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WO2001062947A1 - Fermentation a l'aide de la phytase - Google Patents

Fermentation a l'aide de la phytase Download PDF

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Publication number
WO2001062947A1
WO2001062947A1 PCT/DK2001/000125 DK0100125W WO0162947A1 WO 2001062947 A1 WO2001062947 A1 WO 2001062947A1 DK 0100125 W DK0100125 W DK 0100125W WO 0162947 A1 WO0162947 A1 WO 0162947A1
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WO
WIPO (PCT)
Prior art keywords
protease
fermentation
phytase
alpha
amylase
Prior art date
Application number
PCT/DK2001/000125
Other languages
English (en)
Inventor
Chris Veit
Claus Felby
Larry W. Peckous
Hans Sejr Olsen
Original Assignee
Novozymes A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to AU2001235360A priority Critical patent/AU2001235360A1/en
Priority to EP01907391A priority patent/EP1259630A1/fr
Publication of WO2001062947A1 publication Critical patent/WO2001062947A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/023—Preparation of other alcoholic beverages by fermentation of botanical family Solanaceae, e.g. potato
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a process of fermenting phytic acid-containing materials; a process of alcohol and other fermented compounds production, in particular ethanol production; the use of phytase activity for saccharification and/or fermentation; and a composition suitable for ethanol production.
  • Fermentation processes are used for making a vast number of products of big 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; ketones; amino acids, such as glu- ta ic acid, but also more complex compounds such as antibiotics, such as penicillin, tetracyclin; enzymes; vitamins, such as riboflavin, B ⁇ 2 , beta-carotene; hormones, which are difficult to produce synthetically. Also in the brewing (beer and wine industry) , dairy, leather, tobacco industries fermentation processes are used.
  • the object of the invention is to provide an im- proved method at least including a fermenting step.
  • Fig. 1 shows schematically an ethanol production process of the invention.
  • Fig. 2 shows the results on fermentation of liquefied whole corn mash using AMG and AMG+phytase . C0 loss vs. time
  • Fig. 3 shows the phytin level as g/kg dry matter for the corn substrate and following fermentation with AMG and AMG + phytase .
  • the present invention relates to a process of producing a fermentation product, for instance the ones mentioned in the "Background of the Invention" -section, in particular ethanol, but also beverages, such as beer or wine are contemplated, wherein the fermentation is carried out in the presence of phy- tase activity.
  • a carbohydrate source such as glucose, dextrose, maltose or the like, need to be present during fermentation for the fermenting organism to be able to ferment.
  • the carbohydrate source may be supplied by direct addition of e.g., glucose, or may be supplied as a product of, e.g., (pre-) saccharification step, as will be described further below.
  • the process of the invention may in one embodiment be an ethanol process comprising the below steps, wherein phytase activity is added during pre-saccharification and/or fermentation. It is to be understood that the phytase according to the invention may be added during the propagation of yeast cells and/or later on during the actual fermentation. Beverage production, such as beer or wine production is equally contemplated.
  • Alcohol production, in particular ethanol production, from whole grain can be separated into 4 main steps
  • the (whole) grain is milled in order to open up the structure and allowing for further processing.
  • Two processes are preferred according to the invention: wet and dry milling.
  • Preferred for ethanol production is dry milling where the whole kernel is milled and used in the remaining part of the process.
  • Wet milling may also be used and gives a good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups. Both dry and wet milling is well known in the art of, e.g., ethanol production.
  • milled gelatinized whole grain raw material is broken down (hydrolyzed) into maltodextrins (dextrins) mostly of a DE higher than 4.
  • the hydrolysis may be carried out by acid treatment or enzymatically by alpha-amylase treatment, in particular with a Bacillus alpha-amylase as will be described further below. Acid hydrolysis is used on a limited basis.
  • the raw material is in one embodiment of the invention milled (whole) grain. However, a side stream from starch processing may also be used.
  • enzymatic liquefaction is carried out as a three-step hot slurry process.
  • the slurry is heated to between 60-95°C, preferably 80-85°C (in the Slurry Tank - see Fig. 1), and the enzyme (s) is (are) added to initiate liquefaction (thinning) . Then the slurry is jet-cooked at a temperature between 95-140°C, preferably 105-125°C to complete gelanitization of the slurry. Then the slurry is cooled to 60-
  • the liquefaction process is carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied whole grains are known as mash.
  • the maltodextrin from the liquefaction must be fur- ther hydrolyzed.
  • the hydrolysis is typically done enzymatically by glucoamylases, alternatively alpha-glucosidases or acid al- pha-amylases can be used.
  • a full saccharification step may last up to 72 hours, however, it is common only to do a pre- saccharification of typically 40-90 minutes and then complete saccharification during fermentation (SSF) . Saccharification is typically carried out at temperatures from 30-65°C, typically around 60°C, and at pH 4.5.
  • Fermentation Yeast typically from Sac char omyces spp . is added to the mash and the fermentation is ongoing for 24-96 hours, such as typically 35-60 hours.
  • the temperature is between 26-34°C, in particular about 32°C, and the pH is from pH 3-6, preferably around pH 4 -5.
  • SSF simultaneous saccharification and fermentation
  • fermenting organism such as the yeast, and enzyme (s) is (are) added together.
  • the mash may be dis- tilled to extract the, for instance, ethanol.
  • the end product is ethanol, obtained according to the process of the invention, it may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol.
  • the grain which is typically used for animal feed either in liquid or dried form.
  • the saccharification and fermentation may be carried out simultaneously or separately.
  • the invention relates to a process, wherein phytic acid-containing material is fermented in the presence of a phytase.
  • a phytase during the fermentation (or e.g., a combined or simultaneous fermentation and saccharification step, as will be described further below) results in a number of advantages.
  • the phytase according to the invention may be added during the propagation of yeast cells and/or later on during the actual fermentation. For instance, the addition of phytase results in that more free minerals, e.g., Ca 2+ , are made available to the fermenting organism(s), in particular yeast.
  • Bacillus e.g., BAN or Bacillus licheniformis alpha-amylases
  • Aspergillus e.g., the Asper- gillus oryzae alpha-amylase sold as FUNGAMYLTM from Novozymes A/S alpha-amylases. Therefore, a higher fermentation rate is a result of the
  • phytase is made in combination with a "Carbohydrate-source generating enzyme" .
  • the term "carbohydrate-source generating enzyme” includes gluco-amylases (being a glucose generator) , and beta-amylases and maltogenic amylases (being maltose generators) .
  • the carbohydrate-source generating enzymes are capable of providing energy to the fermenting microorganism (s) in ques- tion.
  • the availability of more free phosphorus minerals and vitamins - as a result of phytin to in ⁇ sitil conversion - improves the yeast growth and viability during fermentation and thus increases the fermentation and ethanol yields. Further, the protein availability is increased.
  • the by-product of an ethanol process may be used as feed
  • the invention relates to a process for the production of ethanol, comprising the steps of: (a) milling whole grains, (b) liquefying the product of step (a) , in the presence of an alpha-amylase,
  • step (e) distilling of the fermented and saccharified material obtained in step (d) .
  • the whole grains in step a) are dry milled, for instance in a hammer mill.
  • the DS% (dry solid percentage) in the slurry tank is in the range from 1-60%, in particular 10-50%, such as 20-40%, such as 25- 35%.
  • the liquefaction step comprising the following sub-steps: bl) the hot slurry is heated to between 60-95°C, preferably 80-85°C, and at least an alpha-amylase is added; b2) the slurry is jet-cooked at a temperature between 95- 140°C, preferably 105-125°C to complete gelanitization of the slurry; b3) the slurry is cooled to 60-95°C and more alpha- amylase is added to finalize hydrolysis.
  • the liquefaction process is in an embodiment carried out at pH 4.5-6.5, in particular at a pH between 5 and 6.
  • Steps (c) and (d) may be carried out either simultaneously or separately/sequential. Further, after step (e) an optional ethanol recovery step may be added.
  • the raw materials contain phytic acids and fermentable sugars or constituents, which can be converted into sugars.
  • This in- elude starch-containing raw materials such as tubers, roots, whole grains, corns, cobs, wheat, barley, rye, milo or cereals, sugar- containing raw materials, such as molasses, fruit materi- als, sugar, cane or sugar beet, potatoes, cellulose-containing materials, such as wood or plant residues.
  • the phytic acid containing material may be the side stream from starch processing, in particu- lar liquefied starch with a DE of 6-20, in particular between 8-10.
  • the microorganism may be a fungal organism, such as yeast or bacteria.
  • filamentous fungi include strains of Penicillium sp .
  • Preferred organisms for ethanol production is yeasts.
  • Preferred yeast according to the invention is baker's yeast, also known as Saccharomyces cerevisiae .
  • the yeast may according to the invention preferably be added before starting the actual fermentation (i.e., during the propagation phase).
  • the yeast cells may be added in amounts of 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially 5xl0 7 viable yeast count per ml of fermentation broth. During the ethanol producing phase the yeast cell count should preferably be in the range from 10 7 to 10 10 , especially around 2 x 10 8 .
  • Example 1 shows a fermentation process of the invention where the yeast is not stressed (yeast count of about 10 10 cells per ml) . Even under such conditions the addition of phytase is shown to improved the fermentation process . 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 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) .
  • Phytases can be classified according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
  • the phytase to be used in the invention may have any specificity, e.g., be a 3 -phytase (EC 3.1.3.8), a 6-phytase (EC 3.1.3.26) or a 5-phytase (no EC number) .
  • the phytase has a temperature op- timum in the range from 25-70°C, preferably 28-50°C, especially
  • the phytase has a temperature optimum above 50°C, such as in the range from 50-70°C. This is advantageous when the phytase is added during (pre- ) saccharification.
  • the dosage of the phytase may be in the range 5.000-250.000 FYT/g DS, particularly 10.000-100.000 FYT/g DS.
  • a preferred suitable dosage of the phytase is in the range from 0.005-25 FYT/g DS, preferably 0.01-10 FYT/g, such as 0.1-1 FYT/g DS.
  • the phytase activity is determined FYT units, one FYT being the amount of enzyme that liberates 1 micromole inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37°C; substrate: sodium phytate (C 6 H 6 0 2 4P6Nai2) at a concentration of 0.0050 mole/1.
  • the phytase may be derived from plants or microorganisms, such as bacteria or fungi, e.g., yeast or filamentous fungi.
  • the plant phytase may be from wheat-bran, maize, soy bean or lily pollen. Suitable plant phytases are described in Thomlinson et al , Biochemistry, 1 (1962) , 166-171; Barrientos et al, Plant. Physiol . , 106 (1994), 1489-1495; WO 98/05785; WO 98/20139.
  • a bacterial phytase may be from genus Bacillus, Pseudomonas or Escherichia, specifically the species B . subtilis or E. coli .
  • Suitable bacterial phytases are described in Paver and Jagannathan, 1982, Journal of Bacteriology 151:1102-1108; Cosgrove, 1970, Australian Journal of Biological Sciences 23:1207-1220; Greiner et al, Arch. Biochem. Biophys . , 303, 107- 113, 1993; WO 98/06856; WO 97/33976; WO 97/48812.
  • a yeast phytase or myo-inositol monophosphatase may be derived from genus Saccharomyces or Schwanniomyces , specifically species Saccharomyces cerevisiae or Schwanniomyces occiden- talis .
  • the former enzyme has been described as a Suitable yeast phytases are described in Nayini et al, 1984, Anlagen Wis- senschaft und Technologie 17:24-26; Wodzinski et al , Adv. Appl . Microbiol .
  • Phytases from filamentous fungi may be derived from the fungal phylum of Asco ycota (ascomycetes) or the phylum Basidiomycota, e.g., the genus Aspergillus, Thermomyces (also called Humicola) , Myceliophthora, Manascus, Penicillium, Penio- phora, Agrocybe, Paxillus , or Trametes, specifically the spe- cies Aspergillus terreus, Aspergillus niger, Aspergillus niger var.
  • Asco ycota ascomycetes
  • Basidiomycota e.g., the genus Aspergillus, Thermomyces (also called Humicola) , Myceliophthora, Manascus, Penicillium, Penio- phora, Agrocybe, Paxillus , or Trametes, specifically the spe-
  • BIO-FEED PHYTASETM PHYTASE NOVOTM CT or L (all from Novozymes)
  • NATUPHOSTM NG 5000 from DSM
  • the liquefaction step may be performed in the presence of an alpha-amylase derived from a microorganism or a plant.
  • Preferred alpha-amylases are of fungal or bacterial origin.
  • Bacil - lus alpha-amylases (often referred to as "Termamyl-like alpha- amylases") , variant and hybrids thereof, are specifically contemplated according to the invention.
  • Well-known Termamyl-like alpha-amylases include alpha-amylase derived from a strain of B . licheniformis (commercially available as TermamyiTM) r B . amy- loliquefaciens , and B.
  • Termamyl-like alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsuka oto et al . , Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.
  • a Termamyl- like alpha-amylase is an alpha-amylase as defined in WO 99/19467 on page 3, line 18 to page 6, line 27.
  • Contemplated variants and hybrids are described in WO 96/23874, WO 97/41213, and WO 99/19467.
  • Contemplated alpha-amylase derived from a strain of Aspergillus includes Aspergillus oryzae and Aspergillus niger alpha-amylases.
  • Commercial alpha-amylase products and products containing alpha-amylases include TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM and SANTM SUPER.
  • Fungal alpha-amylases may be added in an amount of 0.001- 1.0 AFAU/g DS, preferably from 0.002-0.5 AFAU/g DS, preferably 0.02-0.1 AFAU/g DS .
  • Bacillus alpha-amylases may be added in effective amounts well known to the person skilled in the art.
  • the saccharification step (c) or a combined saccharification and fermentation step (SSF step) may be carried out in the presence of a glucoamylase derived from a microorganism or a plant.
  • a glucoamylase derived from a microorganism or a plant.
  • Preferred is glucoamylase of fungal or bacterial origin selected from the group consisting of Aspergillus glucoamy- lases, in particular A . niger Gl or G2 glucoamylase (Boel et al . (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as disclosed in WO 92/00381 and WO 00/04136; the A . awamori glucoamylase (WO 84/02921), A. oryzae (Agric . Biol . Chem. (1991) , 55 (4) , p. 941-949) , or
  • variants include variants to enhance the thermal stability: G137A and G139A (Chen et al . (1996), Prot. Engng. 9, 499-505); D257E and D293E/Q (Chen et al . (1995), Prot. Engng. 8, 575-582); N182 (Chen et al . (1994), Biochem. J. 301, 275-281); disulphide bonds, A246C (Fierobe et al . (1996), Biochemistry, 35, 8698- 8704; and introduction of Pro residues in position A435 and S436 (Li et al . (1997), Protein Engng. 10, 1199-1204.
  • glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448) , Talaromyces leycetta- nus (US patent no. Re. 32,153), Talaromyces duponti , Talaromyces thermophilus (US patent no. 4,587,215).
  • Bacterial glucoamylases contemplated include glucoamylases from the genus Clos- tridium, in particular C. thermoamylolyticum (EP 135, 138) , and C. thermohydrosulfuricum (WO 86/01831).
  • Glucoamylases may in an embodiment be added in an amount of 0.02-2 AGU/g DS, preferably 0.1-1 AGU/g DS, such as 0.2 AGU/g DS
  • the ratio between acidic fungal alpha-amylase activity (AFAU) per glucoamylase activity (AGU) (AFAU per AGU) may in one embodiment be at least 0.1, in particular at least 0.16, such as in the range from 0.12 to 0.30.
  • SANTM SUPER and AMGTM E from No- vozy es
  • OPTIDEXTM 300 from Genencor Int.
  • AMIGASETM from DSM
  • G-ZYMETM G900 from Enzyme Bio-Systems
  • protease increase (s) the FAN (Free amino nitrogen) level and increase the rate of metabolism of the yeast and further gives higher fermentation efficiency.
  • Suitable proteases include 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 de- rived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, En- do thi a, Enthomophtra, Irpex, Penicillium, Sclerotium and Toru- lopsis .
  • proteases derived from As- pergillus niger see, e . g. , Koaze et al . , (1964) , Agr. Biol .
  • Bacterial proteases which are not acidic proteases, include the commercially available products Alcalase ® and Neu- trase ® (available from Novozymes A/S) .
  • Protease (s) may in one embodiment be added in an amount of 10 "7 to 10 "5 gram active protease protein/g DS, in particular 10 " 7 to 5xl0 ⁇ 6 gram active protease protein/g DS
  • Additional enzymes include pullulanases .
  • the invention relates to the use of phytase, in particular the phytases mentioned above for saccharification and/or fermentation (SSF) and for use for ethanol production.
  • SSF saccharification and/or fermentation
  • composition comprising a phytase and at least one carbohydrate-source generating enzyme
  • glucoamylase such as an
  • composition may further comprise a protease, in particular an acid protease, such as an acid fungal protease.
  • Glucoamylase A . niger glucoamylase (available as AMG E (1999- SE-0025) from Novozymes)
  • Alpha-amylase BSG (B. stearothermophilus alpha-amylase which is available from Novozymes as TERMAMYLTM SC)
  • Mash Liquefied whole corn mash prepared by a hot slurry process and Termamyl SC. The mash had a DE of about 17 and a dry substance of 28%.
  • Yeast Dry yeast (Saccharomyces cervisiae)
  • Phadebas ® tablets as substrate.
  • Phadebas tablets (Phadebas ® Amy- lase Test, supplied by Pharmacia Diagnostic) contain a cross- linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and ta- bletted.
  • the measured 620 nm absorbance after 10 or 15 minutes of incubation is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law) .
  • the dilution of the enzyme must therefore be adjusted to fit this criterion.
  • a specified set of conditions temp., pH, reaction time, buffer conditions
  • 1 mg of a given alpha- amylase will hydrolyze a certain amount of substrate and a blue colour will be produced.
  • the colour intensity is measured at 620 nm.
  • the measured absorbance is directly proportional to the spe- cific activity (activity/ g of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
  • Alpha-amylase activity is determined by a method employ- ing the PNP-G7 substrate.
  • PNP-G7 which is a abbreviation for p- nitrophenyl -alpha
  • D-maltoheptaoside is a blocked oligosaccha- ride which can be cleaved by an endo-amylase .
  • Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. No. 1054635) .
  • BM 1442309 To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309) .
  • BM 1462309 To prepare the alpha-Glucosidase one bottle of alpha-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309) .
  • the working solution is made by mixing 5 ml alpha-Glucosidase solution with 0.5 ml substrate .
  • the assay is performed by transforming 20 micro 1 enzyme solution to a 96 well microtitre plate and incubating at 25°C.
  • FAU Acid Amylolytic Activity
  • One Fungal Alpha-Amylase Unit (1 FAU) is defined as the amount of enzyme, which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch 9947275) per hour at Novo Nordisk' s standard method for determination of alpha-amylase based upon the following standard conditions: Substrate Soluble starch
  • Acid alpha-amylase activity is measured in AFAU (Acid Fungal Alpha-amylase Units) , which are determined relative to an enzyme standard, The standard used is AMG 300 L (wild type A . niger Gl AMG sold by Novo Nordisk) .
  • the neutral alpha-amylase in this AMG falls after storage at room temperature for 3 weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.
  • the acid alpha-amylase activity in this AMG standard is determined in accordance with AF 9 1/3 (available from Novo method for the determination of fungal alpha-amylase) .
  • 1 AFAU is defined as the amount of enzyme, which degrades 5.260 mg starch dry matter per hour under standard conditions .
  • Iodine forms a blue complex with starch but not with its degradation products. The intensity of colour is therefore directly proportional to the concentration of starch.
  • Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under specified analytic condi- tions.
  • Iodine (I 2 ) 0.03 g/L CaCl 2 : 1.85 mM pH: 2.50 ⁇ 0.05
  • Enzyme concentration 0.025 AFAU/mL
  • Enzyme working range 0.01-0.04 AFAU/mL
  • AGU Novo Amyloglucosidase Unit
  • AGU is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.
  • a detailed description of the analytical method (AEL-SM-0131) is available on request from Novo Nordisk.
  • the activity is determined as AGU/ml by a method modified after (AEL-SM-0131, available on request from Novo Nordisk) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml .
  • microL substrate 1% maltose in 50 mM Sodium acetate, pH 4.3
  • 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution
  • mash 250 g was filled into a 500 mL blue cap bottle.
  • the pH of the mash was adjusted to 4.5.
  • a pre- saccharification step was carried out by adding the saccharifi- cation enzymes and placing the bottles in a water bath at 60°C for 70 minutes.
  • the bottle was cooled in a water bath for 40 minutes to 30°C and dry yeast was added at a dosage of 0.8 g/bottle (in order to reach 30°C within 40 minutes ice is added to the water bath) .
  • the bottle is closed by a yeast-lock filled with concentrated H 2 S0 4 .
  • the fermentation was continued for 96 hours and the fermentation rate was monitored by weighing the bottle at regular intervals for measuring C0 2 loss.
  • the phytin content was measured for all treatments and the substrate.
  • the phytin content was measured by The Danish Institute . of Agricultural sciences, Tjele, Denmark, according to the method described in: Brooks, J.R. and C.V. Morr. 1984. Phosphorus and phytate content of soybean protein components. J. Ag- ric. Food Chem. 32: 872-874.
  • the results of the fermentations are shown in Fig. 2 and Table 1.
  • the effect of the phytase treatment on the level of phytin is shown in Fig. 3.
  • the phytase efficiently hy- drolyses the phytin below the detection level.
  • Table 1 Fermentation of whole corn mash. C0 2 loss after 96 hour fermentation using AMG E and phytase.

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Abstract

L'invention concerne un procédé de fermentation amélioré dans lequel un matériau contenant de l'acide phytique est mis à fermenter en présence d'une phytase, par exemple, dans une fermentation permettant de produire de l'éthanol.
PCT/DK2001/000125 2000-02-23 2001-02-22 Fermentation a l'aide de la phytase WO2001062947A1 (fr)

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AU2001235360A AU2001235360A1 (en) 2000-02-23 2001-02-22 Fermentation with a phytase
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Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074895A3 (fr) * 2001-03-19 2003-06-12 Novozymes As Procede ameliore de fermentation
WO2005005646A2 (fr) 2003-06-10 2005-01-20 Novozymes North America, Inc. Processus et compositions de fermentation
JP2005278544A (ja) * 2004-03-30 2005-10-13 Asahi Breweries Ltd イノシトール含量の高いビール、発泡酒、麦芽発酵飲料およびその製造方法
WO2007035730A2 (fr) 2005-09-20 2007-03-29 Novozymes North America, Inc. Procédé de production d'un produit de fermentation
WO2008097620A1 (fr) * 2007-02-07 2008-08-14 Danisco Us, Inc., Genencor Division Hydrolyse d'amidon en utilisant une phytase avec une alpha-amylase
WO2008112282A1 (fr) * 2007-03-14 2008-09-18 Danisco Us, Inc., Genencor Division Production d'éthanol à partir d'orge et de ddgs contenant des niveaux réduits de bêta-glucane et d'acide phytique
WO2008116878A1 (fr) 2007-03-26 2008-10-02 Novozymes A/S Phytase de hafnia
WO2008097619A3 (fr) * 2007-02-07 2008-10-16 Danisco Us Inc Genencor Div Variants de la phytase de buttiauxella sp. dotés de propriétés modifiées
WO2009052500A1 (fr) * 2007-10-18 2009-04-23 Novozymes A/S Procédés de production de produits de fermentation
WO2009094614A1 (fr) * 2008-01-25 2009-07-30 Novozymes A/S Production de produits de fermentation en présence d'une aldéhyde déshydrogénase
WO2009121058A1 (fr) * 2008-03-28 2009-10-01 Novozymes A/S Production de produits de fermentation en présence de tréhalase
WO2010086078A1 (fr) * 2009-01-30 2010-08-05 Unilever Nv Utilisation de phytase dans la préparation d'un produit à base de soja fermenté
EP2295545A1 (fr) 2002-09-26 2011-03-16 Novozymes North America, Inc. Procédés et compositions de fermentation
WO2011117397A1 (fr) 2010-03-26 2011-09-29 Novozymes A/S Variantes de phytase thermostables
US8143046B2 (en) 2007-02-07 2012-03-27 Danisco Us Inc., Genencor Division Variant Buttiauxella sp. phytases having altered properties
WO2014001349A1 (fr) * 2012-06-26 2014-01-03 Dsm Ip Assets B.V. Phytase utilisée en production de biogaz
US8877478B2 (en) 2006-09-21 2014-11-04 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
CN105219593A (zh) * 2015-11-13 2016-01-06 绍兴创易科技有限公司 一种以绍酒专用曲为曲种的清爽型米酒酿造工艺
US9670509B2 (en) 2003-03-10 2017-06-06 Novozymes A/S Alcohol product processes
US9695403B2 (en) 2009-05-21 2017-07-04 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
US9856498B2 (en) 2012-03-30 2018-01-02 Novozymes A/S Processes of producing fermentation products
US10227613B2 (en) 2012-03-30 2019-03-12 Novozymes A/S Processes for producing fermentation products
WO2022034211A1 (fr) 2020-08-13 2022-02-17 Novozymes A/S Variants de phytase et polynucléotides les codant
WO2024137246A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 1 d'estérase de glucide (ce1) présentant une activité d'estérase d'acide férulique et/ou d'estérase d'acétyl xylane et polynucléotides codant pour ceux-ci
WO2024137250A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 3 de gludice estérase (ce3) présentant une activité acétyl xylane estérase et polynucléotides codant pour ceux-ci
WO2024137704A2 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédés de production de produits de fermentation faisant appel à des enzymes de dégradation de fibres avec levure modifiée
WO2024137248A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Compositions contenant des arabinofuranosidases et une xylanase, et leur utilisation pour augmenter la solubilisation de fibres hémicellulosiques
WO2024137252A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédé de réduction de la viscosité du sirop à la fin d'un processus de production d'un produit de fermentation
WO2024258820A2 (fr) 2023-06-13 2024-12-19 Novozymes A/S Procédés de fabrication de produits de fermentation à l'aide d'une levure modifiée exprimant une bêta-xylosidase
WO2025128568A1 (fr) 2023-12-11 2025-06-19 Novozymes A/S Composition et son utilisation pour augmenter la solubilisation de fibres hémicellulosiques

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US5756714A (en) * 1995-03-09 1998-05-26 Genencor International, Inc. Method for liquefying starch
US5902615A (en) * 1996-07-18 1999-05-11 Roquette Freres Nutritional composition resulting from maize steeping

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EP0321004A1 (fr) * 1987-11-17 1989-06-21 Dorr-Oliver Incorporated Procédé de trempage de céréales avec une nouvelle préparation d'enzyme
WO1992020777A1 (fr) * 1991-05-17 1992-11-26 Solvay Enzymes, Inc. Procede de production d'ethanol
WO1996013600A1 (fr) * 1994-10-27 1996-05-09 Genencor International, Inc. Procede ameliorant l'utilisation des matieres premieres dans les processus de fermentation
US5756714A (en) * 1995-03-09 1998-05-26 Genencor International, Inc. Method for liquefying starch
US5902615A (en) * 1996-07-18 1999-05-11 Roquette Freres Nutritional composition resulting from maize steeping

Cited By (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074895A3 (fr) * 2001-03-19 2003-06-12 Novozymes As Procede ameliore de fermentation
EP2295545A1 (fr) 2002-09-26 2011-03-16 Novozymes North America, Inc. Procédés et compositions de fermentation
US9670509B2 (en) 2003-03-10 2017-06-06 Novozymes A/S Alcohol product processes
WO2005005646A2 (fr) 2003-06-10 2005-01-20 Novozymes North America, Inc. Processus et compositions de fermentation
JP2005278544A (ja) * 2004-03-30 2005-10-13 Asahi Breweries Ltd イノシトール含量の高いビール、発泡酒、麦芽発酵飲料およびその製造方法
WO2007035730A2 (fr) 2005-09-20 2007-03-29 Novozymes North America, Inc. Procédé de production d'un produit de fermentation
US8216817B2 (en) 2005-09-20 2012-07-10 Novozymes North America, Inc. Process of producing a fermentation product
US8936924B2 (en) 2006-09-21 2015-01-20 Verenium Corporation Methods for using a thermostable phytase in ethanol production
US8877478B2 (en) 2006-09-21 2014-11-04 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
US10196617B2 (en) 2006-09-21 2019-02-05 Basf Enzymes Llc Phytases, nucleic acids encoding them and methods for making and using them
CN101636496B (zh) * 2007-02-07 2014-06-25 丹尼斯科美国公司 具有改变特性的布丘氏菌属菌植酸酶变体
JP2010517573A (ja) * 2007-02-07 2010-05-27 ダニスコ・ユーエス・インク、ジェネンコー・ディビジョン アルファ‐アミラーゼを有するフィターゼを用いるデンプン加水分解
WO2008097619A3 (fr) * 2007-02-07 2008-10-16 Danisco Us Inc Genencor Div Variants de la phytase de buttiauxella sp. dotés de propriétés modifiées
US8143046B2 (en) 2007-02-07 2012-03-27 Danisco Us Inc., Genencor Division Variant Buttiauxella sp. phytases having altered properties
WO2008097620A1 (fr) * 2007-02-07 2008-08-14 Danisco Us, Inc., Genencor Division Hydrolyse d'amidon en utilisant une phytase avec une alpha-amylase
US8399224B2 (en) 2007-03-14 2013-03-19 Danisco Us Inc. Production of ethanol from barley and DDGS containing reduced beta-glucan and phytic acid
WO2008112282A1 (fr) * 2007-03-14 2008-09-18 Danisco Us, Inc., Genencor Division Production d'éthanol à partir d'orge et de ddgs contenant des niveaux réduits de bêta-glucane et d'acide phytique
WO2008116878A1 (fr) 2007-03-26 2008-10-02 Novozymes A/S Phytase de hafnia
US10640794B2 (en) 2007-10-18 2020-05-05 Novozymes North America, Inc. Processes of producing fermentation products
US8273546B2 (en) 2007-10-18 2012-09-25 Novozymes North America, Inc. Processes of producing fermentation products
US8426160B2 (en) 2007-10-18 2013-04-23 Chee Leong Soong Processes of producing fermentation products
US10174344B2 (en) 2007-10-18 2019-01-08 Novozymes North America, Inc. Processes of producing fermentation products
US9359621B2 (en) 2007-10-18 2016-06-07 Novazymes North America, Inc. Processes of producing fermentation products
US8697392B2 (en) 2007-10-18 2014-04-15 Novozymes A/S Processes of producing fermentation products
WO2009052500A1 (fr) * 2007-10-18 2009-04-23 Novozymes A/S Procédés de production de produits de fermentation
US8349592B2 (en) 2008-01-25 2013-01-08 Novozymes North America, Inc. Producing fermentation products in the presence of aldehyde dehydrogenase
WO2009094614A1 (fr) * 2008-01-25 2009-07-30 Novozymes A/S Production de produits de fermentation en présence d'une aldéhyde déshydrogénase
WO2009121058A1 (fr) * 2008-03-28 2009-10-01 Novozymes A/S Production de produits de fermentation en présence de tréhalase
WO2010086078A1 (fr) * 2009-01-30 2010-08-05 Unilever Nv Utilisation de phytase dans la préparation d'un produit à base de soja fermenté
CN102300467B (zh) * 2009-01-30 2013-07-10 荷兰联合利华有限公司 植酸酶在发酵大豆基产品制备中的用途
CN102300467A (zh) * 2009-01-30 2011-12-28 荷兰联合利华有限公司 植酸酶在发酵大豆基产品制备中的用途
US10428340B2 (en) 2009-05-21 2019-10-01 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
US9695403B2 (en) 2009-05-21 2017-07-04 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
US9969992B2 (en) 2009-05-21 2018-05-15 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
EP3101126A1 (fr) 2010-03-26 2016-12-07 Novozymes A/S Variantes de phytase thermostables
WO2011117397A1 (fr) 2010-03-26 2011-09-29 Novozymes A/S Variantes de phytase thermostables
US10954533B2 (en) 2012-03-30 2021-03-23 Novozymes A/S Processes of producing fermentation products
US11987831B2 (en) 2012-03-30 2024-05-21 Novozymes A/S Processes for producing a fermentation product
US10364445B2 (en) 2012-03-30 2019-07-30 Novozymes A/S Processes of producing fermentation products
US10526620B2 (en) 2012-03-30 2020-01-07 Novozymes A/S Processes for producing fermentation products
US9856498B2 (en) 2012-03-30 2018-01-02 Novozymes A/S Processes of producing fermentation products
US10227613B2 (en) 2012-03-30 2019-03-12 Novozymes A/S Processes for producing fermentation products
WO2014001349A1 (fr) * 2012-06-26 2014-01-03 Dsm Ip Assets B.V. Phytase utilisée en production de biogaz
CN105219593A (zh) * 2015-11-13 2016-01-06 绍兴创易科技有限公司 一种以绍酒专用曲为曲种的清爽型米酒酿造工艺
WO2022034211A1 (fr) 2020-08-13 2022-02-17 Novozymes A/S Variants de phytase et polynucléotides les codant
WO2024137246A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 1 d'estérase de glucide (ce1) présentant une activité d'estérase d'acide férulique et/ou d'estérase d'acétyl xylane et polynucléotides codant pour ceux-ci
WO2024137250A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 3 de gludice estérase (ce3) présentant une activité acétyl xylane estérase et polynucléotides codant pour ceux-ci
WO2024137704A2 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédés de production de produits de fermentation faisant appel à des enzymes de dégradation de fibres avec levure modifiée
WO2024137248A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Compositions contenant des arabinofuranosidases et une xylanase, et leur utilisation pour augmenter la solubilisation de fibres hémicellulosiques
WO2024137252A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédé de réduction de la viscosité du sirop à la fin d'un processus de production d'un produit de fermentation
WO2024258820A2 (fr) 2023-06-13 2024-12-19 Novozymes A/S Procédés de fabrication de produits de fermentation à l'aide d'une levure modifiée exprimant une bêta-xylosidase
WO2025128568A1 (fr) 2023-12-11 2025-06-19 Novozymes A/S Composition et son utilisation pour augmenter la solubilisation de fibres hémicellulosiques

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