SE461659B - PROCEDURES FOR PREPARING ENZYMET SPS-AS WITH THE ABILITY TO SUBSTITUTE HIGH-MOLECULE CARBON HYDROGEN - Google Patents

Description

461 659 aunt and was washed (see part I, columns a and b, fig. 1). In this way, a pure residue or residue (called residue I] was isolated (column B, Fig. 1). Part II of the soybean meal was not treated; for the sake of simplicity, the residue in Part II is called residue II (column B in Fig. 1). I remained as Part II using a commercial pectinase, eg PECTINEX (a pectolytic enzyme produced by Schweizerische Ferment A / G, Basel, Scheíz) (see columns b and c in Fig. 1). the undissolved portion of residue I is much smaller than the undissolved portion of residue II, based on the mass balances of nitrogen and dry matter, see Fig. 1, where the dashed areas in column c correspond to insoluble non-protein materials in the above step.

Furthermore, if the supernatant from the pectinase-treated residue I is combined with a soy protein suspension at pH 4.5, a polysaccharide in the supernatant binds to the soy protein. This polysaccharide in the supernatant from residue I, which is part of the residue decomposition product and which is definitely soluble in water in the absence of soy protein, but bound to soy protein at or around the isoelectric point of soy protein, if soy protein is present, is called SPS (§Oluble golysaccharide), see Fig. 1. This SPS has a molecular weight distribution of between 5 x 106 and 4.9 x 104 of the flow chart shown in Fig. 2, which also includes some. The production of isolated SPS is shown by the processes shown in Fig. 1. The problem is thus to find an enzyme which has the ability to decompose SPS in such a way that the SPS decomposition products do not bind soy protein or bind soy protein to a much lesser extent than SPS binds soy protein. Although the description specifically relates to soy protein, the invention is not limited to soy protein but includes all kinds of vegetable proteins, see e.g. the proteins listed in BE patent no. 882,769, page 1.

According to the invention, it has been found that by thinning or so-called screening regarding the ability to decompose soy-SPS it is possible to select or find microorganisms which show the ability to produce a compound which shows such enzymatic efficiency that it effectively decomposes soy-SPS, which in the future for the sake of simplicity will be called SPS-as . Thus, the invention relates to a process for the preparation of an SPS ash, a carbohydrate in useful form and with the ability to decompose soy SPS under suitable conditions into decomposition products, which themselves are coupled to or attached to protein in an aqueous medium to a lesser extent than soy-SPS before decomposition would have done to the same protein under similar conditions.

Furthermore, it has been found that this soy-degrading SPS-as SPS exhibits the ability to degrade polysaccharides, which are similar to SPS and derived from vegetables and fruits, in a more complete way than commercial pectinases and commercial cellulases. The above expression "in useful form" is intended to exclude from the invention e.g. an SPS-as-containing preparation which contains toxic substances or which exhibits low SPS-as activity, which necessitates the use of more than 10% SPS-as-containing preparation, related to the weight of SPS in the substrate, in a reaction carried out for 24 hours and at S0 ° C, at the pH value which is optimal for the SPS-as in question, in order to obtain a decomposition of SPS of any practical significance.

By total or partial elimination of SPS from the final vegetable protein, the purity of the final vegetable protein is necessarily improved as compared with the purity of the final vegetable protein which can be obtained according to the method known from BE patent no. 882,769, since this known vegetable protein product was contaminated with SPS.

At present, it is not known whether the particular SPS-as described below derives its enzymatic activity from a single enzyme or from an enzyme complex comprising at least two enzymes. Some studies seem to indicate that at least two enzymes are responsible for the SPS-as-degradation effect, one of these enzymes exhibiting the ability to perform only a slight decomposition of SPS, after which one or more enzymes exhibit the ability to perform a more extensive degradation of SPS. However, we do not want the invention to be limited by this hypothesis or similar hypothesis.

The SPS ash prepared according to the invention has the ability to decompose soy SPS in an aqueous medium into decomposition products, which themselves adhere to vegetable protein in the aqueous medium to a lesser extent than the soy SPS before decomposition would have made the same vegetable protein in the aqueous medium. The SPS aset exhibits more specific ability to decompose soybean SPS in an aqueous medium having a pH not exceeding 1.5 to 4.5 into decomposition products which themselves adhere to soy protein in the aqueous medium. to a lesser extent than soy-SPS before decomposition would have done with the soy protein in the aqueous medium.

For a preferred embodiment of the process according to the invention, the decomposition products of soybean SPS after complete degradation themselves adhere to the vegetable protein to an extent of less than 50%, especially less than%, compared to what soybean SPS before decomposition would have done on the vegetable protein in the aqueous medium.

The SPS axis produced according to the invention is characterized in that it exhibits a positive SPS axis test, when examined according to the qualitative and quantitative SPS axis determination method described in the present description.

More specifically, the method of the invention is characterized by culturing an SPS-as-producing microorganism belonging to the genus Aspergillus, preferably the group Aspergillus niger, which organism exhibits the ability to grow in a medium containing SPS as the main carbon source in a nutrient medium containing carbon and nitrogen sources and inorganic salts, and that, if necessary, the formed SPS-as is isolated.

According to a preferred embodiment, the SPS-as-producing microorganism is the strain Asp. aculeatus CBS 101.43 or gsp, japonicus IFO 4408. It has been shown that ¿_p. aculeatus CBS 101.43 also produces highly active remanases, cellulases, pectinases and hemicellulases. Furthermore, it has been found that not every tribe belonging to the genus §_p. aculeatus or_A_p. japonicus generates an SPS-axis required by the invention.

Thus, as will be seen later in the present specification (Section 5), it has been shown that the strain Asp, japonicus ATCC 20236 does not produce such amounts of an SPS-as that can be detected by the enzymatic determination of SPS-as described in 461 659 present description.

A further preferred embodiment of the invention means that the produced SPS-axis is immunoelectrophoretically identical to the SRS-axis which can be produced by means of êsp. ggyieatuš cßs 101.43 and is identifiable using the immunoelectrophoretic coating technique, see sections 6 and 7.

When an SPS-as is produced microbially, it is formed in a mixture with several accompanying substances, especially other enzymes.

If desired, the SPS can be purified, e.g. by means of chromatographic separation methods, as will appear below in the description (section 8). ° In Agr.Biol.Chem 40 (1), 87-92, 1976 it is described that one strain of ê_p. japonicus, ATCC 20236, produces an enzyme complex which is capable of carrying out a partial decomposition of an acidic polysaccharide in soy sauce, called APS, of which a fraction is called APS-I. This acidic polysaccharide is not identical to SPS, as will be shown in more detail later in the present description in section 3. Thus, the HPLC gel filtration chromatograms for SPS and APS are clearly different, and in addition, the gel filtration chromatogram for APS is decomposed by the commercial pectinase Pectolyase and for SPS treated with the commercial pectinase Pectolyase clearly different. Furthermore, it does not appear from the article that the acidic polysaccharide is bound to the soy protein and that the decomposed acidic polysaccharide is not bound to the soy protein or is bound to the soy protein to a much lesser extent than the non-decomposed acidic polysaccharide. In addition, it has been shown that this strain does not form SPS-as in amounts that can be detected by the enzymatic determination of SPS-as discussed in the present specification. This created prejudice against that of a tribe of êsp. japonicus could produce an SPS ace, but according to the invention it has now surprisingly been found that certain strains of fi sp. japonicus produces an SPS axis.

The process according to the invention can be carried out on the basis of vegetable crude protein as starting material.

This vegetable crude protein may be defatted soy flour.

The production of this isolated SPS is described above in connection with Figure 2. 461 659 For selection of an SPS-as producing microorganism for the production according to the invention of SPS-as, the tested microorganism is allowed to grow on a fermentation medium, the main carbon source of which is SPS, after which a sample of the fermentation medium is analyzed for SPS-as and the microorganism in question is selected as an SPS-as-producing microorganism, if the analysis for SPS-as is positive.

The cultivation according to the invention can be carried out as a submerged fermentation or as a surface fermentation.

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

A preferred embodiment of the process according to the invention is characterized in that the nutrient medium contains roasted soybean flour. Another preferred embodiment of the process according to the invention is characterized in that the soybean meal is treated with a proteolytic enzyme before it is used as a component in the substrate, preferably the proteolytic enzyme produced microbially using Bacillus licheniformís. Yet another preferred embodiment of the process according to the invention is characterized in that a solution of pectin is added aseptically to the fermentation broth during the cultivation. aculeatus CBS 101.43 also produces highly active residue solubilizing enzymes, cell- It has been found that Asp. lulases, pectinases and hemicellulases in addition to the SPS acetate, and that the enzyme complex produced using Asp. aculeatus CBS 101.43 is particularly well suited as an agent for use in cell wall disintegration of vegetable materials. Thus, the enzyme complex prepared from fi sp. aculeatus CBS 101.43 is used in the food processing industry for the treatment of fruit and vegetable purees and for clarification and viscosity-reducing purposes in the processing or production of juices and wines with excellent results; it can also be used as a dewatering agent (ie a means for decomposing polysaccharides 461 659 and thus for releasing the water bound in the polymer structure of the polysaccharides) in the processing of vegetables.

The SPS-ace prepared according to the invention can thus be used in a method for decomposition of polysaccharides, preferably polysaccharides in plant cell walls, by means of a carbohydrate, where an SPS-as preparation prepared according to the invention in an aqueous medium is introduced into contact with a substrate for said SPS-as preparation.

It has thus been found that SPS-as preparations prepared according to the invention are valuable enzyme preparations for partial or total melting or decomposition of a plurality of materials, preferably vegetable materials, e.g. fruit and plant waste, containing protein, cellulose, hemicellulose (eg glucans, xylan, galactans and araban) rubber materials, pectin, lipids, inulin, polyphenols, starch and alginates, and for related purposes, see the table given later in present description. Examples of such related purposes are all purposes for which commercial pectinases and cellulases are used today. Several examples will be given later in the description.

In connection with the extraction (isolation) process described, for example, in Example 2, it can be noted that the SPS-as preparation is essentially protein-free, depending on the fact that the desired end product, i.e. the protein, otherwise it would be degraded. If one wishes in the same way to extract (isolate) biological materials other than protein from a biological raw material, the SPS-as preparation used should be substantially free of enzyme which degrades this other biological material. Such modified SPS-as preparations can be produced by selective inactivation of the undesired enzyme, by fractionation or by other methods known per se.

Thus, the decomposition may be accompanied by isolation or extraction of a biological material other than soy protein and related vegetable proteins from a biological raw material, whereby the SPS-as preparation is substantially free of enzyme capable of degrading said biological material. According to the invention, it has thus been found that the modified SPS-as preparations (modified in the sense that they are substantially free of enzymes capable of degrading the biological materials to be extracted or isolated) are valuable enzyme preparations for extraction (isolation) of specified biological materials, e.g. starch, lipids, flavorings, colorings and fruit juices, from biological raw materials. Several examples will be given later in the description.

Such use means that one or more of the reaction products (regardless of whether they are desired end products or waste products) are further treated simultaneously with or after the enzyme treatment fl This results in a more flexible and adaptable use.

An alternative use means that the additional treatment is an alcoholic fermentation or fermentation, if one of the reaction products is a fermentable sugar. This provides a simple and inexpensive method for producing alcohol.

In order to further clarify the invention, reference is now made to the following sections 1-10, all of which describe details in connection with the invention: 1. Preparation of SPS. 2. Characterization of SPS, especially its molecular weight distribution. Documentation of the fact that SPS and APS are different associations. 4. Screening of SPS-as-producing microorganisms.

. Characterization of certain SPS-as-forming microorganisms. 6. General description of the coating technique associated with immunoelectrophoresis. Immunoelectrophoretic characterization of SPS-as with polyspecific antibody and coating. Purification of an SPS-as preparation. 9. pH activity dependent, temperature activity dependent and stability of an SPS axis.

. Enzymatic activity determinations. 461 659 SECTION 1.

Ergduction of SPS.

As previously mentioned, the starting material for the production of SPS may be soy residue. The first round therefore describes the production of soy residue.

Soy residue is the protein-free carbohydrate fraction (which in practice may contain small amounts of lignin and minerals) in defatted and peel-free soybean meal, which carbohydrate fraction is insoluble in an aqueous medium at pH 4.5, and it can be prepared in the following way, also referring to flow chart 1. - Degreased soy flour (Soy flour 13 from Aarhus Olíefabrik 'A / S) is suspended in deionized water of 50 ° C in a weight ratio soy flour water = 1: 5 in a tank with pH state and temperature control - ring. The pH is adjusted to 8.0 with 4N NaOH (I). Thereafter, a pH-stat hydrolysis is performed with ALCALASE 0.6 L (a proteolytic enzyme based on B. licheniformis with an activity of 0.6 Anson units / g, whereby the activity is determined according to the Anson method, as described in NOVO 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 one hour of hydrolysis, the sludge is separated by centrifugation (III) and washing (IV), this operation being carried out twice (V, VI, VII). The sludge thus treated is hydrolyzed once more for one hour with ALCALASE 0.6 L (VIII, IX) in the same manner as previously indicated.

The sludge is then separated by centrifugation (X) and washed twice (XI, XII, XIII, XIV), whereby the final washed sludge (6) is spray-dried (XV). The spray-dried powder thus produced is the soybean residue which acts as a raw material for the production of SPS.

SPS is the water-soluble polysaccharide fraction formed by conventional treatment of the above-mentioned soy residue with pectinase. This SPS is produced in the following manner by means of the 14 reaction steps given below, reference also being made to Flowchart 2. 461 659 1.

. The dry matter content of the above soy residue is determined, and the soy residue is diluted with water to 2% dry matter content and kept in suspension at SOOC in a tank with temperature control. The pH is adjusted to 4.50 with 6N NaOH.

Pectinex Super conc. L is added in an amount of 200 g / kg dry material (a commercial pectinase from Swiss Ferment AG, Basel, Switzerland, with a pectinase activity of MOU 750,000, determined in accordance with the publication "Determination of the Pectinase units on Apple"). Juice (MOU) "from 12.6.1981, published by Schweizerische Ferment AG, Basel, Sdnæiz), and also Celluclast 200 L are added in an amount of 20 g / kg dry material (a commercial cellulase described in the brochure NOVO enzymes, information sheet B 153 e-GB 1000 July 1981, published by NOV0 INDUSTRI A / S, Novo Alle, 2880 Bagsvaerd, Denmark).

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

The enzymes are inactivated by raising the pH to 9.0 with 4N NaOH. The reaction mixture is kept for 30 minutes, and the pH is then adjusted back to 4.5 with 6N HCl.

The reaction mixture is centrifuged, and both the centrifugate and the sludge are collected.

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

The control filtrate is ultrafiltered, diafiltered and ultrafiltered once more on a membrane with a cut-off value of 30,000 (DDS GR 60-P from De Danske Sukkerfabrikker), 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 (fl «0 ° Brix). Ultrafiltration to about 15% of the raw material in the concentrate.

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

The ultrafiltered concentrate is cooled to 5 ° C, and an equal volume of 96% ethanol is added.

The pellet is collected by means of a centrifuge. 11. 12. 13. 14. 11 461 659 The precipitate is washed twice with 50% (v / v) ethanol in H 2 O, which corresponds to the volume of the centrifugate from step, ie. two centrifugations are performed.

The washed precipitate is redissolved in water with a volume equal to the volume of the ultrafiltered concentrate from step 9.

The liquid from step 12 is control filtered on a glass filter.

The clear filtrate containing pure SPS is lyophilized. 12 461 659 FLUDESCHEMA No. 1.

Degreased soy flour H29 NaOH¿tí1l pH = 8 I Hydrolysis mixture l I 4 Alcalase 0.6 L> Hydrolysis 1 hour before discharge II E = pH-stat (pH = 8, T = S0 ° C) 4N Nao fl, // ”¿# ~ First Centrifugation III Centrifugation 1 Awfall ------ ä & Slam 1 H20 I First Washing IV> I Second Centrifugation V Centrifugation t 2. ga; Avfa11 I Slam 2 H20 Second wash lvl> I Third centrifugation “VII Centrifuge 3 vfall .________________, Å J, Slam 3 H20> New hydrolysis mixture VIII Na0H to = 8 l F 'Waste Iàll AÄÉÉÄÉÉÉ-QLÉ-ä-> Hydrolysis for 1 hour before ušmatníng. IX 4N NaOH ¿_% pH-stat (pH = 8,0, T = 50 C) Fourth centrifugation X Centrifugation 4 in sludge 4 H20 First wash XI> J »Fifth centrifugation XII Centrifugation 5 VL Slam 5 H20 Second wash [XIII> 4 Sixth Centrifugation XIV J, Slam 6 Spray Drying XV Centrifugation 6 Waste ”461 659 FLOOD SCHEME NO. 2. 1000 g Pectinex Super conc. L 5 kg of egg-dried residue 100 g Celluclast 250 S l 1 Decomposition with pectinase '247 1 H 2 O and cellulase 1, 2, 3, 4, 5 ”__ * _> so ° c; pH = 4.5; t = 24 tins Centrifugation Slam 6 l ss kg> Control filtration- Ultrafiltration diafiltration- 18 1 H20> ring, ultrafiltration 211 1 permeate "___" (GR ooP) (19 - 26%) ----_-> s ä à 33 % Precipitation Supernatant> 9 2 S> l (waste) Centrifugation ___ Centrifugation> 10 l Precipitation 2 x washing l Z x centrifugation> 11 LFä11ning 2 1 H20 [Reconstitution l> [Control filtration of protein sludge> 13 Iavïallï 1 lLyofser10 gSl 461 659 '4 SECTION 2.

Characterization of SPS, especially its molecular weight distribution.

Using gel chromatography on HPLC equipment (Waters pump model 6000, Waters data module 730 and Waters refractometer R 401) the molecular weight distribution of SPS is determined, the preparation of which is carried out as described in the present description (Fig. 4) - Using the same method the molecular weight distribution of the decomposition products of SPS has also been determined by means of SPS-as (Fig. 5). Using the same method, the binding effect between soy protein and SPS (Fig. 6) and the absence of binding effect between soy protein and SPS southernized by means of the agent according to the invention were also demonstrated (Fig. 7).

The calibration curve (the logarithm of the molecular weight plotted against Rf, where the Rf value of glucose is arbitrarily defined as 1 and the Rf value of a specific dextran is defined as the retention time of this dextran divided by the retention time of glucose) has been achieved by several standard dextrans. (T 4, T 10, T 40, T 70, T 110, T S00) from Pharmacia Fine Chemicals AB, Box 175, S-75104 Uppsala, Sweden. The rf value of the maximum for each dextran peak has been determined, and the corresponding molecular weight has been calculated as the MW ~ H n) value and En is the number average molecular weight. 0.1 M NaNO 3 was used as eluent for this chromatographic procedure. The columns used where HW is the weight average molecule in the chromatographic procedure are 60 cm PW S000 followed by 60 cm PW 3000 from Toyo Soda Manufacturing Co., Japan. In this way, the relationship between the molecular weight and Rf of the above-mentioned dextrans was determined, see Figure 3.

On the basis of Fig. 4, it can be calculated that SPS has a molecular weight distribution which gives an HW value of about 5.4 x 105 and an En value of about 4.2 x 104. This figure further shows that the chromatogram shows two clear peaks at the retention time of 34.5 minutes (65), corresponding to a molecular weight of about 5 x 10 6, and the retention time of 47.12 minutes (67%), corresponding to a molecular weight of about 4.9 x 10 4. the curve further shows that a shoulder appears between these two peaks at the retention time of 41.25 minutes (27%), which corresponds to a molecular weight of 2.8 x 105.

After decomposition of SPS with SPS-as membrane membrane, the hydrolysis mixture was filtered, and the filtrate was chromatographed. It was found that about 55% of SPS has decomposed into mono-, di- and trisaccharides, and that the remaining 45% has decomposed into a polymer with three peaks with the following molecular weights: 5 x 104, 104 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 binding effect between soy protein and SPS broken down by means of an SPS axis, the following experiments were performed. 3% SPS in 0.10 M acetate buffer at pH 4.5 is added to a slurry of soy isolate (Purina E 500) in order to form a suspension with an isolate / SPS ratio of 1021. This suspension is incubated in 18 hours in a shaking bath at 50 ° C. After incubation, the suspension is centrifuged, and the clear supernatant is analyzed by HPLC as previously described. From Fig. 6 in comparison with Fig. 4 it appears that SPS is completely adsorbed at the soy isolate.

The same procedure as described in the previous paragraph is performed with a 3% SPS solution hydrolysed with an SPS-as produced using CBS 101.43 (Fig. 7). A comparison between Fig. 7 and Fig. 5 shows that no compound in the hydrolyzed SPS having a molecular weight below about 104 is adsorbed on soy isolate. The hydrolysis reduces the quantitative binding to about 10-15% in relation to the binding of SPS to soy protein.

An NMR analysis for SPS, the preparation of which is carried out as described in the present description, reveals the following approximate composition for the SPS: 1) α-galacturonic acid in an amount of approximately 45%, whereby approximately 40% of the total amount of α-galacturonic acid is present as the methyl ester. 2) ramnopyranose in an amount of approximately 20%, 3) galactopyranose in an amount of approximately 15%, and 4) β-xylopyranose in an amount of approximately 20%.

The components appear to be present in a structure comprising a ramnogalacturon backbone and side chains of xylose and galactose. A complete acid hydrolysis of SPS (8 hours in 1 N H 2 SO 4) and subsequent TLC analysis revealed that even smaller amounts of the monosaccharides fucose and arabinose were present in the hydrolyzed SPS.

An HPLC analysis of SPS decomposed by the SPS-as enzyme complex formed by CBS 101.43 shows a sharp reduction in molecular weight. Accordingly, the NMR spectrum of SPS decomposed as described above shows that the majority of the ester groups have disappeared and that the content of xylose and galactose in the higher molecular weight material has also decreased. The NMR spectrum of the part of the SPS decomposition product which precipitates on addition of a volume of ethanol to a volume of SPS decomposition product is similar to the NMR spectrum of SPS, with the above-mentioned modifications concerning the ester groups and the content of xylose and galactose.

SECTION 3.

Documentation of the fact that SPS and APS are different associations.

APS was prepared as described in Agr.Biol.Chem., Vol. 36, no. 4, pp. S44-550 (1972). This polysaccharide and SPS were hydrolyzed with various enzymes, after which the decomposition mixture was gel chromatographed on HPLC equipment, as described in section 2, "characterization of SPS, especially its molecular weight distribution".

In more detail, the hydrolyses were performed by treating 25 ml of a solution of either 2% APS or 2% SPS in 0.1 M acetate buffer pH 4.5 with 10 mg KRF 68 or 30 mg Pectolyase.

KRF 68 is an SPS-as preparation, the preparation of which is described in Example 1. The results are shown in the following table.

I, Poly-Enzyme HPLC-gel- 'Polysaccharide saccharide chromatogram Decomposed Non-decomposed APS Pectolyase fig. x I APs KRP ss Fig. 9 * x SPS Pectolyase Fig. 10 x SPS KRF 68 figs. 5 x SECTION 4.

Screening for SPS-as-producing microorganisms.

The tested microorganism is incubated on an agar slope substrate with a composition that enables the growth of the microorganism. After initial growth on the agar slope substrate, the microorganism is transferred to a liquid main substrate, in which the main carbon source is SPS (prepared as described), in which the nitrogen source is NO 3 -, NH 4 +, urea, free amino acids, proteins or other nitrogen-containing compounds, smiles a mixture of essential salts and vitamins, preferably in the form of yeast extract. The composition of the main substrate depends on the genus of microorganism, the main requirement being that the main substrate should be able to provide growth and metabolism for the microorganism. When the growth has taken place for a suitable period of time, of the order of 1 to 7 days, depending on the growth rate of the microorganism in question, a sample of the fermentation broth is analyzed for SPS-as according to the enzymatic SPS-as determination described in the present description or according to any other SPS-as determination tailor-made for specific uses of the SPS-ace other than use as a component of a soybean decomposition agent.

In order to provide a more sensitive method for the determination of the enzymatic activity, the temperature can be lowered to 40 ° C and the incubation time increased to 20 hours during the determination of the SPS-as activity, whereby antibiotics should be added to the substrate in order to avoid infection.

By following this test method, other SPS-as-producing microorganisms can be found, both those belonging to the genus Aspergillus and those belonging to other genera.

SECTION S.

Characterization of certain SPS-as-forming microorganisms.

According to the screening method for SPS-as-producing microorganisms described herein, it has been found that the microorganisms listed in the upper part of the following table are SPS-as-producing. The table also contains a tribe belonging to the genus Asp. japonicus, which is not SPS-as-producing.

SPS-as- Släkte Vår identi- Official First producing leading identification deposit - designation be- Year J N A. A. _. _ a e] jâgo- aâgie- “ammg nicus actus X X A 805 CBS 101.43; 1943 DSM 2344 x x A 1443 rFo 4408; DSM 2346 1950 I X A 1384 ATCC 20236; DSM 2345 1969 A brief identification of the above-mentioned strains can be found in the following cultural catalogs: ZS 461 659 W List of Cultures 1978 Centraalbureau voor Schimmelcultures, Baarn, The Netherlands.

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

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

All strains in the above table correspond very closely to the taxonomic description of the strains Asp. japonicus and Asp. aculeatus in The genus Aspergillus of Raper and Fennell, 196š (see especially pp. 327-330). .

SECTION 6.

General description of the coating technique in connection with immunoelectrophoresis.

A method, known as the top agar coating technique, has been developed by us for the identification of individual components in an enzyme complex by cross-linked immunoelectrophoresis with a polyspecific antibody to 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 expressed in another way that the active enzyme site is not identical to the site of the enzyme-antibody binding. The enzyme-antibody complexes precipitate as clear arcs in the gel during electrophoresis. The gel plate is coated or covered with soluble SPS in a top agar. After heating to 45 ° C for 20 hours in an atmosphere with a relative humidity of 10U%, the arc possessing SPS-as activity appears as a clear zone in the SPS cover layer after precipitation with a mixture of equal large volumes of ethanol and acetone, when viewed against a black background. Arcs that do not show any SPS-as activity are left invisible. sexrion 7. 'Immunoelectrophoretic characterization of SPS-as with polyspecific antibody and coating.

Rabbits were immunized with the SPS-as-containing enzyme complex obtained by fermentation of Aspergillus aculeatus CBS 101.43, as described in Example 1 (KRF 68), and the polyspecific antibody was recovered in a manner known per se. Using this polyspecific antibody, cross-linked immunoelectrophoresis of the enzyme complex obtained by fermentation of Asp was performed. aculeatus CBS 101.43, as described in Example 1 (KRF 68), as described in N.H. Axelsen et al, "A Manual of Quantitative Immunoelectrophoresis", 6th edition 1977. We refer to Fig. 11, which shows the arcs corresponding to the different proteins produced by the microorganism. Using the previously described top agar coating technique, it is established that the dashed surface corresponds to SPS-as.

If the previously mentioned hypothesis, which means that it is assumed that SPS-as consists of at least two enzymes, is correct, the dashed area is the area in which all enzymes responsible for SPS-as 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 any mutual surface, part of the SPS-as activity can still be identified by immunoelectrophoresis with a coating with both SPS and a commercial pectinase.

SECTION 8.

Purification of an SPS-as preparation.

The purification of the SPS-as preparation KRP 92 (see Example 1) was performed by ion exchange. The buffer substance is 50 mM Tris (tris-hydroxymethylaminomethane), which is adjusted to pH 7.0 with HCl. The column is K 5/30 from Pharmacia, Sweden. The ion exchange material is DEAE trisacrylic from LKB, Bromma, Sweden (300 ml). The flow rate is 100 ml / hour. g of the SPS-as preparation KRF 92 was dissolved in 450 ml of H 2 O at 600, and all of the following operations were performed at between 600 and 10 ° C. The pH was adjusted to 7.0 with 1 M Tris. The column was equilibrated with the buffer substance, and then the SPS-as sample was introduced on the column. OD280 and the conductivity was measured on the eluate, referring to Fig. 12. Fraction 1 is the eluate which is not bound to the ion exchange material. The column is then washed with 2000 ml of buffer, giving rise to fraction Z. A 0-S00 mM NaCl gradient is now developed, giving rise to fractions 3-9. All nine fractions were concentrated to 200 ml and dialyzed against water to a conductivity of 2 mSi by dialysis (Hollow Fiber DP 2 from Amicon, Massachusetts, USA). The nine fractions were then lyophilized. Only fractions 1 and 2 showed SPS-as activity. 461 659 2 ° Fraction 1 was further purified by gel filtration. 1.5 g of fraction 1 was dissolved in 10 ml of SO 4 mM sodium acetate with pH 4.5 (50 mM KCl). The column is 2.5 x 100 cm from LKB. The gel filter filler material is Sephacryl S-200 from Pharmacia, Sweden.

The flow rate is 30 ml / hour. The fractions containing materials with molecular weights between 70,000 and 100,000, calibrated with globular proteins, contained an enzyme complex called factor G, which cannot decompose SPS when tested according to the qualitative agar test; however, SPS decomposes according to the qualitative agar test when factor G is mixed with a pectinase. Factor G has been found to be capable of cleaving galactose, fucose and some galacturonic acid from SPS, but the main decomposition product according to the HPLC analysis is still a high molecular weight product, which is very similar to SPS.

SECTION 9. pH activity dependence, temperature activity dependence and stability of an SPS axis.

Fig. 13 shows the pH activity dependence of the SPS-as preparation KRF 68. From pH 2.7 to pH 3.5 an formic acid buffer system was used, and from pH 3.7 to 5.5 an acetate buffer system was used.

Pig. 14 shows the temperature activity dependence of the SPS-as preparation KRF 68.

Pig. 15 shows the temperature stability of the SPS-as preparation KRF 68.

SECTION 10.

Enzymatic activity determinations.

The table below is an overview of the various enzyme activity determinations relating to the invention. 21 461 659 IDefinition of activity unit and description of determination of enzymatic activity ä IS1ag of activity, Published Described later in K Enzyme .short name .previously this description Reference SPS-as SPS-as x §Remans-: SRU X_ 1 solubí- _ àšseran § §RUM-Izo x Proteas Y HUT x Cellulas CX x 2 Pu x 3 - f Pekuinas PGE x 4 1 UPTE XS PEE x 6 Hemicellulas VHCU x 7 ¿I The references given in the last column of the above table are reported in more detail in the following table. 461 659 23! Reference can be obtained from NOVO INDUSTRI A / S, Schweizerische Novo Allé, Ferment AG, Reference Identification 2880 Bagsvaerd, Basel, Switzerland A biblio- Nr. ; of reference Denmark tek 1 Analysis Regulation AF 154/4 from 1981- x 12-01 Analytical Biochemistry 84, x 2 522 -532 (1978) Analytical method AF 149/6-GB from x 1981-05-25 Determination of Pectinase Acti - '3 vity with Citrus x Pectín (PU) från 23.3.1976 Viskosirnetrische 4 Polygalacturonase- Bestimnlmg (PGE) x från 10.11 .77 Bestimrrung der S Pectin-trarxseli- ntínase (UPTE / g) x från 24.9.1975 Determinatíon of the Pectinester - 6 ase activity x (undated) with initials NJA / GW 7 Analytical method In connection with the cellulase activity determination, it can be noted that the analysis was performed as described in AF 149/6-GB and that the principles for the determination are explained in Analytical Biochemistry.

SBKTION 1D a.

Enzymatic determination of SPS-as.

The enzymatic determination of SPS-as is performed in two steps, ie. a qualitative agar plate test and a quantitative SPS-as activity determination based on measurement of the total amount of released sugars. If the qualitative agar plate test is negative, the SPS-as activity is zero, regardless of the value derived AF 156 / 1-65 J 23 461 659 from the quantitative SPS-as activity determination. If the qualitative agar plate test is positive, the SPS-as activity is equal to the value derived from the quantitative SPS-as activity determination. “I. Qualitative agar plate test.

An SPS agar plate was prepared as follows. A buffer (B) is prepared by adjusting 0.3 M acetic acid to a pH of 4.5 using 1 N NaOH. 1 g of SPS is dissolved in 20 ml of B. 1 g of agarose (HSB Litex) is mixed with 8% ml of B and heated to boiling point with stirring. When the agarose is dissolved, add the SPS solution slowly. The resulting 1% SPS agarose solution is placed in a 60 ° C water bath. The plates are now cast by holding 15 ml of the 1% SPS agarose solution on a horizontal glass plate measuring 10 cm x 10 cm. Then make nine depressions at a distance of 2.5 cm in the solidified layer of SPS agarose. In each well, 10 μl of a 1% solution of the enzyme protein to be tested for sPs-es activity is introduced. The plate is incubated for 1 hour via the solution with a relative humidity of water. Thereafter, non-decomposed SPS is precipitated with a solution of equal volumes of ethanol and acetone. The SPS-as agar plate test is positive for a sample placed in a specific well, if a clear annular zone appears around that well.

II. Quantitative SPS-as activity determination test.

The purpose of this test is to determine enzyme activities which have the ability to decompose SPS to such an extent that the decomposition products exhibit greatly reduced or no adsorption or binding affinity to soy protein. Experiments have shown that some of the SPS decomposition products, which are not precipitated by a mixture of equal volumes of water and ethanol, have no adsorption or binding affinity to soy protein.

The SPS as determination is based on hydrolysis of SPS under standard conditions followed by a precipitation of the part of SPS that is not hydrolyzed with ethanol. After precipitation, the content of carbohydrate, which has not been precipitated, is determined by quantitative analysis of tou fl t swker (according to AF 169/1, available from 461 659 24 NOVO INDUSTRI A / S, 2880 Bagsvaerd). êëëaêêrêlzetiasëlëêraënërër = Temperature: SOOC pH: 4.5 Reaction time: comparison 210 minutes with substrate only, followed by 2 minutes with added enzyme main value 212 minutes. ëëzuëtainseniuneíaëtër = Shake water bath thermostated to 50 ° C Whirlimixer stirrer Centrifuge Ice water bath BÉê8ÉE§§E_ÉQ§ÉfêÉÉêIl Buffer: 0.6 M acetic acid in demineralized water (a) 1.0 M NaOH (b) of the substrate for: is adjusted to 4.5 with b, and then 4.0 g of SPS is added, and after dissolving the SPS, the pH is readjusted to 4.5, and the volume is adjusted to 100 ml with deionized water. In Stop reagent: Absolute ethanol.

An SPS-as activity unit (SAE or SPSU) is defined as the SPS-as activity which, under the standard conditions specified above, releases an amount of carbohydrate soluble in 50% ethanol equivalent to 1 pmol galactose per minute. Although the initial part of the enzyme standard curve is a straight line, it can be noted that it does not intersect the point (0.0).

SECTION 10 b.

Enzymatic determination of residual solubilizing activity expressed as SRUM 120.

Principle In the method for determining hydrolysis activity, the insoluble part of defatted, de-proteinised and peel-free soybean meal is hydrolyzed under standard conditions. The enzyme reaction is stopped with a stop reagent and the insoluble portion 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. Carbohydrates with endo- and exo-activity are determined according to the method.

The substrate used for this enzymatic assay is identical to the residue substrate described for the SRU method.

The substrate is dissolved as a 3% solution in the citrate buffer listed below: 0.1 N citrate-phosphate buffer pH 4.5, 24 g citric acid 1-hydrate (Merck Art 244) 8.12 g disodium hydrogen phosphate-2-hydrate (Merck Art 6580) Ad 1 l demineralized H 2 O pH 4.5 I 0.05 Stable for 14 days The stop reagent has the following composition: 100 ml 0.5 N NaOH 200 ml 96% ethanol Store in a refrigerator until to be used.

Standard conditions _ Temperature SOOC pH 4,5 Reaction time, sample 120 minutes "" zero sample Unit definition A soybean residue solubilizing unit (SRUM) 120 (M means "manual") is the amount of enzyme which, under the given reaction conditions, releases solubilized polysaccharides equivalent to minutes with one micromole of galactose.

SECTION 10 c.

Enzymatic determination of proteolytic activity.

HUT measurement Method for determining proteinase in an acid medium.

The method is based on digestion of denatured hemoglobin by the enzyme at 40 ° C, pH 3.2, for 30 minutes. The undigested hemo- ZS 461 659 za globin is precipitated with 14% trichloroacetic acid (w / v).

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

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

After stirring for a further 10 minutes, the pH is adjusted to 3.2 with 1N NaOH. the volume of this solution is increased to 200 ml with 0.2 M acetate buffer. This hemoglobin substrate must be placed in a refrigerator, where it is kept for 5 days.

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

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

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

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

Calculations 1 HUT is the amount of enzyme which forms a hydrolyzate for one minute which, in terms of absorbance at 275 nm, is 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, pH 3.2 and for 30 minutes. 27 461 659 sample - zero sample vol in ml HUT = 0.0084 reaction time 1 min. nur = 2I2ïUlU% §% l239ï x yy = (sB) X 43.65 in sB 43 es “UT / g em” = A study of the pH stability dependence of the protea in KRF 68 performed using the HUT analysis with pH values of from 2.0 to 8.0 showed that the stability of the protease above pH 8.0 was very small, see Fig. 16. For the purpose of illustrating the invention, reference is made to the following Examples 1-8, where Example 1 illustrates the preparation of SPS. as, and where examples 2 - 8 illustrate the use of SPS-as with a soy-based raw material to produce a purified vegetable protein. Other applications of SPS-as are listed in the section between Example 8 and the overview of the figures.

Several fermentations with the strains of Asp indicated herein. aculeatus and Asp. japonícus was performed on a laboratory scale.

Thereby was obtained preparations containing SPS-as according to the SPS-as test given herein. However, since relatively large amounts of SPS-as are required to perform application experiments, similar fermentations were performed on a pilot plant scale, see the following Example 1.

Example 1 Production of an SPS axis in pilot plant scale.

An SPS ash produced by submerged fermentation of Aspergillus aculeatus CBS 101.43.

An agar substrate having the following composition was prepared in a Fernbach bottle: Peptone Difco 6 g Aminoline Ortana 4 g Glucose 1 g Yeast extract Dífco 3 g Meat extract Difco 1.5 g KHZPO4 Merck 20 g Malt extract Evers 20 g 1000 ml Jonbytt H20 ad 461 659 28 The pH was adjusted to between 5.50 and 5.35. Then 40 g of Agar Dífco was added, and the mixture was autoclaved for 20 minutes. at 120 ° C (the substrate is called E-agar).

Strain CBS 101.43 was grown on a B agar slope (37 ° C).

The spores from the slope were suspended in sterilized skim milk, and the suspension was lyophilized in test tubes. The contents of a lyophilized test tube were transferred to the Fernbach flask. The flask was then incubated for 13 days at 30 ° C.

A substrate with the following composition was prepared in a 500 liter inoculum fermenter: CaCO3 1.2 kg Glucose 7.2 kg Rofec (corn steep liquor, dry matter) 3.6 kg Soybean oil 1.2 kg Tap water was added to a total volume of about 240 liters. The pH was adjusted to around 5.5 before the addition of CaCO 3. The substrate was sterilized in the inoculum fermentor for 1 hour at 121 ° C. The final volume before inoculation was about 300 liters.

The spore suspension in the Fernbach bottle was transferred to the inoculum. The inoculation conditions were: Fermenter type: Conventional aerated and stirred fermenter with a height / diameter ratio of about 2.3.

Stirringz 300 rpm (two turbine propellers) Aeration: 300 normal liters of air per minute Temperature: 30 - 31 ° C Pressure: 0.5 atö Time: Approximately 28 hours Approximately 28 hours after inoculation, 150 liters are transferred from the inoculum fermenter to the main fermenter.

A substrate with the following composition was prepared in a 2500 liter main fermenter: Roasted soy flour 90 kg KHZPO4 20 kg Pluronic (:) 150 ml Tap water was added to a total volume of about 900 liters.

The roasted soy flour was suspended in water. The pH was adjusted to 8.0 with NaOH, and the temperature was raised to 50 ° C. Approximately 925 ALCALASE 0.6 L Anson units were then added to the suspension. 29 461 659 The mixture was kept for 4 hours at 50 ° C and pH = 8.0 (Na 2 CO 3 to 1 batch) without any aeration, zero atto and 100 rpm stirring. 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 1 hour at 123 ° C. The final volume before inoculation was about 1080 liters.

Then 150 liters of inoculum were added.

The fermentation conditions were: Fermenter type: Conventional aerated and stirred fermenter with a height / diameter ratio of about 2.7.

Stirring: 250 rpm (two turbine propellers) 'Aeration: 1200 normal liters of air per minute Temperature: 30 ° C Pressure: 0.5 atö Time: Approximately 151 hours.

From 24 fermentation hours to about 116 fermentation hours, pectin solution was added aseptically to the main fermenter at a constant rate of about 8 liters per hour. The pectin solution with the following composition was prepared in a 500 liter dosing tank: X) 22 kg 6 kg Pluronic 50 ml X) Genu-pectin (citrus type NF from The Copenhagen pectin factory Ltd.).

Tap water was added to a total volume of about 325 liters.

The substrate was sterilized in the dosing tank for 1 hour at 121 ° C.

The final volume before starting the dosing was about 360 liters. When this portion was finished, another similar- Pectin genu Phosphoric acid, conc. nande portion. The total volume of pectin solution for a fermentation was about 725 liters.

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

The culture fluid was drum filtered on a vacuum drum filter (Dorr Oliver), which was pre-coated with Hy-flo-super-cell diatomaceous earth (filter aid). The filtrate was concentrated by evaporation to about 50% of the volume of the culture broth. The concentrate was filtered on a Seitz filter sheet (type supra 100) with 0.25% Hy-flo-super-cell 461 659 S ”as filter aid (in the following table called filtration I).

The filtrate was precipitated with S61 g of (NH 4) 2 SO 4/1 at a pH of 5.5, and f4% Hy-flo-super-cell diatomaceous earth was added as a filter aid.

The precipitate and the filter aid are separated by filtration on a frame filter. The filter cake is dissolved in water, and insoluble parts are separated by filtration on a frame filter. The filtrate is control filtered on a Seítz filter sheet (type supra 100) with 0.25% Hy-flo-super-cell as filter aid (in the table below referred to as filtration II).

The filtrate is diafiltered on an ultrafiltration apparatus. After diafiltration, the liquid is concentrated to a dry matter content of 12.7% (in the table below referred to as dry matter content in concentrate) ¿An optional base treatment for partial removal of the protease activity can be performed in this step. If the base treatment is used, it is carried out at a pH of 9.2 for one hour, after which the pH value is adjusted to 5.0.

The liquid is then control filtered and filtered in order to achieve germ reduction, and the filtrate is freeze-dried on a freeze-drying equipment from Stokes. Four fermentations were carried out in the manner indicated below, with the strain used for the fermentation, the use of the optional base treatment and other parameters being varied, as shown in the following table.

Kbncentratíon (%) Torr-, av filterhjäänmeâ ñ: ïstanSTAn. del i sambmi ne t i -. . Basëehanëlm fl .. d Pfäpamt 'fiicre-'urfäii- fiitre- koncen- märk- Mikroorganism anvand ej anvan ko ring I ningen ring II trat ning CBS 101.43 x KRF 68 0,5 S 0,2 28 Arcc 20236 x 1 <1u = 74 2.0 4 0.4 7.5 IFO 4408 x KRF 83 1.0 5 0.25 12.4 x) CBS 101.43 x KRF 92 0.25 4 0.25 12.7 x) After seed-reducing filtration, the filtrate is concentrated by evaporation in a ratio of 1: 2.3. A minor portion of the concentrated filtrate was spray-dried, and the remaining portion was lyophilized.

To further reduce the protease activity, some of the above preparations were treated as indicated below, using only one of the three alternatives A, B and C. 31 461 659 A. 100 g of SPS-as preparation are dissolved in 1 liter of deionized water with stirring at 10 ° C at 2 ° C. The pH is adjusted to 9.1 with 4 N NaOH. This basic treatment is carried out for one hour. The pH is then adjusted to 4.5 with glacial acetic acid, and dialysis is performed against ice-cold, deionized water to a conductivity of 3 mSi. Freezing and lyophilization are then performed.

B. 500 g of SPS-as preparation are dissolved in 4 liters of deionized water with stirring at 10 ° C at 2 ° C. The pH is adjusted to 9.1 with 4 N NaOH. This basic treatment is performed for one hour. then add 5.0 g of glacial acetic acid. The resulting material lyophilic pH value is adjusted. C. 50 g of SPS-as preparation are dissolved in 400 ml of deionized water with stirring at 10 ° C in ZOC. The pH is adjusted to 9.1 with 4 N NaOH. This basic treatment is performed for one hour. Thereafter, the pH is reduced to 5.7 with glacial acetic acid. The resulting material is lyophilized.

SPS-as preparations used Use base as starting material for action Preparation code base treatment ABC KRF 68 x KRF 68 BII KRF 68 X KRF 68 BIII KRF 92 X KRF 92 BI The above preparations are characterized by their activities for the enzymes relevant in connection with the invention in the following table. 32 461 65 = °° ~ v> QCQQQHH ° = °°° - Qøomw Qoøøøø fl oøooø flfl Qceøew fl. = U => Qmß Qcca ømw Oßn = ~> .oda Qqm mmm °° -w = °° «« ° = ° - ~ o flfi m fl oomwß Qoßnw øø fi øß nam: oommw ooøøw == @ «@ øe fl v Qoßßß °°° ~» Qøfm fifi. men ocøccwß Qocoøvm ° = ° = ° m> Qøøøww ooøøcßm. Qoocooa øcøøomø fl am ~ m ° ~ ooßm above. ° ~ n «wmmm fvow øøøm xu ßmn Owmm == m ~ H Q fl w fl mmm mø fi ocøßw ~ .fl: Q aa: = ~ = H cqw fi nmß mßm = ~> H owm fl m ~ H ~ ° ~ fl == mw ßmß w fl w www ~ «H _ ~ @« »om ßmß. : mm onw få maa o men .man omm pmm »fi åum fifl uømkfz + + + ... + + +. ammuluum fi m mdm am ~ @ mmm Nm man ng man «> _mma HHHm vw man H fi m æw mm: Q» .mmm w Hmm _ ëmñwwmm 461 659 33 Example Z (Application Example) This example describes the preparation of a pvp from a shell free and degreased soy flour "Sojamel ll" (commercially available from Aarhus Oliefabrik A / S). The dry matter content of this flour was 94.0% and the content of (N x 6.25) calculated as dry matter was 58.7%. The soybean meal was treated with the SPS-as preparations KRF 68 BII (Example 1) as follows: 85.2 g of the soybean meal were suspended and kept under stirring at SOOC in 664.8 g of water, and the pH was adjusted to 4.5 using of 7.5 ml of 6 N HCl. SO g of a solution containing 4200 g of said SPS-as preparation was added, and the reaction mixture was then stirred for 240 minutes at SO ° C. The mixture was then centrifuged in a laboratory centrifuge (Beckman Model J-6B) for 15 minutes at 3000 x g. The supernatant was weighed and analyzed according to Kjeldahl on N and dry matter. The solid phase was then washed with a volume of water equivalent to the supernatant mass obtained by the first centrifugation. This operation was performed twice. The solid phase was then lyophilized, then weighed and analyzed according to Kjeldahl on N and dry matter at Qvíst's laboratory, Marselís Boulevard 169, 8000 Aarhus C, Denmark. This laboratory is approved by the state for analysis of feed and dairy products. The results obtained in the experiment are shown in Table 2.1: Table 2.1 Results obtained component weight N X 6.25%. rorrsux »" yield of Yield of: if __g_ punch% protein% material% Soybean meal 85.2 55.2 94.0 100% 100% SPS-as-prepared 4.00 75.6 - 6.4% - 1 Centrifugate 666 1.50 5.04 21.2% 42.0% pvp 44.5 87.5, 95.7 82.7% 53.Z% Thus a pvp with a protein purity was obtained, i.e. N x 6, Based on dry matter, of 91.4% and with a total protein yield of 83% §x§mpel_§ (Application example) This example was performed in order to compare the protein yields, nutritional quality and certain functional properties of soy protein products produced by the following three procedures: š-n 461 659 34 A: The traditional isoelectric precipitation for the production of soy protein isolates.

B: The traditional isoelectric washing for the production of soy protein concentrate.

C: The isoelectric wash of the invention including a residue solubilizing enzyme to produce pvp.

To enable a real comparison between the process according to the invention (C) and the conventional soy protein processes (A and B), the same raw material has been used in all three cases. Furthermore, the experiments have been performed in such a way that the corresponding temperatures and treatment times are the same in all three cases. Only the pH values were different depending on the fundamental differences between the three experiments.

A. Conventional isoelectric precipitation for the production of soy protein isolates. 425.8 g of soy flour (Soy flour 13 manufactured by Aarhus Oliefabrik A / S) was 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 one hour, the slurry was centrifuged at 3000 x g for 15 minutes using four one liter beakers in a laboratory centrifuge (Beckman Model J-6B], the centrifugate I and the precipitate I were weighed.

The precipitate I was re-extracted with water to a total weight of 4000 g. The temperature was maintained at 50 ° C, then the pH was adjusted to 8 with 4 N NaOH and the slurry was kept stirred for one hour. A centrifugation and weighing of centrifugate II and precipitation II were performed as described above. Samples were taken from centrifuge I and II and precipitate II for Kjeldahl and dry matter determinations. Thereafter, the centrifugates I and II were mixed and kept at 50 ° C. The protein is then precipitated isoelectrically at pH 4.5 using 45 g of 6 N HCl.

After stirring for one hour at SOOC, the protein was recovered by centrifugation at 3000 x g for 15 minutes. Centrifugate III was weighed and analyzed for Kjeldahl-N and dry matter. The solid phase III was weighed and washed with water in an amount corresponding to the weight of centrifugate I. The washing was carried out by stirring for one hour at 50 ° C. The washed protein was recovered by centrifugation at 3000 x g for 15 minutes. The centrifugate IV and the solid phase IV were weighed. Centrifugate IV was analyzed for Kjeldahl-N 461 659 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 kept under stirring for one hour and readjusted to pH = 6.5, if necessary. Finally, the product was subjected to freeze-drying, weighing and analysis regarding Kjeldahl-N and dry matter. The mass balance calculations are reported in Table 3.1.

Table 3.1 Mass balance calculations for the conventional isoelectric precipitation for the production of soy protein isolates.

Fraction Dry- Protein- Toris substance- Operations and fractions weight Protein sub-yield yield 'g% (Nx6.25) punch'%% Extraction: soybean meal 425.8 55.2 94.0 100.0 100.0 water 3574.2 0 0 0 0 4 N NaOH 20.1 0 16.0 0 0.8 1. Centrifugation: Z 4020.1 5.9 10.0 100.9 100.4 centrifugate 1 3141, o 4.4 6.9 58 , 8 54,1 Precipitation I 805,0 - - - ~ Re-extraction: Precipitation I 805,0 - - - - Water 319S, 0 0 0 0 0 2. Centrifugation: Centrifugation II 3104,0 0,5 0,9 6, 6 7.0 Precipitation II 820.0 9.1 17.2 31.7 35.2 Mixing and acidification: Centrifugate I + II 6245.0 - - - - 6 N HCl 45.0 0 21.3 0 2.4 3. Centrifugation: Z 6290.0 Centrifugate III 56S0.0 0.3 1.9 7.2 26.8 Precipitation III 308.0 - - - - 'Wetness Precipitation III 308.0 - - - - Water 3141,0 0 0 0 0 4. Centrifugation: 22 3449.0 Centrifugation IV 3113.0 0.04 0.15 0.5 1.2 Precipitation IV 291.0 - - - - Neutralization: Precipitation IV 291.0 - - - - Water 1550 , 0 0 0 0 0 4 4 NaOH 17.0 0 16.0 0 0, Drying: powder 128.0 93.8 96.3 51.1 30.8 461 659 36 B. Isoelectric washing for the production of soy protein concentrate. 425.6 g of soy flour (Soy flour 13 manufactured by Aarhus-Olíefactory A / S) was washed in 3574 g of water at 50 ° C. The pH was adjusted to 4.5 with 44.8 g of 6 N HCl. The washing was performed for 4 hours by stirring. The slurry was then centrifuged at 3000 x g for 15 minutes in a laboratory centrifuge (Beckman Model J-6B) using four one liter beakers. Centrifugate I was weighed and analyzed for Kjeldahl-N and dry matter.

The solid phase I was weighed and washed again with water to a total weight of 4000 g. The pH was readjusted to 4.5 with 1.7 g of 6 N HCl and the slurry was kept under stirring for 30 minutes at 50 ° C. Centrifugation and weighing of centrifugate II and solid phase II were performed as described above. The solid phase II was resuspended in 1575 g of H 2 O at SOOC and the pH was adjusted to 6.5 with 34.5 g of 4 N NaOH. The mixture was kept under stirring at SOOC for one hour and readjusted to pH = 6.5, if necessary.

Finally, the protein product was subjected to lyophilization, weighing and analysis for Kjeldahl-N and dry matter. The mass balance is reported in Table 3.2.

Table 3.2 37 for the preparation of soy protein concentrate. 461 659 Mass balance calculations for the isoelectric washing Operations and fractions: Fraction- Protein Tbrr ~ Protein- Dry matter weight% (Nx6.25) substance yield yield g%%% Washing: soybean meal 425.8 55.2 94.0 100.0 100.0 Water 3574.0 0 0 0 O 6 N HCl 44.8 0 21.3 0 2.4 1. Centrifugation: Z 4044.6 - - - _ - Centrifugate I 31S0.0 0.6 3.2 8 .0 25.2 Solid phase I 846.0 - - - - New washing: Solid phase I 846.0 - - - - Water 3154.0 0 0 0 0 6 N Hcl 1.7 0 21.3 0 0.1 2. Centrifugation: Z 4001.7 Centrifuge: 11 31s0.0 0.1 0.4 1.3 3.2 Solid phase II 863.0 - - - Neutralization: Solid phase II 863.0 - - - - Water 1575, 0 0 0 0 0 4Nmm MJ 0 mm 0 n Drying: powder 281.0 72.5 98.4 86.7 69.1 C. Isoelectric washing including a residual solubilizing enzyme for the production of pvp. 425.8 g of soy flour (Soy flour 13 manufactured by Aarhus Olíefabrik pH was adjusted to 4.5 A / s) was washed in 1 ssz4, z g of water at s0 ° c. using 43.7 g of 6 N HCl. 24 g of the SPS-as preparation KRF 68 BIII (Example 1) were solubilized in 26 g of water and added to the washing mixture. The washing was then performed for 4 hours by stirring. Thereafter, the purification was performed as described for B, with the amounts of 6 N HCl, 4 N NaOH and water for resuspension being the only deviating parameters. The mass balance is shown in Table 3.3. '40 38 4-6 1 5, _ _ _ _ _. . Table 3.3 Mass balance calculations for isoelectric washing including a residue solubilizing enzyme for pvp production.

Operations and fractions: Fraction- Protein Dry- Protein- Dry weight of the substance Substance yield punch (Nxó, 225] 1 'to exchange in fi äiuüngz Soybean meal 425.8 55.2 94.0 100.0 100.0 Water 3S40 , 2 0 0 0 0 6 N HCl 43.7 0 21.3 0 2.3 SPS axis: KRE 68 BIII 24.0 75.3 96.0 7.7 5.8 1. Centrifugation: 2 4043.7 - - - -_ Centrifugate I 3420.0 1.7 5.2 24.7 44.4 Solid phase I 620.0 - - - - New wash: Solid phase I 620.0 - - - - Water 3380.0 0 0 0 0 6 N HCl 1.3 0 21.3 0 0.1 2. Centrifugation: Z 4001.3 Centrifugate II 3400.0 0.2 0.6 2.9 5.1 Solid phase II 577.0 - - - - Neutralization: Solid phase II 577.0 - - - - Water 1700.0 0 0 0 0 4 N NaOH 25.3 O 16.0 0 1.0 shaking; powder 211.0 87.31) 96.71) 78.2 51.1 86.923 91.02) 1) Analyzed at the Biotechnical Institute, Holbergsvej 10, DK-6000 Kolding, Denmark 2) Analyzed at Qvist's Laboratory, Marselis Boulevard 169, DK-8000, Aarhus C, Denmark Nutritional Cakes Amino Acid Compositions for the three protein products were determined, see table 3.4. The total content of essential amino acids, the chemical grading and the essential amino acid index (EAAI] are calculated using the 1957 FAO reference pattern.

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.0.C.S. is an abbreviation for American Oil Chemists' Society).

The results are reported in Table 3.5, which also contains the nature and the protein / dry matter ratio for the three products. 1 39 461 659 Table 3._4 Amino acid composition and nutritional evaluation of the three protein products A, B and C.

A. Soy protein- B. Soy protein- C. Soy protein- Amino acid isolate concentrate isolate (pvp) g / 16 g N aasn g / 16 g N aasn g / 16 g N aasn l515§: §§§§n'5i§lla = Aspartic acid 12.4 - 11.3 - 11.9 - - serine 4.62 4.69 4.81 Glutamic acid 21.3 - 18.2 - 17.7 - Proline 6.07 - 5.19 - 4.76 - Glycine 4.13 - 4.26 - 4.33 - 41min 5.54 - 4.27 - 4.55 - Hiscidin 2.05 _- 2.78 - 2.50 - Arginm 8.09 - 7.57 - 7 , 04 - §§§§L15i§lla¿ Isoleucine 4.87> 100 4.97> 100 5.19> 100 Leucine 7.80> 100 7.98> 100 8.09> 100 Lysine 6.24> 100 6 , 09> 100 5.57> 100 Phenylalanine 5.47> 100} N01 5.55> 1o01 (> .0 "0'§ 5.17> 100 F00 Tyrosine 5.58> 100 í 5.88> 100; 4 , 44> 100 cysrin 1.29 64.5} 56Ä1.52 66.0} 60.2 | 1.44 12.0 kw r-iethionine 1.08 49.1 * 1.21 55.0 _1.51 59 , 5 Threonine 3.10> 100 3.60> 100 3.97> 100 Tryprofan 1.06 75.7 1.57 97.9 1.52 94.5 water 4.90> 100 5.25> 100 5, 57> 100% total content of essential 38.36 41.31 42.21 amino acids Chemical rating 56.45 60.25 65.55 EAAI 86.711, 90.211 91.311 1) aas = amino acid rating or scoring based on the FAO reference pattern (1957).

ZS 461 659 40 Table 3.5 Process characteristics and trypsin inhibitor content in the three protein products A, Bloch C.

A. Soy protein- B. Soy protein- C. Soy protein isolate concentrate isolate (pvp) Pm- 97.423 75.72 90.096 cess- _ character_ material teris- Protein yield 5l.1% 86.7% 78.2% Trypsin inhibitors - TUI / g product 34,000 21,000 19,000 TUI / g protein 36 250 28 970 21 810 Functional properties §Newability§indgr_ (NSI) was determined in a 1% protein dispersion at pH = 7.0 in 0, 2 M NaCl respectively in distilled water. After stirring for 45 minutes with a magnetic stirrer, the suspension was centrifuged at 4000 x g for 30 minutes, and the supernatant was analyzed for nitrogen. Nitrogen solubility was calculated as (soluble Na / total N%). The results of this evaluation of the three products are presented in Table 3.6. The aggregate capacity was determined three times on each product by a slightly modified Swift titration. 4.0 g of (N x 6.25) of the product was blown into 250 ml of 0.5 M NaCl with a Sorval Omni mixer at low speed. SO ml of the suspension was transferred to a glass mixing bowl and SO ml of soybean oil was added. The total mixture was then weighed. The oil-water mixture was then homogenized at 10,000 rpm with the dish in an ice bath. An additional amount of soybean oil was added at a rate of 0.3 ml per second until the emulsion collapsed. The total amount of oil added before the "end point" was determined by weighing.

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

The average results of the determination of the emulsion capacity of the three products are reported in Table 3.6.

Xigpnigsåeëpa fl aie fl s fl- at pH = 6.5. The tea samples were beaten at speed III for 4 minutes in a Hobart mixer (model N-50) equipped with a wire whisk. Whipping expansion was determined in a 3% protein solution 250 ml of the aqueous dispersion of the portion was calculated according to the formula 461 659 41 V-250 Whipping expansion = 250 x 100%, where V is the final whipping volume in ml.

V was measured by refilling the mixing bowl with water. Duplicate samples were performed for each of the three samples.

The average results are reported in Table 3.6. §Kgm§tabiliteten_ was determined as the ratio between the amount of foam left after defrosting for 30 minutes and the original amount of foam. One gram of foam produced by the above method was inserted into a plastic cylinder (diameter 7 cm, height 9 cm) with a wire mesh with a mesh size of 1 mm x 1 mm. The cylinder was placed on a funnel on top of a glass cylinder and the weight (B) of the liquid which had flowed down into the glass cylinder was determined. The foam stability FS is defined using the equation ÅfB FS = A x 100%.

The results of the determination are shown in Table 3.6.

The strength is defined in the present specification as the Brookfield viscosity measured by means of T-spindles at a Brookfield Helipath position. The gels were prepared by heat treatment of 12% protein suspensions in 0.5 M NaCl. The heat treatment was carried out in closed jars with a diameter of 7.3 cm and a height of 5.0 cm placed in a water bath, which was kept at 80 ° C and 10 ° C, each for 30 minutes. The jars were cooled and thermostated to 20 ° C before being opened and measured. The results of the measurements are reported in Table 3.6. 461 659 42 Table 3.6 Functional properties of the three protein products A, B and C.

- - A. Soy protein- B. Soy protein- C. Soy protein- FunkI1 ° nal1tet isolate concentrate isolate (pvp)% NSI in 0.2 M NaCl 39.5 20.3 25.6% NSI in water 53.9 25.1 28.6 Emulsification capacity: ml oil / g 218 182 354 (N x 6.25) - Whisk expansion% 120 120 340 Foam stability% 50 S0 20 Gel strength Lp] so ° c (0.5 M Nacl) 1.7 x 103 1.2 x 104 3.3 x 102 '100 ° C (0.5 M Nacn 2.0 x 104 4.0 x 104 1.3 x 104 Example 4 (Application Example) A pvp was prepared according to the procedure described in Example SC, with the exception that the cellulase activity was partially derived from Trichoderma reseei, the commercial cellulase preparation CELLUCLAST manufactured by Novo Industri A / S was treated with a base at a low temperature in the following manner.The pH value of a 10% CELLUCLAST solution in water 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 readjusted to 4.7 with 20% acetic acid, this solution was kept at 5 ° C overnight and sterile was then filtered. The filtrate was lyophilized. dried KRF 68 BIII (Example 1). The two enzymes were solubilized in 4 g of the frozen product, together with the SPS-as preparation 172 g of water were added before adding to the washing mixture. The mass balance determinations for this example are shown in Table 4.1.

The experiment shows that this special šPS-as preparation already contains an effective cellulase because the addition of CELLUCLAST does not seem to affect the protein / dry matter ratio. However, other SPS -ase preparations may contain less cellulase, e.g. KRF 92, see the table immediately before example 2. 461 659 43 Table 4.1 Mass balance determinations for the isoelectric washing including an SPS-as preparation and CELLU-CLAST for the production of pvp.

Opefaewwf °° h “šíïtâänen” äte ”ï fi šššans ïíâšïån '“ åšïšíenåïa ffakfwnef i gram (megs) 1 a m1 æ Tvättníng: Sojanüöl 425,8 55,2 94,0 100,1 100,0 Vatten 3546,2 0 0 O 0 6 N HCl 43.1 0 21.3 0 2,3- SPS-axis: KRF-68-B-III 24,0 75,3 96 7,7 5,8 CELLUCLAST 4,0 43,6 96 0,7 1.0 Centrifugation: Z 4043, l - - - - Centrifugate I 3382.0 1.9 5.5 27.3 46.5 Solid material I 661.0 - - - - New washing: Solid material I 661.0 - - - - Water 3339,0 0 0 0 6 N Hcl ooiooo 2nd centrifugation: Z 4000,0 Centrifugate II 3414,0 0,2 0,7 2,9 6,0 Solid material II 582,0 - - - - Neutralization: Solid Material II 582.0 - - - - Water 1691.0 0 0 4 N NaOH 25.3 0 16.0 0 1.0 Tb ü ong: Powder 206.0 88.8 98.9 77.8 50.9 Example 5 C Application Example A pvp was prepared according to the method described in Example 3 C except that all weights were scaled down by a factor of 5 and that the reaction mixture was cooled to about 5 ° C prior to centrifugation. On the basis of the analytical results in relation to the centrifugates, a theoretical yield of precipitated protein is obtained, as shown in Table 5.1. _25 461 659 44 Table 5.1 Theoretical protein yields obtained in the production of PVP Weight Protein Protein- Example 3 C Fractions (Nm6,25) yield Protein Protein- grz (mms) * v yield,'s soybean meal1 85,2 55,2 100 55, 2,100 SPS-as-KRF-68 B-III 4.8 75.3 7.7 75.3 7.7 1st centrifugate 639 0.99 13.5 1.7 24.7 2nd centrifugate 595 0 , 13 1.6 0.2 2.9 pvp - 87.23 92.51 "81.1 80.11" aAverage 87.5 (Biotechnology Institute) and 86.9 (Qvist's Laboratoriunü; dry matter content is 97.6 resp. 98.0% calculated as the total weight of protein - protein loss in centrifugates.

Example 6 (application example) Eenfæ fi ifafäa avnærßzei fl ëiadaiass fl f! §P§ 40 grams of (N x 6.25) from a commercial soy protein isolate (Purina 500 E from Ralston Purína) were dissolved in 680 g of water.

The mixture was heated in a water bath to 50 ° C, and the pH was adjusted to 4.50 with 6 N HCl. was fed to 5 x 250 ml Erlenmeyer flasks, and 10 g of aqueous solutions 90 g of this mixture containing 0 g, 0.2 g, 0.4 g, 0.8 g and 1.6 g of SPS, respectively, produced as described previously in the present description was added. The flasks were then kept under stirring with a magnet in a water bath at SOOC for 240 minutes.

Thereafter, the slurries were centrifuged at 3000 x g for minutes, and the centrifugates I were analyzed for Kjeldahl-N and dry matter. The solid phases were washed in water at room temperature and centrifuged again. This procedure was repeated. Then the solids were dispersed in 50 ml of water, and the pH was adjusted to 6.50 by dropwise addition of 6 N NaOH. The neutralized products were lyophilized and analyzed for Kjeldahl-N and dry matter. Based on the analysis reported in Table 6.1, the protein extraction and the percentage of SPS that have been bound to the protein are calculated using the formulas shown in connection with Table 6.2.

This example shows that SPS bound to the protein, so that the protein / dry matter ratio decreases with increasing content of SPS. An SPS content comparable to about 0.4 g in 10 g of water added to S g of protein isolate is the protein / SPS ratio that applies to soy flour.

The percentage binding of SPS is a calculated value.

The percentage binding of SPS decreases due to the saturation of the protein with respect to SPS at the low protein / SPS ratios.

Table 6.1 Measurements according to Example 6 Ratio Centrifugate In precipitation NK6 25 - protein / SPS 'à N% m' à N 'à N x 6.25% IM' ä °° 0.068 0.62 13.2 82.5 93, 1 88.6 0.045 0.49 13.4 83.8 97.3 80.1 12.5 0.033 0.45 13.0 81.3 97.9 03.0 6.25 0.031 0.45 12.6 78 .8 98.1 80.3 3.125 0.020 0.01 11.0 73.8 97.9 75.3 Table 6.2 Protein recovery and percentage binding of SPS Ratio% recovery)% binding 2) protein / SPS of protein of SPS aa 91 .5 0 94.4 77 12.5 95.3 90 0.25 90.1 70 3.125 90.8 00 l)% recovery of protein = Kl - ÉELÅ-å-§¿å§-1 x 100, where NC l =% N in centrifugate In binding of SPS = x (% recovery_of protein)] x (% recovery of protein) in year (3 P / H), _ (0 PÄD x 100, where lis / sps ratio, where ( % P / H) is the ratio of protein / dry matter in the dried precipitate, and (% P / H) c * §aNser the precipitate without the addition of SPS. 461 659 46 Example 7 (application example) This example describes the preparation of a pvp during use. reversal of the SPS-as preparation KRP 92 BI in a dosage of 5% of the dry material. The preparation method was exactly the same as in Example 3 C, except that all weights were scaled down by a factor S. pvp was analyzed as described in Example Z.

The results obtained in the experiment are shown in Table 7.1.

Table 7.1 Results obtained in Example 7 Weight (Nx6.25) Dry Yield 'Yield K ° p ° nent g% substance% of pro-av-dry tin,% mtrl,% soybean meal 85.2 55.2 94.0 100 ioo Enzyme preparation 4.0 71.2 - 6.1 _ - 1st centrifugate 632 1.88 SA4 25.3 43.0 2nd centrifugate 673 0.30 0.80 4.3 6.7 pvp 39.8 85.6: 98.1: 11.9 48.8 84.4 98.1 aAnalysed at the Biotechnical Institute, Holbergsvej 10, DK-6000 Kolding bAnalysed at Qvist's Laboratory, Marselis Boulevard 169, DK-8000 Aarhus C.

Example 8 (application example) This example shows the effect of pretreatment of the soybean meal by jet cooking before the production of pvp.

Pretreatment A slurry of soybean meal in water consisting of 10 kg soybean meal (Soybean meal 13 manufactured by Aarhus Oliefabrík A / S) per 100 kg was pumped through a steam injector (type Hydroheater B-300) and mixed with steam of 8 bar in such an amount and at such a flow that a final temperature of 150 ° C could be maintained for 25 seconds in a pressurized tube reactor. Then the pressure was relieved in a flame chamber (a cyclone), and from this the slurry was sent through a plate heat exchanger and cooled to about SOOC. The cooled slurry could be used directly for the production of pvp according to the invention, but in this case the slurry 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 S6,9% (N x 6,25). 461 659 47 Production of pvp This production was carried out as follows: 70 g of dry material of the jet-heated and dried soybean meal were suspended and kept under stirring at 50 ° C in 560 g of water, and the pH was adjusted to 4.50 by means of of 6.5 ml of 6N HCl. 6 x 90 g of this suspension were transferred to six 250 ml Erlenmeyer flasks and kept under stirring in a water bath at 50 ° C using a magnetic stirrer. To each flask was added 10 g of a solution containing 0 g, 0.025 g, 0.050 g, 0.10 g, 0.20 g and 0.40 g of sPs-as-preparaue, respectively: KRF-éa-B -III.

The reaction mixtures were then stirred for 240 minutes at 50 ° C.

Then a centrifugation was performed at 3000 x g for 15 minutes.

The supernatant was then analyzed for Kjeldahl-N and the solid phase was washed with the same volume of water and centrifuged. This procedure was performed twice. The solid phase was lyophilized and analyzed for Kjeldahl-N and dry matter.

A similar experiment was performed with an untreated soy flour (Sojamel 13 from Aarhus Oliefabrik A / S) as a starting material. In this case, the enzyme substrate ratios were 0.1%,%, 3%, 4% and 8%.

Based on the protein content of the supernatants, the percentage of protein recovered can be calculated. The protein exchange is based on the assumption that the enzyme product is 100% solubilized after the reaction. The table below shows the results obtained in both experiments. 48 461 659 Table 8.1 Heated soybean meal Unprocessed soybean meal E / 5 z protein exchange protein in dry protein yield protein in dry% material%% material% 0 92.9 70.5 90.7 13.9 0.25 90.1 86, 0 - - 0.50 89.3 88.7 - - 1.0 88.1 89.7 87.1 86.2 2.0 86.6 91.7 85.7 88.1 3.0 - - 84 .3 89.5 4.0 04.1 92.2 02.0 90.9 '8.0 - - 76.2 91.1 Table 8.1 Protein yields and protein dry matter ratio for pvp made from heated or straight soy flour.

With regard to either the extraction (isolation) processes for materials other than proteins or the liquid transfer processes and related processes, reference is made to the general process scheme for applications shown in the form of flow chart no. 3.

The substrate may be one or more carbohydrates present in one raw material, or it may be the whole raw material.

This substrate may be subjected to a pretreatment of a chemical or physical nature, as exemplified below, e.g. acid or alkali treatment, soaking or casting and / or heating with or without steam.

The raw material can be subjected to maceration, chopping, wet grinding and / or homogenization (all these treatments are called homogenization in flow chart no. 3), with or without water additives, and other additives can be added during this step. The homogenization can be carried out with different efficiencies, e.g. For example, different pressures may be only a fraction of the maximum pressure specified for the specific homogenizer used. Various additives may be added before or during homogenization, as shown in symbols b ,; bz, ... bn in flow chart no. 3.

The reaction process including the SPS-as preparation is carried out under specified conditions, e.g. temperature, pressure, time, pH and enzyme dosage; recommendations regarding the used 461 659 49 reactor (eg batch reactor, plug flow reactor) and stirring, if required, are also relevant. A set of additives can be added for different raw materials, which is indicated as c1, cz, ... c in flow chart no. 3.

Furthermore, the separation processes can be performed with different TI efficiencies. During many processes, the separation is omitted or simplified, e.g. when the raw material is completely in liquid form. Various separation equipment can be used (eg centrifuges, filters, ultrafiltration equipment, hydrocyclones, thickeners, sieves or sieves or simple decanters).

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

The resulting liquid or solid phases can be further treated, e.g. concentrated, dried or solvent extracted, for the purpose of removing certain components such as fat or oil, fermented for the production of biomass, alcohol or other products (enzymes, antibiotics or other valuable components).

Furthermore, the products obtained can be returned to repeated treatment by means of the process schedule. 461 659 50 Flow chart no. 3 (fifi om = o - louæ) (Reaction conditions to be specified) Substrate al, V _a2 --__-% _.) Pretreatments _ a ------ ä bl, _; bz _ Homogenization I a bn.

SPS-as- CI production -: L v '--- “- 7 C _________ <> Reaction. 2 r. 3 en.

Homogenization (as above) \ f Separation Liquid Additional Treatments Show? = o -: Loan Solid material (see text) or re-use as substrate 51 461 659 Below are some examples of applications of SPS-as preparation, and an overview of these applications can be found in the following list.

Table I also lists certain characteristics with respect to Flowchart No. 3. 461 659 List of Exemplary Applications of SPS-as Preparations 52 Types of SPS-as- Reference Preparation No. SPS-as-Preparations A 1 Extraction of starch from maize , mainly free wheat and potatoes from one or more A 2 Extraction of lipids from plant undesirable materials enzyme activities A 3 Extraction of essential oils from plant material A 4 Extraction of natural dyes from plant material AS Extraction of rubber from guayulus us - Total Ba l Production of a milk substitute liquid for pets üm fl ß Ba 2 Production of raw materials containing feed saccharified starch or d Ba 3 Total liquid transfer of pears and Harbor e other fruits bdmnd- Ba 4 Production of juice by treatment of fruits and vegetables Ba 5 Treatment with regard to the extraction or pressing of sugar cane or beet Ba 6 Production of soy milk _ Ba 7 Treatment for the purpose of increasing the extraction- Omo. _ Ü. Intensible amount of soluble substances 1 coffee :: §_ Pnmess- Bb 1 Prevent and / or decompose pre_ auxiliary apple turbidity needle Bb 2 Use as a clarifier for paired white wine Bb 3 Production of ISSPH or other vegetable-stable protein hydrolysates Bb 4 in the brewery industry Bb 5 Enzymatic additive for use during beer fermentation and / or storage Bb 6 Almond skin release agents Other Bc l Decomposition of various waste applications Bc Z Saccharification and simultaneous fermentation Bc 3 Decomposition of cellulose Bc 4 Use as a baking aid 5 To improve the alcohol yield and the biomass yield during the fermentation of sulphite liquid from papermaking _ Bc 6 Drainage of biological sludge products Bc 7 Silage aids S3 AÅ1 ARG wouw _ 1u @ »« 11:11? cowum fiflfi uwmu _, 1m fi om W .mn fl c Hw fifl ø V -mm fi onošß hm. »Mä 1 1 o 1 ä fi S: 1 533 33 H å - co fi u Hoz V 1æm fi H ww fifi o uowwn. 1 ~ HHmw> mv 1Hmww «ux oo fi 1 æo fi: omz 1 cwuum> 1Hoxuow m wm co fi um fl wumow Ho: ma» .mz fl c wø fifi ø H> E 1muom 1mm fi HozoxHæ Hmm »www 1 1 o 1 1: omz 1mmEu: øm IPOM wc fi chouzmnmw 1> m .wc fl amnuuwuaox fl m fi e .wc fi næm fi nnummm 1 1 o Q o Hu: 1: wwum> am fi om H mm wc fi cmu fi um osan n: muum> Hø fifl o 1¶¶0nm 1 ~ mH fifi mm 1 w + m N <wcwc Hwco fi u 1nw> u: m fi Ho àämo .w fi a fi åß Gm 1ww: fl: fi øgsmwwohm no fifi o Now 1 1uww> s »mHHm: oc = ~ v om 1 om om 1 QH om 1 ON: omz: mu» m> = wuvm> m fi më H <wmomc weozc wësåc fi co fi umnmmmw cmwc fl v mmm ww MMH 111 1111111111 1111M11 -1111111111MWM 1 1 1 111m .111 kmw fi ää fl wß wäw fl hmsp »wäê Small» Håëmumm nm4 S4 Ame extra n A4.

Extraction of starch from maize, wheat, potatoes and other starchy plants is carried out by one or more of the steps: casting, wet grinding and separation. The use of an SPS-as-preparation without any significant amylolytic activity provides the following advantages, with maize being used as an example: l. Starch release is facilitated during a shorter casting period, 2. Water consumption can be reduced, 3. Release of corn germ material would be facilitated without release of corn germ oil, _ 4. The protein can be obtained with higher purity,. The extraction of maize casting water is simplified.

A 2. Extraction of lipids from plant material.

Because the lipids in plant material are entrapped within the cells and usually bound to proteins, lipids can be extracted in the aqueous phase by treatment with an SPS-as preparation which is substantially free of lipases. Thus, corn germ oil is normally isolated by hexane extraction of the dried corn germs. However, the drying operation is superfluous if wet maize germs are treated with an SPS-as-preparation of the kind indicated above. In the same way, extraction of olive oil in aqueous phase can be improved, if the enzyme used for the enzymatic treatment is an SPS-as preparation of the kind indicated above, see e.g. Food, Pharmaceutical and Bioengineering, No. 172, vol. 74, pp. 93-94. Also water-based extraction of e.g. soybean oil, rapeseed oil and safflower oil can be improved in a similar way.

A 3. Extraction of essential oils from plant material.

If vegetable materials containing essential oils are extracted with an aqueous solution of an SPS-as preparation, which is substantially free of enzymatic activity capable of decomposing or otherwise altering the essential oils, the essential oils will: extracted in high yields at very low, cost.

A 4. Extraction of natural dyes from plant material.

If vegetable materials containing dyes, e.g. beets containing the red dye betanin or the dye in cranberries, treated with an SPS-as preparation, which is substantially free of enzyme activities capable of decomposing or otherwise altering the dyes, the dyes will be recovered in high yields SS 461 659 at a very low cost.

A 5. Extraction of rubber from the guayul bush.

Another example of a substrate for an SPS-as preparation, which is substantially free of enzymatic activity capable of degrading native gum, is the cell wall material in the roots and branches of the guayul bush.

Ba 1. Preparation of a milk substitute for domestic animals, preferably a calf milk substitute.

By total liquid transfer in water-based medium of soybeans, sunflower seeds, cotton seeds, vegetable beans or field peas, a calf milk substitute can be prepared, which is soluble in cold water at a pH value of about 4.5. Using the starchy raw materials such as green beans or field peas, a starch liquid transfer using an alpha-amylase must be performed before, after or simultaneously with a treatment with an SPS-ash, which ultimately solubilizes the non-starch polysaccharides present as structural material. in the cell walls. A detailed example is given below using green beans, and in the case of soybeans, reference is made to Table I. The pretreatment of these soybeans may preferably be a jet cooking, which improves the solubilization of the residue.

Example Ba l.l lS kg of green bean flour (Farine de Feves from GRANDES MINOTERIES A FEVES DE FRANCE, Paris) was suspended in 35 liters of water. 75 g of Thermamyl 60 L and 18 g of CaCl 2 were added. The suspension was heated to 95 ° C using a steam jacket vessel with stirring. The suspension was then treated at this temperature for 60 minutes. Then the pH was adjusted to pH = 4.5, and the product was cooled to 50 ° C. 300 g of the SPS-as preparation KRF 68 were solubilized in 1 liter of water and added.

The reaction was carried out for 440 minutes. When 10 g of Fungamyl 800 L are incorporated, the starch fraction is mainly converted to disaccharide (maltose). Then the reaction mixture was pasteurized at 90 ° C for 2 minutes. A sample of the product was then lyophilized and used for stability testing. The sample was then solubilized at% dry matter content, and the solution of the product could be kept stable without sedimentation for days.

Molten fat or oil can be easily emulsified in the product, whereby a final composition, which is very similar to cow's milk, can be obtained. ZS 461 659 see. An emulsion containing 3.5% oil (soybean oil) could also be kept stable without sedimentation for days.

Example Gel Ba 1.2 soybean meal (soybean meal 13) was crude-heated at 150 ° C for 2 seconds as described in Example 8. The jet-heated soybean meal was spray dried and used for further studies as described in A: below. 50 g of the jet-heated soybean meal was mixed with 450 g of water, and the pH was adjusted to 4.5 with 4.1 ml of 6N HCl. The mixture was then heated to 45 ° C in a water bath, and 0.250 g of the ° SPS -ase preparation KRF-68 was added to the heated mixture, which was then allowed to react for 5 hours with stirring. The mixture was then heat treated at 80 ° C for 2 minutes to inactivate the enzyme. A 100 ml sample was centrifuged at room temperature for minutes at 3000 x g (6 = gravity). The supernatant was subjected to ion exchange and analyzed for carbohydrate composition by HPLC. The supernatant was also analyzed for Kjeldahl-N and the dry matter and nitrogen solubility index (NSI) and solubility index for dry matter (DSI) were calculated; see results in Table Ba I. 100 ml of the reaction mixture cooled to 20 ° C was poured into a 100 ml graduated glass and kept at 4 ° C for two days. The dispersion stability (%) was measured by reading the volume of the dispersions obtained (Table Ba II) after 1 and 2 days.

To 200 ml of the reaction mixture (at 20 ° C) was added 8 g of soybean oil. An emulsion was prepared by mixing for 2 minutes in a Waring mixer. The emulsion stability (%) was measured as described above after 1 and 2 days.

A reaction was carried out as described above, but in this case 1.00 g of the SPS-as preparation were used. The same types of analyzes and stability measurements as described in section A were performed. The results are reported in Tables Ba I and Ba II.

The chemical analysis of the supernatants shows that the values for NSI (%) and DSI (t) obtained in Experiment B are higher than in Experiment A. However, the stability experiments performed on the reaction mixtures show better value for the A-samples. This is probably due to the larger peptide chain length for the proteins in the reaction mixture at the low enzyme dosage.

S7 461 659 From the carbohydrate composition measured by HPLC it appears that mainly mmm and disaccharides are formed. Thus, oligosaccharides, which are known to cause diarrhea and gas when given to calves in excessive amounts, are present only in small amounts.

Table Ba I. Chemical properties of the supernatant.

N _ Enzyme dosage 1 NSI% * DSI%, * HLC-result For- relation to íå H _ (via H _ (neutral sugar-seeking substrate (VP _ P _ species co sition) 2, weight / weight 4-53 45) m) A r / s = o, s% 39,9 62,4 npï + DPZ = ís DP æ DP3: 7,4% DP4: lZ, 2% DP4 +: 8,1% BE / S = 2,0% 57, 0 67.1 DP1 + DP2: 84.4% DP: 6.1% DP:: 3.71.

DP4 + '5.7% * NSI = Nitrogen Solubility Index ** DSI = Dry Material Solubility Index Table Ba II. Stability test for the reaction mixtures.

- - Stability test n En.

Såå- reíšïâgâeïíïïï 1 Without oil With oil substrate Dispersion Dispersion Emulsion Emulsion% w / w 1st day 2nd day 1st day 2nd day 2nd day AE / S = 0.5% 80% 63% 100% 87% BE / S = 2.0% 66% 35% 85% 71% Ba 2. Production of raw materials containing saccharified starch.

In connection with the saccharification of cassava and sweet potatoes as well as other starchy plant materials, it is possible to solve viscosity problems by adding an SPS-as-preparation. By using an SPS-as preparation, it is possible to prepare starch suspensions with a dry matter content of ZS-30%, and after saccharification the mash can be subjected to fermentation, whereby cheap ethanol can be obtained.

Example gel Ba 2.1 On the basis of fresh and grated sweet potatoes (Japanese), 461 659 tablespoons of puree with a dry matter content of 24% were prepared. The starch content of sweet potatoes was found to be approximately 70% of its dry matter content.

A pretreatment for transfer to the liquid state by means of the bacterial amylase Termamyl 60 L in a dose of 0.5 kg / ton of starch was carried out by heating the mash to 90 ° C. The mash was then kept at 90 ° C for 30 minutes. The viscosity n1 of the reaction mixture was then measured by means of a HAAKE spindle at 90 ° C.

Thereafter, the reaction mixture was cooled to 55 ° C, and the pH was adjusted to pH 5.0 with ZN H 2 SO 4. Saccharification was then initiated by the addition of the glucoemylase SAN 150 (trademark of NOV0 INDUSTRI A / S) at a dose of 1.75 liters / ton of starch. The saccharification mixture was then divided into three parts, A, B and C, which were enzyme treated for 15 minutes as reported below before measuring the viscosity: A: This part is the comparison part. The viscosity nz was measured, see Table Ba III B: The Tricoderma viride cellulase Celluclast <) 200 N was added in a dose of 1 kg / ton dry material in sweet potatoes.

The viscosity ns was measured, see Table Ba III.

The C: SPS-as preparation KRF-68 was added in a dose of 0.25 kg / ton dry material in sweet potatoes. The viscosity n4 was measured, see Table Ba III.

Table Ba III: Viscosities.

Reaction Viskosišet Viscosity mixture at 90 C at 55 C Pretreated sweet potato n1 = 770 cp nz = 2190 cp tis for transfer: in liquid: 111- state A - nz = 2190 cp B - ns = 1970 cp C - n4 = 950 cp Sàhnkh.kæ1uan see that the viscosity of the reaction mixture could be effectively reduced with the SPS-ace at low dose compared to SAN 150.

Ba 3. Total transfer to liquid of pears and other fruits.

If whole pears are mechanically crushed and then treated with Celluclast an SPS-as preparation, total transfer to liquid takes place, and a clear pear fruit juice is produced after removal of small amounts of solid material. A similar method can be used in connection with other similar fruits, e.g. apples.

Example Ba 3.1.

Fresh apples were coarsely ground using a "Bucher Central mill". The apple puree was then pasteurized in a heat jacketed tank at 90 ° C for 5 minutes and then cooled to room temperature. The pre-mashed apples were then ground on a Fryma grinder with corundum stone equipment until the mash was even to the touch. The mash was then re-pasteurized at 80 ° C for 10 minutes and cooled to 50 ° C.

Enzyme reactions were now performed at 50 ° C for 30 minutes with Contraves Rheomat 15, stirring and viscosity measurement (relative to a percentage reading on the rheometer at speed 13) being performed simultaneously. After completion of enzyme reactions, 100 g of samples were taken and centrifuged in a graduated tube at 3000 x g for minutes. In this way, the percentage of juice and the percentage of deposited material are measured. The pH and percentage of refractometer-dry material such as ° Brix were also measured. Table Ba IV shows a comparison between the effect of SPS-as, the combination of Celluclast and SPS-as and the combination of Celluclast and Pectinex. The SPS-as preparation KRF-68 was used.

Table Ba IV. Results of total liquid transfer experiments with apple puree at SOOC for 30 minutes SPS-as Celluclastd Pectinex® Final Centrifugation Juice gágsl gšgollmos 3: / h 1 puree ïå: k °: 1-% juice S deposited pH ° Bríx 0 0 100 59 41 3.8 9.7 0 19 81 19 3.5 10.5 50 0 15 79 Zl 3.5 10.7 S0 50 0 4.8 83 l7 3.5 10.8 3.8 83 17 3.1 12.5 0 S0 200 9.5 83 17 3.2 12.9 0 50 2000 4.0 82 18 3.1 13.2 Ba 4. Production of juice through the treatment of fruits and vegetables.

It has been found that SPS-as preparations are well suited for the production of juice by treating a variety of fruits, berries and sweets, e.g. carrots, peas, tomatoes, apples, pears, black 461 659 60 currants, beans and cabbage. This results in improved juice exchange and better extraction of color and flavor components compared to commercially available pectinase and cellulase preparations.

Example gel Ba 4.1 In this case we refer to Example Ba 3.1, where the SPS-as- preparation has been compared with the commercially available convention 200 L 3x. The table shows that the juice yield can be improved slightly with as little as SO g / h l of SPS-as compared to 2000 g / h l of Pectinex, both in combination with 50 g / h l of Celluclast. The viscosity was also slightly lower. Thus, it is believed that the SPS asset is about 40 times more effective than Pectinex.

Ba 5. tional cellulase and pectinase products Celluclast and Pectinex Treatment in connection with extraction or pressing of sugar cane or sugar beet.

It has been found that it is possible to improve the yield with respect to simple extraction processes, if the SPS-as preparation is used for the treatment of sugar cane or sugar beet before and / or during the extraction or pressing thereof. The residue (bagasse) can also be treated with the SPS-as preparation, whereby it is partially converted into fermentable sugars, which can be used as raw material for ethanol fermentation.

Example gel Ba 5.1 kg sugar beet residue (pulp) obtained by continuous countercurrent extraction in a DDS diffuser at Nakskov Sugar Factory was ground twice in a Fryma mill (type MZ-110). Process water was added during the grinding operation. 300 g portions of the pulp were now enzyme treated at 45 ° C for 18 hours using the enzyme doses reported in Table Ba V.

The dry enzyme product (KRF-68) was added to the mass, which was stirred with a stick for the first hour. Thereafter, the mass was transferred in liquid form to such an extent that magnetic stirring could be successfully performed for 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 obtained. Dry matter determinations were performed on the reaction mixtures and on the supernatants. Based on these results, the percentage of solubilized dry matter was calculated. Corrections for soluble dry matter in the enzyme product were made in all calculations. 61,461,659 Supernatants 2, 3 and 4 were ion exchanged and analyzed by HPLC for carbohydrate composition. table Ba V; Results obtained by enzymatic transfer to beet pulp liquid.

Experiments Final measurements ri-% .šïrhå11ande Reaction mixture_ Supernatants ment 1 tOITt _ _ no material, pH% dry% dry% so1ub1l1se ~ E / S% (final) material material rat dry material 1 0 5.5 4.18 0, 0 0.0 2 0.35 3.6 3.85 2.58 66.9 3 0.56 3.5 3.81 2.56 66.2 4 1.02 3.5 3.86 2.73 70 .4 1.58 3.3 3.17 2.34 73.4 6 3.10 3.4 3.23 2.49 76.4 7 7.52 3.4 2.66 2.18 80.5 Reaction conditions : M = 300 g S = 4.18% dry matter E / S as shown above pH not adjusted T = 45 ° ct = 18 hours Table Ba VI. HPLC data.

Sugar type Experiment no (neutral) 2 3 4% of neutral sugars High molecular weight ÜMM +) 43.6 31.9 25.3 Disaccharides 4.6 4.8 - Glucose 20.4 23.7 27.8 Galactose 5.0 5.9 7.3 Fructose / arabinose 26.4 32.2 33.2 Galacturonic acid not measured All sugars formed according to the above Table Ba VI could be fermented into alcohol or used for other purposes.

Ba 6. Production of soy milk.

Soy milk can be easily prepared by total transfer to ground soybean liquid and subsequent homogenization of the resulting mixture. Soy milk is often produced by treating soybeans in boiling water, grinding the soaked beans and extracting water following a separation of insoluble residues, e.g. proteins and polysaccharides. To improve the yield of soy milk, these insoluble residues can be converted to liquid by reaction with the SPS-ace.

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

The enzyme reactions were performed under the following conditions: Substrate: Soy flour with full fat content (Dansk Soja kagefabrik A / S) Weight of the reaction mixture: 220 g Weight of the substrate: 20 g Temperature: 50 ° C pH: 4.5 (6 N HCl) Reaction time: Series A: 1 hour "Series B: 0.5 - 6 hours Enzyme: SPS-as (KRF-68) Enzyme dose: Series A: E / S ratio (w / w): 0 - 8.0% Series B: E / S -ratio (w / w): 1.0%.

After the reaction, the pH was adjusted to 7 with 4 N NaOH, and a separation was performed by centrifugation at 3000 x g for 15 minutes. 461 659 63 m. @ W m. ~ @ @@. Ss @ m.m wß.w m @ .m @ .H o. @ Ss. ~ @ @. oH.m wß.wm @ .mo “H @.« _.ß ~ ~ .mß H @. @ @ ß. ~ æ> .æ m @ .m oH QN m «.mß W ß.æ @ @@ . @ @ m. ~ wß.w «@ .m OH ° .H H.Hß Q.« @ H <. @ @ m. ~ wß.w «@ .m oH m. ° @ .wæ m.æw wo .w @>. » ßm. @ @H. «ow oH @ .mw @.« wm @ .ß @mm no.m Nm.m o. «° .H m.mß m.ßß wH.ß w @. ~ @ m.æ wß.m @. ~ oH <.Hß Hm @ ww. @ w <.N æß.w H> .m @ .H oH <= .oß ~ .m @ w ~. @ H <.N «> .æ æ @ .m mo oH ß.m @ @. @ <~ ß.m> @. ñ = ß_æ m @ .m Q @ .H umëewu HNMPQHNE HwMHOHdE w .DHR w Hmm M Hmm p fi hop w øwøuowm w ph fi ou Q = fi «» o ~ @ ww \ m -mno f ß Mehow xowc fi wuwzww fi MWQ øämuw al hwmäm toilet al cwcm fi nmcowuxmmm wowëzunm nxmwm .cowmwuoumx f m f ew fi if aMwMuma> nOO one VWA vcæpemw H NMWC al cxmamnwcm al mnmwmz .HH> mm Hanna ZS 461 Ba 7. 659 64 treatment in order to increase the recoverable amount of soluble material in coffee.

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 material from coffee. Thus, e.g. malt consumed coffee or green beans are enzymatically treated with favorable results as a result.

Bb 1. Prevention and / or removal of apple or pear turbidity.

After the preparation of apple juice or pear juice and other fruit juices, which must be clear and which have previously been treated with conventional pectinase and cellulase preparations to prevent the appearance of turbidity or turbidity, apple turbidity or turbidity of similar fruits may occur. It has been found that the SPS-as preparations are well suited for the decomposition of such turbidity, which consists mainly of araban bound to proteins.

Example Bb 1 Pear juice concentrate prepared by enzymatic liquid transfer of waste from a pear preservation plant using Celluclast and Pectinex was found to be cloudy when left to stand. The cloudy material was isolated and hydrolyzed with 0.01 N H 2 SO 4 for 24 hours and analyzed by HPLC. The chromatogram showed arabinose and small amounts of oligosaccharides.

By incubating 0.5% w / v of this carbohydrate in 1 mM acetate buffer at 4.5 l for 3 hours at 40 ° C with sps-as (kar-es + KRF-92 1: 1) with an enzyme concentration of 0, 05% w / v, it was found that 84% of the original turbidity carbohydrate (dry matter) was converted to arabinose. Also diluted pear concentrate (200 Bríx) was treated for 2 hours at 40 ° C with an enzyme dose of the above SPS-as at 0.15% w / v or with a commercial product called Clarex at a dose of 1% w / v. It was found that the SPS-ace could reduce the relative HPLC peak area for an araban-like turbidity by 86%, while the corresponding reduction with Clarex (used in a much higher dose than the SPS-as preparation) has only 78% Ä es f 461 659 Bb 2. Use as a clarifier for white wine.

It has been shown that white wines that show a very undesirable turbidity or turbidity can be cleared effectively with the help of SPS-as. It has been shown that the cloudy material consists mainly of arabinogalactans, which are bound to hydroxyproline residues in a structural protein in cell walls.

Bb 3. Preparation of ISSPH or other vegetable protein hydrolysates.

Prior to the separation of ISSPH ("isoelectric soluble soy protein hydrolyzate") or other vegetable protein hydrolysates from the sludge, as described in U.S. Pat. No. 4,100,024 or in Process Biochemistry, Vol. 14, No. 7 (1979), pages 6-8 and 10-11, the reaction mixture can be treated with SPS-as preparations. This results in simpler separation.

Bb 4. Mashing enzyme in the brewing industry.

In the production of beer carbohydrates in the raw materials, e.g. the beta-glucans in malt and barley the viscosity and filterability of the wort. The addition of SPS ~ as during the mashing reduces the wort viscosity and improves the filterability and extract yield.

Furthermore, the addition of SPS-as during the mashing increases the fermentability of the wort and the nitrogen content of the wort.

Example Bb 4.1 In the laboratory, SO g of ground groats consisting of 50% malt and 50% barley were mashed together with 275 g of water (1S% dry matter) according to the following mashing diagram: 52 ° C (60 minutes) / 63 ° C (eo minutes ) / 7e ° c (about minutes).

To demonstrate the effect of the SPS-ace, four experiments were performed, see the table given below, in which the enzymes were added during the mashing (pH of the mash 5.5 - 6.0). 461 659 66 Enzyme Cereflo SPS-as Activity for beta-glucanase / g None (KRF 68) 0 200 BGU 1630 FBG 1630 FBG Dosavemzwnper kg groats Total dose of enzyme activity 0 units per kg in * Filtration rate for the wort after minutes Viscosity for vörten 10 Bal- lung (zs ° c) o 1.5 g 0,05 go, 1s g 300 BGU 80 FBG 300 FBG 120 ml 135 ml 160 ml 170 ml_ 1,52 CP 1,36 CP 1,36 CP 1, CP BGU are beta-glucanase units determined according to the analytical method AF 70/4-GB, which can be obtained from NOVO Industri A / S.

FBGs are fungal beta-glucanase units determined according to the analytical method AF 70.1 / 2-GB, which can be obtained from NOVO Industri A / S.

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

Cereflo is a bacterial beta-glucanase preparation described in information sheet B 214b-GB 1500 July 1981, which can be obtained from NOVO Industri A / S.

Example gel Bb 4.2 In the laboratory, 50 g of ground groats consisting of 40% malt and 60% barley were mashed together with 150 g of water (25% dry matter) according to the following mashing diagram: 45 ° C (60 minutes) / 63 ° C (90 minutes ) / 75 ° C (15 minutes).

In order to demonstrate the effect of the SPS ace, three experiments were performed, see the table given below, in which the enzymes were added during mashing (pH of the mash 5.5 - 6.0). 67 461 659 SPS-as (KRF 68) + Ceremix Enzyme No Ceremix added as in previous experiment Activity for beta-glucam / g zoo BGU 1630 HBG Dose of enzyme per _ kg of groats 1.65 g 0.033 g Total dose of enzyme activity units per kg 0 330 BGU 50 FBG STYH Filtration rate for 48 ml 98 ml lll ml wort after minutes funnel åšallinš 18.6 19.0 19.5 Viscosity of wort lg Bal- 1.72 cP 1.37 cP 1.27 cP ling (25 C) The definition of BGU and FBC is the same as in the context of Example Bb 4.1. given only the activity and the dose derived from the SPS-ace.

Ceremix is a bacterial beta-glucanase preparation described in information sheet B 216 b-GB 1000 feb. 1982, which is available from NOVO Industri A / S.

Bb. 5. Enzymatic additive for use during beer brewing- In the last column of the above table respiration and / or storage.

SPS-as can be added during wort brewing or beer storage in order to reduce the content of beta-glucans and thereby improve beer filtration and beer stability with respect to turbidity. SPS-as also has an effect on the proteins that are responsible for cloudiness in the cold.

Bb 6. Almond peel release agent.

During the mechanical step of removing almond shells after bleaching of almonds, a certain percentage of the almond shells do not come loose. It has been found that enzyme treatment of the tonsils results in a reduction of said percentage.

Bc 1. Decomposition of various waste materials.

In connection with certain manufacturing processes, large amounts of carbohydrate-containing waste materials are formed. For example, this is the case in connection with the production of soy isolate through water extraction 461 659 68 and acid precipitation, soy milk and tofu (a special kind of Japanese cheese). In this context, pulp waste from e.g. apples, pears or citrus fruits. It has been found that an SPS-as preparation can convert this carbohydrate-containing waste material into liquid in a complete manner and produce fermentable sugars which can be used as a starting material for ethanol fermentation.

Example Bc 1.1 In traditional production of soy milk or tofu, soybeans are often treated in boiling water, after which they are ground and extracted with hot water, after which a separation is performed. The residue from this separation is the material used for this experiment. The liquid phase is the soy milk, which can be further processed to make tofu. kg of whole soybeans obtained from Aarhus Oliefabrik A / S were ground simultaneously with 70 liters of boiling water in a Frymafkvarn of type MZ 110. The ground slurry was then kept above 85 ° C for minutes in order to inactivate the natural bean enzymes which produce the well-known weak aroma of soybeans. 5 liters of this soybean slurry were then centrifuged in the laboratory for minutes at 3000 x g (g = gravity). Through analysis, it was found that the residue contained 20.45% and 20.06% dry matter (double determinations, calculated average value 20.26%). 6 N HCl was added slowly and worked into the residue with a spatula until a pH meter showed 4.50, when the electrode was inserted directly into the mass.

Enzyme reactions of 2 x 200 g of the pulp with the two doses of the SPS-acetate (KRP-68) E / S = 0.5% with respect to dry matter and E / S = 3.0% with respect to dry matter were performed in a 500 ml beaker at SOOC. The dry enzyme was added to the mass. During the first 1-2 hours the stirring was carried out with a spatula, and after this the mass was transferred in a liquid state to such an extent that stirring with a magnet could be carried out successfully. The total reaction time was 21 hours. During the reaction, the osmolality was measured with an osmometer (Advanced Digimatic 3DII from Advanced Instruments Inc.). The results in Table Bc I show the course of the reaction. At the end of the experiment, the mixtures were centrifuged at 3000 x g for 15 minutes. A layer of oil appeared on top of the supernatants and the volume thereof was determined. A loose layer of sludge 69,461,659 appeared as the bottom layer. The supernatant including the oil was removed with a pipette. The oil was combined with the clear aqueous phase by homogenization and a sample was taken to determine the dry matter content. The results reported in Table Bc I clearly show that this waste product can be transferred in liquid form by an enzymatic reaction and that crude oil can be produced as discussed in section A4. After extraction of the oil, the solubilized residue can be used in various ways, e.g. for fermentation into valuable compounds or for concentration and drying and subsequent use as feed or food product, or after further purification for the production of valuable products._ Table Bc I. 461 659 70 soy milk and tofu sludge.

Results obtained during liquid transfer of Reaction conditions and results Experiment A Experiment B Weight of the residue 200 g 200 g Weight of SPS-axis (IRF-ÖB) 0.20 g 1.20 g Temperature temperature S 0 ° C 50 ° C PH 4, so 4, so Reaction time 2 1 hours 21 hours Results measured osmo- Aosmo- osmo- Aosmo- reaction process 0 287 0 0 282 0 313 26 10 368 86 Aosmolality is - - - 25 497 215 value corrected 40 391 - 104 45 601 319 for the osmolality 95 501 214 95 718 436 for the mixture at 250 634 347 250 875 593 t = 0 1260 907 620 1260 1145 863 Reaction mixture: Dry matter 20 .3% 20.7% Supernatant: Dry matter 18 .0% 19.4% Supernatant : Oil content 8-10% 8-10% Calculation “of solubilized dry material 88.6% 93.5% Bc 2. Saccharification and simultaneous fermentation.

Carbohydrate-containing plant material, e.g. tuberous plants such as Jerusalem artichokes, potatoes, sweet potatoes, cassawa, or pulp from such tuberous plants, ie the material remaining after removal of the extracted components can be saccharified by treatment with an SPS-as preparation, and at the same time the fermentable saccharides formed can be subjected to fermentation to ethanol. n 461 659 Exemgel Bc 2.1 Production of ethanol by fermentation of the decomposed inulin-containing Jerusalem artichokes was examined on a laboratory scale by simultaneous saccharification with SPS-as'och and inulinase and by four different pretreatments of the Jerusalem artichokes.

SPS-as: The SPS-as preparation KRF-68 was used.

Inulinase: The inulinase was prepared by fermentation of Asp. ficuum (CBS 55 565). Inulinase activity is determined as described in Research Disclosure No. 21234 (December 1981) pages 456-458.

Laboratory fermentation: 150 g portions of the pretreated mash (described later) were subjected to fermentation after the addition of 4: 5 g of baker's yeast and 1 ml of a 4% solution without foaming agent. The fermentation flasks are equipped with CO2 traps containing 98% sulfuric acid, and the fermentation is monitored by measuring the contents of the three flasks of Pluronic as an antidote to the weight loss caused by released CO2. the flasks are stirred during the fermentation carried out at 30 ° C. was used for each parameter studied. In Table Bc II, the weight loss caused by the release of CO 2 is converted to ethanol under the assumption that 1 mole of liberated CO 2 is equivalent to 1 mole of C 2 H 2 OH, i.e. 1 g CO2f * åg g C2H5OH.

Pretreatments of Jerusalem artichokes: Treatment A: 14.1 kg Jerusalem artichokes (22.8% dry matter) Henze-boiled at 140 ° C and 4-S atm. for 20 minutes.

The weight after cooking was 19.0 kg (-vl6.9% dry matter). Fermentations were performed directly on the mash.

Washed and sliced Jerusalem artichokes were mixed with water (lzl) and mixed in a Waríng mixer.

The mash was then heat treated for one hour at ss ° c and pH = 4.5.

As B, but the pH was not adjusted.

As B but without any heat treatment and without any pH adjustment.

Results: In Table Bc II, the results show the effect of adding SPS-as to the pretreated mash on the ethanol yield. A significant improvement in ethanol yield was obtained on all pretreated mosses when SPS-as was added.

Treatment B: Treatment C: Treatment D: 461 659 72 Table Bc II. Fermentation results regarding simultaneous fermentation and enzyme saccharification of Jerusalem artichokes.

Pre- Inulinase- SPS_ Loss of CO 2 (g)% produced units E / S: S after 42-44 ethanol 1 rela- hand-added barrels fermentation tion to dry ling to 1 g material dry mtrl 1.5 0 7, 05 f 0.05 31.5 A 1.5 0.27 8.07 f 0.08 33.2 B 1.5 0 4.85 1 0.03 29.7 - 1.5 0.40 5.41 f 0.03 33.1 1.5 0 5.70 i 0.05 34.8 1.5 0.10 5.97 1 0.01 30.5 c 1.5 0.20 0.13 f 0, 00 37.5 1.5 0.30 0.13 É 0.00 37.5 1.5 0.40 0.18 f 0.05 37, s 1.5 0 5.77 É 0.02 35.3 1.5 0.10 5.89 1 0.00 30.0 0 1.5 0.20 0.04 1 0.11 30.9 1.5 0.30 0.01 3 0.01 30.7 1 .5 0.40 0.02 in 0.03 30.8 0 0.40 5.48 3 0.02 33.5 Bc 3. Decomposition of cellulose.

It has been found that cellulosic materials such as straw, e.g. wheat straw, sawdust, paper and lignocellulose, can be hydrolysed to a greater extent with an SPS-as preparation than with conventional cellulases. This is illustrated by the following example, in which a crystalline cellulosic material (AVICEL) is treated with conventional cellulase Celluclast 200 manufactured by Trichoderma reesei and the SPS-as preparation'KRF 68.

Exqggl Bc 3.1 Avicel was suspended in water (20% dry matter); The pH was adjusted to S, and the temperature was maintained at 0 ° C. After a reaction time of 24 hours, the slurry was filtered, and the content of reducing sugar (mg glucose / g AVICEL) was measured. Using the enzyme doses 5% and 20% of the cellulose content, the following values were obtained: Table Bc III r Enzyme E / S% n glucose / g AVICEL Celluclast S 80 SPS-as 5 200 Celluclast 20 100 SPS- as 20 340 Bc 4. Use as a baking aid.

It has been shown that SPS-as preparations are excellent as baking aids. Thus, when an SPS-as preparation is added to the dry flour, before the dough is prepared, it is possible to obtain a bread of superior quality in terms of volume, crumb and taste. It is thus possible to obtain a high quality bread with low quality wheat flour, if an SPS-as preparation was used as an additive.

Bc 5. Improvement of alcohol exchange and biomass exchange during fermentation of sulphite liquid from papermaking.

It has also been found that the yield of ethanol can be improved if paper sulphite liquid is treated with an SPS-as preparation before it is used as a carbohydrate source for fermentation of ethanol. Paper sulphite liquid can also be used for the production of biomass, e.g. single cell protein, by fermentation, and even in this case the yield of biomass has been improved when the sulphite liquid has previously been treated with an SPS-as preparation. Decomposition due to the presence of the SPS-as preparation and fermentation can also be performed simultaneously.

Bc 6. Drainage of biological sludge products.

During traditional water extraction of many biological materials from plant raw materials, large volumes of insoluble residues are formed consisting of high proportions of swollen polysaccharides. This is e.g. the case when soy milk, tofu or soy isolate is produced by water extraction of soybeans, defatted soy flour or white flakes. The structurally swollen polysaccharide material can then be treated to a small extent with SPS-as, whereby the network structure of the material is opened, and only small amounts of carbohydrates are solubilized. Thereby the material is dewatered, and consequently higher dry matter content in the sludge is obtained in comparison with a product obtained without the enzymatic treatment. Thus, the enzyme process has the advantage of significantly lower energy consumption for removing water by drying, and it also opens up the possibility of producing a cheaper dry animal feed or bulking agent for food applications.

Bc 7. Silage aids .. _ It is known to add enzymatic silage agents to silage in order to increase the speed of the silage process and the degradability of the silage. It has been found that SPS-as preparations are superior to known enzymatic enzymatic aids.

An overview of the figures, to which we have already referred, is given below in order to provide a better overview. gig 'Belongs to Describes 1 The general part Demonstration of binding effect of the description between SPS and soy protein 2 The general part Flow chart describing product- of the description tion of SPS 3 Section 2 Calibration curve for HPLC gel filtration chromatography 4 Section 2 HPLC gel filtration chromatogram for SPS Section 2 HPLC gel filtration chromatogram for SPS disassembled by SPS-as 6 Sections 2 and 3 HPLC gel filtration chromatogram for supernatant from SPS incubated with soy protein 7 Section 2 HPLC gel filtration chromatogram for supernatant from disintegrated SPS incubated with SSapril of Pectolyase 9 Section 3 HPLC gel filtration chromatogram for APS decomposed by SPS-as Section 3 HPLC gel filtration chromatogram for SPS treated with Pectolyase II Section 7 Immunoelectrophoretic peaks including an SPS-as peak identified by ion exchange an SPS axis 13 Sec tion 9 pH activity dependence of an SPS axis 14 Section 9 Temperature activity dependence of an SPS axis Section 9 Temperature stability of an SPS axis 16 Section 10 pH stability of protease in an SPS axis preparation

Claims (6)

75 461 659 PATENT CLAIMS A process for producing SPS-as, which is a carbohydrate capable of producing a clear zone around a depression in an SPS agar plate and which in an aqueous medium having a pH value not exceeding more than 1, 5 from 4.5 exhibits the ability to decompose soybean SPS into decomposition products which bind to protein in an aqueous medium to a lesser extent than soybean SPS before decomposition would bind to the same protein under similar conditions, wherein soybean SPS is the water-soluble polysaccharide fraction in the retentate from ultrafiltration of the supernatant from the soybean meal treated with pectinase where the soybean meal denotes the protein-free carbohydrate fraction in defatted and peel-free soybean meal, characterized by growing an SPS-as microorganism belonging to the genus Aspergillus, which organism has the ability to grow in a medium containing SPS as the main carbon source in a nutrient medium, which contained r carbon and nitrogen sources and inorganic salts, and that if necessary isolate formed SPS-as. Method according to claim 1, characterized in that the SPS-as-producing microorganism is the strain Aspergillus aculeatus CBS 101.43 or Aspergillus japonicus IFO 4408. Process according to Claim 1 or 2, characterized in that the cultivation is carried out as submerged cultivation at a pH of from 3 to 7, preferably from 4 to 6, at a temperature of from 20 to 40 ° C, preferably from tiil ss ° c. A method according to any one of claims 1-3, characterized in that the nutrient medium contains soy flour. 5. A method according to claim 4, characterized in that the soy flour is treated with a proteolytic enzyme before use as a component in the substrate, preferably the proteolytic enzyme which is produced by microbial means by means of Bacillus licheniformis. Process according to one of Claims 1 to 5, characterized in that a sterile solution of pectin is added aseptically to the fermentation broth during the cultivation.