WO1991018977A1 - Process for producing a baking-active pentosanase preparation - Google Patents

Process for producing a baking-active pentosanase preparation Download PDF

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Publication number
WO1991018977A1
WO1991018977A1 PCT/EP1991/000938 EP9100938W WO9118977A1 WO 1991018977 A1 WO1991018977 A1 WO 1991018977A1 EP 9100938 W EP9100938 W EP 9100938W WO 9118977 A1 WO9118977 A1 WO 9118977A1
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Prior art keywords
fermentation medium
net
preparation
baking
xylan
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PCT/EP1991/000938
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German (de)
French (fr)
Inventor
Joachim Schindler
Hans Friedrich Pfeiffer
Horst-Werner Wollenweber
Heinrich Waldhoff
Wolfgang Adams
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Henkel Kommanditgesellschaft Auf Aktien
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Publication of WO1991018977A1 publication Critical patent/WO1991018977A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • the invention relates to a fermentation process for producing a pentosanase preparation with increased baking activity.
  • Pentosanases are enzymes that are able to break down pentosans.
  • the pentosans belong to the henricelluloses.
  • the most important component is arabinoxylosan, which chemically consists of a chain-shaped xylan (polymer of D-xylose) with arabinose in the side chains.
  • Such poiysaccharides can also be found as fiber in cereal flours.
  • US Pat. No. 3,512,992 therefore proposes to use pentosanases and, in particular, xylanases in the bakery when preparing dough. It is known that the dough viscosity can be reduced by using pentosanase. Furthermore, white bread becomes particularly soft and old baking can be delayed. In the case of rye pastries, the elasticity of the crumbs can be improved (C. Garns "The use of microbial enzymes in the bakery in cereals, flour and bread", 30th year, volume 5, May 1976, page 113 ff.). An overview-like description of xylanases and their properties can be found in J. Woodward in "Topics in Enzymology and Fermentation, Biotechnology, Vol. 8 (1984), page 9 ff.
  • inducers such as ⁇ -methyl-xyloside
  • xylan-degrading microorganisms form more than just one type of xylanase, which differ, inter alia, in that they are back-active or back-inactive. It has been shown in food technology practice that the use of pentosanases or Volume enlargement of a biscuit to be achieved correlated directly with a measurable xylanase activity of the baking active xylanase. There is therefore a need for a process which specifically allows enzyme preparations with high baking activity to be produced.
  • the invention therefore relates to a process for producing a baking-active pentosanase preparation, in which microorganisms which are capable of forming pentosanases are grown in a fermentation medium, the cell mass is separated off at the end of the culture period and the enzyme preparation is removed the fermenter broth wins, characterized in that, in addition to or instead of xylan and / or xylan-containing mixtures, the substance ⁇ -methylxyloside is added in quantities of 0.01 to 5% by weight, based on the fermentation medium, to induce baking activity in the fermentation medium.
  • the process according to the invention is suitable for the production of baking-active pentosanase preparations from a large number of microorganism strains of the most diverse genera, for example Aspergillus Rhizopus, Trichodema Bacillus and the like.
  • strains of the genus Aspergillus are preferred, especially Aspergillus niger and Aspergillus awamori and among these Aspergillus awamori DSM 5937.
  • the effect on which the invention is based is that the substrate ⁇ -methylxyloside, as substrate analogs, stimulates the microorganisms in question for the synthesis of pentosanases (xylanases) - in particular also for the formation of the back-active enzyme.
  • the essence of the invention is therefore to add ⁇ -methylxyloside to the fermentation medium in the preparation of the enzyme preparations. Suitable addition amounts are between 0.01 and 5% by weight, in particular between 0.01 and 1% by weight.
  • ⁇ -methylxyloside can be used together with xylan or instead of xylan. It is preferred to use the substance instead of xylan. It is also possible to use ⁇ -methylxyloside together with a component containing xylane, for example together with rye bran, or preferably to add it instead of such a component.
  • Ss-Methylxyloside not only achieves higher xylanase yields, but also significantly reduces the fermentation time until maximum xylanase activity is reached.
  • the fermentation media to be used according to the invention have a composition which is customary in the production of enzymes by microorganisms of the genus Aspergillus, in particular Aspergillus niger or Aspergillus awamori.
  • the fermentation media contain a C source, an N source and trace elements.
  • Products containing polysaccharide are used as the C source, for example corn starch or degraded corn starch or another starch source.
  • Soy flour, but also corn steep liquor can be used as the N source.
  • ammonium salts such as ammonium nitrate
  • the substance used as the C source is present in the fermentation medium in an amount of 0.2 to 5% by weight. The same applies to protein-like substances.
  • the Feraenta tion medium nor the usual trace elements, such as salts of potassium, sodium, magnesium, manganese, iron and the like.
  • the amount of trace elements is less than 1% by weight and is about 0.01 to 0.2% by weight per type of cation, based on the particular cation.
  • the trace elements are preferably present as soluble salts, for example as phosphates, nitrates or sulfates.
  • the combinations are chosen so that precipitation is avoided.
  • the total pH of the fermentation medium is slightly acidic to neutral and can take on values between pH 4.5 and pH 7.5, in particular around pH 6.0.
  • the fermentative production of the pentosanases can be carried out in customary fermentation plants, working under aerobic conditions at room temperature or slightly above.
  • the culture lasts from 24 hours to a few days, and the xylanase formation can be monitored analytically.
  • the fungal mycelium is first separated from the fermenter broth by filtration.
  • the liquid phase obtained in this way can be processed further in various processes. It is thus possible to precipitate the enzyme from this liquid phase, for example with ammonium sulfate, and to further purify or process the precipitated enzyme directly.
  • the pentosanase concentrates produced according to the invention show high pentosanase (xylanase) activity with simultaneously improved baking activity.
  • the xylanase activity can be determined using the dinitrosalicylic acid method as follows:
  • the enzyme-containing solution is incubated in acetate-buffered solution in the presence of approx. 1% xylan at 40 ° C. for 15 minutes. Then an alkaline dinitrosalicylic acid DNA solution (7g DNA, 12g NaOH, 200g potassium sodium tartrate, 5.5g phenol, 5g Na2S2 ⁇ s per 1 l H2O) is added and the color development at 540 nm compared to a xylose standard is measured. The enzyme unit 1 U is present if in 1 min. fragments are released from the xylan under the reaction conditions, the reduction equivalents of which correspond to 1 ⁇ mol of xylose.
  • the increase in volume of rolls is measured from a defined amount of a standard dough. To do this, measure the length, width and height of 30 such buns, add the dimensions in cm and subtract from the number obtained the dimensions of likewise 30 buns which were produced from the same amount of the same dough without the enzyme addition. Examples
  • Vaccine culture To produce vaccine cultures, well-transported agar cultures of strain 5937 (XY1-A007) were washed off with 0.1 PO / j buffer (pH 6.5), the spore suspension was filtered over sterile glass wool, with 10% glycerol as a protective reagent added and stored in portions at -25 ° C.
  • the fungal mycelium was separated by filtration and the cell-free culture filtrate was purified by dialysis and concentrated by ultrafiltration. A freeze-dried powder was obtained from the concentrate, which was mixed with soy flour to a product with 15000 U / g.
  • a dough was produced from 1000 g of wheat flour, type 550, 20 g of salt, 60 g of yeast, 580 g of water and 30 g of baking agent with or without the addition of enzyme. A total of 5 min. kneaded. After a rest period of 5 min. exactly 1500 g are weighed out, this section is kneaded manually and in the following bale oven for a further 10 min. rested.
  • the bale is divided into 50 g dough pieces and knitted round.
  • the round dough pieces are after 11/2 min. Rest time screwed into a »long-knitting machine.
  • Example 1 was repeated, but xylan or rye flour bran was used instead of ⁇ -methylxylitol. The results are shown in the table.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Bakery Products And Manufacturing Methods Therefor (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

In a process for producing a pentosanase preparation, in which a microorganism capable of forming pentosanases is cultured in a fermentation medium, the backing activity of the enzyme obtained is increased. To this end, the baking activity is induced by adding to the fermentation medium 0.01 to 5 wt. %, referred to the fermentation medium, of the substance β-methylxyloside in addition to, or instead of, xylan and/or xylan-containing mixtures.

Description

"Verfahren zur Herstellung eines backaktiven Pentosanase-Präparates" "Process for the Production of a Baking Active Pentosanase Preparation"
Die Erfindung betrifft ein Fermentationsverfahren zur Herstellung eines Pentosanase-Präparats mit erhöhter Backaktivität.The invention relates to a fermentation process for producing a pentosanase preparation with increased baking activity.
Pentosanasen sind Enzyme, die in der Lage sind, Pentosane abzubauen. Die Pentosane gehören zu den Henricellulosen. Die wichtigste Kompo¬ nente ist Arabinoxylosan, das chemisch aus einem kettigenförmigen Xylan (Polymer der D-Xylose) mit Arabinose in den Seitenketten be¬ steht. Derartige Poiysaccharide finden sich auch in Getreidemehlen als Ballaststoffe.Pentosanases are enzymes that are able to break down pentosans. The pentosans belong to the henricelluloses. The most important component is arabinoxylosan, which chemically consists of a chain-shaped xylan (polymer of D-xylose) with arabinose in the side chains. Such poiysaccharides can also be found as fiber in cereal flours.
Das US-Patent 3,512 992 schlägt daher vor, Pentosanasen und insbe¬ sondere Xylanasen in der Bäckerei bei der Zubereitung von Teig ein¬ zusetzen. Es ist bekannt, daß durch den Einsatz von Pentosanase die Teigviskosität gesenkt werden kann. Fernerhin wird Weißbrot beson¬ ders weich und das Altbacken werden kann verzögert werden. Bei Rog¬ gengebäck kann die Krumenelastizität verbessert werden (C. Garns "Der Einsatz von mikrobiellen Enzymen in der Bäckerei in Getreide, Mehl und Brot", 30. Jahrgang, Heft 5, Mai 1976, Seite 113 ff.). Eine übersichtartige Beschreibung über Xylanasen und ihre Eigen¬ schaften findet sich bei J. Woodward in "Topics in Enzymology and Fermentation, Biotechnology, Vol. 8 (1984), Seite 9 ff.US Pat. No. 3,512,992 therefore proposes to use pentosanases and, in particular, xylanases in the bakery when preparing dough. It is known that the dough viscosity can be reduced by using pentosanase. Furthermore, white bread becomes particularly soft and old baking can be delayed. In the case of rye pastries, the elasticity of the crumbs can be improved (C. Garns "The use of microbial enzymes in the bakery in cereals, flour and bread", 30th year, volume 5, May 1976, page 113 ff.). An overview-like description of xylanases and their properties can be found in J. Woodward in "Topics in Enzymology and Fermentation, Biotechnology, Vol. 8 (1984), page 9 ff.
Die Induktion der Xylanasebildung durch Induktoren, wie ß-Methyl- xylosid, ist in der wissenschaftlichen Literatur bereits mehrfach beschrieben worden, so z.B. in den folgenden Artikeln:The induction of xylanase formation by inducers, such as β-methyl-xyloside, has been described several times in the scientific literature, e.g. in the following articles:
M.J. Bailey, K PoutanenM.J. Bailey, K Poutanen
Production of xylanolytic enzymes by strains of Aspergillus Appl.Production of xylanolytic enzymes by strains of Aspergillus Appl.
Microbiol. Biotechnol. (1989) 30:5-10Microbiol. Biotechnol. (1989) 30: 5-10
CT. Kelly, M.R. O'Mahony, W.M. FogartyCT. Kelly, M.R. O'Mahony, W.M. Fogarty
Extracellular xylanolytic enzymes of paecilomyces variotiExtracellular xylanolytic enzymes of paecilomyces varioti
Biotechnology letters, Vol. 11, No. 12, S. 885-890Biotechnology letters, vol. 11, no. 12, pp. 885-890
Morosol , Rolf; Durand, Serge; Letendre, ElaineMorosol, Rolf; Durand, Serge; Letendre, Elaine
Induction of xylanase by beta-methylxyloside in Cryptococcus albidusInduction of xylanase by beta-methylxyloside in Cryptococcus albidus
FEMS Microbiol. Lett., 48 (1-2), S. 261-266FEMS Microbiol. Lett., 48 (1-2), pp. 261-266
C. Royer; J.P. NakasC. Royer; J.P. Nakas
Xylanase production by Trichoderma longibrachiatumXylanase production by Trichoderma longibrachiatum
Enzyme Microb. Technol., 1989, Vol. 11, JulyEnzyme Microb. Technol., 1989, Vol. 11, July
All diese Literaturstellen geben jedoch keinen Hinweis darauf, daß durch ß-Methylxylosid gerade die backaktive Xylanase bevorzugt ge¬ bildet wird.However, none of these references give any indication that ß-methylxyloside in particular forms the backactive xylanase.
Die Mehrzahl der Xylan-abbauenden Mikroorganismen bildet mehr als nur einen Xylanase-Typ, die sich u.a. dadurch unterscheiden, back¬ aktiv oder backinaktiv zu sein. In der nahrungsmitteltechnischen Praxis hat sich gezeigt, daß die beim Einsatz von Pentosanasen bzw. Xylanasen zu erzielende VolumenVergrößerung eines Gebäckstückes direkt mit einer meßbaren Xylanaseaktivität der backaktiven Xylanase korreliert. Es besteht daher der Bedarf nach einem Verfahren, das es gezielt erlaubt, Enzympräparate mit hoher Backaktivität herzustel¬ len.The majority of the xylan-degrading microorganisms form more than just one type of xylanase, which differ, inter alia, in that they are back-active or back-inactive. It has been shown in food technology practice that the use of pentosanases or Volume enlargement of a biscuit to be achieved correlated directly with a measurable xylanase activity of the baking active xylanase. There is therefore a need for a process which specifically allows enzyme preparations with high baking activity to be produced.
Gegenstand der Erfindung ist daher ein Verfahren zur Herstellung eines backaktiven Pentosanase-Präparats, bei dem man Mikroorganis¬ men, die zur Bildung von Pentosanasen befähigt sind, in einem Fer¬ mentationsmedium züchtet, am Ende der Kulturdauer die Zellmasse ab¬ trennt und das Enzympräparat aus der Fermenterbrühe gewinnt, dadurch gekennzeichnet, daß man zur Induktion von Backaktivität dem Fermen¬ tationsmedium neben oder anstelle von Xylan und/oder xylanhaltigen Mischungen die Substanz ß-Methylxylosid in Mengen von 0.01 bis 5 Gew.-%, bezogen auf Fermentationsmedium, zusetzt.The invention therefore relates to a process for producing a baking-active pentosanase preparation, in which microorganisms which are capable of forming pentosanases are grown in a fermentation medium, the cell mass is separated off at the end of the culture period and the enzyme preparation is removed the fermenter broth wins, characterized in that, in addition to or instead of xylan and / or xylan-containing mixtures, the substance β-methylxyloside is added in quantities of 0.01 to 5% by weight, based on the fermentation medium, to induce baking activity in the fermentation medium.
Das erfindungsgemäße Verfahren eignet sich zur Herstellung von backaktiven Pentosanase-Präparaten aus einer Vielzahl von Mikroor¬ ganismenstämmen der verschiedensten Gattungen, so beispielsweise Aspergillus Rhizopus, Trichodema Bacillus und dergleichen . Bevor¬ zugt sind jedoch Stämme der Gattung Aspergillus, speziell von As¬ pergillus niger und Aspergillus awamori und unter diesen Aspergillus awamori DSM 5937. The process according to the invention is suitable for the production of baking-active pentosanase preparations from a large number of microorganism strains of the most diverse genera, for example Aspergillus Rhizopus, Trichodema Bacillus and the like. However, strains of the genus Aspergillus are preferred, especially Aspergillus niger and Aspergillus awamori and among these Aspergillus awamori DSM 5937.
Der der Erfindung zugrundeliegende Effekt besteht darin, daß das Substrat ß-Methylxylosid als Substratanalogen die betreffenden Mi¬ kroorganismen zur Synthese von Pentosanasen (Xylanasen) anregt - insbesondere auch zur Bildung des backaktiven Enzyms.The effect on which the invention is based is that the substrate β-methylxyloside, as substrate analogs, stimulates the microorganisms in question for the synthesis of pentosanases (xylanases) - in particular also for the formation of the back-active enzyme.
Der Kern der Erfindung liegt somit darin, dem Fermentationsmedium bei der Herstellung der Enzymzubereitungen ß-Methylxylosid zuzu¬ setzen. Geeignete Zusatzmengen liegen zwischen 0,01 und 5 Gew.-%, insbesondere zwischen 0,01 und 1 Gew.-%. ß-Methylxylosid kann dabei zusammen mit Xylan oder anstelle von Xylan eingesetzt werden. Be¬ vorzugt ist es, die Substanz anstelle von Xylan einzusetzen. Möglich ist auch, ß-Methylxylosid zusammen mit einer xylanhaltigen Kompo¬ nente, etwa zusammen mit Roggenkleie, einzusetzen oder vorzugsweise anstelle einer solchen Komponente zuzugeben. Durch ß-Methylxylosid werden nicht nur höhere Xylanase-Ausbeuten erzielt, sondern auch die Fermentationszeit bis zum Erreichen maximaler Xylanase-Aktivität deutlich verkürzt.The essence of the invention is therefore to add β-methylxyloside to the fermentation medium in the preparation of the enzyme preparations. Suitable addition amounts are between 0.01 and 5% by weight, in particular between 0.01 and 1% by weight. β-methylxyloside can be used together with xylan or instead of xylan. It is preferred to use the substance instead of xylan. It is also possible to use β-methylxyloside together with a component containing xylane, for example together with rye bran, or preferably to add it instead of such a component. Ss-Methylxyloside not only achieves higher xylanase yields, but also significantly reduces the fermentation time until maximum xylanase activity is reached.
Sieht man von der Mitverwendung von ß-Methylxylosid ab, so weisen die erfindungsgemäß einzusetzenden Fermentationsmedien eine Zusam¬ mensetzung auf, wie sie bei der Herstellung von Enzymen durch Mikro¬ organismen der Gattung Aspergillus, insbesondere Aspergillus .niger oder Aspergillus awamori üblich ist.Apart from the concomitant use of β-methylxyloside, the fermentation media to be used according to the invention have a composition which is customary in the production of enzymes by microorganisms of the genus Aspergillus, in particular Aspergillus niger or Aspergillus awamori.
Im einzelnen erhalten die Fer entationsmedien eine C-Quelle, eine N-Quelle sowie Spurenelemente. Als C-Quelle werden polysaccharid- haltige Produkte eingesetzt, so beispielsweise Maisstärke oder ab¬ gebaute Maisstärke oder eine andere Stärkequelle. Als N-Quelle kön¬ nen Sojamehl, aber auch Maisquellwasser eingesetzt werden. Daneben können auch Ammoniumsalze, etwa Ammoniumnitrat, eingesetzt werden. Die als C-Quelle verwendete Substanz ist im Fermentationsmediuni in einer Menge von 0,2 bis 5 Gew.-% vorhanden. Dasselbe gilt auch für die eiweißartigen Substanzen. Darüber hinaus enthält das Feraenta- tionsmedium noch die üblichen Spurenelemente, wie z.B. Salze von Kalium, Natrium, Magnesium, Mangan, Eisen und dergleichen. Die Menge an Spurenelementen liegt unter 1 Gew.-% und beträgt pro Kationenart etwa 0,01 bis 0,2 Gew.-%, bezogen auf das jeweilige Kation.In detail, the fermentation media contain a C source, an N source and trace elements. Products containing polysaccharide are used as the C source, for example corn starch or degraded corn starch or another starch source. Soy flour, but also corn steep liquor, can be used as the N source. In addition, ammonium salts, such as ammonium nitrate, can also be used. The substance used as the C source is present in the fermentation medium in an amount of 0.2 to 5% by weight. The same applies to protein-like substances. In addition, the Feraenta tion medium nor the usual trace elements, such as salts of potassium, sodium, magnesium, manganese, iron and the like. The amount of trace elements is less than 1% by weight and is about 0.01 to 0.2% by weight per type of cation, based on the particular cation.
Die Spurenelemente liegen vorzugsweise als lösliche Salze vor, so beispielsweise als Phosphate, Nitrate oder Sulfate. Dabei werden die Kombinationen so gewählt, daß Fällungen vermieden werden.The trace elements are preferably present as soluble salts, for example as phosphates, nitrates or sulfates. The combinations are chosen so that precipitation is avoided.
Der Gesamt-pH-Wert des Fermentationsmediums ist schwach sauer bis neutral und kann Werte zwischen pH 4,5 und pH 7,5 einnehmen, insbe¬ sondere um pH 6,0.The total pH of the fermentation medium is slightly acidic to neutral and can take on values between pH 4.5 and pH 7.5, in particular around pH 6.0.
Die fermentative Herstellung der Pentosanasen kann in üblichen Fer¬ mentationsanlagen durchgeführt werden, wobei unter aeroben Be¬ dingungen bei Raumtemperatur oder leicht darüber gearbeitet wird. Die Kulturdauer beträgt 24 h bis einige Tage, wobei die Xylanase- bildung analytisch verfolgt werden kann.The fermentative production of the pentosanases can be carried out in customary fermentation plants, working under aerobic conditions at room temperature or slightly above. The culture lasts from 24 hours to a few days, and the xylanase formation can be monitored analytically.
Zur Aufarbeitung wird das Pilzmyzel zunächst durch Filtration von der Fermenterbrühe abgetrennt. Die so erhaltene Flüssigphase kann in unterschiedlichen Verfahren weiterverarbeitet werden. So ist es mög¬ lich, aus dieser Flüssigphase das Enzym auszufällen, so beispiels¬ weise mit Ammoniumsulfat, und das ausgefällte Enzym weiter zu rei¬ nigen oder direkt zu verarbeiten. Es ist aber auch möglich, die Flüssigphase zunächst durch Ultrafiltration unter Auswaschen (Dia¬ lyse) niedermolekularer löslicher Bestandteile aufzukonzentrieren und danach einen Fällungsschritt durchzuführen oder das Konzentrat auf einen Träger, wie beispielsweise Sojamehl, aufzusprühen und dann zu trocknen. Die erfindungsgemäß hergestellten Pentosanase-Konzentrate zeigen hohe Pentosanase-(Xylanase)-Aktivität bei gleichzeitig verbesserter Backaktivitat.For working up, the fungal mycelium is first separated from the fermenter broth by filtration. The liquid phase obtained in this way can be processed further in various processes. It is thus possible to precipitate the enzyme from this liquid phase, for example with ammonium sulfate, and to further purify or process the precipitated enzyme directly. However, it is also possible first to concentrate the liquid phase by ultrafiltration with washing out (dialysis) of low molecular weight soluble constituents and then to carry out a precipitation step or to spray the concentrate onto a carrier such as soybean meal and then to dry it. The pentosanase concentrates produced according to the invention show high pentosanase (xylanase) activity with simultaneously improved baking activity.
Die Bestimmung der Xylanase-Aktivität kann mit Hilfe der Dinitro- salicylsäure-Methode wie folgt geschehen:The xylanase activity can be determined using the dinitrosalicylic acid method as follows:
Die enzymhaltige Lösung wird in acetatgepufferter Lösung in Gegen¬ wart von ca. 1% Xylan bei 40°C 15 Minuten inkubiert. Anschließend wird eine alkalische Dinitrosalicylsäure-DNS-Lösung (7g DNS, 12g NaOH, 200g Kaliu natriumtartrat, 5,5g Phenol, 5g Na2S2θs pro 1 1 H2O) zugegeben und die Farbentwicklung bei 540 nm im Vergleich zu einem Xylose-Standard gemessen. Die Enzymeinheit 1 U liegt vor, wenn in 1 min. unter den Reaktionsbedingungen aus dem Xylan Bruchstücke freigesetzt werden, deren Reduktionsäquivalente 1 μMol Xylose entspricht.The enzyme-containing solution is incubated in acetate-buffered solution in the presence of approx. 1% xylan at 40 ° C. for 15 minutes. Then an alkaline dinitrosalicylic acid DNA solution (7g DNA, 12g NaOH, 200g potassium sodium tartrate, 5.5g phenol, 5g Na2S2θs per 1 l H2O) is added and the color development at 540 nm compared to a xylose standard is measured. The enzyme unit 1 U is present if in 1 min. fragments are released from the xylan under the reaction conditions, the reduction equivalents of which correspond to 1 μmol of xylose.
Es kann aber auch in der in US-Patent 3 512 992 zitierten Methode gearbeitet werden.However, the method cited in US Pat. No. 3,512,992 can also be used.
Zur Bestimmung der Backaktivität wird die Volumenzunahme von Bröt¬ chen aus einer definierten Menge eines Standardteigs gemessen. Dazu mißt man Länge, Breite und Höhe von 30 derartiger Brötchen, addiert die Maße in cm und zieht von der erhaltenen Zahl die Maße von eben¬ falls 30 Brötchen ab, die aus der gleichen Menge des gleichen Teigs ohne den Enzymzusatz hergestellt worden sind. B e i s p i e l eTo determine the baking activity, the increase in volume of rolls is measured from a defined amount of a standard dough. To do this, measure the length, width and height of 30 such buns, add the dimensions in cm and subtract from the number obtained the dimensions of likewise 30 buns which were produced from the same amount of the same dough without the enzyme addition. Examples
Beispiel 1example 1
Ku1turbedinounqen:Ku1turbedinounqen:
Impfkultur: Zur Herstellung von Impfkulturen wurden gut versporte Agarkulturen des Stammes 5937 (XY1-A007) mit 0,1 PO/j-Puffer (pH 6,5) abgeschwemmt, die Sporensuspension über sterile Glaswolle fil¬ triert, mit 10 % Glycerin als Schutzreagenz versetzt und portions¬ weise bei -25°C gelagert.Vaccine culture: To produce vaccine cultures, well-transported agar cultures of strain 5937 (XY1-A007) were washed off with 0.1 PO / j buffer (pH 6.5), the spore suspension was filtered over sterile glass wool, with 10% glycerol as a protective reagent added and stored in portions at -25 ° C.
Hauptkultur: Zur Gewinnung der Xylanase wurde der o.g. Stamm unter Submersbedingungen in Schüttelkulturen angezüchtet. 100 ml Nährlö¬ sung (in 500 ml Erlenmeyerkolben mit Schikanen) wurde mit 0,1 ml Sporensuspension beimpft und bei 30°C auf die Schüttelmaschine in¬ kubiert (Schüttelfrequenz: 140 U/Min.).Main culture: To obtain the xylanase, the above-mentioned Strain grown in shake cultures under submerged conditions. 100 ml of nutrient solution (in 500 ml Erlenmeyer flasks with baffles) was inoculated with 0.1 ml of spore suspension and incubated at 30 ° C. on the shaker (shaking frequency: 140 rpm).
Medienzusaπroensetzun :Media composition:
Figure imgf000009_0001
1,00 % Maisquellwasser 0,50 % ß-Methylxylosid 1,00 % Maisstärke (abgebaut) pH 6,5
Figure imgf000009_0001
1.00% corn steep liquor 0.50% ß-methylxyloside 1.00% corn starch (degraded) pH 6.5
Zur Isolierung der Pentosanase (Xylanase) wurde das Pilzmyzel durch Filtration abgetrennt und das zellfreie Kulturfiltrat durch Dialyse gereinigt und durch Ultrafiltration aufkonzentriert. Aus dem Konzen¬ trat wurde durch Gefriertrocknung ein Trockenpulver gewonnen, das mit Sojamehl zu einem Produkt mit 15000 U/g aufgemischt wurde.To isolate the pentosanase (xylanase), the fungal mycelium was separated by filtration and the cell-free culture filtrate was purified by dialysis and concentrated by ultrafiltration. A freeze-dried powder was obtained from the concentrate, which was mixed with soy flour to a product with 15000 U / g.
Beispiel 2Example 2
Es wurde ein Teig hergestellt aus 1000 g Weizenmehl, Type 550, 20 g Salz, 60 g Hefe, 580 g Wasser und 30 g Backmittel mit oder ohne En¬ zymzusatz. In einem Spiralkneter wird insgesamt 5 min. geknetet. Nach einer Ruhezeit von 5 min. werden genau 1500 g abgewogen, dieses Teilstück manuell rundgeknetet und in der folgenden Ballengare wei¬ tere 10 min. ruhen gelassen.A dough was produced from 1000 g of wheat flour, type 550, 20 g of salt, 60 g of yeast, 580 g of water and 30 g of baking agent with or without the addition of enzyme. A total of 5 min. kneaded. After a rest period of 5 min. exactly 1500 g are weighed out, this section is kneaded manually and in the following bale oven for a further 10 min. rested.
In einem Teig-Teilungsautomaten wird der Ballen in 50 g schwere Teiglinge geteilt und rundgewirkt. Die runden Teiglinge werden nach 11/2 min. Ruhezeit in ein» Langwirkautomaten eingedreht.In a dough dividing machine, the bale is divided into 50 g dough pieces and knitted round. The round dough pieces are after 11/2 min. Rest time screwed into a »long-knitting machine.
Im Gärschrank bei 30βC und 85 % relativer Luftfeuchte erfolgt die anschließende Stüc gare (35 min.). Bei 230°C werden die Teigstücke dann im Backofen 20 ein. lang gebacken. Nach dem Abkühlen werden 30 Brötchen eines Teigansatzes der Länge, der Breite und der Höhe nach gemessen. Bei einem Zusatz von 1500 U Enzym (entsprechend 0,1g) werden 40 cm mehr gemessen als bei einem Vergleichsteig ohne Enzymzusatz.In the proofer at 30 C β and 85% relative humidity takes place subsequent Stüc gare (35 min.). At 230 ° C the dough pieces are then in the oven 20. baked long. After cooling, 30 rolls of a batch of dough are measured in length, width and height. With the addition of 1500 U of enzyme (corresponding to 0.1 g), 40 cm more are measured than with a comparison tray without the addition of enzyme.
Beispiel 3Example 3
Beispiel 1 wurde wiederholt, jedoch wurde anstelle von ß-Metylxylo- sit Xylan bzw. Roggenmehlkleie eingesetzt. Die Ergebnisse sind in der Tabelle aufgeführt.Example 1 was repeated, but xylan or rye flour bran was used instead of β-methylxylitol. The results are shown in the table.
T a b e l l eTable
InduktorsubstratInductor substrate
Figure imgf000011_0001
Figure imgf000011_0001
Figure imgf000011_0003
Figure imgf000011_0002
Figure imgf000011_0003
Figure imgf000011_0002

Claims

P a t e n t a n s p r ü c h e Patent claims
1. Verfahren zur Herstellung eines backaktiven Pentosanase-Präpa- rats, bei dem man einen Mikroorganismenstamm, der zur Bildung von Pentosanasen befähigt ist, in einem Fermentationsmedium züchtet, am Ende der Kulturdauer das Myzel abtrennt und das Enzympräparat aus der Fermenterbrühe gewinnt, dadurch gekenn¬ zeichnet, daß man zur Induktion von Backaktivität dem Fermen¬ tationsmedium neben oder anstelle von Xylan und/oder xylanhal¬ tigen Mischungen die Substanz ß-Methylxylosid in Mengen von 0,01 bis 5 Gew.-%, bezogen auf Fermentationsmedium, zusetzt.1. Process for the preparation of a baking-active pentosanase preparation, in which a microorganism strain which is capable of forming pentosanases is grown in a fermentation medium, the mycelium is separated off at the end of the culture period and the enzyme preparation is obtained from the fermenter broth, thereby characterized characterized in that for the induction of baking activity the substance ß-methylxyloside is added to the fermentation medium in addition to or instead of xylan and / or xylan-containing mixtures in amounts of 0.01 to 5% by weight, based on the fermentation medium.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß man als Mikroorganismenstamm einen Stamm aus einer der Gattungen Rhi- zopus, Trichodema Bacillus oder Aspergillus, vorzugsweise Asper¬ gillus awamori, insbesondere Aspergillus awamori DSM 5937 (XY1-A007) einsetzt.2. The method according to claim 1, characterized in that a strain from one of the genera Rhizopus, Trichodema Bacillus or Aspergillus, preferably Asper¬ gillus awamori, in particular Aspergillus awamori DSM 5937 (XY1-A007) is used as the microorganism strain.
3. Verfahren nach einem der Ansprüche 1 oder 2, dadurch gekenn¬ zeichnet, daß man ß-Methylxylosid in Mengen von 0,1 bis 1 Gew.-% einsetzt.3. The method according to any one of claims 1 or 2, characterized gekenn¬ characterized in that ß-methylxyloside is used in amounts of 0.1 to 1 wt .-%.
4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekenn¬ zeichnet, daß man zur Gewinnung des Enzympräparats aus der Fer¬ menterbrühe diese durch Ultrafiltration aufkonzentriert und dann das Enzympräparat durch Fällen oder Trocknen auf einem Träger in fester Form gewinnt.4. The method according to any one of claims 1 to 3, characterized gekenn¬ characterized in that to obtain the enzyme preparation from the Fer¬ ferment broth concentrated by ultrafiltration and then the enzyme preparation by precipitation or drying on a support in solid form.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeich¬ net, daß man das Enzympräparat durch Ausfällen abtrennt. 5. The method according to any one of claims 1 to 4, characterized gekennzeich¬ net that the enzyme preparation is separated by precipitation.
6. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeich¬ net, daß das Fermentationsmedium eine C-Quelle, eine N-Quelle und übliche Spurenelemente enthält.6. The method according to any one of claims 1 to 5, characterized gekennzeich¬ net that the fermentation medium contains a C source, an N source and conventional trace elements.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeich¬ net, daß das Fermentationsmedium als N-Quelle einen Eiweißbe¬ standteil und/oder Ammoniumsalze enthält.7. The method according to any one of claims 1 to 6, characterized gekennzeich¬ net that the fermentation medium as an N source contains a protein component and / or ammonium salts.
8. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeich¬ net, daß das Fermentationsmedium als C-Quelle Stärkeprodukte, insbesondere Sojamehl oder Maiswasser enthält.8. The method according to any one of claims 1 to 7, characterized gekennzeich¬ net that the fermentation medium as a C source contains starch products, especially soy flour or corn water.
9. Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeich¬ net, daß das Fermentationsmedium als Spurenelemente Salze des Natriums, Caliums, Magnesiums, Mangans und/oder Eisens enthält. 9. The method according to any one of claims 1 to 8, characterized gekennzeich¬ net that the fermentation medium contains as trace elements salts of sodium, potassium, magnesium, manganese and / or iron.
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WO2002003805A1 (en) 2000-07-06 2002-01-17 Novozymes A/S Method of preparing a dough or a baked product made from a dough, with addition of lipolytic enzymes
WO2004099400A2 (en) 2003-05-09 2004-11-18 Novozymes A/S Variant lipolytic ensymes
EP2270140A2 (en) 2003-05-09 2011-01-05 Novozymes A/S Variant lipolytic enzymes
EP2270139A2 (en) 2003-05-09 2011-01-05 Novozymes A/S Variant lipolytic enzymes
EP2290057A2 (en) 2003-05-09 2011-03-02 Novozymes A/S Variant lipolytic enzymes
EP2428572A2 (en) 2007-03-09 2012-03-14 Danisco US, Inc., Genencor Division Alkaliphilic Bacillus species alpha-amylase variants, compositions comprising alpha-amylase variants, and methods of use
US8318157B2 (en) 2007-03-14 2012-11-27 Danisco Us Inc. Trichoderma reesei α-amylase enhances saccharification of corn starch
US8916369B2 (en) 2007-03-14 2014-12-23 Danisco Us Inc. Trichoderma reesei α-amylase is a maltogenic enzyme
US9040278B2 (en) 2008-06-06 2015-05-26 Danisco Us Inc. Production of glucose from starch using alpha-amylases from Bacillus subtilis
US9040279B2 (en) 2008-06-06 2015-05-26 Danisco Us Inc. Saccharification enzyme composition and method of saccharification thereof
EP3133154A2 (en) 2009-05-19 2017-02-22 DuPont Nutrition Biosciences ApS Amylase polypeptides
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