EP4577664A1

EP4577664A1 - Improved enzymatic splitting of oils and fats to produce free fatty acid

Info

Publication number: EP4577664A1
Application number: EP23856650.9A
Authority: EP
Inventors: Hongbo CUI; Xiangguo ZHAO; Mingjia ZHANG; Yi Cao
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2022-08-24
Filing date: 2023-08-23
Publication date: 2025-07-02
Also published as: WO2024041562A1; CN119816599A

Abstract

The present invention relates to an improved enzymatic splitting method for producing free fatty acid, wherein, fatty acid feedstock is contacted with an enzyme composition comprising lipase and phospholipases to produce a reaction mixture, hydrolyze said mixture for 16 hours, separating the light phase and heavy phase of the reaction mixture and recovering free fatty acid from the light phase. Meanwhile, the present invention also relates to an enzyme composition use to enzymatic splitting soap stock to product free fatty acid, wherein, said enzyme composition comprising a lipase and phospholipases. Said enzyme composition may significantly reduce the mean residence time of oil in any reactor from at least 24hrs to 16hrs and meanwhile achieved desirable acid value.

Description

IMPROVED ENZYMATIC SPLITTING OF OILS AND FATS TO PRODUCE FREE FATTY ACID

FIELD OF INVENTION

The present invention relates to the enzymatic splitting or hydrolysis of oils and fats, i.e., acidic oil, in the presence of water into free fatty acid.

BACKGROUND

Acidified oil refers to the oil obtained by acidifying soap stock, i.e., the by-product of the oil refinery. It is essentially a fatty acid, which also contains pigments, non-acidified triglycerides, diesters, 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.
Enzymatic splitting of acidic oils has been known for a long time and offers, contrary to a pressure splitting mainly practiced in the industry, considerable principal advantages. The enzymatic splitting can be performed at normal pressure and, depending on the enzyme and oil or fat, even at room temperature.
It has also been known for a long time that this method of fat splitting is the gentlest one. The technical progress in industrial biotechnology also provided enzymes that are available now and suitable for fat splitting. However, mainly because of the high enzyme consumption, of the very long splitting times and the resulting low efficiency of the enzymatic fat splitting, the enzymatic splitting did not become an industrial alternative method to the large-scale pressure splitting which is well established in industry.
The enzymatic fat splitting using enzymes, so-called lipases, as biocatalysts acting on a water/oil mixture is known in the art. By means of this splitting technique, the oil or fat, respectively, is split into glycerol and free fatty acids. The glycerol migrates into the water phase whereas the organic phase enriches more and more with free fatty acids until, finally, only the free fatty acids remain in the organic phase.
Current enzymatic splitting process always undergoes a very long reaction time, for example, more than 24 hours in a simple batch system to achieve a desired splitting degree near 100 percent. However, the long reaction time will destroy the enzyme activity. In other words, the activity of the enzyme decreases to some extent with time mainly due to protein denaturation. This reduction can be compensated by further additions of enzyme, which conversely increases costs. Therefore, there are continuous needs to improve the enzymatic splitting process to shorten the reaction time, keep the desired splitting degree and meanwhile still be economic and efficient for use broadly in producing free fatty acid industrially.

SUMMARY OF THE INVENTION

The present invention provides a process for producing free fatty acid comprising: (a) providing fatty acid feedstock, (b) contacting said fatty acid feedstock with an enzyme composition comprising lipase and phospholipases to produce a reaction mixture, (c) hydrolysis of the reaction mixture of step b within 16 hours, (d) separating the light oil phase from the heavy phase of the reaction mixture, (e) recirculating the heavy phase, (f) recovering free fatty acid from the light phase resulting from step d, (g) recirculating unreacted non-volatile glycerides into the reactor of step c. A general objective of the present invention is to provide an enzymatic oil or fat splitting method which allows for a profitable competitive large-scale process.
In another aspect, the present invention provides an enzyme composition for enzymatically splitting fatty acid feedstock to produce free fatty acids, wherein, said enzyme composition comprises lipases and phospholipases.
In one embodiment, the lipase is derived from Mucor miehei, Candida cylindracea, Rhizopus arrhizus, Humicola lanuginosa, and/or Pseudomonas sp, ATCC 21808. In a preferred embodiment, the lipase is derived from Humicola lanuginose, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 1.
In another embodiment, the phospholipase is phospholipase A. In a preferred embodiment, the phospholipase is derived from Talaromyces leycettanus, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 6.
A general objective of the present invention is to provide an improved enzymatic oil or fat splitting method which allows for a profitable competitive large-scale process. This objective is achieved by means of the features of each one of the independent claims. Advantageous further embodiments are defined in the sub-claims. The inventive features provide a short reaction time, a reduced enzyme consumption and a higher glycerol content in the by-product stream.
These and still other objectives and advantages of the present invention will be apparent from the description which follows. In the detailed description below, preferred embodiments of the invention will be described in reference to the accompanying drawings. These embodiments do not represent the full scope of the invention. Rather the invention may be employed in other embodiments. Reference should therefore be made to the claims herein for interpreting the breadth of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a schematic drawing of an example for an industrial process showing an embodiment of enzymatic splitting of soap stock according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for producing free fatty acid comprising: (a) providing fatty acid feedstock, (b) contacting said fatty acid feedstock with an enzyme composition to produce a reaction mixture, (c) hydrolysis of the reaction mixture of step b within 16 hours, (d) separating the light oil phase from the heavy phase of the reaction mixture, (e) recirculating the heavy phase, (f) recovering free fatty acid from the light phase resulting from step d, (g) recirculating unreacted non-volatile glycerides into the reactor of step c.
The present invention refers to a process of producing fatty acids, including adding water or buffer to fatty acid feedstock and allowing the mixture to react in a temperature and pressure condition. 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.
In the present invention, the fatty acid feedstock oil and fat to be hydrolyzed may be vegetable plant, algal, microbial and animal oil and fat and any triglyceride stemming from future sources such as fat-producing genetically manipulated microorganisms or synthetically produced glycerides. Specific examples of fatty acid feedstock is selected from the group consisting of Moringa oil, inacai oil, almond oil, babassu oil, blackcurrant seed oil, borage seed oil, canola oil, cashew oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil, flax seed oil, grape seed oil, hazelnut oil, hempseed oil, jatropha oil, jojoba oil, linseed oil, macadamia nut oil, mango kernel oil, meadowfoam oil, mustard oil, neat’s foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil/ground nut oil, pecan oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil, tsubaki oil and walnut oil or animal derived oil such as tallow, chicken fat, poultry fat fish oil, or fat obtained as by products from processing industries.
Hydrolysis conditions are understood to mean those reaction conditions which bring about at least a partial conversion, preferably a technically or economically relevant conversion of the fatty acid alkyl esters or the fatty acid glycerides (mono-, di-, and tri-glycerides) to free fatty acids. The person skilled in the art will be familiar with hydrolysis conditions known from the prior art select and, if necessary, modify them on the basis of routine tests in order to adapt them to other boundary conditions of the process implementation.
In a preferred embodiment of the process according to the invention, reaction step c is carried out at a temperature of at least 50℃, preferably at least 60℃. These reaction temperatures represent favorable compromises between high reaction rates, and incipient side reactions due to thermal decomposition of the substances involved.
In one aspect, the mean residence time of oil in any reactor is in the range from 0.5 to 16 hours.
In a preferred embodiment of the process according to the invention, when the fatty acid feedstock is reacted with water or buffer in step b, the ratio of water to fatty acid feedstock is at least 1 mol water/1 mol glycerol-bound fatty acid. A ratio up to and beyond 20 mol/ml is a possible but unlikely choice when operating the invention because a key benefit of the invention is the possibility of producing high concentration glycerol byproduct. It has been shown that a favorable compromise between the desired high degrees of conversion and the required reactor volume is achieved in this way.
The terms “light phase” and “heavy phase” refer to the respective density (the “specific weight” ) of the two liquid phases obtained from the hydrolysis under phase separation conditions.
Phase separation conditions are understood to mean all physico-chemical parameters which enable, favor or accelerate the formation of the two liquid phases obtained from the process. Important parameters in this context are the temperature and the strength of the gravitational field (e.g., earth’s gravity or higher gravitational effect, for example during centrifugation) . Thermal separation processes are understood to mean all separation processes which are based on the establishment of a thermodynamic phase equilibrium. In the context of the present invention, this is distillation or rectification, which make use of the evaporation equilibrium of the substances involved.
If it is required that the separation be carried out in such a way that the separation product also contains a proportion of free fatty acids, the person skilled in the art will be able to design the underlying thermal separation process in such a way that this objective is achieved. Thus, when using the distillation, he will select the temperature profiles in the distillation apparatus, the reflux ratio and the mass flows of the light phase and heavy weight accordingly. There is also the case of flash distillation, where the reaction mixture is preheated and pressurized prior to entering a vessel of various designs, operating at a decreased pressure, resulting in sudden evaporation of volatile components.
If the oil holds a significant amount of dissolved water after separation, it is conceivable that a drying step would be required between the separation and distillation-based unit operations. An example of such a drying step would be a vacuum vessel allowing for a sufficient residence time of the oil depending on its moisture level.
In particular when the reaction is carried out continuously, all parts of the process are in fluid connection with one another. A fluid connection between two parts is understood to mean any type of connection that enables a fluid, for example the reaction mixture, the product or the individual separation products, from which one to the other of the two system parts can flow, irrespective of any intermediate areas or components.
The person skilled in the art will select a suitable reaction apparatus as the hydrolysis reactor. In particular, these are reaction apparatus with high mixing or back-mixing. Therefore, in the case of continuous stirred reactors, for example, continuous stirred tank reactors, stirred tank cascades or tower reactors with segmental mixing (split tower) are suitable. These are to be designed in such a way that they are suitable for setting the required pressure, which is achieved, among other things, by selecting appropriate wall thicknesses and providing suitable pressure-maintaining members.
A preferred embodiment of the process according to the invention provides that the separation of the light phase (step d) and/or the recycling of at least part of the second separation product (unreacted non-volatile glycerides) to the reaction step b (from step g) take place in such a way that during the 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 in these free fatty acid concentration ranges there is a favorable compromise between the catalytic acceleration of the reaction on the one hand and the negative influence on the equilibrium position on the other hand.
The process may be carried out by contacting the fatty acid feedstock and water or buffer in a stirred tank with the lipases in native (soluble) form or in immobilized form, or by continuously passing the glycerides and water through one or more columns with a fixed bed of the lipases in immobilized form. The stirred tank may be used continuously. The effluent from the stirred tank may be separated into an oily phase containing triglyceride, monoglyceride, diglyceride and fatty acid, and an aqueous phase containing glycerol and lipase. Water and lipase from the aqueous phase may be recycled, optionally after separation of glycerol, for better utilization of the lipase.
In one aspect, the process may be carried out in presence of buffer, the buffer strength should preferably be sufficient to keep pH within the optimal range throughout the extent of reaction, where pH decreases due to formation of acidic free fatty acids. The optimal range will be lipase specific, with some lipases showing their highest activity at pH above 7.0 and others at lower levels such as pH 4.0. Depending on the fatty acid feedstock the final pH near reaction completion can be as low as pH 3.0, requiring pH control for some enzymes. pH might also be controlled through pH-stat principles, where acid or base such as citric acid or sodium hydroxide is added as reaction progresses.
lipases
Lipase or lipolytic enzyme used in the present invention is one 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 (EC 3.1.1.23) , lysophospholipase, ferulic acid esterase and esterase (EC 3.1.1.1, EC 3.1.1.2) . The numbers in parentheses are the systematic numbers assigned by the Enzyme Commission of the International Union of Biochemistry in accordance with the type of the enzymatic reactivity of the enzyme.
In one aspect, the lipase is of Regio-, and positional specificity/selectivity all relate to the preference of the enzymes towards reacting the 1, 2, and 3 positions of the glycerides.
The lipase used in the present invention may be Regio selective 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, T. lanuginosus (also known as Humicola lanuginosa, EP 305216, US 5869438) , Rhizomucor, R. miehei, Fusarium, F. oxysporum (WO 9826057) , or a lipase variant, e.g., as described in WO 9707202. The specific microbial lipase may also be a cutinase, i.e., an enzyme which also has 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.
In other aspect, the lipase used in the present invention may be eukaryotic, e.g., a fungal lipolytic enzyme such as lipolytic enzymes of the Humicola family.
The Humicola family of lipolytic enzymes consists of the lipase from H. lanuginosa strain DSM 4109 and lipases having more than 50%homology with said lipase. The lipase from H. lanuginosa (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.
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/ferulic acid esterase from A. niger (Y09330) , lipase/ferulic acid esterase from A. tubingensis (Y09331) , lipase from A. tubingensis (WO 98/45453) , lysophospholipase from A. niger (WO 98/31790) , lipase from F. solanii having an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96/18729) .
In one aspect, said lipase is dosed in a total amount corresponding to 0.1-400 mg lipase enzyme protein per kg of oil. Levels of lipase outside this range may be used, as well as different lipase enzymes. The lipase is mixed with water or buffer solution prior to blending with the feedstock.
In another aspect, the preferred lipase is an unspecific enzyme capable of hydrolyzing any glyceride ester bond as quickly as possible on any position and with as little slowdown of reaction speed as possible during the extent of reaction. One may also use a 1, 3-position specific enzyme because the FFA separation step of the invention would promote acyl-migration, resulting in formation of 1-or 3-monoglyceride from a 2-monoglyceride or similar migration of 1, 2-or 2, 3-diglyceride to become 1, 3-diglyceride. Lipase regioselectivity is often fluid, although the concept itself is used in a black and white manner, meaning an enzyme described as 1, 3 specific will often have a high rate of reaction on the 1-and 3-positions while still being able to react the 2-position, albeit significantly slower. As a result of a 1, 3-specific enzyme reaction, the hydrolyzed triglyceride molecule, now a 2-monoglyceride, carries the glycerol portion along in the fat layer instead of the water layer. Use of a chain length or ‘degree of unsaturation’-specific enzyme is conceivable in certain niche applications of the invention. An example being targeted hydrolysis of specific fatty acids, which would allow for simple distillation instead of fractionation. The non-specific enzyme cleaves all three ester linkages in the triglyceride. The chain length-specific enzyme cleaves only those triglycerides having a chain length within a certain range. These types of enzymes also are useful under the teachings of the invention. The selection of lipase specificity will be dependent on the target use of the invention but will in the majority of cases be a fast enzyme preferably with no preference regarding regioselectivity.
In a preferable embodiment, the lipase of the present invention is derived from Humicola lanuginose, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 1.
Phospholipase A
Phospholipase A refers to the enzymes having phospholipase A1 and/or phospholipase A2 activity (A1 or A2, EC 3.1.1.32 or EC 3.1.1.4) , i.e., hydrolytic activity towards one or both carboxylic ester bonds in phospholipids such as lecithin. A phospholipase having both A1 and A2 activity is also referred to as a phospholipase B. It is easy for person skilled in the art to recognize the enzyme with phospholipase A activity by LEU assay. In the LEU assay, the phospholipase A activity is determined from the ability to hydrolyze lecithin at pH 8.0, 40℃. The hydrolysis reaction can be followed by titration with NaOH for a reaction time of 2 minutes. The phospholipase from Fusarium oxysporum (LIPOPAN F) disclosed in WO 1998/26057 has an activity of 1540 LEU/mg enzyme protein and may be used as a standard.
Several types of phospholipases are known which differ in their specificity according to the position of the bond attacked in the phospholipid molecule. Phospholipase A1 (PLA1) removes the 1-position fatty acid to produce free fatty acid and 1-lyso-2-acylphospholipid. Phospholipase A2 (PLA2) removes the 2-position fatty acid to produce free fatty acid and 1-acyl-2-lysophospholipid. The term phospholipase B (PLB) is used for phospholipases having both A1 and A2 activity.
In the context of the present invention, it has been observed that the use of PLA will improve the enzymatic splitting degree of a soapstock, especially when co-using with lipase. In a preferable embodiment, the Phospholipase A of the present invention is derived from Talaromyces leycettanus, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 6. In another preferred embodiment, the Phospholipase A of the present invention is derived from Humicola lanuginosa, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 5.
In one aspect, the total reaction mixture comprises at most a water content in the range of 0.5 –75.0% (w/w) . The oil/water ratio from 1: 2 to 3: 1 (weight/weight) may be above 1: 2, particularly above 1: 1, and may be below 5: 1, particularly below 3: 1 or below 2: 1. The type of water used does not materially affect the reaction. Thus, distilled, tap or deionized water can be used with like effect.
Free fatty acids are then separated from the light organic phase, and the residue which still contains fatty acids chemically bonded as glycerides is fed back or recycled and mixed with fresh oil or fat to be subjected to splitting. In this manner, recycled glycerides are then subjected to a further enzymatic splitting process. If the enzymatic fat or oil splitting is running up to only a splitting degree of about 80 percent, this is possible in a very short time. If afterwards the free fatty acids are extracted from an oil or fat partially split in such a manner and if the chemically bound fatty acids (glycerides) are returned or fed back into the splitting process, the enzyme consumption can be drastically reduced, without having to renounce to a final complete splitting of the oil or fat. The enzymatic splitting process does not get into the above-mentioned process conditions in which the reaction speed, probably due to lack of glycerides at the phase interface between light and heavy phase, is remarkably slowed down in the case of most known lipase enzymes.
The invention in its broader aspects relates to a process of increasing the production of free fatty acids and glycerol from glycerides in a reactor comprising the combining in a first step of the glycerides with a suitable amount of an effective lipase with agitation in the presence of water to partially split the glycerides, and mixing the partially split glycerides in the reactor with water under conditions of temperature and pressure effective to substantially complete the splitting of the glycerides into component free fatty acids and glycerol, wherein the production of the free fatty acids and glycerol from the partially split glyceride is increased relative to a glyceride not treated with the lipase.
The process of the invention can be applied to any oil or fat consisting largely of glycerides (acylglycerols) , e.g., vegetable oils and animal fat, typically containing more than 90% (e.g., more than 95%) by weight of triglycerides. The acyl groups in the triglyceride may be linear fatty acyl groups, typically with 4-24 carbon atoms, particularly 12-22 carbon atoms. They may be saturated or unsaturated containing one or more double bonds. The triglyceride may particularly be a triglyceride of unsubstituted acyl groups, i.e., acyl groups of the general formula R-CO where R is a hydrocarbyl group. Significant amounts of di-and monoglycerides and/or FFA might also be initially present in the feedstock, for example in the case of waste oils or fatty acid distillates. The preferred feedstock would be a largely saturated feedstock such as palm or palm kernel oil, because less yield loss would be expected from the FFA separation unit operation.
The hydrolysis process can be conducted at moderate to high temperatures, and advantageously it can even be applied to thermolabile triglycerides, e.g., triglycerides with polyunsaturated acyl groups. The process leads to a high yield of glycerol and fatty acid which can be separated and purified by conventional methods.
In order to separate the free fatty acids from the partially split process of the enzymatic splitting, preferably a vacuum distillation method is applied, that is, a continuous vacuum column distillation, possibly with the aid of stripping steam and packing material, at mildest possible operating conditions at which at least 80%of the FFA can be removed. Those conditions will depend on the feedstock FFA composition. For example, a deeper vacuum is preferred above increased temperature. In cases where the production volume and product value allow for profitable use of a mild short path distillation (sometimes called molecular distillation) , this would be preferred. In the case of fats and oils with a chain length spectrum of the fatty acids of C10 to C22, as it holds true for most natural fats and oils, the free fatty acids can be separated by distillation from the partially split starting process, i.e., fat or oil, and can be removed without concurrently distilling off any relevant quantities of glycerides or, in other words, any relevant quantities of fatty acids still chemically bonded as glycerides. In the case of splitting degrees between about 80%and 90%, the amounts of fatty acids chemically bonded as mono-and diglycerides are surprisingly small. The main part consists of the not-yet-split triglycerides. Moreover, quantities of free fatty acids remaining in the residue of the distillation step do not lead to a loss, since the residue is recycled into the splitting reaction. In one alternative to applying a distillation, the free fatty acids could be separated by an adsorptive separation method (e.g., column chromatography) .
Examples
Example 1
Figure 1 shown the process of enzymatically splitting feedstock to produce free fatty acid. The amount of free fatty acid (FFA) from soap stock splitting, after incubation with and without enzyme, was measured by lab-scale batch reaction. The feedstock for the reaction was soap stock, which was the by-product of typical chemical soybean oil refining process. The lab-scale batch reaction was generally described that 100 g soap stock was weighed and poured into a blue cap bottle, 15 mL water was added into the bottle and the mixture was stirred at 350 rpm under 85℃, then sulfuric acid was added to adjust pH to 5.0 -5.5 as pretreatment. The bottle was cooled to 55℃. The enzyme compositions were added directly into the soap stock after pretreatment step, to break the emulsion and hydrolyze the oil in the soap stock to generate FFA. The resulting mixtures were stirred at 55℃ for 16 hours for hydrolysis. After the reaction, the bottle with sample was heated up to 85℃ and the pH was then adjusted to 2.0 with sulfuric acid. The mixture was centrifuged for light phase and heavy phase separation. Free fatty acid was mainly recovered in the light phase after separation step, while heavy phase consisted of glycerol, lyso-gum, salt, and water. The yield of FFA was measured by AOCS Official Method Ca 5a-40.
The enzymatic soap stock splitting reactions were performed with individual Lipase and Phospholipase at the dose of 30 ug enzyme protein per gram soap stock (dry substance) , as well as blend consisting of 50%Lipase and 50%Phospholipase at the dose of 60 ug enzyme protein per gram soap stock (dry substance) . For comparison, treatment without enzyme was included. Free fatty acid from soap stock splitting at the specified doses was measured in below Table 1. Wherein, Lipase 1 was derived from Humicola lanuginose with amino acid sequence of SEQ ID NO: 1. Lipase B was derived from Humicola insolens with amino acid sequence of SEQ ID NO: 2. Lipase C was derived from Candida antarctica as disclosed in WO 88/02775, with amino acid sequence of SEQ ID NO: 3. Lipase D was derived from Rhizomucor miehei with amino acid of SEQ ID NO: 4. PLA-A was derived from Thermomyces lanuginosusas as disclosed in EP1131416, with amino acid of SEQ ID NO: 5. PLA-B was derived from Talaromyces leycettanus as disclosed in WO2018171552, with amino acid sequence of SEQ ID NO: 6.
Table 1. enzymatic soap stock splitting reaction
Seen from Table 1, Lipase 1 was more effective than the other lipases in free fatty acid production, and when mixing with PLA-B, the yield of free fatty acid was 186.46. The enzymatic splitting method of the present invention used the effective enzyme composition (such as lipase 1 with PLA) to successfully reduce the mean residence time of oil in any reactor from at least 24hrs to 16hrs and meanwhile achieved desirable acid value.
Example 2
In the example, the amount of free fatty acid (FFA) from soap stock splitting, after incubation with and without enzyme, was measured by lab-scale batch reaction. The feedstock for the reaction was soap stock, which was the by-product of typical chemical soybean oil refining process. The lab-scale batch reaction was generally described that 100 g soap stock was weighed and poured into a blue cap bottle, then stirred at 350 rpm at 85℃, then sulfuric acid was added to adjust pH to 5.0 -5.5 as pretreatment. The bottle was cooled to 55℃, the enzyme was added directly into the soap stock after pretreatment step, to break the emulsion and hydrolyze the oil in the soap stock to generate FFA. The stirring was continued at 55℃ for 16 hours for enzymatic reaction. After the reaction, the bottle with sample was heated up to 85℃and the pH was then adjusted to 2.0 with sulfuric acid. The mixture was centrifuged for light phase and heavy phase separation. Free fatty acid was mainly recovered in the light phase after separation step, while heavy phase consisted of glycerol, lyso-gum, salt, and water. The yield of FFA was measured by AOCS Official Method Ca 5a-40.
The enzymatic soap stock splitting reaction was performed with Lipase with the dose of 30 ug enzyme protein per gram soap stock (dry substance) , as well as blend consisting of 50%Lipase and 50%Phospholipase at the dose of 60 ug enzyme protein per gram soap stock (dry substance) . For comparison, treatment without enzyme was included. Free fatty acid from soap stock splitting at the specified doses was measured in below Table 2. Wherein, Lipase is LP 64 obtained as described in WO 2018/001959. LP-64 is also derived from Humicola lanuginose with at least 97%identity to amino acid of SEQ ID NO: 1.
Table 2. enzymatic soap stock splitting reaction
Seen from Table 2, LP 64 was more effective than the other lipases in free fatty acid production, and when mixing with PLA-A and PLA-B, the yield of free fatty acid was 169. Better than the other lipases (such as Lipase B and Lipase C) , either used alone or combined with PLA. And seen from Table 1 and table 2, lipase derived from Humicola lanuginose shown better performance in enzymatic splitting of soap stock than the other lipases. And when combing Humicola lanuginose with PLA, the mean residence time of oil in any reactor was successfully reduced from at least 24hrs to 16hrs and meanwhile achieved desirable acid value.

Claims

A process for producing free fatty acids comprising:

(a) Providing fatty acid feedstock,

(b) contacting said fatty acid feedstock with enzyme composition comprising lipase and phospholipases to produce a reaction mixture,

(c) hydrolysis of the reaction mixture of step b within 16 hours,

(d) separating the light oil phase from the heavy phase of the reaction mixture,

(e) recirculating the heavy phase,

(f) recovering free fatty acid from the light phase resulting from step d, and

(g) recirculating unreacted non-volatile glycerides into the reactor of step c.
The process of claim 1, wherein said enzyme compositions are dosed in a total amount corresponding to 0.1-400 mg enzyme protein per kg of oil.
The process according to any of the preceding claims, wherein the lipase is derived from Mucor miehei, Candida cylindracea, Rhizopus arrhizus, Humicola lanuginosa, and/or Pseudomonas sp, ATCC 21808.
The process according to any of the preceding claims, wherein the lipase is derived from Humicola lanuginose, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 1.
The process according to any of the preceding claims, wherein the phospholipases is phospholipase A.
The process according to any of the preceding claims, wherein the phospholipase is derived from Talaromyces leycettanus, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 6.
The process according to any of the preceding claims, wherein the phospholipase is derived from Thermomyces lanuginosusas, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 5.
The process according to any of the preceding claims, wherein the free fatty acid content in the mixture after any number of preceding reaction steps is in the range 20-90%, preferably 30-80%and most preferably from 40 to 70%.
The process according to any of the preceding claims, wherein the glycerides in the fatty acid feedstock is a mixture is monoglyceride, diglyceride and/or triglyceride.
The process according to any of the preceding claims, wherein the fatty acid feedstock is a naturally derived oil or fat, or a mixture thereof.
The process according to any of the preceding claims, wherein the fatty acid feedstock is any triglyceride stemming from future sources such as fat-producing genetically manipulated microorganisms.
The process according to any of the preceding claims, wherein the fatty acid feedstock is selected from the group consisting of Moringa oil, acai oil, almond oil, babassu oil, blackcurrant seed oil, borage seed oil, canola oil, cashew oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil, flax seed oil, grape seed oil, hazelnut oil, hempseed oil, jatropha oil, jojoba oil, linseed oil, macadamia nut oil, mango kernel oil, meadowfoam oil, mustard oil, neat's foot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil/ground nut oil, pecan oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil, tsubaki oil, walnut oil or animal derived oil such as tallow, chicken fat, poultry fat fish oil, or fat obtained as by products from processing industries.
An enzyme composition for enzymatically splitting fatty acid feedstock to produce free fatty acids, wherein, said enzyme composition comprising lipases and phospholipases.
The enzyme composition of claim 13, wherein said enzyme compositions are dosed in a total amount corresponding to 0.1-400 mg enzyme protein per kg of oil.
The enzyme composition according to any of claims 13-14, wherein the lipase is derived from Mucor miehei, Candida cylindracea, Rhizopus arrhizus, Humicola lanuginosa, and/or Pseudomonas sp, ATCC 21808.
The enzyme composition according to any of claims 13-15, wherein the lipase is derived from Humicola lanuginose, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 1.
The enzyme composition according to any of claims 13-16, wherein the phospholipases is phospholipase A.
The enzyme composition according to any of claims 13-17, wherein the phospholipase is derived from Talaromyces leycettanus, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 6.
The enzyme composition according to any of claims 13-18, wherein the phospholipase is derived from Thermomyces lanuginosusas, having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%98%, 99%or 100%identity with SEQ ID NO: 5.