depending on its moisture level.

[0025] In particular, when the reaction is carried out continuously, all parts of the process are fluidically connected to each other. A fluid connection between two parts is understood to mean any type of connection that allows a fluid (e.g., a reaction mixture, a product, or a separate separated product) to flow from one of the two system parts to another, regardless of any intermediate regions or components.

[0026] Those skilled in the art will select suitable reaction units as hydrolysis reactors. In particular, these are reaction units with high mixing or back mixing. Therefore, in the case of a continuous stirred reactor, for example, a continuous stirred tank reactor, a stirred tank cascade or a tower reactor (split tower) with section mixing is suitable. These are designed in such a way that they are suitable for setting the required pressure, which is especially achieved by selecting an appropriate wall thickness and providing a suitable pressure retaining member.

[0027] A preferred embodiment of the process according to the invention provides for separation of the light phase (step d) and / or recycling of at least a portion of the second separation product (unreacted non-volatile glycerides) into reaction step b (from step g) in such a way that during reaction step b, the proportion of free fatty acids, based on the proportion of fatty acid alkyl esters or fatty acid triglycerides, is >20 to 90% by weight, preferably 30 to 80% by weight, most preferably 40 to 70% by weight. It has been shown that within these free fatty acid concentration ranges, there is a favorable compromise between a catalytic acceleration of the reaction on the one hand and a negative influence on the equilibrium position on the other hand.

[0028] The method can be carried out by contacting the fatty acid feedstock and water or buffer with a lipase in a native (soluble) form or a fixed form in a stirred tank, or by continuously passing glyceride and water through one or more columns of a fixed bed of a lipase in a fixed form. The stirred tank can be used continuously. The effluent from the stirred tank can be separated into an oil phase containing triglycerides, monoglycerides, diglycerides and fatty acids and an aqueous phase containing glycerol and lipase. The water and lipase from the aqueous phase can be recycled, optionally after separation of the glycerol, to better utilize the lipase.

[0029] In one aspect, the method can be carried out in the presence of a buffer, and the buffer strength should preferably be enough to remain on the pH in the optimum range throughout the reaction process, wherein the pH reduces due to the formation of acidic free fatty acids. The optimum range will be lipase specific, wherein some of these lipases show their highest activity under a pH higher than 7.0, and other lipases show their highest activity under a lower level such as pH 4.0. According to the fatty acid feedstock, the final pH near the reaction is completed and can be as low as pH 3.0, requiring pH control for some enzymes. pH can also be controlled by the pH-stat principle, wherein adding acid or alkali (such as citric acid or sodium hydroxide) along with the reaction.

[0030] Lipase

[0031] The lipase or lipolytic enzyme used in the present invention is an enzyme that can hydrolyze ester bonds. Such enzymes include, for example, lipases such as triacylglycerol lipase (EC 3.1.1.3), lipoprotein lipase (EC 3.1.1.34), monoglyceride lipase (EC3.1.1.23), lysophospholipase, ferulic acid esterase and esterase (EC 3.1.1.1, EC 3.1.1.2). The numbers in brackets are system numbers assigned according to the type of enzymatic reactivity of enzymes by the International Union of Biochemistry (Enzyme Commission of the International Union of Biochemistry).

[0032] In one aspect, lipases have regio- and positional specificities / selectivities, all related to the enzyme's preference for reaction at positions 1, 2, and 3 of glycerides.

[0033] The lipase used in the present invention may be a regioselective microbial lipase: the specific microbial lipase may be fungal or bacterial, e.g., derived from the following genera and species as described in the indicated publications: Thermomyces spp., T. lanuginosus (also known as Humicola lanuginosus, EP 305216, US 5869438), Rhizomucor spp., R. miehei, Fusarium spp., F. oxysporum (WO 9826057), or a lipase variant, e.g., as described in WO 9707202. The specific microbial lipase may also be a Cutinase, ie an enzyme also having Cutinase activity (EC 3.1.1.74), e.g. a Cutinase from Humicola, H. insolens (WO 9613580) or a Cutinase variant, e.g. as described in WO 00 / 34450 or WO 0192502.

[0034] In other aspects, the lipase used in the invention can be eukaryotic, such as a fungal lipolytic enzyme, such as a lipolytic enzyme from the family Humicola.

[0035] The lipolytic enzymes of the Humicolaceae family consist of the lipase from Humicola lanuginosus strain DSM 4109 and lipases having greater than 50% homology thereto. The lipase from Humicola lanuginosus (synonym Thermomyces lanuginosus) is described in EP 258 068 and EP 305 216 and has the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of US 5,869,438.

[0036] The Humicola family also includes the following lipolytic enzymes: lipase from Penicillium camembertii, lipase from Fusarium oxysporum (EP 130064, WO 98 / 26057), lipase from F. heterosporum (R87979), lysophospholipase from Aspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), lipase / feruloyl esterase from A. niger (Y09330), lipase / feruloyl esterase from A. tubingensis (Y09331), lipase from A. tubingensis (WO 98 / 26057), lipase from A. foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), lipase / feruloyl esterase from A. niger (Y09330), lipase / feruloyl esterase from A. tubingensis (Y09331), lipase from A. tubingensis (WO 98 / 26057). 98 / 45453), a lysophospholipase from Aspergillus niger (WO 98 / 31790), a lipase from F. solanii with an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96 / 18729).

[0037] In one aspect, the lipase is administered in a total amount corresponding to 0.1-400 mg lipase protein / kg oil. Lipase levels outside this range as well as different lipases can be used. Prior to blending with the feedstock, the lipase is mixed with water or a buffer solution.

[0038] On the other hand, preferred lipases are non-specific enzymes that are able to hydrolyze any glyceride bond at any position as quickly as possible and with as little slowing of the reaction rate as possible during the course of the reaction. 1,3-position specific enzymes may also be used, as the FFA separation step of the invention may promote acyl migration, resulting in the formation of 1- or 3-monoglycerides from 2-monoglycerides or similar migration of 1,2- or 2,3-diglycerides to 1,3-diglycerides. Lipase regioselectivity is generally not fixed, although the concept itself is used in a black-and-white manner, meaning that an enzyme described as 1,3-specific will generally have high reaction rates at the 1- and 3-positions, while still being able to react at the 2-position, albeit significantly slower. As a result of the 1,3-specific enzyme reaction, the hydrolyzed triglyceride molecule, now a 2-monoglyceride, carries the glycerol moiety in the fat layer rather than the water layer. In certain specific applications of the invention, it is envisioned that chain length or 'unsaturation' specific enzymes may be used. An example is the targeted hydrolysis of specific fatty acids, which would allow simple distillation rather than fractionation. Non-specific enzymes cleave all three ester bonds in a triglyceride. Chain length specific enzymes only cleave those triglycerides with chain lengths within a certain range. These types of enzymes are also useful under the teachings of the present invention. The choice of lipase specificity will depend on the target use of the present invention, but in most cases will be a fast enzyme, preferably with no preference for regioselectivity.

[0039] In a preferred embodiment, the lipase of the present invention is derived from Humicola lanuginosa and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1.

[0040] Phospholipase A

[0041] Phospholipase A refers to an enzyme having phospholipase A1 and / or phospholipase A2 activity (A1 or A2, EC 3.1.1.32 or EC3.1.1.4), i.e., the hydrolysis activity of one or two carboxylic acid ester bonds in phospholipids such as lecithin. Phospholipases having both A1 and A2 activity are also referred to as phospholipase B. It is easy for a person skilled in the art to identify enzymes having phospholipase A activity by LEU determination. In the LEU determination, phospholipase A activity is determined by the ability to hydrolyze lecithin at pH 8.0 and 40°C. After the hydrolysis reaction, titration is performed with NaOH, and the reaction time is 2 minutes. The phospholipase from Fusarium oxysporum (LIPOPANF) disclosed in WO 1998 / 26057 has an activity of 1540LEU / mg enzyme protein and can be used as a standard.

[0042] Several types of phospholipases are known, with specificities that differ according to the position of the bond attacked in the phospholipid molecule. Phospholipase A1 (PLA1) removes fatty acids at the 1-position to produce free fatty acids and 1-lyso-2-acylphospholipids. Phospholipase A2 (PLA2) removes fatty acids at the 2-position to produce free fatty acids and 1-acyl-2-lysophospholipids. The term phospholipase B (PLB) is used for phospholipases that have both A1 and A2 activity.

[0043] In the context of the present invention, it has been observed that the use of PLA will improve the enzymatic decomposition of soap raw materials, especially when used together with lipase. In a preferred embodiment, the phospholipase A of the present invention is derived from Talaromyces rayseidii and has an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6. In another preferred embodiment, the phospholipase A of the present invention is derived from Humicola lanuginosus and has an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5.

[0044] In one aspect, the total reaction mixture comprises a water content in the range of at most 0.5%-75.0% (w / w). The oil / water ratio (weight / weight) of 1:2 to 3:1 may be higher than 1:2, in particular higher than 1:1, and may be lower than 5:1, in particular lower than 3:1 or lower than 2:1. The type of water used does not substantially affect the reaction. Thus, distilled water, tap water or deionized water may be used with similar effects.

[0045] The free fatty acids are then separated from the light organic phase, and the residue that still contains the lipid acid that is chemically bonded to glyceride is sent back or recirculated and mixed with fresh oil or fat to be subjected to decomposition. In this way, the glyceride of recirculation is then subjected to other enzymatic decomposition process. If the enzymatic fat or oil decomposition only reaches about 80% decomposition degree, then this is possible in the very short time. If in this way afterwards from the oil or fat of part decomposition, extract the free fatty acids, and if the lipid acid (glyceride) of chemical bonding is returned or sent back in the decomposition process, then can significantly reduce enzyme consumption, and need not abandon the final complete decomposition of oil or fat. The enzymatic decomposition process does not enter the above-mentioned process conditions, wherein may be owing to lacking glyceride at the phase interface between light phase and heavy phase, the speed of response significantly slows down under the situation of most of known lipases.

[0046] The present invention in its broader aspects relates to a process for increasing the production of free fatty acids and glycerol from glycerides in a reactor, the process comprising combining in a first step the glycerides with a suitable amount of an effective lipase in the presence of water under stirring to partially decompose the glycerides, and mixing the partially decomposed glycerides with water in a reactor under temperature and pressure conditions effective to substantially completely decompose the glycerides into their component free fatty acids and glycerol, wherein the free fatty acids and glycerol produced from the partially decomposed glycerides are increased relative to glycerides not treated with the lipase.

[0047] The method of the present invention can be applied to any oil or fat consisting mainly of glycerides (acylglycerols), for example, vegetable oils and animal fats, typically containing more than 90% (e.g., more than 95%) triglycerides by weight. The acyl groups in the triglycerides can be straight-chain fatty acyl groups, typically having 4-24 carbon atoms, particularly 12-22 carbon atoms. They can be saturated or unsaturated, containing one or more double bonds. The triglycerides can be particularly triglycerides of unsubstituted acyl groups, i.e. acyl groups with the general formula R-CO, wherein R is a hydrocarbon group. Significant amounts of diglycerides and monoglycerides and / or FFAs may also initially be present in the feedstock, for example in the case of waste oils or fatty acid distillates. Preferred feedstocks will be mostly saturated feedstocks, such as palm oil or palm kernel oil, because the yield loss of the FFA separation unit operation is expected to be small.

[0048] The hydrolysis process can be carried out at moderate to high temperatures, and advantageously it can be applied even to thermolabile triglycerides, e.g., triglycerides with polyunsaturated acyl groups. The process produces high yields of glycerol and fatty acids, which can be separated and purified by conventional methods.

[0049] In order to separate free fatty acids from the partial decomposition process of enzymatic decomposition, it is preferred to apply a vacuum distillation method, that is, under the mildest possible operating conditions that can remove at least 80% of FFA, it is possible to carry out continuous vacuum column distillation with the help of stripping steam and packing materials. Those conditions will depend on the raw material FFA composition. For example, a deeper vacuum is preferred compared to the temperature of the increase. In the case where production volume and product value allow the advantageous use of gentle short-range distillation (sometimes referred to as molecular distillation), this will be preferred. In the case of fats and oils with a chain length spectrum of fatty acids of C10 to C22, as most natural fats and oils, free fatty acids can be separated from the initial process of partial decomposition (i.e. fat or oil) by distillation, and any relevant amount of glycerides can be distilled out at the same time, or in other words, any relevant amount of fatty acids still chemically bonded to glycerides will be removed. In the case of a degree of decomposition between about 80% and 90%, the amount of fatty acids chemically bonded to monoglycerides and diglycerides is unexpectedly small. The main part is composed of triglycerides that have not yet been decomposed. Furthermore, the amount of free fatty acids remaining in the residue of the distillation step does not result in losses, since the residue is recycled to the decomposition reaction. In an alternative to the application of distillation, the free fatty acids can be separated by adsorptive separation methods (eg column chromatography).

[0050] Examples

[0051] Example 1

[0052] Figure 1 The process of enzymatically decomposing raw materials to produce free fatty acids is shown. The amount of free fatty acids (FFA) decomposed from soap raw materials after incubation with and without enzymes was measured by a laboratory-scale batch reaction. The raw material for the reaction is soap raw material, which is a byproduct of a typical chemical soybean oil refining process. The laboratory-scale batch reaction is generally described as weighing 100g of soap raw material and pouring it into a blue-capped bottle, adding 15mL of water to the bottle and stirring the mixture at 85°C at 350rpm, and then adding sulfuric acid to adjust the pH to 5.0-5.5 as a pretreatment. The bottle is cooled to 55°C. After the pretreatment step, the enzyme composition is added directly to the soap raw material to break the emulsion and hydrolyze the oil in the soap raw material to produce FFA. The resulting mixture is stirred at 55°C for 16 hours for hydrolysis. After the reaction, the bottle containing the sample is heated to 85°C, and then the pH is adjusted to 2.0 with sulfuric acid. The mixture is centrifuged to separate the light phase and the heavy phase. After the separation step, free fatty acids are recovered mainly in the light phase, while the heavy phase consists of glycerol, lysate, salts and water. The yield of FFA is measured by AOCS official method Ca 5a-40.

[0053] Enzymatic soap stock decomposition reactions were performed with individual lipases and phospholipases at a dosage of 30 ug enzyme protein / gram soap stock (dry matter), and a blend consisting of 50% lipase and 50% phospholipases at a dosage of 60 ug enzyme protein / gram soap stock (dry matter). For comparison, a treatment without enzymes was included. The free fatty acids from soap stock decomposition at the specified dosages are measured in Table 1 below. Wherein lipase 1 is derived from Humicola lanuginosus, having an amino acid sequence of SEQ ID NO: 1. Lipase B is derived from Humicola insolens, having an amino acid sequence of SEQ ID NO: 2. Lipase C is derived from Candida antarctica as disclosed in WO 88 / 02775, having an amino acid sequence of SEQ ID NO: 3. Lipase D is derived from Rhizomucor miehei, having an amino acid sequence of SEQ ID NO: 4. PLA-A is derived from Thermomyces lanuginosus as disclosed in EP1131416, having an amino acid sequence of SEQ ID NO: 5. PLA-B is derived from Talaromyces retsei as disclosed in WO 2018171552 and has the amino acid sequence of SEQ ID NO:6.

[0054] Table 1 Enzymatic soap raw material decomposition reaction

[0055]

[0056] As can be seen from Table 1, Lipase 1 is more effective than other lipases in free fatty acid production, and when mixed with PLA-B, the yield of free fatty acids is 186.46. The enzymatic decomposition method of the present invention uses an effective enzyme composition (e.g., Lipase 1 with PLA) to successfully reduce the average residence time of oil in any reactor from at least 24 hours to 16 hours, and at the same time achieve the desired acid value.

[0057] Example 2

[0058] In the example, the amount of free fatty acids (FFA) decomposed from soap raw materials after incubation with and without enzymes was measured by a laboratory-scale batch reaction. The raw material for the reaction is soap raw material, which is a byproduct of a typical chemical soybean oil refining process. The laboratory-scale batch reaction is generally described as weighing 100g of soap raw material and pouring it into a blue cap bottle, then stirring at 350rpm at 85°C, and then adding sulfuric acid to adjust the pH to 5.0-5.5 as a pretreatment. The bottle is cooled to 55°C, and the enzyme is directly added to the soap raw material after the pretreatment step to break the emulsion and hydrolyze the oil in the soap raw material to produce FFA. Stirring is continued at 55°C for 16 hours to carry out the enzymatic reaction. After the reaction, the bottle containing the sample is heated to 85°C, and then the pH is adjusted to 2.0 with sulfuric acid. The mixture is centrifuged to separate the light phase and the heavy phase. After the separation step, the free fatty acids are mainly recovered in the light phase, while the heavy phase consists of glycerol, hemolysate, salt and water. The yield of FFA was measured by AOCS official method Ca5a-40.

[0059] The enzymatic soap raw material decomposition reaction was carried out with a lipase at a dosage of 30ug enzyme protein / gram soap raw material (dry matter), and a blend consisting of 50% lipase and 50% phospholipase at a dosage of 60ug enzyme protein / gram soap raw material (dry matter). For comparison, a treatment without enzyme was included. The free fatty acids from the decomposition of soap raw materials at the specified dosages are measured in Table 2 below. Among them, the lipase is LP 64 obtained as described in WO 2018 / 001959. LP-64 is also derived from Humicola lanuginosus and has at least 97% identity with the amino acid of SEQ ID NO: 1.

[0060] Table 2 Enzymatic soap raw material decomposition reaction

[0061]

[0062]

[0063] As can be seen from Table 2, LP 64 is more effective than other lipases in free fatty acid production, and when mixed with PLA-A and PLA-B, the yield of free fatty acids is 169. Whether used alone or in combination with PLA, it is superior to other lipases (such as lipase B and lipase C). And as can be seen from Tables 1 and 2, the lipase derived from Humicola lanuginosus shows better performance than other lipases in the enzymatic decomposition of soap raw materials. And when Humicola lanuginosus is combined with PLA, the average residence time of oil in any reactor is successfully reduced from at least 24 hours to 16 hours, and at the same time, the desired acid value is achieved.

Claims

1. A method for producing free fatty acids, the method comprising: (a) providing fatty acid raw materials, (b) contacting the fatty acid feedstock with an enzyme composition comprising a lipase and a phospholipase to produce a reaction mixture, (c) hydrolyzing the reaction mixture of step b within 16 hours, (d) separating the light oil phase from the heavy phase of the reaction mixture, (e) recycling the heavy phase, (f) recovering free fatty acids from the light phase obtained in step d, and (g) recycling the unreacted non-volatile glyceride to the reactor in step c.

2. The method of claim 1, wherein the enzyme composition is administered in a total amount corresponding to 0.1-400 mg enzyme protein / kg oil.

3. The method of any one of the preceding claims, wherein the lipase is derived from Mucor miehei, Candida columnaris, Rhizopus arrhizus, Humicola lanuginosa, and / or Pseudomonas sp. ATCC 21808.

4. The method of any one of the preceding claims, wherein the lipase is derived from Humicola lanuginosa having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

1.

5. The method of any one of the preceding claims, wherein the phospholipase is phospholipase A.

6. The method of any of the preceding claims, wherein the phospholipase is derived from Talaromyces retsei and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

6.

7. The method of any of the preceding claims, wherein the phospholipase is derived from Thermomyces lanuginosus and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

5.

8. A process as claimed in any preceding claim, wherein the free fatty acid content in the mixture after any number of the preceding reaction steps ranges from 20% to 90%, preferably from 30% to 80%, and most preferably from 40% to 70%.

9. The process of any one of the preceding claims, wherein the glycerides in the fatty acid feedstock are a mixture of monoglycerides, diglycerides and / or triglycerides.

10. A process as claimed in any preceding claim, wherein the fatty acid feedstock is an oil or fat of natural origin, or a mixture thereof.

11. The method of any of the preceding claims, wherein the fatty acid feedstock is any triglyceride from a future source, such as a genetically manipulated lipid-producing microorganism.

12. The method of any one of the preceding claims, wherein the fatty acid feedstock is selected from the group consisting of moringa oil, acai oil, almond oil, babassu oil, black currant seed oil, borage seed oil, canola oil, cashew oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil, linseed oil, grapeseed oil, hazelnut oil, hemp seed oil, jatropha oil, jojoba oil, linseed oil, macadamia oil, mango kernel oil, Limnanthes oil, mustard oil, neatsfoot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil / groundnut oil, pecan oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil, camellia oil, walnut oil or animal derived oils such as tallow, chicken fat, poultry fat fish oil, or fats obtained as by-products from processing industries.

13. An enzyme composition for enzymatically decomposing a fatty acid feedstock to produce free fatty acids, wherein the enzyme composition comprises a lipase and a phospholipase.

14. The enzyme composition of claim 13, wherein the enzyme composition is administered in a total amount corresponding to 0.1-400 mg enzyme protein / kg oil.

15. The enzyme composition of any one of claims 13-14, wherein the lipase is derived from Mucor miehei, Candida columnaris, Rhizopus arrhizus, Humicola lanuginosa, and / or Pseudomonas sp. ATCC 21808.

16. The enzyme composition of any one of claims 13-15, wherein the lipase is derived from Humicola lanuginosa and has an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

1.

17. The enzyme composition of any one of claims 13-16, wherein the phospholipase is phospholipase A.

18. An enzyme composition as described in any of claims 13-17, wherein the phospholipase is derived from Talaromyces retsei and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

6.

19. The enzyme composition of any one of claims 13-18, wherein the phospholipase is derived from Thermomyces lanuginosus and has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5.

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