IE911797A1

IE911797A1 - A process for the production of a baking-active pentosanase¹preparation

Info

Publication number: IE911797A1
Application number: IE179791A
Authority: IE
Original assignee: Henkel Kgaa
Priority date: 1990-05-28
Filing date: 1991-05-27
Publication date: 1991-12-04
Also published as: DE4017150A1; WO1991018977A1

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 beta-methylxyloside in addition to, or instead of, xylan and/or xylan-containing mixtures. [WO9118977A1]

Description

A process for the production of a baking-active pentosanase preparation This invention relates to a fermentation process for the production of a pentosanase preparation having increased baking activity.

Pentosanases are enzymes which are capable of degrad5 ing pentosans. Pentosans are homocelluloses. Their most important component is arabinoxylosan which, chemically, consists of a chain-like xylan (polymer of D-xylose) with arabinose in the side chains. Polysaccharides such as these are also to be found as ballast in cereal flours.

Accordingly, it is proposed in US-PS 3,512,992 that pentosanases and, in particular, xylanases be added during the preparation of dough for baking. It is known that the viscosity of dough can be reduced by using pentosanase. In addition, white bread is made particularly soft and staling can be delayed. In the case of rye bread, crumb elasticity can be improved (C. Gams Der Einsatz von mikrobiellen Enzymen in der Backerei in Getreide, Mehl und Brot, Vol. 30, Number 5, May 1976, pages 113 et seq).

A synoptic desciption of xylanases and their proper20 ties can be found in J. Woodward, Topics in Enzymology and Fermentation, Biotechnology, Vol. 8 (1984), pages 9 et seg) .

The induction of xylanase formation by inductors, such as B-methylxyloside, has already been repeatedly described in the scientific literature, for example in the following D 8974 2 Articles: M.J. Bailey, K Poutanen Production of xylanolytic enzymes by strains of Aspergil5 lus, Appl. Microbiol Biotechnol. (1989), 30:5-10 C.T. Kelly, M.R. O'Mahony, W.M. Fogarty Extracellular xylanolytic enzymes of paecilomyces varioti, Biotechnology Letters, Vol. 11, No. 12, pages 885-890 Morosoli, Rolf; Durand, Serge; Letendre, Elaine Induction of xylanase by B-methylxyloside in Cryptococcus albidus, FEMS Microbiol. Lett., 48 (1-2) pages 261-266 C. Royer; J.P. Nakas Xylanase production by Trichoderma longibrachiatum Enzyme Microb. Technol., 1989, Vol. 11 July However, there is nothing in any of these literature references to show that the baking-active xylanase is preferentially formed by β-methylxyloside.

The majority of xylan-degrading microorganisms form more than just one type of xylanase which differ from one another inter alia in the fact that they are either active or inactive in the baking process. It has been found in the food industry that the increase in volume of a bakery product which can be obtained where pentosanases or xylanases are used correlates directly with a measurable xylanase activity of the baking-active xylanase. Accordingly, there was a need for a process which would enables enzyme preparation of high baking activity to be specifically produced .

Accordingly, the present invention relates to a process for the production of a baking-active pentosanase preparation, in which microorganisms capable of forming Ϊ911797 D 8974 3 pentosanases are cultured in a fermentation medium, the cell mass is removed at the end of the culture period and the enzyme preparation is recovered from the fermenter broth, characterized in that, to induce baking activity, the substance β-methyl xyloside is added to the fermentation medium in quantities of 0.01 to 5% by weight, based on the fermentation medium, in addition to or instead of xylan and/or xylan-containing mixtures.

The process according to the invention is suitable for 10 the production of baking-active pentosanase preparations from a plurality of microorganism strains belonging to various species, including for example Aspergillus rhizopus, Trichodema bacillus and the like. However, preferred microorganism strains are strains of the aspergillus species, particularly Aspergillus niger and Aspergillus awamori and, more particularly, Aspergillus awamori DSM 5937.

The effect on which the invention is based is that the substrate β-methyl xyloside stimulates the particular microorganisms as substrate analogs to synthesize pentosanases (xylanases) and, in particular, to form the baking-active enzyme.

Accordingly, the crux of the invention is that Bmethyl xyloside is added to the fermentation medium in the production of enzyme preparations. Suitable additions are from 0.01 to 5 % by weight and, more particularly, from 0.01 to 1 % by weight. B-Methyl xyloside may be used together with or instead of xylan. The substance is preferably used instead of xylan. The B-methyl xyloside may also be used together with a xylan-containing component, for example together with rye bran, or may preferably be added instead of such a component. Not only are higher xylanase yields obtained by the B-methyl xyloside, the fermentation time required to reach maximum xylanase activity is also distinctly shortened. ΪΕ 911797 D 8974 4 In the absence of β-methyl xyloside, the fermentation media to be used in accordance with the invention would have the composition typically encountered in the production of enzymes by microorganisms of the aspergillus species, more particularly Aspergillus niger or Aspergillus awamori.

More particularly, the fermentation media contain a carbon source, a nitrogen source and trace elements. Polysaccharide-containing products, for example cornstarch or degraded cornstarch or any other starch source, are used as the carbon source. Soybean flour and also corn steep liquor may be used as the nitrogen source. Ammonium salts, for example ammonium nitrate, may also be used as the nitrogen source. The substance used as the carbon source is present in the fermentation medium in a quantity of 0.2 to 5 % by weight. The same applies to the protein-like substances. In addition, the fermentation medium contains the usual trace elements, for example salts of potassium, sodium, magnesium, manganese, iron and the like. The quantity of trace elements is below 1 % by weight and amounts - per cation type - to between about 0.01 and 0.2 % by weight, based on the particular cation.

The trace elements are preferably present as soluble salts, for example as phosphates, nitrates or sulfates.

The combinations are selected so that precipitation is avoided.

The overall pH value of the fermentation medium is mildly acidic to neutral and may assume values of pH 4.5 to pH 7.5 and, more particularly, of the order of pH 6.0.

The fermentative preparation of the pentosanases may be carried out in standard fermentation units operated under aerobic conditions either at or slightly above room temperature. The culture time is 24 h to a few days, during which the formation of xylanase can be followed by analysis.

D 8974 5 For working up, the mycelium is first removed from the fermenter broth by filtration. The liquid phase thus obtained may be further processed in various ways. Thus, the enzyme may be precipitated from the liquid phase, for example with ammonium sulfate, and the enzyme precipitated may be further purified or directly processed. However, the liquid phase may first be concentrated by ultrafiltration with removal of low molecular weight solid constituents by washing (dialysis) and may subsequently be sub10 jected to precipitation or the concentrate may be sprayed onto a support, such as soybean flour for example, and subsequently dried.

The pentosanase concentrates prepared in accordance with the invention show high pentosanase (xylanase) ac15 tivity coupled with improved baking activity.

The xylanase activity may be determined by the dinitrosalicylic acid method as follows: The enzyme-containing solution is incubated for 15 minutes at 40°C in acetate-buffered solution in the pres20 ence of approximately 1 % xylan. An alkaline dinitrosalicylic acid/DNS solution (7 g DNS, 12 g NaOH, 200 g potassium sodium tartrate, 5.5 g phenol, 5 g Na2S2O5 per 1 H20) is then added and color development is measured at 540 nm by comparison with a xylose standard. The enzyme unit 1 U is present if fragments of which the reduction equivalent corresponds to 1 Mmol xylose are released from the xylan in 1 minute under the reaction conditions.

However, the method described in US-PS 3,512,992 may also be used.

Baking activity is determined by measuring the increase in volume of bread rolls produced from a defined quantity of a standard dough. To this end, the length, width and height of 30 such bread rolls are measured, the dimensions in cm are added together and the dimensions of bread rolls produced from the same quantity of the same D 8974 6 dough without the added enzyme are subtracted from the figure obtained.

Examples Example 1 Culture conditions: Seed culture; To prepare seed cultures, thoroughly spored agar cultures of the strain 5937 (XY1-A007) are floated off with 0.1 m P0(, buffer (pH 6.5), the spore suspension is filtered through sterile glass wool, 10% glycerol is added as protective reagent and the suspension is stored in portions at -25 °C.

Main culture: To recover the xylanase, the above-mentioned strain was cultured under submersion conditions in shaking cultures. 100 ml nutrient solution (in 500 ml Erlenmeyer flask with chicanes) were inoculated with 0.1 ml spore suspension and incubated at 30°C in the shaking machine (shaking frequency: 140 r.p.m.).

Media composition: 0.15 % NaH2PO4 0.10 % K2HPOa 0.05 % MgSO4 · 7H2O 0.02 % MnSO<, · 4H2O 0.001 % FeSO4 · 7H2O 0.20 % (NHJNO3 0.50 % soybean flour 1.00 % corn steep liquor 0.50 % β-methyl xyloside 1.00 % cornstarch (degraded) PH 6.5 D 8974 7 To isolate the pentosanase (xylanase), the mycelium was removed by filtration and cell-free culture filtrate was purified by dialysis and concentrated by ultrafiltration. A dry powder was recovered from the concentrate by freeze drying and was mixed with soybean flour to form a product containing 15,000 U/g.

Example 2 A dough was made from 1,000 g wheat flour, type 550, 10 20 g salt, 60 g yeast, 580 g water and 30 g baking powder with or without added enzyme. The dough was kneaded for a total of 5 minutes in a spiral kneader. After standing for 5 minutes, exactly 1,500 g were weighed out, manually kneaded until round and then left standing for another 10 minutes in the following ball cooker.

In an automatic dough divided, the ball is divided into 50 g portions which are then kneaded until round. After standing for 1.5 minutes, the round dough portions are kneaded in an automatic long kneader.

The dough portions are then placed in a proving cabinet for 35 minutes at 30°C/85 % relative air humidity. They are then oven-baked for 20 minutes at 230°C.

After cooling, 3 0 bread rolls from the same dough batch are measured for length, width and height. For an addition of 1,500 U enzyme (corresponding to 0.1 g), 40 cm more are measured than in the case of a comparison dough with no added enzyme.

Example 3 The procedure was as in Example 1, except that xylan or rye bran was used instead of B-methyl xyloside. The results are shown in the following Table.

D 8974 8 Table Inductor substrate Cone. used Xylanase activity (U/ml) Baking activity (baking points/5 ml) Culture time (h) Xylan (98%) 2 % 500 29 110 Rye bran 4 % 253 20 120 β-Methyl xyloside 0.5 % 570 40 72

Claims

1. A process for the production of a baking-active pentosanase preparation, in which microorganisms capable of forming pentosanases are cultured in a fermentation medium, 5 the cell mass is removed at the end of the culture period and the enzyme preparation is recovered from the fermenter broth, characterized in that, to induce baking activity, the substance β-methyl xyloside is added to the fermentation medium in quantities of 0.01 to 5% by weight, based on 10 the fermentation medium, in addition to or instead of xylan and/or xylan-containing mixtures.

2. A process as claimed in claim 1, characterized in that a strain from one of the species rhizopus, trichodema bacillus or aspergillus, preferably Aspergillus awamori 15 and, more particularly, Aspergillus awamori DSM 5937 (XY1A007), is used as the microorganism strain.

3. A process as claimed in claim 1 or 2, characterized in that β-methyl xyloside is used in quantities of 0.1 to 1% by weight. 20

4. A process as claimed in any of claims l to 3, characterized in that to recover the enzyme preparation from the fermenter broth, the fermenter broth is concentrated by ultrafiltration and the enzyme preparation is subsequently recovered in solid form by precipitation or drying on a 2 5 support.

5. A process as claimed in any of claims 1 to 4, characterized in that the enzyme preparation is separated by precipitation.

6. A process as claimed in any of claims 1 to 5, charac30 terized in that the fermentation medium contains a carbon source, a nitrogen source and typical trace elements.

7. A process as claimed in any of claims 1 to 6, characterized in that the fermentation medium contains a protein constituent and/or ammonium salts as the nitrogen source. 35

8. A process as claimed in any of claims 1 to 7, characIE 911797 D 8974 terized in that the fermentation medium contains starch products, more particularly soybean flour or corn liquor as the carbon source.

9. A process as claimed in any of claims 1 to 8, charac5 terized in that the fermentation medium contains salts of sodium, potassium, magnesium, manganese and/or iron as trace elements.

10. A process for the production of a baking-active pentosanase preparation, substantially as described herein by way of Example.

11. A baking-active pentosanase preparation, prepared by a process according to any of Claims 1 to 10.