SI8212812A8 - PROCESS FOR PREPARING OF SPS-ase - Google Patents

Abstract

The enzyme, which is able to decompose a high molecular carbohydrate, abbreviated SPS (soluble polysaccaride), is designated SPS-ase. The SPS-ase is able to decompose SPS into decomposition products which attach themselves to protein in an aqueous medium to a lesser extent than the SPS prior to decomposition. The method for selection of SPS-ase producing microorganisms is based on the fact that the main carbon source of a growth medium is SPS and on a qualitative SPS-agar plate test. A method for production of an SPS-ase by means of a deposited strain of Asp. aculeatus is described. The SPS-ase also has utility in the fruit and vegetable industry and for production of juice and wine.

Description

Improvements in the enzyme for the degradation of high molecular weight carbohydrate and related, isolated high molecular weight carbohydrate, the process for selecting the microorganism producing such enzyme and the process for producing such enzyme.

In Belgian patent no. No. 882,709 describes a process for the production of purified vegetable protein product (p.v.p., purified vegetable protein product) by enzymatic removal of the residue without dissolving and re-staining the protein. Pvp purity obtainable by known

- 2 process is not satisfactory and could be improved. In cases, the purity of pvp is demonstrated to be about 85%. Even if it is possible to obtain it by a known pvp process with a purity of about 90%, this can only be achieved with certain pre-treated starting materials, e.g. with soy protein concentrate. It would be desirable to achieve a pvp purity of about or above 90% with a much wider range of starting materials, in particular peeled and degreased soybean flour.

The invention is based on the surprising finding that a certain part of the degradation product of a residue, as it appears in the enzyme treatment mentioned above, that is, a water-soluble, high molecular weight carbohydrate, binds to a part of a plant protein, as will be explained later in detail. This, of course, results in lower protein purity. We have also found that this high molecular weight carbohydrate has the ability to bind to proteins of animal origin.

Thus, the purpose of the invention is to provide an enzyme for the degradation of the above high molecular weight carbohydrate that will allow the production of pvp with improved purity and a process for producing such an enzyme.

The basis of the invention can be described in the following manner, referring to Figure 1, which shows only material consisting of undissolved solids, while all supernatants are omitted. The soybean meal batch was divided into two parts, part I and part II (column a in Figure 1).

- 3 Part I was proteolytically degraded at a pH of about 8 by ALCALASE (a proteolytic enzyme produced by

B.licheniformis sold by NEW INDUSTRI A / S, 2880 Bagsvaerd, Denmark) and then rinsed at pH about 8 to remove the total amount of protein; the remainder was separated from the supernatant and washed (see part I, columns a and b, Fig. 1).

In this way, we isolated the pure residue (called the residue)

I) (column b in Fig. 1). Part II of soybean meal was not processed; for the sake of brevity, we call the remainder in Part II the remainder II (column b in Fig. 1). We now degrade both residue I and part II with the help of market pectinase, e.g. FECTINEXA (pectolytic enzyme produced by Schveizerische Ferment A / G, Basel, Switzerland) (see columns b and c in Fig. 1). Surprisingly, we found that the undissolved part of residue I was much smaller than the undissolved part of residue II, based on mass balances of nitrogen and dry matter, see Figs. 1, where the dashed areas in column c correspond to the insoluble non-proteinaceous materials in the above step. Further, when the supernatant of pectinase treated residue I is brought into contact with the soy protein suspension at pH 4.5, the polysaccharide in the supernatant binds to the soy protein. This polysaccharide in the supernatant of residue I, which is part of the degradation product of the residue and which, in the absence of soy protein, is soluble in water but binds to soy protein at or around the isoelectric point of soy protein, is called SPS (Soluble Folysaccharide), θ see fig. 1. A SPS has a molecular weight distribution between 5 x 10

- 4 4 _ and 4.9 χ 10. The production of isolated SrS is evident from the technological scheme in FIG. 2, which also comprises some of the processes shown in FIG. 1. Therefore, the problem is to find an enzyme capable of breaking down the SPS into. only way that SFS degradation products do not bind soy protein or to bind soy protein under much smaller amounts than bind soy 5 protein SPS.

Although the description explicitly refers to soy protein, the invention is not limited to soy protein, but encompasses all types of plant proteins, see e.g. proteins listed in Belgian patent no. 882.769, p. 1.

According to the invention, we have now found that by selecting the ability to degrade soybean SPS, we can select microorganisms that are capable of producing a compound that exhibits enzymatic activity that effectively degrades soybean SPS, which is hereinafter referred to as SPS ezes .

Thus, the first aspect of the invention encompasses SFS-azo, a carbohydrate in a useful form, which is capable of degrading under suitable ppg soybean SPS into degradation products that bind to a protein in aqueous medium to a lesser extent than soybean SPS would itself bind before degradation. to the same protein under appropriate conditions.

We further found that this SPSase, which is capable of degrading soybean SFS, is capable of degrading SPS-like polysaccharides that originate from lentils and fruits, more fully than market pectinase and market cellulase.

- 5The expression above, in useful form, is intended to exclude from the invention e.g. a preparation containing SPSase containing toxic substances or showing such low SPSase activity that it requires the use of a preparation containing more than 10% SPSase by weight of SPS in the substrate for a 24 hour metabolism and at 50 [deg.] C. and the pH optimum of said SPSase to achieve any substantially significant degradation of SPS.

By completely or partially removing SFS from the final plant protein, the purity of the final plant protein is in comparison with the purity of the final plant protein obtainable according to the method known from Belgian patent no. 882.76% necessarily improved as this known plant protein product was contaminated with SPS.

It is currently unknown whether the enzyme activity of a single SPSase, described below, originates from a single enzyme or from an enzyme complex comprising at least two enzymes.

It seems the natfco's non-catches show decomposition; the effect of SPS-aase is responsible for at least two enzymes, with one of the two enzymes being able to carry out only a slight degradation of SPS, and then one or more enzymes capable of carrying out more extensive degradation of SPS. However, the applicant does not wish to be restricted by such a hypothesis or similar hypotheses.

The preferred form of SPS aase of the invention is characterized in that the SPS aase is capable of degrading soybean SPS in aqueous

- 6 media into degradation products that bind to the plant protein in the aqueous medium to a lesser extent than the SFS soybean would bind to the same plant protein in the aqueous medium before degradation.

The preferred form of SPS-aase of the invention is characterized in that the SPS-aase is capable of breaking down soybean SPS in aqueous medium with a pH value not exceeding 1.5 by 4.5 into degradation products which they bind to soy protein in aqueous medium to a lesser extent than soy SFS would bind to soy protein in aqueous medium before degradation.

The preferred form of SPSase according to the invention is characterized in that the degradation products of soybean SPS bind non-vegetable protein to the extent of less than 50%, especially less than 20%, than the soybean SPS would bind to the vegetable protein after degradation. in aqueous medium.

The preferred form of SPSase according to the invention is characterized in that the SPSase exhibits a positive test for SPSase when examined according to the method for qualitative and quantitative determination of SPSase described herein.

A preferred form of SPS-aza of the invention is characterized in that SPS-azo was produced by a microorganism belonging to the genus Aspergillus, preferably to the Aspergillus niger group.

The preferred form of SPSase according to the invention is characterized in that the SPSase is derived from enzymes that can be produced by Asp. aculeatus CBS 101.4-3. The same SPS azo can be produced by Asp. japonicus IFO 4408. We have been found to produce Asp. aculeatus CBS 101.4-3 also very effective remanase, cellulase, pectinase and hemicellulase.

Furthermore, we found that not every strain belonging to Asp species produces just that. aculeatus or Asp. japonicus, the SPS-aze needed for the invention, We have thus demonstrated, as it is shown later in the description (Chapter 5), that the Asp strain japonicus ATCC 20236 does not produce such quantities of SPS-sze that can be determined by the enzymatic determinations of SFS aase described herein.

A preferred form of the SPSase of the invention is characterized in that the SPSase is immunoelectrophoretically identical to the SFSase produced by Asp. aculeatus CBS 101.43, and identified by immunoelectrophoretic overlay technique, see sections 6 and 7.

When microbial SPS is produced, it forms in a mixture with several accompanying substances, in particular other enzymes. If desired, said SPSase can be purified if e.g. using chromatographic separation methods, as will be seen later in this description (Chapter 8).

In Agr.Biol.Chem 40 (1), 87 to 92, 1976 it is described that strain Asp. japonicus ATCC 20236 produces an enzyme complex capable of partially degrading an acid polysaccharide in a soy sauce called APS, part of which is called

- 8 AFS-I. These acid polysaccharide are not identical to SPS, as we will show in more detail in this description later in Chapter 3. Thus, the HPLC (high-pressure liquid chromatography) gel filtration chromatograms of SPS and APS are clearly different and, in addition, the APS gel filtration chromatograms degraded by market pectinase Pectolyase, and SPS treated with Pectolyase market pectinase are clearly different. In addition, the article does not show that the acid polysaccharide is bound to soy protein and that the degraded acid polysaccharide is not bound to soy protein or that it is bound to soy protein to a much lesser extent than undigested acid polysaccharide. It has also been shown that this strain does not form SPS-aase in such quantities as could be ascertained by the enzymatic determination of SPS-aase described herein. This created the premise that no strain of Asp. japonicus does not produce SPS-aza, and it has been surprisingly found in accordance with the invention that some strains of Ashp. japonicus produce SPS-azo.

Another aspect of the invention comprises an isolated SPS produced on the basis of vegetable crude protein as starting material.

The preferred form of isolated SFS of the invention is characterized in that ds is vegetable crude protein cleansed soybean meal. The production of this insulated SPS is described in the foregoing with reference to FIG. 2.

A third aspect of the invention is a process for selecting a SPS-producing microorganism for the production of SPS a

- 9 according to the invention, characterized in that we grow the microorganism to be tested on a medium whose main source of carbon & SPS, after which the sample medium is analyzed for SPS-azo, and δ, β is an analysis for SPS-azo positive, said microorganism is selected as the microorganism that produces SPS-aase.

A fourth aspect of the invention is a process for the production of SPS aase, characterized in that the strain which can be selected in accordance with the above method for selecting a microorganism that produces SPS aase is grown on a culture medium. Cultivation can be performed as submerged fermentation or as surface fermentation.

A preferred form of the process of the invention is characterized in that all Asp is grown on the culture medium. aculeatus CBS 101.43 or Asp. japonicus IFO 4408.

A preferred form of process according to the invention is characterized in that it is grown as submerged cultivation at a pH in the range of 3 to 7, preferably 4 to 6, at a temperature in the range of 20 to 40 ° C, preferably of 25 to 35 ° θ, wherein the medium contains a source of carbon and nitrogen and inorganic salts.

A preferred form of the process of the invention is characterized by the fact that the medium contains roasted soya flour.

A preferred form of the process of the invention is characterized in that soybean meal is treated, before it is used as a substrate component, by a proteolytic enzyme, preferably by a proteolytic enzyme produced microbially by Bacillus licheniformis.

The preferred form of the process according to the invention is characterized in that the pectin solution is aseptically added to the fermentation broth during cultivation.

We found that it produces Asp. aculeatus CBS 101.45, in addition to SPS aase, also has highly effective enzymes that solubilize the residue, cellulases, pectinases, and hemicellulases, and that the enzyme complex produced by Asp. aculeatus CBS 101.45, ideally suited for use in the decomposition of cell walls of plant materials. Thus, the enzyme complex that can be produced by Asp. aculeatus CBS 101.45, used with excellent results in the food processing industry for the processing of fruit and vegetable pulp and for the purposes of clarifying and reducing the viscosity of juice and wine processing; it can also be used as a dehydrating agent (that is, a decomposition agent for polysaccharides and therefore for the release of water bound in the polymer structure of polysaccharides) in the processing of plant foods.

The fifth aspect of the invention involves the use of an SPSase or a process for the degradation of polysaccharides, preferably plant cell wall polysaccharides, by carbohydrate, characterized in that the SPSase preparation of the invention is contacted with a substrate for this SPS preparation in aqueous medium. -aze.

Thus, in accordance with the invention, we have found that SPSase preparations are valuable enzyme preparations for partial or complete

- 11 liquefaction sounds the decomposition of various materials, preferably plant materials, e.g. fruits and vegetable wastes containing protein, cellulose, hemicellulose (eg glucans, isylans, galactens and arsban) "> gum Is, pectin, lipids, inulin, polyphenols, starch and alginates, or for related purposes, see table shown later in the description. As examples of such related purposes, we can mention all the purposes for which pectinases and cellulases are used today. We will provide more examples later in this description.

With respect to the extraction (isolation) process described e.g. in Example 2, we note that the SPS-aase preparation is substantially free of proteinase due to the fact that otherwise the desired end product, i.e. the protein, would be degraded. Similarly, the preparation of SPS-aza must be used if extracting (isolating) from raw biological material biological materials other than protein is essentially free of any enzyme that degrades this other biological material. Such modified SPSase preparations can be produced by selective inactivation of the undesired enzyme by fractionation or other methods known per se.

Thus, the preferred use of the invention is characterized by the fact that the degradation is accompanied by the isolation or extraction of biological material other than soy protein and related plant proteins from crude biological materials, wherein the SPSase preparation does not substantially contain any enzyme which is able to degrade this biological material.

Thus, in accordance with the invention, we have found that modified SPS-aase preparations (modified in that they do not essentially contain enzymes capable of breaking down biological material to be extracted or isolated) are valuable enzyme extraction preparations (isolation) of said biological materials, e.g. starch, lipids, flavorings, colorings and juices, of crude biological materials. We will provide more examples later in this description.

A preferred form of use according to the invention is characterized in that it further treats one or more reaction products (whether end products or waste products are desirable), simultaneously with or after enzymatic treatment.

This ensures a more flexible and flexible use.

A preferred form of use according to the invention is characterized in that the further treatment, in case one of the reaction products is fermentable sugar, is alcoholic fermentation. This provides an easy and affordable alcohol production process.

tel «

- 13 To refer to Chapter 1 of the invention.

in order to clarify the nature of the invention, we refer to the following up to 10, which all describe the details of

1. Production of SPS.

2. Characterization of SPS, in particular its molecular weight distribution.

3. Documentation of the fact that SPS and AFS are different compounds.

4. Selection of SPS-producing microorganisms.

5. Characterization of some SPS-forming microorganisms.

6. General description of the immunoelectrophoresis-related overlay technique.

7. Immunoelectrophoretic characterization of SPSase with a semi-specific antibody and a coating. 'U ·'

8. purification of the SPS-aze preparation.

9. Activity dependence on pH, activity dependence on temperature and stability of SPS aase.

10. Enzyme activity determinations

- 14 CHAPTER 1

PRODUCTION OF SPS

As mentioned earlier, the starting material for the production of SPS soy may be a residue. Therefore, we first describe the production of the soy residue.

Soybean residue is a protein-free carbohydrate fraction (which may in practice contain smaller amounts of lignin and minerals) in flour from soybean and peeled soybeans; this carbohydrate fraction is insoluble in aqueous medium with a pH of 4.5 and can be produced in the following way, also referring to Scheme 1.

The degreased soybean meal (Soymmel 15, manufactured by Aarhus Oliefabrik A / S) was suspended in deionized water at 50 ° C in a soybean meal to water ratio: water = 1: 5 in a pH stat tank and temperature control. adjusted from 4 N NaOH to 8.0 (I).

We now perform pH-hydrolysis with ALCALASE 0.6 L (B. licheniffirmis-based proteolytic agent with an activity of 0.6 Anson units / g, determining the activity according to the Anson method as described in NEW ΕΝΖΥΜΕ INFORMATION IB Νό · 058 e-GB), where the enzyme / substrate is equal to 4% of the amount of protein in soybean meal (II). After 1 hour of hydrolysis, the slurry was separated by centrifugation (III) and washing (IV), performing this operation twice (V, VI, VII). The slurry thus treated is re-hydrolyzed for 1 hour with ALCALASE 0.6 L (VIII, IX),

- 15 similar to what we stated earlier. The slurry is then separated by centrifugation (X) and washed twice (XI, XII, XIII, XIV), whereby the finally washed slurry (6) is spray dried (XV).

The spray dried powder thus produced is a soy residue that serves as a raw material for the production of SIS.

SIS is a water-soluble polysaccharide fraction, which is formed by the conventional treatment of the soy residue mentioned above with pectinase. The SIS is produced as follows using the 14 reaction steps given below, also referring to Scheme 2.

1. Determine the dry matter content of the soybean residue mentioned above and dilute the soybean residue with water to 2% dry matter and maintain in suspension at 50 ° C in a temperature control vessel.

2. Adjust the pH from 6 N NaOH to 4.50.

3. Add Iectinex Super Zone. L in an amount of 200 g / kg of dry matter (market pectinase ^ manufactured by Schweizerische Ferment AG, Basel, Switzerland) with a pectinase activity of 75 750,000 MOU determined in accordance with the Prospectus Determination of the lectinase units on Apple Juice (MOU) of 12/06/1981 , obtainable from Schweizerische Ferment AG, Basel, Switzerland), and Celluclast 200 L was added in an amount of 20 g / kg of dry matter (market cellulase described in

- 16 prospectus NEW enzymes, .information sheet B 153 e-GB 1OOO July, 1981, obtainable from NOVO INDUSTRI A / S, Novo Alle, 2880 Bagsverd, Denmark).

4. Maintain the contents of the vessel while stirring at 50 ° C for 24 hours.

5. Enzymes are inactivated by raising the pH from 4 N NaOH to 9 »0. The reaction mixture was maintained for 30 minutes and then pH adjusted again with 6 N HCl to 4.5.

6. Centrifuge the reaction mixture and collect both the centrifuge and the slurry.

7 · The control of the centrifuge from step 6 is filtered on a filter press (the filter is washed with water before the control filtration).

8. The control filtrate is ultrafiltered, diafiltered and re-ultrafiltered on a membrane with a limit value of 50,000 (DDS GR 60-P from De Danske Sukkerfabrikker), using the following parameters:

1. Ultrafiltration corresponding to six times the volume concentration.

2. Diafiltration until the percentage of dry matter in the permeate is 0 (0 ° Brix).

3. Ultrafiltration 80 about 15% of the dry matter in the concentrate.

The temperature is 50 ° C, the pH is 4.5 and the average pressure is 3 bar.

- 17 9 ·, Cool the ultrafiltrate concentrate to 5 ° C and add the same volume of 9θ% ethanol.

10. Collect the precipitate by means of a centrifuge.

11. Wash both © and © twice with §0% v / v ethanol in EL ^ O, which corresponds to the volume of the centrifuge from step 10, that is, perform two centrifugations.

12. Re-dissolve the washed precipitate in water with a volume equal to that of the ultrafiltration concentrate from step 9 ·

15 · Filter the liquid from step 12 on a glass filter.

14. A clear filtrate containing pure SPS was lyophilized.

- 18 ί 5. µ. 99

TECHNOLOGICAL SCHEME ABOUT M.

degreased soybean mokt H ~ O NaOH ¹ and that pH = δ

Alcalasa 0.6 L (E / S = 4%)!

j 4N NaOH

Hydrolysis mixture

Hydrolysis of 1'uro before release of pH-stat (pH »8, T = 50 ° G

II

H-0

- 1. centrifugation III Sludge 1 s, l.washing 2 centrifugation m v .. Gn & h? and 2 3 2. s piran j e 5. centrifugation VII -

Centrifuge 2 s, Waste

-Centrifugate - 3 Waste £ nana 3

VIII

Centrifuge _] - _> Waste

NaOK «o r.HMH ^Mov8 hydrolysis mixture

- - p

Alcalasa- 0.6 L

4N NaOH ϊ Hydrolysis 1 hour before release of _% _pH-stat (pH = 8.0, rn - ^r '

IX

T = 50 C)

4. centrifugation

Centrifuge 4 Waste

Sludge 4

l.spi

-H. ₂ ^g - ^ Ί · spiranj9 χχ

5. centrifugation χχχ

Centrifuge. 5 $ & p © dek «—2 ^ 2 — s 2. s pirsnj e

XIII

S. centrifugation

XIV

Centrifuge g §4 $ g4ek

Sludge 6

Hard drying

XV. / f

TECHNOLOGICAL SCHEME OF ST. 2

1000 g Pectinex SuperkcPnc.L 5 kg spray dried residualfc

100 g CelluclastaSSOcg

247 1 H ₂ O

Decomposition by _aase -jj pectinase and cellulase

50 ° C; pH = 4.5; t = 24 and "ur" -------- <- 1, 2, 3, 4, 5

Centrifugation

Control filtration kg

1 H ₂ O _;

Ultrafiltration, diafiltration, ultrafiltration (GR 60P) (19 - 26 C) .2J1 ..... 1 ΡίθΡτι \ | Z

^ Staining n. 1 Centrifugation e

seate 8

Supernatant ((idpsdek)

Centrifuge

Precipitation x Wash x Centrifuge - p.1

Precipitation

2-.1 P ₂ ° 3

Again ra zt * p | .j lamb

Control filter-CLj z

Protein sludge ^ 3 (waste ^)

Lyophilization

310 g lyophilized g SPS

5. ^1f

5. H. 92

- 20 CHAPTER 2

CHARACTERIZATION OF THE SPS, IN PARTICULAR ITS DISTRIBUTION OF THE MOLECULAR MASS

Using gel chromatography on HPLC equipment (Waters pump model 6000, Waters data modules 730 and Waters refractometer R 401), the molecular weight distribution of the SPS is determined, the production of which is carried out as indicated in this description (Fig. 4). Using the same method, we also determined the molecular weight distribution of the degradation | hh products of SPS using SPS-aase (Fig. 5) · We proved the binding effect between soy protein and SPS (Fig. 6) using the same method and the absence of a binding effect between soy protein and SPS digested by the agent of the invention (Fig. 7).

The calibration curve (the logarithm of the molecular weight plotted against R ^, where we define the R ^ value for glucose arbitrarily as 1, and R _{f the} value for a given dfextBn defined as the retention time for that dextran divided by the retention time for glucose) was determined by several standard known molecular weight dextran (T 4, T 10, T 40, T 70, T 110, T 500) by Pharmacia Pine Chemicals AB, Box 175>

S-75104, Uppsala, Sweden. We determined the R _f -value for the maximum of each dextran peak and calculated the corresponding molecular weight as ^ M ^. where M _{w is the} average value of the molecular weight in accordance with the mass and M _{q the} average value of the mo- 21 molecular weight in accordance with the number. 0.1 MaNS ^ O ^ was used as the eluent for this chromatographic process. The columns used in the chromatographic process are 60 cm PW 5000 and then 60 cm PW 5θ00 from Toyo Soda Manufacturing Co., Japan. In this way, the above-mentioned dextra molecular weight ratios were determined for R _f , see FIG. 3.

Based on FIG. 4, we can calculate that the SPS has a molecular weight distribution that results in a value of M _w about 5 »4 x 10 * and a value of M _q around 4.J2 χ 1θ \ It can also be seen from this figure that the chromatogram shows two distinct dots at a retention time of 34.5 minutes ($%) »corresponding to a molecular weight of about 5 x 10 ^, and a retention time of 47.12 minutes (67%), which Zl corresponds to a molecular weight of about 4.9 x 10. It is also clear from this curve that between these two points of the shoulder at a retention time of 41.25 minutes (27%), "which corresponds to a molecular weight of 2.8 x 10 ^.

After the decomposition of SPS with SPSase, the hydrolysis mixture was filtered membrane and the filtrate was chromatographed. We found that about 55% of the SPS is degraded to ralo-, di-, and trisharides, and that the remaining 45% decomposes into a three-dot polymer with the following molecular weights: 5 x 1θ \ 10 ^ and 4.4 χ 1θ \ see FIG. 5.

The following experiments were performed to demonstrate the binding effect between soy protein and SPS and to significantly reduce the binding effect between soy protein and SPS degraded by SPS aase.

- 22 5% SPS in 0.10 M acetate buffer with pH 4.5 was added to the soybean isolate broth (Purina E 5θ0) to give a suspension with an isolate / SPS ratio of 10: 1. This suspension was incubated for 18 hours on an agitator at 50 ° C. After incubation, the suspension was centrifuged and the clear supernatant analyzed by HFLC as described in the yarn. From FIG. 6 in comparison with FIG. 4 shows that SPS completely adsorbed on soybean isolate.

The iCfifet procedure as mentioned in the previous paragraph is performed with a 3% SPS solution hydrolyzed with SPS aase produced by CBS 101.45 (Fig. 7). The comparison between FIG. 7 and FIG. 5 shows that no compound in the hydrolyzed SPS with a molecular weight below about 10 is adsorbed on soybean isolate. Hydrolysis reduces the quantitative binding to about 10 to 15% relative to the binding of SPS to soy protein.

NMR analysis of the SPS, the production of which is carried out as indicated in the present description, shows the following approximate composition of the SPS:

1) aagalacturonic acid in an amount of about 45%, with about 40% of the total amount of α-galacturonic acid present as methyl ester ^

2) ramnopyranose in the amount of about 20%,

5) galactopyranose in an amount of about 15% and

4) β-xylopyranose in an amount of about 20%.

The ingredients appear to be present in a structure comprising the ramnogalacturon framework and the xylose and galactose side chains.

Complete acid hydrolysis of SPS (8 hours in 1 N ^ SO ^) and subsequent TLC (thin layer chromatographic) analysis showed that smaller amounts of fucose and arabinose monosaccharides were also present in the hydrolyzed SPS.

HPLC analysis of SPS degraded by the CBS 101-enzyme SPS-enzyme complex showed a strong decrease in molecular weight. Accordingly, the NMR spectra of SPS decomposed as shown above show that the major part of the ester groups has disappeared and that the share of xylose and galactose in the higher molecular weight material has also decreased. The NMR spectra of that portion of the SPS degradation product that precipitates from the addition of one volume of ethanol to one volume of the SPS degradation product are similar to the SPS NMR spectra, with the above changes in ester groups and xylose and galactose content.

11. 5. 92

- *

CHAPTER 3

DOCUMENTATION WITH THE FACT THAT SPS AND APS ARE DIFFERENT COMPOUNDS

APS was prepared as indicated in Agr.Biol.Ghem., Vol. 36, No. 1 4, p. 544 - 550 (1972).

We have now hydrolyzed this polysaccharide and SPS with various enzymes, after which the degradation of the mixture was gel chromatographed on HPLC equipment, as indicated in Chapter 2, Characterization of SPS, in particular its molecular weight distribution.

Specifically, ', hydrolysis was performed by treating 25 ml of 2% APS or 2% SPS solution in 0.1 M acetate buffer with pH

4.5 with 10 mg KRF 68 or 3θ mg Pectolyase. KRF 68 is a preparation of SPS-aze, the preparation of which is described in Example 1. The results are shown in the following table.

 fr »>» HPLC ge ^ & 'chromatogram 'Polysaccharide. 4 APS Pectolyase lij δ X • APS · KRF 68 W & 9 X SPS Pectolyase 10 X SPS KRF 68 5 X

5. «92 - 25 *

CHAPTER 4

SELECTION OF MICRO-ORGANISMS, EC MANUFACTURING SPS-AZO

The microorganism to be tested is incubated on an oblique agar substrate with a composition that allows the microorganism to grow. After initial growth on an oblique agar substrate, the microorganism is transferred to a liquid main substrate in which the major carbon of the SPS (prepared as indicated) containing the nitrogen source is N0 ^ ~, NH ^ ⁺ , urea, free amino acids, proteins or other nitrogen-containing compounds containing, in addition, a mixture of the necessary salts and vitamins, preferably in the form of a yeast extract. The composition of the main substrate depends on the genus of the micro-organism, the main problem being that the main substrate must be able to support the growth and metabolism of the micro-organism. When there has been growth in the order of magnitude from 1 to 7 days, depending on the growth rate of said microorganism, a sample of fermentation broth per SPS-azo is analyzed according to the enzymatic determination of SPS-aase described herein. or in accordance with any other determination of SPS-aza adapted to other specific uses of SPS-aza and not used as a constituent of a soy residue decomposition agent.

To obtain a more sensitive method for the determination of enzyme activity, the temperature may be lowered to 40 ° C during the determination of SPS-aase activity and the incubation time extended to 20 hours at

5. It 92 to which antibiotics should be added to the substrate to avoid infection.

v

If we adhere to this test method, can we find other SPS-producing microorganisms belonging to the genus Aspergillus _? in such _? belonging to other genera.

5. It 92

CHAPTER 5

CHARACTERIZATION OF SOME MICRO-ORGANISMS PRODUCING SPS-AZO

In accordance with the selection of SPS-aase-producing microorganisms listed here, we have determined that the microorganisms listed in the upper section of the following table are SPS-aase producers. The table also contains a strain belonging to species Asp. japonicus, which is not a producer of SPS aze.

Producer SPS aze Species Our identification mark identification designation First deponi- wounding they are flying Det. m - Asp. japo- nicus Asp. acule- atus s- X X X X X X A 805 A 1443 A 1384 CBS 101.43; P5M 1344 IFO 4408; 23V6 ATCC 20236; DS / 1 23 * 15 ^ 9

A brief identification of the above strains can be found in the following crop catalogs.

List of Cultures 1978 Centraalbureau voor Schimmelcultures, Baarn, The Netherlands.

Osaka Fermentation Institute, List of Cultures, 1972, 5th edition, 17 - 85, Juso-honmachi 2-chome, lodogana-ku, Oshka 532, Japan.

5. H 92

-a »*

The American Type Culture Collection The Catlpgne of Strains I, 14th edition 1980, 12301 Parklawn Drive, Rockville, Maryland 20852.

All the strains in the table above almost exactly fit the taxonomic description of species Asp. japonicus and Asp. aculeatus published in The genus of Aspergillus Raper and Fennell, 1965 (see in particular pages 327 - 550).

5. II 92 • Mb

CHAPTER 6

GENERAL DESCRIPTION OF IMMUNOELECTRIC RELATED TECHNIQUES

The applicant has developed, for the identification of individual components of a cross-immunoelectrophoresis enzyme complex with a semi-specific antibody against all enzyme components in an enzyme complex, a method called top agar overlay technique. The method is based on the fact that even after specific binding of the enzyme to the antibody, the enzymes are still active, or in other words, that the active site of the enzyme is not identical to the site of binding of the enzyme with the antibody. The enzyme complexes with the antibody precipitate during electrophoresis as distinct arcs in the gel. Cover the gel plate with soluble SPS in top agar. After heating at 45 ° C for 20 hours in an atmosphere with a relative humidity of 100%, the arc having SPS-aase activity will emerge as a clearing zone in the overlying SPS after precipitation with a mixture of ethanol and acetone volumes, when viewed against black background. Locs that do not have SFS activity remain invisible.

² 5. 11 92

- 30 CHAPTER 7

IMMUNOELECTROPHORETIC CHARACTERIZATION OF SPS-AZE WITH POLISPECIFIC ANTIBODIES AND CROSS

The rabbits were immunized with an enzyme complex containing SPS-aase obtained by fermentation of Aspergillus aculeatus CBS 101.43 as shown in Example 1 (KRF 68 and the semi-specific antibody was obtained in a manner known per se.

Cross-linked immunoelectrophoresis of the enzyme complex obtained by the fermentation of Asp was performed by means of this semi-specific antibody. aculeetus CBS 101.43 as indicated in Example 1 (KRF 68) as described in N.H. Axelsen et al.,

· Reference is made to Figure 11, which shows the arcs corresponding to the various proteins produced by the microorganism. Using the above-described top agar overlay technique, it is found that it corresponds to the dashed area of the SPS-azi.

If the above hypothesis, comprising the assumption that SPSase consists of at least two enzymes, is correct, the slash area is the area in which all the enzymes responsible for SPSase activity are present. If these enzymes were separated by other forms of enzyme by immunoelectrophoresis in such a way that they did not cover any common area, part of the activity of SPS-aase could still be identified by immunoelectrophoresis with Trekrrfc but also by market pectinase.

11. 5. 92

- 2Λ2 -

CHAPTER 8

CLEANING THE SPS-AZE PREPARATION

Purification of the KRF 92 SPSase preparation (see Example 1) was performed by ion exchange. The buffer is 50 mM Tris (trichydroxymethylaminomethane), which is adjusted with HCl to pH 7 »0.

The column is K 5/50 of Pharmacia, Sweden. The ion exchange m8teri8l is a DEAE-trisacryl of LKB, Bromma, Sweden (300 ml). The flow rate is 100 ml / h.

g of the KRF 92 SPSase preparation was dissolved in 4-50 ml of H ^ O at 6 ° C and all the operations described below were performed between 6 ° C and 10 ° C. The pH was adjusted to 7.0 with 1 M Tris. The column was equilibrated with buffer and then a SPSase sample was added to the column. The optical density at 280 nm and the conductivity were measured in the eluate, referring to Figure 12. Fraction 1 is the eluate which does not bind to the ion exchange material. The column was then washed with 2000 ml of buffer, ksr to give a fraction of 2. Now I introduce a gradient of 0 to 500 mM Above, giving fractions of 3 to 9 ·

All 9 fractions were concentrated with 200 ml and dialyzed against water by dialysis (Hollow Fiber DP 2 from Amicon, Massachusetts, U.S.A.) to a conductivity of 2 mS. Then, all nine fractions were lyophilized. Only fractions 1 and 2 showed SPS-aase activity.

Fraction 1 was partially purified by gel filtration.

From fraction 1, it was dissolved in 10 ml of 50 mM sodium acetate

5. Ji 32

- 53 with pH 4,5 (500 mM KC1). The column is 2.5 x 100 cm by LKB. The gel filtration filler material is Seph8cryl S-200 from Pharmacia, Sweden. The flow rate is 50 ml / h. Fractions containing materials with molecular weights between 70,000 and 100,000 calibrated with globular proteins contained an enzyme complex called factor G, which cannot degrade SPS when tested according to a qualitative Sgar test, but which SPS decomposes according to qualitative agsr assay when factor G is mixed with pectinase. Factor G has been found to be able to cleave galactose, fucose and some galacturonic acid from SPS, but the major degradation product according to HPLC analysis is still a high molecular weight product, which is very similar to SPS.

11. 5. 92

CHAPTER 9

PH-dependent activity, temperature-dependent activity and SPS-AZE stability

Figure 13 shows the dependence of the activity of the KRF 68 SPSase preparation on pH. A pH buffer system with formic acid was used from pH 2.7 to pH 3.5, and an acetate buffer system was used from pH 3.7 to 5 »5.

Figure 14 shows the dependence of the activity of the KRF 68 SPSase preparation on temperature.

Figure 15 shows the temperature stability of the SPSase preparation

KRF 68.

5. H. 92

- & CHAPTER 10

DETERMINATIONS OF ENZYMIC ACTIVITY

The table below is an overview of the various determinations of enzyme activity of the invention.

Definition of activity unit and description of determination of enzyme activity sjitipoz- the way Public disposition ložljivo Described i later in this description '' -— Reference SPS-W: X Solubilizing zirj.e Γ Residues SRU X 1 SRUM-120 ' X Protease and HUT X Oelulazaa ^c x X 2 • Pektinsz® PU X 3 PGE X 4 UPTE X 5 PEE 6 Hemicegrv iulaza * ' VHCU X 7

The references given in the last column of the table above are explained in detail in the table below.

and ' The reference is to get from NEW INDUSTRI A / S, New Alle, 2880 Bagrsvsrd, Denmark Schvzei- zerische Ferment AG, Basel, Switzerland • Reference no. Identif icat <and I references bookstores 1 Analyseforskrift AF 154/4; dated 1981-12-0 1 x 2 Analytical Biochemistry 84, 522 - 532 (1973) X Analytical raethod AF 149/6-GB, dated 1981-05-25 X 3 Determination of Pectinase Activity with Citrus Pectin ^ dated « 3/23/1976 > X 4 Viscosimetric Polygalacturonase-Bestim- * 4a tiraj ». 7? X 5 Bestimmung der Pectintrans el imin a s e ^ UKTE / g) X 6 Determination of the Pectinesterase activity ^ undated) <fcar chetniks WJA / GV ji / • X '7 Analytical method AF 156/1-GB X

With regard to the determination of cellulase activity, we note that the analysis was performed as described in AF 149/6-GB and that the principles of determination were explained in Analytical Biochemistry.

11. 5. 92

- JjB -

CHAPTER 10 a

ENCYMIC DETERMINATION OF SPS-AZE

The enzymatic determination of SPA-aase is performed in two stages, that is, by a qualitative test on an agar plate and a quantitative determination of SPS-aase activity based on measuring the amount of total sugars released, if the qualitative test on an agar plate is SPS-aase activity 0 , regardless of the value derived from the quantitative determination of SPS-aase activity. if the qualitative test on the agar plate poses positive, the SPS-aase activity is of the same value resulting from the quantitative determination of the SPS-aase activity.

I. Qualitative test not agar plate

The SPS agar plate was prepared as follows.

Buffer (B) was prepared by adjusting 0.3 M acetic acid to pH 4.5 using 1 N NaOH. Dissolve 1 g of SPS in 20 ml of B. 1 g of agarose (HSB Litex) is mixed with 80 ml of B and warm to boiling point. When the agarose dissolves, SPS solution is slowly added. The resulting 1% SFS agarose solution was placed in a 60 ° C water bath. Now pour the plates by pouring 15 ml of a 1% SPS agarose solution onto a 10 cm x 10 cm horizontal glass plate. Then embedded in a solidified layer of SPS-agarose 9 wells at a distance of .2.5 cm.

To each well was placed 10 / ul 1% solution of enzyme protein ^

With tl 92 -Wiki we test it for SPS-aase activity. The plate was incubated for 18 hours at 50 ° C and 100% relative humidity. We now precipitate the undiluted SPS with a solution of equal volumes of ethanol and acetone.

II. Activity quantification test

SPS aze

The purpose of this assay is to determine the enzymatic activities that are capable of degrading SPS to such an extent that the degradation products exhibit very reduced or no adsorption or binding affinity for soy protein. Fossils have shown that part of the SPS degradation products that is not precipitated by a mixture of equal volumes of water and ethanol has no adsorption or binding affinity for soy protein.

Determination of SPS-based is not hydrolysis of SPS under standard conditions, followed by precipitation of that portion of SPS that is not hydrolyzed with ethanol. After precipitation, determine the proportion of non-precipitated carbohydrate by quantifying the total sugars $ ® (according to AP 169/1, available at NOVO INDUSTRI A / S 2880 Bagsvaerd).

§Standard conditions:

temperature: 50 ° C pH: 4.5 reaction Salt control 210 minutes with substrate only, then 2 minutes with enzyme added: main value 212 minutes

Equipment_of range:

shaking water bath thermostated at 50 ° G swirling centrifuge ice water bath.

R £ 8genti_include:

buffer: 0.6 M acetic acid in demineralized wound water (a)

1.0 M NaOH (b) substrate: Adjust the pH value of 50 ml a from b to 4.5, then add 4.0 g of SPS and after dissolving the SPS, adjust the pH to 4.5 again and adjust the volume with deionized water to 100 ml.

interrupting reagent: absolute ethanol.

- £ $ 1 unit of SPSase activity (SAE or SPSU) is defined as SPSase activity which, under the above standard conditions, releases an amount of carbohydrate soluble in 50% ethanol equivalent to 1 micromole of galactose per minute .

Even if the initial part of the enzyme standard curve is a straight line, we note that it does not intersect point (0,0).

_ 40 ¹ ,

CHAPTER 10 b

ENZYME DETERMINATION OF SOLUBILIZING ACTIVITY FOR THE STAY EXPRESSED AS SRUM 120 Princi p

In the method for the determination of hydrolysis activity, the insoluble portion of flour is hydrolyzed from disposed, deproteinized and husked soybeans under standard conditions. The enzyme reaction is quenched with an interrupting reagent and the insoluble portion is filtered off. The amount of dissolved polysaccharides is determined spectrophotometrically after acid hydrolysis in accordance with AS 169/1, available from NOVO INDUSTRI A / S 2880 Bagswaerd.

According to the method, carbohydrates with endo- as well as exo-activity are determined.

The substrate for this enzyme determination is identical to the substrate residue described for the SRU method. Dissolve the substrate as a 3% solution in the citrate buffer below:

0.1 N citrate phosphate buffer pH 4.3

5.24 g citric acid monohydrate (Merck Art 244)

8.12 g of disodium hydrogen phosphate dihydrate (Merck Art 6580) demineralized water to 1 1 pH 4.5 + 0.05 stability for 14 days

5. Η. 92 -Μ;? - The interrupting reagent has the following composition:

100 ml of 0.5 N NaOH

Store 200 ml of 96% ethanol in the refrigerator until use.

Standard conditions temperature ................. 50 ° C pH .......................... 4.5 reaction time, sample ....... 120 minutes

- and - control * ·· «· 5 minutes.

Unit definition

One unit of solubilizing activity for soybean residues (SRUM) 120 (M for manual) is the amount of enzyme which, at the indicated reaction conditions, releases solubilized polysaccharides equivalent to one micromole of galactose per minute.

5. r / ₉₂

CHAPTER 10 c

ENZYME DETERMINATION OF FROTEOLITE ACTIVITY

HUT MEASUREMENT

Method for the determination of proteinase in acidic medium.

The method is based on 3θ minute digestion of denatured hemoglobin with the enzyme at 40 ° C and pH 3.2. Undigested hemoglobin was precipitated with 14% trichloroacetic acid (w / v%).

All enzyme samples were prepared by dissolving them in 0.1 M acetate buffer, pH 3.2.

The hemoglobin substrate is prepared using 5 0 0 g of lyophilized bovine hemoglobin powder, preserved with 1% Thiomersalate and 100 ml of demineralized water, which is stirred for 10 minutes, then adjusted to pH from 0.33 H HCl to 1.7.

After further 10 minutes of stirring, the pH was adjusted to pH 3.2 with 1 N NaOH. Increase the volume of this solution with 0.2 M acetate buffer per 200 ml. This hemoglobin substrate should be refrigerated for 5 days.

The hemoglobin substrate is brought to room temperature.

At time 0, a tube containing 1 ml of Bciam »5 ml of substrate is added. After shaking for 1 second, place the tube for 30 minutes in a 40 ° C water tank. After exactly 3θ minutes, 5 ml of 14% trichloroacetic acid was added to the tube and then shaken and brought to room temperature for 40 minutes.

For control, bring the hemoglobin substrate to room temperature ² ≤ 92. At time O, 5 ml of substrate is added to a tube containing 1 ml of enzyme. After shaking for 1 second, place the tube in a tube of 5 ml of 14% trichloroacetic acid, and then shake and return to room temperature for 40 minutes.

After 40 minutes, the control and samples are shaken, filtered once or twice through a Berzelius filter no. 0 and placed in a spectrophotometer. The sample was read against the control at 275 nm, and the spectrophotometer was adjusted against water.

As the tyrosine absorbance at 275 nm is a known factor, it is not necessary to determine the tyrosine standard curve unless necessary to control the Beckms spectrophotometer.

Calculation

HUT is the amount of enzyme that forms a hydrolyzate in 1 minute, which in its absorbance at 275 nm is equivalent to a solution of 1.10 / Ug / ml tyrosine in 0.006 N HCl. This absorbance value is 0.0084. The reaction should be carried out within 30 minutes at 40 ° C and pH 3.2.

ΗϋΦ _ Sample-Control _χ vol, in ml. _ _{___}

0,0084 reaction time in min

HUT =

Sample-Control

0,0084

X ji = (V-K) x 45.65

HUT / g enzyme = ·, ^& g enzyme in 1 ml

5. fi 32

- 44 The fresco dependence of the ^ pH stability of the protease in KRF 68, performed by means of HUT analysis with pH values from 2.0 to 8.0, showed that the stability of the protease above pH 8.0 was very low, see Fig. 16.

- 45 To illustrate the invention, reference is made to the following Examples 1 to 8, which illustrates Example 1 production of SPS aase and where Examples 2 to 8 illustrate the use of SPS aase on a soybean-based feedstock to produce purified plant protein. Other uses of SPSase are mentioned in the section between Example 8 and the overview of the figures.

Several fermentations were performed with the Asp strains listed here. aculeatus and Asp. japonicus on a laboratory scale. Preparations containing SFS-aza in accordance with the test for SPS-aase mentioned herein were obtained. However, since rather large amounts of SPS aase are required to perform the use trials, similar fermentations were performed at the pilot plant scale, see the following example 1.

EXAMPLE 1

Pilot plant production of SPS.

SPS azo was prepared by submerged fermentation of Aspergillus aculeatus CBS 101.43.

An agar substrate was prepared in Pernbach flask with the following composition:

Pepton Difco 6 g

Aminolin Ortana 4 g

Glucose 1 g

Difco yeast extract 3 g

Difco meat extract 1.5 g

KH ₂ F04 Merck 20 S Sweet extract Evers 20 S Ionic exchanged H ₂ c ad 1000 ml. We are pH balanced on 5 » 50 to 5,55 . We then added

g of Difco agar and the mixture was autoclaved for 20 minutes at 120 ° C (the substrate is called E-agar).

The CBS 101.45 strain was cultured on slanted E-agar (57 ° ^C ). The spores from the slanted medium were suspended in sterilized skim milk and the suspension lyophilized in bottles. The contents of one freeze-dried bottle were transferred to a Fernbach flask. The flask was then incubated for 15 days at 50 ° C.

A V050® liter inoculation fermenter was prepared with the following composition:

CaCO ^ 1.2 kg

Glucose 7> 2 kg

Sofec (corn macerate dry matter) 5.6 kg

Soybean oil 1.2 kg

We added tap water to the total volume around

240 liters. Fred was adjusted to pH 5 5 5 5 Sub5 str by the addition of CaCO by sterilization in an inoculation fermenter for 1 hour at 521 ° C. The final volume before inoculation was about 50θ liters.

The spore suspension from the Fernbach flask was transferred to an inoculation fermenter. The conditions of inoculation fermentation were:

5. 11 92 - 4? Fermenter type: conventional stirring and ventilation ferroenter, height / diameter ratio about 2.3

300 rpm (two turbine mixers) 300 normal liters of air per minute 30 to 31 ° C

1.5 bars for about 28 hours.

Mixing: Aeration: Temperature:

Pressure:

Time:

About 28 hours after inoculation, 150 liters were transferred from the inoculation fermenter to the main fermenter.

A substrate with the following composition was prepared in a 2500 liter main fermenter:

Roasted soybean meal 90 kg

KH ₂ P0 ₄ 20 kg

Pluronic® 150 ml

We added tap water to the total volume around

900 liters. The soybean flour was suspended in water. The pH was adjusted to 8.0 with NaOH and raised to 50 ° C.

Thereafter, about 925 Anson ALCALASE® 0.6 L units were added to the suspension. The mixture was maintained for 4 hours at 50 ° C and pH = 8.0 (addition of Na 2 CO 4) without ventilation, without overpressure, and stirred at 100 rpm./ mi: We then added the remaining constituent constituents and adjusted the pH with phosphoric acid to about 6.0. The substrate was sterilized in the main fermenter for 123 hours at 123 ° C. Final volume before

- 48 * 4 ίί. 92 inoculation was about 1080 liters.

Then, 150 liters of inoculation culture was added. The fermentation conditions were:

Type of fermenter: conventional fermenter with aeration and mixing, height / diameter ratio about 2.7. Mixing: 250 rpm (two turbine mixers) Radiation: 1200 normal liters of air at a minute Temperature: 30 ° C Pressure: 1.5 bar time: about 151 hours. Between 24 hours fermentation to about 116 yes? fermentation

we aseptically added to the main fermentor a pectin solution at a constant rate of about 8 liters per hour. The pectin solution of the following composition was prepared in a 500 liter dosage container:

-. . x) rectm genu ^J 22 kg Phosphoric acid, conc. 6 kg Pluronic ® 50 ml

^χ ) Genu pectin (citrus type NF by The Copenhagen pectin factory Ltd.)

Tap water was added to a total volume of about 325 liters. The substrate was sterilized in a dosing vessel for 1 hour at 121 ° C. The final volume before dosing started was around

- 49 360 liters. When that portion went, we made another similar portion. The total volume of the pectin solution for one fermentation was about 725 liters.

After about 151 hours of fermentation, the fermentation process was stopped. About 1850 liters of culture broth were cooled to about 5 ° C and enzymes were prepared according to the following method.

The culture broth was drum-filtered on a vacuum drum filter (Dorr Oliver), which was previously coated with Hy-flo-super-whole diatomaceous earth (filtered device). The filtrate was concentrated by evaporation to about 15% by volume of the culture broth. The concentrate was filtered on a Seitz filter (supra 100 type) with 0.25% Hy-flo-super-whole as a filter aid (called filtration I in the following table). The filtrate was precipitated with 561 g (NH2), / 30 ^ / 1 at pH 5.5, and 4% Hy-flo-super-whole diatomaceous earth was added as a filter aid. The precipitate and filter aid are separated by filtration on a filter with a frame. The filter cake is dissolved in water and the insoluble parts are separated by filtration on the frame filter. The filtrate was filtered under control on a Seitz filter (supra 100 type) with 0.25% Hy-flo-super-whole as a filter aid (called filtration II in the following table). The filtrate was filtered off on an ultrafiltration apparatus. After diafiltration, concentrate the liquid to a dry matter content of 12.7 ° / ° (called the dry matter content of the concentrate in the following table).

At this stage, facultative base treatment can be performed to partially remove protease activity. In order to use basic treatment, it is carried out at pH 9.2 for 1 hour and then adjusted to pH 5.0.

Now the liquid is filtered and filtered to minimize germs, and the filtrate is lyophilized on Stokes freeze drying equipment.

Four fermentations were performed in the manner described below, and, as indicated in the following table, the strain used for fermentation, the use of facultative basic treatment and other parameters were varied.

Concentration (%) of filter aid in

Proportion of dry sno-

related s you v code con- Basic processing fil- obar- fil- cen- up- neupo-  pnp- gossip- • Jan- gossip- tra- Note · Used microorganism used straight jo I i eat jo II there be CBS 101.43 X KRF 68 0.5 5 0.2 28 ATCC 20236 X KRF 74 2.0 4 0.4 7.5 IFO 4408 X KRF 83 1.0 5 0.25 12,4 x) CBS 101.43 x KRF 92 0.25 4 0.25 12.7

X *) ^J To concentrate the filtrate to reduce the call, concentrate the filtrate by evaporation in a ratio of 1: 2.3. A small portion of the concentrated filtrate is spray dried and the remainder lyophilized.

To further reduce protease activity, some of the above preparations were treated as indicated below, using only one of the three alternatives A, B, and C.

11. 5. 92

A. Dissolve 100 g of the SFS-aase preparation in 1 liter of deionized water while stirring at 10 ° G + 2 ° C. pH was adjusted from 4 N NaOH to 9.1 · This basic treatment was carried out for 1 hour. The pH was then adjusted to 4.5 with glacial acetic acid and dialyzed against ice-cold, deionized water to a conductivity of 3 mS. If we freeze and lyophilize.

B. Dissolve 5θθ g of the SFS-aase preparation in 4 liters of deionized water while stirring at 10 ° G + 2 ° C. pH was adjusted from 4 N NaOH to 9.1 · This basic treatment was carried out for 1 hour. The pH was then adjusted with glacial acetic acid to 5.0. The resulting material was lyophilized.

C. Dissolve 50 g of SFS-8ze in 400 ml of deionized water while stirring at 10 ° C + 2 ° C. pH is adjusted from 4 N NaOH to 9.1 »

This basic processing is performed for 1 hour. The pH was then reduced with glacial acetic acid to 5.7 · The resulting material was lyophilized.

SPS-aze preparation used as starting material for basic processing Basic processing applied Product code A B C KRP 68 X KRP 68 Bill KRP 68 X KRP 68 BIH KRP 92 X KRP 92 BI

The above preparations are indicated by their enzyme activities which are important for the invention in the following table.

5.! 5. 92

Η fO CM Yes fc < + Oh m Γ n r- Oh m o »H r * cn n CM cn Oh m 7600000 Oh Oh CO., LTD CD ID Oh o CM VD s o YES Oh o o CM • Wed. C CM YES δ X + \ o r> kD CM Oh Oh ΧΓ ^ 0 rH o co Yes and o o Γ * n o o o o o xr ω Oh Oh Oh o ID o o o 'T -T o o o »—1 o o o o o r-i rH CO., LTD 00 δ « + co <£> r — 1 n CD ID CO., LTD n r- o z> CD CM and — 1 Oh o 00 o o o o o m r- Oh o «<3 * <0 o o o Γ— CM m o YES ID Oh Oh Oh Oh o CM CM • Μ · Γ- Μ I o CM r — 1 CD r- and Oh CD ID • H o CM m r — i o o o o D · CD o o rH -r o m r-i un r-ί Oh n- CO., LTD Oh Oh Oh and AD Η Η Η 03 CO., LTD d fc. « Κ + ΟΊ m 1-1 co • sr o CM Γ i—! Yes n n <O σ> co σΐ O o o o o. CD CO., LTD o o Γ- Γ- r- o o Yes AD Γ- o r ~ r ~ o o o o o o rH Η Η m CD ΑΟ & 4 K K + “—And oh CO., LTD o n Oh D m r-t and o and — 1 •? R o 00 Oh o o o o o YES o o o CM r ~ o o r- ~ n CD o rH YES Oh Oh Oh Oh o rH rH CD MO + Oh and CD γ- γο C and CM rH CM o o o c- ID o o o CD o o o o o CO., LTD Oh rH Oh o σ \ rH i-H o o rH CD Γ * Oh • 3 · CO., LTD Oh o o o o AD rH • P CD CQ 2 00 0) • rl KJ > ra o rH P « CD P P 00 Φ EH • rl £ • H -P CD +3 • rl -P P CD £ Oh CM CM n m X ·· s • rl -rl bO O -P P rM CD H rod w < ω X Would W rH 2 x Would CO., LTD a E-i x X X in X B w 0 B ω H B X M W B X at ⁵

11. 5. 92

-5Φ EXAMPLE 2 (use case),

This example describes the production of p.v.p. from husked and ground soybean meal, Soyamel 13 (commercially available from Aarhus Oliefabrik A / S). The dry matter content of this flour was 94.0% and the dry matter basis (N x 6.25) was 58.7%. Soybean meal was treated with the preparations of SPS-aza KRP 68 Bil (Example 1) as follows:

85.2 g of soybean meal were suspended and stirred at 50 ° C in 664.8 g of water, and pH 7 was adjusted to 4.5 with 7 5 5 ml of 6 N HCl. A 50 S solution containing 4.00 g of said SPS-aase preparation was added and the reaction mixture was then stirred for 240 minutes at 50 ° C. The mixture was then centrifuged on a laboratory centrifuge (Beckman Model J-6B) for 15 minutes at 3000 x g. The supernatant was weighed and analyzed on Kjeldahl N and dry matter. The solid phase was then washed with a volume of water equivalent to the mass of the supernatant obtained in the first centrifugation. We performed this operation twice.

The solid phase was then lyophilized, weighed and analyzed for Kjeldahl N and dry matter at Qvist's Laboratorium,

Marselis Boulevard 169 »8000 Aarhus C, Denmark. This laboratory is authorized by the state for the analysis of feed and dairy products. The results obtained from the experiment are shown in Table 2.1:

5. H, g2

- 54 Table 2.1 Results obtained

Ingredient Mass ⁶ N x 6.25% Dry matter % Profit protein % Dry matter yield % Soybean meal 85,2 55,2 94.0 100% 100% Preparation 6.4% SPS aze 4,00 75,6 - - 1. Centrifuge 666 1.50 5.04 21.2% 42,0% p.v.p. 44.5 87.5 95,7 82.7% 53,2% So we got p .v.p. with protein purity , that is

N x 6.25 on a dry matter basis, 91.4% and a total protein yield of 85%.

HRIMER 5 (use case)

This example was performed to compare protein yields, nutritional quality, and some functional properties of soy protein products made using the following three procedures:

A: Traditional isoelectric precipitation for the production of soy protein isolate.

B: Traditional isoelectric rinsing for the production of soy protein concentrate.

C: Isoelectric washing according to the invention, which includes an enzyme for solubilizing the residue, for the production of p.v.p

5. II. 92

- 55 In order to truly compare the process according to the invention (C) with the conventional soy protein (A and B) methods, the same raw material was used in all three cases.

The experiments were also conducted in such a way that the corresponding temperatures and processing times were the same in all three cases.

Only the pH values were different due to the fundamental differences between the three processes.

A. Traditional isoelectric precipitation for the production of soy protein isolate

425.8 g of soybean meal (Soyamel 13, manufactured by Aarhus Oliefabrik A / S) was extracted into 3574.2 g of tap water at 50 ° C. The pH was adjusted from 20.1 g of 4 N NaOH to 8.0. After stirring for 1 hour, the slurry was centrifuged at 3000 xg for 15 minutes in a laboratory centrifuge (Beckman Model J-6B) using 4 one-liter beakers. The centrifuge I and the precipitate I were weighed. The precipitate I was again extracted with water to a total mass of 4000 g. The temperature was maintained at 50 ° C, the pH was adjusted from 4 N NaOH to 8 and the slurry was stirred for another 1 hour. As above, centrifugation and weighing of centrifugate II and precipitate II were performed. Samples for Kjeldahl and dry matter determination were collected from centrifuges I and II and precipitate II. The centrifuges I and II were then mixed and maintained at 50 ° C. The protein was then precipitated isoelectrically at pH 4.5 with 45 g of 6 N HCl. For 1 hour stirring at 50 ° C, the protein was obtained by centrifugation at 3000 xg for 15 minutes. Centrifugate III was weighed and analyzed 5. The 32-56 centrifugate was weighed on Kjeldahl N and ₍ dry substance. Solid phase III was weighed and washed with water in an amount corresponding to the weight of the centrifugate I. The rinsing was carried out by stirring at 50 ° C for one hour. The washed protein was obtained by centrifugation at 5000 xg for 15 minutes Centrifugate IV and solid phase IV were weighed Centrifugate IV was analyzed for Kjeldahl N and dry matter The solid phase was suspended in 1550 g of water at 50 ° C and pH adjusted 17 g of 4 ^ N NaOH at 6.5 · The mixture was stirred for 1 hour and re-adjusted to pH = 6.5 if necessary. Finally, the product was lyophilized, weighed and analyzed for Kjeldahl N and dry matter. in Table 5 · 1 ·

- ^r 57 Table 3 · 1 Calculation of the mass balance of traditional isoelectric precipitation for the production of soy protein isolate.

Operations and fractions Mass fractions Protein Dry Substance sr % Profit 1 protein% Profit dry matter% Mr Rücker % (Nx6.25) Extraction: Soybean meal 425,8 55,2 94.0 100,0 100,0 Water 5574,2 0 0 0 0 4 N NaOH 20,1 0 16.0 0 0.8 1. Centrifugation: 4020,1 . 5.9 10,0 100,9 100,4 Centrifuge I 3141,0 4.4 6,9 58,8 54,1 Precipitation I 805,0 - - Extractions again: • Precipitation I 805,0 - - - - Water 3195,0 0 0 0 0 2. centrifugation: Centrifugst II 3104,0 0.5 0.9 6.6 7.0 Precipitation II 820,0 9.1 17.2 31.7 35,2 Mixing and acidification: I + Centrifuges II 6245,0 - - - - 6 N HC1 45,0 0 21.3 0 2.4 3. centrifugation: Z 6290,0 Centrifuge III 5650,0 0.3 1.9 7.2 26,8 Precipitation III 308,0 - Rinse: - - ] Precipitation ΪΙΙ 308,0 - - - - Water 3141,0 0 0 0 0 4. centrifugation: Σ 3449,0 Centrifuge IV 3113,0 0.04 0.15 0.5 1,2 Precipitation IV 291,0 - - Neutralization: Precipitation IV 291,0 Water 1550,0 0 0 0 0 4 N NaOH 17,0 0 16.0 0 0.7 Drying: Powder 128,0 93,8 96.3 51,1 30,8

5, Η. 92

- 58 B. Isoelectric rinsing for the production of soy protein concentrate

425.6 g of soybean meal (Soyamel 13, manufactured by Aarhus Oliefabrik A / S) was washed in 3574 g of water at 50 ° C. The pH was adjusted to 4.5 with 44.8 g of 6 N HCl. The rinsing was performed for 4 hours with stirring. The slurry was then centrifuged at 3θθθ x g for 15 minutes in a laboratory centrifuge (Beckman Model J-6B) using 4 one-liter beakers. Centrifugate I was weighed and analyzed on Kjeldahl N and dry matter. The solid phase I was weighed and washed again with water to a total mass of 4000 g.

With 1.7 g of 6 N HCl the pH was adjusted again to 4.5 and the slurry was stirred for an additional 3θ minutes at 50 ° C. As above, centrifugation and weighing of centrifugate II and solids II were performed. Solid phase II was resuspended in 1575 g 1 ^ 0 with 50 ° C and s

34.5 g of 4 N NaOH were adjusted to pH 6.5. The mixture was stirred at 50 ° C for 1 hour and adjusted to pH = 6.5 if necessary.

Finally, the protein product was lyophilized, weighed and analyzed for Kjeldahl N and dry matter. The mass balance is shown in Table 3.2.

- 59 Table 3 · 2 Calculation of isoelectric leaching mass balance for the production of soy protein concentrate

Operations and fractions Mass fractions Protein Dry substance % Profit protein % Dry matter yield % S % (Nx6.25) Rinse: Soybean meal 425,8 55,2 94.0 100,0 100,0 Water 3574,0 0 0 0 0 6 N HCl 44,8 0 21.3 0 2.4 1. centrifugation: 4044,6 3150,0 Centrifuge I 0.6 3.2 8.0 25,2 Solids I 846,0 - - - Backwash: Solids I 846,0 - - - - Water 3154,0 0 0 0 0 6 N HCl 1.7 0 21.3 0 0.1 2. Centrifugation: 4001,7 Centrifuge II 3130,0 0.1 0.4 1.3 3.2 Solids II 863,0 - - - Neutralization: Solids II 863,0 - - - - Water 1575,0 0 0 0 0 4 N NaOH 34.5 0 16.0 0 1.4 Drying: Powder 281,0 72.5 98.4 86,7 69,1

² 5. II. 32 - SOi *.

C. Isoelectric flushing, including an enzyme for solubilizing the residue, to produce p.v.p.

425.8 g of soybean meal (Soyamel 13, manufactured by Aarbus Oliefabrik A / S) were washed in 3524.2 g of water at 5O ° C. The pH was adjusted to 4.5 using 43.7 g of 6 N HCl. 24 g of the KRP 68 BIH SPSase preparation (Example 1) was solubilized in 26 g of water and added to the spiral mixture. The rinsing was then performed for 4 hours with stirring. Then, purification was performed as described for B, with the only parameters with different values of the amount of 6 N HC1, 4 N NaOH and water for resuspension. The mass balance is shown in Table 3 · 5 ·

5. II. 32 - 61 Table 3 · 3 Calculation of the mass balance of isoelectric washes, including an enzyme for solubilizing the residue, for the production of p.v.p.

Operations and fractions ilasa fractions Protein Dry substance % Profit protein % Profit dry matter% 6 % (Nx6.25) Rinse: Soybean meal 425,8 55,2 94.0 100,0 100,0 Water 5540,2 0 0 0 0 6 N HG1 45,7 0 21.3 0 2.3 SrS-azaiKSP 68 Bill 24,0 75,3 96.0 7.7 5.8 1. centrifugation: 4043,7 - - - Centrifuge I 3420,0 1.7 5.2 24.7 44,4 Solids I 620,0 - - Rinse: Solids I 620,0 - - - - Water 3380,0 0 0 0 0 6 N HCl 1.3 0 21.3 0 0.1 2. centrifugation: 4001,3 Centrifuge II 3400,0 0.2 0.6 2.9 5.1 Solids II 577,0 - Neutralization: Solids II 577,0 - - - - Water 1700,0 0 0 0 0 4 N NaOH 25.5 0 16.0 0 1.0 Drying: Powder 211,0 87,3 ^ 96.7 ^Ό 78,2 51,1 86,9 ¹⁾ 97, ² '

1). .

Analyzed at the Biotechnical Institute, Holbergsvej 10, DK-6000 Kolding, Denmark

p)

Analyzed in Qvist's Laboratorium, Marselis Boulevard 169, DK-8000, Aerhus C, Denmark

Nutritional properties

The amino acid compositions of the three protein products were determined, see Table 3-4. Using the 1957 FAO reference sample, the total essential amino acid content, chemical value, and essential amino acid index (EAAI) are calculated.

The trypsin inhibitor content of all three products was determined using the method described in A.O.G.S. Tentative Method Ba 12 - 75 (A.O.G.S. is an abbreviation for the American Oil Chemists' Society). The results are shown in Table 3.5, which also includes the yields and the protein / dry ratio of all three products.

5. It 32

- 63 Table 3.4 Amino acid composition and nutritional assessment of the three protein products A, B and C.

Amino acids A. Sojin,; pro.tein-B. Soy protein- C.-Soy protein isolate '(n.v. p.) ski. cijzo ^ at ski concentrate g / 1 6 g N 1). aas g / 16 g n 1) aas g / 1 6 g N 1) aas Non-essential έιΐϊίηέ ¹¹⁸ ^ · ⁸ 12,4 - 11.3 - 11.9 - Serini 4,62 - 4.69 - 4.81 - Glutamine acid 21.3 - 18,2 - 17.7 Prolin ' 6.07 - 5.19 - 4.76 - Glycine 4.13 - 4.26 - 4.33 - Alanin 3.54 - 4.27 4,55 - Histidine 2.83 - 2.78 - 2.50 Arginine 8.09 - 7.57 - 7.04 - Essential Isoleucine 4.87 > 100 4.97 > 100 5.19 > 100 Levcin 7,80 > 100 7.98 > 100 · 8.09 > 100 Lysine 6.24 > 100 6.09 > 100 5.57 > 100 Phenylalanine 5.47 > 100) 5.35 > 100) 5.17 > 100 ·) /> 100 ^> 100 4,44 /> 100 Tyrosine 3,38 > 100) 3.88 > 100) > 100) Gistin 1.29 64,5} 1.32 66,0} 1.44 72,0) Me you they n 56,4  6C £ 7 65.5 1.08 49, lJ ' 1.21 55,0. 1.31 59,5J ' Threonine 3.10 > 100 3.60 > 100 3.97 > 100 Tryptophan, 1.06 75,7 1.37 97, 9 1.32 94,3 Valin 4.90 > 100. 5,23 > 100 5.57 > 100 Ίο the entire de- lies the essential them amino 38, 36 41 , 31 42.21 acids Chemical value 56, 4% 69.2% 65,5% EAAI 86, 7% 90 , 2% 91, 3%

aas = amino acid value based on reference sample ΈΆΟ (1957)

1)

- 64 Table 5-5 Process Characteristics and Trypsin Inhibitor Proportion of the Three Protein Products A, B, and C.

A. Soy protein- B. ski isolate Soy protein concentrate C. Soy Protein Isolate (p.v.p. Process characters - ^dry 97.4% 75.7% 90.0% ristics Win protein 51.1% 86.7% 78.2% Trypsin TUI / g product inhibitors 54,000 21,000 19,000

TUI / g protein

56.250

28.970

21,810 th most common

Functional properties

The nitrogen solubility index (USI) was determined in 1% protein dispersion at pH = 7 »θ in 0.2 M NaCl or. in distilled water. After 45 minutes of magnetic stirring, the suspension centers were grouted at 4000 x g for 30 minutes and the supernatant analyzed for nitrogen. Nitrogen solubility was calculated as (soluble N% / total N%). The results of this evaluation of all three products are shown in Table 3 · 6 ·

The emulsifying ability was determined by slightly modified Swift titration on each product three times. 4.0 g (N x 6.25) of the product were mixed with Sorval Omni mi xrp at low speed in 250 ml of 0.5 M NaCl. 50 ml of the suspension was transferred into a glass mixing glass and 50 ml of soybean oil were added and the whole mixture was weighed. The mixture of oil and water was then homogenized at 10,000 rpm, leaving the glass in an ice bath. Then, at a rate of 0.3 ml / sec. add extra soybean oil until the emulsion has collapsed. The total amount of oil added before the “end point” was determined by weighing.

Emulsifying capacity was calculated as ml of oil per gram of protein (N x 6.25). The oil density was taken as 0.9 g / ml.

- 66 '-

The average results of determining the emulsifying capacity of the three products are shown in Table 3.6.

*

Expansion by whipping was determined in 3% protein solution at pH = 6.5 · 250 ml of aqueous dispersion of protein samples was whipped at a rate of III for 4 minutes in a Hobart mixer (model N-50) with a mounted wire broom. Expansion at whipping was calculated in accordance with the formula expansion at whipping = ^x 100%, where V is the final volume in ml.

V was measured by refilling the mixing glass with water. Two determinations were made for each of the three samples. The average results are shown in Table 3 · 6.

Foam stability was defined as the ratio of the amount of foam remaining after 3θ minute drainage to the original amount of foam. A gram of foam produced by the above method was placed in a plastic cylinder (diameter 7 o "height 9 cm) with a wire mesh with a mesh size of 1 mm x 1 mm. The rolls were placed on the funnel at the top of the glass cylinder and the mass (B) of the drained liquids in the glass cylinder was determined. The foam stability of FS is defined by the equation fs = —A- = A— _{x 1O} o%

A

The results of the determination are shown in the table

3.6.

Gel strength is defined in this description as Brookfield viscosity, as measured by a T-spindle on a Brookfield Helipath stand. Gels were prepared by heat treatment of a 12% protein suspension in 0.5 M NaCl. The heat treatment was carried out in sealed cans with a diameter of 7, 3 cm and a height of 5, θ cm, which were placed in a water bath maintained at 80 ° C and 100 ° C each time for $ 0 minutes. The cans were cooled and thermostatically cooled to 20 ° C before being opened and measured. The measurement results are shown in Table 3 * 6.

Table 3 · 6 Functional properties of the three protein products A, B, and C.

Functionality A. Soy Protein B. Soy Pro- C. Soy

isolate teinski concentrate proteii ski izc lat (p.v.p.) % NSI in 0.2 M NaCl 39.5 20.3 25.6 % NSI in water 53,9 25.1 28.6 Emulsion capability: ml Oil / g (N x 6.25) 218 182 354 Expansion at stepping% 120 120 340 Foam stability% 50 50 20 Gel strength; [Pa.s] 80 ° C (0.5 M NaCl) 1.7 χ 10 ² 1.2 χ 10 ⁵ 3.3 100 ° C (0.5 M NaCl) 2.0 χ 10 ⁵ 4.0 χ 10 ⁵ 1.3 x 10 *

5. II. $ 32 - $ 6 -

EXAMPLE 4 (use case)

We prepared pvp according to the mode of operation described in Example 3θ, except that cellulase activity originated in part from Trichoderma reseei. The CELLUCLAST cellulase preparation prepared by Novo Industri A / S was treated with a low temperature base as follows. The pH of the 10% solution of CELLUCLAST in water was adjusted with NaOH to 9.2 and the solution thus obtained cooled to 5 ° θ · After 1 hour at this pH and at this temperature the pH was adjusted again with 20% acetic acid. at 4.7. This solution was stored overnight at 5 ° ^G and then sterile filtered. The filtrate was lyophilized. 4 g of lyophilized product was added to the preparation of SES-aase KRP 68 BIH (Example 1).

Both enzymes were solubilized in 172 g of water before being added to the rinsing mixture. The mass balance determinations of this example are shown in Table 4.1.

The experiment demonstrates that this particular SPS-aase preparation already contains effective cellulase, since the addition of GELLUOLAST clearly does not affect the protein / dry ratio. However, they may contain other SPSase preparations, e.g. KRP 92, less cellulase; see table just before example 2.

^{2 ;;} · Π, 92

Table 4.1 Determinations of the mass balance of iso-electric flushes, including the preparation of SES-aza and CELLUCLAST ®, for the production of p.v.p.

Operations and fractions Mass fraction gram Protein % .0Lx.6.25) Dry matter % Profit protein .......% .......... Dry matter yield .....%. Rinse: Soybean meal 425,8 55,2 94.0 100,0 100,0 Water 3546,2 0 0 0 0 6 N BOI 43,1 0 21.3 0 2.3 SES: KRE-68-B-III 24,0 75,3 96 7.7 5.8 CELLUCLAST 4.0 43,6 96 0.7 - 1,0 Centrifuges, wound: 4043,1 - Centrifuge I 3382,0 1.9 5.5 27.3 46.5 Solids I 661,0 - Rinse: Solids I 661,0 - - - - Water 3339,0 0 0 0 0 6 N BOI 0 0 0 0 0 2, centrifuge- 5 ~ grinding: 4000,0 Centrifuge II 3414,0 0.2 0.7 2.9 6.0 Solids II .....- ^ 8.2xQ_ «M - - -

S. r <;

- 70 - Continued of Table 4.1 Neutralizations: Solids II 582, 0 - - - Water 1691,0 0 0 0 0 Zl W 25.3 0 16.0 0 1.0

Drying:

Dust_206.0 88.8 98.9 77.8 50.9

EXAMPLE 5 (use case)

We prepared p.v.p. according to the method described in Example 3C, except that all masses were reduced by a factor of 5 and the reaction mixture was cooled to about 5 ° C before centrifugation. Based on the analytical results compared to the centrifuges, the theoretical yield of the precipitated protein was obtained, as shown in Table 5 · 1 ·

Table 5 · 1 · Theoretical protein gains achieved during production

p.v.p.

Fractions Mass Mr Rücker Protein (H x 6.25) % Protein yield % Example 3 0 Protein (H x 6.25) % Profit protein, % Soybean meal 85,2 55,2 100 55,2 100 SPS aza KRP-68-B-III - 4,8 75,3 .. 7.7 75,3 7.7 1st centrifuge 639 0.99 13.5 1.7 24.7 2nd centrifuge 595 0.13 1.6 0.2 2.9 p.v.p. 87,2 ^a 92.6 ^b 87,1 80,1 ¹³

5. ίί. 92

-ffEP average of 87.5 (Biotechnical Institute) and 86.9 (Qvist's Laboratory); the dry matter is 97 »6 oz. 98.0%,

b) Calculated as total weight of protein - protein lost in centrifugatib.

Proof of protein binding to SPS g (N x 6.25) of soybean protein isolate market (Ralston Purina Purina 500 E) was dissolved in equilibration from 6 N HCl to 4.50. 90 g of this mixture was transferred to five 250 ml Erlenmeyer flasks and 0 g, 0.2 g, 0.4 g, 0.8 g, respectively. 1.6 g of SPS produced as described earlier in this specification. The flasks were then maintained under magnetic stirring in a water bath for 240 minutes at 50 ° θ.

The slurries were then centrifuged at 5000 x g for 15 minutes and centrifugates I were analyzed for Kjeldahl N and dry matter. The solid phases were washed in water at room temperature and centrifuged again. We repeated this process. The solids were then dispersed in 50 ml of water and the pH adjusted to 6.50 by dropwise addition of 6 N NaOH. The neutralized products were lyophilized and analyzed for Kjeldahl N and dry matter. Based on the analysis shown in Table 6.1, we have used the formulas shown in Table 6.1

5. 9?

-72link to Table 6.2, calculate protein gain and percentage of SPS bound to protein. '

This example demonstrates that the SES is firmly bound to the protein in such a way that the increasing proportion of SPS decreases the protein / dry ratio. The proportion of SPS comparable to about 0.4 g in 10 ml of water added to the 5 S protein isolate is the protein / SPS ratio present in soybean meal.

% SPS binding is the calculated value. % SIS binding decreases due to protein saturation relative to SPS at low protein / SPS ratios.

Table 6.1 Measurements according to Example 6

The ratio protein / SPS Centrifuges I Dried precipitation % N % dry matter % N x 6.25 % dry «/ N x 6.25 substances ^Λ dry sno-v CX) 0.068 0.62 13.2 82.5 93,1 88.6 25 0.045 0.49 13,4 83,8 97.3 86,1 12.5 0.038 0.45 13,0 81,3 97.9 83,0 6.25 0.031 0.45 12.6 78,8 98.1 80,3 3,125 th most common 0,026 th most common 0.61 11,8 73,8 97.9 75,3 Table 6.2 Protein yield and% of the SPS The ratio % protein gain % SPS binding protein / SPS OO 91,5 0 25 94,4 77 12.5 95,3 90 6.25 96.1 70 3,125 th most common 96.8 60

1) ^J % protein gain gg 1 =% N in the centers of fugate I _η . gg V 6.25 5 x 100 where

2)% SPS binding

X (% protein gain) 5 x (% protein gain) ^L 1¾P / 5) _ (% P / B) ^ _ J [5 / SPS ratio] x 100, where (% P / H) is protein / dry ratio in the dried precipitate and (% P / H) is for the precipitate without the addition of SPS.

- 74 EXAMPLE 7 (use case)

This example describes the production of p.v.p. using a SES aase preparation KRF 92 B-I at a dose of 5% dry matter. The mode of production was exactly as in the case of 3 C, except that we reduced all masses by a factor of 5 · P.v.p. were analyzed as described in Example 2. The results obtained in the experiment are shown in Table 7.1.

Table 7 · 1 Results obtained in Example 7

Ingredient Mass S (N x 6.25) Dry matter %% Profit protein, % Dry matter yield, % Soybean meal 85,2 55,2 94.0 100 100 An enzyme preparation 4.0 71,2 - 6,1 - 1st centrifuge 632 1.88 5.44 25.3 43,0 2. centrifuge 673 0.30 0.80 4.3 6,7 p.v.p. 39,8 85, e ^a 98.1 ^a 71,9 48,8 84.4 ^b 98.1 ^b

^a Analyzed at the Biotechnical Institute, Holbergsvej 10, DK-6000 Kolding ^b Analyzed at Qvist's Laboratorium, Marselis Boulevard 169, DK-8000 Aarhus C

5. ίί 9?

EXAMPLE 8 (use case)

This example demonstrates the effect of pre-treatment of soybean meal by cooking in a steam ejector before production

p.v.p.

Pre-treatment

Soybean flour in water containing 10 kg of soybean meal (Sojamel 13 »manufactured by Aarhus Oliefabrik A / S) per 100 kg was pumped through a steam ejector (type Hydroheater B-300) and mixed with steam of 8 bars in such quantity and with a flow rate such that the final temperature of 150 ° 0 was maintained in the tube pressure reactor for 25 seconds. The pressure was then reduced in the decompression chamber (cyclone) and from there the slurry was passed through a plate heat exchanger and cooled to about 50 ° C. The cooled slurry could be used in accordance with the invention directly for the production of p.v.p., but in this case the slurry was spray dried at an inlet temperature of 200 ° 0 and an outlet temperature of 90 ° 0. The pre-treated product was found to have a solids content of 96.5% and a protein content of 56.9% (N x 6.25).

The preparation of the p.v.p.

We prepared this preparation as follows:

g of dry matter in the steam ejector of boiled and dried soybean meal was suspended and stirred at 50 ° δ in 560 g of water and adjusted to 4.50 with 6.5 ml of 6 N HOl. 6 x 90 g of this suspension were transferred to six 250 ml Erlenmeyer flasks and

Ί November 5, 92

-76'- stirred in a 5θ ° θ water bath using magnetic stirrers. To each flask was placed 10 g of a solution containing 0 g,

0.025 g, 0.050 g, 0.10 g, 0.20 g oz. 0.40 g of SES-aza KRE-68-B-III preparation. The reaction mixtures were then stirred for 240 minutes at 50 ° C. Then, 15 min centrifugation was performed at 3000 x g.

The supernatant was then aB & lysed on N after Kjeldahl and the solid phase was washed with water at equal volumes and centrifuged. We performed this procedure twice. The solid phase was then lyophilized and analyzed for Kjeldahl N and dry matter.

A similar experiment was performed with untreated soybean meal (Soyamel 13, manufactured by Aarhus Oliefabrik A / S) as starting material. In this case, the enzyme / substrate ratios were 0, 1%, 2%, 3%, 4%, and 8%.

Based on the protein content of the supernatants, the percentages of protein obtained can be calculated. The protein yield is based on the assumption that the enzyme product is 100% solubilized after reaction. The table below shows the results obtained in both experiments.

Table 8.1 Protein yields and p.v.p. protein / dry ratio prepared from cooked or raw soybean meal

Soybean meal cooked Raw soybean meal E / S% Profit protein % Protein in dry matter Profit protein % Protein in dry matter % 0 92,9 76,5 90.7 m 75,9 0.25 90.1 86.6 - - 0.50 89,3 88,7 - - 1.0 88.1 89,7 87,1 86,2 2.0 86.6 91,7 85.7 88.1 3.0 - - 84,3 89.5 4.0 84.7 92,2 82.6 90,9 8.0 - - 76,2 91,1

- 79 5 · H 92

With reference to extraction (isolation) processes for materials other than proteins, as well as to liquefaction processes and related processes, we refer to the general process scheme for uses as shown in technology scheme no. 3.

The substrate may be one or more carbohydrates present in the feedstock, or it may be the entire feedstock.

This substrate may be subjected to a pre-treatment of a chemical or physical character, as subsequently exemplified, e.g. acidic or alkaline treatment, soaking or watering and / or steaming with or without steam.

The raw material can be macerated, cut, wet milled and / or homogenized (all these treatments are designated as homogenisation in technological scheme # 3), with or without water additives, and other additives can be added during this step. Homogenization can be carried out with different efficiencies, e.g. different pressures which are only part of the maximum pressure indicated for the particular homogenizer used. Various additives can be added before or during homogenization, as presented in the technological scheme no. 3 by means of the symbols b ^, br>, ... b ^.

The reaction process involving the preparation of SlS-aza is carried out under specified conditions, e.g. at temperature, pressure, time, pH and doses of the enzyme; also guidance on the reactor used (eg batch, plug flow) and mixing, if necessary, are useful. For various raw materials,

5. «92

- S® is added to the set of accessories listed. · In the technological scheme no. 3 as ^c 1 » ^c 2 ··· ' ^c n'

Separation processes can also be performed with different efficiencies. Among many processes, separation is eliminated or facilitated, e.g. If the raw material is completely liquefied. Various separation devices (eg centrifuges, filters, ultrafiltration equipment, hydrocyclones, thickeners, sieves or nets or simple decanters) can be used.

Separation efficiency is defined as the ratio of the absolute proportion of slurry in the solid phase to the absolute proportion of slurry in the reaction mixture.

The resulting liquid or solid phases can be further processed, for example, concentrated, dried or solvent extracted to remove certain ingredients, such as fat or oil, fermented to produce biomass, alcohol or other products (enzymes, antibiotics or other valuable ingredients).

The obtained products can also be returned for reprocessing according to the process scheme.

- π. 92

- 80 Technological scheme no. 3

= O - 100%) = 0 - 100%) (Specify reaction conditions)

Further processing (see text) or reuse as substrate

The following are some examples of uses of SPS preparation and an overview of these uses can be seen in the following list.

Table I also lists some of the features related to technology scheme no. 3.

- 83 List of examples of used uses of SPS-aze preparations

Type of SPS preparation

Reference no.

An SPS-aase preparation that is substantially free of one or more undesirable enzyme activities

A 1

A 2 A 3 A 4

Extraction of starch from corn, wheat and potatoes Lipid extraction from plant material Extraction of essential oils from plant material Extraction of natural dyes from plant material

Rubber extraction from gua ^ ule bush (Parthenium argentatumj

Unmodified preparations

SPS aze

Full Liquidation · Doing or similar treatment

Ba

Ba

Ba

Ba

Ba

Ba

Ba

7

Production of milk substitutes for pets Production of raw materials containing saccharified starch Full liquefaction of pears and other fruits Production of juice by treatment of fruit and lentils Processing in connection with the extraction or squeezing of cane or beet production Soya milk Processing to increase the solubility of coffee, obtainable

Processing Bb Gadgets

Bb

Bb

Bb

Bb

Bb

Ί

Prevention and / or degradation of apple opacity Use as white wine clarifying agent Production of 1SSPH or other] vegetable protein hydrolysates

The stinging enzyme in brewery štv

Enzyme additive for use during fermentation and / or storage of beer Almond peeling agent continued ϊ 5. n 92

Other uses

Bc Ί Decomposition of various waste materials

Bc 2 Saccharification and simultaneous fermentation

Bc 3 Breakdown of cellulose

Bc 4 Use as a baking aid Bc 5 Improvement of alcohol and biomass yield during fermentation of sulphite liquor from paper production

Bc 6 Removal of water from biological sludge products

Bc 7 Silencing aid

- $ 3 TABLE I

Φ δ rH Φ rt rt ta · ο rt rt «P Φ > o rt o $ 4 rd Φ 1 1 <» ** Φ 1 rt to p • ra pl TO A rt o rt o rt-d rt ta rt A H-P-H rt d «» φ φ rt φ O d H O A * oo, A ^{, | -} 3 rt HHr: rt O »H rt o rt +> rt ta da Φ -rl o m d φ A d o ci «* o o • MT-3 3 o flj 5 this d -H Φ p 'd > this one A p Ρ, ‘γο g a a m a rt A 3 Φ g d g d cape cape 03 cape cape A ο -j rt rt o rt φ > φ φ k Φ A 3 A Pi, y rt who Λ5 Ad H A A φ CŠIv- / • Ο rt 1 • ra 1 1 1 rt Α rt rt φ rt rt A φ d f 3 « rt -o rt rt Pr ~> n: 3 ta Φ «H« h -n d U 1 1 ΟΛΙ 1 rt ίΐ · 2 AO * Ad O Φ Φ O 03 v-zd d - ”- cape φ -c rt ® ^ ^- q | H + 1 ro rt • ra φ d A o 1 rtA > o rt Λ rt a φζ-s φ A p d rt A> o rt o φ • n> rt> o 03 rt A N φ A A d τ: H d KO Φ 1 1 • h ta | T rt gSS-dS Oh Cha H-H v ^ t >> o rt rMA s-z Oj t> Φ · Γ3 * sO R rt > o o Oh φ o | o o o Oh o > ο A K ~ v ο o Α K \ rt • γ: 1 ·. H o Ο ο K \ a « a ο this one o 1 o o 1 1 1 Α · ι 4 trt • φ o Γ CM 60 | Ο 1 a φ a R σ \ ο this one o Α · ι j cape M 1 Oh o ! d • ra 1 o o • Φ ac ^ o v • r V 600 CM trt A trt H S.HH With AA r- | • rl Oh O A A Ž3B Mr Rücker Ž3B ! 25 «18 S rt rt a • d 1 rtA 1 • rl o | © k 1 3J A l> h a α rt rt rt rt rt d rt d d d d Ο d CM o o Oh o Α oo i> A 1 k t> t> % 1 _ • n d rt Α rt 3-rt rt rtz ~> rt • rl A rt rt O 03 35 g 1 rt this one this Jm cape S cape .M A • M Φ m a rt A rt rt d> o • H rt N rt 3 a Yes d g a 44 d α rt rt -h • ra ^ S ro φ cape a rt fl Φ -ra ® m Ji o .srt o o rt> o A ft r ~ 4 rt ort OH A 03 a ^ KO 03 5 03 d a A <n _ a • «Η v CM V. CM ir \ v Φ Α -4 <4 rt rt rt o A A A A

A 1. Extraction of starch from maize, wheat and potatoes *

Starch extraction from maize, wheat, potatoes and other starch-containing plants is carried out by one or more of the following measures: irrigation, wet milling and separation. The use of the SES preparation, which is substantially free of amylolytic activity, will provide the following advantages, using maize as an example:

1. Starch release will be facilitated during short irrigation times.

2. Water consumption can be reduced.

3. The release of corn germs will be facilitated without the release of corn germ oil.

4. Protein can be obtained with greater purity.

5. Recovery of corn irrigation water will be facilitated.

A 2. Lipid extraction from plant material

Because the lipids in the plant material are enclosed within the cells and usually bound to proteins, the lipids in the aqueous phase can be extracted by treatment with a substantially lipase-free SPS preparation. Thus, corn germ oil is usually isolated by extraction of dried maize germ with hexane. However, the drying operation is unnecessary if wet maize germs are treated with the SPS-aza preparation of the above species. Similar things can be improved

-9 & olive oil extraction in aqueous phase if the enzyme used for enzymatic treatment is an SES preparation of the above species, see e.g. Food, Pharmaceutical and Bioengineering, No. 172, vol. 74, p. 93 - 94. Similarly, aqueous extractions can be improved e.g. soybean oil, rapeseed oil and sunflower oil.

A 3. Extraction of essential oils from plant materials

If we extract plant materials containing essential oils with an aqueous solution of the SPS-aze preparation, which is substantially free of enzymatic activity, capable of degrading or otherwise altering the essential oils, we will obtain essential oils at great low cost.

A 4. Extraction of natural dyes from plant material

If we treat plant materials containing dyes, e.g. beets containing betanin red or cranberry dye with SPS-aase preparation essentially free of enzymatic activities capable of breaking down. e or otherwise it changes the dyes, we will obtain dyes with great profits and at very low cost

A 5- Extraction of rubber from guayula bush

Another example of a substrate for the preparation of SPS-aza, essentially devoid of enzymatic activity capable of degrading natural rubber, is cell wall material in the roots and branches of guayula bush.

- ί®?

Ba 1. Production of milk replacer for domestic animals, preferably milk replacer for calves With complete liquefaction of soybeans, sunflower seeds, cotton seeds, beans or grabs in aqueous medium, a milk replacer for cold-water soluble calves at pH can be produced. values of about 4.5 · When using starch-containing raw materials such as beans or peas, starch liquefaction must be performed using oc-amylase before, after or at the same time as treatment with SPS-aase, which finally snlubilizes non-starch polysaccharides, which are present as a support material in cell walls. Below is a detailed example of using a bean, with regard to soybeans, referring to Table I. Pre-treatment of this soybean may be preferred cooking in a steam ejector, which improves the solubilization of the remainder.

EXAMPLE We suspended 1.1 kg of bean meal (Parine de Peves from GRANDES MINOEERIIB A PEVES DE PRANCE, Paris) in 55 l of water. We took 75 S of Termamyla 60 L and 18 g of GaCl ^. The suspension was heated to 95 ° ^C using a steam jacket while stirring. · The suspension was then treated at this temperature for 60 minutes. The pH was then adjusted to pH 4.5 and the product was cooled to 5θ ° θ · 500 g of the KRP 68 SPSase preparation was solubilized in 1 L of water and added. We are metabolized

- 88 in 440 minutes. If 10 g of Fungamyla 800 1 is injected, the starch fraction will be converted mainly to disaccharide (maltose). The reaction mixture was then pasteurized for 2 minutes at 90 ° C. An aliquot of the product was then lyophilized and used for stability tests. The sample was then solubilized in 10% dry matter and the product solution was able to remain stable without sedimentation for several days.

The melted fat or oil can be easily emulsified into the product, yielding a final composition that is very similar to cow's milk. Also, an emulsion containing 3.5% oil (soybean oil) can easily be kept stable without sedimentation for several days.

EXAMPLE Ba 1.2

Soybean meal (Soyamel 13) was boiled in a steam ejector at 150 ° θ for 25 seconds as described in Example 8. The steamed soybean meal was spray dried and used for the further studies described below.

A g in the steam ejector of cooked soybean meal was mixed with 450 g of water and adjusted to pH 4.5 with 4.1 ml of 6 N HOl. The mixture was then heated in a water bath at 45 ° C and 0.250 g of the KRF-68 SPS-aase preparation was added to the heated mixture and then digested with stirring for 5 hours. The mixture was then heat treated for 2 minutes at 80 ° C to inactivate the enzyme. The 100 ml sample was centrifuged at room temperature for 15 minutes at 3000 x g (g = weight). Supema-89 & tant ion was exchanged and analyzed with the HELC composition of carbon t

hydrates. The supernatant was also analyzed for Kjeldahl N and dry matter and the nitrogen solubility index (NSI) and dry matter solubility index (BSI) were calculated, see results in Table Ba I. 100 ml of the reaction mixture cooled to 20 ° C was poured into 100 ml measuring cylinder and maintained at 4 ° C for 2 days. Dispersion stability (%) was measured by reading the volume of dispersions obtained (Table Ba II) after 1 and 2 days.

8 g of soybean oil were added to 200 ml of the reaction mixture (at 20 ° C). The emulsion was prepared by stirring for 2 minutes in Waring Brender. The emulsion stability (%) was measured as above after 1 and 2 days.

B_

The metabolism was performed as above, except that 1.00 g of the SPSase preparation was used in this case. The same types of stability analyzes and measurements were performed as described in Chapter A. The results are shown in Tables Ba I and Ba II.

The chemical analysis of the supernatants shows that the values for NSI (%) and DSI (%) obtained in Experiment B are greater than in Experiment A. However, the? Stability tests performed with the reaction mixtures show a better value for samples A. This is probably due to the longer length of the peptide chain of the proteins in the reaction mixture with a small dose of enzyme.

The composition of the carbohydrates, as measured by HECiC, shows that they are mainly formed by mono- and disaccharides. Therefore, oligosaccharides known to be responsible for diarrhea and flatulence when given to calves in excessive amounts are only present in small quantities.

- 91 -

Table Ba I Kerni Properties of the Supernatant

Give it a try i ϊ Dose of enzyme relative to substrate% m / m NSI,% ^K (at pH = 4.5) DSI,% ** (at pH = 4.5) HELC results (composition of neutral sugars) } '! j ^A I s 1 E / S = 0.5% 39,9 62,4 DPzj + DP ₂ ^DP 3 ^DP 4 ^BP 4+ 79.7% 7.4% 12,2% 8.1% 1 1 B i j E / S = 2.0% 57,0 67.1 DP.J + dp ₂ ^DP 3 ^DP 4 ^PP 4+ 84.4% 6.1% 3.7% 5.7%

^H HBI = nitrogen solubility index ** DSI = dry solubility index

Table Ba II Stability tests of reaction mixtures

Give it a try Dose of enzyme relative to substrate% m / m Stability tests' Without ol Lja With oil Dispersion Day 1 Dispersion Day 2 Emulsion Day 1 Emulsion Day 2 A E / S = 0.5% 80% 63% 100% 87% B E / S = 2.0% 66% 35% 85% 71%

-9Κ5Ba 2. Production of saccharified starch from raw materials containing starch

With regard to saccharification of cassava and sweet potatoes <£ and other plant materials containing starch, the addition of the SPS-aase preparation is capable of solving viscosity problems. Using the SPSase preparation, starch suspensions with a solids content of 25 to 3θ% can be prepared, and after saccharification, the Žonta can be fermented, thereby obtaining cheap ethanol.

EXAMPLE Ba 2.1

On the basis of fresh and grated sweet potatoes (Japanese), Zonta was made with a solids content of 24%. We found that the dry matter of potato mulch contains about 70% starch. Pre-fluidization with bacterial amylase from Termamyla® 60 L at a dose of 0.5 kg / tonne of starch

The AV was performed by heating the Zonto to 90 C. The zonto was then maintained at 90 ° 0 for 30 minutes. The viscosity of the reaction mixture was then measured using a HAAKE spindle at 9θ ° θNa the reaction mixture was cooled to 55 ° ^c and the pH adjusted: from 2 NH 2 SO 4 to pH 5.0. Then, with the addition of gluco-amylase san 1] (brand name, N0V0 INDUSTRI A / S) at a dose of 1.75 liters / ton <

starch triggered saccharification. The saccharification mixture was then divided into three parts A, B and 0, which were treated with enzyme for 15 minutes before measuring the viscosity, as shown below:

At: This part is a control, we measured the viscosity ^ 2 * See Table Ba III.

B: We added Tricoderma viride cellulase from

Celluclasta® 200 N at a dose of 1 kg / tonne of dry matter of sweet potatoes. Viscosity was measured, see Table Ba III.

C: We added the preparation of SPS aase KRF-68 at a dose of 0.25 kg / tonne of dry matter of sweet potatoes. Viscosity was measured, see Table Ba III.

Table Ba III: Viscosities

Reaction mixture ^ §Bg ^8t ^ 8g ^3t

Pre-liquid / n _n sweet potato ^C 1 ^{B x}

A

B

C

10 " ¹ Pa.s" 21.90 x 10 " ¹ Pa.s ^ 2 = 21.90 χ 10 ¹ Pa.s = 19.70 x 10" ¹ Pa.s = 9.50 χ 10 ' ¹ Pa. s

It can therefore be seen that the viscosity of the reaction mixture can be effectively reduced by a small dose of SPSase compared to Celluclast® and SAN 150.

- 94 ^

Ba 3. Complete liquefaction of pears and other fruits

If whole pears, which are crushed mechanically, are then treated with the SPS-aze preparation, complete liquefaction occurs and, after removal of small quantities of solids, clear pear juice is produced. A similar method can be used in relation to other similar fruits, e.g. apples.

EXAMPLE Ba 3.1

We ground the fresh apples with the help of a Bucher Central mill. The apple cider was then pasteurized in a heating jacket for 5 minutes at 90 ° C and then cooled to ambient temperature. The pre-milled apples were then ground in a Fryma mill with corundum elements until the broth was smooth to the touch. The slurry was then re-pasteurized at 80 ° C for 10 minutes and cooled to 50 ° C.

The enzymatic metabolism was now carried out at 50 ° C for 50 minutes, while simultaneously mixing and measuring the viscosity with Contraves Rheomat 15 (compared to the percentage reading on the Rheometer at speed 13). After enzymatic digestion was completed, 100 g samples were collected and centrifuged in a graduated tube at 3000 x g for 15 minutes. Thus, the percentage of juice and the percentage of sediment were measured. The pH and percentage of refractometric dry matter as ° Brixam were also measured. Table Ba IV shows a comparison between the effect of SPS-aase, a combination of Celluclast and SPS-aase, and a combination of Celluclast and Pectinex. We used the SPSase preparation KRP-68.

- 95 Table Ba IV Results of complete liquefaction experiments with apple splashes at 50 ° C for 30 minutes

sps-aza g / hl broth Celluclast '200 1 (r) Pectinex ^v —'3x g / hi without ge The final one viscose- nnsf Centrifuged- ae Juice g / hi broths % juice; %> used · not pH ° Brix 0 0 0 100 59 41 3.8 9.7 25 0 0 19 81 19 3.5 10.5 50 0 0 15 79 21 3.5 10.7 50 50 0 4.8 3.8 83 83 17 17 3.5 3.1 10.8 12.5 0 50 200 9.5 83 17 3.2 12.9 0 50 2000 4.0 82 18 3.1 13.2

- 96 Ba 4. Production of juice by treatment of fruit and lentils

K

We have found that SPS-aza preparations are very suitable for the production of juice by processing various fruits, berries and lentils, e.g. carrots, peas, tomatoes, apples, pears, black currants, beans and cabbage. This results in better juice yield and better extraction of coloring and flavoring ingredients compared to commercially available prectinase and cellulase preparations.

EXAMPLE Ba 4.1

We refer to the example Ba 3 * 1 where we are a preparation

SPS aases were compared to commercially available conventional cellulase products. pectinases Celluclast 200 L and Pectinex 3x ·

The table shows that the juice yield is only slightly improved with only 5θ g / hl of SPSase compared to 2000 g / hl of Pectinexa®, (g) in combination with 50 g / hl of Celluclast ^. The viscosity was also slightly lower. Thus, SPSase appears to be about 40 times more effective than Pectinex.

Ba 5 · Processing in connection with the extraction or compression of sugar cane or beet

It has been found that the yield pertaining to simple extraction processes can be improved by using the SPS preparation for the treatment of sugar cane or beet

- 97 and / or during their extraction or compression. The bagaso can also be treated with the SPS-aase preparation, thereby partially converting it into fermentable sugars that can be used as a feedstock for ethanol fermentation.

EXAMPLE Ba 5.1 kg of residual sugar beet (pulp) obtained from continuous countercurrent Extraction in a DDS diffuser at Nakskov Sugar Pactory was ground twice in a Pryma mill (type MZ-110). Process water was added during the milling operation.

Sections of 3θθ S pulp were now enzymatically treated at 45 ° C for 18 hours using the doses of the enzyme shown in Table Ba V. The dry enzyme product (KRP-68) was added to the pulp, which was stirred for the first hour with a stick. The pulp was then liquefied to such an extent that magnetic stirring was then possible during the remaining time. At the end of the metabolism, the pH was measured (pH was not corrected during the onset of metabolism) and the reaction mixture was centrifuged until a clear supernatant was obtained.

Determination of dry matter was performed in the reaction mixtures and in the supernatants. Based on these results, the percentages of solubilized dry matter were calculated. For all calculations, corrections were made for the soluble dry matter of the enzyme product.

Supernatante no. 2, 3 and 4 were ion-exchanged and the composition of the carbohydrates analyzed by BPLC.

- 98 Table Ba V Results obtained by enzymatic liquefaction of beet pulp

Try it no. Dose of enzyme based on dry matter E / S% Final measurements The reaction mixture Supernatants pH (final) % dry substances % dry substances % solubilized dry ^{, e} dreams 1 0 5.5 4,18 0.0 0.0 2 0.35 3.6 3.85 2.58 66,9 3 0.56 3.5 3.81 2.56 66,2 4 1.02 3.5 3.86 2.73 70.4 5 1.58 3.3 3.17 2,34 73,4 6 3.10 3.4 3.23 2.49 76,4 7 7.52 3.4 2.66 2.18 80.5 Reactionary conditions: M = 300 g S = 4.18% dry substances

E / S as above pH was not adjusted T = 45 ° 0 t = 18 hours

- 99 Table Ba VI HPLC data

Type of sugar (neutral) Try it No. 2 5 4 % of neutral sugars High Molecular (DP4 +) 45,6 51.9 25.5 Disaccharides 4.6 4,8 - Glucose 20,4 25.7 27.8 Galactose 5.0 5.9 7.5 Prukt o z a / ar abinoz a 26,4 32,2 33,2 Galacturonic acid we did not measure

All sugars produced according to Table VI Ba above can be fermented into alcohol or used for other purposes.

Ba 6. Soy milk production

Soy milk can be easily produced by the complete liquefaction of ground soybean and the subsequent homogeneous mixture of the resulting mixture. Soy milk is often produced by soaking soy in boiling water, grinding soaked grain and extracting it with water, followed by separation of insoluble residues, e.g. proteins

- 100 and polysaccharides. In order to improve the yield of soy milk, these insoluble residues can be liquefied by digestion with SPS.

Example Ba 6.1

The procedure for soy milk is illustrated by the following series of enzymatic metabolites, showing the calculations for the protein solubility index (PSI,%) and the solubility index of the substance (DSI,%) yields obtained after separation at pH = 7 (see Table Ba VII). The enzymatic metabolisms were performed under the following conditions:

Substrate: Full fat soybean meal (Dansk Sojakagefabri

A / S)

Mass of reaction mixture: 220 g

Substrate mass:

Enzyme:

Enzyme dose:

g ° C

4.5 (6 N H01)

Series A: 1 hour

B Series: 0.5 - 6 hrs of SPS (KEP-68)

Series A: E / S ratio (m / m):

- 8,0%

Series B: E / S ratio (m / m): 1.0%

After metabolism, pH was adjusted with 4 N NaOH to pH a 7 and was performed separately by centrifugation at 3000 x g for 15 minutes.

- 101 Table Ba VII Calculation of the mass balance in relation to the enzyme process for soy milk • H • P ra o

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- 102 Ba 7 · Processing to increase the amount of soluble coffee matter obtainable.

We have found that the treatment of coffee beans in different stages of instant coffee production results in an increased yield of soluble coffee. Thus, e.g. spent coffee or green beans are enzymatically treated with favorable results.

- 103 Bb 1. Prevention and / or decomposition of apple or pear *

turbidity

After the production of apple juice or pear juice and other fruit juices, which must be clear and which have been pretreated with conventional pectinase and cellulase preparations to prevent turbidity, apple turbidity or similar fruit turbidity may occur. We have found that SPSase preparations are very suitable for the breakdown of such turbidity, consisting mainly of protein-bound araban.

Example Bb 1

We have found that the pear juice concentrate produced by the liquefaction of pear canning waste using Celluclast® and Pectinex® has become disturbed. Turbidity was isolated and hydrolyzed for 24 hours with 0.01 N H ^ SO ^ and analyzed with WHITE. The eromatogram showed arabinose and small amounts of oligosaccharides.

We found that by incubating 0.5% w / v of this carbohydrate in 3 mM acetate buffer at pH 4.5 at 40 ° 0 with SPS aase for 3 hours (ERP-68 + ERP-92 1: 1) ) with an enzyme concentration of 0.05% w / v converted 84% of the original carbohydrate turbidity (dry matter) into arabinose.

The diluted pear concentrate (20 ⁰ Brix) was also treated for 2 hours at 40 ° C with an enzyme dose of 0.15% w / v in the above SPS aases or with a market product called Clarex,

- 104 at a dose of 1% w / v. We found that SPSase was able to reduce the relative HPLC surface area of araban-like turbidity by 86%, while correspondingly reducing (S) with Clarexoffl (used at a much higher dose than the SPSase preparation) only 78% .

Bb 2. Use as a clarifying agent for white wine

We have found that white wines that exhibit highly unwanted turbidity can be effectively clarified using SPS aza; it has been shown that the turbid material consists mainly of arabinogalactans bound to the hydroxyproline residues of the cell wall structural protein.

Bb 3 · Production of ISSPH or other plant protein hydrolysates

Prior to separation of ISSPH (isoelectrically soluble soy protein protein hydrolyzate) or other plant protein hydrolysates from the slurry as described in U.S. Pat.

100 024 or in Process Biochemistry, vol. 14, No. 1 7 (1979), pages 6 to 8 and 10 to 11, the reaction mixture can be treated with SES preparation. This makes separation easier.

Bb 4. Brewery Enzymatic Enzyme

Beer production is influenced by carbohydrates in raw materials, e.g. β-glucans in malt and barley, on the viscosity and filterability of barley. The addition of SPS aze during grunting will reduce the viscosity of the barley and improve the filterability and yield of the extract. In addition, the addition of SPS-aze during harvesting will increase the fermentability of barley and the nitrogen content of barley.

- 105 Example Bb 4,1

In the laboratory, 50 g of ground meal consisting of 50% malt and 50% barley were sacrificed, together with 275 g of water (15% dry matter) according to the following diagram and 52 ° C (60 minutes) / 63 ° C ( 60 minutes) / 76 ° C (30 minutes).

In order to prove the effect of SPS-aza, 4 tests were performed, see table below, with enzymes added during sacrificing (pH Bands 5.5 to 6.0).

- 106 -

Enzyme Nothing Cereflo SPS aza (KRF 68) Activity 0 beta-glucanase / g 200 BGU 1630 FBG 1630 FBG Dose of enzymes 0 kg per kg of semolina 1.5 S 0.05 S 0.18 g Full 0 dose of enzyme activity units per kg of meal 300 BGU 80 FBG 300 FBG Filtration rate of 120 ml of barley tration after 10 minutes 135 ml 160 ml 170 ml Viscosity 1,25.10 'Barley Pa.s. 10 ° Balling (.25 ° g? ” ⁵ 1,36.10-5 Pa.s. 1,36.10 ^-5 Pa.s. 1,30.10 ^-5 Pa.s.

BGUs are beta-glucanase units determined in accordance with the AF 70/4-GB analytical method obtainable from NOVO Industri A / S.

FBGs are units of fungal beta-glucanase, determined in accordance with the AF 70.1 / 2-GB analytical method, obtainable from NOVA Industri A / S.

The only difference between BGU and FBG is the pH at which the enzymatic determination is performed: pH 7.5 for BGU and pH 5.0 for FBG.

Cereflo is a bacterial beta-glucanase preparation described in Fact Sheet B 214b-GB 1500, July, available from NOVO Industri A / S.

- 107 Example Bb 4.2

In the laboratory, 50 g of ground meal consisting of 40% malt and 60% barley were harvested, along with 150 g of water (25% dry matter) according to the following charting; 45 ° C (60 minutes) / 63 ° 0 (90 minutes) / 75 °° (15 minutes). In order to demonstrate the effect of SPS-aza, three tests were performed, see table below, with enzymes added during sacrifice (pH Bands 5.5 to 6.0).

Enzyme Nothing Ceremix SPS-aza (KRP 68) + Ceremix added as in the previous test Betaglucanase activity / g • - 200 BGU 1630 PBG Dose of enzyme per kg of meal - 1.65 g 0,035 g Total dose of enzyme activity units per kg of meal 0 330 BGU 50 PBG The barley filtration rate recovers after 30 minutes 48 ml 98 ml 111 ml Extract, ° Balling 18.6 19,0 19.5 Viscosity barley 10 ° Balling (25 ° g) 1,72.1cr ⁵ Pa.S. 1,37.10 ^-5 Pa. s. 1,27.10- ⁵ Well. s.

The definition of BGU and PBG is given in Example Bb 4.1.

The last column of the table above lists only the activity and dose originating from the SPS.

- 108 Ceremix is a preparation of bacterial beta-glucanase, *

described in Fact Sheet B 216 b-GB 1000, Feb. 1982, available from NOVO Industri A / S.

Bb 5. Enzymatic additive for use during fermentation and / or storage of beer

SPS-azo can be added during barley fermentation or beer storage to reduce the β-glucan content and thus improve beer filtration and beer stability for turbidity. SPSase will also have an effect on proteins that are responsible for turbidity.

Bb 6. Almond peeling agent

During the degree of mechanical peeling of the almonds following the blanching of the almonds, a certain percentage of almond peels are not eliminated. We have found that enzymatic processing of almonds results in a decrease in the percentage mentioned above.

Bc 1. Decomposition of various waste materials

With respect to certain production processes, large quantities of waste materials containing carbohydrates are generated. Thus, e.g. with regard to the production of soybean isolate by extraction with water and by precipitation with acid, soy milk and tofu (a special type of Japanese cheese). In this connection we can also mention waste pulp e.g. apples, pears or citrus fruits. We have found that the SPS aze preparation is capable of

- 109 completely liquefies this waste material containing carbohydrates and produces fermentative sugars that can be used as starting material for ethanol fermentation.

Example Bc 1.1

In the traditional production of soy milk or tofu, soy is often soaked in boiling water, ground and extracted with hot water and then separated. The residue from this separation is the material used for this experiment. The liquid phase is soy milk that can be further processed to produce tofu.

kg of whole soybean grains obtained from Aarhus Oliefabrik A / S were ground simultaneously with 70 l of boiling water in Pryma mill, type MZ 110. The ground slurry was then maintained for 15 minutes above 85 ° C to inactivate natural soybean enzymes that develop known bad soy aroma. 5 1 of these soybean slurries were then grouted in the laboratory for 15 minutes at 3000 ^x g (g = weight). The analysis revealed that the remainder contained

20.45% and 20.06% of dry matter (twice determined, calculated average 20.26%). 6 N HCl was slowly added and embedded in the residue with a spatula until the pH meter showed 4.50 if the electrode was inserted directly into the mass.

The enzymatic metabolism was carried out at 2 x 200 g mass with two doses of SPS-aase (KRF-68) E / S = 0.5% by weight of the dry matter. E / S = 3,0% by dry matter, in a 500 ml beaker at 50 ° 0. A dry enzyme was added to the mass. During the first 1 to 2 hours blending was carried out with a trowel, and then the mass was liquefied to such an extent that the mixing with the magnet was then successful. The total reaction time was 21 hours.

- 110 Osmolality with an osmometer (Advanced Digimatic 3DII, from Advanced Instruments Inc.) was measured during the reaction. The results in the table Bel show the course of metabolism. At the end of the experiment, the mixture centers were grouted for 15 minutes at 3000 x g. An oil layer appeared on the surface of the supernatant and its volume was determined. A slight layer of sludge appeared as a layer at the bottom. The supernatant, including the oil, was removed by pipette. The homogenization of the oil was combined with the clear aqueous phase and a dry matter sample was taken. The results shown in Table Bc I clearly demonstrate that by enzymatic digestion this waste product can be liquefied and that crude oil can be produced as mentioned in Chapter A 4. After the oil has been obtained, the solubilized residue can be used in various ways, e.g. for fermentation into valuable compounds, or for concentrating and drying, and thereafter used as feed or food, or after further purification to produce valuable products.

- 111

Table Bc I. Results »obtained from liquefied soy milk and tofu slurry

Reaction conditions and results Experiment A Experiment B The mass of the residue SPS weight (ERF-68) Temperature pH Reaction time 2 00 g 0.20 g 50 ° C 4,50 21 hours 200 g 1.20 g 50 ° c 4,50 21 hours Results measured on an osmometer during the course of metabolism . t min. eighth- lality mOsm Zk> we lality mOsm t min. L osmolality and mol. ' mOsm | nose tj < ⁽ mOsm Aosmolality is the value corrected for the osmolality of the mixture at t = 0 0 10 40 95 250 I260 287 m 391 501 634 907 0 26 104 214 347 620 0 10 25 45 95 250 1260 282 368 497 601 718 875 1145 0 86 215 319 436 593 863 Reaction mixture: Dry matter Supernatant: Dry matter Supernatant: Oil content Calculating% solubilized solids 20,3% 18.0% 8 - 10% 88.6% 20,7% 19.4% 8 - 10% and 95.5%

- 112 Bc 2. Saccharification and simultaneous fermentation

Vegetable materials containing Carbohydrates, e.g. tubers ^ kbt lash, potatoes, sweet potatoes, shortage or pulp of such tubers, i. material remaining after the extracted components are removed can be saccharified by treatment with the SPS-aase preparation, and at the same time the resulting fermentative saccharides can be fermented into ethanol.

Example Bc 2.1

At the laboratory scale, we examined the production of ethanol by fermentation of decomposed Lug beet containing inulin, with simultaneous saccharification with SPS aase and inulinase, and by four different Lug beet pretreatments. SPSase: We used the preparation of SPSase KRP-68.

Inulinase: Inulinase was produced by fermentation of Asp. ficuum (CBS 55 5θ5) · The inulinase activity was determined as described in Research Disclosure No. 21254 (December 1981) pages 456 to 458.

laboratory fermentation: 15θ From a portion of pretreated broth (described later) was fermented after the addition of 4.5 g of baker's yeast and 1 ml of 4% Pluronica solution as an antifoaming agent. The fermentation flasks were equipped with COg traps containing 98% sulfuric acid, and the fermentation was monitored by measuring the weight loss due to the released CC ^. The contents of the pumpkins were during fermentation carried out at

- 115 50 ° C, stirred. Three pumpkins were used for each parameter studied.

Table Bc II shows weight loss due to relaxation

CO2 converted to ethanol assuming that 1 mole of COg released is equivalent to a mole of CO ^ eOH, i.e.

g 00 ₂ ~ -Tft- E ° 2H ₅ 0H Pre-treatment of turnips:

Treatment A: 14.1 kg of beet (22.8% dry matter) was boiled in

Henze boiler at 140 ° C and 4 to 5 bar for 20 minutes. The weight after cooking was 19.0 kg (^ 16.9% of the dry matter of the Fermentation was carried out directly on Žonta.

Treatment B: The washed and chopped turnips were mixed with water (1: 1) and then mixed in a Waring mixer. The zonto was then heat treated for 1 hour at 85 ° 0 and pH = 4,

Treatment C: As B, but we did not adjust the pH.

Treatment D: As B, but without heat treatment and regulation, pH.

Results: Table Bc II shows the results of the effect of the addition of SPS-aza to pre-treated Zonta on ethanol yield. Significant improvement of the uninolol yield was obtained for all pre-treated Zonta if SPS azo was added.

114

Table Bc II Results of fermentation versus simultaneous fermentation and enzymatic sabarification of lash

Pre- hodna processing- va Inulinase units added to 1 g of dry matter SPS A / E% Loss of COp (g) after 42-44 hours of fermentation s ». % of ethanol produced, based on dry matter A b ⁵ b ⁵ 0 0.27 7.65 + 0.05 8.07 + 0 _f 08 · 31.5 33,2 B 1 | 5 ^ -r ⁵ 0 0.40 4.85 + 0.03 5.41 + 0.03 29.7 33.1 C . b ⁵ v · M M V ⁵ 0 0.10 520 0.30 0.40 5.70 + 0.05 5.97 +0.01 6.13 + 0.06 6.13 + 0.00 .6.18 + 0.05 34.8 36,3 • 37.5 37.5 37,8 * D * lr5 ^X f ⁵ M M 1.5 0 0 0.10 0.20 0.30 0.40 0.40 5.77 + 0.02 5.89 + 0.00 6.04 + 0.11 6.01 + 0.01 6.02 + 0.03 5.48 + 0.02 35,3 36,0 36,9 36.7 36.8 33.5

- 115 Bc 3 · Decomposition of cellulose.

We have found that cellulose-containing materials, such as e.g. straw, e.g. Cereal straw, sawdust, paper and lignocellulose are hydrolyzed to a greater extent with SPS preparation than with conventional cellulases. This is illustrated by the following example in which crystalline cellulose material (AVICEL) is treated using the conventional Celluclast ® 200 cellulase produced by Trichoderma reesei and the KRP 68 SPSase preparation.

Example Bc 5.1

Avicel was suspended in water (20% dry matter); The pH was adjusted to 5 and the temperature was maintained at 50 ° C.

After a reaction time of 24 hours, the slurry was filtered and the proportion of reductive sugar (mg glucose / g AVICELA) was measured. The use of enzyme doses of 5% and 20% by weight of cellulose revealed the following values:

Table Bc III

Enzyme E / S% mg glucose / g AVICELA Celluclast 5 80 SPS aza ...... 5. 200 Celluclast 20 100 SPS aza 20 340

Bc 4. Use as a baking accessory

We have found that SPS-aza preparations are excellent as baking utensils. if we add dry flour

- 116 bread can be obtained prior to the preparation of the SPS preparation test

Towards better quality in volume, core and taste. Thus, high quality wheat flour of poor quality can be obtained by using the SPS aze preparation as an additive.

Bc 5. Improvement of alcohol and biomass yields during fermentation of sulphite effluent from paper production

We have also found that ethanol yield can be improved by treating paper sulphite lye with a SPS-aase preparation before using it as a carbohydrate source for ethanol fermentation. Paper waste sulfite liquor can also be used to produce biomass, e.g. of the single-cell protein, by fermentation, and even in this case, the biomass yield was improved if the sulphite liquor was pretreated with the preparation of SPS aase. Decomposition in the presence of an SPS-preparation and fermentation can also be carried out simultaneously.

Bc 6. Removal of water from biological sludge products

During the traditional aqueous extraction of many biological materials from plant raw materials, large volumes of insoluble residue are formed, consisting of large proportions of swollen polysaccharides. Thus, e.g. in the production of soy milk,

- 1-17 tofu or soybean isolate with aqueous soy extraction, degreased

To soy flour or white flakes. The structural swelling of the polysaccharide material can then be treated to a slight degree with SPS aase, thereby opening the network structure of the material and solubilizing only small amounts of carbohydrates. This removes the water from the material and, consequently, a higher proportion of dry matter is obtained in the slurry as compared to the product obtained without enzyme treatment.

Thus, the enzyme process shows the advantage of significantly lower energy consumption for dehydrated water, and also opens up the possibility of producing cheaper dry material for animal feed or fillers for food use.

Bc 7 · Silencing aid

It is known that, due to the increase in the speed of the silage process and the digestibility of the silage, enzymatic silencing aids are added to the silage. We have found that SPS-aza preparations exceed the known enzyme silencing aids.

- 118 An overview of the images previously referenced is provided below in order to provide a more complete overview.

Image no. It belongs to It describes 1 to the general, part of the description evidence of a binding effect between SPS and soy protein 2 the general part of the description technological scheme describing the preparation of the SAA 3 Chapter 2 calibration curve for HPLC gel filtration chromatography 4 Chapter 2 HPLC gel chromatogram filtration SPS 5 Chapter 2 HPLC chromatogram of SPS gel filtration degraded by SPS aase 6 Chapters 2 and 3 HPLC chromatogram of gel filtration of supernatant from SPS incubated with soy protein 7 Chapter 2 HPLC chromatogram of supernatant gel filtration from digested SPS incubated with soy protein 8 Chapter 3 HPLC chromatogram of APS gel filtration digested with Pectolvas · 9 Chapter 3 HPLC chromatogram of SPSase-digested APS gel filtration 10 Chapter 3 HPLC chromatogram of Pectolvas-treated gel filtration SPS

- 119 -

Image no. It belongs to It describes 11 Chapter 7 immunoelectrophoretic spots, including the SPS-aase peak, identified. with overlay technique 12 Chapter 8 SPS-ion ion exchange chromatogram 13 Chapter 9 pH dependence of SPS-aase activity 14 Chapter 9 temperature dependence of SPS activity 15 Chapter 9 temperature stability SPS aze 16 Chapter 10 Protease pH stability in SPS-aase preparation

- 120 -

Claims (21)

PATENT APPLICATIONS 1. SPSase, a carbohydrate in useful form, capable of degrading under suitable conditions soybean SPS into degradation products that bind to a protein in aqueous medium to a lesser extent than would bind SPS prior to degradation to the same protein under appropriate conditions. SPSase according to claim 1, characterized in that the SPSase is capable of breaking down the soybean SPS in the aqueous medium into degradation products that bind to the plant protein in the aqueous medium to a lesser extent than the soybean SPS prior to degradation bound itself to the same plant protein in aqueous medium. SPSase according to claim 1 or 2, characterized in that the SPSase is capable of decomposing soybean SPS in aqueous medium with a pH not exceeding 1.5 by 4.5 into degradation products , which bind to soy protein in aqueous medium to a lesser extent than soybean SPS would, prior to degradation, bind itself to soy protein in aqueous medium. SPSase according to Claims 1 to 3, characterized in that the degradation products of soybean SPS bind to the plant protein to the extent of less than 50%, especially less than 20%, than the soybean SPS would bind before decomposition. plant protein in aqueous medium. - 121 SESase according to claims 1 to 4, characterized in that the SESasease shows a positive test for SESasease when tested according to the method for qualitative and quantitative determination of SESasease. SESase according to claims 1 to 5, characterized in that the SESasease was produced by a microorganism belonging to the genus Aspergillus, preferably to the Aspergillus niger group. 7. The SESase according to claims 1 to 6, characterized in that the SESasease is derived from the enzymes which can be produced by Asp. aculeatus CBS 101.43. 8. The SESase according to claims 1 to 7, characterized in that the SESasease is immunoelectrophoretically identical to the SESasease produced by Agp. aculeatus CBS 101.43, and identified by immunoelectrophoretic overlay technique. 9. Isolated SES, characterized in that the isolated SES is produced on the basis of vegetable crude protein as a raw material. An isolated SES according to claim 8, characterized in that the vegetable crude protein is degreased soybean meal. A method for selecting an SES-producing microorganism for the production of an SES according to claims 1 to 8, characterized in that the micro-organism to be tested is grown on a medium whose main carbon source is SES according to claim 9, or 10 - 122 then analyze the sample medium for SPS-azo and, if the SES-azo assay is positive, the said micro-organism is selected as the micro-organism producing SES-azo. 12. A method for producing a SPS aase according to claims 1 to 8, characterized in that the strain which can be selected in accordance with the selection process according to claim 11 is grown on a culture medium. 13 A method according to claim 12, characterized in that the strain Asp is grown on the culture medium. aculeatus CBS 101.43 or Asp. japonicus IPO 4408. A method according to claim 12 or 13, characterized in that the cultivation is carried out as dry-growing at a pH in the range from 3 to 7, preferably from 4 to 6, at a temperature in the range from 20 to 40 ° G, preferably from 25 to 35 ° C, containing the medium as carbon and nitrogen sources and inorganic salts. Method according to claim 12 to 14, characterized in that the medium contains soya flour. Process according to claim 15, characterized in that soybean flour is treated, before being used as a substrate component, with a proteolytic enzyme, preferably with a proteolytic enzyme produced microbially by Bacillus licheniformis. The method of claim 12 to 16, characterized in that the sterile pectin solution is aseptically added to the fermentation broth during cultivation. 18. A process for the degradation of polysaccharides, preferably polysaccharides of plant cell walls, by carbohydrate, characterized in that the preparation of SPS aase according to claims 1 to 8 in aqueous medium is contacted with a substrate for - 123 this SES preparation. K A method for the degradation of polysaccharides according to claim 18, characterized in that the degradation is accompanied by the isolation or extraction of biological material other than soy protein and related plant proteins from the raw biological material, wherein the SPSase preparation does not contain substantially any enzyme which is able to degrade this biological material. A process for the degradation of polysaccharides according to claim 18 or 19, characterized in that one or more reaction products (regardless of whether they are desirable end products or waste products) are treated simultaneously with or after enzymatic treatment. 21. The process for the decomposition of polysaccharides according to claim 20, characterized in that the further treatment in case one of the reaction products is fermentable sugar is alcoholic fermentation.