NL8204924A - IMPROVEMENTS IN AND CONCERNING AN ENZYME FOR THE DECOMPOSITION OF A LARGE MOLECULAR WEIGHT CARBON HYDROGATE, THE LARGE MOLECULAR WEIGHT CARBON HYDROGATE, A METHOD FOR SELECTING A SIMILAR ENERGY ENERGY, AND SUCH A GREAT PROPERTY. - Google Patents

Description

V- V, 1 * 1 * "• Improvements in and just relating to an enzyme for the decomposition of a high molecular weight carbohydrate, the isolated high molecular weight carbohydrate, a method of selecting a microorganism that produces such an enzyme and a method of preparing such an enzyme.

A process for the preparation of a purified vegetable protein product (PVP) by enzymatic removal of the remanence without solution and reprecipitation of the protein is described in Belgian patent 882,769. The purity of the pvp obtainable by the known method is not satisfactory and is therefore open to improvement. In the examples, a purity of the PVP of about 85% was demonstrated. Even if it is possible to obtain a pvp with a purity of about 90% by the known method, it is only available with certain pre-treated starting products, for example soy protein concentrate. It would be desirable to be able to achieve a pvp purity of about or greater than 90% with a much wider spectrum of starting materials, especially husked and defatted soybean meal.

The invention is based on the surprising finding that a certain part of the retentive decomposition product, as it occurs during the above-mentioned enzymatic treatment, ie a water-soluble high molecular weight carbohydrate, itself attaches to part of the vegetable protein, as will be explained in detail later. Obviously, this results in a lower purity of the protein. It has also been found that this high molecular weight carbohydrate has an ability to attach itself to proteins of animal origin.

Therefore, an object of the present invention is to provide an enzyme for the decomposition of the above-mentioned high molecular weight carbohydrate, which will open the possibility for the preparation of a pvp of improved purity and a method of preparing such a enzyme.

The basis of the invention can be described in the following manner with reference to Fig. 1, in which only material which exists as non-dissolved solids, with all of the above layers omitted, is described. An amount of soybean meal was divided into two equal parts, Part I and Part II (column a in Fig. 1).

Part I was proteolytically decomposed at a pH of about 8 by ALCALASE (a proteolytic enzyme prepared by 8204924 il 2 B. lichenlformis and marketed by NOVO INDUSTRI A / S, 2880 Bagsvaerd, Denmark) and then further washed at about pH 8 in order to eliminate the total amount of protein and the romance was separated from the supernatant and washed (see 5 part I, columns a and b, fig. 1). In this manner, a pure remanence (referred to as remanence I) was isolated (column b in Figure 1). Part II of the soybean meal was not treated; for brevity, the remanence in part II is referred to as remanence II (column b in fig. 1). Now both remanence I and part II are decomposed by a commercial pasease, for example PECTINEX (a pectolytic enzyme prepared by Schweizerische Ferment AG, Basel, Switzerland) (see columns b and c in Fig. 1). Surprisingly, it was found that the undissolved portion of retentivity I is much smaller than the undissolved portion of retentivity II based on nitrogen and dry matter mass balances, see Figure 1, where the shaded areas in column c correspond to the insoluble non-protein materials at the above step. Furthermore, when the supernatant from the pectinase-treated remanence I is brought together with a soy protein suspension at pH 4.5, a polysaccharide in the supernatant layer is bound to the soy protein. This polysaccharide in the above layer of remanence I, which is part of the remanence decomposition product and which is clearly soluble in water in the absence of soy protein, however, is bound to soy protein at or about the isoelectric point of soy protein, when soy protein is present is referred to as SPS (soluble polysaccharide), see Figure 1. The SPS has a molecular weight distribution between 5 x 10 ^ and 4.9 x 10 ^. The production of isolated SPS is evident from the flow chart shown in FIG.

2, which also includes part of the processes described in FIG. 1. Therefore, the problem is to find an enzyme capable of decomposing the SPS in such a way that the SPS decomposition products do not bind soy protein or bind soy protein to a much lesser degree than SPS binds soy protein.

Although the description specifically refers to soy protein, the invention is not limited to soy protein, but includes all types of vegetable proteins, see for example the proteins mentioned in Belgian patent 882,769 on page 1.

According to the present invention, it has now been found that by comparative research on the ability to decompose soybean SPS, it is possible to select mirco-organisms capable of producing a compound 40 which exhibits an enzymatic activity to efficiently 8204924 3, f '. to analyze soybean SPS, In the following for brevity, just SPS-ase.

Thus, the present invention relates in a first aspect to a SPSase, a carbohydrase In a suitable form and in soy SPS is capable of decomposing under suitable conditions into decomposition products which are themselves less of protein in an aqueous medium. then attach the soybean SPS prior to decomposition itself to the same protein under similar conditions.

\

It has further been found that this SPSase, which is capable of degrading soybean SPS, is capable of degrading polysaccharide similar to SPS and derived from plants and fruits more than commercial pectinases and cellulases.

The expression "in a suitable form" indicated above aims to exclude from the invention, for example, an SPS-ase which contains a composition containing toxic substances or which exhibits such a low SPS-ase activity that the use of a composition containing more than 10 2 SPS-ase, based on the weight of SPS in the substrate, is required in a reaction for 24 hours and carried out at 50 ° C at the pH optimum of the respective SPS-ase, in order to decompose of SPS of any practical importance.

By total or partial elimination of the SPS from the final vegetable protein, the zeal of the final vegetable protein is necessarily improved as compared to the purity of the final vegetable protein, which is obtainable according to the known method of the Belgian patent specification. 882,769, since this known vegetable protein product was contaminated with SPS.

It is currently unknown whether the particular SPSase described in the following derives its enzymatic activity from a single 30-fold enzyme or from an enzyme complex containing at least two enzymes. Some studies seem to indicate that at least two enzymes are responsible for the SPSase degradation effect, one of which is capable of only minor decomposition of SPS, after which one or more enzymes are capable of a more extensive 35 carry out the breakdown of the SPS. However, applicants do not wish to be limited by such a hypothesis or similar hypotheses.

A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS-ase is capable of decomposing soybean SPS In 40 in an aqueous medium into decomposition products, which in themselves contain 8204924 * y 4 vegetable protein in the aqueous medium. attach less than the soybean SPS for decomposition would adhere to the same vegetable protein in the aqueous medium.

A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS-ase is capable of decomposing soybean SPS in an aqueous medium with a pH value that does not deviate more than 1.5 from 4.5. to decomposition products which themselves adhere to soy protein in the aqueous medium to a lesser extent than the soy SPS would decompose to the soy protein in the aqueous medium before decomposition. A preferred embodiment of the SPS-ase according to the invention is characterized in that the decomposition products of soy SPS themselves adhere to the vegetable protein to a degree of less than 50%, in particular less than 20%, after completed degradation. the soybean SPS would decompose to the vegetable protein in the aqueous medium before decomposition itself.

A preferred embodiment of the SPS-ase according to the invention is characterized in that the SPS-ase shows a positive SPS-ase test when examined according to the qualitative and quantitative SPS-ase determination method as described herein. .

A preferred embodiment of the SPS ase according to the invention is characterized in that the SPS ase was produced by a microorganism belonging to the genus Aspergillus, preferably belonging to the Aspergillus niger group.

A preferred embodiment of the SPS ase according to the invention is characterized in that the SPS ase is derived from the enzymes which can be produced by means of Asp. aculeatus CBS 101.43. The same SPS axis can be produced using Asp. japoni-cus IF0 4408. It was found that Asp. aculeatus CBS 101.43 also produces very potent remanas, cellulases, pectinases and hemicellulases. Furthermore, it was found that not every and every strain belong to the species Asp. aculeatus or Asp. japonicus develops an SPS axis necessary for the invention. Therefore, as will be seen later in the description (section 5), the strain Asp. japonicus ATCC 35 10236 does not generate such amounts of an SPS-ase as can be determined by the enzymatic determination of SPS-ase described herein.

A preferred embodiment of the SPS-ase according to the invention is characterized in that the SPS-ase is immuno-electrophoretic 40 identical to the SPS-ase, which can be produced by means of 8204924 r 5 -vule CBS. 101.43 and Identifiable by the lmmuno-electrophoretic coating technique, see sections 6 and 7.

When a SPSase has been microbially generated, it is mixed with various accompanying substances formed, especially other enzymes. If desired, the SPS axis in question can be purified, for example, by chromatographic separation methods, as will be seen later in the description (section 8).

Agr Biol.Chem 40 (1), 87-92, 1976 discloses that a strain of Asp, japonlcus ATCC 20236, produces an enzyme complex capable of partial degradation of an acidic polysaccharide in soy sauce , called APS, a fraction designated APS-I. This acidic polysaccharide is not identical to SPS, as will be shown in more detail in section 3 later in the description. Thus, the HPLC gel filtration chromatograms of SPS and APS are clearly different, and further, the gel filtration chromatograms of APS decomposed by the commercial spectinase Pectolyase and of SPS treated with the commercial spectralase Pectolyase are distinctly different. Furthermore, it does not appear from the article that the acidic polysaccharide is bound to the soy protein and that the decomposed polysaccharide is not bound to the soy protein or is bound to the soy protein to a much lesser degree than the undissected acid polysaccharide. It has also been shown that this strain does not form SPS-ase in amounts that can be determined by the enzymatic determination of SPS-ase as described herein. This created a bias against every strain of Monkey, japonlus as a producer of an SPS-ase, but surprisingly, according to the invention, some strains of Monkey, japonlus were producers of an SPS-ase.

In a second aspect, the invention includes the isolated SPS produced on the basis of a vegetable crude protein as a starting product.

A preferred embodiment of the isolated SPS of the invention is characterized in that the vegetable crude protein is defatted soybean meal. The production of this isolated SPS has been described above in connection with Figure 2.

The invention includes in a third aspect a method of selecting an SPS-ase producing microorganism for producing the SPS-ase according to the invention, wherein the microorganism to be tested is grown in a fermentation medium, the main carbon source of which SPS, after which a sample of the fermentation medium on SPS-ase is analyzed and the relevant micro-organism 8204924 6 is selected as an SPS-ase-producing micro-organism, when the analysis on SPS-ase is positive

The invention includes in a fourth aspect a method of preparing SPS-ase, wherein a strain selectable according to the previous selection method of an SPS-ase-producing microorganism is grown in a food medium. The culture can be carried out as a submerged fermentation or as a surface fermentation.

A preferred embodiment of the method according to the invention is characterized by the fact that the strain Asp. aculeatus CBS 10 101.43 or Asp, japonlcus IFO 4408 is grown in a food environment.

A preferred embodiment of the method according to the invention is characterized in that the culture is carried out as a submerged culture at a pH in the range of 3 to 7, preferably of 4 to 6, at a temperature in the range of 20 up to 40 ° C, preferably 25 to 35 ° C, the food medium containing carbon and nitrogen sources and inorganic salts.

A preferred embodiment of the method according to the invention is characterized in that the food medium contains heated soybean meal.

A preferred embodiment of the method according to the invention is characterized in that the soybean meal has been treated with a proteolytic enzyme before use as a component of the substrate, preferably the proteolytic enzyme microbially produced by means of Bacillus lichenlformis.

A preferred embodiment of the method according to the invention is characterized in that a pectin solution is added aseptically to the fermentation broth during the culture.

Asp. aculeatus CBS 101.43 also produces very potent remanence solubilizing enzymes, cellulases, pectinases and hemi-cellulases in addition to the SPSase and that the enzyme complex is produced by means of Asp. aculeatus CBS 101.43 is excellent as an agent for use in cell wall degradation of plant materials. Therefore, the enzyme complex which can be generated by Asp. aculeatus CBS 101.43 are used in the food processing industry for the treatment of fruit and vegetable batters and for clarification and viscosity-reducing purposes in the processing of juice and wine with excellent results; it can also be used as a dewatering agent (i.e., a decomposing agent for 40 polysaccharides and, consequently, for releasing the water bonded within the polymeric structure of the polysaccharides 8204924r) in the processing of vegetables.

Therefore, in a fifth aspect, the invention includes the use of an SPSase or a method of decomposing polysaccharides, preferably polysaccharides from the cell wall of plants, by means of a carbohydrase, wherein an SPSase preparation of the present invention is contacted in an aqueous medium with a substrate for this SPS-ase preparation, except for the decompositions already described in Danish patent application 5691/81.

Therefore, according to the present invention, it has been found that SPSase preparations are valuable enzyme preparations for partial or total liquefaction or decomposition of various materials, preferably vegetable materials, for example fruits and plant waste, containing protein, cellulose, hemicellulose (eg glucans, xylan, galactans and araban), gums, pectin, lipids, inulin, polyphenols, starch and alginates and for related purposes, see the table mentioned later in the description. As examples of such related purposes, all purposes for which commercial prospects and cellulases are used today may be mentioned. Some examples will be given later in the description.

In connection with the extraction (isolatle) process described in, for example, Example II, it is noted that the SPSase preparation is essentially proteinase-free, due to the fact that the desired end product, ie the protein, would otherwise be degraded. Also, if it is desired to extract (isolate) biological materials other than proteins from an impure biological product, the SPS-ase preparation used should be substantially free of any enzyme that degrades this other biological material. Such modified SPSase preparations can be prepared by selective inactivation of the undesired enzyme, by fractionation or by other methods known per se.

Thus, a preferred embodiment of the use of the present invention is characterized in that the decomposition is accompanied by the isolation or extraction of a biological material other than soy protein and related vegetable proteins from an impure biological material, wherein the SPS- the preparation is essentially free of any enzyme capable of degradation of the biological material.

Thus, according to the present invention, it has been found that the 8204924 i • 8 modified SPS-ase preparations (modified in that they are essentially free of enzymes capable of degrading the biological material to be extracted or isolated) yielding enzyme preparations are for extraction (isolation) of specified biological materials, for example starch, lipids, flavors, dyes and juices, from impure biological materials. Several examples will be given later in the description.

A preferred embodiment of the use of the present invention is characterized in that one or more of the reaction products (irrespective of whether they are desired end products or semi-products) are further treated simultaneously with or after the enzyme treatment. A more flexible and adaptable use is hereby provided.

A preferred embodiment of the use according to the invention is characterized in that the further treatment is an alcoholic fermentation in case one of the reaction products is a fermentable sugar. Hereby a simple and inexpensive method is provided for the preparation of alcohol.

In order to illustrate the nature of the invention, reference is made to the following sections 1-10, which describe all details associated with the invention: 1. Production of SPS.

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

25 3. Documentation that SPS and APS are different connections.

4. Comparative study for SPSase-generating micro-organisms.

5. Characterization of some SPS-ase forming microorganisms.

30 6. General description of the coating technique associated with immunoelectrophoresis.

7. Immunoelectrophoretic labeling of SPSase with polyspecific antibody and coating.

8. Purification of an SPS-ase preparation.

35 9. ρΗ activity dependence, temperature dependence and stability of an SPS axis.

10. Enzymatic activity determinations.

SECTION 1.

PREPARATION OF SPS.

As mentioned above, the starting material for the preparation 8204924 r 9 r of SPS may be soy manhood * Therefore, the preparation of soy manhood is described first.

Soy manhood is the protein carbohydrate fraction (which may contain minor amounts of lignin and minerals in practice) 5 in defatted and husked soybean meal, which carbohydrate fraction is insoluble in an aqueous medium at pH 4.5, and can also be obtained in the following manner, also reference is made to Fig. 1.

Defatted soybean meal (Sojamei 13 from Aarhus Oliefabrik A / S) is suspended in deionized water at 50 ° C in a weight ratio of soybean meal: water 10 * 1: 5 in a tank with pH condition and temperature control. The pH is adjusted with 4 N NaOH (I). Now a pH-state hydrolysis is performed with ALCALASE 0.6 L (a proteolytic enzyme based on B. lichenlformis with an activity of 0.6 Anson units / g, the activity being determined according to the Anson method as described in NOVO 15 ENZYME INFORMATION IB No. 058 e-GB), the enzyme / substrate ratio being equal to 4% of the amount of protein in the soybean meal (II). After 1 hour hydrolysis, the sludge is separated by centrifugation (III) and washing (IV), this operation being performed twice (V, VI, VII). The sludge thus treated is hydrolysed once more for 1 hour with ALCALASE No. 0.6 L (VIII, IX) similar as indicated above. The sludge is then separated by centrifugation (X) and washed twice (XI, XII, XIII, XIV), the final washed sludge (VI) being subjected to a spray-drying treatment (XV). The powder thus produced by spray drying is the soy mane, which serves as the raw material for the production of SPS.

SPS is the water-soluble polysaccharide fraction, which is formed by the usual treatment of the above-mentioned soy mane with pectinase. The SPS is generated in the following manner by the 14 reaction steps indicated below, also referring to Figure 2.

1. The dry matter content in the aforementioned soy mania is determined and the soy mana is diluted with water to 2% dry matter and kept in suspension in a tank under temperature control at 50 ° C.

2. The pH value is adjusted to 4.50 with 6 N NaOH.

3. Pectlnex Super cone. L is added in an amount of 200 g / kg of dry matter (a commercial spectinase from Schwelzerische Ferment AG, Basel, Switzerland with a pectinase activity of 750,000 MOU, 40 as determined according to the brochure "Determination of the Pectinase units 8204924 10

* J

% V

on Apple Juice (HOU) "of 12.6.1981, available from Scfteizerische Ferment AG, Basel, Switzerland) and also Celluclast 200 L is added in an amount of 200 g / kg dry matter, a commercial cellulose described in the brochure NOVO enzymes , information sheet B 153 e-GB 5 1000 July, 1981, available from NOVO INDUSTRI A / S, Novo Alle, 2880

Bagsvaerd, Denmark).

4. The contents of the tank are kept at 50 ° C with stirring for 24 hours.

5. The enzymes are activated by increasing the ρΉ value to 10 9.0 with 4 N NaOH. The reaction mixture is kept for 30 minutes and the pH value is adjusted again to 4.5 with 6 N HCl.

6. The reaction mixture is centrifuged and both the centrifuge product and the sludge are collected.

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

8. The control filtrate is subjected to ultrafiltration, diafiltration and further ultrafiltration on a membrane with a limit value of 30,000 (DDS GR 60-P by De Danske Sukkerfabrikker), 20 using the following parameters: 1. Ultrafiltration corresponding to a volume concentration of 6.

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

25 3. Ultrafiltration to about 15% dry matter in the concentrate.

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

9. The ultra-filtered concentrate is cooled to 5 ° C and an equal volume of 96% ethanol is added.

10. The precipitate is collected by centrifuge.

11. The precipitate is washed twice with 50% by volume ethanol in water, corresponding to the volume of the centrifuge product of step 10, i.e. two centrifugations are performed.

12. The washed precipitate is redissolved in water with a volume equal to the volume of the ultra-filtered concentrate from step 9.

13. The liquid from stage 12 is filtered under control over a glass filter.

6204924 11. The transparent filtrate containing pure SPS is lyophilized.

8204924 12 \ i

Flow chart No. 1 Degreased soybean meal H2O NaOH to pH 8 I I 1

Hydrolysis mixture_I_ - - - - - * r— - - - -------- T ----------.

Alcalase 0.6 Hydrolysis 1 hour before release II

(E / S = 4%) pH state (pH = »8, T a 50 ° C) __ 4N NaOH_ <f le centrifugation III centrifuge product 1 sludge waste 1 __

H9O? le was IV

v _ 2nd centrifugation V centrifuge product 2 ^ sludge waste 2

H9O 2nd was VI

\ t 3rd centrifugation VII centrifuge product 3 x sludge waste 3 _S '__

H9O New hydrolysis mixture VIII

NaOH to pH »8 t

Alcalase 0.6 L ,. Hydrolysis 1 hour before release IX 4N NaOH_ pH state (pH »8.0, T * 50 ° C

8204924. 13

Flow drum no. 1 (cont.) 4th spin X centrifuge product 4 sludge waste 4 _Jc_

It was IV

___ 5th centrifugation XII centrifuge product 5 sludge waste 5 __2t_

H 2 O 2nd was XIII

_itt___ 6th spin | XIV centrifuge product 6 waste sludge 6 _I--

spray drying XV

8204924; ·· '

Flow chart No. 2 1000 g Pectin * Super cone. L 5 kg spray dried remanentle

100 g Celludast 250 S.

V_ik_ ¥ _

Decomposition with pectase 247 1H 2 O 4 and cellulase 1, 2, 3, 4, 5 50 ° C; pH = 4.5; t 24 hours_ Y /

Spin sludge ^ 6 3 8 kg

V

Control filtration 7 _I_

Ultrafiltration, diafiltration, 18 1 H9O ultrafiltration 241 1 permeate ^ (GR 60P) (19 - 26 °) _ 8 _ik_________ 5 1 50% CpHqOH ^ Precipitation ^ above layer (waste)

Spin centrifuge product 10 precipitate I 2 x Wash 2 x centrifuge product 11 precipitate 8204924

* I

15

Flow Diagram No. 2 (Continued) 2 1 Η? 0 ^ Redissolve 12

Control filtration__proteinllb ^ 13 (waste)

Lyoflllserlng

310 g of lyophilized SPS

»8204924 · SECTION 2.

CHARACTERISTICS OF SPS, IN PARTICULAR ITS MOLECULAR WEIGHT DISTRIBUTION.

The molecular weight distribution of the SPS, the production of which is carried out as indicated in this description, is determined by gel chromatography on HPLC equipment (Waters pump 5 model 6000, Waters data module 730, and Waters refractometer R 401) (Fig. 4). The molecular weight distribution of the decomposition products of SPS is also determined by SPS-ase by the same method (Fig. 5). Furthermore, by the same method, the binding effect between soy protein and SPS (Fig. 6) and the absence of binding effect between soy protein and SPS decomposed by the agent of the present invention (Fig. 7) is demonstrated.

The calibration curve (the logarithm of the molecular weight versus 15 Rf, where the Rf value for glucose is arbitrarily defined as 1 and the Rf value for a specific dextran is defined as the retention time for this dextran divided by the retention time for glucose) determined by some standard dextrans with known molecular weights (T 4, T 10, T 40, T 70, T 110, T 500) from 20 Pharmacia Fine Chemicals AB, Box 175, S-75104, Uppsala, Sweden. The Rf value for the maximum of each dextran peak is found and the corresponding molecular weight is calculated as ^ Mw · Mn »where Mw is the average value of the molecular weight by weight and Mn is the average value of the molecular weight by number. 0.1 M NaNO 3 is used as an eluent for this chromatographic method. The columns used in the chromatographic method are 60 cm PW 5000 followed by 60 cm PW 3000 from Toyo Soda Manufacturing Co., Japan. In this way, the relationship between the molecular weight and the Rf for the aforementioned dex-30 tears is established, see Fig. 3.

Based on Figure 4, it can be calculated that SPS has a molecular weight distribution giving rise to a value of about 5.4 x 10 ^ and a value of Mn of about 4.2 x 10 ^. It also appears from this figure that the chromatogram 35 shows two different peaks at a retention time of 34.5 min. (6%) corresponding to a molecular weight of about 5 x 10 ^ and a retention time of 47.12 min. (67%) corresponding to a molecular weight of about 4.9 x 10 ^. It also appears from this curve that a shoulder exists between these two peaks at a retention time of 41.25 40 min. (27%) corresponding to a molecular weight of 2.8 × 10 4.

8204924 • τ 17

After decomposition of SFS with SPS-ase, the hydrolysis mixture was filtered through a membrane and the filtrate was chromatographed. It was found that about 55% SPS is decomposed into mono-, dl and tri-saccharides and the remaining 45% is decomposed into a three-peak polymer 5 with the following molecular weight: 5 x 10 ^, 10 ^ and 4, 4 x 10 ^, see fig. 5.

In order to demonstrate the binding effect between soy protein and SPS and the significant reduction of the binding effect between soy protein and SPS decomposed by means of an SPS ase, the following 10 experiments were performed.

3% SPS in 0.10 M acetate buffer at pH 4.5 is added to an isolated soy suspension (Purina E 500) to produce a suspension with an isolated product / SPS ratio of 10: 1.

This suspension is incubated on a shaking bath at 50 ° C for 18 hours. After the incubation, the suspension is centrifuged and the transparent supernatant is analyzed by HPLC as described above. Fig. 6 shows from comparison with Fig. 4 that the SPS is completely adsorbed to the isolated soy product.

The same method as indicated in the previous paragraph 20 is performed with a 3% SPS solution hydrolyzed with an SPS-ase generated by CBS 101.43 (Fig. 7). A comparison between Fig. 7 and Fig. 5 shows that no compound is adsorbed in the hydrolyzed SPS with a molecular weight below about 10 4 to the isolated soy product. The hydrolysis reduces the quantitative binding to about 10-15% in relation to the binding of SPS to soy protein.

An NMR analysis of the SPS, the preparation of which has been carried out as indicated in the present description, gives the following approximate composition of the SPS: 1) or-galacturonic acid in an amount of about 45%, with about 40% of the total amount of orgalacturonic acid is present as the methyl ester, 2) rhamnopyranose in an amount of about 20%, 3) galactopyranose in an amount of about 15% and 4) 0-xylopyranose in an amount of about 20%.

The constituents appear to be in a structure consisting of a rhamnogalacturon backbone and xylose and galactose side chains.

Complete acid hydrolysis of SPS (8 hours in 1 N sulfuric acid) and 40 subsequent thin layer chromatographic analysis indicated that only minor amounts of the monosaccharides glucose and arabinose were present in the hydrolyzed SPS.

An HPLC analysis of the SPS decomposed by the SPSase enzyme complex formed by CBS 101.43 shows an impressive reduction in molecular weight. Accordingly, the NMR spectrum of the SPS decomposed as indicated above shows that most of the ester groups have disappeared and that the xylose and galactose content in the higher molecular weight product has also decreased. The NMR spectrum of the part of the SPS decomposition product which precipitates by adding 1 part by volume ethanol to 1 part by volume SPS decomposition product is similar to the NMR spectrum of the SPS, with the modifications indicated above, with with regard to the ester groups and the content of xylose and galactose.

SECTION 3 15 DOCUMENTATION ON THE FACT THAT SPS AND APS ARE DIFFERENT CONNECTIONS.

APS was prepared as indicated in Agr. Biol. Chem., 36_, No. "4," (1972) 544-550.

Now this polysaccharide and SPS were hydrolyzed with various enzymes, after which the decomposition mixture was gel chromatographed on HPLC equipment, as indicated in section 2, "Characterization of SPS, in particular its molecular weight distribution".

In more detail, the hydrolyses were performed by treating 25 ml of solution of either 2% APS or 2% SPS in 0.1 M acetate buffer of pH 4.5 with 10 mg of KRF 68 or 30 mg of Pectolyase. KRF 68 is an SPS-ase preparation, the preparation of which is described in Example I. The results are shown in the following table.

8204924 19 19 r

Poly- HPLC gel Polysaccharide saccharide Enzyme chromatogram not decomposed 5 ____; ____ APS Pectolyase fig. 8 x APS KRF 68 fig. 9 x 10 SPS Pectolyase fig. 10 x SPS KRF 68 fig. 5 x 15 SECTION 4 COMPARISON FOR SPS- ASE PRODUCING MICROORGANISMS.

The microorganism to be examined is incubated on a slanting agar substrate with a composition that allows the growth of the microorganism. After initial growth on the angled agar substrate, the microorganism is transferred to a main liquid substrate, wherein the main carbon source is SPS (prepared as indicated), wherein the nitrogen source is ΝΟβ ", NH 2, urea, free amino acids, proteins or other nitrogen. containing compounds and which further contains a mixture of necessary salts and vitamins, preferably in the form of yeast extract The composition of the main substrate depends on the microorganism species, the main point of which is that the main substrate must be capable of to support its growth and metabolism of the microorganism When growth has taken place in an appropriate period of time, on the order of magnitude of 1 to 7 days, depending on the growth rate of the particular microorganism, a sample of the fermentation broth is analyzed for SPS-ase according to the enzymatic SPS-ase determination described in the present description or according to any an Any SPS-ase assay adapted to other specific uses of the SPS-35 ase than use as a constituent of a soy mania decomposition agent.

In order to achieve a more sensitive method for the determination of the enzymatic activity, the temperature can be lowered to 40 ° C and the incubation time can be increased to 20 hours during the determination of the SPS-ase activity, where antibiotics have to be added to the substrate 8204924. are added to avoid infection.

By following this test method, other SPSase-producing microorganisms can be found, both belonging to the genus Aspergillus or to other genera.

5 SECTION 5

CHARACTERISTICS OF SOME SPS ASE FORMING MICROORGANISMS

According to the comparative study for SPSase producing microorganisms indicated here, it was found that the microorganisms listed in the top part of the following table are SPSase products. The table also contains a strain belonging to the species Asp. japonicus, which is not an SPS-ase producer.

SPS-ase Our iden- Official first 15 producer__Type of identification- identifi- depone- yes no Asp. Asp. indication of year of citation japo acule designation nicus atus 20_____J___ x x A 805 CBS 101.43; DSM 2344 1943 x x A 1443 IFO 4408; DSM 2346 1950 25 x x A 1384 ATCC 20236; DSM 2345 1969

A brief identification of the strains indicated above can be found in the following breeding catalogs: 30 List of Cultures 1978 Central Agency for Fungal Cultures, Baarn,

The Netherlands.

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

The American Type Culture Collection Catalog of Strains I, 14th 35 edition 1980, 12301 Parklawn Drive, Rockville, Maryland 20852.

All strains in the above table closely correspond to the taxonomic description of the Asp species. japonicus and Asp. aculeatus, which appear in The genus Aspergillus of Raper and Fennell, 1965 (see, for example, pages 327-330).

40 SECTION 6 8204924 21 GENERAL DESCRIPTION OF COATING TECHNIQUE RELATED TO IMMUNO ELECTROPHORESIS.

A method referred to as the top-agar coating technique has been developed by applicants for the identification of individual components of an enzyme complex by cross-immunoelectrophoresis with a polyspecific antibody against all enzyme components in the enzyme complex. The method is based on the fact that enzymes are still active after the specific enzyme-antibody binding or, in other words, that the active enzyme site is not identical to the enzyme-antibody site. The enzyme-antibody complexes deposit as separate arcs in the gel during electrophoresis. The gel plate is covered with soluble SPS in a top agar. After heating at 45 ° C for 20 hours in an atmosphere with a relative humidity of 100%, the arc, which has SPS-ase activity, will appear as a brightening zone in the SPS-cover after precipitation with a mixture of equal volumes of ethanol and acetone when looking at it against a black background. Arcs that do not have SPS-ase activity remain invisible.

SECTION 7 IMMUNO ELECTROPHORETIC CHARACTERISTICS OF SPS ASE WITH POLY-SPECIFIC ANTI-20 BODY AND COATING.

Rabbits were immunized with the SPSase containing enzyme complex obtained by fermentation of Aspergillus aculeatus CBS 101.43 as indicated in Example I (KRF 68) and the polyspecific antibody recovered in a known manner. By means of this polyspecific antibody, a crossed immuno-electrophoresis of the enzyme complex was obtained by fermentation of Asp. aculeatus CBS 101.43, as indicated in Example I, KRF 68, performed as described in NH Axelsen et al., "A Manual of Quantitative Immunoelectrophoresis", 6 'printing 1977. Reference is made to Fig. 11, which shows the bo-30 gene. corresponding to different proteins produced by the microorganism By means of the above-described top-agar coating technique, it was found that the hatched region corresponds to SPSase.

When the aforementioned hypothesis, which assumes that SPSase consists of at least two enzymes, is correct, the shaded region is the region in which all enzymes responsible for the SPSase activity are present. If, in other embodiments of the invention, these enzymes were to be separated by immunoelectrophoresis in such a way that they do not cover each reciprocal region, some of the SPSase activity may be valid 8204924. It is immunized by electrolysis with a coating of both SPS and a commercial spectrum.

SECTION 8

PURIFICATION OF A SPS-ASE PREPARATION

The purification of the SPS-ase preparation KRF 92 (see example I) was carried out by ion exchange. The buffer is 50 mmol Tris (tris-hydroxymethylaminoethane) adjusted to pH 7 with HCl. The column is K 5/30 from Pharmacia, Sweden. The wage exchange material is DEAE-trisacryl from LKB, Bromma, Sweden (300 ml). The flow rate 10 is 100 ml / hour.

15 g of the SPS-ase preparation KRF 92 were dissolved in 450 ml of water at 6 ° C and all of the following operations were performed between 6 ° C and 10 ° C. The pH was adjusted to 7.0 with 1 M Tris. The column was equilibrated with the buffer and then the SPS-ase 15 sample was loaded onto the column. 0D and the conductivity were measured at the eluate, referring to Fig. 12. Fraction 1 is the eluate which is not bound to the wage exchange material. The column is then washed with 2000 ml of buffer to obtain fraction 2. A 0-500 mmol NaCl gradient is now established to yield fractions 3-9. All nine fractions were concentrated to 200 ml and dialyzed against water to a conductivity of 2 M mSi by dialysis (Hollow Fiber DP 2 from Amicon, Massachusetts, U.S.A.). The nine fractions were then subjected to a freeze-drying treatment. Only the 25 fractions 1 and 2 showed SPS-ase activity.

Fraction 1 was further purified by gel filtration. 1.5 g of fraction 1 were dissolved in 10 ml of 50 mmol of sodium acetate with a pH of 4.5 (500 mmol of KCl). The column is 2.5 x 100 cm from LKB. The gel filtration filler material is Sephacryl S-200 from Pharmacia, Sweden. The flow rate is 300 ml / hour. The fractions, containing materials with molecular weights between 70,000 and 100,000, calibrated with spherical proteins, contain a factor G designated as an enzyme complex, which SPS cannot decompose when tested according to the qualitative agar test; however, SPS is decomposed according to the qualitative agar test when factor G 35 is mixed with a pectinase. It has been found that factor G is able to cleave galactose, fucose and some galacturonic acid from SPS, but the major decomposition product according to the HPLC analysis is still a high molecular weight product very similar to SPS.

SECTION 9

40 pH ACTIVITY DEPENDENCE, TEMPERATURE ACTIVITY DEPENDENCE AND

8204924 23

Jp STABILITY OF AN SPS ASE.

Fig. 13 shows the pH activity dependence of the SPS-ase preparation KEF 68. A formic acid buffer system was used from pH 2.7 to pH 3.5 and an acetate buffer system 5 was used from pH 3.7 to 5.5.

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

Fig. 15 shows the temperature stability of the SPS-ase preparation KRF 68.

10 SECTION 10 ENZYMATIC ACTIVITY PROVISIONS.

The table below is a summary of the various enzymatic activity assays pertaining to the invention.

15

Definition of activity unit and description of the enzymatic activity _______ type 20 activity, publicly available later in the short description description

Enzyme described reference SPS-ase SPS-ase__x__ 25 Remanence SRU x 1 soluble- SRUM-120 x making____

Protease HUT__x__

Cellulase _C ^ __x____2_ 30 _Pu____x___3 PGE__x _4

Pectinase UP TE_ x__5 _ PEE___x___6

Heml- cellulase VHCU χ 7

The references indicated in the last column of the previous table are detailed in the following table.

8204924. : '

Reference can be obtained from i___NOVO INDUS- Schwei- 5 Refe- TRI A / S, zerische ren- Novo Alle, Ferment AG, a tie Identification of the 2880 Bagsvaerd, Basel, library no. Reference Denmark Switzerland theek 10 1 Analytical report AF 154 / 4 or 1981-12-01 x 2 Analytical Biochemistry 84, 522-532 (1978) ____ x 15 Analytical method AF 149/6-GB or 1981-05-25 x -.

3 Determination of Pectinase 20 Activity with Citrus Pectin (PU) or 23.3.1976 x 4 Viscosimetric Poly- [galacturonase-Bestimmung 25 (PGE) or 10.11.77 'x 5 Bestimmung der Pectin-transeliminase (UPTE / g) or 24.Sept .1975 x 30_____ 6 Determination of the Pectinesterase activity (undated) with initials WJA / GW x 35 ______ 7 J Analytical method AF 156/1-GB x 40 In connection with the cellulase activity determination, please note that 82Ö4924 25 '' the analysis was performed as indicated in AF 149/6-GB and that the principles of the assay are explained in Analytical Biochemistry.

SECTION 10a 5 ENZYMATIC DETERMINATION OF SPS ASE.

The enzymatic determination of SPS-ase is performed in two steps, i.e. a qualitative agar plate test and a quantitative SPS-ase activity determination based on the measurement of the amount of total released sugars. When the qualitative agar plate test is nega-10 tlef, the SPS-ase activity is zero, without regard to the value derived from the quantitative SPS-ase activity determination. When the qualitative agar plate test is positive, the SPS-ase activity is equal to the value obtained from the quantitative SPS-ase activity determination.

15 I. Qualitative agar plate test.

An SFS agar plate was prepared in the following manner. A buffer (B) is prepared by adjusting 0.3 M acetic acid to 4.5 with 1 N NaOH. 1 g SPS is dissolved in 20 ml B.

1 g of agarose (HSB Litex) is mixed with 80 ml of B and heated to boiling point with stirring. When the agarose is dissolved, the SPS solution is added slowly. The obtained 1% SPS agarose solution is placed in a water bath at 60 ° C. The plates are now poured by pouring 15 ml of the 1% SPS agarose solution onto a horizontal glass plate measuring 10 cm x 10 cm. Then, 25 places with a distance of 2.5 cm are punched out in the solidified layer of SPS agarose. 10 µl of a 1% solution of the enzyme protein to be tested for SPSase activity is placed in each site. The plate is incubated at 50 ° C and a relative humidity of 100% for 18 hours. SPS not yet decomposed is precipitated with a solution of equal parts by volume of ethanol and acetone. The SPS-ase agar plate test is positive for a sample placed in a specific location when a transparent annular zone appears around this location.

II. Quantitative SPS-ase activity determination test

The purpose of this test is to determine enzymatic activites capable of decomposing SPS to such an extent that the decomposition products exhibit greatly reduced or no adsorption or binding affinity for soy protein. Experiments have shown that that portion of the SPS decomposition products, which are not precipitated by a mixture of equal volumes of water and ethanol, have no adsorption-binding activity for soy protein.

8204924

The SPS-ase assay is based on a hydrolysis of SPS under standard conditions followed by a precipitation of that portion of SPS that is not hydrolyzed with ethanol. After precipitation, the non-precipitated carbohydrate content is determined by quantitative analysis on total sugar (according to AF 169/1, available from NOVO INDUSTRI A / S, 2880 Bagsvaerd).

The standard conditions are temperature: 50 ° C pH: 4.5. Reaction time: control 210 minutes with substrate only, followed by 2 minutes with added enzyme reaction time: main value 212 minutes.

The equipment includes: a shaking water bath with a thermostat controlled at 50 ° C. A Whirlimixer mixer centrifuge ice water bath. The reagents include; buffer: 0.6 M acetic acid in demineralized water (a) 20 1.0 M NaOH (b) substrate: the pH value of 50 ml of a is adjusted to 4.5 with b, then 4.0 g SPS are added and after dissolution of the SPS, the pH is adjusted again to 4.5 and the volume is adjusted to 100 ml with deionized water, stop reagent: absolute ethanol.

One SPS-ase activity unit SAE or SPSU is defined as the SPS-ase activity, which releases an amount of 50% ethanol soluble carbohydrate equivalent to 1 µmol galactose per minute under the standard conditions indicated above.

Even if the standard enzyme curve is a straight line in the initial section, it should be noted that it does not intersect point (0.0).

SECTION 10 b 35 ENZYMATIC DETERMINATION OF REMANIENCY SOLUBILIZING ACTIVITY EXPRESSED AS SRUM 120.

Principle

In the method of determining hydrolysis activity, the insoluble portion of degreased, deprotolized and cap-free soybean meal is hydrolyzed under standard conditions. Enzyme reaction 8204924 27 * 'is stopped with stop reagent and the insoluble part is filtered off. The amount of dissolved polysaccharides is determined spectrophotometrically after acid hydrolysis according to AF 169/1, available from NOVO INDUSTRI A / S, 2880 Bagsvaerd.

Carbohydrases with both endo and exo activity are determined by the method.

The substrate related to this enzymatic assay is identical to the remanence substrate described for the SRU method.

The substrate is dissolved as a 10% solution in the citrate buffer indicated below: 0.1 N citrate-phosphate buffer pH 4.5 5.24 g citric acid 1-hydrate (Merck type 244) 8.12 g disodium hydrogen phosphate 2-hydrate (Merck type 6580) to 1 1 demineralized H2O 15 pH 4.5 + 0.05 stable for 14 days

The stop reagent has the following composition: 100 ml 0.5 N NaOH 200 ml 96% ethanol 20 Store in a refrigerator until use.

Standard temperature conditions 50 ° C

pH 4.5 reaction time, sample 120 min.

25 reaction time, blank 5 min.

Definition of the unit

A soybean solvent solubilizing unit (SRUM) 120 (M for hand) is the amount of enzyme which liberates solubilized polysaccharides equivalent to 1 ml-30 cromol-galactose per minute under the given reaction conditions.

SECTION 10 c ENZYMATIC DETERMINATION OF PR0TE0LYTIC ACTIVITY.

HUT MEASUREMENT

Method for the determination of proteinase in an acidic environment.

The method is based on the digestion of denatured hemo globin by the enzyme at 40 ° C, pH 3.2 for 30 minutes. The digested hemoglobin is precipitated with 14% w / v trichloroacetic acid.

All enzyme samples are prepared by dissolving in 0.1 M acetate-40 buffer at a pH of 3.2.

8204924 * l * 28

The hemoglobin substrate is prepared using 5.0 g of lyophilized bovine hemoglobin powder, preserved with 1% thiomer salate and 100 ml of demineralized water, which is stirred for 10 min, after which the pH is adjusted to 1.7 with 0.33 N HCl .

After stirring for an additional 10 min., The pH is adjusted to 3.2 with 1 N NaOH. The volume of this solution is increased to 200 ml with 0.2 M acetate buffer. This hemoglobin substrate should be kept in a refrigerator for 5 days.

The hemoglobin substrate is brought to room temperature. At time zero, 5 ml of substrate are added to a test tube containing 1 ml of enzyme. After shaking for 1 second, the tube is placed in a water bath at 40 ° C for 30 min. After exactly 30 min, 5 ml of 14% trichloroacetic acid are added to the reaction tube, which is then shaken and brought to room temperature for 40 ml. For the blank test, the hemoglobin substrate is brought to room temperature. At time zero, 5 ml of the substrate are added to a test tube containing 1 ml of enzyme. After shaking for 1 second, the tube is placed in a water bath at 40 ° C for 5 min. After exactly 5 min, 5 ml of 14% trichloroacetic acid are added to the reaction tube 20, which is then shaken and brought to room temperature for 40 min.

After 40 min., The blank and the samples are shaken, filtered once or twice through Berzelius filter No. 0 and placed in a spectrophotometer. The sample is read against the blank test at 275nm while the spectrophotometer is adjusted against water.

Since the absorbance of tyrosine at 275 nm is a known factor, it is not necessary to make a standard curve for tyrosine unless it is necessary to check the Beckman spectrophotometer.

Calculations 1 HUT is the amount of enzyme which forms a hydrolyzate in absorbance at 275 nm in 1 min equivalent to a solution of 1.10 micrograms / ml tyrosine in 0.006 N HCl. This absorbance value is 0.0084. The reaction should take place at 40 ° C, a pH of 3.2 and 35 in 30 minutes.

8204924. "Γ * f 29 ___ sample blank vol. In ml HUT * - x _ 0.0084 reaction time in min.

5 sample bianco 11 cc HUT »__ x _» (S-B) x 43.65 0.0084 30 10 HDT / g «*. - <S ~ °> ^ 43'65 g of enzyme in 1 ml

An investigation on the pH stability dependence of the protease in KRF 68 conducted by the HUT analysis with pH values from 2.0 to 8.0 showed that the stability of the protease above pH 8 , 0 was very small, see fig. 16.

In order to illustrate the invention, reference is made to the following Examples 1 to VIII, wherein Example I illustrates the production of SPS-20 ase and wherein Examples II to VIII illustrate the use of SPS-ase with a soy-based raw material to provide a purified planter Protein. Other uses of SPSase are given in the section between Example VIII and the overview of the figures.

Different fermentations with the Asp.

aculeatus and Asp. japonicus were performed on a laboratory scale.

Hereby, preparations containing SPS-ase according to the SPS-ase test indicated here were obtained. Since relatively large amounts of SPS-ase are required to perform the application tests, similar fermentations were carried out on a pilot scale, see the following Example I.

Example I

Preparation of SPS-ase on a pilot scale.

An SPS ase was prepared by immersion fermentation of Asper-35 gillus aculeatus CBS 101.43.

An agar substrate of the following composition was prepared in a Fernbach flask: 8204924-430

Pepton Difco 6 g

Aminolin Ortana 4 g

Glucose X g 5 Glass extract Difco 3 g

Meat extract Difco 1.5 g KH2PO4 Merck 20 g

Malt extract Evers 20 g

H2O 1000 ml treated with ion exchanger 10

The pH was adjusted between 5.30 and 5.35. Then 40 g of Agar Difco were added and the mixture was autoclaved at 120 ° C for 20 min (the substrate is called E-agar).

The CBS 101.43 strain was grown on an inclined E-agar 15 (37 ° C). The traces of the skewed agar were suspended in sterilized skim milk and the suspension lyophilized in vials. The contents of a lyophilized vial were transferred to the Fernbach flask. The flask was then incubated at 30 ° C for 13 days.

A substrate of the following composition was prepared in a seed fermentation machine:

CaCOj 1.2 kg

Glucose 7.2 kg 25 Rofec (dry stopper of corn steep liquor) 3.6 kg soybean oil 1.2 kg

Tap water was added to a total volume of about 240 L. The pH was adjusted to about 5.5 before CaCO 3 was added. The substrate was sterilized in the seed fermenter at 121 ° C for 1 hour. The total volume for the inoculation was about 300 l.

The spore suspension from the Fernbach flask was transferred to the seed germination apparatus. The germ fermentation conditions were: 8204924 * *. > 31

Type of conventional aerated and stirred fermenter: fermenter with a height / diameter ratio of about 2.3. Stirring: 300 rpm. (two turbine blades)

Aeration: 300 normal liters of air per minute

Temperature: 30-31 ° C

Pressure: 50 kPa

Time: about 28 hours 10

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

A substrate of the following composition was prepared in a 2500 L main fermenter: 15

Heated soy flour 90 kg KH2P04 20 kg

Pluronic ® 150 ml 20 Tap water was added to a total volume of approximately 900 l. The heated soybean meal was suspended in water. The pH was adjusted to 8.0 with NaOH and the temperature was raised to 50 ° C.

About 925 Anson units of ALCALASE® 0.6 L were then added to the suspension. The mixture was kept at 50 ° C and pH 8.0 for 4 hours (addition of Na 2 CO 3) without aeration, without pressure and at 100 rpm. stir. Then the remaining substrate components were added and the pH was adjusted to about 6.0 with phosphoric acid. The substrate was sterilized in the main fermenter for 90 min at 123 ° C. The final volume for the inoculation was approximately 1080 30 1.

Then 150 l of seed culture were added.

The fermentation conditions were: 8204924.

Type of conventional aerated and stirred fermenter: fermenter with a height / diameter ratio of about 2.7. Stirring: 250 rpm. (two turbine blades)

Aeration: 1200 normal liters of air per minute

Temperature: 300C

Pressure: 50 kPa

Time: about 151 hours 10

From 24 fermentation hours to about 116 fermentation hours, a pectin solution was aseptically added to the main fermenter at a constant rate of about 8 L per hour. The pectin solution of the following composition was prepared in a 15 500 L dosing tank:

Pectin genu * 22 kg

Conc. phosphoric acid 6 kg

Pluronic® 50 ml 20 * Genu pectin (citrus type NF from The Copenhagen pectin factory Ltd.)

Tap water was added to a total volume of about 325 L. The substrate was sterilized in the dosing tank for 1 hour at 121 ° C. The final volume before the start of dosing was about 360 l. When this portion was discarded, another similar portion was prepared. The total volume of the pectin solution for a fermentation was about 725 L.

After about 151 hours of fermentation, the fermentation process was stopped. The about 1850 L of broth was cooled to about 5 ° C and the enzymes were recovered by the following method.

The culture fluid was filtered through a vacuum drum filter (Dorr Oliver), which was pre-coated with Hy-flo super-cell diatomaceous earth (filtration aid). The filtrate was concentrated to about 15% of the volume of the broth by evaporation. The concentrate was filtered through a Seitz filter sheet (type supra 100) using 0.25% Hy-flo super cell as a filtration aid (referred to as filtration I in the following table). The filtrate was precipitated with 561 g (Nfy ^ SO ^ / l at a pH of 5.5 and 4% Hy-flo-super-cell diatoms-40 soil is added as a filtration aid. The precipitate and the filtration aid 8204924 Γ 33 «> are separated by filtration over a frame filter. The filter cake is dissolved in water and insoluble parts are separated by filtration over a frame filter. The filtrate is filtered for control over a Seitz filter sheet (type supra 5 100) with 0.25% Hy-flo- supercell as filtration aid (cited in the following table as filtration II) The filtrate is diafiltrated on an ultrafiltration apparatus After the diafiltration the liquid is concentrated to a dry matter content of 12.7% (in the following table cited as dry matter content in concentrate 10).

Optional base treatment for partial removal of the protease activity can be carried out at this step. In case the base treatment is carried out, it is carried out at a pH of 9.2 for 1 hour, after which the pH value is adjusted to 5.0.

The liquid is then filtered for control and filtered for the purpose of germination and the filtrate is subjected to a freezing treatment on a freeze-drying equipment from Stokes.

Four fermentations were carried out in the manner described below, varying the strain used for the fermentation, use of the optional base treatment and other parameters, as indicated in the following table.

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

5 A. 100 g of SPS-ase preparation are dissolved in 1 l of deionized water with stirring at 10 ° C + 2eC. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The pH is then adjusted to 4.5 with glacial acetic acid and dialyzed against ice-cold deionized water to a conductivity of 3 mSi. The freezing treatment and lyofilling are then carried out.

B. 500 g of SPS-ase preparation are dissolved in 4 l of deionized water with stirring at 10 ° C + 2eC. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The pH value is adjusted to 5.1 with glacial acetic acid. The obtained material is lyophilized.

C. 50 g of SPS-ase preparation are dissolved in 400 ml of deionized water with stirring at 10 ° C + 2eC. The pH is adjusted to 9.1 with 4N NaOH. This base treatment is carried out for 1 hour.

Then the pH is reduced to 5.7 with glacial acetic acid. The material obtained is lyophilized. SPS-ase preparation used used as starting material for preparation with code preparation

base ABC

30 KRF 68 x KRF 68 Bil

KRF 68 x KRF 68 EXT

KRF 92 x KRF 92 BI

35 _L___L_I_

The aforementioned preparations are characterized by their activities of the enzymes with respect to the invention in the following table.

8204924 ’36

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Example II (application example)

This example describes the production of a p.v.p. soybean meal, "Sojamei 13" (commercially available from Aarhus Oliefabrlk A / S), freed and degreased from caps. The dry matter content of this flour 5 was 94.0% and the (N x 6.25) dry matter content was 58.7Z. The soybean meal was treated with the SPS-ase preparations KRF 68 B II (Example I) in the following manner: 85.2 g of the soybean meal were suspended and stirred at 50 ° C in 664.8 g of water and the pH was adjusted by means of from 7.5 ml 6 N HCl to 4.5 in 10. 50 g of a solution containing 4.00 g of the SPS-ase preparation were added and the reaction mixture was then stirred at 50 ° C for 240 min. The mixture was then centrifuged in a laboratory centrifuge (Beekman Model J-6B) for 15 min at 3000 x g. The supernatant was weighed and analyzed for 15 Kjeldahl nitrogen and dry matter. Then the solid base was washed with a volume of water equivalent to the mass of the supernatant obtained from the first centrifugation. This treatment was performed twice. The solid base was then lyophilized, weighed and analyzed for Kjeldahl N and dry matter at Qvist's Laboratory, Marselis Boulevard 169, 8000 Aarhus C, Denmark. This laboratory is authorized by the state for analyzes on feed and milk products. The results obtained in this experiment are shown in Table 2.1.

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8204924 39 '

Therefore, a p.v.p. obtained with a protein purity, i.e. N x 6.25 on a dry matter basis, of 91.4% and with a total protein yield of 83%.

Example III (Application Example) This example was performed to compare the protein yields, food quality and some functional properties of soy protein products prepared by the following three methods: A: The traditional isoelectric precipitation for the preparation of isolated soy protein.

10 B: The traditional isoelectric washing treatment for the preparation of concentrated soy protein.

C: The isoelectric washing treatment of the invention including a remanence solubilizing enzyme for the preparation of p.v.p.

In order to develop a true comparison of the method of the invention (C) with the conventional soy protein processes (A and B), the same raw material has been used in all cases. Also, the experiments were conducted in such a way that corresponding temperatures and treatment times are the same in all three cases.

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

A. The traditional isoelectric precipitation for the preparation of isolated soy protein.

425.8 soybean meal (Soybean egg 13 prepared by Aarhus Oliefabrik A / S) were extracted in 3574.2 g of tap water at 50 ° C. The pH was adjusted to 8.0 with 20.1 g of 4 N NaOH. After stirring for 1 hour, the suspension was centrifuged at 3000 x g for 15 min using 4 1 L beakers in a laboratory centrifuge (Beckman Model J-6B). The centrifuge product I and the precipitate I were weighed. The precipitate I was again extracted with water to a total weight of 4000 g. The temperature was kept at 50 ° C, the pH was adjusted to 8 with 4 N NaOH and the suspension was stirred for 1 hour. Centrifugation and weighing of centrifuge product II and precipitate II were performed as before. Samples were withdrawn from the centrifuge products I and II and the precipitate II for Kjeldahl and dry matter determinations. The centrifuge products I and II were then mixed and kept at 50 ° C. The protein was then isoelectrically precipitated at pH

4.5 by means of 45 g of 6 N HCl. After stirring at 50 ° C for 1 hour, the protein was collected by centrifugation at 3000 x g for 15 min.

The centrifuge product III was weighed and analyzed for Kjeldahl N

8204924. "40 and dry matter. The solid phase III was weighed and washed with water in an amount corresponding to the weight of centrifuge product I. The washing treatment was carried out by stirring at 50 ° C for 1 hour. The washed protein was collected by centrifugation at 3000 x g for 15 min. The centrifuge product IV and the solid phase IV were weighed. Centrifuge product IV was analyzed for Kjeldahl N and dry matter. The solid phase was suspended in 1550 g of water at 50 ° C and the pH was adjusted to 6.5 with 17 g of 4 N NaOH. The mixture was stirred for 1 hour and adjusted to pH 6.5 again if necessary.

Finally, the product was subjected to a freeze-drying treatment, weighed and analyzed for Kjeldahl N and dry matter. The mass balance calculations are listed in Table 3.1.

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8204924. è 43% B. The isoelectric wash for the preparation of soy protein concentrate. 425.6 g of soybean meal (Soybean egg 13 prepared by Aarhus Oliefabrik A / S) were washed in 3574 g of water at 50 ° C. The pH was washed with 44.8 g 6 N 5 HCl adjusted to 4.5 The wash was performed by stirring for 4 hours The suspension was then centrifuged at 3000 xg for 15 min using 4 1 L beakers in a laboratory centrifuge (Beckman Model J -6B) Centrifuge product I was weighed and analyzed for Kjeldahl N and dry matter Solid phase I 10 was weighed and washed again with water to a total weight of 4000 g The pH was re-heated with 1.7 g of 6 N HCl 4.5 and the suspension was stirred at 50 ° C for 30 min. Centrifugation and weighing of centrifuge product II and solids II was carried out as before. The solid phase II was resuspended in 1575 g of water at 50 ° C. C and the pH was adjusted to 6.5 with 34.5 g of 4 N NaOH. The mixture was stirred at 50 ° C for 1 hour and readjusted to pH 6.5 if necessary. Finally, the protein product was subjected to a freeze-drying treatment, weighed and analyzed for Kjeldahl N and dry matter. The mass balance is shown in table 3.2.

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8204924 * ♦ \ 46 C. The isoelectric washing treatment including a remanence solubilizing enzyme for the preparation of p.v.p.

425.8 g of soybean meal (Soybean egg 13 prepared by Aarhus Oliefabrik A / S) was washed in 3524.2 g of water at 50 ° C. The pH was adjusted to 4.5 using 43.7 g of 6 N HCl. 24 g of the SPS-ase preparation KRF 68 B III (Example I) were dissolved in 26 g of water and added to the wash mixture. The wash was then carried out with stirring for 4 hours. Then purification was performed as described for B, with the amounts of 6 N HCl, 4 N NaOH and water for resuspending being the only parameters with different values. The mass balance is given in table 3.3.

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8204924 Μ '’

Nutritional properties

The amino acid compositions of the three protein products were determined, see Table 3.4. Total essential amino acid content, chemical state and essential amino acid index (EAAI) is calculated using the FAO reference pattern from 1957.

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

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8204924 * * 53

Functional features

The nitrogen soluble heroin index (MSI) was determined in a 1% protein dispersion at pH 7.0 in 0.2 M NaCl bar. in distilled water. After stirring for 45 min with a magnetic stirrer, the suspension 5 was centrifuged at 4000 x g for 30 min and the supernatant was analyzed for nitrogen. The nitrogen solubility was calculated as (soluble NZ / total N%). The results of this valuation for the three products are shown in Table 3.6.

The emulsifying power was determined three times for each product according to a slightly modified Swift titration. 4.0 g (N x 6.25) of the product were mixed in 250 ml 0.5 M NaCl with a Sorval Omni mixer at low speed. 50 ml of the suspension was transferred to a glass mixing cup and 40 ml of soybean oil was added. The total mixture was then weighed. The oil-water mixture was then run at 10,000 rpm. homogenized with the beaker in an ice bath. An additional amount of soybean oil was added at a rate of 0.3 ml per sec. until the emulsion collapsed. The total amount of added oil for the "endpoint" was found by weighing.

The emulsifying power was calculated as ml oil per g protein 20 (N x 6.25). The density of the oil was taken as 0.5 g / ml.

The average results of the determination of the emulsifying power of the three products are shown in Table 3.6.

The beaten expansion was determined in a 3% protein solution at pH 6.5. 250 ml of the aqueous dispersion of the protein samples 25 were beaten at speed III for 4 min in a Hobart mixer (model N-50) equipped with a wire whipper. The beaten expansion was calculated according to the formula of beaten expansion - x x 1001, where V * is the beaten final volume in ml.

V was measured by refilling the mixing cup with water.

Duplicates were run for each of the three samples. The average results are shown in Table 3.6.

The foam stabilizer was determined as the ratio between the amount of foam remaining after 30 minutes run-out and the original amount of foam. 1 g of foam obtained by the previous method was placed in a plastic cylinder (diameter 7 cm, height 9 cm) with a wire net with a mesh size of 1 mm x 1 mm. The cylinder 40 was placed on a funnel on top of a glass cylinder and the 8204924.

weight (B) of liquid spilled into the glass cylinder is determined. The foam stability FS is defined by the equation FS = * A- ~ - ~ x 100%

a

5

The results of the determination are shown in Table 3.6.

The gel strength is defined in this specification as the Brookfield viscosity measured by T-axes on a Brookfield Helipath tripod. The gels were prepared by heat treatment of 12% protein suspensions in 0.5 M NaCl. The heat treatment was carried out in closed jugs with a diameter of 7.3 cm and a height of 5.0 cm placed in a water bath, maintained at 80 ° C and 100 ° C for 30 min each. The jugs were cooled and thermostat-controlled at 20 ° C before opening and measuring. The results of the measurements are shown in Table 3.6.

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Example IV (application example)

A p.v.p. was prepared according to the method described in Example III C, except that the cellulase activity was partially derived from Trichoderma reseei. The commercially available cellulase preparation 5 CELLUCLAST prepared by Novo Industrie A / S was treated with a low temperature base in the following manner. The pH of a 10% aqueous CELLUCLAST solution was adjusted to 9.2 with NaOH and the resulting solution was cooled to 5 ° C. After 1 hour at this pH and this temperature, the pH was reset to 4.7 with 20% acetic acid. This solution was kept at 5 ° C overnight and then sterile filtered. The filtrate was subjected to a freeze-drying treatment. 4 g of the product obtained by freeze-drying were added together with the SPS-ase preparation KRF 68 B III (example I). The two enzymes were solubilized in 172 g of water before addition to the wash mixture. The mass balance determinations of this example are given in Table 4.1.

The experiment shows that this particular SPSase preparation already contains an effective cellulase since addition of CELLUCLAST does not seem to affect the protein / dry matter ratio. However, other SPSase preparations may contain less cellulase, for example KRF 92, see the table immediately preceding Example II.

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Example V (application example)

According to the method described in Example III C, a p.v.p. except that all weights were reduced by a factor of 5 and the reaction mixture was cooled to about 5 ° C prior to centrifugation. Based on the analysis results related to the centrifuge products, a theoretical yield of precipitated protein is obtained, as stated in table 5.1.

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Example VI (application example)

Demonstration of the protein binding of SPS

40 g (N x 6.25) of an isolated commercial soy protein product (Purina 500 E from Ralston Purina) were dissolved in 680 g of water.

The mixture was heated to 50 ° C in a water bath and the pH was adjusted to 4.50 with 6 N HCl. 90 g of this mixture were transferred to five 250 ml Erlenmeyer flasks and 10 g of aqueous solutions, respectively. 0g, 0.2g, 0.4g, 0.8g and 1.6g of the SPS, prepared as described earlier in the description, were added. The flasks were then stirred with a magnet in a water bath at 50 ° C for 240 min.

After this, the suspensions were centrifuged at 3000 x g for 15 min and the centrifuge products I were analyzed for Kjeldahl N and dry matter. The solid phases were washed in water at room temperature and recentrifuged. This method was repeated. Then the solids were dispersed in 50 ml of water and the pH was adjusted to 6.50 by the dropwise addition of 6 N NaOH. The neutralized products were subjected to a freeze-drying treatment and analyzed for Kjeldahl N and dry matter. Based on the analyzes listed in Table 6.1, the protein recovery and the percentage of SPS bound to the protein are calculated using the formulas listed in Table 6.2.

This example shows that the SPS is tightly bound to the protein, so that the protein / dry matter ratio decreases with increasing SPS content. An SPS content comparable to about 0.4 g in 10 g of water added to 50 g of isolated protein product is the protein / SPS ratio present in the soybean meal.

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

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Example VII (application example)

This example describes the preparation of a p.v.p. using the SPS-ase preparation KRF 92 B1 in a dose of 5% of the dry matter. The method of preparation was exactly as in Example III C, 5 except that all weights were reduced by a factor of 5. The p.v.p. was analyzed as described in Example II. The results obtained in the experiment are shown in Table 7.1.

8204924 65

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Example VIII (application example)

This example illustrates the effect of pretreatment of soybean meal by jet cooking prior to the preparation of p.v.p.

Pre-treatment

A suspension of soybean meal in water consisting of 10 kg of soybean meal (Soybean 13 prepared by Aarhus Oliefabrik A / S) per 100 kg was pumped through a steam injector (type Hydroheater B-300) and with steam of 8 bar in such an amount and with a flow mixed such that a final temperature of 150 ° C for 25 sec. could be maintained in a tubular pressurized reactor. After this, the pressure was released in a relaxation room (a cyclone) and from there the suspension was passed through a plate heat exchanger and cooled to about 50 ° C. The cooled suspension could be used directly for the preparation of p.v.p. according to the invention, but in this case, the suspension was spray dried at an inlet temperature of 200 ° C and an outlet temperature of 90 ° C. The pretreated product was found to have a dry matter content of 96.5% and a protein content of 56.9% (N x 6.25).

Preparation of p.v.p.

This preparation was carried out in the following manner: 70 g of dry matter of the jet-boiled and dried soybean meal was suspended and stirred in 560 g of water at 50 ° C and the pH was adjusted to 4 by 6.5 ml of 6 N HCl at 4 , 50 set. 6 x 90 g of this suspension were transferred to six 250 ml Erlenmeyer flasks and stirred on a water bath at 50 ° C by magnetic stirrers. 10 g of a solution were added to each flask, which were resp.

0 g, 0.025 g, 0.050 g, 0.10 g, 0.20 g and 0.40 g of the SPSase preparation KRF 68 B III. The reaction mixtures were then stirred at 50 ° G for 240 min. Centrifugation was then performed at 3000 x g for 15 min.

The supernatant was then analyzed for Kjeldahl N and the solid phase was washed with water of equal volumes and centrifuged. This method was performed twice. The solid phase was then subjected to a freeze-drying treatment and analyzed for Kjeldahl M and dry matter.

A similar experiment was performed with an untreated soybean meal (Sojamei 13 from Aarhus Oliefabrik A / S) as a starting product. In this case, the enzyme substrate ratios were 0%, 1%, 2%, 3%, 4% and 8%. 40 Based on the protein content of the above layers, the 8204924 * 67 percentage of protein recovered can be calculated. The efficiency of the protein 1a based on the assumption that the enzyme product 1001 is solubilized after the reaction. The table below shows the results obtained by both experiments.

5

Table 8.1 Protein efficiency and protein dry matter ratio for p.v.p. prepared from cooked and untreated soy flour.

y ...... .............. 1 1 ......... ................. Γ "'.............. ........... ......... 1 _cooked soybean meal__ untreated soybean meal 10 E / SZ Protein Protein Protein Protein

yield Z dry matter Z yield Z dry matter Z

0 92.9 76.5 90.7 73.9 0.25 90.1 86.6 15 0.50 89.3 Λ, 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 20 8.0 - - 76.2 91.1 8204 924 9 68

In connection with either the extraction (isolation) processes for materials other than proteins or the liquefaction processes and related processes, reference is made to the general process scheme for applications as indicated in flow diagram No. 3.

The substrate can be one or more carbohydrates present in a raw material or it can be the total raw material.

This substrate can be subjected to a chemical or physical pretreatment, as will be explained later, for example, treatment with acid or alkali, soaking or soaking and / or boiling with or without steam.

The raw material can be soaked, chopped, wet milled and / or homogenized (all these treatments are referred to as homogenized in flow diagram No. 3) with or without water additives and other additives can be added during this step. The homogenization can be carried out with different efficiency, for example, different pressures are only a fraction of the maximum pressure given for the specific homogenizing device used. Various additives can be added before or during homogenization, as represented by b ^, 1> 2, ...

20 bn in flow diagram no. 3.

The reaction process, which includes the SPS-ase preparation, is carried out under specified conditions, for example, temperature, pressure, time, pH and enzyme dosage; all recommendations regarding the reactor used (eg discontinuous, plug flow) and if necessary stirring are relevant. A set of additives can be given for different raw materials, indicated as q, C2, ... cn in flow diagram no. 3.

Also, the separation processes can be performed with different efficiency. The separation is omitted or facilitated during many processes, for example when the raw material has been completely liquefied. Different separation equipment can be used (e.g. centrifuges, filters, ultrafiltration equipment, hydrocyclones, thickeners, sieves or simple decanters).

The separation efficiency is defined as the ratio between the absolute sludge content in the solid phase and the absolute sludge content of the reaction mixture.

The resulting liquid or solid phases can be further treated, for example, concentrated, dried or solvent-extracted, to remove certain components such as fat or oil, 8204924 ♦.

69 fermented for the preparation of biomass, alcohol or other products (enzymes, antibiotics or other valuable components).

The products obtained can also be recycled for repeated treatment through the process scheme.

8204924 70

Flowchart No. 3 Substrate ai -> * - £ 2 - ^ Pretreatments 2n- »I - Z 1 bi-» * - b2- »Homogenize ^ * ÏHom ^ 100%) bn —-7 - SPS-ase preparation ci - »-i - * - c„, (response to be specified 2 - $ Response c ^ _ ^ circumstances) _ £ _

Homogenize (as above) _i_. , (0 = 0- 100%) separation lSep liquid solids 4, *

Further treatments (see text) and reuse as substrate 8204924. 71

In the following some examples of applications of SPS-ase preparations are given and an overview of these applications is shown in the following list.

Also, accompanying Table A lists certain features related to flow chart No. 3.

9 8204924 m 72

List of examples of applications of SPS-ase preparations.

Type Reference SPS ase preparation no. [_______ 5 SPS ase pre- A 1 Extraction of starch from tender, wheat and potatoes prepared in A 2 Extraction of lipids from plant material the special A 3 Extraction of essential oils from plant material free of A 4 Extraction of natural dyes from plants - one or more material 10 unwanted A 5 Extraction of rubber from the guayul plantation enzyme activity___ total Ba 1 Production of a milk substitute for pets liquid Ba 2 Production of sugared starch containing soil - fabrics making Ba 3 Total liquefaction of pears and other fruits not or Ba 4 Production of juice by treatment of fruits and vegetables possible Ba 5 Treatment in connection with extraction or pressing of 20 di-treated sugar cane or sugar beet fi Ba 6 Production of soymilk ceer- Ba 7 Treatment to increase the recoverable amount of soluble coffee constituents_ SPS- ver Bb 1 Prevention and / or decomposition of ap pelt clouding 25 ase wer- Bb 2 Use as a clarifier for white wine sermons Bb 3 Production of ISSPH or other vegetable hydroplaysed protein products ras Bb 4 Mixing enzyme in the brewery in parts Bb 5 Enzyme additive before use during beer fermentation and / or storage _Bb 6 Release agent from almond flakes other Be 1 Decomposition of different waste materials> add Bc 2 Sugaring and simultaneous fermentation pass Bc 3 Decomposition of cellulose 35 singen Bc 4 Application as baking aid

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Φ cO Ü _cd ta__cq_ 75 A. 1 Extraction of starch from tender, wheat and potatoes.

Extraction of starch from tender, wheat, potatoes and other plants containing starch is carried out by one or more of the steps: soaking, wet grinding and swearing. Using an SPS-ase pre-5 preparation with substantially no amylolytic activity will provide the following advantages, using as an example tender: 1. The release of starch will be facilitated for a short soaking period, 2. The water consumption can be reduced , 3. The release of maize germ will be facilitated without release of maize germ oil. 4. The protein can be obtained with greater purity.

5. The recovery of maize soaking water will be facilitated.

15 A. 2 Extraction of lipids from plant material.

Since the lipids in plant materials are entrapped in the cells and usually bound to proteins, lipids can be extracted into an aqueous phase for treatment with an SPSase preparation which is substantially free of lipases. Therefore, corn germ oil is usually isolated by hexane extraction from dried corn germ. However, the drying treatment becomes superfluous when the wet corn germs are treated with an SPS-ase preparation of the type indicated above. Also, the extraction of olive oil in an aqueous phase can be improved if the enzyme used for the enzymatic treatment is an SPS-ase preparation of the type indicated above, see for example Food, Pharmaceutical and Bioengineering, No. 172, 74, 93-94. Also, the aqueous extraction of, for example, soybean oil, rapeseed oil and sunflower oil can be similarly improved.

30 A. 3 Extraction of essential oils from plant materials.

When plant materials containing essential oils are extracted with an aqueous solution of an SPS-ase preparation which is substantially free of enzymatic activity capable of decomposing or otherwise altering the essential oils, the essential oils will in high yields at very little cost.

A. 4 Extraction of natural dyes from plant material.

When vegetable materials containing dyes, for example carrot roots, containing the red dye betanin 40 or the dye in cranberries, are treated with an SPS-ase preparation, which is essentially free of enzyme activities, which able to decompose or otherwise change the dyes, the dyes with high yields will be recovered at very little cost.

A.5 Extraction of rubber from the guayul plantation.

Another example of a substrate for an SPS-ase preparation, which is essentially free of enzymatic activity, which is capable of decomposing natural rubber, is the cell wall material in roots and branches of the guayul plantation.

Ba 1 Production of a milk substitute for pets, preferably a calf milk substitute.

By total liquefaction in an aqueous medium of soybeans, sunflower seeds, cotton seeds, faba beans or peas, a calf milk substitute which is soluble in cold water at a pH value of about 4.5 can be produced. Using the starch-containing raw materials such as faba beans or peas, a starch liquefaction must be carried out by an α-amylase before, after or simultaneously with a treatment with an SPS-ase, which finally contains the non-starch polysaccharides as the structural material solubilizes in the cell walls. A detailed example 20 is given below using faba beans, and with regard to soybeans, reference is made to Table A. The pretreatment of these soybeans may preferably be a jet cooking treatment which improves remanence dissolution.

Example Ba 1.1

25 15 kg faba bean flour (Farine de Feves from GRANDES MINOTERIES A

FEVES DE FRANCE, Paris) were suspended in 35 l of water. 75 g of Termyl 60 L and 18 g of CaCl 2 were added. The suspension was heated to 95 ° C using a steam jacketed kettle with stirring. The suspension was then treated at this temperature for 60 minutes. The pH was then adjusted to 4.5 and the product was cooled to 50 ° C. 300 g of the SPS-ase preparation KRF 68 were solubilized in 1 l of water and added. The reaction was carried out for 440 min. When 10 g of Fungamyl 800 L is included, the starch fraction will be converted mainly to disaccharide (maltose). After this, the reaction mixture was pasteurized for 2 min at 90 ° C. A fraction of the product was then subjected to a freeze-drying treatment and used for stability tests. Then the sample was solubilized at 10% dry matter and the solution of the product could be kept stable for days without sedimentation.

8204924 'k77

Molten fat or oil can be easily emulsified in the product, yielding an elemental composition very similar to cow's milk. An emulsion containing 3.5Z oil (soybean oil) could also be kept stable for days without sedimentation.

Example Ba 1.2

Soybean meal (Soybean 13) was stirred for 25 sec. cooked at 150 ° C with a jet as described in Example VIII. The jet-cooked soybean meal was spray dried and used for further studies as described below.

A: 50 g of the jet boiled soybean meal were mixed with 450 g of water and the pH was adjusted to 4.5 with 4.1 ml of 6 N HCl. The mixture was then heated in a water bath at 45 ° C and 0.250 g of the SPS-ase preparation KRF 68 was added to the heated mixture, which was then reacted with stirring for 5 hours. The mixture was then heat treated at 80 ° C for 2 ml to inactivate the enzyme. A 100 ml sample was centrifuged at ambient temperature for 15 min at 3000 x g (g gravity). The supernatant was subjected to an ion-exchange treatment and analyzed for carbohydrate composition by high pressure liquid chromatography. Also the above layer was analyzed for Kjeldahl N and dry matter and the nitrogen solubility index (NSI) and the dry matter solubility index (DSI) were calculated; see the results in table Ba 1. 100 ml of the reaction mixture cooled to 20 ° C was poured into a 100 ml calibrated glass and kept at 4 ° C for two days. Dlips stability (%) was measured by reading the volume of the dispersions obtained (Table Ba II) after one and two days.

To 100 ml of the reaction mixture (at 20 ° C), 8 g of soybean oil was added. An emulsion was prepared by mixing in a Waring blender for 2 min. The emulsion stability (X) was measured after one and two days as before.

I!

A reaction was carried out as before, however 1.00 g of the SPSase preparation was used in this case. The same types of analyzes and stability measurements as described in section A were performed. The results are shown in Tables Ba I and Ba II.

It appears from the chemical analysis of the above layers that the values for NSI (Z) and DSI (%) obtained in the B experiment are higher than for the A experiment. However, the stability tests 8204924 w 78 performed in the reaction mixtures show a better value for the A samples. This is probably due to the longer length of the peptide chain of the proteins in the low enzyme dose reaction mixture.

The carbohydrate composition measured by high pressure liquid chromatography shows that mainly mono- and disaccharides are produced. Therefore, oligosaccharides, which are known to be responsible for diarrhea and flatulence when administered to calves in excessive amounts, are present only in small amounts.

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Ba 2 Preparation of sugared starch-containing raw materials.

In connection with the dispersion of cassava and sweet potatoes and other starch-containing plant materials, the addition of an SPS-ase preparation is able to solve viscosity counting problems. By using an SPS-ase preparation it is possible to produce starch suspensions with a dry matter content of 25-30% and after sugaring the batter can be fermented, whereby a cheap ethanol can be obtained.

Example Ba 2.1 10 On the basis of fresh and grated sweet potatoes (Japanese), a batter with a dry matter content of 24% was produced. The starch content of sweet potatoes was found to be about 70% of its dry matter content. A prior liquefaction by means of the bacterial amylase of Termamyl® 60 L in a dose of 0.5 kg / tonne of starch was carried out by heating the batter to 90 ° C. The batter was then kept at 90 ° C for 30 min. The viscosity of the reaction mixture was then measured by means of a HAAKE axis at 90 ° C.

The reaction mixture was then cooled to 55 ° C and the pH was adjusted to 5.0 with 2 N H 2 SO 4. Subsequently, a saccharification was initiated by adding the glucoamylase SAN 150 (trademark of NOVO INDUSTRI A / S) at a dose of 1.75 L / ton starch. The saccharification mixture was then divided into three parts, A, B and C, which were enzyme treated for 15 min as indicated below before viscosity measurement took place: 25 A: This part is the control part. The viscosity was μ2

measured, see Table Ba III

B: The Trlchoderma viride cellulase of Celluclast® 200 N was added at a dose of 1 kg / ton of sweet potato dry matter. The viscosity μβ was measured, see table Ba III 30 C: The SPS-ase preparation of KRF 68 was added in a

dosage of 0.25 kg / ton of sweet potato dry matter. The viscosity was measured, see Table Ba III

Table Ba III Viscosities_

Reaction mixture Viscosity Viscosity

35__ at 90 ° C__ at 55 ° C

pre-liquefied sweet potatoes μ ^ * 770 cp V1 2 * 2190 cp A - μ 2 * 2190 cp B - μ 3 * 1970 cp 40 C - μ 4 * 950 cp 8204924> 82

Therefore, it can be seen that the viscosity of the reaction mixture can be effectively reduced with the low dose SPSase as compared to Celluclast® and SAN 150.

Ba 3 Total liquefaction of pears and other fruits.

When whole pears, which have been mechanically pressed, are subsequently treated with an SPS-ase preparation, a total liquefaction takes place and a transparent pear juice is produced after removal of minor amounts of solid. A similar method can be used in connection with other similar fruits, eg apples.

Example Ba 3.1

Fresh apples were coarsely ground using a Bucher Central grinder. The apple batter was then pasteurized in a jacketed vessel for heating at 90 ° C for 5 min and then cooled to ambient temperature. The pre-crushed apples were then ground on a corundum stone Fryma mill until the batter was smooth to the touch. The batter was then pasteurized for 10 min at 80 ° C and cooled to 50 ° C.

Enzyme reactions were now performed at 50 ° C for 30 min with the Contraves Rheomat 15, with agitation and viscosity measurement (related to a percentage reading on the rheometer at speed 13) being performed simultaneously. After completion of the enzyme reactions, 100 g samples were withdrawn and centrifuged at 3000 x g in a calibrated tube for 15 min. The percentage of juice and the percentage of deposition are measured here. The pH and percentage of dry matter refractometer as ° Brix were also measured. Table Ba IV shows a comparison between the effect of SPS-ase, the combination of Celluclast and SPS-ase and the combination of Celluclast and Pectinex. The SPSase preparation KRF 68 was used.

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Ba 4 Preparation of juice by treatment of fruits and vegetables.

It has been found that SPS-ase preparations are well suited for the preparation of juice by treating various fruits, berries and vegetables, for example carrots, peas, tomatoes, apples, pears, black currants, beans and cabbage. Hereby, an improved juice yield and a better extraction of color and flavor components are achieved compared to commercially available pectinase and cellulase preparations.

Example Ba 4.1 Reference is made to Example Ba 3.1, in which the SPSase preparation has been compared with the commercially available conventional cellulase and pectinase products Celluclast® 200 L and Pectinex® 3x. The table shows that the juice yield can be slightly improved with only 50 g / hl SPS-ase compared to 2000 g / hl Pectinex®, 15 rings in combination with 50 g / hl Celluclast®. The viscosity was also slightly lower. Thus, it appears that the SPS axis is about 40 times more effective than Pectinex.

Ba 5 Treatment in connection with extraction or pressing of sugar cane or sugar beet.

It has been found that it is possible to improve the efficiency in connection with simple extraction processes when the SPS-ase preparation is used for the treatment of sugar cane or sugar beet before and / or during its extraction or pressing. The remanence (the ampas) can also be treated with the SPS-ase preparation, partly converting it into fermentable sugars, which can be used as raw material for ethanol fermentation.

Example Ba 5.1 10 kg of sugar beet remanence (pulp) obtained from continuous counter-current extraction in a DDS diffuser at Nakskov Sugar Factory 30 was ground twice in a Fryma mill (type MZ-110). Process water was added during the grinding operation.

Portions of 300 g of the pulp were now enzyme-treated at 45 ° C for 18 hours using the enzyme dosages given in Table Ba V. The dry enzyme product (KRF 68) was added to the pulp 35, which was stirred with a bar for the first hour.

The pulp was then liquefied to such an extent that magnetic stirring can be carried out gradually over the remaining time. At the end of the reaction, the pH was measured (no pH corrections were made during the start of the reaction) and the reaction mixture was centrifuged until a clear supernatant was added 8204924 * * (no pH corrections were made during the start of the reaction). the reaction was carried out) and the reaction mixture was centrifuged until a clear supernatant was obtained. Dry matter determinations were made on the reaction mixtures and on the supernatants. Based on these results, the percentages of solubilized dry matter were calculated. Corrections for the soluble dry matter of the enzyme product were made in all calculations.

The above layers 2, 3 and 4 were treated with an ion exchanger and analyzed by high pressure liquid chromatography on the carbohydrate composition.

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5 _ Experiment no_ sugar type _2__3__4_ (neutral)% of neutral sugars high molecular weight (DP4 +) 43.6 31.9 25.3 10_____ disaccharides 4.6 4.8 glucose 20.4 23.7 27.8 15 galactose 5.0 5.9 7.3 fructose / arabnose 26.4 32.2 33.2 galacturonic acid not measured 20 __

All sugars formed according to the preceding Table Ba VI could be fermented to alcohol or used for other purposes.

25 Ba 6 Preparation of soy milk.

Soy milk can be easily produced by total liquefaction of ground soybeans and subsequent homogenization of the resulting mixture. Soy milk is often produced by soaking soybeans in boiling water, grinding the soaked beans and extracting with water followed by separation of insoluble residues, for example, proteins and polysaccharides. To improve the efficiency of the soymilk, these insoluble residues can be liquefied by reaction with the SPSase.

Example Ba 6.1 35 The soy milk process is illustrated by the following series of enzyme reactions, where calculations of the protein solubility index (PSI, Z) and the dry matter solubility index (DSI,%) illustrate the yields obtained after separation at pH 7 (see Table Ba VII). The enzyme reactions were conducted under the following conditions: 8204924

Substrate: complete fat soybean meal (Dansk Sojakagefabrlk A / S)

Reaction mixture weight: 220 g 5 Substrate weight: 20 g

Temperature: 50eC

pH: 4.5 (6 N HCl)

Response time: series A: 1 hour

Response time: series B: 0.5-6 hours 10 Enzyme: SPS-ase (KRF 68)

Enzyme Dosage: Series A: E / S Ratio (W / W): 0-8.0% Series B: E / S Ratio (W / W): 1.0% 15

After the reaction, the pH was adjusted to 7 by 4 N NaOH and a separation was performed by centrifugation at 3000 x g for 15 min.

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Ba 7 Treatment in order to increase the recoverable amount of soluble coffee constituents.

It has been found that the treatment of coffee beans at different stages during the production of instant coffee results in an increased yield of soluble coffee ingredients. Thus, for example, spent ground coffee products or green beans with enzyme can be treated with favorable results.

Bb 1 Prevention and / or decomposition of apple or pear clouding.

After the production of apple or pear juice and other fruit juices, which must be transparent, and which have been pretreated with conventional pectase and cellulase preparations to avoid cloudiness, apple cloudiness or similar fruit cloudiness may appear. It has been found that the SPSase preparations are very suitable for decomposition of such haze consisting mainly of protein-bound araban.

Example Bb 1

Concentrated pear juice prepared by enzymatic liquefaction of pear canned waste using Celluclast® and Pectinex® was found to be cloudy upon storage. The cloudiness was eliminated. 20 isolated and hydrolysed with 0.01 N H 2 SO 4 for 24 hours and analyzed by high pressure liquid chromatography. The chromatogram showed arabinose and small amounts of oligosaccharides.

By incubating 0.5 w / v% of this carbohydrate in 1 mmol acetate buffer at pH 4.5 for 3 hours at 40 ° C with SPS-ase (KRF 68 + 25 KRF 92 1: 1) with an enzyme concentration of 0.05 w / v%, found that 84% of the original turbid carbohydrate (dry matter) had been converted to arabinose.

Dilute per concentrate (20 ° Brix) was also treated for 2 hours at 40 ° C with an enzyme dose of the aforementioned SPS-30 ase of 0.15 w / v% or with a commercial product Clarex® in a dose of 1 wt ./full.%. It was found that the SPS ase was able to reduce the relative HPLC peak region of an araban-like haze by 86%, while the corresponding reduction with Clarex® (used in a much higher dose than the SPS-ase preparation) was only 35 78 % used to be.

Bb 2 Use as a preparation for white wine.

It has been found that white wines, which exhibited highly undesirable cloudiness, can be effectively cleared by SPS-ase. It is shown that the turbid material mainly consists of arabinogalactans, which are bound to hydroproline residues in a structural protein of the cell wall.

Bb 3 Production of ISSPH or other vegetable protein hydrolysates.

Prior to the separation of ISSPH (Ioelectrically-soluble soy protein hydrolyzate) or other vegetable protein hydrolyzates from the sludge, as described in U.S. Patent 4,100,024 or in Process Biochemistry, 14 No. 7 (1979), 6-8 and 10-11, the reaction mixture can be treated with an SPS-ase preparation.

An easier separation is hereby obtained.

10 Bb 4 Batter enzyme in the industry lndustrle.

In the preparation of beer, carbohydrates from the raw materials, for example the β-glucans of malt and barley, influence the viscosity and the filterability of the wort. Addition of SPS-ase during mashing will reduce the viscosity of the wort and improve fll-15 digestibility and extract yield. Furthermore, adding SPS-ase during the mashing will increase the fermentability of the wort and the nitrogen content in the wort.

Example Bb 4.1

In the laboratory, 50 g of milled grains, consisting of 50% malt and 50% barley, were battered together with 275 g of water (15% dry matter) according to; the following batter flag: 52eC (60 min.) / 63 ° C (60 min.) / 76 ° C (30 min.).

To illustrate the effect of the SPS axis, four experiments were performed, see the table below, adding the enzymes during the batter (batter pH 5.5-6.0).

* 8204924 t 92 «

Enzyme none Cereflo SPS-ase (KRF 68) 5 Activity of

beta-glucanase / g 0 200 BGU 1630 FBG 1630 FBG

Dosage of enzyme per kg of granules 0 1.5 kg 0.05 g 0.18 g 10_____

Total dosage of enzyme activity units

present per kg of grains 0 300 BGU 80 FBG 300 FBG

15 Rapid filtration of wort after 10 minutes 120 ml 135 ml 160 ml 170 ml

Viscosity of wort

10 ° Balling (25eC) 1.52 cP 1.36 cP 1.36 cP 1.30 cP

20 ____ BGU is β-glucanase units determined by the AF 70/4-GB analytical method available from NOVO INDUSTRI A / S.

FBG is fungus β-glacanase units determined by the analytical method AF 70.1 / 2-GB available from NOVO INDUSTRI A / S.

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

Cereflo is a bacterial β-glucanase preparation described in information bulletin B 214b-GB 1500, July 1981 available from NOVO IN-30 DUSTRI A / S.

Example Bb 4.2

In the laboratory, 50 g of milled grains, consisting of 40% malt and 60% barley, were battered together with 150 g of water (25% dry matter) according to the following batter diagram: 45 ° C (60 min.) / 63 ° C (90 35 min.) / 75 ° C (15 min.).

In order to demonstrate the effect of the SPS axis, three tests were performed, see the table below, adding the enzymes during the batter (pH of the batter 5.5-6.0).

8204924 r% 93

Enzyme no Ceremlx SPS-ase (KRF 68) +

Ceremix added as 5 ____________________________ in previous trial_

Activity of

beta-glucanase / g - 200 BGU 1630 FBG

Dosage of 10 enzyme per kg of granules - 1.65 kg 0.033 g

Total dose of enzyme activity unit

present per kg of grains 0 330 BGU 50 FBG

15 ______

Filtration rate of wort after 30 minutes 48 ml 98 ml 111 ml

Extract, ° Balling 18.6 19.0 19.5 20 Wort viscosity

10 ° Balling (25 ° C) 1.72 cP 1.37 cP 1.27 cP

The definition of BGU and FBG is as indicated in example Bb 25 4.1. In the last column in the previous table, only the activity and the dosage from SPS-ase are indicated.

Ceremix is a bacterial β-glucanase preparation described in information bulletin B 216d-GB 1000, February 1982, available from NOVO INDUSTRI A / S.

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

SPS-ase can be added during wort fermentation or beer storage to reduce the β-glucans content thereby improving beer filtration and beer stability with respect to cloudiness. SPS-ase will also affect proteins responsible for cold turbidity.

Bb 6 Release agent for almond peels.

During the mechanical removal step of almond shells after peeling almonds, a certain percentage of the almond shells are not loosened. It has been found that treatment of the 8204924 t 94% almonds with enzyme results in a decrease of the aforementioned percentage.

Be 1 Decomposition of various waste materials.

In connection with certain manufacturing processes, large amounts of waste carbohydrate-containing materials are formed. For example, this is the case in connection with the production of isolated soy product by extraction with water and precipitation with acid, soyulk and tofu (a special kind of Japanese cheese). Waste pulp from, for example, apples, pears or citrus fruits can also be mentioned in this connection. It has been found that an SFSase preparation is capable of completely liquefying this carbohydrate-containing waste material and producing fermentable sugars, which can be used as a starting product for ethanol fermentation.

Example Bc 1.1 15 In the usual production of soy milk or tofu, soybeans are usually immersed in boiling water, ground and extracted with warm water, after which a separation is carried out. The residue from this separation is the material used for the experiment. The liquid phase is the soy milk, which can be further treated to produce tofu.

10 kg of whole soybeans obtained from Aarhus Oliefabrik A / S were simultaneously ground with 70 l of boiling water in a Fryma mill type MZ 110. The ground suspension was then held above 85 ° C for 15 min to inactivate the natural bean enzymes, which develop the known unpleasant soybean odor. 5 L of this soybean suspension were then centrifuged at 3000 x g (g * gravity) in the laboratory for 15 min. Analysis found that the remanence contained 20.45% and 20.06% dry matter (duplicate determinations, calculated average 20.26%). 6N HCl was added slowly and processed in the retentivity with a spatula until a pH meter showed the value 4.50 when the electrode was directly introduced into the mass.

Enzyme reactions on 2 x 200 g of the mass with the two doses of the SPS-ase (KRF 68), E / S = 0.5% relative to dry matter and E / S * 3.0% relative to dry matter were carried out in a 500 ml beaker at 50 ° C. The dry enzyme was added to the mass. During the first 1-2 hours, the stirring treatment was carried out with a spatula, after which the mass was liquefied to such an extent that magnetic stirring could be successfully performed. The total reaction time was 21 hours. During the reaction, osmolality was measured with an osmo-40 meter (Advances Digimatic 3DII from Advanced Instruments Ine.).

8204924 "4 * 95

The results in Table Be 1 show the progress of the reaction. At the end of the experiment, the mixtures were centrifuged at 3000 x g for 15 ml. A layer of oil appeared on top of the above layers and its volume was determined. The supernatant was removed with a pipette, including the oil. The oil was combined with the transparent aqueous phase by homogenization and a sample was taken for dry matter determinations. The results shown in Table Be I clearly show that this waste product can be liquefied by an enzymatic reaction and that crude oil can be produced as stated in section A 4. After recovery of the oil, the solubilized remanence can be used in various ways, for example for fermentation into valuable compounds or for concentrating and drying and subsequent use as feed or food product or after further purification to produce valuable products.

8204924 t 1 Λ * 96

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8204924% ^ 97

Be 2 Sugaring and simultaneous fermentation.

Carbohydrate-containing plant materials, eg tubers such as Jerusalem artichokes, potatoes, sweet potatoes, cassava or pulp of such tubers, ie the material remaining after removal of the extracted components, can be sweetened by treatment with an SPS-ase preparation and simultaneously the fermentable saccharides formed are fermented to ethanol.

Example Bc 2.1

The preparation of ethanol by fermentation of the decomposed inulin 10-containing Jerusalem artichokes was examined on a laboratory scale by simultaneous saccharification with SPS-ase and inulinase and by four different pretreatments of the Jerusalem artichokes.

SPS axis:

The SPSase preparation KRF 68 was used.

15 Inulinase:

The inulinase was generated by fermentation of Asp. ficuum (CBS 55565). The inulinase activity is determined as described in Research Disclosure No. 21234 (December 1981), pp. 456-458.

Laboratory Fermentation: 20 aliquots of 150 g of the pretreated batter (described later) were fermented after adding 4.5 g of baker's yeast and 1 ml of a 4% Pluronic solution as an anti-foaming agent. The ferm-tattoo flasks are supplied with CO2 traps containing 98% sulfuric acid and the fermentation is followed by measurement of the weight loss due to released CO2 · The contents of the flasks are stirred during the fermentation carried out at 30 ° C . Three flasks were used for each parameter examined.

In Table Be II, the weight loss due to CO2 release is converted to ethanol assuming 1 mole of released CO2 is equivalent to 1 mole of C2H5OH, i.e.

1 g CO2 / w ^ 8 C2H5OH.

Pre-treatments of the artichokes:

Treatment A: 14 kg artichokes (22.8% dry matter) were cooked according to Henze at 140 ° C and 400-500 kPa for 20 35 min. The weight after cooking was 19.0 kg (/ v 16.9% dry fabric). Fermentations were performed directly on the batter.

Treatment B: Washed and cut artichokes were mixed with water (1: 1) and mixed in a Waring blender. The batter was then treated at 85 ° C at 8204924 * 98 for 1 hour at a pH of 4.5.

Treatment C: Like B, but the pH was not adjusted.

Treatment D: Like B, but no heat treatment and no pH control.

5 Results:

In Table Be II, the results show the effect of adding SPS-ase to the pretreated batter on the ethanol yield. A significant improvement in ethanol efficiency was obtained with all pretreated batters when SPS-ase was added.

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Be 3 Decomposition of cellulose.

It has been found that cellulose-containing materials such as straw, for example wheat straw, sawdust, paper and lignocellulose, can be hydrolyzed to a greater degree with an SPSase preparation 5 than with conventional cellulases. This is illustrated by the following example wherein a crystalline cellulose material (AVICEL) is treated by a conventional cellulose Celluclast® 200 produced by Trlchoderma reesei and the SPS-ase preparation KRF 68. Example Bc 3.1 10 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 suspension was filtered and the reducing sugar content (mg glucose / g AVICEL) was measured. Using enzyme dosages of 5% and 20% of the cellulose content, the following values were found:

Table Be III

Enzyme E / S% mg glucose / g AVICEL

20_____

Celluclast 5 80 SPS-ase 5 200

Celluclast 20 100 25 SPS-ase 20 340

Bc 4 Use as a baking aid.

It has been found that SPSase preparations are excellent as baking aids. Thus, when an SPS-ase preparation is added to the dry flour prior to making the dough, it is possible to obtain a bread of better quality in volume, crumb and flavor. Thus, it is possible to obtain a high quality bread with a low quality wheat flour when using an SPS-ase preparation as an additive.

Bc 5 Improvement of the alcohol yield and the biomass yield during the fermentation of sulphite waste liquor from papermaking.

It has also been found that the ethanol efficiency can be improved when paper sulfite waste liquor is treated with a 3204924 * 101 SPS-ase preparation before being used as a carbohydrate source for ethanol fermentation. Paller sulfite waste liquor can also be used for the manufacture of biomass, for example single cell protein, by fermentation and in this case too the biomass yield is improved when the sulflet waste liquor has been pre-treated with an SPS-ase preparation. Also, decomposition due to the presence of the SPSase preparation and fermentation can be performed simultaneously.

Bc 6 Dewatering of biological sludge products.

During the usual extraction with water of many biological materials from plant raw materials, large volumes of insoluble residue, consisting of large amounts of swollen polysaccharides, are formed. This is the case, for example, when soy milk, tofu or isolated soy product is produced by water extraction of soybeans, degreased soybean meal or white flakes. The structural, swollen polysaccharide material can then be treated to a small extent with SPS-ase, opening the network structure of the material and dissolving only small amounts of carbohydrates. The material is thereby dewatered and consequently a higher dry matter content in the sludge is obtained compared to a product without the enzymatic treatment. Therefore, the enzyme process exhibits the advantage of a significantly lower energy consumption for water removal by drying and also opens the possibility for the production of a cheaper dry animal feed material or bulk material for food applications.

Bc 7 Silage Aid.

It is known to add enzymatic silage aids to silage in order to increase the ensilage speed and digestibility of the silage. It has been found that SPS-30 ase preparations are better compared to the known enzymatic coloring aids.

8204924 * ♦% * 102

An overview of the figures already referred to is given below for the purpose of providing a more concise overview.

Fig. Belongs To Describes 5 No.

1 the general part demonstrating the binding effect of the description between SPS and soy protein 10 2 the general part the flow chart, which describes the preparation of the description SPS 3 section 2 the calibration curve for HPLC gel filtration chromatography 15___

4 section 2 the HPLC gel filtration chromatogram of SPS

5 section 2 the HPLC gel filtration chromatogram of SPS

decomposed by SPS-ase 20___ 6 sections 2 and 3 the HPLC gel filtration chromatogram of the above layer from SPS incubated with soy protein 7 section 2 the HPLC gel filtration chromatogram of the above layer from decomposed SPS incubated with soy protein

8 section 3 the HPLC gel filtration chromatogram of APS

Decomposed by pectolyase

9 Section 3 The HPLC Gel Filtration Chromatogram of APS

decomposed by SPS-ase

35 10 section 3 the HPLC gel filtration chromatogram of SPS

treated with pectolyase 11 section 7 the immunoelectrophoretic peaks including an SPS-ase peak identified 40 by a coating technique 8204924 * · 103 (continued) 12 section 8 the ion-exchange chromatogram of an SPS-ase 5___ 13 section 9 the pH activity dependence of an SPS-ase 14 section 9 the temperature activity dependence 10 of an SPS-ase 15 section 9 the temperature stability of an SPS-ase 16 section 10 the pH stability of protease in an SPS-15 ase preparation 8204924

Claims (21)

1. SPS-ase, a carbohydrase in a useful form and capable of decomposing soybean SPS under decomposition products, which, in an aqueous medium, themselves attach to a lesser degree of protein than the soybean SPS before decomposition itself to the same protein would adhere under similar conditions. 2. SPS-ase according to claim 1, characterized in that the SPS-ase is capable of decomposing soybean SPS in an aqueous medium into decomposition products, which themselves adhere to a lesser degree of vegetable protein in the aqueous medium than the soy SPS before decomposition would attach itself to the same vegetable protein in the aqueous medium. SPS-ase according to claim 1 or 2, characterized in that the SPS-ase is capable of decomposing soybean SPS in an aqueous medium with a pH value which does not deviate more than 1.5 from 4.5. decomposition products which themselves adhere to a lesser degree of soy protein in the aqueous medium than the soy SPS for decomposition would themselves adhere to the soy protein in the aqueous medium. SPS-ase according to claims 1 to 3, characterized in that the decomposition products of soy SPS, after complete degradation, adhere themselves to the vegetable protein to a degree of less than 50%, in particular less than 20%, than the soy SPS would attach to the vegetable protein in the aqueous medium before decomposition itself. SPS axis according to claims 1 to 4, characterized in that it 25 SPS-ase shows a positive SPS-ase test when examined according to the qualitative and quantitative SPS-ase determination method. SPS ase according to claims 1 to 5, characterized in that the SPS ase is produced by means of a microorganism® belonging to the genus Aspergillus, preferably belonging to the Aspergillus 30 niger group. SPS ase according to claims 1 to 6, characterized in that the SPS ase is derived from the enzymes which can be produced by means of Asp. aculeatus CBS 101.43. SPS axis according to claims 1 to 7, characterized in that it SPS-ase is immunoelectrophoretic identical to the SPS-ase that can be produced by Asp. aculeatus CBS 101.43 and identifiable by the immunoelectrophoretic coating technique. 9. Isolated SPS, characterized in that the isolated SPS is produced on the basis of vegetable impure protein as raw material. Insulated SPS according to claim 8, characterized in that the 8204924 4 'm vegetable impure protein is non-fat soybean meal. 11. A method for selecting an SPS-ase producing microorganism for the preparation of the SPS-ase according to claims 1 to 8, characterized in that the micro-organism to be tested is grown on a fermentation medium, the main carbon source of which is SPS according to claim 9 or 10, after which a sample of the fermentation medium is analyzed on SPS-ase and the micro-organism concerned is selected as an SPS-ase producing micro-organism, when the analysis on SPS-ase is positive * Process for preparing SPS-ase according to claims 1 to 8, characterized in that a strain which is selectable according to the selection method of claim 11 is cultivated in a food medium. Process according to claim 12, characterized in that the strain Asp. aculeatus CBS 101.43 or Asp, japonicus IFO 4408 cultures in a nutritional environment. Method according to claim 12 or 13, characterized in that the culture treatment is carried out as an immersion culture treatment at a pH in the range from 3 to 7, preferably from 4 to 6, in a temperature in the range from 20 to 40 ° C, preferably from 25 to 35 ° C, the food medium containing carbon and nitrogen sources and inorganic salts. Process according to claims 12 to 14, characterized in that a food medium containing soy flour is used. 16. A method according to claim 15, characterized in that the soybean meal is treated with a proteolytic enzyme prior to its use as a component of the substrate, preferably the proteolytic enzyme, which is produced microbially by Bacillus licheniformis. 17. Process according to claims 12 to 16, characterized in that a sterile pectin solution is added aseptically to the fermentation broth during the cultivation treatment. 18. A process for decomposing polysaccharides, preferably polysaccharides from plant cell walls, by means of a carbohyse, characterized in that an SPS-ase preparation according to claims 1 to 8 is contacted with a substrate for the SPS-ase preparation, except for the decompositions already described in Danish patent application 5691/81. Process for decomposing polysaccharides according to claim 18, characterized in that the decomposition is accompanied by isolating or extracting a biological material other than 8204924 V Η * soy protein and related vegetable proteins with an impure biological material. wherein the SPSase preparation is substantially free of any enzyme capable of degrading the biological material. Process for the decomposition of polysaccharides according to claim 18 or 19, characterized in that one or more of the reaction products (irrespective of whether these are desired end products or waste products) is further simultaneously with or after the enzymatic treatment. Process for decomposing polysaccharides according to claim 20, characterized in that an alcoholic fermentation is used as further treatment in case one of the reaction products is a fermentable sugar. 8204924