WO2017182667A1

WO2017182667A1 - Enzyme-assisted oil extraction using sterilizer condensate

Info

Publication number: WO2017182667A1
Application number: PCT/EP2017/059579
Authority: WO
Inventors: Martin Rushworth
Original assignee: Novozymes A/S
Priority date: 2016-04-22
Filing date: 2017-04-21
Publication date: 2017-10-26
Also published as: AU2017252228A1; WO2017182665A1; MY197084A; AU2017252228B2; AU2017253501A1; AU2017253501B2

Abstract

The present invention relates to a process for extraction of crude palm oil, comprising contacting palm fruit mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition, and adding an oil-containing aqueous composition to dilute said mass passing to digester (MPD), fruit mash and/or crude oil. The invention also relates to a crude palm oil. Finally, 5 the invention relates to a palm oil milling line comprising sterilizer, a thresher, a digester and a press, such as a screw press and means for dosing an enzyme composition. The palm oil milling line is equipped with pibes, tubes or hoses which allow an oil-containing aqueous composition to be combined with mass passing to digester (MPD), fruit mash and/or crude oil.

Description

ENZYME-ASSISTED OIL EXTRACTION USING STERILIZER CONDENSATE

Field of the invention

The present invention relates to processes for extraction of crude palm oil using enzymes. More particularly, the present invention relates to a process for extraction of crude palm oil, comprising contacting palm fruit mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition, and adding an oil-containing aqueous composition to dilute said mass passing to digester (MPD), fruit mash and/or crude oil.

The invention also relates to a crude palm oil,

Finally, the invention relates to a palm oil milling line comprising sterilizer, a thresher, a digester and a press, such as a screw press and means for dosing an enzyme composition. The palm oil milling line is equipped with pibes, tubes or hoses which allow an oil-containing aqueous composition to be combined with mass passing to digester (MPD), fruit mash and/or crude oil. Description of the Related Art

Palm oil production has huge economic implications in many developing countries in Asia, Africa and Latin America as large land banks are being devoted for growing the crop in recent years. Palm fruits or fruitlets grow in large bunches. Crude palm oil can be extracted from the palm fruitlets after the fresh fruit bunches (FFB) have gone through a sterilization and separation process. In general, the crude palm oil extraction process is characterised by the following major steps:

• sterilization of the fresh palm fruit bunches process; e.g. to arrest oil quality deterioration due to enzymatic activity, and facilitate the stripping of fruits from bunch stalks and the extraction of oil;

· threshing or stripping to remove fruitlets from the bunch stalk;

• discharging the fruitlets into vessels commonly referred to as digesters and digesting the fruitlets to produce a digested mash under controlled temperature;

• pressing of the digested mash; e.g. using a screw press, for subsequent recovery of oil;

• subjecting the pressed liquor, also referred to as crude oil, to screening, e.g. to remove coarse fibre, and then to a clarification process to separate oil from water, cell debris, and any remaining fibrous material.

Conventional methods for oil palm processing requires pressurized steam for heating whole fresh fruit bunch (FFB) whereby the whole FFB gradually heats up from outer palm fruitlets to inner layer and center core of the FFB. This conventional sterilization process is a wet process which generally requires high pressure steam. The process is carried out on a batch basis where the FFB are loaded onto a cage and pushed into the sterilizer or in a continuous process. The conventional sterilization requires large amount of water to generate steam. The steam also condenses on the oil palm fruitlets, softens them and inactivates the lipase present in the fruitlet. Crude palm oil (CPO) is obtained through pressing of the mass passing to digester (MPD) by a press; e.g. a screw press. Along with the crude palm oil, water and solids are also expressed in this pressing and must subsequently be separated from the crude oil.

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.

During operation of the sterilizing unit an aqueous condensate is formed. It is well known that this condensate also contains oil, mainly because the palm fruits "bleed" during sterilization, due to the heat treatment and mechanical impact during conveyance. The amount of oil in this sterilizer condensate may be substantial, depending e.g. on the ripeness of the fruit and the amount of loose fruit. If the sterilizer condensate cannot be brought back into the extraction process, it is discharged as part of the Palm Oil Mill Effluent (POME) and the oil in the sterilizer condensate is lost.

In the palm oil milling process, oil is also lost in the empty fruit bunches, which are produced when the wet, sterilized fruit bunches are threshed or stripped to remove fruitlets from the bunch stalk. The oil content of the empty fruit bunches mainly come from palm fruitlets remaining attached to bunch stalks after the threshing or stripping procedure. To reduce the amount of oil lost with the empty fruit bunches, the bunches may be subject to pressing, and the resulting fruit bunch press liqueur, which contains oil and large amounts of water, then must be brought back into the milling process.

In palm oil milling, the crude oil released by pressing the digested fruit mash is generally diluted with hot water typically in the exit from the screw press, to reduce its viscosity for more effective oil and water separation. Instead of using preheated tap or well water for dilution, the mills generally have an interest in using oil-containing aqueous waste streams, such as sterilizer condensate or fruit bunch press liqueur, in order to bring the waste streams back into the process and minimize the oil loss.

There have been several reports indicating that enzymes, in particular cellulolytic enzymes, may be used successfully in palm oil milling to increase the oil yield (e.g. WO 2012/01 1 130). In particular, enzymes have been shown to facilitate the separation of oil from water, fibre and sludge in the crude pressed oil, and to minimize the amounts of oil lost in the sludge. However, it has also been reported that the positive effects of the enzymes on oil separation and oil lost to the sludge are compromised by dilution of the crude oil. Therefore, when applying enzymes in the milling process, the total water content of the crude oil or dilute crude oil should be kept as low as possible and should in any event not exceed 40% by weight (AU2015101377, WO 2016/097266). Considering the fact that palm fruitlets contain on average 24% water, a requirement for such very low water content effectively prevents bringing any aqueous waste streams back into the process, as this would increase the water content of the dilute crude oil, in most instances to far more than 40% (w/w).

Hence, there is still a need for developing effective palm oil milling processes, which benefit from the use of enzymes and at the same time minimizes the oil lost in waste streams.

Summary of the Invention

In a first aspect, the invention provides a process for extraction of crude palm oil, comprising contacting palm fruit mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition, and adding an oil-containing aqueous composition to said mass passing to digester (MPD), fruit mash and/or crude oil.

In a further aspect, the present invention relates to a crude palm oil, which is obtainable by the process according to the invention.

Finally, the invention provides a palm oil milling line comprising sterilizer, a thresher, a digester and a press, such as a screw press and means for dosing an enzyme composition, and pibes, tubes or hoses which allows a condensate formed in the sterilizer to be combined with MPD, palm fruit mash in the digester or with press liquid exiting the press.

Definitions

Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esterase activity can be determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01 % TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 μηηοΐβ of p-nitrophenolate anion per minute at pH 5, 25°C.

Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1 .139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. Alpha-glucuronidase activity can be determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase equals the amount of enzyme capable of releasing 1 μηηοΐβ of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40°C.

Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1 .21 ) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta- D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1 .0 μηηοΐβ of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20.

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 μηηοΐβ of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D- xyloside 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-D-glucosidic 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 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 .

Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic enzyme activity include: (1 ) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481 . Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1 -50 mg of cellulolytic enzyme protein/g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3-7 days at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnS0₄, 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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 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 ei a/., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-1 18, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier ei a/., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, 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. In particular, the term "crude oil" refers to the effluent from the screw press of a palm oil mill; i.e. to the mixture of oil and water pressed out of the palm fruit mash, before it has been subject to clarification and separation of oil from water.

Endoglucanase: The term "endoglucanase" means a 4-(1 ,3;1 ,4)-beta-D-glucan 4- glucanohydrolase (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: 452- 481 ). 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.

Feruloyi esterase: The term "feruloyi esterase" means a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyi) groups from esterified sugar, which is usually arabinose in natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyi esterase (FAE) is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. Feruloyi esterase activity can be determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. One unit of feruloyi esterase equals the amount of enzyme capable of releasing 1 μηιοΐβ of p-nitrophenolate anion per minute at pH 5, 25°C.

Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyi esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates for these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. C em. 59: 1739-1752, at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.

Hemicellulosic material: The term "hemicellulosic material" means any material comprising hemicelluloses. Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. These polysaccharides contain many different sugar monomers. Sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses contain most of the D-pentose sugars. Xylose is in most cases the sugar monomer present in the largest amount, although in softwoods mannose can be the most abundant sugar. Xylan contains a backbone of beta-(1 -4)-linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1 -4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L- galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1 -67. Hemicellulosic material is also known herein as "xylan-containing material".

Sources for hemicellulosic material are essentially the same as those for cellulosic material described herein.

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).

Polygalacturonases: The term "polygalacturonases" (EC 3.2.1.15) are pectinases that catalyze random hydrolysis of (1 ,4)-alpha-D-galactosiduronic linkages in pectate and other galacturonans. They are also known as pectin depolymerase. Polygalacturonase hydrolyses the alpha-1 ,4- glycosidic bonds in polygalacturonic acid with the resultant release of galacturonic acid. This reducing sugar reacted then with 3,5-dinitrosalicylic acid (DNS). The colour change produced due to the reduction of DNS is proportional to the amount of galacturonic acid released, which in turn is proportional to the activity of polygalacturonase in the sample. One polygalacturonase unit (PGNU) is defined as the amount of enzyme which will produce 1 mg of galacturonic acid sodium salt under standard conditions (acetate buffer, pH 4.5, 40°C, 10 min reaction time, 540 nm).

Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1 -4)-linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1 -4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L- arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1 -67.

Amylase: For the purpose of the present invention, "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.

Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1 ) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta- xylosidases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchard, 2006, Journal of the Science of Food and Agriculture 86(1 1 ): 1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601 ; Herrimann et al., 1997, Biochemical Journal 321 : 375-381 .

Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. A common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey et al., 1992, I nterlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. Xylanase activity can also 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 μηηοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p- hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

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 μηηοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Pectin methylesterase (PME), EC 3.1 .1 .1 1 , is an enzyme that acts mainly in the hydrolysis of methyl ester groups in pectin chains to form carboxylate groups, releasing methanol and H30+ (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 determined by different methods and thermal inactivation of exogenous pme in mango juice. Ciena agrotec. vol.35 no.5 Lavras Sept./Oct. 201 1

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

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 to Fresh fruit bunch (FFB) times 100. According to this definition, the mathematical formula is:

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

Temperature optimum: In the context of the invention, the term "temperature optimum" refers to the temperature at which an enzyme's catalytic activity is at its greatest. Below the temperature, reacting molecules have more and more kinetic energy as the temperature rises. This increases the chances of a successful collision and so the rate increases. Above temperature optimum the enzyme structure begins to denature since at higher temperatures intra- and intermolecular bonds are broken as the enzyme molecules gain even more kinetic energy.

A temperature optimum may be determined by assessing the enzyme activity; e.g. the cellulase activity, of a purified enzyme, a crude extract of the enzyme or an enzyme in a whole broth, over a range of temperatures (e.g. 40 to 90°C) at a relevant pH (e.g. pH 5) and for an appropriate incubation period; e.g. for a period of 5-60 minutes, such as 5-30 minutes, 10-30 minutes or 20- 30 minutes, or 20-25 minutes. For determination of cellulase activity, the buffers, substrate and assay principle disclosed below, in the definition of "thermostability" may be used in a determination of temperature optimum.

Thermostability: As used herein, "thermostability" refers to the stability of an enzyme when the enzyme is tested or left at a specific high temperature, such as 70°C. The thermostability may be determined by incubating the enzyme in an appropriate buffer (e.g. 0.1 M Na-OAc buffer pH 5.0) at an elevated temperature, e.g. 70 degrees Celsius for varying time periods (i.e. 0 min, 20 min, 40 min and 60 minutes) followed by transfer of the samples to ice, and then determine the residual enzyme activity. For instance, residual cellulase activity may be determined on Konelab using the following protocol:

Substrate: Carboxymethyl cellulose (CMC), 5 g/L in Na-AOc buffer pH 5.0

Sample dissolution and dilution buffer: 0.1 M Na-OAc buffer pH 5.0

Stop and detection reagent: PAH BAH, 50 g/L K-Na-tartrate, 20 g/L PAH BAH, 5.52g/L Bismuth(lll)-acetate, 0.5 M NaOH

Assay principle: Cellulose is hydrolyzed and form reducing carbohydrate. The substrate carboxymethyl cellulose (CMC) is a substituted form of cellulose. The reaction is stopped by an alkaline reagent containing PAH BAH and Bismuth that forms complexes with reducing sugar. The complex formation results in colour production which can be read at 405 nm by a spectrophotometer. The produced colour is proportional to the cellulose activity.

The residual activity is calculated and plotted against time of heat treatment In the present application, reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X".

As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that the aspects of the invention described herein include "consisting" and/or "consisting essentially of" aspects.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Description of the figures

Figure 1 : Schematic representation of a production line in a commercial palm oil mill from thresher to screw press. The production line includes: Sterilizer (A), Thresher (B), Conveyor(s) (C), (D) and (E), Digester (F) and Screw press (G). Enzyme application is shown at points (H), (I) and (J); including water dosing (L) and enzyme dosing (K). Dilute crude oil (DCO) exit from screw press is shown as (N). Pibes, hoses or tubes allowing condensate formed in the Sterilizer (A) to flow or be pumped and be combined with mass passing to digester (MPD) at point (O), fruit mash in the digester at point (P) or press liquid from the press (G) at point Q) are shown.

Figure 2: A schematic representation of a production line as in Figure 1 , also showing pibes, hoses or tubes allowing a fruit bunch press liqueur from a fruit bunch press (R) to flow or be pumped and be combined with mass passing to digester (MPD) at point (S), fruit mash in the digester at point (T) or press liquid from the press (G) at point (U) are shown.

Figure 3: A schematic representation of a production line as laid out in figure 2, wherein a pre- cooker (E1 ) is included in connection with the digester (E2).

Detailed Description of the Invention

The present inventors have found that the water content of the dilute crude oil (DCO) obtained in palm oil milling may be increased to a large extent, without any negative impact on oil yield obtained in enzyme-assisted thermo-mechanical palm oil extraction processes. In particular, the water content may be increased much beyond the 40% (w/w) which has previously been reported as being the maximum acceptable water content in DCO. This observation is of high technical and commercial relevance, because it allows the palm oil mills to run more wet processes, for instance by bringing oil-containing aqueous waste streams back into the milling process. This enables the mills to recover the oil that would otherwise be lost in the aqueous waste streams, even if the mills use enzymes, e.g. cellulolytic enzymes, to increase the oil extraction rate (OER). The aqueous waste streams include condensate formed in the sterilizers (hereinafter referred to as "sterilizer condensate") and liqueur obtained from pressing empty fruit bunches after threshing or stripping (hereinafter referred to as fruit bunch press liqueur).

The inventors have also found that there is no significant increase in the amounts of free fatty acids with recycling of sterilizer condensate (SC) for dilution of undilute crude oil (UDCO) to produce dilute crude oil (DCO). In particular, the inventors have found that the % FFA in the final oil is less than 5%.

Hence in a first aspect, the present invention provides a process for extraction of crude palm oil, comprising contacting palm fruit mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition, and adding an oil-containing aqueous composition to said mass passing to digester (MPD), fruit mash and/or crude oil.

The said oil-containing aqueous composition may in particular be an oil-containing aqueous waste stream, such as a waste stream obtained by sterilization of palm fruit bunches and/or a waste stream obtained by threshing or stripping of sterilized palm fruit bunches.

In particular embodiments, the oil-containing aqueous composition is selected from the group consisting of a sterilizer condensate and fruit bunch press liqueur.

The process according to the invention may in particular comprise

i) Sterilizing bunches of palm fruitlets to provide sterilized bunches of palm fruitlets and a sterilizer condensate,

ii) Separating the palm fruitlets from bunch stalks, e.g. by mechanical stripping, to provide MPD, and empty fruit bunches,

iii) Optionally subjecting the empty fruit bunches to pressing, to provide a fruit bunch press liqueur,

iv) Subjecting the MPD to a digestion procedure to produce fruit mash;

wherein

said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD and/or the fruit mash,

the fruit mash is subjected to pressing to produce DCO comprising oil and water, together with cell debris, and/or fibrous material, and

the oil in said DCO is separated from the water and from the cell debris, and/or fibrous material

or the fruit mash is subjected to pressing to provide crude oil comprising oil and water, together with cell debris and/or fibrous material,

said sterilizer condensate and/or said fruit bunch press liqueur is added to the crude oil to provide DCO, and

the oil in said DCO is separated from the water, and from the cell debris, and/or fibrous material.

The process according to the invention may also comprise removing coarse fibers from said DCO; e.g. by passing it through one or more screens.

The process according to the invention comprises in some embodiments clarifying said DCO to separate oil from water, cell debris, fibrous material and/or non-oily solids.

The said DCO may be clarified by sedimentation.

In particular embodiments, the said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD, the digested fruit mash and/or the crude oil composition in such amounts that the water content of the MPD, the digested fruit mash and/or said dilute crude oil composition exceeds 35%, 38% or 40% (w/w).

In further embodiments, the said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD, the digested fruit mash and/or the crude oil composition in such amounts that the water content of the MPD, the digested fruit mash and/or said dilute crude oil composition is 90% (w/w) or less, such as 85% (w/w) or less, 80% (w/w) or less, 82% (w/w) or less, 75% (w/w) or less, 73% (w/w) or less, 70% (w/w) or less, 65% (w/w) or less, 64% (w/w) or less. 60% (w/w) or less, 55% (w/w) or less, 50% (w/w) or less or such as 46% (w/w) or less.

In still further embodiments, the said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD, the digested fruit mash and/or the crude oil in such amounts that the water content of said DCO, while exceeding 40% (w/w) is at the most 90% (w/w); e.g. within the range of 41 -90% (w/w), 41 -85% (w/w), 41 -82% (w/w), 41 -75% (w/w), 41 -73% (w/w), 41 -70% (w/w) 41 -65% (w/w) 41 -64% (w/w) 41 -60% (w/w) 41 -55% (w/w) 41 -50% (w/w), 41 -46% (w/w), 43-90% (w/w), 43-85% (w/w), 43-82% (w/w), 43-75% (w/w), 43-73% (w/w), 43-70% (w/w) 43-65% (w/w) 43-64% (w/w) 43-60% (w/w) 43-55% (w/w) 43-50% (w/w), or such as within the range of 43-46% (w/w).

In the process according to the invention, the said MPD, said digested fruit mash, said crude oil and/or said DCO may be contacted with an enzyme composition comprising one or more hydrolases.

The enzyme composition may in particular comprise one or more cellulases and/or one or more amylases and/or one or more hemicellulases and/or one or more pectinases. In some embodiments, the one or more cellulases are present in the enzyme composition or are added to said MPD, said digested fruit mash, said crude oil in combination with one or more hemicellulases, one or more pectinases, one or more amylases, or a combination thereof.

In one embodiment, the enzyme composition used in the process of the invention comprises a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP (Genencor Int.), ACCELERASE™ TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Rohm GmbH), or ALTERNAFUEL® CMAX3® (Dyadic International, Inc.).

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691 ,178, U.S. Pat. No. 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care and whiteness maintenance benefits. Examples of such cellulases are cellulases described in EP 0 531 372, WO 96/1 1262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299. Commercially used cellulases include Renozyme®, Celluzyme®, Celluclean®, Endolase® and Carezyme®. (Novozymes A/S), Clazinase®, and Puradax HA®. (Genencor Int. Inc), and KAC- 500(B)™ (Kao Corporation).

In currently preferred embodiments, the enzyme composition comprises Palmora® OER, which is commercially available from Novozymes A/S.

Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME™ (Novozymes A/S), CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC® HTec3 (Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK). The bunches of palm fruitlets may be sterilized in a batch sterilizer or in a continuous sterilizer.

In particular, the process according to the invention may comprise the steps of:

a. Contacting the palm fruitlets, the MPD, the fruit mash and/or the crude oil with said enzyme composition at a temperature of above 65°C;

b. Extracting the CPO.

In the process according to the invention, the palm fruitlets, the MPD, the fruit mash and/or the crude oil may be contacted with said 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, or 70-85°C.

The enzyme composition may be dosed in amounts corresponding to 20-1000 mg enzyme protein/kg palm fruitlet, MPD, fruit mash, or crude oil, 20-750 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, such as 20-450 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-400 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-350 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-300 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-250 mg enzyme protein/kg palm fruitlet, MPD or crude oil, 20-200 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-150 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-100 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-75 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 20-50 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 40-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 50-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 75-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 100-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 150-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 200-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 250-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 300-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 350-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 400-500 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 200-1000 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 200-750 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30-400 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30-300 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30- 200 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30-150 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30-100 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, 30-75 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil, or such as 30-50 mg enzyme protein/kg palm fruitlet, MPD, fruit mash or crude oil.

The enzyme composition may be dosed in amounts corresponding to 5-200 mg enzyme protein per metric ton of fresh fruit bunches (FFBs) entering the oil extraction process, such as 10-200 mg enzyme protein, such as 20-200 mg enzyme protein, such as 5-150 mg enzyme protein, such as 5-100 mg enzyme protein, such as 5-50 mg enzyme protein, such as 10-200 mg enzyme protein, such as 10-100 mg enzyme protein, such as 10-75 mg enzyme protein, or such as 10-50 mg enzyme protein per metric ton of fresh fruit bunches (FFBs) entering the oil extraction process.

In some embodiments, the palm fruitlets, the MPD, the fruit mash and/or the crude oil is contacted with the enzyme composition for a period of 5-60 minutes, such as for a period of 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.

The digestion procedure may comprise retaining the MPD and/or the fruit mash 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.

The enzymes in said enzyme composition, such as the one or more cellulases, one or more hemicellulases, one or more pectinases, and/or one or more amylases, has a temperature optimum in the range of 60-85°C, such as in the range of 65-85°C, 66-85°C, 67-85°C, 68-85°C, 69-85°C, 70-85°C, 65-79°C, 65-80°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, and 70-85°C.

In some embodiments, the one or more cellulases, one or more hemicellulases, one or more pectinases and/or one or more amylases, are thermostable to such an extent that at least 15% of the enzyme activity (i.e. the cellulase, hemicellulase, amylase and/or pectinase activity) is retained after incubation at 70°C for 20 minutes, to such an extent that at least 20% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 25% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 30% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 35% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 40% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 50% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 60% of the enzyme activity is retained after incubation at 70°C for 20 minutes, or to such an extent that at least 70% of the enzyme activity is retained after incubation at 70°C for 20 minutes.

The thermostability may in particular be determined by incubation at 70°C for 20 minutes in a 0.1 M Na-OAc buffer pH 5.0, followed by transfer to ice and determination of residual enzyme activity (i.e. residual cellulase, hemicellulose, amylase and/or pectinase activity) on Konelab by a method comprising: hydrolyzing substrate (e.g. carboxymethyl cellulose (CMC) form reducing carbohydrate; stopping the hydrolyzation by an alkaline reagent containing PAHBAH and Bismuth, which that forms complexes with reducing sugar; and measuring color production by complex formation at 405 nm in a spectrophotometer.

The process according to the invention may, as illustrated in figure 2, also comprise the steps of:

i) Sterilizing bunches of palm fruitlets in a sterilizer (A) to provide sterilized fruitlets and a sterilizer condensate,

ii) Subjecting bunches of palm fruitlets to threshing or stripping in a thresher or stripper

(B), to provide palm fruitlets and empty fruit bunches, and optionally subjecting the empty fruit bunches to pressing in a press (R), to provide a fruit bunch press liqueur iii) discharging the stripped fruitlets or MPD to a conveyor; e.g. a screw conveyor (C), iv) Conveying the stripped fruitlets or MPD to a digester (F) on one or more conveyors; e.g. conveyors (C), (D) and/or (E);

Or

conveying the stripped fruitlets or MDP from the thresher to a pre-cooker, retaining said stripped fruitlets or MDP in the pre-cooker to produce precooked fruitlets or MPD, and then conveying or discharging the precooked fruitlets or MPD into a digester (F)

v) Retaining the stripped fruitlets or MPD or the precooked fruitlets or MPD in the digester (F) to produce fruit mash, and

vi) Pressing the fruit mash in a press (G), such as a screw press, to extract CPO; wherein said sterilizer condensate and/or said fruit bunch press liqueur is added to the stripped fruitlets or MPD in step iii), in step iv) or v) and/or to the CPO extracted in step vi).

The process according to the invention may, as illustrated in figure 2 and 3, also comprise the steps of:

ii) Subjecting bunches of palm fruitlets to threshing or stripping in a thresher or stripper (B), to provide palm fruitlets and empty fruit bunches, and optionally subjecting the empty fruit bunches to pressing in a press (R), to provide a fruit bunch press liqueur iii) discharging the stripped fruitlets or MPD to a conveyor; e.g. a screw conveyor (C), iv) Conveying the stripped fruitlets or MPD to a digester (F) on one or more conveyors; e.g. conveyors (C), (D) and/or€ as shown in figure 2;

Or

conveying the stripped fruitlets or MDP from the thresher to a pre-cooker (F1 ), retaining said stripped fruitlets or MDP in the pre-cooker to produce precooked fruitlets or MPD, and then conveying or discharging the precooked fruitlets or MPD into a digester (F2)

v) Retaining the stripped fruitlets or MPD or the precooked fruitlets or MPD in the digester to produce fruit mash, and

vi) Pressing the fruit mash in a press (G), such as a screw press, to extract CPO; wherein

said sterilizer condensate and/or said fruit bunch press liqueur is added to the stripped fruitlets or MPD in step iii), in step iv) or v) and/or to the CPO extracted in step vi).

In some embodiments of the invention, the one or more enzymes are applied in step iii), such as by application onto the stripped fruitlets or MPD, while the stripped fruitlets or MPD is/are conveyed to a digester (F), optionally via a pre-cooker, on one or more conveyors; e.g. conveyors (C), (D) and/or (E).

In these and other embodiments, the one or more enzymes; e.g. one or more enzymes as defined above, are applied in step iv).

According to further embodiments, the one or more enzymes; e.g. one or more enzymes as defined above, are applied after step v) and before clarification of the DCO. The palm fruitlets or MPD may be retained in said pre-cooker for a period of 15 - 120 minutes, such as 15-60 minutes, such as 30-120 minutes, or such as for a period of 30-60 minutes.

In some embodiments according to the invention, the retention time in the pre-cooker may be even shorter. Hence, the palm fruitlets or MPD may be retained in said pre-cooker for a period of 2-20 minutes, such as 3-20 minutes, such as 5-20 minutes, such as 2-15 minutes, such as 3- 15 minutes, such as 3-10 minutes, such as 2-100 minutes, or such as for a period of 5-10 minutes.

The palm fruitlets or MPD may be retained in said pre-cooker for an amount of time, which is sufficient to provide a total retention time of 15 minutes or more, such as 20 minutes or more, such as a retention time of 15 minutes - 1 hour, such as a retention time of 20 minutes - 1 hour, such as a retention time of 20 - 45 minutes or such as a retention time of 20 - 30 minutes; the retention time being calculated as the time from application or dosing of enzyme to the time point at which the fruit mash is subject to pressing.

According to still further embodiments, step iv) comprises retaining the stripped fruitlets or MPD in the digester at temperatures above 65°C and up to 85°C from 10-30 minutes, such as from 10-28 minutes, 15-28 minutes, 12-30 minutes, 12-28 minutes or 12-25 minutes; e.g. by controlling the speed of the screw press (G) and/or by controlling steam supply (K).

In further embodiments step iv) comprises retaining the stripped fruitlets or MPD in the digester at temperatures between 55 and 90°C, such as between 60 and 90°C, between 60 and 85°C, between 60 and 80°C, between 65 and 85°C, or such as between 65 and 80°C from 7-30 minutes, from 10-30 minutes, such as from 7-28 minutes, from 10-28 minutes, 15-28 minutes, 12- 30 minutes, 12-28 minutes, from 7-25 minutes, from 10-25 minutes, from 7-20 minutes, from 10- 20 minutes or 12-25 minutes; e.g. by controlling the speed of the screw press (G) and/or by controlling steam supply (K).

A second aspect of the invention provides a crude palm oil, which is obtainable or obtained by the process according to the invention.

A further aspect of the invention pertains to a palm oil milling line comprising sterilizer, a thresher, a digester and a press, such as a screw press and means for dosing an enzyme composition, and pipes, tubes or hoses which allows a condensate formed in the sterilizer to be combined with MPD, palm fruit mash in the digester or with press liquid exiting the press.

As illustrated in figure 1 , the palm oil milling line according to the invention may comprise a sterilizer (A), a thresher or stripper (B), conveyors (C), (D) and/or (E), a digester (F) and in a press (G) and pibes, tubes or hoses which allows a condensate formed in the sterilizer (A) to be combined with MPD at point (O), palm fruit mash in the digester at point (P) or with press liquid from the press (G) at point (Q). In further embodiments, as illustrated in Figures 2 and 3, the palm oil milling line according to the invention comprises a sterilizer (A), a thresher or stripper (B), a fruit bunch press (R), conveyors (C), (D) and/or (E), a digester (F) and in a press (G) and pibes, tubes or hoses which allows a fruit bunch press liqueur formed in fruit bunch press (R) to be combined with MPD at point (S), palm fruit mash in the digester at point (T) or with press liquid from the press (G) at point (U).

The sterilizer, the thresher or stripper, the digester, the press and optionally the pre-cooker are connected in series. The serial connection allows fruit bunches to be conveyed via the sterilizer into the thresher or stripper, palm fruitlets or "mass passing to digester" to be conveyed from the thresher or stripper to the digester, optionally via the pre-cooker, and transport of fruit mash from the digester into the screw press. In further embodiments, the exit of the screw press is fluidically connected to downstream equipment for separation of oil from water and sludge.

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

Examples

Example 1 : Oil yield in enzyme assisted oil extraction, with different amounts of moisture in fruit/MPD.

Samples were prepared by making 120g and 10Og fruits up to 200g with water to attain the final %M of the mash to 40 and 50% Moisture, respectively during mashing. The palm fruit was mashed with enzymes at 70°C/30min followed by extraction by parapress and clarification.

Results are presented in Table 1 below.

Table 1 :

Example 2: Effect of dilution on Oil recovery in downstream from UDCO.

Experimental procedure:

Approximately 2Kg of UDCO was collected from Screw Press during peak processing hou and was mixed properly. The UDCO (28 %moisture and 65%oil) was aliquoted in 50ml graduated Falcon tubes and diluted with hot water to make final volume to 40ml such that the final sample %Moisture ranged from 18% to 82% as shown in table 2.

Table 2:

Enzymes were added (Comprising Cellic CTech 2 and Cellic Htech) at 0.25 ml dose for each (corresponding to 45 mg active enzyme protein for Cellic® CTec 2; and to 28.75mg active enzyme protein for Cellic®Htec). The entire mass was mixed thoroughly and incubated at 65°C (±3°) with intermittent mixing for 2h. Clarification was done at 90°C for 1 h in static condition and the clarified extract was centrifuged at 4000 rpm for 5min to separate oil from the sludge. Oil, emulsion, water and sludge quantities were estimated.

Results:

The results are presented in Table 3 below.

The results show that Oil Extraction efficiency from UDCO is maintained with enzyme treatment even at higher %Moisture >55% compared to control. Emulsion% decreases with enzyme treatment at higher %Moisture indicating that enzyme is able to break down emulsion even at higher %Moisture, especially from 46-64%Moisture.

Table 3:

25 6,75 15 0,75 22,5 55%

4 40,75

20 5,4 20 0,75 26,15 64%

5 40,75

6 15 4,05 25 0,75 29,8 73% 40,75

10 2,7 30 0,75 33,45 82%

7 40,75

Table 3, cont'd:

Table 3, cont'd:

5

13 92% 98% 6%

6 9,75 92% 97% 5%

7

6,5 94% 100% 6%

Example 3. Recycling of sterilizer condensate at a 30 metric ton/hour palm oil mill Trial design:

Dosing of cellulolytic enzymes to increase oil yield was tested at a Malaysian palm oil mill operating at 30 metric tonnes FFB per hour and with a continuous sterilizer. The mill was fitted for recycling of fruit bunch press liquor (EFB liquor (EFBL) and sterilizer condensate (SC) to dilute the crude oil (UDCO).

The enzyme (Palmora®OER, commercially available from Novozymes A/S) was dosed in amounts corresponding to 330 or 750 ppm, by spraying onto the mass passing to digester (MPD) during conveyance from thresher to pre-cooker and digester.

The total retention time from dosing of enzyme to pressing was estimated to be 22 minutes. The temperature during enzyme incubation ranged from 60-90°C; the pre-cooker being operated with temperatures ranging from 65-80, the digester being operated with temperatures ranging from 60-90°C.

Determination of water content in UDCO and DCO samples performed on samples having an initial weight of 20 g. The samples were dried at 105°C for 12h.

30g for liquid samples were taken for soxhlet extraction for measuring oil content. Samples were prepared by first drying them in hot air oven for 8-12 hrs at 105°C. Soxhlet extraction was carried out by taking dried samples into cellulose thimbles, followed by extraction using 200 ml of hexane for 4 hrs until the thimble was colourless. The extracted oil samples were dried in hot air oven at 105°C for 2 hrs for removal of residual hexane and kept in desiccator until constant weight. After cooling, the samples were weighed and the weight of the extracted oil was noted.

Results:

Results are shown in table 4 below. The data confirm that clarification/separation of oil from water was efficient, even though the water content of the dilute crude oil was above 60% by weight. Further, it is notable that the %FFA (4.25%) is less than the maximum permissible limit of 5% despite recycling the SC.

Table 4:

index (DOBI)

Table 4, cont'd:

n ex

Example 4. Recycling of sterilizer condensate at a 40 metric ton/hour palm oil mill Trial design:

Dosing of cellulolytic enzymes to increase oil yield was tested at a Malaysian palm oil mill operating at 40 metric tonnes FFB per hour, with a batch sterilizer. The mill was fitted for recycling of fruit bunch press liquor (EFB liquor (EFBL) and sterilizer condensate (SC) to dilute the crude oil (UDCO).

The enzyme (Palmora®OER, commercially available from Novozymes A/S) was dosed in amounts corresponding to 330 or 750 ppm, by spraying onto the mass passing to digester (MPD) during conveyance from thresher to digester.

The amounts of free fatty acids (FFA) were estimated following the standard titration protocol: 4.0g (±0.01 g) of liquid crude palm oil sample was transferred to a 250ml conical flask. 30ml neutralized iso-propanol was added into it followed by hitting to dissolve the oil completely. After that it was titrated with 0.05N NaOH until the colour changed to brick red; indicator used was phenolphthalein.

Results:

Results are shown below in tables 5 and 6. The moisture content of the DCO sample was estimated by oven drying method as described before and it was found in the range of 43-45%.

Table 5:

Table 5, cont'd:

Table 6: CPO quality-%FFA

Example 5: Feasibility study for 100% recovery of sterilizer condensate in enzymatic crude palm oil extraction Experimental design:

Enzyme-assisted crude palm oil was performed as described in Example 4.

The freshly generated SC (~2L) was collected from the out let pipe of sterilizer condensate during the batch sterilization of FFB at a Malaysian palm oil mill operating at 40 metric tonnes FFB per hour, with a batch sterilizer. The UDCO (~2L) was collected from the press. Both were mixed thoroughly as per the volume given in the following table to make dilutions from 0-100%. The SC diluted DCO was taken in screw capped glass bottles at 85±5°C for 4h at static condition to mimic the industrial conditions. The CPO was then separated by centrifugation at 4000rpm for 5min.

The amounts of FFA were estimated as described in Example 4.

Results:

Results are shown below in table 7. The results show that there is a linear relationship between % SC dilution and % FFA as well as DOBI of production oil.

The FFA Content is found to be less than 5.0% when the %FFA of SC-oil (as such) is around 17% (avg.) and dilution is 20%.

At 20% SC dilution, the DOBI is decreased by 0.028% compared to DOBI of oil without any SC dilution. 20% SC dilution is found to have no negative impact on DOBI. 20% SC dilution is representative of approximately 80-100% SC recycling/day.

Table 7:

Claims

A process for extraction of crude palm oil, comprising contacting palm fruit mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition, and adding an oil-containing aqueous composition to said mass passing to digester (MPD), fruit mash and/or crude oil.

The process according to claim 1 , wherein said oil-containing aqueous composition is an oil-containing aqueous waste stream, such as a waste stream obtained by sterilization of palm fruit bunches and/or a waste stream obtained by threshing or stripping of sterilized palm fruit bunches.

The process according to claim 1 or 2, wherein said oil-containing aqueous composition is selected from the group consisting of a sterilizer condensate and fruit bunch press liqueur.

The process according to any of the preceding steps, comprising

iv) Subjecting the MPD to a digestion procedure to produce fruit mash; wherein

or

the fruit mash is subjected pressing to provide crude oil comprising oil and water, together with cell debris and/or fibrous material,

the oil in said DCO is separated from the water, and from the cell debris, and/orfibrous material.

5. The process according to claim 4, comprising removing coarse fibers from said DCO by passing it through one or more screens.

6. The process according to claim 4 or 5, comprising clarifying said DCO to separate oil from water, cell debris, fibrous material and/or non-oily solids.

7. The process according to any of claims 4-6, wherein said DCO is clarified by sedimentation.

8. The process according to any of claims 3-7, wherein said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD, the digested fruit mash and/or the crude oil composition in such amounts that the water content of the MPD, the digested fruit mash and/or said dilute crude oil composition exceeds 35%, 38% or 40% (w/w).

9. The process according to any of claims 3-8, wherein said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD, the digested fruit mash and/or the crude oil composition in such amounts that the water content of the MPD, the digested fruit mash and/or said dilute crude oil composition is 90% (w/w) or less, such as 85% (w/w) or less, 80% (w/w) or less, 82% (w/w) or less, 75% (w/w) or less, 73% (w/w) or less, 70% (w/w) or less, 65% (w/w) or less, 64% (w/w) or less. 60% (w/w) or less, 55% (w/w) or less, 50% (w/w) or less or such as 46% (w/w) or less.

10. The process according to any of any of claims 3-9, wherein said sterilizer condensate and/or said fruit bunch press liqueur is added to the MPD, the digested fruit mash and/or the crude oil in such amounts that the water content of said DCO, while exceeding 40% (w/w) is at the most 90% (w/w); e.g. within the range of 41 -90% (w/w), 41 -85% (w/w), 41 - 82% (w/w), 41 -75% (w/w), 41 -73% (w/w), 41 -70% (w/w) 41 -65% (w/w) 41 -64% (w/w) 41 - 60% (w/w) 41 -55% (w/w) 41 -50% (w/w), 41 -46% (w/w), 43-90% (w/w), 43-85% (w/w), 43- 82% (w/w), 43-75% (w/w), 43-73% (w/w), 43-70% (w/w) 43-65% (w/w) 43-64% (w/w) 43- 60% (w/w) 43-55% (w/w) 43-50% (w/w), or such as within the range of 43-46% (w/w).

1 1 . The process according to any of the preceding claims, wherein said MPD, said digested fruit mash, said crude oil and/or said DCO is/are contacted with an enzyme composition comprising one or more hydrolases.

12. The process according to claim 1 1 , wherein said enzyme composition comprises one or more cellulases and/or one or more amylases and/or one or more pectinases.

13. The process according to claim 12, wherein said one or more cellulases is/are in combination with one or more hemicellulases.

14. The process according to any of the preceding claims, wherein said bunches of palm fruitlets are sterilized in a batch sterilizer or in a continuous sterilizer.

15. The process according to any of the preceding claims, comprising the steps of:

a. Contacting the palm fruitlets, the MPD, the fruit mash and/or the crude oil with said enzyme composition at a temperature of above 50°C, such as above 55°C, such as above 60°C, or such as above 65°C;

b. Extracting the CPO.

16. The process according to any of the preceding claims, wherein the palm fruitlets, the MPD, the fruit mash and/or the crude oil is contacted with said 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, or 70-85°C.

17. The process according to any of the preceding claims, wherein the enzyme composition is dosed in amounts such that the amount of enzyme protein corresponds to 100-1000 ppm, such as 200-1000 ppm, 100-500 ppm, such as 200-500 ppm, 250-400 ppm or 350- 1000 ppm relative to the amount of palm fruitlet, MPD, fruit mash or crude oil.

18. The process according to any of the preceding claims, wherein the palm fruitlets, the MPD, the fruit mash and/or the crude oil is contacted with said enzyme composition for a period of 5-60 minutes, such as for a period of 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.

19. The process according to any of the preceding claims, wherein the digestion procedure comprises retaining the MPD and/or the fruit mash 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.

20. The process according to any of the preceding claims, wherein the enzyme, such as the one or more cellulases, one or more hemicellulases, one or more pectinases, and/or one or more amylases, has a temperature optimum in the range of 60-85°C, such as in the range of 65-85°C, 66-85°C, 67-85°C, 68-85°C, 69-85°C, 70-85°C, 65-79°C, 65-80°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, and 70-85°C.

21 . The process according to any of the preceding claims, wherein the one or more cellulases, one or more hemicellulases, one or more pectinases and/or one or more amylases, are thermostable to such an extent that at least 15% of the enzyme activity (i.e. the cellulase, hemicellulose, amylase and/or pectinase activity) is retained after incubation at 70°C for 20 minutes, to such an extent that at least 20% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 25% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 30% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 35% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 40% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 50% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 60% of the enzyme activity is retained after incubation at 70°C for 20 minutes, or to such an extent that at least 70% of the enzyme activity is retained after incubation at 70°C for 20 minutes.

22. The process according to any of the preceding claims comprising the steps of:

ii) Subjecting bunches of palm fruitlets to threshing or stripping in a thresher or stripper (B), to provide palm fruitlets and empty fruit bunches, and optionally subjecting the empty fruit bunches to pressing in a press (R), to provide a fruit bunch press liqueur

iii) discharging the stripped fruitlets or MPD to a conveyor; e.g. a screw conveyor (C),

iv) Conveying the stripped fruitlets or MPD to a digester (F) on one or more conveyors; e.g. conveyors (C), (D) and/or (E),

Or

conveying the stripped fruitlets or MDP from the thresher to a pre-cooker, retaining said stripped fruitlets or MDP in the pre-cooker to produce precooked fruitlets or MPD, and then conveying or discharging the precooked fruitlets or MPD into a digester (F) v) Retaining the stripped fruitlets or MPD or the precooked fruitlets or MPD in the digester (F) to produce fruit mash, and

23. The process according to claim 22, wherein one or more enzymes; e.g. one or more enzymes as defined in claim 12 or 13, are applied in step iii), such as by application onto the stripped fruitlets or MPD, while the stripped fruitlets or MPD is/are conveyed to a digester (F) on one or more conveyors; e.g. conveyors (C), (D) and/or (E).

24. The process according to claim 22, wherein one or more enzymes; e.g. one or more enzymes as defined in any of claims 12 and 13, are applied in step iv).

25. The process according to claim 22, wherein one or more enzymes; e.g. one or more enzymes as defined in any of claims 12 and 13, are applied after step v) and before clarification of the DCO.

26. The process according to any of claims 22-25, wherein step iv) comprises retaining the stripped fruitlets or MPD in the digester at temperatures above 65°C and up to 85°C from 10-30 minutes, such as from 10-28 minutes, 15-28 minutes, 12-30 minutes, 12-28 minutes or 12-25 minutes; e.g. by controlling the speed of the screw press (G) and/or by controlling steam supply (K).

27. A crude palm oil, which is obtainable by the process according to any of the preceding claims.

28. A palm oil milling line comprising sterilizer, a thresher, a digester and a press, such as a screw press and means for dosing an enzyme composition, and pibes, tubes or hoses which allows a condensate formed in the sterilizer to be combined with MPD, palm fruit mash in the digester or with press liquid exiting the press.

29. The palm oil milling line according to claim 28, comprising a sterilizer (A), a thresher or stripper (B), conveyors (C), (D) and/or (E), a digester (F) and in a press (G) and pibes, tubes or hoses which allows a condensate formed in the sterilizer (A) to be combined with MPD at point (O), palm fruit mash in the digester at point (P) or with press liquid from the press (G) at point (Q).

30. The palm oil milling line according to claim 28 or 29, comprising a sterilizer (A), a thresher or stripper (B), a fruit bunch press (R), conveyors (C), (D) and/or (E), a digester (F) and in a press (G) and pibes, tubes or hoses which allows a fruit bunch press liqueur formed in fruit bunch press (R) to be combined with MPD at point (S), palm fruit mash in the digester at point (T) or with press liquid from the press (G) at point (U).