DE4017150A1 - PROCESS FOR PREPARING A BACKACTIVE PENTOSANASE PREPARATION - Google Patents

Description

The invention relates to a fermentation process for the preparation a pentosanase preparation with increased baking activity.

Pentosanases are enzymes that are able to break down pentosans. The pentosans belong to the hemicelluloses. The most important compo is arabinoxylosan, which is chemically chain-shaped Xylan (polymer of D-xylose) with arabinose in the side chains be stands. Such polysaccharides are found in cereal flours as fiber.

US Patent 35 12 992 therefore proposes pentosanases and esp special xylanases in the bakery in the preparation of dough enforce. It is known that by the use of pentosanase the Dough viscosity can be lowered. Furthermore, white bread is special soft, and staling can be delayed. At Rog The crumb elasticity can be improved (C. Gams "Der Use of microbial enzymes in the bakery in cereals, flour and Bread ", Volume 30, Issue 5, May 1976, page 113 ff.).  

An overview of xylanases and their properties J. Woodward in "Topics in Enzymology and Fermentation, Biotechnology, Vol. 8 (1984), page 9 et seq.

The majority of xylan-degrading microorganisms produce more than only a xylanase type that u. a. thereby different, back to be active or backinactive. In the food industry Practice has shown that in the use of pentosanases or Xylanases to achieve volume increase of a pastry piece directly with a measurable xylanase activity of the back-acting xylanase correlated. There is therefore a need for a method that it specifically allowed to produce enzyme preparations with high baking activity len.

The invention therefore relates to a process for the preparation a back-active pentosanase preparation containing microorganisms which are capable of forming pentosanases in a Fer mentation medium breeds the cell mass at the end of the culture period separates and the enzyme preparation from the fermenter broth wins, thereby characterized in that to induce baking activity to ferment tion medium next to or instead of xylan and / or xylanhaltigen Blends the substance β-methylxyloside in amounts of 0.01 to 5 wt .-%, based on fermentation medium, added.

The inventive method is suitable for the production of back-active pentosanase preparations from a variety of microor hives of the most diverse genera, such as Aspergillus rhizopus, Trichodema bacillus and the like. before however, there are strains of the genus Aspergillus, especially of As pergillus niger and Aspergillus awamori and among these Aspergillus awamori DSM 5937.  

The effect underlying the invention is that the Substrate β-methylxyloside as substrate analogues concerned Mi stimulates microorganisms for the synthesis of pentosanases (xylanases) - In particular, for the formation of the back-active enzyme.

The core of the invention thus lies in the fermentation medium in the preparation of the enzyme preparations β-methylxyloside added put. Suitable additional amounts are between 0.01 and 5 wt .-%, in particular between 0.01 and 1 wt .-%, β-methylxyloside can thereby used together with xylan or instead of xylan. Be It is preferable to use the substance instead of xylan. Possible is also β-methylxyloside together with a xylan-containing compo nente, about together with rye bran, use or preferably instead of adding such a component. By β-methylxyloside Not only are higher xylanase yields achieved, but also the Fermentation time to reach maximum xylanase activity significantly shortened.

Apart from the concomitant use of β-methylxyloside, so have the fermentation media to be used according to the invention are a co composition, as used in the production of enzymes by micro organisms of the genus Aspergillus, in particular Aspergillus niger or Aspergillus awamori is common.

In detail, the fermentation media receive a C source, a N source and trace elements. As a C source are polysaccharide containing products such as corn starch or from built cornstarch or other source of starch. As N-source k soybean meal, but also corn steep liquor. Besides It is also possible to use ammonium salts, for example ammonium nitrate. The substance used as C source is in the fermentation medium in in an amount of 0.2 to 5% by weight. The same applies to the proteinaceous substances. In addition, the Fermenta contains  tion medium nor the usual trace elements such. B. salts of Potassium, sodium, magnesium, manganese, iron and the like. The amount of trace elements is less than 1 wt .-% and is per cation species about 0.01 to 0.2 wt .-%, based on the respective cation.

The trace elements are preferably present as soluble salts, so for example, as phosphates, nitrates or sulfates. Here are the Combinations chosen so that precipitations are avoided.

The total pH of the Fementationsmediums is slightly acidic to neutral and can take values between pH 4.5 and pH 7.5, esp especially around pH 6.0.

The fermentative preparation of pentosanases can be carried out in conventional Fer mentation plants are carried out, under aerobic Be conditions at room temperature or slightly above. The culture period is 24 h to several days, with the xylanase education can be followed analytically.

For workup, the fungus mycelium is first filtered by filtration the fermenter broth separated. The liquid phase thus obtained can in be processed in different ways. That's the way it is to precipitate the enzyme from this liquid phase, for example with ammonium sulfate, and the precipitated enzyme continues to tear or to process directly. But it is also possible that Liquid phase first by ultrafiltration with washing (Dia lysis) of low molecular weight soluble constituents and then carry out a precipitation step or the concentrate on a carrier, such as soybean meal, spray and then to dry.  

The pentosanase concentrates prepared according to the invention show high pentosanase (xylanase) activity with simultaneously improved Back activity.

The determination of xylanase activity can be made with the help of dinitro salicylic acid method done as follows:

The enzyme-containing solution is in acetate-buffered solution in counter were about 1% xylan incubated at 40 ° C for 15 minutes. Subsequently is an alkaline Dinitrosalicylsäure-DNS solution (7 g of DNA, 12 g NaOH, 200 g of potassium sodium tartrate, 5.5 g of phenol, 5 g Na₂S₂O₅ per 1 l H₂O) was added and the color development at 540 nm compared to measured according to a xylose standard. The enzyme unit 1 U is present when in 1 min under the reaction conditions from the xylan fragments whose reduction equivalents 1 μMol xylose equivalent.

It can also be used in the method cited in US Pat. No. 3,512,992 to be worked.

To determine the baking activity, the volume increase of bread measured from a defined amount of a standard dough. To If one measures length, width and height of 30 such rolls, added Measures in cm and draws from the received number the dimensions from just if there are 30 rolls made from the same amount of the same dough have been prepared without the enzyme additive.

Examples Example 1 Culture conditions inoculum

For the production of inoculants were well talked about Agar cultures of strain 5937 (XY1-A007) with 0.1 M PO₄ buffer (pH 6.5), the spore suspension filament sterile glass wool fil trated, mixed with 10% glycerol as a protective reagent and portions stored at -25 ° C.

main Culture

To obtain the xylanase, the o. G. Trunk under Submersbedingungen grown in shake cultures. 100 ml nutrient solution solution (in 500 ml Erlenmeyer flasks with baffles) was mixed with 0.1 ml Inoculated spore suspension and at 30 ° C on the shaker in cubed (shaking frequency: 140 rpm).

media composition 0.15% NaH₂PO₄ 0.10% K₂HPO₄ 0.05% MgSO₄ · 7H₂O 0.02% MnSO₄ · 4H₂O 0.001% FeSO₄ · 7H₂O 0.20% (NH₄) NO₃ 0.50% soy flour 1.00% Corn steep liquor 0.50% β-Methylxylosid 1.00% Corn starch (mined) pH 6.5

To isolate the pentosanase (xylanase) the fungus mycelium was Filtration separated and the cell-free culture filtrate by dialysis cleaned and concentrated by ultrafiltration. From the Konzen By freeze drying, a dry powder was obtained which was mixed with soy flour to a product of 15000 U / g.

example 2

It was a dough made from 1000 g of wheat flour, type 550, 20 g Salt, 60 g of yeast, 580 g of water and 30 g of baking agent with or without ene zymzusatz. In a spiral kneader is kneaded for a total of 5 min. After a rest period of 5 minutes exactly 1500 g are weighed, this Section manually kneaded and white in the following Ballengare rest for 10 minutes.

In a dough divider, the bale weighs 50 g Dough pieces divided and round worked. The round dough pieces are after 1 ½ min rest time screwed in a Langwirkautomaten.

In the proofer at 30 ° C and 85% relative humidity, the subsequent piece cooking (35 min). At 230 ° C, the dough pieces then baked in the oven for 20 minutes.  

After cooling, 30 rolls of a Teigansatzes of length, the width and the height measured. With an addition of 1500 U Enzyme (corresponding to 0.1 g) is measured 40 cm more than one Comparison route without enzyme addition.

example 3

Example 1 was repeated, but instead of β-methylxylo sit xylan or rye flour bran used. The results are in listed in the table.

table

Claims (9)

1. A process for the preparation of a back-active pentosanase Präpa rats, in which a microorganism strain capable of forming pentosanases is grown in a fermentation medium, at the end of the culture period separates the mycelium and wins the enzyme preparation from the fermenter broth, characterized marked , that for the induction of baking activity of the fermentation tationsmedium next to or instead of xylan and / or xylanhal term mixtures the substance β-methylxyloside in amounts of 0.01 to 5 wt .-%, based on fermentation medium, added. 2. The method according to claim 1, characterized in that as Microorganism strain a strain of one of the genera Rhi zopus, Trichodema bacillus or Aspergillus, preferably Asper gillus awamori, in particular Aspergillus awamori DSM 5937 (XY1-A007). 3. The method according to any one of claims 1 or 2, characterized marked characterized in that β-methylxyloside in amounts of 0.1 to 1 wt .-% starts. 4. The method according to any one of claims 1 to 3, characterized marked characterized in that to obtain the enzyme preparation from the Fer Menterbrühe this concentrated by ultrafiltration and then the enzyme preparation by precipitation or drying on a support in solid form wins. 5. The method according to any one of claims 1 to 4, characterized net, that separates the enzyme preparation by precipitation.   6. The method according to any one of claims 1 to 5, characterized gekennzeich net, that the fermentation medium is a C source, an N source and contains common trace elements. 7. The method according to any one of claims 1 to 6, characterized gekennzeich net, that the fermentation medium as N source a Eiweißbe contains constituent and / or ammonium salts. 8. The method according to any one of claims 1 to 7, characterized net, that the fermentation medium as C source starch products, especially soybean meal or corn water. 9. The method according to any one of claims 1 to 8, characterized net, that the fermentation medium as trace elements salts of Sodium, calcium, magnesium, manganese and / or iron.