Title of Invention

A METHOD OF PRODUCING ETHANOL

Abstract

The present invention relates to a method of producing ethanol, said method comprising the steps of: a. providing a mash comprising a starch containing material and water; b. pre liquefying the mash of step (a) in the presence of a beta-glucanase; c. gelatinizing the mash of step (b) by jet cooking; d. liquefying the mash of step (c) in the presence of an alpha-amylase, a beta-glucanase and a xylanase_and e. saccharifying and fermenting the mash of step (d) to produce ethanol. f. recovering the ethanol, wherein the beta-glucanase and xylanase present in step (d) is added following step (c).

Full Text

HELD OF THE INVENTION The invention relates to a process for producing a fermentation product wherein the viscosity of the mash is reduced by application of beta-glucanase and xylanase activity. BACKGROUND OF THE INVENTION Fermentation processes are used for making a vast number of products of commercial interest. Fermentation is used in industry to produce simple compounds such as alcohols (in particular ethanol); acids, such as citric acid, itaconic acid, lactic acid, gluconic acid, lysine; ketones; amino acids, such as glutamic acid, but also more complex compounds such as antibiotics, such as penicillin, tetracyclin; enzymes; vitamins, such as riboflavin, B12l beta-carotene; hormones, such as insulin which are difficult to produce synthetically. Also in the brewing (beer and wine industry), dairy, leather, tobacco industries fermentation processes are used. There is a large number of disclosures concerning production of fermentation products, e.g. ethanol, among which is WO2002038787A2. There is a need for further improvement of fermentation processes and for improved processes including a fermentation step. Accordingly, the object of the invention is to provide an improved method of fermentation processes for producing e.g., ethanol. SUMMARY OF THE INVENTION The present invention relates to an improved process of producing a fermentation product, in particular ethanol, but also for instance the products mentioned in the "Background of the lnvention"-section. Also beverage production, such as beer production is contemplated according to the invention. The invention provides in a first aspect a method of producing a fermentation product, said method comprising preliquefaction of non-starch polysaccharides in the presence of a beta-glucanase, followed by jet cooking and liquefaction in the presence of a thermostable beta-glucanase and a xylanase. Provided in a second aspect is a method of producing a fermentation product, said method comprising the steps of: (a) providing a mash comprising a starch containing material and water; (b) preliquefying the mash of step (a) in the presence of a beta-glucanase; (c) gelatinizing the mash of step (b); (d) liquefying the mash of step (c) in the presence of a alpha-amylase and a beta-glucanase and a xylanase; and (e) saccharifying and fermenting the mash of step (d) to produce the fermentation product. Provided in a second aspect is a use of beta-glucanase and xylanase in a process for producing ethanol. The application of thinning enzymes such as beta-glucanase and xylanase in the process of the invention degrades glucan and xylan thereby reducing the viscosity of the mash. The reduced viscosity resutts in increased flow rates of the liquefied mash, thereby increasing the capacity of the production plants, especially by improving heat transfer and facilitating passage of the liquefied mash through the mash coolers. Thus the process of the invention facilitates the use of higher dry matter percentage in the fermentation while still securing an efficient cooling and a correct and uniform temperature of the mash delivered to the fermentation tanks. The effect on the distillation process of the prior hydrolysis of non-starch polysaccharides like arabinoxylan and beta-glucans is an overall increased capacity and better heat transfer and phase transfer. The effect on the by-products, such as the distiller's dry grain, of the prior hydrolysis of the non-starch polysaccharides is an overall improved feed conversion and better digestibility of the nutrients like minerals, protein, lipids and starch. DETAILED DESCRIPTION OF THE INVENTION The process of the invention may be used in the production of a large number of fermentation products comprising but not limited to alcohols (in particular ethanol); acids, such as citric acid, itaconic acid, lactic acid, gluconic acid, lysine; ketones; amino acids, such as glutamic acid, but also more complex compounds such as antibiotics, such as penicillin, tetracyclic enzymes; vitamins, such as riboflavin, B12, beta-carotene; hormones, such as insulin. Preferred is drinkable ethanol as well as industrial and fuel ethanol. Raw material Any suitable starch containing material may be used as raw material in the process of the present invention. In one embodiment, the starch containing material is whole grain obtained from cereals, preferably selected from the list consisting of corn (maize), wheat, barley, oat, rice, cassava, sorghum, rye, milo, and millet. Furthermore the starch containing material may be obtained from potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane. Sugar cane or sugar beet may be utilized as described in e.g. GB 2115820 A and US4886672A1. Preferred for the process of the invention are cereals, such as wheat, barley, oat, triticale, especially oat and barley, as well as malt derived from cereals, such as wheat, barley, oat, triticale, especially oat and barley. Slurries made from wheat, barley, oat and triticale are highly viscous why thinning is advantageous. The main process steps of the present invention may in one embodiment be described as separated into the following main process stages: (a) mash formation; (b) preliquefaction; (c) gelatinization; (d); liquefaction; and (e) saccharification and fermentation, wherein the steps (a), (b), (c) and (d) is performed in the order (a), (b), (c), (d) and (e). Step (e) may be performed as a simultaneous saccharification and fermentation (SSF) or as two separate sub steps. The individual process steps of alcohol production may be performed batch wise or as a continuous flow. For the invention processes where all process steps are performed batch wise, or processes where all process steps are performed as a continuous flow, or processes where one or more process step(s) is(are) performed batch wise and one or more process step(s) is(are) performed as a continuous flow, are equally preferred. The cascade process is an example of a process where one or more process step(s) is(are) performed as a continuous flow and as such preferred for the invention. For further information on the cascade process and other ethanol processes consult The Alcohol Textbook. Ethanol production by fermentation and distillation. Eds. T.P. Lyons, D.R. Kesall and J.E. Murtagh. Nottingham University Press 1995. Milling In a preferred embodiment of the process of the invention, the starch containing material is milled cereals, preferably barley, and the method comprises a step of milling the cereals before step (a), in other words, the invention also encompasses processes of the invention, wherein the starch containing material is obtainable by a process comprising milling of cereals, preferably dry milling, e.g. by hammer or roller mils. Grinding is also understood as milling, as is any process suitable for opening the individual grains and exposing the endosperm for further processing. Two processes of milling are normally used in alcohol production: wet and dry milling. The term "dry milling" denotes milling of the whole grain. In dry milling the whole kernel is milled and used in the remaining part of the process Mash formation The mash may be provided by forming a slurry comprising the milled starch containing material and brewing water. The brewing water may be heated to a suitable temperature prior to being combined with the milled starch containing material in order to achieve a mash temperature of 45 to 70CC, preferably of 53 to 66°C, more preferably of 55 to 60°C. The mash is typically formed in a tank known as the slurry tank. Typically the dry solids% (dry solid percentage) in the slurry tank (containing milled whole grain) is in the range from 1-60%, in particular 10-50%, such as 20-40%, such as 25-35%. rreiiqueTacilon In the preliquefaction step the starch containing material (front end mash) is held in the presence of a thinning enzyme, such as a beta-glucanase or a xylanase, preferred are a beta-glucanase, at a temperature of 45 to 70°C, more preferably to 53 to 66°C, most preferably to 55 to 60°C, such as 58°C. The duration of the preliquefaction step is preferably 5 to 60 minutes, and more preferably 10 to 30 minutes, such as around 15 minutes. Gelatinization During the gelatinization step the starch is gelatinized. Gelatinization may be achived by heating the starch containing slurry to a temperature above the gelatinization temperature of the particular starch used. Gelatinization is preferably by jet-cooking at appropriate conditions, such as, e.g. at a temperature between 95-140oC, preferably 105-125°C, such as 120°C to complete gelatinization of the starch. Also preferred is gelatinization by non-pressure cooking. During gelatinization the enzymes added in the preliquefaction step will be subjected to elevated temperatures and may be fully or partly inactivated. Thus according to the invention new thinning enzymes are preferably added following the gelatinization step. The liquefaction process is in an embodiment carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. During jet cooking the enzymes added in the preliquefaction step will be subjected to elevated temperatures and may be fully or partly inactivated. Thus according to the invention new thinning enzymes are preferably added following the jet cooking step. Liquefaction In the liquefaction step the gelatinized starch (down stream mash) is broken down (hydrolyzed) into maltodextrins (dextrins). To achieve starch hydrolysis a suitable enzyme, preferably an alpha-amylase, is added. According to the invention a beta-glucanase and a xylanase are added to the mash. In an embodiment further an endo-glucanase is added. The temperature during the liquefaction step is from 60-95°C, preferably 80-90°C, preferably at 70-80°C such as 85°C, for a period of 1-120 min, preferably for 2-60 min, such as 12 min. It is surprising that the enzymes functions at these high temperature applied during the liquefaction step. In one embodiment, the liquefaction in step (d) is performed at a pH in the range of about pH 4-7, preferably pH about 4.5-6.5. In a preferred embodiment, the pH during the liquefaction is at most about 5. The pH of the slurry may by adjusted or not, depending on the properties of the enzymes used. Thus, in one embodiment the pH is adjusted, e.g. about 1 unit upwards, e.g. by adding NH3. The adjusting of pH is advantageously done at the time when the alpha-amylase is added. In yet another embodiment, the pH is not adjusted and the alpha-amylase has a corresponding suitable pH-activity profile, such as being active at a pH about 4. In an embodiment of the invention the thinning enzyme(s) is(are) added to the gelatinized mash together with an alpha-amylase. Sacchariftcation and fermentation The saccharification step and the fermentation step may be performed as separate process steps or as a simultaneous saccharification and fermentation (SSF) step. The saccharification is carried out in the presence of a saccharifying enzyme, e.g. a glucoamylase, a beta-amylase or maltogenic amylase. Optionally a phytase and/or a protease is added. The fermenting organism may be a fungal organism, such as yeast, or bacteria. Suitable bacteria may e.g. be Zymomonas species, such as Zymomonas mobilis and E colL Examples of filamentous fungi include strains of Penicillium species. Preferred organisms for ethanol production are yeasts, such as e.g. Pichia or Saccharomyces. Preferred yeast according to the invention is Saccharomyces species, in particular Saccharomyces cerevisiae or bakers yeast. The yeast cells may be added in amounts of 105 to 1012, preferably from 107 to 1010, especially 5x107 viable yeast count per ml of fermentation broth. During the ethanol producing phase the yeast cell count should preferably be in the range from 107 to 1010, especially around 2 x 108. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The alcohol Textbook" (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference The microorganism used for the fermentation is added to the mash and the fermentation is ongoing until the desired amount of fermentation product is produced; in a preferred embodiment wherein the fermentation product is ethanol to be recovered this may, e.g., be for 24-96 hours, such as 35-60 hours. The temperature and pH during fermentation is at a temperature and pH suitable for the microorganism in question and with regard to the intended use of the fermentation product, such as, e.g., in an embodiment wherein the fermenting organism is yeast and the product is ethanol for recovery the preferred temperature is in the range about 26-34°C. e.g. about 32°C, and at a pH e.g. in the range about pH 3-6, e.g. about pH 4-5. In another embodiment wherein the fermenting organism is yeast, and the fermented mash is to be used as a beer, the temperature of the mash the preferred temperature is around 12-16°C, such around 14°C. in a preferred embodiment, a simultaneous saccharification and fermentation (SSF) process is employed where there is no holding stage for the saccharification, meaning that yeast and saccharification enzyme is added essentially together. In one embodiment, when doing SSF a pre-saccharification step at a temperature above 50°C is introduced just prior to the fermentation. Distillation The method of the invention may further comprise recovering of the fermentation product, i.e. ethanol; hence the alcohol may be separated from the fermented material and purified. Thus, in one embodiment, the method of the invention further comprises the step of: (f) distillation to obtain the ethanol. Bv-products from distillation The aqueous by-product (Whole Stillage) from the distillation process is separated into two fractions, for instance by centrifugation: 1) Wet Grain (solid phase), and 2) Thin Stillage (supernatant). The Wet Grain fraction is dried, typically in a drum dryer. The dried product is referred to as "Distillers Dried Grain", and can be used as animal feed. The Thin Stillage fraction may be evaporated providing two fractions: - condensate fraction of 4-6% dry solids (mainly of starch, proteins, and cell wall components), and - syrup fraction, mainly consisting of limit dextrins and non fermentable sugars, which may be introduced into a dryer together with the Wet Grain (from the Whole Stillage separation step) to provide a product referred to as "Distillers Dried Grain", which can be used as animal feed. "Whole Stillage' is the term used in the art for the side-product coming from the distillation of fermented mash. 'Thin Stillage" is the term used in the art for the supernatant of the centrifugation of the Whole Stillage. Typically, the Thin Stillage contains 4-6% dry solids (mainly starch and proteins) and has a temperature of about 60-90°C. Thin Stillage is viscous and difficult to handle. Thin Stillage is normally kept in a holding tank for up to a few hours before recycling to the slurry tank. The stillage may be thinned with suitable enzymes, such as beta-glucanase and xylanase, before recycling. Further details on how to carry out liquefaction, saccharification, fermentation, distillation, and recovering of ethanol are well known to the skilled person. Use of the products produced by the method of the invention In embodiments wherein the fermentation product is ethanol, the ethanol obtained by the process of the invention may be recovered from the fermented mash and used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits, or industrial ethanol, including fuel additive. In embodiments wherein the fermentation product is ethanol, and the ethanol obtained by the process of the invention is not recovered from the fermented mash the mash comprising the ethanol may be used as a beer. The beer may be any beer including ales, strong ales, bitters, stouts, porters, lagers, export beers, malt liquors, barley wine, Commercial products include SAN™ SUPER™ and AMG™ E (Trom Novozymes M/O;. Glucoamylases may in an embodiment be added in the saccharification and fermentation step (e) in an amount of 0.02-2 AGU/g dry solids, preferably 0.1-1 AGU/g dry solids, such as 0.2 AGU/g dry solids. Protease Addition of protease(s) in the saccharification step, the SSF step and/or the fermentation step increase(s) the FAN (Free amino nitrogen) level and increase the rate of metabolism of the yeast and may increase the fermentation efficiency. Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7. Suitable acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Torulopsis. Especially contemplated are proteases derived from Aspergillus niger (see, e.g., Koaze et al., (1964), Agr. Biol. Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida, (1954) J. Agr. Chem. Soc. Japan, 28, 66), Aspergillus awamori (Hayashida et al., (1977) Agric. Biol. Chem., 42(5), 927-933, Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepA protease; and acidic proteases from Mucor pusillus or Mucor miehei. ALCALASE™ is a Bacillus licheniformis protease (subtilisin Carlsberg). ALCALASE™ may according to the invention preferably be added is amounts of 10'7to 10"8 gram active protease protein/g dry solids, in particular 10"6 to 10"4 gram active protease protein/g dry solids, or in amounts of 0.1-0.0001 AU/g dry solids, preferably 0.00025-0.001 AU/g dry solids. FLAVOURZYME™ (available from Novozymes A/S) is a protease preparation derived from Aspergillus oryzae. FLAVOURZYME™ may according to the invention preferably be added in amounts of 0.01-1.0 LAPU/g dry solids, preferably 0.05-0.5 LAPU/g dry solids. A suitable dosage of the protease is in the range in an amount of 10'7to 10*3 gram active protease protein/g dry solids, in particular 10* to 10"4 gram active protease protein/g dry solids Phvtase: The phytase used according to the invention may be any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) or from any salt thereof (phytates). A suitable dosage of the phytase is in the range from 0.005-25 FYT/g dry solids, preferably 0.01-10 FYT/g, such as 0.1-1 FYT/g dry solids enzymes were tested in a slurry liquefied with bacterial alpha-amylase. The 28% dry solids sluny was DE 16 and pH 5.0. The slurry was portioned to 1 litre flasks and maintained at 84°C in a temperature controlled water bath. Different enzyme combinations were tested in dosages according to table 3. The viscosity was measured as a function of time, using a Haake Viscotester VT-02, see table 4. The combinations of betaglucanase + xylanase II + endo-glucanase worked more effectively that the product GC 880 or beta-glucanase alone or xylanase II + endo-glucanase. Example 3 1 kg slurries of 30 % grain dry matter were prepared by stirring milled rye into the corresponding amount of water, which had a temperature of 55°C. The pH was adjusted to 5.0 with sulphuric acid. The final temperature of the slurry was 50°C. Enzymes were added at t=0 minutes and mixed into the slurry by 3 minutes of stirring. The viscosity was measured, using a Haake viscotester VT-02.. The combinations of betaglucanase + xylanase II and xylanase II + endo-glucanase resulted in a higher viscosity reduction that beta-glucanase or xylanase alone. Example 4 To simulate downstream processes in laboratory 1 kg slurries of 20 % rye dry matter were prepared by stirring milled rye into the corresponding amount of water. Using a dosage of 0.5 kg Termamyl SC /tons of grain (as is) liquefaction was performed at 75°C. Afterwards the slurries were treated at 70°C with viscosity reducing enzymes and measured at 70°C, pH=6-6.5. This was done as a model test in order to test the effect of the viscosity reducing enzymes. Industrially Termamyl + the non-starch polysaccharide degrading viscosity reducing enzymes should operate simultaneously. The viscosity measurements were made using HAAKE Viscotester VT-02 was taken at t=3, 15, 30, 60 minutes as above, but at 70°C. The temperature was measured on the samples after the viscosity measurements. The combinations of beta-glucanase + xylanase II and endo-glucanase + xylanase II resulted in a high viscosity reduction at 70°C. Claims; 1. A method of producing ethanol, said method comprising the steps of: a. providing a mash comprising a starch containing material and water; b. preliquefying the mash of step (a) in the presence of a beta-glucanase; c. gelatinizing the mash of step (b) by jet cooking; d. liquefying the mash of step (c) in the presence of an alpha-amylase, a beta- glucanase and a xylanase; and e. saccharifying and fermenting the mash of step (d) to produce ethanol. f. recovering the ethanol. 2. The method of claim 1, further comprising a pre-saccharification step which is performed after the liquefaction step (d) and before step (e). 3. The method of any of claims 1 or 2, wherein the xylanase is derived from a strain of Aspergillus sp., preferably from a strain of A. Aculeatus. 4. The method of any of claims 1-3, wherein the beta-glucanase is derived from a strain of Bacillus sp., preferably from a strain of S. amyloliquefaciens. 5. The method of any of claims 1-4, wherein also an endo-glucanase is present in the liquefaction step (d), said endo-glucanase preferably derived from a strain of Tricho-derma sp., preferably from a strain of T.reesei. 6. The method of any of claims 1-5, wherein the starch containing material is obtained from cereals and/or tubers. 7. The method of any of claims 1-6, wherein the starch containing material is selected from the groups consisting of maize, wheat, barley, rye, millet, sorghum, and milo. 8. The method of any of claims 1-7, wherein the starch containing material is selected from the groups consisting of potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and sugar cane. 9. The method of any of claims 1-8, wherein the fermentation in step (e) is performed using a micro-organism, such as bacteria and fungi (including yeasts), e.g. Zymomo-nas species and Sacharomyces species, such as e.g. Saccharomyces cerevisiae. 10. The method of any of claims 1-9, wherein the fermentation is carried out in the presence of phytase and/or protease. 11. The method of any of claims 1-10, wherein preliquefaction in step (b) is performed at a temperature of 45 to 70°C, more preferably to 53 to 66°C, most preferably to 55 to 60°C, such as 58°C for a period of 5 to 60 minutes, and more preferably 10 to 30 minutes, such as around 15 minutes. 12. The method of any of claims 1-11, wherein the liquefaction in step (d) is performed at 60-95°C, preferably 80-90°C for 10-120 min, more preferably at 83-85 °C for 15-80 min.

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2526-CHENP-2005 ABSTRACT.pdf

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2526-CHENP-2005 CORRESPONDENCE OTHERS.pdf

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2526-CHENP-2005 DESCRIPTION (COMPLETE).pdf

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2526-chenp-2005 abstract granted.pdf

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