AU2018344497A1 - Enzyme assisted crude palm oil extraction - Google Patents

Abstract

The present invention relates to a process for extraction or separation of crude palm oil (CPO), comprising contacting a substrate comprising palm oil with an enzyme composition, extracting or separating the crude palm oil (CPO) and wherein the water content of the substrate comprising palm oil is above 40% w/w.

Description

Enzyme assisted crude palm oil extraction

Reference to a Sequence Listing

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

Field of the Invention

The present invention relates to a process for extraction or separation of crude palm oil and the crude palm oil obtainable by the process.

Description of the Related Art

Palm oil is an edible vegetable oil which is obtained from the mesocarp of palm fruits. Palm fruits orfruitlets grow in large bunches. The palm fruitlets are stripped from the fruit bunches after being sterilized. The high temperature causes the enzymes naturally occurring in the palm fruits to denature and facilitates stripping of the fruits from the bunch stalks. The palm fruitlets are discharged into vessels commonly referred to as digesters, whereby a digested mash of palm fruits is produced under controlled temperature. The digested mash is then pressed, e.g. by using a screw press for subsequent recovery of palm oil. The crude palm oil may be subjected to screening, e.g. to remove coarse fibers, and then to a clarification process to separate oil from water, cell debris and any remaining fibrous material.

Palm fruit mesocarp contains large amounts of oil present as oil droplets within the mesocarp cells. Generally, the oil extraction rate (OER), which is a measure of the amount of extracted oil relative to the weight of the palm fruits is within the range of 20-24%, depending e.g. on fruit quality, and is subject to seasonal variation. In general, the palm oil milling process has been carefully optimized at each mill in order to minimize oil losses to the extent possible but there is still a strong incentive to improve the OER.

International patent application WO 2012/011130 (Advanced enzyme technologies Ltd.) concerns an enzyme composition (with exocellulolytic, pectinolytic, mammanolytic and glucanoloytic activity) used in a process for palm oil extraction. WO2016/097266 (Dupont) concerns a method for improving palm oil yields by mixing an enzyme composition having cellulase activity with palm fruit or palm fruit liquid having at the most 40% water.

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Summary of the Invention

The present invention relates to a process for extraction or separation of crude palm oil (CPO), comprising the steps of:

i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil (CPO) wherein the water content of the substrate comprising palm oil is above 40% w/w. The invention further relates to a crude palm oil obtainable by the above process.

Definitions

Amylolytic enzyme or Amylase: For the purpose of the present invention, “Amylolytic enzyme” or “amylase” refers to an enzyme that catalyses the hydrolysis of starch into sugars. Amylase activity may be determined as described by Joseph D. Teller, Measurement of amylase acitivity, J. Biol. Chem. 1950, 185:701-704.

Beta-glucanase: Beta-glucanase (E.G. 3.2.1.4). The beta-glucanase may be of microbial origin, such as derivable from a strain of a bacteria (e.g. Bacillus) or from a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium). A beta-glucanases to be used in the processes of the invention may be an endo- glucanase, such as an endo-1 ,4-beta-glucanase. Beta-glucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). Beta-glucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure andAppl. Chem. 59: 257-268, at pH 5, 40°C.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1—>4)-xylooligosaccharides to remove successive D-xylose residues from non-reducing termini. Beta-xylosidase activity can be determined using 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20 at pH 5, 40°C. One unit of beta-xylosidase is defined as 1.0 pmole of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-Dxyloside in 100 mM sodium citrate containing 0.01% TWEEN® 20.

Cellobiohydrolase: The term “cellobiohydrolase” means a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1,4-beta-Dglucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,

1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity can be determined

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PCT/EP2018/077095 according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Endoglucanase: The term “endoglucanase” means a 4-(1,3;1,4)-beta-D-glucan 4glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452481). Endoglucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.

Pectinase: The term pectinase is defined as any enzyme that degrades pectic substances. Pectic substances include homogalacturonans, xylogalacturonans, and rhamnogalacturonans as well as derivatives thereof. Pectinase treatment may be achieved by one or more pectinases, such as two or more pectinases of the same type (e.g., two different pectin methylesterases) or of different types (e.g., a pectin methylesterase and an arabinanase). The pectinase may, for example, be selected from the group consisting of arabinanase (catalyses the degradation of arabinan sidechains of pectic substances), arabinofuranosidase (removes arabinosyl substituents from arabinans and arabinogalactans), galactanase (catalyses the degradation of arabinogalactan and galactan sidechains of pectic substances), pectate lyase (cleaves glycosidic bonds in polygalacturonic acid by beta-elimination), pectin acetylesterase (catalyses the removal of acetyl groups from acetylated pectin), pectin lyase (cleaves the glycosidic bonds of highly methylated pectins by beta-elimination), pectin methylesterase (catalyses the removal of methanol from pectin, resulting in the formation of pectic acid, polygalacturonic acid), polygalacturonase (hydrolyses the glycosidic linkages in the polygalacturonic acid chain), rhamnogalacturonan acetylesterase (catalyses the removal of acetyl groups from acetylated rhamnogalacturonans), and rhamnogalacturonase and rhamnogalacturonan lyase (degrade rhamnogalacturonans).

Pectin methylesterase (PME), EC 3.1.1.11, is an enzyme that acts mainly in the hydrolysis of methyl ester groups in pectin chains to form carboxylate groups, releasing methanol and H3O+ (Jayani, R.S.; Saxena, S.; Gupta, R. Microbial pectinolytic enzymes: a review. Process Biochemistry, London, v.40, p.2931-2944, 2005). Pectin methyl esterase activity may be determined e.g. as described by Lemke Gonzalez et al., Pectin methylesterase activity

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PCT/EP2018/077095 determined by different methods and thermal inactivation of exogenous pme in mango juice. Cienc. agrotec. vol.35 no.5 Lavras Sept./Oct. 2011

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Xyloglucanase: Xyloglucanses are capable of catalyzing the solubilization of xyloglucan to xyloglucan oligosaccharides. Some xyloglucanases only exhibit xyloglucanase activity, whereas others exhibit both xyloglucanase and cellulase activity. Xyloglucanses may be classified in EC 3.2.1.4 or EC. 3.2.1.151. Enzymes with xyloglucanase activity are for example described in Vincken et al. (1997) Carbohydrate Research 298(4):299-310, wherein three different endoglucanases Endol, EndoV and EndoVI from Trichoderma viride (similar to T. reesei) are characterized. Endol, EndoV and EndoVI belongs to family 5,7 and 12 of glycosyl hydrolases, respectively, see Henrissat, B. (1991 ) Biochem. J. 280: 309-316, and Henrissat, B. and Bairoch,

A. (1993) Biochem. J. 293: 781-788.

Glucoside hydrolase families GH5, GH8, GH10, GH11, GH12, GH16, GH30, GH42, GH44, GH55, GH74: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase (GH) famiies 5, 8, 10, 11, 12, 16, 30, 42, 44, 55 or 74, respectively, in the database of Carbohydrate-Active enZYmes (CAZymes) available at http://www.cazy.org/. (Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P. M.; Henrissat,

B. (21 November 2013). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Research. 42 (D1): D490-D495; Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (January 2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37 (Database issue): D233-8).

Cellulosic material: The term “cellulosic material” means any material containing cellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses

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PCT/EP2018/077095 usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp. 23-40, SpringerVerlag, New York). It is understood herein that the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix. In one aspect, the cellulosic material is any biomass material. In another aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.

Crude oil: The term “crude oil” (also called a non-degummed oil) refers to a pressed or extracted oil or a mixture thereof. In the present context, it is to be understood that the oil is palm oil, in particular un-refined palm oil. The crude palm oil is also designated CPO.

Digestion: The term “digestion” refers to a process where the substrate comprising palm oil is kept at a temperature in the range of 65-85°C for disintegrating the substrate and releasing palm oil from the mesocarp. The digestion can be carried out in a digestion tank and/or a precooker tank equipped with baffles. During the digestion, the substrate comprising palm oil e.g. the palm fruitlets are disintegrated and oil released from the mesocarp.

Oil extraction rate (OER): For the purpose of the present invention, “Oil extraction rate (OER)” may be defined as by Chang et al., oil palm Industry economic journal, volume 3, 2003[9]. Chang et al. defines the Oil extract rate as ratio of oil recovered and Fresh fruit branch (FFB) times 100. According to this definition, the mathematical formula is:

OER = (weight of oil recovered/weight of FFB processed) x 100

Palm oil mill effluent (POME): Palm oil mill effluent (POME) is the waste water discharged e.g. from the sterilization process or crude oil clarification process.

Palm press liquid: The term “palm press liquid” refers to the liquid discharged from the pressing of the substrate comprising palm oil. The palm press liquid may comprise palm oil, water and impurities. Palm press liquid is not a crude palm oil and water has not been separated from the palm press liquid.

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Detailed Description of the Invention

The present invention concerns a process for enzyme assisted extraction of crude palm oil from a substrate comprising palm oil. The substrate comprising palm oil can be selected from the group consisting of palm fruitlets, pressed palm fruit liquid, mashed or partly mashed palm fruitlets. The inventors have found that by combining the use of enzymes and controlling the water content of the substrate comprising palm oil, the oil extraction rate (OER) can be increased. Reference is made to example 1, where the volume of oil extracted from palm fruits is higher when enzymes are used at a water content of 47% w/w compared to volume of oil obtained at a water content of 39.4%.

The invention concerns a process for extraction or separation of crude palm oil (CPO), comprising the steps of:

i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil (CPO) wherein the water content of the substrate comprising palm oil is above 40% w/w. In one embodiment the water content of the substrate comprising palm oil is in the range of40 to 56% w/w, such as in the range of 41 to 55%, in the range of 41 to 50%, in the range of 41 to 49%, in the range of 41 to 48%, in the range of 41 to 47%, in the range of 42 to 47%, in the range of 43 to 47%, in the range of 44 to 47%, in the range of 45 to 47%, in the range of 46 to 47%.

In other embodiments, the water content of the substrate comprising palm oil is above 40%, such as in the range from 40 to 70%, such as in the range of 40 to 65% w/w, such as in the range 40 to 63 % w/w, such as in the range of 40 to 57%, such as in the range of 40 to 56 % w/w; such as in the range of 40 to 50 % w/w; such as in the range of 40 to 47 % w/w; or for example in the range of 41 to 70%, such as in the range of 41 to 65% w/w, such as in the range 41 to 63 % w/w, such as in the range of 41 to 57%, such as in the range of 41 to 56 % w/w; such as in the range of 41 to 50 % w/w; such as in the range of 41 to 47 % w/w; or for example in the range of to 70%, such as in the range of 42 to 65% w/w, such as in the range 42 to 63 % w/w, such as in the range of 42 to 57%, such as in the range of 42 to 56 % w/w; such as in the range of 42 to 50 % w/w; such as in the range of 42 to 47 % w/w; or for example in the range of 43 to 70%, such as in the range of 43 to 65% w/w, such as in the range 43 to 63 % w/w, such as in the range of to 57%, such as in the range of 43 to 56 % w/w; such as in the range of 43 to 50 % w/w; such as in the range of 43 to 47 % w/w; or for example in the range of 44 to 70%, such as in the range of 44 to 65% w/w, such as in the range 44 to 63 % w/w, such as in the range of 44 to 57%, such as in the range of 44 to 56 % w/w; such as in the range of 44 to 50 % w/w; such as in the range of 44 to 47 % w/w; or for example in the range of 45 to 70%, such as in the range of 45 to 65% w/w, such as in the range 45 to 63 % w/w, such as in the range of 45 to 57%, such as in the

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PCT/EP2018/077095 range of 45 to 56 % w/w; such as in the range of 45 to 50 % w/w; such as in the range of 45 to 47 % w/w.

In one embodiment of the invention, the substrate comprising palm oil is palm fruitlets, which comprise oil in the mesocarp of the fruit. The palm fruitlets are contacted with the enzyme composition. In one embodiment the substrate is palm fruitlets, which are mashed or partly mashed and contacted with the enzyme composition. This increases availability of mesocarp cells and thereby enhances enzyme activity on the mesocarp cells. In one embodiment the substrate comprising palm oil is crude palm oil which is contacted with the enzyme composition. In the various aspects and embodiments of the invention, the substrate, which comprises palm oil, may be a substrate which also comprises fiber, in particular fiber from the mescocarp of palm fruitlets.

In one embodiment of the invention, the substrate comprising palm oil is sterilized before being contacted with the enzyme composition. Palm fruits grow in large bunches and need to be stripped from the bunch stalks before being contacted with the enzyme composition. Steam sterilization of the fresh fruit bunches facilitates fruits being stripped from bunches to give the palm fruitlet. The sterilization step has several advantages one being that it softens the fruit mesocarp for subsequent digestion. A further advantage is that the quality of the final palm oil product is better if the palm fruits are stripped from the bunch stalks.

The sterilization can be a batch sterilization or a continuous sterilization. The sterilization process can be carried out at a temperature of 100°C-150°C. In one embodiment of the invention, the pressure is reduced during the sterilization procedure.

After the sterilization the palm fruitlets are stripped from the bunch stalks. Stripping or threshing can be carried out in a mechanized system having a rotating drum or fixed drum equipped with rotary beater bars which detach the fruit from the bunch and leave the spikelets on the stem. The stripped palm fruitlets can be contacted with the enzyme composition according to the invention.

In one embodiment of the invention, the substrate comprising palm oil is subjected to digestion before extracting the crude palm oil. The stripped palm fruitlets can be transported into a digester by one or more transportation means, e.g. a conveyor belt. In the digester, the fruitlets are further heated in order to loosen the pericarp. The digester is typically a steam heated vessel, which has rotating shafts to which are attached stirring arms or it is equipped with baffles. The fruitlets are rotated, causing the loosening of the pericarps from the nuts and degradation of the mesocarp. The digester is a continuous process where the digester is kept full and as the digested fruit is drawn out, freshly stripped fruits are brought in.

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In one embodiment of the invention, the first part of the digestion is carried out in a precooker. The substrate may be kept at a temperature within the range of 65-85°C for some time and then be transferred to the digester tank.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature of above 50°C and the crude palm oil is then extracted.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature within the range of 55-90°C, such as a temperature within the range of 55-85°C, 55-80°C, 60-90°C, 60-85°C, 60-80°C, 66-90°C, 67-90°C, 68-90°C, 69-90°C, 70-90°C, 66-85°C, 66-80°C, 67-80°C, 66-79°C, 66-78°C, 66-77°C, 66-76°C, 66-75°C,

66- 74°C, 66-73°C, 66-72°C, 66-71 °C, 67-80°C, 67-79°C, 67-78°C, 67-77°C, 67-76°C, 67-75°C,

67- 74°C, 67-73°C, 67-72°C, 67-71 °C, 68-79°C, 68-78°C, 68-77°C, 68-76°C, 68-75°C, 68-74°C,

68- 73°C, 68-72°C, 68-71 °C, 69-79°C, 69-78°C, 69-77°C, 69-76°C, 69-75°C, 69-74°C, 69-73°C,

69- 72°C, 69-71 °C, 70-90°C, 70-89°C, 70-88°C, 70-87°C, 70-86°C, or70-85°C.

The substrate comprising palm oil is contacted with the enzyme composition for a period of 5-120 minutes, such as a period of 20-120 minutes, 25-120 minutes 5-60 minutes,20-60 minutes, 25-60 minutes, 30-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes,30-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes,20-30 minutes, 25-28 minutes, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes,20-28 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature of above 50°C, such as a temperature within the range of 55-90°C for a period of 5-120 minutes and the crude palm oil is then extracted.

The digested substrate is passed into a press, e.g. a screw press, from which the palm press liquid is discharged. The palm press liquid is a mixture of oil, water, press cake/fibre and nuts. The palm press liquid is delivered from the press to a clarification tank forfurther processing.

In one embodiment of the invention, the digestion comprises retaining the substrate comprising palm oil at temperatures above 65°C and up to 85°C for 10-30 minutes, such as for 10-28 minutes, 15-28 minutes, 12-30 minutes,12-28 minutes or 12-25 minutes.

It is to be understood that the enzyme composition used according to the invention may be applied at any point in the crude palm oil extraction process, after the palm fruit bunches have been sterilized and until the oil is separated from water, the water from cell debris and fibrous material, which is also present in the liquid which is obtained by pressing of the mashed palm fruitlets. In particular, the substrate may be selected from the group consisting of palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.

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It will be well within the capacity of the skilled person to optimize the dosing of the enzyme composition in view of other process parameters. In particular embodiments of the invention, the enzyme composition is dosed in amounts corresponding to 10-500 mg enzyme protein/kg FFB (fresh fruit bunch) comprising palm oil, such as 10-450 mg enzyme protein/kg FFB comprising palm oil, 10-400 mg enzyme protein/kg FFB comprising palm oil, 10-350 mg enzyme protein/kg FFB comprising palm oil, 10-300 mg enzyme protein/kg FFB comprising palm oil, 10-250 mg enzyme protein/kg FFB comprising palm oil, 10-200 mg enzyme protein/kg FFB comprising palm oil, 10-150 mg enzyme protein/kg FFB comprising palm oil, 10-100 mg enzyme protein/kg FFB comprising palm oil, 10-75 mg enzyme protein/kg FFB comprising palm oil, 1050 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB comprising palm oil, 100500 mg enzyme protein/kg FFB comprising palm oil, 150-500 mg enzyme protein/kg FFB comprising palm oil, 200-500 mg enzyme protein/kg FFB comprising palm oil, 250-500 mg enzyme protein/kg FFB comprising palm oil, 300-500 mg enzyme protein/kg FFB comprising palm oil, 350-500 mg enzyme protein/kg FFB comprising palm oil, 400-500 mg enzyme protein/kg FFB comprising palm oil, 30-400 mg enzyme protein/kg FFB comprising palm oil, 30-300 mg enzyme protein/kg FFB comprising palm oil, 30-200 mg enzyme protein/kg FFB comprising palm oil, 30-150 mg enzyme protein/kg FFB comprising palm oil, 30-100 mg enzyme protein/kg FFB comprising palm oil, 30-75 mg enzyme protein/kg FFB comprising palm oil, or such as 30-50 mg enzyme protein/kg FFB comprising palm oil..

According to some embodiments of the invention, the enzyme(s) are dosed at amounts corresponding to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm, 40-1000 ppm, 50-1000 ppm, 100-1000 ppm, 200-1000 ppm, 100-750 ppm, such as 200-750 ppm, 250-750 ppm, 350750 ppm or 350-500 ppm relative to the amount of substrate comprising palm oil.

In one embodiment of the invention, the enzyme composition comprises cellobiohydrolase wherein the amount of the cellobiohydrolase corresponds to 15-30% (w/w), such as to 21.7-26.0% (w/w), of the total amount of enzyme protein in said enzyme composition. In one embodiment of the invention, the cellobiohydrolase is selected from a group consisting of a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII) and combinations thereof. In one embodiment of the invention, the substrate comprising palm oil is contacted with said cellobiohydrolase, such as said cellobiohydrolase I, in amounts corresponding to 10-45 mg enzyme protein/kg substrate comprising palm oil, such as 12.5-15.0 mg enzyme protein/kg substrate comprising palm oil.

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In one embodiment of the invention, the enzyme composition comprises endoglucanase, wherein the amount of endoglucanase corresponds to 0.5-5% (w/w), such as to 1.52-2.36% (w/w) of the total amount of enzyme protein in said enzyme composition. In one embodiment of the invention the endoglucanase is selected from a group consisting of an endoglucanase I (EGI), an endoglucanase II (EGII), an endoglucanase III (EGIII), an endoglucanase IV (EGIV), an endoglucanase V (EGV), an endoglucanase (EGVI) and combinations thereof. In one embodiment of the invention, the substrate comprising palm oil is contacted with said endoglucanase in amounts corresponding to 0.5-5 mg enzyme protein/kg substrate comprising palm oil, such as 0.9-1.4 mg enzyme protein/kg substrate comprising palm oil.

In one embodiment of the invention, the enzyme composition comprises beta-glucanase, wherein the amount of beta-glucanase corresponds to 0.01-0.1% (w/w), such as to 0.02-0.06% (w/w), of the total amount of enzyme protein in said enzyme composition. In one embodiment of the invention, the beta-glucanase is selected from a group consisting of a GH5 beta-glucanase, a GH42 beta-glucanase, a GH12 beta-glucanase, a GH55 beta-glucanase and a GH16 betaglucanase and combinations thereof. In one embodiment of the invention, the substrate comprising palm oil is contacted with said beta-glucanase in amounts corresponding to 0.0050.075 mg enzyme protein/kg substrate comprising palm oil, such as 0.015 - 0.045 mg enzyme protein/kg substrate comprising palm oil.

In one embodiment of the invention, the ratio of the amount of cellobiohydrolase expressed as mg enzyme protein to the amount of beta-glucanase expressed as mg enzyme protein is within the range of 1:0.001 to 1: 0.0008.

In one embodiment of the invention, the enzyme composition comprises xylanase, wherein the amount of xylanase corresponds to 30-45% (w/w), such as to 37.0-41.0% (w/w), of the total amount of enzyme protein in said enzyme composition. In one embodiment of the invention, the substrate comprising palm oil is contacted with said xylanase in amounts corresponding to 15.0-30.0 mg enzyme protein/kg substrate comprising palm oil, such as 21.524.0 mg enzyme protein/kg substrate comprising palm oil. In one embodiment of the invention the xylanase is selected from a group consisting of a GH5 xylanase, a GH8 xylanase, a GH10 xylanase, a GH11 xylanase, a GH30 xylanase and combinations thereof.

In one embodiment of the invention, the enzyme composition comprises beta-xylosidase, wherein the amount of beta-xylosidase corresponds to 15-30% (w/w), such as to 21.8-26.2% (w/w), of the total amount of enzyme protein in said enzyme composition. In one embodiment of the invention, the substrate comprising palm oil is contacted with said beta-xylosidase in amounts corresponding to 10.0-20.0 mg enzyme protein/kg substrate comprising palm oil, such as 12.515.5 mg enzyme protein/kg substrate comprising palm oil.

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In one embodiment of the invention, the enzyme composition further comprises one or more enzymes selected from the group consisting of: a xyloglucanase, a pectinase and an amylolytic enzyme. In one embodiment of the invention, the xyloglucanase is selected from a group consisting of GH5, GH44 or GH74 and combination thereof. In one embodiment of the invention, the amylolytic enzyme is selected from a group consisting of a gluco-amylase, an alpha-amylase or a beta-amylase and combination thereof.

In one embodiment of the invention the ratio of Cellobiohydrolase : Xylanase : Betaxylosidase : Endoglucanase : Xyloglucanase : Amylolytic is 1 : 1.641 ±0,8 : 1.01510,5 : 0.08410.04 : 0.054± 0.03 : 0.0004±.0.0002, such as 1 : 1.64110,4 : 1.01510,25 : 0.08410.02 : 0.0541 0.015 :0.00041.0.0001.

In one embodiment of the invention, the enzyme composition comprises at least one xyalanse, such as at least one GH10 xylanase.

In one embodiment, the at least one GH10 xylanase is selected from the group consisting of:

a) the mature polypeptide ofSEQ ID NO: 1, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1,

b) the mature polypeptide of SEQ ID NO: 2, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 2, and

c) the mature polypeptide of SEQ ID NO: 4 or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 4.

In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1.

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In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 2.

In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 4.

In one embodiment of the invention, the enzyme composition further comprises one or more enzymes obtained from Trichoderma reesei. The enzymes are substantially inactive at a temperature of about 70°C. The enzymes can be cellulases. In one embodiment of the invention, the enzymes are GH62 arabinofuranosidases.

In one embodiment of the invention, the enzyme composition further comprises a GH62 arabinofuranosidase.

Preferred embodiments of the aspect of the invention relating to the GH62 polypeptide having arabinofuranosidase activity are disclosed herein below. Additional details of preferred GH62 polypeptides having arabinofuranosidase activity are found in PCT/CN2015/071015 filed 19 January 2015.

In one embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 3 of at least 80%, e.g., 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%.

In one aspect the invention concerns a crude palm oil, which is obtainable by the process according to any of the process of the invention.

Examples

Example 1

Palm press liquid obtained by pressing the digested palm fruitlets is a rather viscous liquid. It is therefore normal procedure to dilute the pressed palm liquid with water before going to the clarifier in order to reduce viscosity. The purpose of this study was to examine the effect on viscosity of treatment with enzyme compositions as well as water dilution. The effect on solubilisation of insoluble dry matter and amount of bound oil in the sediment from pressed palm liquid was also tested.

Viscosity reduction of palm press liquid after treatment with enzyme composition and water dilution.

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Palm press liquid with 24% moisture was increased to 27% and 30% moisture respectively as indicated in the table. To the palm press liquid were added at 750 ppm of enzyme composition and incubated at 60°C for 5 hours under continuous agitation. The experimental procedure is described below. Viscosity was measured at the end of 5 hours as mentioned below.

Material

The substrate comprising palm oil was palm press liquid obtained from a conventional oil mill. The palm press liquid was obtained by sterilizing the palm fruitlets, treshing, passing to digester, digested at 70-85°C for about 12-15 minutes and then pressed in a screw press. The palm press liquid obtained from the screw press was used directly in this experiment.

Enzyme compositions:

Laminex® (DuPont Industrial Biosciences), 10166 CMC- DNS/g, 185 MVU/g

Palmora® OER 1.0 (Novozymes A/S). Palmora OER 1.0 is a commercially available product comprising xylanase (Aspergillus fumigatus GH10 xylanase, SEQ ID NO. 4).

Enzyme composition A: Enzyme composition comprises a GH10 Xylanase (SEQ ID NO. 1) and a GH62 arabinofuranosidase SEQ ID NO. 3).

Experimental:

gram Pressed Palm Liquid with and without enzyme composition was incubated with magnetic stirring at 60 °C for 4 hours in an Alum cup fitted for Rapid Visco Analyser. Description of the experimental setup can be found in table 1.

After incubation the viscosity of the sample was immediately measured using a Rapid Visco Analyzer using the following program: Agitation 400 rpm, Time: 1-120 seconds: temperature setting 60°C, Time 121-300 seconds: Increase temperature from 60°C to 80°C, Time 301-480 seconds: keep 80°C. The viscosity at 60°C was calculated as the average of measurements between 61 and 120 seconds. Viscosity at 80°C was calculated as average measurements between 301 and 360 seconds. The sample was then transferred to a 50-mL centrifuge tube and centrifuged at 4180 rpm for 10 minutes at 60 °C. The upper oil layer and water was removed.

Palm Pressed liquid, gram	30	30	27	27	24	24
Water, ml	0	0	3	3	6	6
Water added %	0	0	20	20	30	30
Water in sample %	0	0	10	10	20	20
Enzyme dosage, ppm	0	750	0	750	0	750
Reaction time, hours	1	1	1	1	1	1
Reaction temperature, °C	60	60	60	60	60	60

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			Viscosity in cP
SI no	Sample details	Enzyme dosage ppm	60°C	80 °C
1	Control 0%water		108	77
2	Palmora® OER 1.0	750	78	60
4	Enzyme composition A	750	83	60
3	Laminex	750	73	59
5	Control 10%water		85	68
6	Palmora® OER 1.0	750	82	65
7	Enzyme composition A	750	90	64
8	Laminex	750	79	60
9	Control 20%water		100	75
10	Palmora® OER 1.0	750	88	68
11	Enzyme composition A	750	93	72
12	Laminex	750	90	67

Example 2

Effect of moisture on oil separation and viscosity reduction

Palm press liquid obtained by pressing the digested palm fruitlets is a rather viscous liquid. It is therefore normal procedure to dilute the pressed palm liquid with water before going to the clarifier to reduce viscosity. The purpose of this study was to examine the effect on viscosity of enzyme treatment with Palmora® OER 1.0 and Laminex as well as water dilution. The impact on oil separation with the mentioned enzymes and water dilution was also investigated.

In this experiment, three different palm press liquids were tested to study variations in raw material with different water content.

A palm press liquid with 24% moisture was increased up to 62% moisture as indicated in the table. Enzyme composition was added to the palm press liquid in a concentration of 750ppm each and incubated at 60°C for 1 hour under continuous agitation on a magnetic stirrer. Viscosity was measured at the end of 1 hour as described below.

Material

The substrate comprising palm oil was palm press liquid obtained from a conventional oil mill.

The palm press liquid was obtained by sterilizing the palm fruitlets, treshing, passing to digester,

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Enzyme compositions:

Laminex® (DuPont Industrial Biosciences), 10166 CMC- DNS/g, 185 MVU/g

Palmora® OER 1.0 (Novozymes A/S) Palmora OER 1.0 is a commercially available product comprising xylanase (Aspergillus fumigatus GH10 xylanase, SEQ ID NO 4).

Enzyme composition A: Enzyme composition comprises a GH10 Xylanase (SEQ ID NO. 1) and a GH62 arabinofuranosidase SEQ ID NO. 3).

Experimental:

gram Pressed Palm Liquid or 30 g diluted Pressed Palm Liquid and enzyme was incubated with magnetic stirring at 60 °C for 1 hour in an Alum cup fitted for Rapid Visco Analyzer.

After incubation the viscosity of the sample was immediately measured using a Rapid Visco Analyzer using the following program: Agitation 500 rpm, temperature setting 60°C for 5 minutes, increase temperature to 70°C, keep 70°C for 4 minutes, increase temperature to 80°C and keep 80°C for 4 minutes, increase the temperature to 90°C and keep 90°C for 4 minutes. Cool down to 50°C for 2 minutes.

The viscosity was calculated as the average of measurements for 1 minute at each temperature.

The sample was then transferred to a 50-mL centrifuge tube and heated to 70 °C for 10 minutes, and then turned upside down 10 times. The oil separation of the sample was followed visually for 10 minutes. The oil separation for the sample was measured as the volume % of upper oil layer after 10 minutes.

Palm Pressed liquid	gram	30	30	24	24	21	21	18	18	15	15
Water	ml	0	0	6	6	9	9	12	12	15	15
Water added	%	0	0	20	20	30	30	40	40	50	50
Water in sample	%	24.3	24.3	39.4	39.4	47.0	47.0	54.6	54.6	62.2	62.2
Reaction time	hour	4	4	4	4	4	4	4	4	4	4
Reaction temperature	°C	60	60	60	60	60	60	60	60	60	60

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Results are shown in below table.

% Moisture in samples	Sample details	Enzyme dosage PPm	Volume of oil (%)
24,3 %	Control	0	25
Palmora® OER 1.0	750	28
Enzyme composition A	750	33
Laminex®	750	30
39,4%	Control	0	25
Palmora® OER 1.0	750	32
Enzyme composition A	750	38
Laminex®	750	33
47%	Control	0	25
Palmora® OER 1.0	750	33
Enzyme composition A	750	42
Laminex®	750	37
56,4%	Control	0	17
Palmora® OER 1.0	750	20
Enzyme composition A	750	23
Laminex®	750	20
63%	Control	0	8
Palmora® OER 1.0	750	15
Enzyme composition A	750	23
Laminex®	750	12

The oil separation of the control and enzyme treated samples was followed visually over

10 minutes. Enzyme treatment had strong improvement on oil separation, it was however observed that water dilution with the enzyme treatment did not negatively impact oil separation.

Increased water addition to the control samples did not lead to enhanced oil separation but led to a decrease in oil separation.

Claims (17)

1. Claims

   1. A process for extraction or separation of crude palm oil (CPO), comprising the steps of:

   i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil (CPO) wherein the water content of the substrate comprising palm oil is above 40% w/w.
2. 2. A process according to claim 1, wherein the water content of the substrate is in the range of 40 to 56% w/w, such as in the range of 41 to 55%, in the range of 41 to 50%, in the range of 41 to 49%, in the range of 41 to 48%, in the range of 41 to 47%, in the range of 42 to 47%, in the range of 43 to 47%, in the range of 44 to 47%, in the range of 45 to 47%, in the range of 46 to 47%.
3. 3. The process according to any of the preceding claims, wherein the enzyme composition comprises endoglucanase, wherein the amount of endoglucanase corresponds to 0.5-5% (w/w), such as to 1.52-2.36% (w/w) of the total amount of enzyme protein in said enzyme composition.
4. 4. The process according to any of the preceding claims, wherein the enzyme composition comprises beta-glucanase, wherein the amount of beta-glucanase corresponds to 0.01 0.1% (w/w), such as to 0.02-0.06% (w/w), of the total amount of enzyme protein in said enzyme composition.
5. 5. The process according to any of the preceding claims, wherein the enzyme composition comprises xylanase, wherein the amount of xylanase corresponds to 30-45% (w/w), such as to 37.0-41.0% (w/w), of the total amount of enzyme protein in said enzyme composition.
6. 6. The process according to any of the preceding claims, wherein the enzyme composition comprises beta-xylosidase, wherein the amount of beta-xylosidase corresponds to 1530% (w/w), such as to 21.8-26.2% (w/w), of the total amount of enzyme protein in said enzyme composition.
7. 7. The process according to any of the preceding claims, wherein the ratio of the amount of cellobiohydrolase expressed as mg enzyme protein to the amount of beta-glucanase expressed as mg enzyme protein is within the range of 1:0.001 to 1: 0.0008.
8. 8. The process according to any of the preceding claims, wherein the enzyme composition comprises at least one xylanase, such as at least one GH10 xylanase.
9. 9. The process according to claim 8 wherein the at least one xyalanse is selected from the group consisting of:

   a) the mature polypeptide of SEQ ID NO: 1, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at

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   PCT/EP2018/077095 least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1,

   b) the mature polypeptide of SEQ ID NO: 2, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 2, and

   c) the mature polypeptide of SEQ ID NO: 4 or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 4.
10. 10. A process according to any of the preceding claims, wherein the substrate comprising palm oil is selected from the group consisting of palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.
11. 11. The process according to any of the preceding claims, wherein the substrate comprising palm oil is subjected to sterilization before being contacted with the enzyme composition.
12. 12. The process according to any of the preceding claims, wherein the process comprises contacting the substrate to the enzyme composition at a temperature of above 50°C before extracting the crude palm oil (CPO).
13. 13. The process according to any of the preceding claims, wherein the substrate contacted to the enzyme composition is subjected to digestion, before extracting the crude palm oil (CPO).
14. 14. The process according to claim 12, wherein the temperature is within the range of 5590°C, 55-85°C, 60-90°C, 60-85°C, 60-80°C, 66-90°C, 67-90°C, 68-90°C, 69-90°C, 7090°C, 66-85°C, 66-80°C, 67-80°C, 66-79°C, 66-78°C, 66-77°C, 66-76°C, 66-75°C, 66-

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    74°C, 66-73°C, 66-72°C, 66-7ΓΟ, 67-80°C, 67-79°C, 67-78°C, 67-77°C, 67-76°C, 6775°C, 67-74°C, 67-73°C, 67-72°C, 67-7ΓΟ, 68-79°C, 68-78°C, 68-77°C, 68-76°C, 6875°C, 68-74°C, 68-73°C, 68-72°C, 68-7ΓΟ, 69-79°C, 69-78°C, 69-77°C, 69-76°C, 6975°C, 69-74°C, 69-73°C, 69-72°C, 69-7ΓΟ, 70-90°C, 70-89°C, 70-88°C, 70-87°C, 7086°C, or70-85°C.
15. 15. The process according to any of the preceding claims, wherein the enzyme composition is dosed in amounts corresponding to 10-500 mg enzyme protein/kg FFB (fresh fruit bunch) comprising palm oil, such as 10-450 mg enzyme protein/kg FFB comprising palm oil, 10-400 mg enzyme protein/kg FFB comprising palm oil, 10-350 mg enzyme protein/kg FFB comprising palm oil, 10-300 mg enzyme protein/kg FFB comprising palm oil, 10-250 mg enzyme protein/kg FFB comprising palm oil, 10-200 mg enzyme protein/kg FFB comprising palm oil, 10-150 mg enzyme protein/kg FFB comprising palm oil, 10-100 mg enzyme protein/kg FFB comprising palm oil, 10-75 mg enzyme protein/kg FFB comprising palm oil, 10-50 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB comprising palm oil, 100-500 mg enzyme protein/kg FFB comprising palm oil, 150500 mg enzyme protein/kg FFB comprising palm oil, 200-500 mg enzyme protein/kg FFB comprising palm oil, 250-500 mg enzyme protein/kg FFB comprising palm oil, 300-500 mg enzyme protein/kg FFB comprising palm oil, 350-500 mg enzyme protein/kg FFB comprising palm oil, 400-500 mg enzyme protein/kg FFB comprising palm oil, 30-400 mg enzyme protein/kg FFB comprising palm oil, 30-300 mg enzyme protein/kg FFB comprising palm oil, 30-200 mg enzyme protein/kg FFB comprising palm oil, 30-150 mg enzyme protein/kg FFB comprising palm oil, 30-100 mg enzyme protein/kg FFB comprising palm oil, 30-75 mg enzyme protein/kg FFB comprising palm oil, or such as 30-50 mg enzyme protein/kg FFB comprising palm oil.
16. 16. The process according to any of the preceding claims, wherein the enzyme composition is dosed such that the amount of enzyme protein corresponds to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm, 40-1000 ppm, 50-1000 ppm, 100-1000 ppm, 200-1000 ppm, 100-750 ppm, such as 200-750 ppm, 250-750 ppm, 350-750 ppm or 350-500 ppm relative to the amount of substrate comprising palm oil.
17. 17. A crude palm oil, which is obtainable by the process according to any of the preceding claims.