BRPI0819896B1 - PROCESS TO CONVERT VEGETABLE MATERIAL DERIVED FROM A ARACACEAE SP. Containing manana in an alcohol - Google Patents

Abstract

process for converting a plant material from an aracaceae sp. The present invention relates to processes comprising the enzymatic degradation of mannan-containing materials to produce a hydrolyzate. The invention also relates to processes for producing a fermentation product from mannan containing materials.

Description

“PROCESS TO CONVERT A VEGETABLE MATERIAL DERIVED FROM ARACACEAE SP. CONTAINING MANANA IN AN ALCOHOL ”

TECHNICAL FIELD

The present invention relates to processes that comprise the enzymatic degradation of mannan-containing materials to produce a hydrolyzate. The invention also concerns processes for producing a fermentation product from materials containing mannan. BACKGROUND OF THE INVENTION

Mannan is a non-starch polysaccharide that is a polymer of the mannose monosaccharide. Manana is found in plant, fungal and bacterial cell walls. Manana is present in significant amounts in certain vegetable residues, such as, for example, palm kernel cake and palm kernel flour.

If the substantially complete enzymatic hydrolysis of mannan and cellulose in the palm kernel cake could be used as a suitable substrate for the production of bio-ethanol, since the yeast Saccharomyces cerevisiae is capable from fermenting mannose and glucose to ethanol. The residue obtained after enzymatic hydrolysis and fermentation would furthermore make up an animal feed with a higher protein content than untreated palm kernel cake / flour.

The processes that allow partial hydrolysis of palm kernel flour are disclosed in EP1345499, US6797292, and in Düsterhõft et al (J. Sei. Food Agric. 63, 211-220, 1993) in which 40 to 50% of mannan are solubilized. The aim of the present invention is to provide improved processes, which include fermentation processes, which comprise the enzymatic hydrolysis of both mannan and cellulose present in palm kernel cake / flour and similar mannan-rich substrates. SUMMARY OF THE INVENTION

The purpose of the present invention is to provide processes

Petition 870180149158, of 11/08/2018, p. 8/13 improved, which include fermentation processes, which include the enzymatic hydrolysis of both mannan and cellulose present in palm kernel cake and similar mannan-rich substrates.

In a first aspect the invention relates to a process for converting a mannan-containing material into a fermentation product, the process comprising: a) forming an aqueous slurry comprising the mannan-containing material, b) contacting the slurry with a composition enzyme comprising the cellulase, mannanase, and mannosidase enzyme activities and producing a soluble hydrolyzate, c) contacting the soluble hydrolyzate with a fermentation organism to produce a fermentation product.

In other aspects the invention concerns a composition comprising a cellulase, a mannanase, and a mannosidase, as well as the use of such a composition in a process for the hydrolysis of a material containing mannan which comprises contacting said material with said composition.

DETAILED DESCRIPTION OF THE INVENTION

Materials containing manana

The term "mannan-containing material" used herein refers to a material that comprises a significant amount of mannan. Any material containing mannan is considered in accordance with the present invention. In a preferred embodiment the material containing mannan contains at least 1% by weight, preferably at least 5% by weight, more preferably at least 10% by weight, even more preferably at least 15% by weight of mannan, even more preferably at least 20% by weight, or most preferably at least 25% by weight. It should be understood that the mannan-containing material may also comprise other constituents such as cellulosic material, which include cellulose and / or hemicellulose, and may also comprise other constituents such as proteinaceous material, starch, sugars, such as fermentable sugars and / or non-sugars fermentable.

Mannan and galacto-mannan are found in cell walls in plant, fungal and bacterial cell walls. The mannan-containing material is generally found, for example, in the stems, leaves, fruits, bark, pods, and cobs of plants or leaves, branches, and wood from trees. It is understood that the material containing mannan may be in the form of plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.

The material containing mannan can be selected from the list consisting of herbaceous and / or woody crops, agricultural food and feed crops, animal feed products, tubers, roots, stems, vegetables, cassava husks, cocoa pods, husks and / or rice pods, rice bran, cobs, straw, husks, pods, sugar beet pulp, locust bean pulp, vegetable cake, agricultural crop residue, straw, stalks, leaves, corn bran, husks, cobs, crusts, shells, pods, wood waste, tree bark, chips, sawdust, wood pulp, wood pulp manufacturing liquid, waste paper, cardboard, wood waste, industrial or municipal waste water solids, manure, manufacturing by-product fermented drink and / or fermentation processes, moist distillers grain, dry distillers grain, spent grain, vinasse and bagasse.

In a preferred embodiment the material containing mannan is derived from a plant within an Aracaceae sp. such as a genus selected from the group consisting of Cocos, Elaeis, Coffea, and Cyamopsis. In a more preferred embodiment the material containing mannan is derived from an Aracaceae sp. such as a species selected from the group consisting of Cocos nucifera (coconut), Elaeis guineensis, Elaeis malanococca (palm), and Coffea sp. (coffee) and Cyamopsis tetragonoloba (guar beans)

The oil palm tree (Elaeis sp.) Is known for its fruits that each comprise an oily, fleshy outer layer (the pericarp), with a single seed (the core). The oil can be extracted from both the pericarp and the stone. The extraction of palm oil from palm kernels can be done by mechanical extraction or solvent extraction. The residue obtained after mechanical extraction is called palm kernel cake and the residue obtained after solvent extraction is called palm kernel flour.

Palm kernel cake contains approximately 50% by weight of polysaccharides other than starch, approximately 20% by weight of protein, and approximately 12% by weight of lignin. The main polysaccharides in palm kernel cake are mannan (78% of the polysaccharides other than total starch), cellulose (12%), arabinoxylan (3%), and 4-O-methylglycuronoxylan (3%).

Copra flour is the residue remaining after the coconut oil is pressed from the copra, the endosperm of Cocos nucifera (expelled copra flour) or the residue that remains after the extraction of coconut oil using solvents (extracted copra flour ). Copra flour contains 25 to 32% mannan.

Both oil palm residues and coconut residues are highly suitable as substrates for the present invention.

Pre-treatment

The mannan-containing material can optionally be pretreated before hydrolysis and / or fermentation. The pretreatment goal is to separate and / or release constituents other than mannan such as cellulose, hemicellulose and / or lignin and thus improve the hydrolysis rate of the cellulose and hemicellulose compounds. Pretreatment methods such as wet oxidation and alkaline pretreatment target lignin, while dilute acid and auto5 hydrolysis target hemicellulose.

The mannan-containing material can be chemically, mechanically and / or biologically pre-treated before hydrolysis and / or fermentation. Mechanical treatment (often referred to as physical treatment) can be used alone or in combination with subsequent or simultaneous hydrolysis, especially enzymatic hydrolysis.

Preferably, chemical, mechanical and / or biological pretreatments are carried out before hydrolysis and / or fermentation. Alternatively, chemical, mechanical and / or biological pretreatments can be carried out simultaneously with hydrolysis, such as simultaneously with the addition of one or more of the enzymatic activities mentioned below, to release, for example, fermentable sugars, such as mannose , glucose and / or maltose.

The term “chemical treatment” refers to any chemical pretreatment that promotes the separation and / or release of cellulose, 15 hemicellulose and / or lignin. Examples of suitable chemical pretreatments include treatment with; for example, dilute acid, lime, alkali, organic solvent, ammonia, sulfur dioxide, carbon dioxide. In addition, wet oxidation and pH-controlled hydrothermolysis are also considered chemical pre-treatment.

The chemical pretreatment can be an acidic treatment, more preferably, a diluted and / or continuous mild acidic treatment, such as treatment with sulfuric acid, or another organic acid, such as acetic acid, citric acid, tartaric acid, acid succinic, hydrogen chloride or mixtures of these. Other acids can also be used. Soft acid treatment means that the treatment pH is in the range of 1 to 5, preferably pH 1 to 3.

Alkaline chemical pretreatment based, for example, on NaOH, Na ₂ CO ₃ and / or ammonia or the like, is also considered according to the invention. Pretreatment methods using ammonia are described, for example, in WO 2006/110891, WO 2006/11899, WO 2006/11900, WO

2006/110901 (which are hereby incorporated by reference).

Wet oxidation techniques involve the use of oxidizing agents, such as: sulfite-based oxidizing agents or the like. Examples of solvent pretreatments include treatment with DMSO (Dimethyl Sulfoxide) or the like. Chemical pre-treatment is generally carried out for 1 to 60 minutes, such as 5 to 30 minutes, but can be carried out for shorter or longer periods of time depending on the material to be pre-treated.

Other examples of suitable pretreatment methods are described by Schell et al. (2003) Appl. Biochem and Biotechn. 105-108, p. 69-85, Mosier et al. Bioresource Technology 96 (2005) 673-686, Ahring et al. in W02006032282 and WO200160752, Foody et al. in W02006034590, and Ballesteros et al. in US publication No. ² 2002/0164730, references which are hereby incorporated by reference.

The term "mechanical pre-treatment" refers to any mechanical (or physical) treatment that promotes the separation and / or release of cellulose, hemicellulose and / or lignin from the material containing mannan. For example, mechanical pretreatment includes various types of grinding, irradiation, steam entrainment / explosion, wet oxidation, and other hydrothermal treatments.

Mechanical pretreatment includes comminution (mechanical size reduction). Comminution includes dry grinding, wet grinding and vibrating ball grinding. Mechanical pretreatment may involve high pressure and / or high temperature (vapor explosion). In one embodiment of the invention, high pressure means pressure in the range of 300 to 600 psi (2070 to 4140 kPa), preferably 400 to 500 psi (2760 to 3450 kPa), such as around 450 psi (3105 kPa). In an embodiment of the invention, high temperature means temperatures in the range of about 100 to 300 ° C, preferably about 140 to 235 ° C. In a preferred embodiment the mechanical pretreatment is a batch process, system steam gun hydrolyzer that uses high pressure and high temperature as defined above. A Sunds Hydrolyzer (available from Sunds Defibrator AB (Sweden) can be used for this.

As used in the present invention the term "biological pretreatment" refers to any biological pretreatment that promotes the degradation of mannan-containing material. Biological pretreatment techniques may involve applying microorganisms that solubilize lignin (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in the Handbook on Bioethanol: Production and Utilization, Wyman, CE, ed., Tailor & Francis, Washington, DC, 179-212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic / microbial conversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, JD, 1994, Pretreating lignocellulosic biomass: a review, in the Enzymatic Conversion of Biomass for Fuels Production, Himmel, Μ. E., Baker, JO, and Overend, RP, eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15; Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol production from renewable resources, no Advances in Biochemical Engineering / Biotechnology, Scheper, T., ed., Springer -Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson, L., and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander, L., and Eriksson, K.-E. L., 1990, Production of ethanol for lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

In a preferred embodiment in which the mannan-containing material is palm kernel flour or palm-kernel cake the mannan-containing material is not subjected to a pretreatment.

Hydrolysis

The mannan-containing material is hydrolyzed to break down mannan, cellulose and hemicellulose into sugars and / or oligosaccharides. Hydrolysis can be carried out before and / or simultaneously with a fermentation step.

According to the invention the material containing mannan to be hydrolyzed is contacted by a cellulase, a mannanase and a mannosidase. The mannan-containing material is converted into a soluble hydrolyzate, in a process which comprises a) forming an aqueous slurry comprising the biomass substrate, b) contacting the slurry with an enzyme composition comprising the enzymatic activities cellulase, mannanase, and mannosidase and producing a soluble hydrolyzate.

In one embodiment one or more enzymes of galactomannanase, beta-xylosidase, endo-1,4-beta-xylanoase, beta-glucosidase, alpha-galactosidase, alpha-L-arabinofuranosidase, acetyl xylan esterase, ferulic acid esterase and alpha-glucuronidase are present. Also alpha-amylase, glycoamylase and / or the like may be present during hydrolysis and / or fermentation since the mannan-containing material may include some starch. Galactomannanase is preferably applied if the substrate comprises galactomannan.

The enzyme (s) used for hydrolysis are (are) capable of directly or indirectly converting mannan and other carbohydrate polymers into fermentable sugars that can be fermented into a desired fermentation product, such as ethanol. Suitable enzymes are described in the “Enzymes” section below.

The dry solids content during hydrolysis can be in the range of at 50% by weight, preferably from 10 to 40% by weight, preferably from 20 to 30% by weight. Hydrolysis, in a preferred embodiment, can be carried out as a batch fed process where the mannan-containing material (substrate) is gradually fed to an enzyme-containing hydrolysis solution. The mannan-containing material can be supplied to the enzyme-containing hydrolysis solution in one or more distinct batches, as one or more distinct continuous streams or as a combination of one or more distinct batches and one or more distinct continuous streams.

The appropriate hydrolysis time, temperature and pH conditions can be easily determined by a person skilled in the art. The temperature must be decided with respect to the optimum temperatures of the applied enzymes. In general a temperature between 25 ° C and 70 ° C, preferably between 40 ° C and 60 ° C, more preferably between 45 ° C and 55 ° C, especially around 50 ° C will be suitable. The process is preferably carried out at a pH in the range of 3 to 8, preferably pH 4 to 6, especially around pH 5, for example, in the range of pH 4.5 to 5.5. Preferably, the hydrolysis is carried out between 12 and 144 hours, preferably from 16 to 120 hours, more preferably between 24 and 96 hours, such as between 32 and 48 hours.

Preferably the hydrolysis in step b) is allowed to process for a time and under conditions that allow at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the mannan content of the mannan-containing material are degraded to mannose and / or for a time and under conditions that allow at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, or at least 95% of the cellulose content of the mannan-containing material is degraded to glucose.

During the hydrolysis of substrates such as oil palm or palm kernel cake, palm kernel flour and / or residual copra pie oil is released and can be easily isolated from the soluble hydrolyzate. Thus in a preferred embodiment the process comprises isolating an oily fraction from the soluble hydrolyzate, for example, where the mannan-containing material is derived from a plant comprising vegetable oil, such as a residue produced in a vegetable oil extraction process.

Fermentation

According to the invention the hydrolyzed material containing mannan is fermented by at least one fermentation organism capable of fermenting fermentable sugars, such as mannose, glucose, and galactose directly or indirectly in a desired fermentation product. Consequently, in one embodiment a material containing mannan is converted into a soluble hydrolyzate, in a process which comprises a) forming an aqueous slurry comprising the biomass substrate, b) contacting the slurry with an enzyme composition comprising the enzymatic activities cellulase, mannanase, and mannosidase and produce a soluble hydrolyzate, and c) contact the soluble hydrolyzate with a fermentation organism to produce a fermentation product.

Preferred for ethanol fermentation is the yeast of the Saccharomyces cerevisiae species, preferably strains that are resistant to high levels of ethanol, that is, up to, for example, about 10, 12 or 15% by vol. of ethanol or more, such as 20 vol%. of ethanol. The fermentation is preferably continuous between 8 and 96 hours, preferably from 12 to 72, more preferably from 24 to 48 hours.

In one embodiment, fermentation is carried out at a temperature between 20 and 40 ° C, preferably from 26 to 34 ° C, in particular around 32 ° C. In one embodiment the pH is from pH 3 to 6, preferably around pH 4 to 5.

According to the invention, hydrolysis in step (b) and fermentation in step (c) can be carried out simultaneously or sequentially. In one embodiment there is no separate containment stage for hydrolysis, meaning that the hydrolyzation enzyme (s) and the fermentation organism are added together. When fermentation (for example, ethanol fermentation using the yeast Saccharomyces) is carried out simultaneously with hydrolysis the temperature is preferably between 26 ° C and 35 ° C, more preferably between 30 ° C and 34 ° C, as in 32 ° C. A temperature program comprising at least two containment stages at different temperatures can be applied according to the invention.

The process of the invention as well as the individual steps can be carried out as a batch process, batch process or as a continuum. Preferably the fermentation step is carried out as a continuous fermentation.

Recovery

Subsequent to fermentation, the fermentation product can be separated from the fermentation broth. The broth can be distilled to extract the fermentation product or the fermentation product can be extracted from the fermentation broth using microfiltration or membrane filtration techniques. Alternatively, the fermentation product can be recovered by stripping. Recovery methods are well known in the art. Fermentation products

The process of the invention can be used to produce any fermentation product. Especially considered fermentation products include alcohols (for example, ethanol, methanol, butanol); organic acids (for example, citric acid, acetic acid, itaconic acid, lactic acid, glyconic acid); ketones (for example, acetone); amino acids (for example, glutamic acid); gases (for example, H ₂ and CO ₂ ); antibiotics (for example, penicillin and tetracycline); enzymes; vitamins (for example, riboflavin, BI2, beta-carotene); and hormones.

In a preferred embodiment the fermentation product is an alcohol, especially ethanol. The fermentation product, such as ethanol, obtained according to the invention, can preferably be fuel alcohol / ethanol. However, in the case of ethanol it can also be used as potable ethanol.

Fermentation organism

The term "fermentation organism" refers to any organism, which includes bacterial and fungal organisms, suitable for producing a desired fermentation product. Especially suitable fermentation organisms according to the invention are capable of fermenting, i.e. converting, C6 sugars, such as mannose and glucose, directly or indirectly into the desired fermentation product. Also suitable are fermentation organisms capable of converting C5 sugars such as xylose to a desired fermentation product. Examples of fermentation organisms include fungal organisms, especially yeast. The preferred yeast includes strains of Saccharomyces spp., In particular a strain of Saccharomyces cerevisiae or Saccharomyces uvarum ', a strain of Pichia, preferably Pichia stipitis, such as Pichia stipitis CBS 5773; a strain of Candida, in particular a strain of Candida utilis, Candida diddensii, or Candida boidinii. Another yeast considered includes strains of Zymomonas', Hansenula, in particular H. anômala ', Klyveromyces, in particular K. fragilis', and Schizosaccharomyces, in particular S. pombe.

Commercially available yeast includes, for example, yeast ETANOL RED® (available from Fermentis / Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), fresh yeast SUPERSTART and THERMOSACC® (available from Ethanol Technology, WI, USA) , BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERM1OL (available from DSM Specialties). ANQI Yeast (available from Anqi Yeast (CHIFENG) CO., LTD, China).

Compositions

The present invention also relates to compositions suitable for use in the processes of the invention, said compositions comprising a cellulase, a mannanase, and a mannosidase. The composition can comprise additional enzymatic activities, such as acetylxylan esterase, alpha-glucuronidase, arabinofuranosidase, aminopeptidase, amylase, carboidrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyrribonuclease , beta-galactosidase, betaxilosidase, glycoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, transcriptase, proteinase xylase .

Enzymes

Although not specifically mentioned in the context of a process of the invention, it should be understood that enzymes (as well as other compounds) are used in an "effective amount".

Cellulases

The term “cellulases” as used herein is understood to comprise cellobiohydrolases (EC 3.2.1.91), for example, cellobiohydrolase I and cellobiohydrolase II, as well as endo-glycanases (EC 3.2.1.4) and beta-glucidases (EC 3.2.1.21) .

In order to be efficient, the digestion of cellulose and hemicellulose requires several types of enzymes acting cooperatively. At least three categories of enzymes are needed to convert cellulose into fermentable sugars: endo-glycanases (EC 3.2.1.4) that cut cellulose chains at random; cellobiohydrolases (EC 3.2.1.91) that cleave cellobiosil units at the ends of the cellulose chain and beta-glycosidases (EC 3.2.1.21) that convert soluble cellobiose and cellodextrins into glucose. Among these three categories of enzymes involved in cellulose biodegradation, cellobiohydrolases are the key enzymes for the degradation of native crystalline cellulose. The term "cellobiohydrolase I" is defined herein as a cellulose 1,4-beta-cellobiosidase activity (also referred to as exo-glycanase, exocelobiohydrolase or 1,4-beta-cellobiohydrolase), as defined in the enzyme class

EC 3.2.1.91, which catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose and cellotetraose, by releasing cellobiosis from the non-reducing ends of the chains. The definition of the expression “cellobiohydrolase activity

II ”is identical, except that cellobiohydrolase II attacks from the reducing ends of the chains.

Endoglycanases (EC No. 3.2.1.4) catalyze endo hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenine, beta-1 bonds, 4 in mixed beta-1,3 glycans such as cereal beta-D-glycans or xyloglycans and other plant material containing cellulosic parts. The authorized name is endo1,4-beta-D-glycan 4-glycan hydrolase, but the abbreviated term endoglycanase is used in this specification.

Cellulases can comprise a carbohydrate binding module (CBM) that enhances the enzyme's binding to a cellulose-containing fiber and increases the effectiveness of the active catalytic part of the enzyme. A CBM is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme with a discrete fold having carbohydrate-binding activity. For more information on CBMs see the internet server CAZy (Supra) or Tomme et al., (1995) in Enzymatic Degradation of Insoluble Polysaccharides (Saddler, JN & Penner, M., eds.), Cellulose-binding domains: classification and properties. pp. 142-163, American Chemical Society, Washington.

Cellulase activity, in a preferred embodiment, can be derived from a fungal source, such as a strain of the genus Trichoderma, preferably a strain of Trichoderma reeser, a strain of the genus Humicola, such as a strain of Humicola insolens ¹ , or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense.

In a preferred embodiment the cellulases can be a preparation as defined in co-pending application US 60 / 941.251, which is hereby incorporated by reference. In a preferred embodiment the cellulase preparation which comprises a polypeptide having cellulolytic enhancing activity (GH61A), preferably that disclosed as SEQ ID NO: 2 in WO 2005/074656. The cellulase preparation can further comprise a beta-glycosidase, such as the fusion protein disclosed in US 60 / 832,511. In one embodiment, the cellulase preparation also comprises a CBH II, preferably Thielavia terrestris cellobiohydrolase II CEL6A. In one embodiment, the cellulase preparation also comprises an enzymatic cellulase preparation, preferably that derived from Trichoderma reesei. In a preferred embodiment the cellulase preparation is the cellulase A preparation used in Example 1 and disclosed in US co-pending application US 60 / 941,251.

In one embodiment the cellulase preparation is the commercially available product CELLUCLAST® 1.5L or CELLUZYME® (Novozymes A / S, Denmark).

Cellulase can be dosed in the range of 0.1 to 100 FPU per gram of dry solids (DS), preferably from 0.5 to 50 FPU per gram of DS, especially from 1 to 20 FPU per gram of DS.

Mananases

In the context of the present invention a mannanase is a betamananase and defined as an enzyme belonging to EC 3.2.1.78.

Mannanases have been identified in several Bacillus organisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol. 56, N ⁵ 11, pp. 3505-3510 (1990) describe a beta-mannanase derivative of Bacillus stearothermophilus having an optimal pH of 5.5 to 7.5. Mendoza et al., World J. Microbiol. Biotech., Vol. 10, N ² 5, pp. 551-555 (1994) describes a beta-mannanase derivative of Bacillus subtilis having an optimal activity at pH 5.0 and 55 ° C. JP-03047076 discloses a beta-mannanase derivative of Bacillus sp., Having an optimal pH 8 to 10. JP-63056289 describes the production of an alkaline, thermostable beta-mannanase. JP-08051975 discloses alkaline beta-mannanases of alkalophilic Bacillus sp. AM-001. A purified Bacillus amyloliquefaciens mannanase is disclosed in WO 97/11164. WO 94/25576 discloses an Aspergillus aculeatus enzyme, CBS 101.43, which exhibits mannanase activity and WO 93/24622 discloses an isolated mannanase from Trichoderma reesei.

The mannanase can be derived from a strain of the genus Bacillus, such as the amino acid sequence having the sequence deposited as GENESEQP accession number AAY54122 and shown here as SEQ ID NO: 1 or an amino acid sequence that is homologous to this sequence of amino acid.

A suitable commercial mannanase preparation is Mannaway® produced by Novozymes A / S.

Manosidases

In the context of the present invention a mannosidase, such as a beta-mannosidase, is defined as an enzyme belonging to EC 3.2.1.25.

A suitable mannosidase preparation can be obtained from Aspergillus niger, such as for example a mannosidase preparation comprising mannosidase having the amino acid sequence deposited as Swiss-Prot accession number A2QWU and shown here as SEQ ID NO: 2., or an amino acid sequence that is homologous to this amino acid sequence.

Hemicellulases

Any hemicellulase suitable for use in hydrolyzing hemicellulose can be used. Preferred hemicellulases include xylanoases, arabinofuranosidases, acetyl xylan esterase, feruloyl esterase, glucuronidases, endo-galactanase, endo or exo arabinases, exo-galactanases, and mixtures of two or more of these. Preferably, the hemicellulase for use in the present invention is an exo-acting hemicellulase, and more preferably, the exo-acting hemicellulase that has the ability to hydrolyze hemicellulose under acidic conditions below pH 7, preferably pH 3 to 7.

An example of hemicellulase suitable for use in the present invention includes

ULTRAFLO L (available from Novozymes A / S, Denmark).

Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of non-terminally reducing alpha-L-arabinofuranoside residues in alpha-Larabinosides.

Galactanase (EC 3.2.1.89), arabinogalactan endo-1,4-betagalactosidase, catalyzes the endo-hydrolysis of 1,4-D-galactoside bonds in arabinogalactans.

Pectinase (EC 3.2.1.15) catalyzes the hydrolysis of 1,4alpha-D-galactosiduronic bonds in pectate and other galacturonans.

Xyloglycanase catalyzes the hydrolysis of xyloglycan.

The hemicellulase can be added in an amount effective to hydrolyze hemicellulose, such as in amounts from about 0.001 to 0.5% by weight of dry solids (DS), more preferably from about 0.05 to 0.5% by weight. DS weight.

Alpha-Amylase

According to the invention an alpha-amylase can be used. In a preferred embodiment the alpha-amylase is an acidic alpha-amylase, for example, fungi acidic alpha-amylase or bacterial acidic alpha-amylase. The term "acid alpha-amylase" means an alpha-amylase (EC 3.2.1.1) that added in an effective amount has optimal activity in a pEl in the range of 3 to 7, preferably from 3.5 to 6, or more preferably from 4 to 5. Bacterial Alpha-Amylase

According to the invention, the bacterial alpha-amylase is preferably derived from the genus Bacillus.

In a preferred embodiment, Bacillus alpha-amylase is derived from a strain of B. licheniformis, B. amiloliquefaciens, B. subtilis or B. stearothermophilus, but can also be derived from other Bacillus sp. Specific examples of alpha-amylases considered include Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, Bacillus amyloliquefaciens alphaamylase from SEQ ID NO: 5 in WO 99/19467 and alpha-amylase of Bacillus stearothermophilus shown in SEQ ID NO: 3 in WO 99/19467 (all sequences hereby incorporated by reference). In an embodiment of the invention the alpha-amylase can be an enzyme having a degree of identity of at least 60%, preferably at least 70%, more preferred at least 80%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with any of the sequences shown in SEQ ID NOS: 1, 2 or 3, respectively, in WO 99/19467.

Bacillus alpha-amylase can also be a variant and / or hybrid, especially those described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents are hereby incorporated by reference). Alpha-amylase variants specifically considered are disclosed in US Patents No. 6,093,562, 6,297,038 or US Patent No. ² 6,187,576 (hereby incorporated by reference) and include variants of Bacillus stearothermophilus alpha-amylase (alpha (BSG amylase) having a deletion of one or two amino acids at positions R179 to G182, preferably a double deletion disclosed in WO 1996/023873 - see for example, page 20, lines 1 to 10 (hereby incorporated by reference) , preferably corresponding to delta (181-182) compared to the amino acid sequence of wild-type BSG alpha-amylase shown in SEQ ID NO: 3 disclosed in WO 99/19467 or the deletion of amino acids R179 and G180 using SEQ ID NO: 3 in WO 99/19467 for numbering (this reference is hereby incorporated by reference). Even more preferred are Bacillus alpha19 amylases, especially Bacillus stearothermophilus alpha-amylase, which have a delta double deletion (181182) and further comprising an N193F substitution (also indicated 1181 * + G182 * + N193F) compared to amino acid sequence of wild-type BSG alpha-amylase shown in SEQ ID NO: 3 disclosed in WO 99/19467.

Fungal Alpha-Amylase

Fungal alpha-amylases include alpha-amylases derived from a strain of the genus Aspergillus, such as, alpha-amylases from Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii.

A preferred acidic fungal alpha-amylase is a Fungamil-like alpha-amylase that is derived from a strain of Aspergillus oryzae. According to the present invention, the expression "Fungamil-like alpha-amylase" indicates an alpha-amylase that exhibits a high identity, ie at least 70%, at least 75%, at least 80%, at least 85% at least least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strain of Aspergillus niger. In a preferred embodiment the acidic fungal alpha-amylase is that of A. niger disclosed as “AMYA ASPNG” in the Swiss-prot / TeEMBL database under primary access N ² P56271 and described in WO 89/01969 (Example 3 ).

Other wild-type alpha-amylases considered include those derived from a strain of the genus Rhizomucor and Meripilus, preferably a strain of Rhizomucor pusillus (WO 2004/055178 incorporated by reference) or Meripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al. J. Ferment. Bioeng. 81:

292-298 (1996) “Molecular-cloning and determination of the nucleotide sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii.”; and also as EMBL: # AB008370.

Glycoamylases

A glycoamylase used according to the invention can be derived from any suitable source, for example, derived from a microorganism or a plant. The preferred glycoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glycoamylases, in particular A. niger G1 or G2 glycoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097- 1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glycoamylase disclosed in WO 84/02921, A. oryzae glycoamylase (Agric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof. Other variants of Aspergillus glycoamylase include variants with enhanced thermal stability: G137AeG139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E / Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulfide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 86988704; and introduction of Pro residues at positions A435 and S436 (Li et al. (1997), Protein Eng. 10,1199-1204.

Other glicoamilases include Athelia rolfsii glucoamylase (previously designated Corticium rolfsii) (see US Patent No. 4,727,026 ^and and (Nagasaka, Y. Et al. (1998) "Purification and properties of the raw-amidedegrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50: 323-330), Talaromyces glycoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (US patent N® Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (US patent N ² 4,587 .215).

The bacterial glycoamylases considered include glycoamylases of the genus Clostridium, in particular C. thermoamilolyticum (EP 135,138), and C. thermoidrosulfuricum (WO 86/01831) and Trametes cingulata disclosed in WO 2006/069289 (which is hereby incorporated by reference).

Hybrid glycoamylases are also considered according to the invention. Examples of hybrid glycoamylases are disclosed in WO 2005/045018. Specific examples include the hybrid glycoamylases disclosed in Tables 1 and 4 of Example 1 (hybrids which are hereby incorporated by reference).

Glycoamylases which exhibit a high identity to any of the aforementioned glycoamylases are also considered, ie at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity with the mature enzyme sequences.

Commercially available compositions comprising glycoamylase include AMG 200L; AMG 300 L; SAN® SUPER, SAN® EXTRA L, SPIRIZYME® PLUS, SPIRIZYME® FUEL, SPIRIZYME® B4U and AMG® E (from Novozymes A / S); OPTIDEX® 300 (from Genencor Int.); AMIGASE® and AMIGASE® PLUS (from DSM); G-ZYME® G900, G-ZYME® and G990 ZR (from Genencor Int.).

The glycoamylases, in one embodiment, can be added in an amount of 0.02 to 20 AGU / g DS, preferably 0.1 to 10 AGU / g DS, especially between 1 and 5 AGU / g DS , such as 0.5 AGU / g DS.

Animal food

The solids that remain after enzymatic hydrolysis and fermentation have an improved nutritional value as an animal feed compared to material containing untreated mannan, for example, untreated palm kernel cake / flour. During hydrolysis and fermentation the protein content is increased by removing polysaccharides merely because the digestibility of the remaining polysaccharides is increased. In addition, protein composition is improved by yeast residues.

Consequently, the solids that remain after the process of the first aspect are very suitable as an animal feed or for inclusion in an animal feed composition.

The term animal includes all animals, which include humans. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, horses, and cattle, for example beef cattle, cows, and young calves. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include monogastric animals, for example pigs or pigs (which include, but are not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chickens (which include but are not limited to chickens, layers); young calves; and fish (which include but are not limited to salmon, trout, tilapia, catfish and carp; and crustaceans (which include but are not limited to shrimp and pitus).

The terms feed or feed composition mean any compound, preparation, mixture, or composition suitable for, or intended for, ingestion by an animal.

MATERIALS & METHODS

Chemicals

Arabinose, galactose, glucose, xylose, mannose, and succinic acid were purchased from Merck (Darmstadt, Germany). Sulfuric acid was from Bie & Bemtsen A / S (Rodovre, Denmark).

Substrate

Palm kernel pie was from Indonesia and had a dry matter content of 96% by weight.

Enzymes

The cellulase A composition is a cellulolytic composition comprising a polypeptide having cellulolytic enhancing activity (GH61A) disclosed as SEQ ID NO: 2 in WO 2005/074656; a beta-glucosidase from Aspergillus oryzae (in the fusion protein disclosed in US 60 / 832,511), and a preparation of cellulolytic enzymes derived from Trichoderma reesei comprising cellobiohydrolase I and cellobiohydrolase II, endo-glycanase and beta-glucosidase, as well as endo1 side activities , 4-beta-xylanoase and beta-xylosidase.

The mannanase B composition comprises a Bacillus mannanase having the sequence deposited as GENESEQP accession number AAY54122 and shown here as SEQ ID NO: 1 (NP001840).

The mannosidase C composition comprising a mannosidase derivative of Aspergillus niger and having the sequence deposited as SWISSPROT accession number A2QWU9 and shown here as SEQ ID NO: 2.

Identity determination

The relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

The degree of identity between two amino acid sequences can be determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE® MEGALIGN® software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Interval penalty of 10 and interval length penalty of 10. Paired alignment parameters are Ktuple = 1, interval penalty = 3, windows = 5, and diagonals = 5.

The degree of identity between two nucleotide sequences can be determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983,

Proceedings of the National Academy of Science USA 80: 726-730) using LASERGENE® MEGALIGN® software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Interval penalty of 10 and gap length penalty of 10. The paired alignment parameters are Ktuple = 3, gap penalty = 3, and windows = 20.

Amino acid sequences or nucleotide sequences that exhibit a high identity to a particular amino acid or nucleotide sequence, that is, at least 60%, at least 70%, at least 75%, at least 80%, at least 85% by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity, are considered homologous to that particular amino acid or nucleotide sequence.

High Performance Anion Exchange Chromatography (HPAEC)

Hydrolysates (10 μΐ) were applied in a Dionex BioLC system adapted with a Dionex CarboPac® PAI analytical column (4 x 250 mm) (Dionex Corporation, Sunnyvale, CA, USA) combined with a CarboPac® PAI pre-column (4 x 50 mm) . The monosaccharides were separated isocratically with 0.001 M KOH for 40 min, flow: 1 mL-min ' ¹ . Monosaccharides were detected by a pulsed electrochemical detector in the pulsed amperiometric detection mode. The electrode potential was programmed to +0.1 V (t = 0 to 0.4 s) to -2.0 V (t = 0.41 to 0.42 s) to 0.6 V (t = 0, 43 s) and finally -0.1 V (t = 0.44 to 0.50 s), while integrating the signal resulting from t = 0.2 to 0.4 s. A mixture of arabinose, galactose, glucose, xylose, and mannose (concentration of each component: 0.0025 to 0.075 g-L ' ¹ ) was used as a standard.

EXAMPLES

Example 1

Determination of Composition and Monosaccharide Content

The palm kernel cake was degraded using sulfuric acid hydrolysis following the standard procedure of the US National Renewable Energy Laboratory (NREL) (Analytical Lab Procedure 002 (1996)). The samples were then centrifuged (20800 g, 10 min), and the composition and monosaccharide content (g-L ' ¹ ) were determined by HPAEC, see above. The values are the average of three determinations and are reported in g / kg of palm kernel cake DM.

The polysaccharide material in the palm kernel cake was composed primarily of mannose and glucose, with the liquid hydrolysis yield of mannose and glucose being 358 g / kg DM and ~ 86.6 g / kg DM, respectively (Table 1) . The sum of the monosaccharides that result after acid hydrolysis indicated that carbohydrates made up> 51% by weight of the DM palm kernel cake (Table 1).

Table 1. Monosaccharide composition of palm kernel cake determined by acid hydrolysis and HPAEC. The results are expressed as g kg ' ¹ of palm oil cake DM ± SD Arabinose Galactose Glucose Xylose Mannose 14.6 ± 1.97 24.9 ± 3.03 87.8 ± 2.71 33.0 ± 6.10 358 ± 32.8 Example 2 Enzymatic hydrolysis of stone cake of palm

The palm kernel cake was incubated with a mixture of cellulase A preparation, mannanase B preparation and mannosidase C preparation added at a total I / O level of 2.5% by weight (25 mg EP / g of DM) per test at pH 5 and 50 ° C. The I / O ratio refers to the weight of enzyme protein (E) added in percent by weight of the DM (S) palm kernel cake. The substrate level was always 5% by weight of dry matter (DM), and the total reaction volume was kept constant in all experiments. To assess possible synergistic between enzyme preparations, 1: 1 mixtures and 1: 1: 1 mix of enzyme preparations were added at a total I / O level of 5% by weight of I / O and 7.5% by weight of I / O, respectively. These hydrolysates were compared to those obtained with the addition of 2.5% by weight of I / O of the individual enzyme preparations under the same reaction conditions. All samples were taken after exactly 24 h of reaction and immediately heated to 100 ° C for 10 min to stop the enzyme reaction. The samples were then centrifuged (20800 g, 10 min), and the levels of arabinose, galactose, glucose, xylose, and mannose were determined by HPAEC analysis. The values are the average of three determinations and are reported in g / kg of DM palm kernel cake.

The 1: 1 mixtures of the cellulase A preparation: mannanase B preparation induced a significantly high glucose release when compared to both the corresponding individual enzyme treatments and the hypothetical sum, calculated from the glucose release catalyzed by the individual preparations. After 24 hours of treatment, -95% by weight of the available glucose, it was released which was -71% by weight more than the situation of no theoretical interaction, and thus there was a synergistic effect between the enzymatic activities in the two enzyme preparations ( Table 2).

The 1: 1: 1 mixture of cellulase A preparation: mannanase B preparation: mannosidase C preparation also induced a significantly high glucose release compared to the corresponding individual enzyme treatments and the hypothetical calculated glucose sum. After 24 hours the mixture released> 100% by weight of the glucose released by acid hydrolysis with H2SO4.

Table 2. Glucose released from palm kernel cake after treatments with 2.5% by weight (25 g EP / kg DM) from cellulase A preparation, mannanase B preparation and mannosidase C preparation alone or in mixtures 1: 1, or a 1: 1: 1 mixture for 24 hours at pH 5, 50 ° C. Results are expressed as g-kg ' ¹ of palm oil cake DM ± SD Enzyme Name Hypothetical sum Experimentally obtained No enzyme - 0.00 Prep. of Cellulase A - 20.6 ± 1.7 Prep. from Mananase B - 0.00 ± 0.0 Prep. of Manosidase C - 11.2 ± 0.6

Prep. of Mannanase B: prep. of Manosidase C 11.2 22.7 ± 1.5 Prep. of Cellulase A: prep. of Manosidase C 31.8 33.5 ± 7.8 Prep. of Cellulase A: prep. from Mananase B 20.6 82.5 ± 2.0 Prep. of Cellulase A: prep. of Mannanase B: prep. of Manosidase C 31.8 88.1 ± 1.6

The 1: 1 mixtures of the mannosidase C preparation: mannanase B preparation, mannosidase C preparation: Cellulase A preparation, and Cellulase A preparation: Mannanase B preparation all induced a significantly high release of mannose when compared to both corresponding individual enzyme treatments and the hypothetical sum, calculated from the mannose release catalyzed by the individual preparation. After 24 hours of treatment, the mixture of the preparation of mannanase B: preparation of mannosidase C released ~ 83% by weight of the available mannose, which was - 11% by weight more than the theoretical non-interaction situation, and so on. there was a synergistic effect between mannan degradation activities in the two enzyme preparations (Table 3).

The 1: 1: 1 mixture of cellulase preparation A: mannanase preparation B: mannosidase C preparation induced a significantly high release of mannose compared to the corresponding individual enzyme treatments and the hypothetical sum, calculated from mannose. But the mannose released by this mixture was not significantly higher than the amount of mannose released by the 1: 1 mixture of the mannanase B and mannosidase C preparation (Table 3).

Table 3. Mannose released from palm kernel cake after treatments with 2.5% by weight (25 g EP / kg DM) of cellulase A preparation, mannanase B preparation and mannosidase C preparation alone or in mixtures 1: 1, or a 1: 1: 1 mixture for 24 hours at pH 5, 50 ° C The results are expressed as g kg ' ¹ of palm oil cake DM ± SD Enzyme Name Hypothetical Sum Experimental and Obtained No enzyme - 0.00 ± 0.0 Prep. cellulase A - 18.5 ± 0.6 Prep. of mannanase B - 161 ± 29 Prep. of mannosidase C - 136 ± 1.5 Prep. Mannanase B: Prep. of mannosidase C 297 334 ± 1.5 Prep. cellulase A: Prep. of mannosidase C 154.5 210 ± 15 Prep. cellulase A: Prep. of mannanase B 179.5 201 ± 11

Prep. cellulase A: Prep. Mannanase B: Prep. of mannosidase C 315.5 335 ± 6.5

Example 3

Saccharification and enzymatic fermentation of palm kernel cake

A suspension of palm kernel cake (PKC) at 35% by weight of dry matter D (M) was incubated 24 hours at pH 5 and 50 ° C with various mixtures of mannanase B and mannosidase C preparation. experiments the cellulase A preparation was also added. After this hydrolysis, baker's yeast was added to the samples and the mixture was fermented for 156 hours. The other part of the samples was hydrolyzed for 156 hours without added yeast.

The fermented samples were removed after 156 hours of fermentation and centrifuged at 20800 g, for 10 min. The ethanol level was determined by HPLC. The values are the average of three determinations and are reported in g / kg of DM palm kernel cake.

All hydrolyzed samples were taken after exactly 24 + 156 hours of reaction and heated to 100 ° C for 10 min to stop the enzymatic reaction. The samples were then centrifuged (20800 g, 10 min), and glucose and mannose levels were determined by HPAEC analysis. The values are the average of three determinations and are reported in g / kg of DM palm kernel cake. The results are shown in the table

4.

Table 4. Comparison of sugars released by palm kernel cake hydrolysis alone and ethanol formed by simultaneous hydrolysis / saccharification and fermentation Enzyme measurement; mg of EP / kg of PKC DM Mannose formed after 180 hours of hydrolysis g / kg of PKC DM Glucose formed after 180 hours of hydrolysis g / kg of PKC DM Ethanol formed after 24 hydrolysis + 156 hours of fermentation g / kg PKC DM preparation of mannanase B preparation of mannosidase C preparation of cellulase A 171 85 0 240 5 120 171 85 324 240 20 130 171 255 0 295 5 145 171 255 324 300 20 160 342 510 648 320 40 180

Table 4 shows that, like glucose, mannose is completely fermentable by baker's yeast and that the formation of ethanol in all cases is close to the theoretically possible yield (51% of released monosaccharides).

Mannanase is the main responsible for the solubilization of the mannan of the palm kernel cake, otherwise insoluble, where the 5 mannosidase prefers the solubilized mannose oligomers as substrate. But the results indicate synergy between mannanase and mannosidase as an increase in mannosidase results in a greater increase in solubilized mannan than an increase in mannanase.

Claims (5)

1. Process for converting a plant material derived from Aracaceae sp. containing mannan in an alcohol, the process characterized by the fact that it comprises a) form an aqueous slurry comprising mannan-containing plant material, b) contacting the slurry with an enzyme composition that comprises the enzymatic activities: (i) cellulase comprising cellobiohydrolase belonging to EC 3.2.1.91, endoglucanase belonging to EC 3.2.1.4 and beta-glycosidase belonging to EC 3.2.1.21, in which cellulase is derived from Trichoderma reesei, (ii) mannanase belonging to EC 3.2. 1.78, in which mannanase is derived from Bacillus sp., And has the sequence of SEQ ID NO: 1, and (iii) mannosidase belonging to EC 3.2.1.25, in which mannosidase is derived from Aspergillus niger, and presents the sequence SEQ ID NO: 2, producing a soluble hydrolyzate, c) contacting the soluble hydrolyzate with a Saccharomyces cerevisiae to produce an alcohol, such as ethanol. 2. Process according to claim 1, characterized by the fact that the plant material containing mannan derived from an Aracaceae sp. is selected from the group consisting of Cocos mucifera, Elaeis guineenses and Elaeis malanococca. Process according to any one of claims 1 to 2, characterized in that the mannan-containing material comprises palm kernel cake, palm kernel flour and / or copra cake. 4. Process according to any of the claims of 1 to 3 characterized by the fact that it comprises isolating an oily fraction from the Petition 870180149158, of 11/08/2018, p. 9/13 5. Process according to any of the claims of 1 to 4, characterized by the fact that it comprises the recovery of the fermentation product from the fermentation medium that results from step (c), by distillation. 6. Process according to any of the claims of 1 to 5, characterized by the fact that the pre-treatment is the acid pre-treatment is carried out using an organic acid selected from the group consisting of soluble hydrolyzate. sulfuric acid, acetic acid, citric acid, tartaric acid, succinic acid, and mixtures thereof.