WO2017182665A1 - Extraction d'huile de palme à l'aide d'enzymes et d'un stérilisateur continu - Google Patents

Extraction d'huile de palme à l'aide d'enzymes et d'un stérilisateur continu Download PDF

Info

Publication number
WO2017182665A1
WO2017182665A1 PCT/EP2017/059576 EP2017059576W WO2017182665A1 WO 2017182665 A1 WO2017182665 A1 WO 2017182665A1 EP 2017059576 W EP2017059576 W EP 2017059576W WO 2017182665 A1 WO2017182665 A1 WO 2017182665A1
Authority
WO
WIPO (PCT)
Prior art keywords
minutes
enzyme
process according
oil
digester
Prior art date
Application number
PCT/EP2017/059576
Other languages
English (en)
Inventor
Martin Rushworth
Original Assignee
Novozymes A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to AU2017253501A priority Critical patent/AU2017253501B2/en
Publication of WO2017182665A1 publication Critical patent/WO2017182665A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/04—Pretreatment of vegetable raw material
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/06—Production of fats or fatty oils from raw materials by pressing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to a process for extraction of crude palm oil.
  • the present invention relates to a process for extraction of crude palm oil, comprising the use of a continuous sterilizer and an enzyme composition.
  • the invention also relates to a palm oil obtainable by the process and to a milling line comprising a continuous sterilizer and means for dosing an enzyme composition.
  • 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:
  • Palm fruit mesocarp contains large amounts of oil present as oil droplets within the mesocarp cells.
  • 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.
  • OER oil extraction rate
  • the palm oil milling process has been carefully optimized at each mill in order to minimize oil losses to the extent possible.
  • sterilization of the FFB is carried out in a batch process, in which the fruit bunches are loaded into cages or carriages, which run on railway tracks and are pushed into and out of the sterilizers, either manually or by use of winches.
  • the sterilizers are large pressure chambers in which the fruit bunches are cooked in batches using steam at approximately 40 psi. After batch sterilization, the fruit bunches are conveyed to the thresher or stripper.
  • the traditional batch sterilization process is problematic for several reasons: in particular the batch sterilizers, the necessary railway tracks and the equipment for loading and unloading of the carts or carriages are space consuming and may occupy as much as 70% of the entire floorage of a palm oil mill.
  • the process is also labor-consuming and has several safety hazard due to the use of pressurized steam.
  • the equipment requires frequent shut-downs for maintenance and safety inspections.
  • continuous sterilizers also leads to a more wet milling process: first of all, the use of lower temperature and ambient pressure increases the water content of the sterilized fruits as compared to batch sterilization. Furthermore, continuous sterilizers do not increase the "stripability" of the palm fruit bunches to the same extent as batch sterilizers, so the number of fruitlets remaining attached to the fruit bunch stalk after threshing or stripping is relatively high. As a consequence, one must subject the empty fruit bunches coming out of the thresher or stripper to pressing, to recover oil that would otherwise be lost with the empty fruit bunches. Pressing of the empty fruit bunches, produces a liqueur containing oil and large amounts of water, which then needs to be brought back into the milling process, if the oil is to be recovered.
  • the present invention relates to palm oil milling using enzymes and milling equipment comprising a continuous sterilizer.
  • the present invention provides a process for extraction of crude palm oil CPO, comprising sterilizing fresh fruit bunches (FFB) in a continuous sterilizer and contacting mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition.
  • FFB fresh fruit bunches
  • MPD digester
  • the invention provides a palm oil obtainable by the process according to the invention.
  • the invention provides a palm oil milling line comprising a continuous sterilizer and means for dosing an enzyme composition.
  • 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 % TWEENTM 20 (polyoxyethylene sorbitan monolaurate).
  • One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • 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 ⁇ of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40°C.
  • 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.
  • beta-glucosidase is defined as 1 .0 ⁇ 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 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.
  • beta-xylosidase is defined as 1.0 ⁇ 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 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.
  • E.C. 3.2.1 .91 and E.C. 3.2.1.176 catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer
  • 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 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.
  • PCS pretreated corn stover
  • Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnS0 4 , 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 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.
  • the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.
  • the cellulosic material is any biomass material.
  • the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.
  • Crude oil refers to a pressed or extracted oil or a mixture thereof.
  • the oil is palm oil, in particular un-refined palm oil.
  • 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 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.
  • CMC carboxymethyl cellulose
  • 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 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • 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.
  • 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.
  • 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.
  • GHs glycoside hydrolases
  • CEs carbohydrate esterases
  • 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.
  • 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
  • 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 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 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, poly
  • 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).
  • DNS 3,5-dinitrosalicylic acid
  • 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 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 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 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 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).
  • 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, Interlaboratory 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 ⁇ 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.
  • PBAH p- hydroxybenzoic acid hydrazide
  • 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 ⁇ 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 is the waste water discharged e.g. from the sterilization process, crude oil clarification process.
  • Oil extraction rate (OER):
  • Oil extraction rate (OER) may be defined as by Chang et al., oil palm Industry economic journal, volume 3, 2003[9].
  • 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.
  • the buffers, substrate and assay principle disclosed below, in the definition of "thermostability" may be used in a determination of temperature optimum.
  • 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.
  • an appropriate buffer e.g. 0.1 M Na-OAc buffer pH 5.0
  • time periods i.e. 0 min, 20 min, 40 min and 60 minutes
  • 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
  • CMC carboxymethyl 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.
  • FIG. 1 Schematic representation of a continuous sterilizer.
  • the continuous sterilizer comprises a crusher (A), depicted as a double roll crusher, a conveyor (B), a first steam lock (C), a sterilizing chamber (D) which has conveying means and steam injection nozzles (E) and (F), a second steam lock (G) and a discharge conveyor (H).
  • A crusher
  • C first steam lock
  • D sterilizing chamber
  • E steam injection nozzles
  • G second steam lock
  • H discharge conveyor
  • FIG. 2 Schematic representation of a production line in a commercial palm oil mill from thresher to screw press.
  • the production line includes: Thresher (I), Conveyor(s) (J), (K) and (L), Digester (M) and Screw press (N).
  • Enzyme application is shown at points (O); including water dosing (S) and enzyme dosing (R).
  • Steam supply to the digester is shown as (P) and dilute crude oil (DCO) exit from screw press is shown as (Q).
  • FIG. 3 Palm oil milling equipment configuration comprising pre-cooker.
  • the equipment comprises a thresher or stripper (A), a pre-cooker (R), such as a vertical pre-cooker, a digester (M) with steam supply (P), such as a vertical digester, and a screw press (N), connected in a series.
  • A thresher or stripper
  • R pre-cooker
  • M digester
  • P steam supply
  • N screw press
  • Palm oil milling equipment comprising a continuous sterilizer, which comprises a crusher (A), such as a double roll crusher, a conveyor (B), such as a chain-type conveyor, a first steam lock
  • C sterilizing chamber
  • D sterilizing chamber
  • E sterilizing means and steam injection nozzles
  • G second steam lock
  • H discharge conveyor
  • I thresher or stripper
  • M digester
  • P steam supply
  • N screw press
  • FIG. 5 Palm oil milling equipment comprising a continuous sterilizer and pre-cooker.
  • the equipment comprises a continuous sterilizer, which comprises a crusher (A), such as a double roll crusher, a conveyor (B), such as a chain-type conveyor, a first steam lock (C), a sterilizing chamber
  • A crusher
  • B conveyor
  • C first steam lock
  • the present invention relates to a process for extraction of crude palm oil using enzymes, and in addition, the process according to the invention involves sterilizing fruit bunches in a sterilizer using steam at low or atmospheric pressure such as a continuous sterilizer.
  • the present inventors have surprisingly found that oil extraction efficiency is maintained with enzyme treatment even at a relatively high content of moisture in the fruit mash or press liquid (dilute crude oil (DCO)). Furthermore, the inventors found that amount of emulsion formed during clarification decreases with enzyme treatment at higher %moisture, indicating that enzymes is able to break down emulsion even at relatively high %moisture, especially from 46- 64 %moisture
  • 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).
  • SC sterilizer condensate
  • UDCO undilute crude oil
  • DCO dilute crude oil
  • the inventors have found that the % FFA in the final oil is less than 5%.
  • Sterilization is the first step in the process, which is crucial to the final oil quality as well as the strippability of fruits. Steam sterilization of the FFBs 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.
  • the palm fruits are subject to stripping, digestion and pressing. Stripping or threshing is generally carried out in a mechanized system having a rotating drum or fixed drum equipped with rotary beater bars detach the fruit from the bunch, leaving the spikelets on the stem.
  • the palm fruitlets also referred to as "mass passing to digester" (“MPD")
  • M mass passing to digester
  • the fruitlets are further reheated to loosen the pericarp.
  • the digester is typically a steam heated vessels, which has rotating shafts to which are attached stirring arms. The fruitlets are rotated about, causing the loosening of the pericarps from the nuts and degradation of the mesocarp.
  • the digester is kept full and as the digested fruit is drawn out, freshly stripped fruits are brought in.
  • the digested mass is passed into a screw press, from which a mixture of oil, water, press cake or fibre and nuts are discharged.
  • the mixture of oil, water and solids (Undiluted Dilute Crude Oil, (UDCO) or Diluted Crude Oil (DCO)) from the fruitlets is delivered from the press to a clarification tank for further processing.
  • UDCO Undiluted Dilute Crude Oil,
  • DCO Diluted Crude Oil
  • the present invention provides a process for extraction of crude palm oil CPO, comprising sterilizing fresh fruit bunches (FFB) in a continuous sterilizer and contacting mass passing to digester (MPD), fruit mash and/or crude oil with an enzyme composition.
  • FFB fresh fruit bunches
  • MPD digester
  • the FFBs are subject to conditioning prior to sterilization, so as to loosen the arrangement of spikelets in the FFB; e.g. by use of a crusher or bunch splitter, such as a double-roll crusher.
  • the FFB is then fed into the Continuous Sterilizer, for instance via a sealed scrapper bar type feeding conveyor.
  • This conveyor may be designed to take the advantage of excess steam from the Continuous Sterilizer to preheat as well as to evacuate the air from the conditioned FFB. In doing so, it contains and controls the excessive steam from leaking into the atmosphere and pre-heats the conditioned FFB at the same time.
  • the said continuous sterilizer comprises one or more non- pressurized chamber(s) fitted with steam injection nozzles and means for conveying the FFB through the chamber.
  • the said means for conveying may in particular be a chain-type conveyor.
  • the continuous sterilizer has a configuration as shown in Figure 1 , comprising a crusher/bunch splitter (A), such as a double roll crusher, one or more conveyor(s) (B), such as one or more chain-type conveyor(s), a first steam lock (C), one or more sterilizing chamber(s) (D) with conveying means and steam injection nozzles (E) and (F), a second steam lock (G), and optionally a discharge conveyor (H).
  • A crusher/bunch splitter
  • B such as one or more chain-type conveyor(s
  • C first steam lock
  • D sterilizing chamber(s)
  • E sterilizing chamber(s)
  • G second steam lock
  • H optionally a discharge conveyor
  • the crude oil may in particular be extracted from sterilized fruit using milling equipment configured as shown in Figure 2, said equipment comprising a thresher or stripper (I), a digester (M) with steam supply (P), such as a vertical digester, and a screw press (N), connected in a series.
  • a thresher or stripper I
  • M digester
  • P steam supply
  • N screw press
  • the crude oil is extracted from sterilized fruit using milling equipment configured as shown in Figure 3, said equipment comprising a thresher or stripper (A), a pre-cooker (R), such as a vertical pre-cooker, a digester (M) with steam supply (P), such as a vertical digester, and a screw press (N), connected in a series.
  • a thresher or stripper A
  • R pre-cooker
  • M digester
  • P steam supply
  • N screw press
  • the crude oil is extracted using milling equipment configured as shown in Figure 4, said equipment comprising a continuous sterilizer, which comprises a crusher (A), such as a double roll crusher, a conveyor (B), such as a chain-type conveyor, a first steam lock (C), a sterilizing chamber (D) with conveying means and steam injection nozzles (E) and (F), a second steam lock (G), and optionally a discharge conveyor (H), linked to a thresher or stripper (I), a digester (M) with steam supply (P), such as a vertical digester, and a screw press (N), which are serially connected; e.g. by means of conveyors (J), (K), and/or (L).
  • a crusher such as a double roll crusher
  • a conveyor such as a chain-type conveyor
  • a first steam lock (C) a sterilizing chamber (D) with conveying means and steam injection nozzles (E) and (F)
  • a second steam lock G
  • 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.
  • the exit of the screw press is fluidically connected to downstream equipment for separation of oil from water and sludge.
  • the crude oil may be extracted using milling equipment configured as shown in Figure 5, said equipment comprising a continuous sterilizer, which comprises a crusher (A), such as a double roll crusher, a conveyor (B), such as a chain-type conveyor, a first steam lock (C), a sterilizing chamber (D) with conveying means and steam injection nozzles (E) and (F), a second steam lock (G), and optionally a discharge conveyor (H), linked to a thresher or stripper (I), a pre-cooker (T), such as a vertical pre-cooker, a digester (M) with steam supply (P), such as a vertical digester, and a screw press (N), which are serially connected; e.g. by means of conveyors (J), (K), and/or (L).
  • a crusher such as a double roll crusher
  • a conveyor such as a chain-type conveyor
  • a first steam lock (C) a sterilizing chamber (D) with conveying means and steam injection nozzles
  • the sterilization in said continuous sterilizer may proceed from 60 to 120 minutes, such as from 80 to 120 minutes, from 80-1 10 minutes, from between 80 minutes to 100 minutes, or such as from 80-90 minutes.
  • the enzyme composition used in the process according to the invention may comprise one or more hydrolases, such as one or more cellulases.
  • the cellulases are optionally in combination with one or more hemicellulases, one or more pectinases, one or more amylases, or a combination thereof.
  • the enzyme composition used in the process of the invention comprises a commercial cellulolytic enzyme preparation.
  • 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), CELLUCLASTTM (Novozymes A/S), NOVOZYMTM 188 (Novozymes A/S), SPEZYMETM CP (Genencor Int.), ACCELERASETM TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENTTM 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.
  • 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.
  • Renozyme® Commercially used cellulases include Renozyme®, Celluzyme®, Celluclean®, Endolase® and Carezyme®. (Novozymes A/S), Clazinase®, and Puradax HA®. (Genencor Int. Inc), and KAC- 500(B)TM (Kao Corporation).
  • 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, SHEARZYMETM (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), DEPOLTM 333P (Biocatalysts Limit, Wales, UK), DEPOLTM 740L. (Biocatalysts Limit, Wales, UK), and DEPOLTM 762P (Biocatalys
  • the said enzyme composition may comprise one or more cellulases and/or one or more amylases and/or one or more pectinases.
  • the process according to the invention comprises
  • 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 and from cell debris and fibrous material, which is also present in the liquid which is obtained by pressing of the mashed palm fruitlets.
  • the enzyme composition may be applied to a substrate selected from the group consisting of palm fruitlets, mass passing to digester (MPD), mashed or partly mashed palm fruitlets or MPD, and palm press liquid, such as DCO or UDCO.
  • the enzyme composition or the one or more enzymes may be 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 (I) on one or more conveyors; e.g. conveyors (J), (K) and/or (L) in figure 2.
  • a digester e.g. conveyors (J), (K) and/or (L) in figure 2.
  • the enzyme composition or the one or more enzymes are applied in step iv).
  • the enzyme composition or the one or more enzymes are applied after step v) and before the oil in said crude oil is separated from the water and from the cell debris, and/or the fibrous material.
  • 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 (N) and/or by controlling steam supply (P).
  • 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 (N) and/or by controlling steam supply (P).
  • N screw press
  • P controlling steam supply
  • the process according to the invention may comprise the steps of:
  • 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,
  • 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
  • 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.
  • FFBs fresh fruit bunches
  • the enzyme composition may also be dosed 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.
  • the palm fruitlet, MPD, fruit mash or 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 process may in some embodiments comprise retaining the palm fruitlets or MPD, fruit mash or crude 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.
  • the process according to the invention comprises retaining the palm fruitlets or MPD in a pre-cooker at a temperature between 40 and 85°C, such as between 50 and 85°C, such as between 65 and 85°C, such as between 60 and 85°C, or such as between 70 and 80°C.
  • the palm fruitlets or MPD are retained in a precooker together with enzyme at a temperature between 65 and 75°C.
  • 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.
  • the retention time in the pre-cooker may be even shorter.
  • 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.
  • the enzyme or enzyme composition such as the one or more cellulases, one or more hemicellulases, one or more pectinases, and/or one or more amylases, may have 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
  • the one or more cellulases, one or more hemicellulases, one or more pectinases and/or one or more amylases may be 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.
  • 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, 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
  • substrate e.g. carboxymethyl cellulose (CMC) form reducing carbohydrate
  • a further aspect of the invention provides palm oil obtainable by the method according to the invention.
  • a still further aspect of the invention provides a palm oil milling line comprising a continuous sterilizer, e.g. a continuous sterilizer as defined in any of the above embodiments, and means for dosing an enzyme composition.
  • the means for dosing an enzyme composition are for dosing the enzyme composition to MPD being conveyed to a digester, for dosing the enzyme composition into a digester and/or for dosing the enzyme composition to a crude oil, such as an undiluted crude oil (UDCO) or a diluted crude oil (DCO).
  • a crude oil such as an undiluted crude oil (UDCO) or a diluted crude oil (DCO).
  • the palm oil milling line according to the invention is configured as shown in Figure 4, said milling line comprising
  • a continuous sterilizer which comprises: i) A crusher or bunch splitter (A), such as a double roll crusher,
  • a conveyor (B) such as a chain-type conveyor,
  • the palm oil milling line is configured as shown in Figure 5, said milling line comprising
  • a continuous sterilizer which comprises:
  • a conveyor (B) such as a chain-type conveyor,
  • a pre-cooker such as a vertical pre-cooker
  • Example 1 Effect of dilution on Oil recovery in downstream from UDCO. Experimental procedure:
  • the UDCO (28 %moisture and 65%oil) was aliquoted in 50ml graduated Falcon tubes and diluted with hot water (HW) to make final volume to 40ml such that the final sample %Moisture ranged from 18% to 82% as shown in table 1 .
  • Enzymes were added (Comprising Cellic® CTec 2 and Cellic®Htec, both commercially available from Novzymes A S) at 0.25ml 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
  • Example 2 Use of cellulolytic enzymes at a continuous sterilizer palm oil mill.
  • the enzyme (Palmora®OER, commercially available from Novozymes A/S) was dosed in amounts corresponding to 37.95 mg Enzyme protein per metric ton of FFB by spraying onto the mass passing to digester (MPD) during conveyance from thresher to pre-cooker and digester.
  • MPD mass passing to 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.
  • Oil content in underflow and heavy phase was determined by Soxhiet analysis according to the following procedure:
  • Samples were prepared by first drying them in hot air oven for 8-12 hrs at 105°C. Soxhiet 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.

Abstract

La présente invention concerne un procédé d'extraction d'huile de palme brute consistant à utiliser un stérilisateur continu et une composition enzymatique. L'invention concerne également une huile de palme pouvant être obtenue par ledit procédé et un circuit de broyage comprenant un stérilisateur continu et un moyen pour doser une composition enzymatique.
PCT/EP2017/059576 2016-04-22 2017-04-21 Extraction d'huile de palme à l'aide d'enzymes et d'un stérilisateur continu WO2017182665A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2017253501A AU2017253501B2 (en) 2016-04-22 2017-04-21 Enzyme assisted palm oil extraction with continuous sterilizer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
MYPI2016701498 2016-04-22
MYPI2016701497 2016-04-22
MYPI2016701498 2016-04-22
MYPI2016701497 2016-04-22

Publications (1)

Publication Number Publication Date
WO2017182665A1 true WO2017182665A1 (fr) 2017-10-26

Family

ID=58638847

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2017/059576 WO2017182665A1 (fr) 2016-04-22 2017-04-21 Extraction d'huile de palme à l'aide d'enzymes et d'un stérilisateur continu
PCT/EP2017/059579 WO2017182667A1 (fr) 2016-04-22 2017-04-21 Extraction d'huile assistée par enzyme utilisant un condensat de stérilisateur

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/059579 WO2017182667A1 (fr) 2016-04-22 2017-04-21 Extraction d'huile assistée par enzyme utilisant un condensat de stérilisateur

Country Status (3)

Country Link
AU (2) AU2017253501B2 (fr)
MY (1) MY197084A (fr)
WO (2) WO2017182665A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019209100A1 (fr) * 2018-04-25 2019-10-31 Sime Darby Plantation Intellectual Property Sdn. Bhd. Procédé de production d'huile brute de fruit de palmier
WO2019216757A1 (fr) * 2018-05-08 2019-11-14 Sime Darby Plantation Intellectual Property Sdn. Bhd. Appareil d'extraction d'huile intégré pour stériliser, digérer et presser des fruits égrapés de palmier à huile
WO2020141966A1 (fr) * 2019-01-03 2020-07-09 Palmite Process Engineering Sdn Bhd Procédé de stérilisation continue de régimes de noix fraîches de palmier à huile
WO2020225801A1 (fr) * 2019-05-03 2020-11-12 Carlos Enrique Rey Melendez Équipement pour l'extraction d'huile de palme par un stérilisateur/digesteur continu

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022182225A1 (fr) * 2021-02-26 2022-09-01 Sime Darby Plantation Intellectual Property Sdn Bhd Procédé de broyage d'huile de palme pour observer, annoter, classifier et séparer des grappes non dénudées
CN113026409A (zh) * 2021-03-29 2021-06-25 边静 一种利用棕榈纤维制生物无硫半化学浆的方法

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
US5039455A (en) * 1989-07-31 1991-08-13 Kooi Boon Lam Process for continuous extraction of palm oil or vegetable edible oil
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
EP0531315A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Enzyme capable de degrader la cellulose ou l"hemicellulose.
WO1994007998A1 (fr) 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1995024471A1 (fr) 1994-03-08 1995-09-14 Novo Nordisk A/S Nouvelles cellulases alcalines
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO1998012307A1 (fr) 1996-09-17 1998-03-26 Novo Nordisk A/S Variants de cellulase
WO2009017389A2 (fr) * 2007-08-01 2009-02-05 Malaysian Palm Oil Board Procédé d'extraction d'huile de palme brute
WO2012011130A2 (fr) 2010-07-23 2012-01-26 Advnaced Enzyme Technologies Ltd. Composition enzymatique et procédé pour l'extraction d'huile à partir des fruits du palmier à huile
AU2015101377A4 (en) 2014-12-19 2015-11-05 Dupont Nutrition Biosciences Aps Method of treating a palm fruit or a palm fruit liquid
WO2016097266A1 (fr) 2014-12-19 2016-06-23 Dupont Nutrition Biosciences Aps Procédé pour améliorer les rendements en huile de palme obtenue à partir du fruit du palmier ou d'un liquide provenant du fruit du palmier
WO2016162507A1 (fr) * 2015-04-08 2016-10-13 Novozymes A/S Procédé d'extraction d'huile de palme à l'aide d'enzymes

Patent Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691178A (en) 1988-03-22 1997-11-25 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
US5776757A (en) 1988-03-24 1998-07-07 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase and method of making thereof
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
US5039455A (en) * 1989-07-31 1991-08-13 Kooi Boon Lam Process for continuous extraction of palm oil or vegetable edible oil
US5686593A (en) 1990-05-09 1997-11-11 Novo Nordisk A/S Enzyme capable of degrading cellulose or hemicellulose
EP0531315A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Enzyme capable de degrader la cellulose ou l"hemicellulose.
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
US5763254A (en) 1990-05-09 1998-06-09 Novo Nordisk A/S Enzyme capable of degrading cellulose or hemicellulose
US5457046A (en) 1990-05-09 1995-10-10 Novo Nordisk A/S Enzyme capable of degrading cellullose or hemicellulose
WO1994007998A1 (fr) 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1995024471A1 (fr) 1994-03-08 1995-09-14 Novo Nordisk A/S Nouvelles cellulases alcalines
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO1998012307A1 (fr) 1996-09-17 1998-03-26 Novo Nordisk A/S Variants de cellulase
WO2009017389A2 (fr) * 2007-08-01 2009-02-05 Malaysian Palm Oil Board Procédé d'extraction d'huile de palme brute
WO2012011130A2 (fr) 2010-07-23 2012-01-26 Advnaced Enzyme Technologies Ltd. Composition enzymatique et procédé pour l'extraction d'huile à partir des fruits du palmier à huile
AU2015101377A4 (en) 2014-12-19 2015-11-05 Dupont Nutrition Biosciences Aps Method of treating a palm fruit or a palm fruit liquid
WO2016097266A1 (fr) 2014-12-19 2016-06-23 Dupont Nutrition Biosciences Aps Procédé pour améliorer les rendements en huile de palme obtenue à partir du fruit du palmier ou d'un liquide provenant du fruit du palmier
WO2016162507A1 (fr) * 2015-04-08 2016-10-13 Novozymes A/S Procédé d'extraction d'huile de palme à l'aide d'enzymes

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
BAILEY ET AL.: "Interlaboratory testing of methods for assay of xylanase activity", JOURNAL OF BIOTECHNOLOGY, vol. 23, no. 3, 1992, pages 257 - 270, XP023704921, DOI: doi:10.1016/0168-1656(92)90074-J
BIELY; PUCHARD, JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, vol. 86, no. 11, 2006, pages 1636 - 1647
CHANG ET AL., OIL PALM INDUSTRY ECONOMIC JOURNAL, vol. 3, 2003
DE VRIES, J. BACTERIOL., vol. 180, 1998, pages 243 - 249
EBRINGEROVA ET AL., ADV. POLYM. SCI., vol. 186, 2005, pages 1 - 67
GHOSE, PURE AND APPL. CHEM, vol. 59, 1987, pages 257 - 268
GHOSE, PURE APPL. CHEM., vol. 59, 1987, pages 257 - 268
GHOSE; BISARIA, PURE & APPL. CHEM, vol. 59, 1987, pages 1739 - 1752
HERRIMANN ET AL., BIOCHEMICAL JOURNAL, vol. 321, 1997, pages 375 - 381
JAYANI, R.S. ET AL: "Microbial pectinolytic enzymes: a review", PROCESS BIOCHEMISTRY, LONDON, vol. 40, 2005, pages 2931 - 2944, XP025306710, DOI: doi:10.1016/j.procbio.2005.03.026
JOSEPH D. TELLER: "Measurement of amylase acitivity", J. BIOL. CHEM., vol. 185, 1950, pages 701 - 704
LEMKE GONZALEZ ET AL.: "Pectin methylesterase activity determined by different methods and thermal inactivation of exogenous pme in mango juice", CIENC. AGROTEC., vol. 35, no. 5, September 2011 (2011-09-01)
LEVER ET AL., ANAL. BIOCHEM., vol. 47, 1972, pages 273 - 279
LEVER, ANAL. BIOCHEM., vol. 47, 1972, pages 273 - 279
LYND, APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 24/25, 1990, pages 695 - 719
MOSIER ET AL.: "Advances in Biochemical Engineering/Biotechnology", vol. 65, 1999, SPRINGER-VERLAG, article "Recent Progress in Bioconversion of Lignocellulosics", pages: 23 - 40
SHALLOM; SHOHAM, CURRENT OPINION IN MICROBIOLOGY, vol. 6, no. 3, 2003, pages 219 - 228
SPANIKOVA; BIELY, FEBS LETTERS, vol. 580, no. 19, 2006, pages 4597 - 4601
TEERI ET AL., BIOCHEM. SOC. TRANS., vol. 26, 1998, pages 173 - 178
TEERI, TRENDS IN BIOTECHNOLOGY, vol. 15, 1997, pages 160 - 167
TOMME ET AL., EUR. J. BIOCHEM., vol. 170, 1988, pages 575 - 581
VAN TILBEURGH ET AL., FEBS LETTERS, vol. 149, 1982, pages 152 - 156
VAN TILBEURGH; CLAEYSSENS, FEBS LETTERS, vol. 187, 1985, pages 283 - 288
VENTURI ET AL., J. BASIC MICROBIOL, vol. 42, 2002, pages 55 - 66
WISELOGEL ET AL.: "Handbook on Bioethanol", 1995, TAYLOR & FRANCIS, pages: 105 - 118
WYMAN, BIORESOURCE TECHNOLOGY, vol. 50, 1994, pages 3 - 16
ZHANG ET AL., BIOTECHNOLOGY ADVANCES, vol. 24, 2006, pages 452 - 481

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019209100A1 (fr) * 2018-04-25 2019-10-31 Sime Darby Plantation Intellectual Property Sdn. Bhd. Procédé de production d'huile brute de fruit de palmier
WO2019216757A1 (fr) * 2018-05-08 2019-11-14 Sime Darby Plantation Intellectual Property Sdn. Bhd. Appareil d'extraction d'huile intégré pour stériliser, digérer et presser des fruits égrapés de palmier à huile
WO2020141966A1 (fr) * 2019-01-03 2020-07-09 Palmite Process Engineering Sdn Bhd Procédé de stérilisation continue de régimes de noix fraîches de palmier à huile
WO2020225801A1 (fr) * 2019-05-03 2020-11-12 Carlos Enrique Rey Melendez Équipement pour l'extraction d'huile de palme par un stérilisateur/digesteur continu

Also Published As

Publication number Publication date
AU2017252228B2 (en) 2021-12-02
AU2017252228A1 (en) 2018-10-25
AU2017253501B2 (en) 2022-01-20
WO2017182667A1 (fr) 2017-10-26
MY197084A (en) 2023-05-24
AU2017253501A1 (en) 2018-10-25

Similar Documents

Publication Publication Date Title
AU2017253501B2 (en) Enzyme assisted palm oil extraction with continuous sterilizer
US11180786B2 (en) GH10 xylanase, GH62 arabinofuranosidase, milling process and other application
CN102791853B (zh) 新型糖基水解酶及其用途
US11649298B2 (en) Wet milling process
EP3511418B1 (fr) Corollospora maritima mannanase et son utilisation
US20170327855A1 (en) Milling Process
US20220297171A1 (en) Processes For Solubilizing Municipal Solid Waste With Enzyme Compositions Comprising Protease And Enzyme Compositions Thereof
AU2016245218B2 (en) Process for extraction of palm oil using enzymes
AU2016101771A4 (en) Extraction of crude palm oil using enzymes
AU2016245215B2 (en) Process for extraction of palm oil using enzymes
US20230405653A1 (en) Method for sanitizing waste
AU2018344497A1 (en) Enzyme assisted crude palm oil extraction
EP3390650A1 (fr) Procédé de production de sucre réducteur à partir de substrats lignocellulosiques
AU2018319349B2 (en) Enzyme assisted crude palm oil extraction
WO2017182666A1 (fr) Utilisation de la phospholipase c pour le broyage d'huile de palme
WO2018233559A1 (fr) Procédé pour augmenter le pourcentage de xylose d'hydrolysat
WO2021198077A1 (fr) Procédé pour récupérer de l'huile de palme brute
EP4176068A1 (fr) Procédé pour améliorer le rendement en huile à partir de germe dans un procédé de broyage humide

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017253501

Country of ref document: AU

Date of ref document: 20170421

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17720422

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17720422

Country of ref document: EP

Kind code of ref document: A1