WO2016162510A1 - Procédé d'extraction d'huile de palme à l'aide d'enzymes - Google Patents

Procédé d'extraction d'huile de palme à l'aide d'enzymes Download PDF

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WO2016162510A1
WO2016162510A1 PCT/EP2016/057809 EP2016057809W WO2016162510A1 WO 2016162510 A1 WO2016162510 A1 WO 2016162510A1 EP 2016057809 W EP2016057809 W EP 2016057809W WO 2016162510 A1 WO2016162510 A1 WO 2016162510A1
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minutes
palm
enzyme
fruitlets
process according
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PCT/EP2016/057809
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Arnab GUHA
Aindrila Dasgupta
Harsha Ramakrishna
Harinee Desikan
Martin Rushworth
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Novozymes A/S
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    • 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 processes for extraction of palm oil from palm fruit bunches using enzymes.
  • the present invention relates to a process for extraction of palm oil (such as crude palm oil) from fruit bunches with an enzyme composition.
  • the present invention relates to a process for extraction of palm oil from fruit bunches with an enzyme composition at low temperature.
  • the invention also relates to such compositions, for use in processes for extraction of crude palm oil.
  • Crude 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.
  • Crude palm oil extraction process can be generalized by the following major steps i.e. sterilizing the fresh palm fruit bunches, conveying the sterilized fruits in cages and subsequently discharging them into vessels commonly referred to as digesters, digesting the fruits to produce a digested mash under controlled temperature, pressing of the digested mash by screw press for subsequent recovery of oil and subjecting the pressed liquor to vibration screen and then the clarification process for production of crude palm oil (CPO).
  • CPO crude palm oil
  • the expressed crude palm oil from the screw press is pumped via vibrating screens, a decanter and sometimes a skimmer tank to a clarification tank. During this part of the process more hot water is added, typically in the exit from the screw press, to reduce viscosity for more effective oil and water separation.
  • the large amount of water needed in the conventional crude palm oil processing causes environmental concerns.
  • 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 industry has long been in pursuit of a higher oil yield.
  • mass balance calculations performed by the industry have repeatedly indicated that conventional processes for crude palm oil extraction are highly efficient and that less than 1 % oil remains and is lost in various side- or waste streams from the extraction process, including the palm oil mill effluent (POME).
  • POME palm oil mill effluent
  • the main object of the present invention is to provide a process for extraction of palm oil that would improve the yield of crude palm oil by enzymatic treatment to ensure higher recovery of oil from fresh fruit bunches.
  • Another important object of the present invention is to improve the oil and water separation efficiency and reduce the consumption of steam and power during the oil milling process, which can results in energy savings and a higher yield for the same process.
  • the present invention relates to a process for extraction of palm oil from palm fruitlets using an enzyme composition.
  • the invention provides a process for extraction of crude palm oil from palm fruitlets comprising the steps:
  • the enzyme composition comprises one or more cellulase(s) and a protease.
  • the invention relates to use of an enzyme composition comprising one or more cellulase(s) for extraction of crude palm oil from palm fruitlets.
  • the invention provides a crude palm oil obtained by a process or method according to the invention.
  • 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 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.
  • 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 et al., 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 (also called a non-degummed oil) a pressed or extracted oil or a mixture thereof from. In the present context it is to be understood that the oil is palm oil, in particular un-refined palm oil..
  • 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
  • Feruloyl esterase means a 4-hydroxy-3- methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy-3- methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).
  • Feruloyl esterase (FAE) is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II.
  • Feruloyl esterase activity can be determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0.
  • One unit of feruloyl 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 feruloyl 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. Chem.
  • 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.
  • protease means a polypeptide having protease activity.
  • protease activity is defined herein as a proteolytic activity which catalyzes the hydrolysis of the peptide bond connecting two amino acids in a peptide. Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question.
  • protease further includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof, these enzymes being in the following referred to as "belonging to the EC 3.4. -.-group").
  • the EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1 -5 published in Eur. J. Biochem. 1994, 223: 1 -5; Eur. J. Biochem. 1995, 232: 1 -6; Eur. J. Biochem. 1996, 237: 1 -5; Eur. J. Biochem. 1997, 250: 1 -6; and Eur. J. Biochem. 1999, 264: 610-650; respectively.
  • the nomenclature is regularly supplemented and updated; see, e.g., the World Wide Web at www.chem.qmw.ac.uk/iubmb/enzyme/index.html .
  • 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),
  • 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, andbeta-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 (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 and Fresh fruit branch (FFB) times 100. According to this definition, the mathematical formula is:
  • OER (weight of oil recovered/weight of FFB processed) x 100
  • 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 or the protease 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
  • elevated temperature e.g. 70 degrees Celsius
  • 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
  • CMC carboxymethyl cellulose
  • the reaction is stopped by an alkaline reagent containing PAHBAH 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.
  • reference to "about” a value or parameter herein includes aspects that are directed to that value or parameter per se.
  • description referring to "about X” includes the aspect "X”.
  • Figure 1 Mass balance data of control run in a commercial palm oil mill.
  • FIG. 2 Schematic representation of a production line in a commercial palm oil mill from thresher to screw press.
  • the production line includes: Thresher (A), Conveyor(s) (B), (C) and (D), Digester (E) and Screw press (F).
  • Enzyme application is shown at point (G); including water dosing (H) and enzyme dosing (I).
  • Steam supply to the digester is shown as (J) and dilute crude oil (DCO) exit from screw press is shown as (K).
  • the present invention relates to a process for extraction of palm oil from palm fruitlets using an enzyme composition.
  • palm oil is extracted from fresh fruit bunches (FFB) by a thermo- mechanical process.
  • a typical mill has many operations which include sterilization, stripping, digestion and pressing, clarification, purification, drying and storage.
  • the present inventors have surprisingly found that despite the steep temperature gradients in industrial palm oil milling processes, and the high temperatures reached, the temperatures are in fact approaching the ideal conditions for many hydrolytic enzymes (approximately 70°C; e.g. 65-80°C) for the majority of the period during which palm fruit is passed from the thresher, to the digester and through the digester to reach the screw press.
  • 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.
  • stripping, digestion and pressing may be performed using equipment in a configuration as outlined in Figure 1 .
  • Stripping or threshing is 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 (A).
  • the palm fruitlets also referred to as "mass passing to digester" (“MPD")
  • MPD 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 (F), 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.
  • the fruitlets coming out of the sterilization step have a temperature of up to 95°C; in some instances 95°C or above.
  • the palm fruitlets start to cool and the temperature of the fruitlets decreases to about 45-65°C; e.g. 50-65°C or 45-55°C, as the threshed mass of fruitlets or mass passing to digester (MPD) passes towards the digester by means of one or more conveyors.
  • the digester is supplied with continuous steam where the fruitlets are again reheated to temperatures of above 90°C for extraction of oil.
  • the temperature in the upper one third of digester is about 45-65°C; e.g. 50-65°C or 45-55°C; in the middle one third of digester it is about 75°C and in the lower one third of the digester it is above 90°C.
  • the present inventors have observed that when carefully determining the mass balance of palm oil extraction, for instance by performing double Soxtherm analyses to determine the amount of oil lost in various side- or waste streams, it appears that the oil loss in a conventional palm oil extraction process is substantially greater than previously believed. Hence, contrary to what is currently accepted in the field of palm oil extraction there is indeed a potential for increasing the oil extraction rate by further improving the process of extracting oil from the palm fruitlet mesocarp.
  • the inventors have found that, despite the steep temperature changes and the high temperatures reached in palm oil milling the temperatures during conveyance from the thresher to the digester and through the digester to the screw press do in fact approach the ideal conditions for most enzyme products that would be useful in palm oil extraction.
  • palm oil is highly viscous and the requirement for high temperatures in the digester zone is partly in order to reduce the viscosity of the oil during pressing and subsequent separation of oil from water.
  • the present invention relates to a process for extraction of palm oil from palm fruitlets comprising steps of: contacting the palm fruitlet with an enzyme composition at a temperature of above 65°C and extracting the crude palm oil.
  • the invention provides a process for extraction of crude palm oil from palm fruitlets comprising the steps:
  • the enzyme composition comprises one or more cellulase(s) and a protease.
  • the enzyme composition may further comprises a hemicellulase, an amylase, a pectinase, or combination thereof.
  • the oil palm is from the genus Elaeis.
  • the inventors have found that extraction of oil with an enzyme composition is achieved at a temperature of about 65°C to about 85°C, such as a temperature within the range of 65°C to 85°C at the lower one third of the digester, and that this is very effective in providing a high oil yield compared to the conventional process of extraction of oil.
  • the palm fruitlets coming out of sterilization step have a temperature of up to 95°C (in some instances 95°C or above).
  • the palm fruitlets start to cool and the temperature of the fruitlets cools down to about 45-65°C; such as in the range of 45-55°C; e.g. in the range of 50-60°C or typically 55°C, as the threshed mass of fruitlets passes into the digester by means of a conveying system.
  • the digester is supplied with continuous steam where the fruitlets are again reheated for extraction of oil.
  • the temperature in the upper one third of digester is about 45- 55°C; such as in the range of 45-55°C, e.g.
  • the temperatures ranges in each zone within the digester may be controlled as needed, by injecting more or less steam.
  • the process of contacting the palm fruitlet with an enzyme composition is done at a temperature of above 65°C, which would be the temperatures reached in the middle and lower third of the digester.
  • contacting of palm fruitlet with an enzyme composition can be at a temperature of 66-90°C, such as at a temperature of 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, 67-74°
  • contacting of palm fruitlet with an enzyme composition is done in presence of water.
  • the water used during the contact of palm fruitlet with an enzyme composition may comprise distilled water, sterilized water and/ or liquid condensate. In preferred embodiments, however, the water used in the process is tap water. In the process according to the invention, the water is preferably pre-heated to a temperature within the range of 65-80°C.
  • the enzyme composition used in the process according to the invention is preferably an aqueous formulation.
  • the enzyme composition is heated to 50-70°C, such as to 55-70°C, to 50-65°C, or such as to 55-65°C, prior to contacting it with the palm fruitlets or MPD.
  • the enzyme composition is dosed in amounts corresponding to 20-500 mg enzyme protein/kg palm fruitlet, such as 20-450 mg enzyme protein/kg palm fruitlet, 20-400 mg enzyme protein/kg palm fruitlet, 20-350 mg enzyme protein/kg palm fruitlet, 20-300 mg enzyme protein/kg palm fruitlet, 20-250 mg enzyme protein/kg palm fruitlet, 20-200 mg enzyme protein/kg palm fruitlet, 20-150 mg enzyme protein/kg palm fruitlet, 20-100 mg enzyme protein/kg palm fruitlet, 20-75 mg enzyme protein/kg palm fruitlet, 20-50 mg enzyme protein/kg palm fruitlet, 30-500 mg enzyme protein/kg palm fruitlet, 40-500 mg enzyme protein/kg palm fruitlet, 50-500 mg enzyme protein/kg palm fruitlet, 75-500 mg enzyme protein/kg palm fruitlet, 100-500 mg enzyme protein/kg palm fruitlet, 150-500 mg enzyme protein/kg palm fruitlet, 200- 500 mg
  • 20-500 mg enzyme protein/kg palm fruitlet such as 20-450 mg enzyme protein/kg palm fruitlet, 20-400 mg enzyme protein/kg
  • the enzyme(s) are dosed at amounts corresponding to 100-500 ppm, such as 200-500 ppm or 250-400 ppm.
  • contacting of palm fruitlet with an enzyme composition is done for a period of 5 minutes or above.
  • contacting is done for a period of less than 3 hours.
  • contacting of palm fruitlet with an enzyme composition is done 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, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 20-28 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes.
  • the contacting of mash palm fruitlet with an enzyme composition can be performed before or during the loading of palm fruitlet or MPD into the digester.
  • palm fruitlets may be bruised during threshing and conveyance, but any further disintegration, such as disintegration during maceration/pre-cooking step is preferably avoided, such that approximately 80%, preferably in the range of 30-90% of the palm fruitlets are substantially intact when arriving at the digester.
  • Contacting with enzyme composition onto substantially intact palm fruitlets i.e. "coating" the palm fruitlets with enzyme) reduces the phosphorous/phospholipid content in the crude oil as compared to mixing the enzyme composition with macerated fruitlets.
  • enzymes are mixed with substantially intact palm fruitlets; i.e. palm fruitlets which have not been subjected to maceration, less phospholipids are liberated together with the oil fraction as compared to treatment of palm fruitlets which have been subject to maceration before being contacted with enzyme and arriving at the digester.
  • the contacting of palm fruitlet or MPD with an enzyme composition is done when the mash, fruitlet or MPD is conveyed towards the digester.
  • the enzyme may alternatively be dosed directly into the digester, such that it is first contacted with the palm fruitlets in the upper one third of the digester.
  • the process comprises steps of: sterilizing and threshing fresh palm fruit bunches to provide palm fruitlets; and conveying the palm fruitlets into a digester.
  • the palm fruitlets are threshed and conveyed from threshing to a digester without being subject to disintegration other than the disintegration, which occurs during threshing and conveyance, such as without being subject to maceration/pre- cooking/mashing.
  • contacting e.g. contacting of palm fruitlets or MPD with enzyme, is done before or in the digester.
  • the palm fruitlets are contacted with the enzyme during conveyance from threshing to the digester, such as during transport of the fruitlets on a conveyer belt, in a screw conveyor or auger conveyor.
  • the palm fruitlets are contacted with the enzyme by distributing the enzyme onto the surface of the palm fruitlets, such as by sprinkling or spraying the enzyme onto the fruitlets, during conveyance.
  • enzymes are sprinkled or sprayed onto the palm fruitlets during conveyance to the digester, which leads to improved exposure of the palm fruitlets to enzyme and a more homogenous mixture as compared to mixing within the digester.
  • the skilled person would by default add the enzymes in the digester, without having realized that more even distribution of enzyme on the fruitlet surface could be obtained by sprinkling or spraying enzyme onto the fruitlet prior to entry into the digester.
  • a further advantage of applying the enzyme composition during conveyance of the fruitlets or MPD toward the digester is early penetration of the enzyme into the mesocarp through scratches or bruises on the exocarp. This helps in "positioning" the enzyme in the mesocarp and further reduces the reaction time needed when appropriate temperatures are reached in the digester.
  • the palm fruitlets are contacted with the enzyme for 1 -15 minutes, such as for 2-10 minutes during conveyance to the digester, such as for 3-7 minutes or such as for 4- 6 minutes during conveyance to the digester.
  • the palm fruitlets are contacted with the enzyme in the digester for 10 minutes or above.
  • the palm fruitlets are contacted with the enzyme in the digester for less than 40 minutes.
  • the palm fruitlets must be retained within the digester for sufficient time to allow the enzymes to act e.g. on the cellulosic matter of the palm fruitlets.
  • the exact retention time needed in the digester will depend on the exact conditions, and whether the enzyme composition is dosed directly in the digester or onto the palm fruitlets or MPD while being conveyed to the digester. Dosing the enzymes upstream of the digester onto the palm fruitlets or MPD while they are transported to the digester will generally lower the minimum retention time needed in the digester i.e.
  • the palm fruitlets are contacted with the enzyme in the digester for 15-60 minutes, such as for 20-60 minutes, 25-60 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 40-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20-30 minutes, 25-30 minutes, 15-25 minutes or such as for 15-20 minutes.
  • the temperature in the upper one third of digester may be in the range of 45-55°C, e.g. in the range of 55-65°C; in the middle one third of the digester it may be about 65°C, such as in the range of 55-65°C, in the range of 60-70°C or in the range of 65-70°C; and in the lower one third of the digester it may be about 85°C, such as in the range of 70-85°C, typically about 80°C.
  • contacting or incubation of the palm fruitlet or MPD with an enzyme composition is done 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, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes; the time period being calculated as the time from which the enzyme composition is applied onto the palm fruitlets or MPD and until the digested fruit is discharged into the press.
  • the contacting or incubation of the palm fruitlet or MPD with an enzyme composition is done for a period of 25-35 minutes, more preferably a period of 25-30 minutes, most preferably a period of 25-28 minutes.
  • the enzymes used in the process according to the Invention may be inactivated, or at least substantially inactivated, when the digested fruit is pressed, due to the high temperatures reached in the screw press.
  • the incubation time or retention time of the palm fruit mash at temperatures above 65°C and up to 85°C is from 10-30 minutes, such as from 10-28 minutes, 15-28 minutes, 12-30 minutes, 12-28 minutes or 12-25 minutes.
  • Retention time at temperatures above 65°C and up to 85°C may be controlled according to need; e.g. by increasing the digester volume, or by slowing down the screw press. Also, throughout the milling process retention time at temperatures close to 65C may be increased by the use of a predigester, and/or by use of a slow conveyor method.
  • contacting with an enzyme composition is done at one or more contact points.
  • contacting is done at least at one or more points, such as two or more points, which are then spaced at least 0.1 -4.0 feet apart from each other, such as 0.1 -3.0 feet, 0.1 -2.5 feet, 0.1 -2.0 feet, 0.1 -1 .5 feet, 0.1 -1 feet, 0.2-4.0 feet, 0.5-4.0 feet, 1 .0-4.0 feet, 1.5-4.0 feet, 2.0-4.0 feet, 2.5-4.0 feet, 3.0-4.0 feet, 0.5-2 feet, 0.5-1 foot or 1 .0-2.0 feet apart from each other, during conveyance of MPD from thresher to digester.
  • points such as two or more points, which are then spaced at least 0.1 -4.0 feet apart from each other, such as 0.1 -3.0 feet, 0.1 -2.5 feet, 0.1 -2.0 feet, 0.1 -1 .5 feet, 0.1 -1 feet, 0.2-4.0 feet, 0.5-4.0 feet, 1 .0-4.0 feet, 1.5-4.0 feet, 2.0-4.0 feet, 2.5-4.0 feet, 3.0-
  • the mass of palm fruitlet are conveyed into the digester by means such as but not limited to screw-conveyer or auger conveyor or belt conveyor or roller conveyor or skate-wheel conveyor or chain conveyor or bucket elevator.
  • the palm fruitlets are subject to temperatures during passage through the digester, which increase from 45-85°C,such as from 45-90°C, from 50-85°C or such as from 50-90°C.
  • the palm fruitlets or MPD is/are contacted with said enzyme composition under conditions which allow the enzyme(s) to weaken the mesocarp cell wall structure and thereby to reduce the number of cells, which are left intact and containing oil droplets when having been subject to screw pressing or hydraulic pressing.
  • the reduced number of intact cells is clearly visible on photomicrographs of samples taken from POME, such as the ones disclosed in the examples herein.
  • the palm fruitlets or MPD is/are contacted with said enzyme composition under conditions which allow the enzyme(s) to weaken the mesocarp cell wall structure and thereby to reduce the number of cells, which are left intact and containing oil droplets when having been subject to screw pressing or hydraulic pressing, while the average fibre length is reduced by no more than 50%, such as by no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, or such as no more than 2%,
  • the palm fruitlets or MPD is/are contacted with said enzyme composition at a dosage and under conditions which allow the enzyme composition to reduce the number of cells, which are left intact and containing oil droplets after having been subject to screw pressing or hydraulic pressing, thereby reducing the number of unbroken mesocarp cells in the palm oil mill effluent (POME) by at least 5%, such as by at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, or such as at least 80%.
  • POME palm oil mill effluent
  • enzymes which have a temperature optimum close to, but below the processing temperature in the digester, such as the processing temperature in the lower third of the digester.
  • the palm fruitlets are substantially disintegrated as they break when approaching the bottom of the digester due to top load of fruit in the digester; hence the interior mesocarp is mainly exposed to enzyme when the temperature is approaching 70°C at the bottom of the digester and close to screw pressing.
  • the palm fruitlets are subject to increasing temperatures during passage through the digester, reaching temperatures with the range of 70-85°C in the lower one third of the digester.
  • the process is a batch process, continuous and/or semi-continuous.
  • the enzyme has a temperature optimum within the range of 65-85°C.
  • the one or more cellulases, one or more hemicellulases, one or more protease, one or more pectinases, one or more amylases, one or more pectin methyl esterases, one or more polygalacturonases may each have a temperature optimum in the range of 65- 85°C, such as in the range of 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
  • the one or more cellulases, one or more hemicellulases, one or more proteases, one or more pectinases, one or more amylases are thermostable to such an extent that at least 15% of the enzyme activity (i.e.
  • the cellulase, hemicellulose, protease, 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, hemicellulase, protease, 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 PAH BAH 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
  • the process of the invention comprises the steps of:
  • an enzyme composition e.g. an enzyme composition as defined above, onto the stripped fruitlets or MPD, while the stripped fruitlets or MPD is conveyed to a digester (E) on one or more conveyors; e.g conveyors (B), (C) and/or (D);
  • the extracted crude palm oil is subsequently refined.
  • the yield of the crude palm oil is improved by at least 0.4% compared to a process done in the absence of the added enzymes.
  • the ratio of fruitlets to water is in the range of 1 :0.001 to 1 :1.1 during digestion and pressing, preferably in the range of 1 :0.007 to 1 :1 ; more preferably in the range of 1 :0.08 to 1 :0.80.
  • the ratio of pressed mass to water is in the range of 1 :0.6 to 1 : 1.6, preferably in the range of 1 :0.45 to 1 :1 .45, more preferably in the range of 1 :0.4-1 :1 .04.
  • Water is added to facilitate the extraction during the contacting step where the water aids in dissolution of enzyme(s) which act on the fruit mash.
  • the pressing using screw press or hydraulic press is done at temperature of above 65°C.
  • the diluted pressed mass is clarified by heating to at least 85°C for a minimum of 30 minutes.
  • the enzyme inactivation is due to the heat exposure of diluted pressed mass in clarification.
  • the enzyme is in form of a liquid or a granulate.
  • the percent of free fatty acid (FFA) in extracted oil in the present invention is equal to the percent of FFA in oil extracted by a conventional method.
  • the Deterioration of Bleachability Index (DOBI) of the extracted oil in the present invention is equal to the percent of DOBI in oil extracted by a conventional method.
  • the phosphorous/phosphatide content is reduced by at least 0.1 % compared to a process done in the absence of the added enzymes.
  • the phosphorous content in the crude palm oil may in particular be reduced by at least 0.1 %, such as by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25% or at least 30%, such as from 1 - 50%, from 1 -40%, from 1 -30%, from 1 -20%, from 1 -10%, from 2-50%, from 2-40%, from 2-30%, from 2-20%, from 2-10%, from 2-5%, from 5-50%, from 5-40%, from 5-30%, from 5-20%, from 5-10%, from 10-50%, from 10-40%, from 10-30%, from 10-20%, from 20-50%, from 20-40% or such as from 20-30%, compared to a process wherein palm fruitlets are processed in the digester, e.g. for 30 minutes, at a temperature reaching 90°C, in the absence of
  • the process comprises i) contacting the palm fruitlets or MPD with an enzyme composition; e.g an enzyme composition as defined above, at a temperature of above 65°C, and ii) extracting the crude palm oil; wherein the palm fruitlets or MPD is/are contacted with said enzyme composition under such conditions that the phosphorous content in the crude palm oil is reduced by at least 20% compared the phosphorous content of crude palm oil obtained by a process wherein palm fruitlets are processed in the digester at a temperature reaching 90°C, in the absence of the added enzymes.
  • an enzyme composition e.g an enzyme composition as defined above, at a temperature of above 65°C
  • extracting the crude palm oil e.g an enzyme composition as defined above
  • the phosphorous/phosphatide content of a palm oil sample may be determined using the official method of the American Oil Chemists' Society (AOCS), Ca 12-55, according to which the phosphorous content is determined by charring and ashing the oil sample with zinc oxide (ZnO), followed by the calorimetric measurement of phosphorus as blue phosphomolybdic acid.
  • AOCS American Oil Chemists' Society
  • ZnO zinc oxide
  • the process improves the oil extraction so as to add at least an additional 0.4% to the oil extraction rate (OER), such as an additional 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1 .1 %, 1 .2%, 1.3%, 1 .4%, 1 .5%, 1.6%, 1 .7%, 1 .8%, 1 .9% or at least an additional 2% to the OER, compared to a process, wherein palm fruitlets are processed in the digester for 30 minutes at a temperature reaching 90°C, in the absence of added enzymes.
  • OER oil extraction rate
  • the clarification time is reduced by at least 0.5% compared to a process done in the absence of the added enzymes.
  • the crude palm oil or dilute crude oil (DCO) clarification time may be reduced by at least 0.5%, such as by at least 1 %, 5%, 10%, 20%, 30%, 40% or at least 50% compared to a process, wherein palm fruitlets are processed in the digester for 30 minutes at a temperature reaching 90°C in the absence of added enzymes.
  • the process may comprise extracting the crude palm oil and subjecting it to clarification at a temperature between 85°C and 95°C.
  • the process may also comprise clarification of extracted oil at approximately 90°C for about 3 hours to inactivate residual enzyme.
  • the quantity of steam passed into the digester is reduced by at least 1 .5% compared to a process done in the absence of the added enzymes.
  • the quantity of steam passed into the digester is reduced by at least 1.5%, such as by at least 2%, 3%, 4%, 5% or by at least 10%, compared to a process wherein palm fruitlets are processed in the digester for 30 minutes at a temperature reaching 90°C in the absence of the added enzymes.
  • the total energy consumption to process one ton of FFB is reduced by at least 0.2%, such as by at least 1 %, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or such as by at least 50%, compared to a process wherein palm fruitlets are processed in the digester for 30 minutes at a temperature reaching 90°C in the absence of the added enzymes.
  • the total retention time for treatment of Palm oil mill effluent is reduced by at least 0.5% compared to the total retention time for treatment of POME generated using a process without addition of added enzymes.
  • the total retention time for treatment of POME e.g. the time required for anaerobic fermentation of POME
  • the process comprises contacting the palm fruitlets with an enzyme composition comprising one or more cellulases.
  • the enzyme composition further comprises a hemicellulase, protease, an amylase, a pectinase, or combination thereof.
  • the enzyme composition comprises one or more (e.g., several) cellulases.
  • the enzyme composition comprises or further comprises one or more (e.g., several) proteins selected from the group consisting of a cellulase, a hemicellulase, a protease, a pectinase, an amylase, or combination thereof.
  • one or more proteins selected from the group consisting of a cellulase, a hemicellulase, a protease, a pectinase, an amylase, or combination thereof.
  • the one or more (e.g., several) cellulolytic enzymes comprise 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).
  • 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 MS), PULPZYME® HC (Novozymes MS), 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 (Biocatalysts Limit,
  • pectinase of the invention may comprise a single activity or at least two different activities.
  • Pectinases of the invention may be obtained by fermentation of organisms. Fermentation of organisms to produce enzymes is known in the art. There are different kinds of fermentation including but not limited to submerged fermentation (SmF) and surface fermentation (SSF). Submerged fermentation (SmF) is known in the art and includes a process of growing a microorganism in a liquid medium.
  • SmF submerged fermentation
  • SSF surface fermentation
  • Submerged Fermentation is also alternatively known as Submerged Liquid Fermentation or submerse fermentation
  • SSF solid-state fermentation
  • Most of the SSF processes are aerobic and so the term fermentation in the context of SSF is meant to mean the "controlled cultivation of organisms”.
  • Processes and apparatus for solid state fermentation are known in the art. For example, a useful reference is Mitchell D.A. et al, 2006, Solid-State Fermentation Bioreactors, published by Springer Berlin Heidelberg.
  • the pectinase is obtained from a non-genetically modified organism. In another aspect, the pectinase is obtained from a genetically modified organism.
  • Pectinase producing organisms are known in the art. They include microorganisms and higher plants. The microorganisms include bacteria, yeast and fungi. For example, Aspergillus, Rhizopus, Bacillus, Pseudomonas, Fusarium, Penicillium, Saccharomyces, Erwinia etc., are all known to produce pectinase enzymes. The procedures for carrying out the submerged and solid state fermentations for many of these organisms are well known in the art.
  • the effective amount of cellulase, hemicellulase, protease, pectinase, or amylase used in embodiments of the invention will vary with the type of enzymes used in the process, the ultrastructure and composition of the cell wall (which varies by plant type), the pretreatment or pre-processing step, and well as the as the desired yield. Commercial enzymes may be used according to their manufacturer's instructions.
  • the method for extraction of crude palm oil from palm fruitlets comprises contacting the palm fruitlets with an enzyme composition comprising one or more cellulase(s).
  • the method for extraction of crude palm oil comprises contacting the palm fruitlets with an enzyme composition, which further comprises a hemicellulase, a protease, an amylase, a pectinase or combination thereof.
  • the method comprises the steps as described above.
  • the invention provides use of an enzyme composition comprising one or more cellulase(s) for extraction of crude palm oil from palm fruitlets.
  • the invention comprises the use of an enzyme composition further comprises a hemicellulase, a protease, an amylase, a pectinase or combination thereof.
  • the invention provides a crude palm oil obtained by the process according to the invention.
  • Enzyme compositions suitable for use in the present invention include composition comprising cellulase, hemicellulase, pectinase, and/or amylase or combinations thereof.
  • the cellulase were obtained from commercial products such as CeremixTM, ViscoflowTM, (Novozymes A/S, Denmark) and an experimental product, and are hereinafter referred to as "cellulase complex".
  • Protease was obtained from the commercial product ProtamexTM (Novozymes A/S, Denmark Enzymes were used alone or in combination.
  • Palm fruitlet mainly consists of thin outer layer (exocarp), oil rich mesocarp and inner nut which have shell and kernel.
  • the palm fruitlets are highly non-homogenous. Due to natural variation in the nut:fruit ratio, there is difference in the actual palm mesocarp (containing CPO) in various treatment even from the same batch of fruit. Therefore, a nut factoring system was used wherein, the amount of mesocarp is normalized for each treatment in order to understand the effect of enzymes on CPO extraction from small sample size of 1 kg segregated palm fruitlets irrespective of variation in nut:fruit ratio. The amount of mesocarp of palm fruitlet is estimated by negating the inherent weight of the nuts from the total amount of 1 kg segregated palm fruitlet.
  • the inherent weight of nut in fruit is back-calculated by adding its inherent moisture to the 60°C / 48 hours dried nut. Further, the mesocarp content across all the treatments in normalized to check the oil extracted. Then the CPO from the normalized mesocarp is then compared for % increase in CPO yield due to enzyme treatment.
  • Inherent moisture of the nut generally present have been analyzed in lab and fixed. Inherent moisture in Indian fruitlet was 10%; Indonesian fruitlet was 5%. Amount of mesocarp generally present have been analyzed in lab. Amount of mesocarp in Indian and Indonesian palm fruitlet fruit was 770g and 650g respectively.
  • Example 1 Crude palm oil Yield from Indian Palm Fruit
  • the DCO was further centrifuged at 5000 rpm (1558g) for 10 minutes using a Beckman Coulter centrifuge (model - Avanti JE). After the centrifugation step, three layers were formed - oil floating at the top, POME separated at the middle, and solid sludge accumulated at the bottom.
  • the liquid stream (oil and POME) was decanted in a glass separating funnel. All the centrifuge tubes were rinsed without disturbing the solid sludge pellet with 600g of boiling water. This water used for rinsing, was added to the separating funnel.
  • the oil (top) and POME (bottom) phases separate after keeping the whole mass stationary for at least 2 minutes.
  • the POME fraction was drained from the bottom of the separating funnel and total weight of the POME was measured.
  • the top oil layer was collected for determining the yield of crude palm oil from 1 kg palm fruitlets. Keeping all parameters same as above described process, the control crude palm oil extraction (without added enzymes) was performed at condition of 90°C for 15 minutes.
  • Palm oil extraction was performed essentially as described in Example 1.
  • the hand pressed mass extract i.e DCO - Dilute crude oil
  • mesh number # 8 2000 micron
  • 600g of boiling water was used for initial wash of the fiber and mashing bath vessel; the resultant fiber was further washed with 300g boiling water. Palm nuts were also washed with 300g of boiling water.
  • the bucket, cylinders (used for collecting DCO) and sieve were all washed with 300g of boiling water to ensure consistency of mass balance.
  • the separated, water washed, pressed fibers and palm nuts are subjected to drying at 60°C for 48 hours and then their respective weights were measured.
  • the DCO was kept for clarification in a hot air oven for 30 minutes at 90°C.
  • the DCO was further centrifuged at 5000 rpm (1558g) for 10 minutes using a Beckman Coulter centrifuge (model - Avanti JE). After the centrifugation step, three layers were formed - oil floating at the top, POME separated at the middle, and solid sludge accumulated at the bottom.
  • the liquid stream (oil and POME) was decanted in a glass separating funnel. All the centrifuge tubes were rinsed without disturbing the solid sludge pellet with 600g of boiling water. This water used for rinsing, was added to the separating funnel.
  • the oil (top) and POME (bottom) phases separate after keeping the whole mass stationary for at least 2 minutes.
  • the POME fraction was drained from the bottom of the separating funnel and total weight of the POME was measured.
  • the top oil layer was collected for determining the yield of crude palm oil from 1 kg palm fruitlets. Keeping all parameters same as above described process, the control crude palm oil extraction (without added enzymes) was performed at condition of 90°C for 15 minutes.
  • Table 3 indicate that there is an increase percent of crude palm oil yield by enzymatic treatment at 65°C or above temperature for 30 minutes compared to control at 90°C for 15 minutes.
  • Example 5 Measurement of Oil losses in waste stream - Pressed fibre, solid sludge and POME and effect of enzyme in oil loss reduction from waste streams
  • Example 6 Crude Palm Oil Extraction in a Commercial Palm Oil Mill using enzyme
  • Fresh fruit bunches (FFB) procured from nearby palm plantations were autoclaved in vertical sterilizer (10 tons capacity) at steam pressure (2 kg/cm2), temperature (130-150 °C) and sterilization time of 40-80 minutes.
  • MPD mass passing to digester
  • the sterilized FFBs were passed at throughput rate of 5 tons per hour for control run through thresher (stripper) having horizontal rotating drum.
  • the resultant empty fruit bunches (EFBs) were collected seprately and weighed.
  • the mashed MPD from the digester is passed through the screw-press and 2 fractions are obtained; i.e, total extract and pressed cake (pressed fibres and palm nuts). The total extract is passed through vibratory sieve separator and the resultant mass is DCO (dilute crude oil) which is pumped to the vertical clarifier.
  • the temperature in the vertical clarifier is maintained at 90°C for minimum 2 hours.
  • the clarified CPO at the top is skimmed out to the CPO tank.
  • the remaining heavy fraction was centrifuged through sludge centrifuge separator and the CPO fraction is pumped to the CPO tank while the POME stream is passed to the POME pit.
  • the OER is measured as percentage of total CPO extracted in the CPO tank to the total FFB processed.
  • throughput of the FFB was decreased to 2.5 tons per hour; digester temperature maintained at 65-70°C; enzyme concentration in enzyme dilution was 1 % and flowrate of enzyme dilution was 82.5 litre per hour. Rest of the procedure was similar to the control run.
  • the results from Table 6 indicate that there is increase percent of crude palm oil yield by enzyme at 65°C or above temperature for 30 minutes compared to control at 90°C for 15 minutes.

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Abstract

La présente invention concerne un procédé d'extraction d'huile de palme brute à partir de petits fruits de palmier comprenant la mise en contact des petits fruits de palmier avec une composition enzymatique à une température supérieure à 65 °C et l'extraction de l'huile de palme brute. L'invention porte également sur un procédé d'extraction d'huile de palme brute comprenant la mise en contact des petits fruits de palmier avec des compositions enzymatiques et sur l'utilisation de telles compositions enzymatiques pour l'extraction d'huile de palme brute.
PCT/EP2016/057809 2015-04-08 2016-04-08 Procédé d'extraction d'huile de palme à l'aide d'enzymes WO2016162510A1 (fr)

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WO2017202983A1 (fr) * 2016-05-24 2017-11-30 Novozymes A/S Appareil et procédé d'application d'une préparation enzymatique
WO2018135937A1 (fr) * 2017-01-20 2018-07-26 Sime Darby Plantation Sdn. Bhd. Système d'extraction d'huile de palme à partir d'une pluralité de noix de palme
WO2019245358A1 (fr) * 2018-06-22 2019-12-26 Sime Darby Plantation Intellectual Property Sdn. Bhd. Procédé de réduction de la formation d'acides gras libres dans des fruits de palmier à huile et des plants d'olivier

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CN104818107B (zh) * 2015-04-15 2018-05-18 山东中爱农林科技开发有限公司 一种牡丹籽油的制备工艺

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WO2017202983A1 (fr) * 2016-05-24 2017-11-30 Novozymes A/S Appareil et procédé d'application d'une préparation enzymatique
AU2017270259B2 (en) * 2016-05-24 2021-11-18 Novozymes A/S Apparatus and method for applying an enzyme preparation
WO2018135937A1 (fr) * 2017-01-20 2018-07-26 Sime Darby Plantation Sdn. Bhd. Système d'extraction d'huile de palme à partir d'une pluralité de noix de palme
WO2019245358A1 (fr) * 2018-06-22 2019-12-26 Sime Darby Plantation Intellectual Property Sdn. Bhd. Procédé de réduction de la formation d'acides gras libres dans des fruits de palmier à huile et des plants d'olivier

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