DD208821A5 - PROCESS FOR MANUFACTURING PLC ASE - Google Patents

Abstract

The invention relates to a process for the preparation of SPSase for the degradation of a high molecular weight carbohydrate. The object of the invention is to provide a novel enzyme which can easily and economically produce a protein product of high purity. can be. According to the invention, a novel microorganism capable of forming SPS ase, a carbohydrase, is selected for the degradation of soybean SPS into degradation products which attach themselves to protein in a moderate medium only to a small extent, in such a way that the microorganism to be tested is cultivated on a remote fermentation medium whose main carbon source is SPS, which is formed on the basis of vegetable crude protein, preferably defatted soybean meal, as starting material, whereupon a sample of the fermentation medium is assayed for SPS-ase and the microorganism in question as SPS-ase forming microorganism, if the analysis is positive for SPS-ase.

Description

The invention relates to the improvement of an enzyme for degrading a high molecular weight carbohydrate, a method for selecting a microorganism which forms such an enzyme, and a process for producing such an enzyme.

The novel enzyme provided according to the invention is used in the food industry, for example in the total liquefaction of vegetables, in the juice and wine industry, in sugar production, in beer production * for bakery production, in alcohol production and for many other purposes.

Characteristic of the known technical solutions

A method for producing a purified vegetable protein product (pvp) by enzymatic removal of the stain without dissolving and reprecipitating the protein is described in Belgian Patent 882,769. However, the purity of the pvp obtainable by the known process is unsatisfactory and therefore should be improved. In the examples, a purity of the pvp of about 85 % is stated. Even if it is possible, a pvp having a purity of about 90 % after this is only known

2SiIPR 1933 * 086331

24 617 5 2

_ ₂ _

possible using certain pretreated starting substances, for example of soy protein concentrate. It would be beneficial if it were possible to obtain a pvp purity of about or above 90% with a much broader spectrum of starting materials, especially from hulled and defatted soybean meal.

The invention is based on the surprising discovery that a certain part of the residue decomposition product, as occurs during the above-mentioned enzymatic treatment, ie. a water-soluble high molecular weight carbohydrate partially attaches to the vegetable protein (binds), as will be explained later. This naturally leads to a lower purity of the protein. It has also been found that this high molecular weight carbohydrate has the ability to attach to proteins of animal origin.

Object of the invention

The object of the invention is to provide an enzyme for degrading a high molecular weight carbohydrate, which offers the possibility of producing a pvp with improved purity.

Explanation of the essence of the invention

The object of the invention is to provide a method for producing such an enzyme.

The principle of the invention can be described as follows

61 626 12

246 17 5 2. ".

with reference to Pig "1, in which only products which are present as undissolved solids are indicated while all supernatants (and solutes therein) have been omitted. A quantity of soy flour was divided into two equal parts, part I and Part II (column a in Fig. 1). Part I was digested proteolytically at a pH of about 8 with the aid of ALCALASE (a proteolytic enzyme that has been produced by B. licheniformis and marketed by NOVO INDUSTRI A / S) and then further at about pH 8 to remove the total amount of protein, and the residue was separated from the supernatant liquid and washed (see Part I, columns a and b of Figure 1). Thus, a pure residue (referred to as residue I) was obtained. ieolated (column b in Fig. 1) · Part II of the soy flour was not treated ·

O / C 17 G Ί 61626 12

24 0 17 D Z _.3-

The residue in Part II is referred to as residue II (column b in Pig. 1). Now, both residue I and part II were degraded using a commercial pectinase (see columns b and c in Pig · 1).

Surprisingly, it has been shown that the undissolved part of the residue I is substantially lower than the undissolved part of the residue II on the basis of the nitrogen and dry substance balance, see Pig. 1, where the shaded areas in column c denote the insoluble Protein substances in the above-mentioned stage correspond. Moreover, when the supernatant fluid of the pectinase-treated residue I is brought together with a soy protein suspension at pH 4.5, a polysaccharide in the supernatant fluid is bound to the soy protein. This polysaccharide in the supernatant of the remainder I, which is a part of the remainder of the degradation product, which is clearly soluble in water in the absence of soy protein but is bound to it at or about the isoelectric point of the soy protein, if any, is termed SPS (Stolublβ polysaccharides) (see FIG. 1). The SPS has a molecular weight distribution between 5 × 10 and 4 × 9 × 10 ×. The formation of isolated SPS is evident from the scheme given in FIG. 2, which also comprises a part of the method indicated in FIG. Thus, the problem is to find an enzyme capable of degrading the SPS in such a way that the SPS degradation products

does not attach to soy protein or bind to soy protein to a much lesser extent than SPS. . ' .. '. , '-. / ^: -

Although the specification specifically mentions soy protein, the invention is not limited to soy protein, but includes all types of vegetable protein, see e.g. the proteins given in Belgian Pat. No. 882,769, page 1.

. ' ¹ . '

It has now been found, according to the invention, that it is possible to reduce its ability to degrade soybean SPS by selecting microorganisms capable of forming a compound which has enzymatic activity and is capable of degrading soybean SPS and hereinafter referred to as SPS -ase is called.

The invention therefore relates primarily to a SPS ase, a carbohydrase (glycosidase) in a suitable form, which is capable of degrading soybean SPS under corresponding conditions to products which attach themselves to protein to a lesser extent in aqueous medium than the soybean. SPS would have attached to the same protein under appropriate conditions prior to degradation.

It has also been found that this SPSase, which is capable of degrading soybean SPS, is capable of degrading more completely polysaccharides, similar to SPS and derived from plants (vegetables and fruits), than conventional pectinases and common cellulases.

The term "in a suitable form" as used above is understood to exclude, for example, an SPS ase containing agent containing toxic substances or having so little SPS activity ^: that more than 10 % of SPS ase used agent

24617 5 2-5-

must be based on the weight of the SPS in the substrate in a reaction which is carried out at 50 C for 24 h at the pH optimum of the relevant SPS ase to achieve a reduction of SPS of practical importance.

By completely or partially separating the SPS from the obtained vegetable protein, the purity of the obtained vegetable protein is improved in comparison with the purity of vegetable protein obtained by the method known from the BE-PS 882 769, since this vegetable protein product with PLC was contaminated.

It is not known at this time whether the special SPS ase described below has its enzymatic value; activi-

15 did or derives from a single enzyme

Enzyme complex comprising at least two enzymes. Some studies seem to indicate that at least two enzymes are responsible for the degradation effect of SPS ase, one of which can cause only a small degradation of the SPS, whereupon one or more enzymes are capable of further degrading the SPS. However, the invention should not be limited by this hypothesis.

The invention preferably relates to a SPSsese capable of degrading soybean SPS in an aqueous medium to products which attach less to vegetable protein in the aqueous medium than the soybean SPS prior to degradation. Preferably, the SPS ase invention is capable of degrading soybean SPS in an aqueous medium having a pH which does not deviate more than 1.5 from 4.5. The inventive

after completion of dismantling

SPS Ase leads to degradation products which accumulate preferably less than 50 %, in particular less than 20 %, based on the soybean SPS prior to degradation of vegetable protein in an aqueous medium positive SPS ase ^ test, when qualitatively and quantitatively determined by the method explained later, The SPSse is preferably formed by means of a microorganism belonging to the genus Aspergillus, preferably to the species Aspergillus niger SPS ase derived from enzymes that can be formed using Asp. Aculeatus CBS 101.43 · The same SPS ase can be formed using Asp. Japonicus IFO 4408. Asp. Aculeatus CBS 101.43 has also been shown to be very In addition, it has been found that not every strain belonging to the species Aspergillus aculeatus or Arpergillus japonicus produces a As can be seen later in the description (Section 5), it can be shown that the strain Asp. Japonicus ATCC 20 236 does not form such amounts of SPSase with Help the enzymatic determination of SPS-ase, as described later, can be detected. The SPSase according to the invention is preferably immunoelectrophoretically identical to the SPSase, which can be formed by Asp. Aculeatus CBS 101.43 and can be identified by immunoelectrophoresis overlay.

30 (see sections 6 and 7) ·

When a SPS seed is formed microbiologically, it is formed in admixture with various accompanying substances, in particular other enzymes.

- 7 -

24b17b L

If desired, the SPS Ase in question may be purified, for example by chromatographic separation techniques, as described in more detail later (Section 8).

In Agr. Biol. Chem. 40 (1), 87-92, 1976, it is described that a strain of Asp. Japonicus, ATCC 20236, forms an enzyme complex capable of undergoing a specific degradation of an acidic polysaccharide in soy sauce, referred to as APS. is called to evoke, a part of which is called APS-I. This acidic polysaccharide is not identical to SPS, as discussed in more detail later in Section 3. Thus, the HPLC-GeIfiltrations-Chroiaatogramme of SPS and APS are significantly different and also the gel filtration chromatograms of APS, which has been degraded using commercially available pectinase, pectolyase, and SPS, which has been treated with their usual pectinase, pectolyase _f is, clearly different. Moreover, it is not apparent from the article that the acidic polysaccharide is bound to the soybean protein and that the degraded acidic polysaccharide is not bound to the soybean protein or to a much lesser degree to the soybean protein than the undegraded acidic polysaccharide. In addition, it could be shown that this strain does not form SPS ase in such amounts as can be detected by the enzymatic determination of SPSase, as described in more detail later. This led to a prejudice against the use of any strain of Asp. Japonicus as a SPS ase-former. But surprisingly, it has been found in accordance with the invention that some strains of Asp. Japoriicus are SPS-ase generators. The invention also relates to the isolated SPS which has been formed on the basis of crude vegetable protein as the starting material.

I / J

In a preferred isolated

SPS is the vegetable raw protein

defatted soy flour. , The manufacture of the isolated SPS has been described above with reference to FIG.

The invention further relates to a method for selecting a SPS-ase producing microorganism to form the SPS-ase invention, wherein the microorganism to be examined is grown on a growth medium whose main carbon source is SPS, after which a sample of the fermentation medium analyzed for SPS-ase and the microorganism in question is selected as a SPS ase producing microorganism, if the analysis

15 PLC-ase is positive.

The invention further relates to a process for forming SPS ase, in which a selectable according to the above selection method for a SPS ase forming microorganism strain is grown in a nutrient medium. The cultivation can be carried out as a submerged culture or as a surface culture.

In the method according to the invention, preferably the strain Asp. Aculeatus CBS 101.43 or Asp. Japonicus IFO 4408 is grown in a nutrient medium. It is further preferred that the cultivation is carried out as a submerged culture at a pH in the range of 3 to 7, preferably 4 to 6 at a temperature in the range of 20 to 40, preferably 25 to 35 C, wherein the nutrient medium carbon and nitrogen sources and inorganic salts. Conveniently, the nutrient medium additionally contains roasted soybean meal. The soy flour

246175 2

is preferably treated prior to use as a component of the substrate with a proteolytic enzyme, preferably one which has been formed microbiologically, with the aid of Bacillus licheniformis. It is also preferred that a solution of pectin is added aseptically to the fermentation broth during the culture.

It has been found that Asp. Aculeatus CBS 101 .43 also forms very effective coagulation-solubilizing enzymes, cellulases, pectinases and hemicellulases in addition to SPS ase, and that the enzyme complex formed by Asp. Aculeatus GBS 101.43 is extremely well suited as a means of cell wall destruction of plant materials. Thus, the complex formed by Asp. Aculeatus CBS 101.43 can be used in the food processing industry for the treatment of fruit and vegetable mash and for clarification and viscosity reduction in the processing of fruit juice and wine with excellent results. It can also be used as a dehydrating agent (ie, means for breaking down polysaccharides and thus releasing the water bound in the polymeric structure of the polysaccharides (in the processing of vegetables) .The invention further relates to the use of a SPS ase or a method of degradation of polysaccharides, preferably plant cell wall polysaccharides, by means of a carbohydrase, in which an SPS ase-containing agent according to the invention is combined in an aqueous medium with a substrate for the SPS ase preparation.

30 is brought, · ·,

Thus, according to the invention it has been found that SPS ase additions provide valuable enzyme preparations for the partial or complete liquefaction or decomposition of various enzyme preparations.

- 10 -

246 17 5 2-

ro -

various materials, preferably plant materials, ζ. E. Fruit and plant wastes containing protein, cellulose, hemicellulose (eg glucans, xylan, galactans and araban), gums, pectin, lipids, inulin, polyphenols, starch and alginates are and for related purposes (see later table). As examples of such related purposes, there may be mentioned all purposes in which commercial pectinases and cellulases are used today. Some examples are given later.

In connection with the extraction (insulating) method, the z. As described in Example 2, it was found that the SPS-ase preparation is substantially proteinase-free, due to the fact that the desired end product, that the protein would _(otherwise degraded. Similarly, it should, if other biological materials as protein extracted from a biological raw material (isolated), the SPS ase preparation used will be substantially free of enzymes which degrade these other biological materials. Such modified SPS ase preparations may be formed by selective inactivation of the undesired enzyme, darCh Fractionation or other methods known per se.

Thus, a preferred use of the invention is characterized by the fact that degradation is achieved by isolating or extracting a biological material other than soybean protein and related plant proteins from a biological

- 11 -

24617 5 2-

see raw material wherein the SPS-ase preparation is substantially free of enzymes capable of degrading the biological material.

Thus, according to the invention it has been found that the modified SPS preparations (modified in the sense that they are substantially free of enzymes which are capable of degrading the biological material to be extracted or isolated) are valuable enzyme preparations Extraction (isolation) of special biological materials eg. As starch, lipids, flavors, dyes and juices from biological raw materials. Various examples are given later.

; .: Y. '. '-. ''.

According to the invention, it is preferred that one or more of the reaction products (regardless of whether they are desired end products or by-products) be treated simultaneously with or after the enzyme treatment.

This allows for a more flexible and adaptable way of working. This further treatment is preferably an alcoholic fermentation when one of the reaction products is a fermentable sugar. This gives a cheap process for the production of alcohol.

In order to explain the essence of the invention in more detail, reference is made to the following sections 1 to 10, each of which describes details in connection with the invention:

1. Production of SPS.

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

: '·. '' - ^'': ': ^·;..' .- - 12 -...

24 617 5 2

Λ O -

3. Proof that SPS and APS are different compounds.

4. Selection of SPS-forming microorganisms.

- ^: · ^;: . , '. ; ''. ^: '':

5. Characterization of some SPS-ase forming microorganisms.

6. General Description of Superposition Technique 10 in Immunoelectrophoresis.

7. Immunoelectrophoretic characterization of SPS ase with polyspecific antibody and overlay.

8. Cleaning a. · PLC as.e preparation.

9. Dependence of the SPS ase activity on pH value and temperature as well as stability of the SPS ase.

10 .. Enzymatic activity determinations.

Part 1

Production of PLC

As mentioned above, the starting material for the production of SPS 3a may be jarüdcstand (ingredients other than protein). Therefore, the production of soy residue is described in the first place.

Soy residue is the protein-free carbohydrate fraction (which in practice may contain small amounts of lignin and minerals) in defatted and

- 13 -

246 175 2, ι, - ^6162612

Soybean Flour · This carbohydrate fraction is insoluble in an aqueous medium at pH 4.5 and may be prepared in the following manner, with reference to Flow Sheet 1.

Defatted soy flour is in deionized water at 50 ⁰ C in a weight ratio of soy flour to water = 1: 5 suspended in a tank with a pH-stat under temperature control _9, the pH value (I) is adjusted to 8.0 with 4N NaOH · now is determined by means of Alkalase 0.6 L (a B. licheniformis proteolytic enzyme having an activity of 0.6 Anson units / g.sup.2 according to the Anson method described in Novo Enzyme Information IB ETr.058 e After a hydrolysis time of 1 h, the slurry is centrifuged off (III) and washed (IV.). Hydrogenysis under constant pH is carried out, the ratio enzyme: substrate being 4% of the amount of protein in the soybean meal (II) These measures are repeated twice (V, VI, VII). The slurry thus obtained is hydrolyzed once more for 1 h with Alkalse 0.6 L (VIII, IX), similarly as above. Then the slurry is centrifuged off (X. ) and twice gewa (XI, XII, XII, XIV), and the slurry (6) obtained after the last washing step is spray-dried (XV). The spray-dried powder thus obtained is the soy residue used as starting material for the production of SPS.

SPS is the water-soluble polysaccharide fraction obtained by conventional treatment of the above soy residue with pectinase. The SPS is obtained in the following manner using the 14 reaction steps given below.

/ C 1 7 C-O 61626 12

4 b I / 5. 2 - ¹⁴ -

1 "The content of the dry ingredients in the above-mentioned soy residue is determined and the soybean residue is diluted with water to a content of 2% and Peststoffe fehalten in a tank with temperature control at 50 ⁰ C.

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

3. Peetinex Super Cone · L is added in an amount of 200 g / kg solids (a commercial pectinase with a pectinase activity of 750 000 MOU, determined in accordance with the prospectus "Determination of the Pectinase unite on Apple Juice (MOU)" of 12. 6. 1981). Ee is further Celluclast 200 L added in an amount of 20 g / kg of dry matter (a commercial cellulae, described in the prospectus NOVO enzymes, information sheet B 153 e-GB 1000, July 1981).

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

5 · The enzymes are inactivated by raising the pH to 9.0 with 4N NaOH. Daa reaction mixture is allowed to stand for 30 min and the pH is then adjusted again to 4 »5 with 6N HCl.

6 · The reaction mixture is centrifuged and both the centrifuges (supernatant) and the residue (sludge) are collected »

2U175 2 -ι ₅ - ^6162612

7. The tetrastrotate from step 6 is filtered through a filter press (the filter was washed with water before filtration)

8. The filtrate is ultrafiltered, diafiltered, and re-ultrafiltered over a membrane having a 30,000 Trenh value using the following parameters:

1. ultrafiltration to a volume concentration of 6.

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

3 · ultrafiltration to about 15 % solids content in the concentrate ·

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

9. The ultrafiltered concentrate is cooled to 5 ⁰ C and a volume of 9 & gleicheβ% ethanol added "

10. The precipitate is collected by means of a centrifuge ·

11 · The precipitation is repeated twice with 50 % (Vol / Vol)

Ethanol in H "0, corresponding to the volume [of the centrifugate from stage 10, ie two centrifugation stages are carried out.

D 17 ί)

- ID -

12. The washed precipitate is redissolved in water, with a volume corresponding to the volume of the ultrafiltered concentrate of Stage 9.

13. Level 12 fluid is filtered through a glass filter for testing.

1.4. The clear filtrate containing pure SPS is lyophilized.

- 17 -

- - »WI ^ J

Flow chart Γ defatted soy flour H ₃ O NaC) H to pH

= S

J hydrolysis mixture

Alcalase O, 6 LJ _hydrolysis χ _{h VX) for} discharging

pH-stat (pH = 8, T = 50 ° C)

(E / S = 4 4n NaOH

II

1. centrifugation

III

Zentrifugati

j On slumber

1. ^ e. _:

IV

' Centrifugation

ν.

waste

centrifugate

  waste

H-O 2 .. wash V > Digestion 2 VII VIII Center i - · * ·> step VI On schlmniunc /. ' \ fnaat '^ \ Hycrolysegeinisch Hydrolysis 1 h before discharge:. , - pH - & tat (pH = 8.0, Waste ? ³ T = 50 C) .New H-O '' 3. centrifugation NaOK ^to pH - ^ S ' ÄiraTa ^ e O 6 ί. , , IX 4N NaOK

4. 'Centrifugation

Cirrus fugat

H-O

J l. Vjasch- A level

Aufschlärrnnung

XI

- "Z entrif ugat ion

XII

centrifugate

HO J ? .. Wasph-

waste

On ρ ch la mrau jig

XIII

, centrifugation

XIV

Centrifugate ·

slurry

spray drying

XV

FIi e scheme II

1000 g Pectinex Super cone. f spray-dried L 5 kg ter residue ' 1 »2. _f 3, 4, 5 100 σ Cellucl branch 250 S ' Rait degradation and cellulase 50 ⁰ C; pH = 4 _T 5? s Pectinase t = 24 h 24 7 1 H, 0   ν · J. .- · /

centrifugation , On schlaiaijnc · Check filtration     , supernatant 241 I ^ centrifugation Centrifugate ^ precipitation Protein on £ Lyöphilisierung 3b kg Ultrafiltration, diafiltration, ultrafiltration (GR 60P) (19 - 26 ° C) Permeate · 2x ', What _C hen I 2 χ Centrifugate ^ ichlmrrvung _v > : Bourgeoisie (Waste) / (Waste) precipitation 18 1 Η_0. precipitation redissolving S 1 50% C_H-OH. Check "filtration   > 2 1 Η_0 ν

310 g lyophilized SPS

λ , ra nc ο ^{61 626 12}

2Λ 6 17 5 2 -19-

Section 2!

Characterization of SPS and especially molecular weight distribution

By gel chromatography using an HPLC apparatus (Waters Pump Model 6000, Waters Data Module 730 and Waters Refractometer R 401), the molecular weight distribution of the SPS, whose preparation was carried out as described in the description, was determined (Pig x 4) Using the same method, the molecular weight distribution of the degradation products of SPS obtained with SPSase was also determined (Pig × 5). In addition, by the same method, the attachment effect between soybean protein and SPS (Pig × 6) and the absence of the attachment effect were inferred Soy protein and SPS, which had been degraded using the agent according to the invention (Pig 7) shown.

The calibration curve (the logarithm of the molecular weight against R 1, wherein the R 1 value for glucose is arbitrarily defined as 1 and the R 1 value for a specific dextran as the retention time for this dextran divided by the retention time for glucose, is defined) using various standard dextrans of known molecular weight (T 4, T 10, T 40, T 70, T 110, T 500). The R ^ value for the maximum of each dextran peak was determined and the corresponding

the molecular weight calculated as V® _w * _n * where the weight average molecular weight and SL is the number average molecular weight. The eluent used for this gyration procedure was 0.1 mM solution of the solution. The columns used for the chromatography

246 17 5 2

61 626 12 - 20 -

were 60 cm PW 5000 and then 60 cm PW 3000. In this way, the relationship between the molecular weight and the Rp value for the above dextranes was set up, see Pig · 3 ·

On the basis of Pig * 4, it can be calculated that SPS has a molecular weight distribution leading to a value of M__ of approximately 5.4 x 10 and a value of approximately 4.2 x 10 * # This figure also goes show that the chromatogram shows two distinct peaks at retention times of 34.5 min (6 ^, corresponding to a molecular weight of approximately 5 x 10 and a retention time of 47 to 12 min (67 %), corresponding to an apparent molecular weight of approximately 4.9 x 10 ' From the curve, it can be seen that there is a shoulder between these two peaks, with a reaction time of 41.2!> min (27 %) t, corresponding to a molecular weight of Ji, 8 χ 10 ·

After degradation of the SPS with SPS ase, the hydrolysis mixture was filtered through a membrane and the filtrate chromatographed. It was found that about 55 % of the SPS was degraded to mono-, di- and trisaccharides, d and that the remaining 45 % was of a polymer having three peaks with the following molecular weights: 5 × 10 ⁴ , 10 × and 4.4 × 10 ³ , see Pig × 5.

In order to show the attachment effect between soy protein and SPS and the substantial reduction of the soy protein-SPS attachment effect, which had been degraded by means of SPS ase, the following experiments were carried out s.

3 % SPS in 0.10m acetate buffer at pH 4.5 became too

2Λ617 5 2-

a slurry of soy isolate (Purina 35 500) to obtain a suspension with an isolate to SPS ratio of 10: 1. This suspension was incubated for 18 h on a shaking bath at 50 ^° C. After incubation, the suspension was centrifuged and the clear supernatant liquid analyzed by HPLC as described above. From a comparison of Fig. 6 with Fig. 4 shows that the SPS is completely adsorbed to the soy isolate.

io. · ·. '. ^; ;. ;;. ν-. , ';..:.; ; '''·.

The same procedure as given in the previous paragraph was carried out with a 3% SPS solution hydrolysed with a SPS ase formed from CBS 101.43 (Figure 7).

A comparison between Fig. 7 and Fig. 5 shows that no compound in the hydrolyzed SPS having a molecular weight below about 10 was adsorbed to the soy isolate *. Hydrolysis reduces the quantitative addition to about 10 to 15%, based on the investment.

20 PLC from soy protein.

The NMR analysis of the SPS, whose preparation was performed as indicated in the description, shows the following approximate composition of the SPS. .25 / · ^; ....

1) dC-galacturonic acid in an amount of about 45%, with about 40 % of the total amount of the oC-galacturonic acid being present as methyl ester.

30 2) Rhamnopyranose in an amount of about 20 %.

3) galactopypyranose in an amount of about 15% and

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

- 22 -

2A6 17 5 2

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

Complete acid hydrolysis of SPS (8h in 1 η HpSO,) and subsequent analysis by thin layer chromatography (TLC) showed that even minor amounts of the monosaccharides fucose and arabinose in the _; Jiydrolysierten PLC were present.

: '. ; HPLC analysis of the SPS degraded by the SPS ase enzyme complex formed by CBS 101.43 showed a strong reduction in molecular weight. Consistent with this, the NMR spectrum of the SPS degraded as indicated above indicates that most of the ester groups had disappeared and that the levels of xylose and galactose in the higher molecular weight material had also decreased. The NMR spectrum of the portion of the SPS degradation product precipitated by addition of one volume of ethanol to one volume of SPS degradation product is similar to the NMR spectrum of the SPS with the above-mentioned modifications to the ester groups and the content of xylose and galactose.

23 -

24617 5 2

Section 3

Documentation of the fact that PLC and APS are different connections.

APS was produced as in Agr. Biol. Chem., Vol.36, No.4 pp. 544-550 (1972).

This polysaccharide and SPS were now hydrolyzed with different enzymes, after which the digestion mixture was assayed by gel chromatography using HPLC equipment as indicated in Section 2 "Characterization of SPS, especially Molecular Weight Distribution".

, , ^'': '' - ^'.:;'. '...''· .. ^..:': ': More specifically, the hydrolyses were carried out by treating 25 ml solution of either 2% or 2% APS SPS in 0.1 M acetate buffer, pH 4.5 with 10 mg KRF 68 or 30 mg pectolyase KRF 68 is a SPS ase preparation, the preparation of which is described in Example 1 The results are shown in the following table.

Polysac- · enzyme HPLC gel      - polysaccharide light degraded CharID. Pectolyase chromatogram , reduced APS KRF 68 Fig. 8 X hJPS Pectolyase Fig. 9 X X SPS KRF 68 Fig. 10 SPS Fig. 5 χ

- 24 -

246 17 5 2

Section 4 ί

Selection of SPS-forming microorganisms.

The microorganism to be examined is incubated on an agar slant substrate with an agent which allows the growth of the microorganism. After initial growth on the agar slant substrate, the microorganism is transferred to a main liquid substrate in which the main carbon source is SPS (prepared as indicated) in which the nitrogen source is N0, ~, NH ^ ⁺ , urea, free amino acids, The composition of the main substrate depends on the nature of the microorganism, the most important factor being that the main substrate should be capable of being formed to support the growth and metabolism of the microorganism. When the growth has taken place for a corresponding time of the order of 1 to 7 days, depending on the growth rate of the microorganism concerned, a sample of the fermentation broth is placed on SPS ase after the enzymatic SPS ase determination as indicated in this specification , analyzed or for any other SPS ase determination that is "tailor-made" for other specific uses of SPS ase than use as part of a soy residue remover.

In order to achieve a more sensitive method for determining the enzymatic activity, the temperature should be lowered to 40 ^° C. and the incubation time should be increased

24 617 5 2 - ₂₅ -

20 hours during the determination of the SPSase activity, antibiotics should be added to the substrate to avoid infection.

By following this test procedure, other SPS ase-forming microorganisms belonging to both the species Aspergillus and other species can be found.

. '. Section 3 . ' . ^:: ''. :. ,

io. . .. '' \ _'':::. ·. ^: ';' . ". '': ' '

Characterization of some SPS-ase forming microorganisms |. '/ I .. ''.''. . ' .. '

According to the search method described here for SPS-ase-forming microorganisms, it has been found that the microorganisms indicated in the upper part of the following table are SPS-ase-formers. The table also contains a strain belonging to the species Asp. Japonicus, but not a SPS ase-bildner.

PLC-Ase Bildner .No."'. kind Asp. • .japoenicus Asp. Aculatus Identification Nuirans Deposit number Deposit year .Yes χ X X A 805 A 1443 A 1384 CBS 101.43; P ^ M l "3" fH IFO 4408; o? M. 23 * 6 ATCC 2023 6; DSM 2.JV-5 " 1943 1950 1969 χ χ

246 17 5 2

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

List of Cultures 1978 Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands

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

;; ^; ;,;.; , , '.. -' .. '-' _/:.: The American Type Culture Collection Catalog of Strains I, 14th edition 1980, 12301 Parklawn Drive, Rockville, Maryland 20852..

All of the above strains correspond well to the taxonomic description of the species Asp. Japonicus and Asp. Aculeatus as reported in The genus Aspergillus of Raper and Fennell, 1965 (see especially pp. 327-330).

'20 ·. ν i- ';:': ''. '. · · .. · · ·; . ''.; Section 6

General description of the overlay method in immunoelectrophoresis.

It has been referred to as a top agar overlay technique for identifying individual components of an enzyme complex by cross-immunoelectrophoresis

Developed 30 poly-specific antibodies against all enzyme components in the enzyme complex. The method is based on the fact that enzymes are still active after specific enzyme-antibody binding, or in other words, that the active enzyme sites are not identical.

35 tables are with the sites of enzyme-antibody binding.

246 17 5 2- ²⁸ -

The enzyme-antibody complexes are precipitated as distinct arcs during electrophoresis in the gel. The gel plate is covered with soluble SPS in agar. After heating for 20 hours at 45 ° C in an atmosphere with a relative humidity of 100 % , the arc with SPS ase activity appears as a clear zone in the SPS cover after precipitation with a mixture of equal volumes of ethanol and acetone when viewed against a black background. Bows that have no SPS ase activity remain invisible.

Section 7

Immunoelectrophoretic characterization of SPS ase with polyspecific antibodies and overlay.

Rabbits were immunized with the SPS-ase containing enzyme complex obtained by fermentation of Aspergillus aculeatüs CBS 101.43 as indicated in Example 1 (KiIF 68) and. Polyspecific antibodies were obtained in a manner known per se. Using this polyspecific antibody, cross-immunoelectrophoresis of the enzyme complex obtained by fermentation of Asp. Aculeatüs CBS 101.43 as indicated in Example 1 (KRF 68) was performed as described by NH Axelsen et al., In A Manual of Quantitative Immunocelectrophoresis ", 6th Edition 1977. Reference is made to Figure 11, which shows the arcs corresponding to the different proteins produced by the microorganism. With the aid of the agar overlay method described above, it has been found that the shaded areas correspond to the SPS axis.

If the above hypothesis that the SPS ase

- 29 -

24 6 17 5 2 -

61 626 12

29 W-. .-- .. '- ..,. ; '-.

is true, the hatched area is the area where all the enzymes responsible for the SPS ase activity are present. When these enzymes are separated by immunoelectrophoresis in other ways according to the invention in such a way In order to avoid covering a common area, some of the SPS ase activity can still be identified by immunoelectrophoresis overlaid with both SPS and a commercial pectinase.

Section 8

Cleaning a PLC ase preparation

The purification of the SPS ase preparation KRP 92 (see Example 1) was carried out by ion exchange. The buffer was 50 mM Iris (tris-hydroxymethylaminomethane) adjusted to pH 7.0 with HCl. The column was a Κ-5 / 30 column · The ion exchange material was DEAE-Trisacryl (300 ml) · The flow rate was 100 ml / h.

15 g of the SPS ase preparation KRP 92 were dissolved in 450 ml HpO at 6 ⁰ G and carried out all other specified process steps between 6 and 10 ⁰ C · The pH was adjusted to 7.0 with 1 M Tris. The column was equilibrated with the buffer and then the SPS ase sample was applied to the column. The optical density (ODoqq) and conductivity were measured on the eluate (see Pig. 12). The fraction 1 was the eluate which was not was bound to the ion exchange material. Then the column was filled with 2000 ml of buffer

0 / C17C 1 61626 12

246 1 7 5 2 -3 ° -

Then, a 0-500 mM NaCl gradient was formed to give fractions 3 to 9. All 9 fractions were concentrated to 200 ml and dialyzed against water to a conductivity of 2 mSi. Then the 9 fractions were freeze-dried. Only fractions 1 and 2 showed SPS ase activity.

Fraction 1 was further purified by gel filtration. 1.5 g of fraction 1 were dissolved in 10 ml of 50 mM sodium acetate, pH 4.5 (500 mM KCl). The column was a 2.5 x 100 cm column. The gel filtration filler was Sephacryl S-200. The Durshflußgeschwindigkeit was 30 ml / h. The fractions containing substances with molecular weights between 70,000 and 100,000, calibrated with globular proteins, contained an enzyme complex referred to as factor G which, upon assaying after the qualitative agar test, can not degrade SPS. However, SPS degrades after the qualitative agar test when the factor G is mixed with a pectinase. It has been found that the factor G is able to cleave galactose, fucose and part of the galacturonic acid from SPS, but according to the HPLC analysis the main degradation product is still a high molecular weight product which is very similar to SPS.

Section 9,

Dependence of the activity of pH and temperature as well as stability of a SPS-ase

Fig. 13 shows the pH dependence of the activity of the SPS ase preparation KRF 68 · from pH 2.7 to pH 3.5

2Λ6 17 5 2

For example, a formic acid buffer was used, and from pH 3.7 to 5.5, an acetate buffer.

FIG. 14 shows the dependence of the activity on the temperature of the SPS ase preparation KRF 68.

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

- 32 -

 24.6.! 7: 5- 2

Section 10

Determination of enzyme activity.

The table below is a summary of the various enzyme activity determinations with reference to

The invention. - - - -.:. ,

 r Short description of the activity Definition of the activity unit and description of the determination of enzyme activity Described in the description later Literature no. enzyme SPS-ase Generally accessible '-! ·'' ^X -.'' SPS-ase SRU •. '·'. ^! '.- "''· 1 Residue-dissolving £ RUM-120 .. ^x X protease CAP cellulase ^G x 2 pectinase PU * 3 Hemicel Jlulase PGE X 4. UPTE X 5 PEE X • 6 VHCU X 7 X

The literature given in the above table is explained in more detail in the following table.

2Λ6 17 5 2

  Identification of the reference received from swine; Ferrari AG, Ba sel, Swit zerland book trade Litera door no Analytical Research AF 154/4 of 1981-12-01 NOVO INDUSTRI h / S, Novo Alle, 2880 Bagsvsrd, Denmark 1 Analytical Biochemistry 84, 522-532 (1973) X X 2 Analytical method AF 149/6-GB.of 1981-05-25. 3 Determination of Pectinase Activity with Cetrus Pectin (PU) of March 23, 1976 X X 4 Viscosimetric Polygalacturonase Determination (PGE) of 10.11.77 X 5 Determination of pectin transeliminase (UPTE / g) of 24.Sept.1975 χ Determination of the pectin esterase acti vity (undated) with initials WJA / GW X 7 Analytical method AF 156/1-GB

With particular reference to the determination of cellulose activity, it should be noted that the analysis was carried out as indicated in AF 149/6-GB and that the principle of determination is explained in Analytical Biochemistry.

O / C 17 C 0 6162612

7 k BI 7 5 2 -34-

Section 10 a

Determination of the enzyme activity of SPS-ase

The determination of the enzyme activity of SPS-ase was carried out in two stages, ie a qualitative agar plate test and a quantitative determination of the SPS ase activity, which is based on the measurement of the total amount of released sugars. If the qualitative agar plate If the qualitative agar plate assay is positive, the SPS ase activity is zero, regardless of the value obtained in the quantitative determination of the SPS ase activity. If the qualitative agar plate assay is positive, the SPS ase activity is equal to the value obtained in the quantitative determination of the SPS ase activity.

I · Qualitative agar plate test

A SPS agar plate was prepared as follows: Buffer B was prepared by adjusting 0.3 M acetic acid to pH 4 »5 with 1 N NaOH. 1 g of SPS is dissolved in 20 ml of B · 1 g of agarose (HSB Litex) is mixed with 80 ml of B and heated with stirring to the boiling point. When the agarose has dissolved, the SPS solution is slowly added · The 1% SPS-agarose Iösung obtained is placed in a water bath at 60 ⁰ C · The plates are now by pouring 15 ml of the 1 jSigen PLC agarose -Losung made on a horizontal glass plate of 10 χ 10 cm · Then nine holes at a distance of 2.5 cm punched into the solidified layer of the SPS agarose · In each well 10 / Ul of a 1 $ Lgen solution of the PLC ase activity to be examined enzyme protein given «The plate is 16 h

246175 2

61 626 12

35 -

at 50 ⁰ CJund a relative humidity of 100% inkubieri · now not degraded SPS is precipitated with the aid of a solution of equal volumes of ethanol and acetone. The SPS Ase agar plate test is positive for a sample in a particular well when a clear annular zone is formed around this well. . ' , . ' / \ ·: '. , :. 'v;',

II. Test for the quantitative determination of the SPS ase activity

The purpose of this experiment is to determine the enzyme activity capable of degrading SPS to such an extent that the degradation products show greatly reduced or no adsorption or binding affinity to soybean protein. Tests have shown that the portion of the SPS degradation products, the is not precipitated by a mixture of equal volumes of water and ethanol, has no adsorption or binding affinity to soy protein.

The SPS ase determination is based on the hydrolysis of SPS under standard conditions and subsequent precipitation of the part of the SPS which has not been hydrolysed with ethanol. · After filling, the content of non-precipitated carbohydrates is determined by quantitative analysis on the total sugar content.

The standard conditions are:

24 b 1 / t> L -36-

Temperature: 50 ° C

pH: ' : "': - ;. ·.; - / 4, '5 ·>: ·· = :: - ^: ..:",' /. Reaction time: equal to 210 min only with substrate, then 2 min with added ' . '' · ,, : / Enzyme ',; -y, - ·''... · -v' - ";'.-.,.'r'. ,

Main value 212 min.

The device comprises:

Thermostatically controlled vibrating water bath of 50 ⁰ C magnetic pulser centrifuge

Ice-water bath

The reagents include:

Buffer: 0.6 M acetic acid in defloccinated

Water (a) 15 1.0 M NaOH (b)

Substrate: The pH of 50 ml a is raised to b

4.5 are added, then 4.0 g of SPS are added and after dissolution of the SPS, the pH is readjusted to 4.5 and the volume is treated with deionized water.

_? ... brought to 100 ml.

Stop Reagent: Absolute Ethanol.

A SPSase unit (SAE or SPSU) is defined as the SPS ase activity which liberates an amount of 50 % ethanol soluble carbohydrate, corresponding to 1 / μM galactose per minute, under the standard conditions given above.

Even if the initial part of the enzyme standard curve is even, it is noted that it is the (0x0) point

- 37 -

24617 5 2 -37-

does not intersect · section TO b

Enzymatic determination of residue-solubilizing activity expressed as SRtJM 120

Principle:; '/,' '. "..'. '- \

In the method for the determination of the hydrolysis activity, the insoluble portion of degreased deproteinized and uncovered soybean meal is hydrolyzed under standard conditions. The enzyme reaction is started with the demolition reagent and the insoluble portion is filtered. The amount of dissolved polysaccharides is determined spectrophotometrically after acid hydrolysis. .-: '.;' - ....... ν ^; ^ '·' '' ..

According to the method, carbohydrases with endo- and exo-activity are determined ·

The substrate for this enzyme determination is identical to the residue substrate described for the SRU process. The substrate is dissolved as a 3% solution in the below-mentioned octitrate buffer;

0.1 η citrate-phosphate buffer pH 4f5

5.24 g citric acid 1 hydrate (Merck Art 244) 8.12 g disodium hydrogen phosphate 2 hydrate (Merck Art 6580)

ad 11 demineralised water pH 4.5 ± 0.05 14 days stable ·

H UI / O / - 38 -

The termination reagent has the following composition:

100 ml of 0.5 N NaOH

200 ml of 96 % ethanol

Store in the refrigerator until use.

standard conditions

temperature 50 ⁰ C pH 4.5 Reaktionszeil 120 min 5 min ;, Sample blank Definition of the unit

A soy residue solubilizing unit (SRUM) 120 (M for manual) is the amount of enzyme that releases dissolved (solubilized) polysaccharides per minute, corresponding to 1 / μmole galactose, under the given reaction conditions.

Section 10c

Enzymatic determination of proteolytic activity.

HAT measurement

Method for the determination of proteinase in acidic medium.

The method is based on the degradation (digestion) of denatured hemoglobin by the enzyme at 40 ⁰ C, pH 3.2 within 30 min. The undegraded hemoglobin is precipitated with 14% trichloroacetic acid (w / v).

- 39 -

246 17

All Erizympröben are prepared by dissolving in 0.1 M acetate buffer pH 3.2.

The hemoglobin substrate is prepared using 5.0 g lyophilized bovine hemoglobin powder preserved with 1 % thiomersalate and 100 mL of defibrillated water and stirred for 10 minutes, then the pH is adjusted to 1.7 with 0.33 η HCl. will ... Λ.:. ; ' ^: ::: "' -, ' .' . .. · ~ ^": V. , '. ''

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

The hemoglobin substrate is brought to room temperature. At time zero, 5 ml of substrate are added to a test tube containing 1 ml of enzyme. After 1 s after shaking the tube for 30 min placed in a water bath at 4O ⁰ C. After exactly 30 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.

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

30 becomes.

- 40 -

SJ I f JC -40-

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

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

calculations

1 HUT is the amount of enzyme that forms a hydrolyzate in 1 min, which, when absorbed at 275 nm, corresponds to a solution of 1.10 / μg / ml tyrosine in 0.006 η HCl. This absorption value is 0.0084. The reaction should proceed at 40 ^° C. and pH 3.2 for 30 minutes.

HUT = sample blank _χ vol. In ml

0.0084 reaction time in min

HAT = _χ Λ ± _{= (p} _ _{B) χ} ^ ^

0.0084 30

(P-B) χ 43,65

HAT / g enzyme =

The study of the dependence of protease stability in KRF 68 on pH by HUT analysis with pH values of 2.0 to 8.0 showed that stability

- 41 -

6 17 5 2;

the protease was very low above pH 8.0, see Pig. 16th

embodiment

For a more detailed explanation of the invention, reference is made to the following Examples 1 to 8, wherein Example 1 illustrates the preparation of SPS ase and Examples 2 to 8 illustrate the application of SPS ase to a soy source to add a purified vegetable protein receive. Other applications of SPS-ase are given in the section between example 8 and the summary of the figures.

Several fermentations were carried out on a laboratory scale with the strains of Aspacleatus and Asp. Japonicus indicated herein. This gave an agent containing SPS ase, according to the here indicated SPS ase test. However, since relatively large amounts of SPS are required to run application trials, similar pilot scale experiments were performed, see Example 1 below.

Example 1

Production of SPS-ase in a pilot plant

An SPS ase was prepared by submerged fermentation of Aspergillus aculeatus GBS 101 * 43.

In a Fernbach flask, an agar substrate of the following composition was prepared:

Peptone Difco 6g

Aminolin Ortana 4g

Glucose 1g

Yeast Extract Difco 3g

248175 2

Meat Extract Difco 1,5g

KH ₂ PO ^ Merck 20 g

Malt extract Evers 20 g

demineralized H ₂ O ad 1000 ml

The pH was adjusted between 5.3 and 5.35. Then, 40 g of agar (Difco) were added and the mixture was treated for 20 min in an autoclave at 12O ⁰ C. The substrate is called E agar.

The strain CBS 101.43 was cultivated on an E-agar slant (37 ⁰ C). The spurs from the slope were suspended in sterilized skimmed milk and the suspension was lyophilized in glasses. The contents of a vial of lyophilized contents were placed in the Fernbach flask. The flask was then incubated at 30 ^° C. for 13 days.

A substrate having the following composition was prepared in a 500 1 seeding fermentor.

CaCO2, 1.2 kg

Glucose 7.2 kg

20 Rofec (maize water solids) 3.6 kg

Soy 1,2kg

Tap water was added to a total volume of approximately 240 liters. The pH was adjusted to about 5.5 before the addition of CaCO3. The substrate was sterilized in the seed fermenter for ¹ h at 121 ^° C. The final volume before inoculation was about 300 l.

The spore suspension from the Fernbach flask was in

246175 2

transferred the vaccine fermenter. The conditions of the seed fermentation were:

Fermenter type: conventional aerated and stirred fermenter with a ratio height: diameter of approximately. ^: ''.-2.3. ' ^r ' ν · '. ". ' -. "'^;

Stirring: 300 rpm (two turbine propeller stirrers);

Ventilation: 300 normal liters of air per minute Temperature: 30 to 31 ⁰ C

Pressure: 1.5 bar (0.5 ato) time: about 28 h

Approximately 28 hours after inoculation 150 μl were transferred from the seed fermenter to the main fermenter.

In a 2 500 liter main fermentor, a substrate of the following composition was prepared:

Toasted Soy Flour 90 kg KH ₂ pp ^. 20 kg

Pluronic 150 ^

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 raised to 50 ⁰ C. Thereafter, approximately 925 Anson units of Alcalase 0.6 L were added to the suspension. The mixture was maintained at 50 ^° C. for 4 h and pH 8.0 (Na ₂ CO, addition) without aeration under normal pressure (zero ato) and stirring at 100 rpm. Subsequently, the remaining components of the substrate were added and the pH was adjusted to about 6.0 with phosphoric acid. The substrate was in the main fermenter for 1.5 h

- 44 -

2 k 6 ι 7 5 2 - 44 -

sterilized at 123 ° C. The final volume before inoculation was approximately 1080 1x.

Then 150 μl of seed culture were added. The conditions of the fermentation were:

Type of per- menter: Usual aerated and stirred permen-

height with a diameter of approximately 2.7 l

Stirring: 250 rpm (two turbine propeller stirrers)

Ventilation: 1200 normal liters of air per minute Temperature: 30 ⁰ C Pressure: 1.5 bar (0.5 ato) Time: about 151 h

Between about 24 and about 116 hours of fermentation, a pectin solution was aseptically added to the main fermenter at a constant rate of about 8 L / hr. The pectin solution of the following composition was prepared in a 500 L dosage tank:

Enjoy pectin 22 kg Phosphoric acid, conc " 6 kg Pluronic 50 ml

^x ' Genu pectin (Citrus type HP)

Tap water was added to a total volume of about 325 L. The substrate was sterilized in the dosing tank for 1 h at 121 ^° C. The volume of volume

246 17 5 2-45-

Before the start of dosing or addition was about 360 1 * After expiration of this amount, a second similar approach was prepared. The total volume of pectin solution for fermentation was approximately 725 l.

After about 151 hours of fermentation, the fermentation was stopped. The approximately 1850 L of culture broth was cooled to approximately 5 ° C by the following procedure.

were cooled to about 5 C and the enzymes after

The culture broth was filtered using a vacuum drum filter (Dorr Oliver) coated with filter aid (Hy-flosuper-cel diatomaceous earth). The filtrate was concentrated by evaporation to about 15% of the volume of the culture broth. The concentrate was filtered through a Seitz filter film (Type supra 100) with 0.25 % Hy-flo-super-cel as a filter aid (referred to as Filtration I in the following table). The filtrate was precipitated with 561 g (NH ₂ ) ₂ SO 4 / l at pH 5.5 and 4 % Hy-flo-super-cel diatomaceous earth added as a filter aid. The precipitate and filter aid were separated by filtration through a frame filter. The filter cake was dissolved in water and insoluble constituents were filtered through a frame filter. The filtrate was filtered (check) over a Seitz filter film (type supra 100) with 0.25 % Hy-flo-super-cel as a filter aid (referred to as Filtration II in the following table). The filtrate was diafiltered through an ultrafiltration device. After diafiltration, the liquid was concentrated to a solids content of 12.7 # (referred to as solids in the concentrate in the following table).

- 46 -

24617

An optional base treatment to partially remove the protease activity may be performed at this time. In the case where the base treatment is applied, it is carried out at pH 9.2 for 1 hour, whereupon the pH is adjusted to 5.0.

Now the liquid is (check) filtered and filtered for the purpose of germ reduction and the filtrate is freeze-dried over a Stokes freeze-drier.

Four fermentation runs were carried out in the manner indicated below with the strain used for the fermentation altering the performance of the optional base treatment and other parameters. As in the following table

given.

Base treatment. No Code no. Concentration (%) of financial aid precipitation Filtra tion H Solids content in the concentrate Remarks Microorganism Yes X KRF 68 Filtra tion I 5 0.2 28 CBS 101.43 X KRF 74 0.5 4 0.4 7.5 ATCC 20236 X KRF 83 2.0 5 0.25 12.4 X) IPO 4408 KRF 92 I ₃ O  4 ..; 0.25 12.7 CBS 101.43 X 0.25

After filtration for germ reduction, the concentrate becomes

concentrated in the ratio 1: 2.3. A minor portion of the concentrated filtrate was spray dried and the remainder was freeze-dried.

- 47 -

24617 5 2-

To further reduce protease activity, some of the above formulations were treated as indicated below with only one of the three alternatives A, B and C carried out

A. 100 g of SPS-ase preparation are dissolved in 1 l of deionized water while stirring at 10 ⁰ C - 2 ⁰ C. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The pH is then adjusted to 4.5 with glacial acetic acid and the mixture is dialyzed against ice-cold deionized water to a conductivity of 3 mSi. Then (the product) is frozen and lyophilized.

, "* '.

B. 500 g of SPS ase preparation are dissolved in 4 l of deionized water with stirring at ⁰ ° C.-2 ° C. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 h. The pH is adjusted to 5.0 with glacial acetic acid. The resulting material is lyophilized.

C. 50 g of SPS-ase preparation are dissolved in 400 ml of deionized water with stirring at 10 ⁰ C - 2 ° C dissolved. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 h. Then the pH is reduced to 5.7 with glacial acetic acid. The resulting material is lyophilized.

-48 -

246 17 5 2

As starting substance for the base treatment applied SPS-ase-Präparurig Base treatment B C Code no. KRF 68 A KRF 68 BII KRF 68 X X KRF 68 BIII KRF 92 y X KRF 92 BI

The preparations indicated above are characterized in the following table by their enzyme activities according to the invention.

-49 -

Enzyme activity per g Disk test SRU. KRF 68 350 KRF 68 BII KRF 68 BIII KRF 74 ⁰ KRF 83 KRF 92 KRF 92 BI SAE Quantitative r test ^SRUM 120 + 737 , + ';' '142 HAT pH 3.2 2125 301 34 9 578 168 476 430 ^C x 67000 507 481 1630 683 626 757 PU 8000 1560 1720 1320 753 1640 1030 PGE 10300000 105 339 840000 128 UO 5960 397 ÖPTE • 119400 8044 9396 4100 8040 5700 3092 PEE 78100 9000000 8800000 15130 7500000 8400000 7600000 VHOJ 840 72000 77700 370 64600 60000 68800 1600000 83700 76900 65000 327000 44000 62400 910 770 690 1000 790 1100000 1000000 2200000 1100000 742000

246 175 2 - B eispje 1 2 (Application example)

This example describes the formation of a ρ · ν · ρ. from a hulled and defatted soybean meal "Sojamel 13" · The level of pesticides of this flour was 94.0 %, and the content of (N σ 6.25) on the basis of pesticides was 58.7 % · The soya flour was made with the SPS-ase preparation KRP 68 BII (Example 1) is treated in the following way:

85.2 g of the soy flour was suspended in 664.8 g of water and kept under stirring at 50 ⁰ C and the pH-value by means of 7.5 ml of 6 η HCl adjusted to 4.5 x 50 g of a solution containing 4 , 00 g of the mentioned SPS ase preparation were added and the reaction mixture was then stirred for 240 min at 50 ^° C. The mixture was then centrifuged in a laboratory centrifuge (Beckmann Model J-6B) for 15 min at 3000 g. The supernatant was weighed and examined for Kjeldahl on N as well as on the pesticide content. The solid phase was then washed with one volume of water corresponding to the amount of supernatant liquid obtained in the first centrifugation. This operation was carried out twice. The solid phase was then freeze-dried, weighed and examined for Kjeldahl to N and for the pesticide content. The results obtained in the experiment are shown in Table 2 · 1:. ''^{'-: -.}

246 17 5 2

Table 2.1 Results

component g N χ 6.25 (%) Solids (%) Yield. to Prcteia (%) Yield of solids. (%) Soya flour SPS-ase-. , , .-Preparation-.· 85.2 4.00 55.2 75.6 94.0 100% 6.4% 100% 1. Centrifugation pv.p. y _r 666 44.5 1.50 87.5 5.04 95.7 21.2% 82.7% 42.0% 53 _; 2%

This gave a p <, v _o p. with a protein purity, ie Nx 6.25, based on the solids, of 91.4 % and with a total protein yield of 83 %.

B e i le 3 (application example)

This example was performed to compare protein yields, nutritional quality, and some functional properties of soy protein products prepared by the following three methods.

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

B. Conventional isoelectric washing process for the production of soy protein concentrate.

C · Isoelectric washing process according to the invention, comprising a residue-dissolving enzyme for the formation of pvp _s

- 52 -

λ, λ _Π γ - 61 626 12

24 6 1 7.5 2 -52-

In order to obtain a true comparison of process (C) according to the invention with the usual processes for soybean protein (A and B), the same starting material was used in all three cases. The experiments were also carried out in such a way that corresponding 5 'emperatures and treatment times in all only the pH values were different due to the fundamental differences between the three experiments

A · Typical isoelectric precipitation for the production of soy protein isolate

425 »8 g of soy flour were in 3574 _f 2 g of tap water at 50 ⁰ C extrahiert4 the pH was adjusted with 20.1 g of 4N NaOH to 8.0 · After stirring for 1 hour, the slurry was χ at 3000 g for 15 min using Centrifuge 41-1 beakers in a laboratory centrifuge (Beckmann Model J-6B). Centrifuge I and Precipitate I were weighed. Precipitate I became like; The temperature was maintained at 50 ^° C., the pH adjusted to 8 with 4N NaOH and the slurry stirred for 1 h. The mixture was centrifuged and the centrifugate II and the precipitate II as weighed from above. Centrifuges I and II and precipitate II were sampled for Kjeldahl and analyzed for solids content. Subsequently, centrifugates I and II were mixed and kept at 50 ^° C. The protein was then isoelectrically carried at pH 4.5 Aid of 45 g of 6N HCl was precipitated. After stirring for ¹ hour at 50 ° C., the protein was recovered by centrifugation at 300,000 g for 15 minutes. The centrifugate IH was weighed and analyzed according to Kjeldahl on N and on the solid.

24617

analyzed. The solid phase III was weighed and washed with water in an amount corresponding to the weight of the centrifugate I. The washing was carried out by stirring at 50 ^° C. for 1 hour. The washed protein was recovered by centrifuging at 3000 g for 15 minutes. Centrifuge IV and solid phase IV were weighed. "Centrifuge IV was analyzed for Kjeldahl to N and for solids content. The solid phase was suspended in 1550 g of water at 5O ⁰ C and the pH is adjusted with 17 g of 4 η NaOH adjusted to 6.5. The mixture was stirred for 1 h and the pH adjusted back to 6.5, if necessary. Finally, the product was freeze-dried, weighed and weighed according to Kjeldahl on N and on

Analyzed solids content. The calculations of the men-; Genetic balance is given in Table 3.1. ;

Table 3.1 -

£ <t, .U I / J

Table 3.1 Calculation of the mass balance in the usual way

isoelectric precipitation for the production of soy protein isolate.

Operations and! fractions r Crowd of Fraiction g Protein% (Nx6 _; 25) Solids · (%) Yield of protein (%) Yield to solids (%) E> rtxaktion: Sajarnehl Kasser 4 .hNaOH 425.8 3574.2 20.1 55.2 0 0 94.0 0 16.0 100.0 0 10O _n O 0 0.8 1. Centrifugation: X 4020.1 5.9 10.0 100.9 100.4 Centrifugate I, precipitation I 3141.0 805.0 4.4 6.9 58.8 54.1 805.0 3195.0 0 0 0 0 Re-extraction: precipitation I- i water 3104.0 820,0- 0.5 9.1 17.2 6,6 .31,7 35.2 2. Centrifuge tion: ^ entrifugat II Precipitation II 6245.0 45.0 , 0 21.3 0 2 ₇ i4 Mixing and acidification Centrifugate I, + II 6 ri HG 6290 ^ 0 5650.0 308.0 0.3 # '' 26.8 3. Z-centrifugation: Σ. Centrifugation in precipitation _ IH I; 308.0 3141.0   _. -i 0 0 I 0 0 I do not wash them. water 3449.0 3113.0 291 ₁ 0 0.04 0 _T 15 0.5 ¹ J ² 4. "'Centrifuge: 2Γ' Centrifugate IV Precipitation IV 291.0 1550 0 17.0 '0 0 0 16.0 0 0 0 0.7 Neutralization:; Never hit JS ! , Water 4 .η NaOH 128.0 93.8 96 _? 3 51.1 I 30.8 TiDeckne η *. powder

- 55 -

2U175 2

B. Isoelectric Washing Process for the Preparation of Sojaprotein Concentrate

". ' '.' '.' '. j

425.6 g of soybean meal were washed in 3574 g of water at 50 ^° C. The pH was adjusted to 4.5 with 44.8 g of 6N HCl. The washing was carried out under rinses for 4 h. The slurry was then centrifuged at 3000 g for 15 minutes in a laboratory centrifuge (Beckman Model J-6B) The centrifuges I were weighed and analyzed for Kjeldahl to .Bf and for the pesticide content. The solid phase I was weighed and washed again with water to a total weight of 4000 g. The pH became again adjusted to 4.5 with 1.7 g of 6N HCl and the slurry stirred at 50 ^° C. for 30 min. The mass was centrifuged and the centrifuge II and the solids; II were weighed as above * The solid phase II was called on-j neut suspended in 1575 g HgO at 50 ⁰ C and the pH is adjusted with 34.5 g of 4N NaOH to 6.5. The mixture was stirred for 1 h at 50 ⁰ C and the pH, if necessary, adjusted to 6.5 again. Finally, the protein product was freeze-dried, weighed and analyzed for Kjeldahl on Έ and for the level of pesticide. The mass balance is given in Table 3 * 2.

Table 3 · 2 Calculation of the quantity balance in isoelectric washing for the production of soy protein concentrate

Operations and fractions Amount of fraction g Protein % (Nx6.25) Solids % Yield of protein % Yield of solids * ^: - Wash soy flour water 6n HCl 425.8 3574.0 44.8 55.2 0 94.0 0 21.3 100.0 0 0 100.0 0 2.4 1 · Centrifugation : Σ centrifugate I pesticides I 4044.6 3150.6 846.0 0.6 3.2 8.0 25.2 rinse again solids. I Wash 6n HCl 846.0 3154.0 1.7 0 p " mm. 0 21,3 0 0 0 0.1 2 · 2entrifugation: £? Centrifugate II solids II 4001.7 3130.0 863.0 0.1 0.4 1.3 3.2 Neutralization: solids II water 4N NaOH 863.0 1575.0 34.5 0 0 0 '": 16.0 ' 0 o - ^: o "1.4 Dry s powder 281.0 72.5 98.4 86.7 69.1

2Λ617

61 626 12

C. Isoelectric washing process using a residue-solubilizing enzyme to form p, v "p,

425.8 g of soya flour were washed in 3524.2 g of water at 50 ⁰ C, the pH was adjusted with the aid of 43.7 g of 6N HCl to 4.5 x 24 g of the SPS-ase preparation KKF 68 BIII ( Example 1) were dissolved in 26 g of water and added to the washing mixture

24 617 5 2

- 57 -

Washing was then carried out with stirring for 4 hours. Subsequently, the purification was carried out as described in B, with the amounts of 6 η HCl, 4 η NaOH and water for resuspension being the only parameters with different (different) values. The mass balance is given in Table 3.3

Table 3.3

Calculation of the mass balance in the isoelectric washing, including the residue-solubilizing enzyme for the preparation of p.v.p.

Operations and fractions I Amount of fraction g Protein% (N χ 6.25, 2 3 Solids 3 Yield of proteir, (%,) ' Yield of solids (%) Washing: - soybean water 6 η HCl SPS ase .: KKF 68 BHI I 425.8 354O ₇ 2 43.7 24.0 55 _T 0 0 75, r 94 0 21 96 ' ⁶ 100,0 0 0 7) 7 10O ₁ O 0 2.3 5.8 1. Centrifugation: Σ. Centrifugate solid? I H II 4043.7 3420.0 620.0 5 f ° 24.7 44 _; 4 Wash again Solids Water 6 η HCl II 620 0 3380.0 1.3 0 0 2 0 21 , 7 " 0 0 0 O ₁ I 2. Zantrifugation: X Zjentrifugafc. solids 4001.3 3400Jo O, 0 y 5.1 Neutralization: solids water 4 η NaOH 577 _T 0 1700 ^ 0 25.3 0 · 0 ³ 2) 0 16 0 0 0 ifo Drying: powder 211.0 ⁸⁷ I 96 78 _; 2 51.1

1) Analysis, Bioteknisk Institute, Holbergsvej 10, DK-6000 Kolaing, Denmark

2) Analysis of Qvist's Laboratory, Marseiis Boulevard 169, DK-8000, Aärhus C, Denmark

246175 2 -

nutritional properties

The amino acid compositions of the three protein products were determined, see Table 3.4. The total content of essential amino acids, the chemical rating and the index of essential amino acids (EAAI) were calculated according to the FAO reference pattern of

The content of trypsin inhibitor of the three products was determined by means of the method described in A.O.C.S. Tentative Method Ba 12-75 (A.O.C.S. is an abbreviation for American Oil Chemists * Society) method described. The results are shown in Table 3.5, which also shows the yields and the protein to solids ratio of the three products.

- Tables 3e4 and 3.5 -

U 6 17 5 2 - 59 - of the three protein products I 12.4 aas ¹ > Eg soy protein aas ^1} i, B and C. (p.v.p in - Table 3.4 A. So, japarinary · 4.62         , isolate aas .) - isolate 2I ₇ 3   , - g / 16 gN D - g / 16 g N 6.07 - - - Amino acid Composition of the amino acid and nutrient • 4.13 :. - - n _n 3  ., -.-; C. soybean - > 100 rating 3.54 - 4.69 I solat - Vloo Nonessential 2.83 - *  18.2 .. '. " g / 1 6 g N - > 100 Aspartic acid " 8.09 - 5.19 - - > ιοο) 100 i Serine. - 4.26 11 _T 9 ¹ e 59,5J glutamic acid 4.87 - 4.27 '.' '-' ·; 4.81 > 100 proline 7 _r 80 2.78 ¹⁷ I ⁷ 94 _T 3 glycine 6.24 > 100 7.57 > 100 4.76 > 100 alanine 5.47 3.38 > 100 > ioo 4.33 21 histidine 1.29 1.08 > 100 4.97 > 100 4.55 5% arginine 3.10 > 100 ) >>100> iooj 7.98 aoo) |> 100> 100) 2.50 3% Essential l _T 06 64.5]> 56.4 49.IJ 6 _; 09 66, Ol '? 6Q, 2 55 ₇ 0j 7 _T ° 4 isoleucine 4.90 > 100 5 ₇ 35 O DD J _r OO > 100 Leucine ' 38 75.7 1.32 1 _; 21 97 _; 9 5.19 lysine • ⁵ p > 100 3.60 > 100 8.09 Phenylalanine Tyrosine ' 86 36 1.37 41.31 5.57 Cystine Methionine 4% 5 _r 23 60.2% 5 _T 17 4 _r 44 threonine  7% 90.2% 1.44 1.31 tryptophan 3.97 valine ¹ T ³² % Total, salary. the essential amino acid 5 _r 57 Chemical evaluation 42 ₅ EAAI .- ⁶⁵ ; 91st

aas = evaluation of the amino acid based on the FAO reference pattern (1957)

2A6175 2

- 60 -

Table 3.5 Tracing Characteristics and Trypsin Inhi-

bitor content of the three protein products A, B and C.

Protein in the solids A. Soy Protein Isolate B. Soy Protein Concentrate C. soy protein-I solate (p.v.p.) Procedural characteristics Protein yield 97.4% 73.7% , 90.0% Trypsin inhibitor TUI / g product ⁵¹ ; ^{1 %} 86.7% 78.2% Tül / g protein 34,000 21,000 19,000 36 250 28,970: 21 810     '- ...

Functional properties

The index of nitrogen solubility (NSI) was determined in a 1% protein dispersion at pH 7.0 in 0.2 M NaCl solution or in distilled water. After stirring for 45 minutes with a magnetic stirrer, the suspension was stirred for 4,000 min Centrifuged und g and the supernatant solution examined for nitrogen. The nitrogen solubility was calculated as (soluble N 2 / total N %). The results of this calculation for the three products are given in Table 3.6.

- 61 -

24617

- 61 -

Emulsifying ability was determined three times on each product by a slightly modified Swift titration. 4.0 g (N χ 6.25) of the product was mixed in 250 ml of 0.5 M NaCl with a Sorval Omnimixer at low speed. 50 ml of the suspension was added to a mixing glass and 50 ml of soybean oil was added. Subsequently, the entire mixture was weighed. The oil-water mixture was then homogenized at 10,000 rpm, with the glass standing in an ice bath. An additional amount of soybean oil was added at a rate of 0.3 ml / s until the emulsion collapsed. The total amount of oil added before the "end point" was determined by weighing. i

Emulsifying ability was calculated as ml of oil per gram of protein (N χ 6.25). The density of the oil was assumed to be 0.9 g / ml.

The mean values of the determinations of the emulsifying ability of the three products are given in Table 3 × 6.

The whippability was determined in a 3% protein solution at pH 6.5. 250 ml of the aqueous dispersion of protein samples were whipped at speed III for 4 minutes in a Hobart mixer (Model N-50) equipped with a wire whisk. The Aufschlagbarkeit or expansion at impact was calculated according to the formula

V-250 expansion at impact = 250 χ 100%,

where V is the final volume of the product in ml.

246 17 5 2

-62 -

V was measured by refilling the mixer glass with water. There were duplicate experiments for each of the three samples. The obtained mean values!

 '' '·' · '' '. j

are given in Table 3.6, j

   '. , ..- '. '' '. '. The foam stability was measured as the ratio between djer

Amount of foam that fades after 3 minutes of running and determines the original amount of foam. 1 g of the foamed or whipped product according to the above-mentioned method was placed in a plastic cylinder (diameter 7 cm, height 9 cm) with a wire mesh with a mesh size of 1 χ 1 mm. The cylinder was placed on a funnel on a glass cylinder and the weight (B) of the elapsed liquid in the glass cylinder was determined. The foam stabilizer

15 FS is defined by the equation

FS = ^A T ^B x 100 %.

The results of this determination are given in Table 3.6.

in this specification defined as the Brookfield viscosity measured by T-spindles in a Brookfield Helipath stand. The gels were prepared by heat treatment of 12% protein suspensions in 0.5 M NaCl. The heat treatment was carried out in closed cans with a diameter of 7 »3 cm and a height of 5 cm placed in a water bath at 80 ^° C and 100 ^° C

carried out

was maintained, each within 30 to miny cans were cooled and opened and before it (the content) was measured, thermostatically controlled at 20 ⁰ C einge-

- 63 -

24 617 5 2

- 63 -

provides. The results of the measurements are given in Table: 3.6.

Table 3.6 Functional properties of, three protein products. ^:::

function A. soy protein I. sola t B. Soy Protein Concentrate C. Soybean protein isolate (p.v.p. % KSI in 0 _; 2 m NaCl% NSI in water 39.5 53.9 20 ₇ 3 ²⁵ yi 25.6 28 y 6 Emulsifying ability: ml oil / g (Nx 6 _, 25) 218 182 : 354 On impactibility /.(%) 120 120 ³⁴⁰ ^, Foam stability (%) 50 20 Gel strength (dPa.s) 80 ° C (0 _; 5m NaCl) 10 ° C (0.5m NaCl) 1.7x 10 ³ 2, Ox 10 ⁴ 1.2 χ TO ⁴ 4 4.0x10 3 _T 3x 10 ² 1.3 10 ⁴ χ

Example 4 (application example)

A p.v.p. was prepared according to the procedure described in Example 3c with the exception that the gellulase activity was partly derived from Trichoderma reseei. The commercially available cellulase preparation was

2Λ6 17 5 2

 - 64 -

CELLUCLAST

was mixed with a base at low temperature

treated the following way. The pH of a 10%

NaOH on Celluclast solution in water was washed with -v9.2.

and the resulting solution is cooled to 5 ° C. After 1 hour at this pH and this temperature, the pH was again with 20% acetic acid adjusted to 4.7 The solution was kept overnight at 5 ⁰ C and then sterile filtered. The filtrate was freeze-dried. 4 g of the freeze-dried product are added together with the SPS ase preparation KRF 68 BIII (Example 1). The two enzymes were dissolved in 172 g of water prior to addition to the wash mixture. The determination of the mass balance is given in Table 4.1

15 given.

  ': ..' .. I

The experiment shows that this particular SPS-ase preparation already contains an effective cellulase, since the addition of Celluclast does not affect the protein: solids ratio. However, other SPS ase preparations may contain less cellulase, e.g. KRF 92, see the table immediately before Example 2.

Table 4.1 -

24 6 175 2

- 65 -

Table 4.1 Calculation of the balance of quantities in isoelectric washing, including. SPS-ase preparation and Celluclast ^ for the production

';. '. .'.... '.' Of p, v. P. , ^: .-. ·; '.:'.,. , ../. '

Operations and Amount of protein Solids · go back to ' yield fractions fraction (%) (%). protein : on solid matter he G- (Nx 6.25) (%) (%) To wash soy flour 425.8 55.2 94 _T 0 iOOyO lOOjO water 3,546.2 0 .0 0 ο 43.1 0 21 ₇ 3 0 ² I ³ 6 η HCl 24 _? 0 75.3 96 ⁷ > ⁷ 5.8 PLC-ase: KRF-68-B-III 4.0 43 _; 6 96 0.7 1.0 CELLUCLAST 4,043.1 - -.   ; - - Centrifugate Σ 3,382.0 ⁵ ; ^{5 27} T ³ 46 _; 5 Centrifugate I 661.0 ^: - I. - Solids I _m renewed washing: 661.0 - -. '. - ' Solids I 3,339.0 0 0 0 0 water 0 0 0 0 0 6 η HCl 4000.0 2. Centrifugation: Σ 3,414.0 0.2 ⁰ T? 2.9 6.0 Centrifugate II 582.0 -. -. Solids II Neutralization: 582.0 - - - - Solids Il 1,691.0 0 ο 0 0 water 25.3 0 16 ₇ O 0  1.0 4 η NaOH Dry: 206 _r 0 88.8 98.9 77 _? 8th 50 _; 9 powder

2Λ6175 2

- 66 -

B ice ρ ie 1 5 (application example)

A PVP was prepared according to the procedure described in Example 3c procedure with the exception that all quantities were smaller by a factor of 5, and that the iReaktionsgemisch was cooled prior to centrifuging at about 5 ⁰ C. Based on the analysis results related to the centrifugates, a theoretical yield of protein was obtained, as shown in Table 5.1.

Table 5.1 In the production of pvp obtained ^theoretical: protein yield. '

fractions amount Protein - (N χ 6.25); (%) Rape of protein (%) Example 3 C Protein,% Soya flour SPS-ase ^; KPP-68 B-III 85.2 4.8 55.2 ⁷⁵ r ³ ; loo Protein (N χ 6.25)% 100 ' )'. 1. Centrifuge 2 .. centrifugate p.v.p. 639 595 0 _? 99 0 _r 13 8? _r 2 ^a 13.5 - ¹ V ⁶ 92, 6 ^b 55.2 75.3 24 ^ 7 ² ' ⁹ b 80, l ^b V ⁰ T ² 87 _; 1

Average of 87.5 (Bioteknisk Institute) and 86.9 (Qvist's Laboratory); corresponding to solids of 97.6 and 98.0%.

Calculated as the total amount of protein - protein loss in the centrifugates.

246175 2 - «-

For s ρ ie 1 6 (application example) Detection of protein binding by SPS

40 g (N χ 6.25) from a commercially available soy protein isolate - were dissolved in 680 g of water. The mixture was heated in a water bath to 50 ° C and the pH adjusted to 4.5 with 6 η HCl. 90 g of this mixture were placed in 5/250 ml Erlenmeyer flasks and 10 g of aqueous solutions containing 0, 0.2, 0.4, 0.8 and 1.6 g of SPS as previously ι described in the description were added. The flasks were then stirred for 240 minutes with a magnetic stirrer in a water bath at 50 ⁰ C. !

The slurries were then centrifuged at 3000 g / min for 15 min and Kjeldahl centrifuges I analyzed for N and solids. The solid phases were washed in room temperature water and then recentrifuged. This method j was repeated. Then the solids were dispersed in 50 ml of water and the pH was adjusted to 6.50 by the dropwise addition of 6N NaOH. The neutralized products were freeze-dried and analyzed for Kjeldahl to N and for solids content. Based on the analysis given in Table 6.1, the protein recovered and the percentage of SPS bound to the protein were calculated using the formulas given in Table 6.2.

This example shows that the SPS is tightly bound to the protein, so that the ratio of protein solids decreases with increasing content of SPS. SPS content / approximately 0.4 g in 10 g water,

- 68 -

24617 5 2

- 68 -

added to 5 g of protein isolate, is the protein: SPS ratio present in the soy flour.

The percent binding of SPS is a calculated value. The percent binding of SPS decreases due to the saturation of the protein in relation to SPS at low protein: SPS ratios.

- Table 6.1 -

2Λ617 5 2

- 69 -

Table 6.1 Measurements according to Example 6

Ratio protein / SPS Centrifugate I % DM dry precipitate % Nx6 _; 25 % DM _o N χ 6.25 25 12.5 6.25 3.125 % N 0.62 '0.49 0.4 5 0.45 0.61 % N ⁸² \ ^s . 83.8 I 81.3 78.8 73.8 93.1 ⁹⁷ T ³ 97 j 9 98 _; 1 97 _; 9 , DM 'Ό, Ό68 0.045 0 _; 038 0.031 0.026 13.2 13.4 13.0 12 _} 6 88 _f 6 B6,1 - 83,0 80,3 75,3

Table 6.2 Protein recovery and percent binding of SPS

Ratio protein / SPS % Protein recovery 2)% binding of SPS OO 25 91.5 94.4 0 77 12.5 95.3 90 6.25 96 _; 1 70th 3 _; 125 96 y 8 60

1)% protein recovery ₌ fi - NC 1x6.2.51

• NC 1 =% N in centrifugate I "* 5 *.

^ _where at

2)

% Binding of SPS =

Back in

5x (% protein gain) (% ^ -

return

5 χ (% Protei n -'vlewinnunq. (%

Γ 5 / quantity

SP Sj

* (% P / H) ratio of protein / solid in dry precipitate and

applies to precipitation without addition of SPS

24817

- 70 -

At ep le 1

J. (application example)

This example describes the preparation of a pvp using the SPS ase preparation KRF 92 BI in an amount of 5 %, based on the solids. The manner of preparation was exactly the same as that of Example 3c, except that all amounts were reduced by a factor of 5. The pvp was analyzed as described in Example 2. The results obtained in this experiment are shown in Table 7.1.

Table 7.1 results obtained in Example 7

Components · Quantity:,.: S; (Nx 6 _; 25) (*) Solids (%) ' Yield of protein, (%). Yield of solids (%) Soybean meal enzyme preparation 85 _T 2 4.0 SS ₁ 2 71.2 94.0 100 ⁶ I ¹ 100 1 . centrifu.gat 2. - Zen-tr-if-ugat .pvV.-p.'V / 632 · £ 73 39} 8 1.88 O ₁ 30 ⁸⁵ T ⁶ b 84 | 4 ^b 5 _V 44 0.80 Ö8jl ^b 25.3 4.3 71.9 43.0 6.7 48.8

^a Analysis of the · Bioteknisk Institut , Holbergsvej 10, DK-6000 Kolding Analysis of the Qvist's Laboratory, Marseiis Boulevard 169, DK-8000

246 17 5 2

- 71 -

For s ρ I el 8 (example of application)

This example shows the effect of pretreatment of soybean meal by steam jet treatment prior to the preparation of p.v.p.

5 pretreatment

A slurry of soy flour in water, consisting of 10 kg of soybean meal (Sojamel 13 manufactured by Aarhus Oliefabrik A / S) per 100 kg was pumped through a steam jet (Hydroheater B-300 type) and with steam of 8 bar in such an amount and with mixed such a flow rate that a final temperature of 150 ⁰ C 25 s could be maintained in a tubular pressure reactor. Subsequently, the pressure in a relaxation chamber (a cyclone) was released and from here the slurry was passed through a plate heat exchanger and cooled to about 50 ° C. The cooled slurry could be used directly for the preparation of pvp according to the invention but in this case the slurry was at an inlet temperature of 200 ° C

and an outlet temperature of 90 ⁰ C spray-dried. It was found that the pretreated product had a solids content of 96.5 % and a protein content of 56.9 % (N χ 6.25).

, ι. . ·

25 Production of pvp «

The preparation was carried out in the following manner:

70 g of solids of the steamed and dried soybean meal were suspended in 560 g of water and

24617 5 2

- 72 -

stirred at 5O ⁰ G and the pH was adjusted to 4.50 with the aid of 6.5 ml of 6 η HCl. 6 χ 90 g of this suspension were placed in 6 250 ml Erlenmeyer flask and stirred on a water bath of 50 ⁰ C using magnetic stirrers. To each flask was added 10 grams of a solution containing 0, 0.025, 0.050, 0.10, 0.20 and 0.40 grams of SPS Ase formulation KRF 68 BIII, respectively. The reaction mixtures were then stirred at 50 ^° C. for 240 minutes. Then, the mixture was centrifuged at 3000 g for 15 minutes.

The supernatant was then analyzed for Kjeldahl to N and the solid phase washed with equal volumes of water and centrifuged. This procedure was carried out twice. The solid phase was then freeze-dried and analyzed for Kjeldahl on N and on the solids content. "

A similar experiment was carried out with untreated soybean meal as starting material. In this case the ratios were enzyme: substrate 0, 1, 2, 3, 4 and 8 % , respectively .

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

Table 8.1 -

246 17 5 2

- 73 -

Table 8.1

Protein yield ratio and protein of the solids of pvv.p., made from steamed or raw soybean meal.

Steamed soybean meal -. · '· .. · Protein of the solids% "untreated soy flour Protein def solids ..% E / S% 'Protein solids'% 76.5 86.6 88.7 89 _; 7 91.7 92.2. Protein solids% 73.9 86.2 88.1 89.5 90.9 0 0.25 0.50 2.0 3.0 4.0 8.0 .92 _V 9. 90.1 89.3 88 ^ 1 86.6 '.'. · 84.7 9O ₇ 7 87.1 85.7 84.3 82.6 76.2

In connection with either the extraction (isolation) procedure for materials other than proteins or with the liquefaction processes and related processes, reference is made to the general process scheme for the application as indicated in Flow Sheet 3.

The substrate may be one or more carbohydrates in a starting material or it may be the whole

- 74 -

246 17 5 2 be starting material.

- 74 -

This substrate may undergo a chemical or physical pretreatment, as exemplified later, ζ.Β. an acid or alkali treatment, soaking, wetting and / or cooking with or without steam.

The starting material may be soaked (macerated), chopped, wet ground and / or homogenized (all of these treatments are referred to as homogenization in Flow Chart 3), with water and other additives added or can not be added during this stage. The homogenization can be carried out with different effects, e.g. at different pressures, which are only a fraction of the maximum pressure specified for the particular homogenizer. Different additives can be added before or during the homogenization, as indicated in flow chart 3 by b> |, bg... · B.

The reaction process, including SPS-ase preparation, is carried out under special conditions, e.g. of temperature, pressure, time, pH and enzyme amount. Also, recommendations are made regarding the reactor used (e.g., batch work or piston flow reactors) and stirring, if necessary. A number of additives can be given for different starting materials as indicated by c, j, C £ ... c in Flow Chart 3.

The separation processes can also be different

- 75 -

246 17 5 2

- 75 -

Effects are carried out. In many processes, the separation is omitted or facilitated, e.g. when the starting material is completely liquid (liquefied). Different separation devices may be used (e.g., centrifuges, filters, ultrafiltration devices, hydrocyclones, thickeners, sieves, or simple decanting devices).

The separation effect is defined as the ratio between the absolute solids (sludge) content in the solid phase and the sludge content of the reaction mixture.

The resulting solid or liquid phases may be further treated, for example concentrated, dried, extracted with solvent to remove certain components such as grease or oil, fermented to biification biomass, alcohol or other products (enzymes, antibiotics or other valuable ingredients). ,

The products obtained can also be recycled for repeated treatment according to the process scheme »

- 76 -

24617 5 2

- 7 6 -

Formula I III

substratum

2   ν. η 7 Λ 1 λ

SPS-ase

preparation

homogenization

reaction

homogenization

separation

 ν

liquid horn

= Ό - 100%)

(special reaction conditions)

September

. = '0 - 100%)

solids

Further treatment (see text) oner re-use as a substrate

2Λ6 17 5 2-

77 -

The following are some examples of the use of SPS ase preparations and an overview of these applications is shown in the following list. , · · ·. · '. .... ' : '. , '' '

Also in the attached Table I, various characteristics are summarized together with the flow chart 3.

- 78 -

246 175 2

- 78 -

Set up of possible applications of SPS ase preparations

Type of PLC ase preparation Full-standing liquefaction or similar Treatment Reference no. Extraction of starch from maize, wheat and potatoes Extraction of lipids from vegetable materials Extraction of essential oils from vegetable materials Extraction of natural dyes from vegetable materials Extractions of rubber from the guayul bush SPS ase preparation, which is essentially free of one or more interfering with the enzyme activities Machining assistance A 1 A 2 A 3 A 4 A 5 Production of milk substitute for pets Production of saccharified starch containing raw materials Complete liquefaction of pears and other fruits Production of juice by treatment of fruits and vegetables Treatment in connection with the extraction or pressing of sugar cane or sugar beet Production of soya milk Treatment for Increase the profitable amount of soluble coffee ingredients unmodified PLC acse loading Ba 1 Ba 2 Ba 3 Ba 4 Ba 5 Ba 6 Ba 7 Prevention and / or degradation of "slices" in apple juice Use for clarifying white wine Production of ISSPH or other vegetable protein hydrolysates Mash enzyme in the brewery Enzyme supplement in beer fermentation and / or storage Agent for removing almond shells clean up Bb 1 Bb 2 Bb 3 Bb 4 Bb 5 Bb 6

246175 2 -78a

Type of Other loading Degradation of different waste PLC ase- To whom train- products preparation types of training No. Saccharification and simultaneous fermentation Bc 1 Degradation of cellulose Use as a baking aid not modifiable Bc 2 Improvement of alcohol yield and ornamental- Bc 3 Yield of biomass in the Fer te PLC Bc 4 mentation of sulphite lye from ase- Bc 5 papermaking To- Drainage of biological sludge loading Silage Help pure do-qen Bc 6 Bc 7

/ 79

Sub strat. ··. additions ^b i ^c l   Table I: 2 ¹ Homo genization »2 Horn * 0 Separation ^ Sep ^% liquid phase * - further treatments I ί United phase (no separation) Lit. Corn water NaOH or HCl 1 'homo genization * l Horn * 10-30 - 30-50 (contains 'germs)' washing, oil gum cup Crystalization solid phase a i Corn-r ₍ germ Water like this ₂ - NaOH or. HCl 20-50 0   - 100 - - Washing / process     ; - A I Soy flour (def. Tet) ' - - HCl 0 - 0 Pasteurization Concentration spray drying Ba 1 sweet potatoes water Termyl NaOH or HCl 0 0 ; - Yeast for alcohol fermentation, distillation Ba 2 Sugar beets < water - NaOH or HCl - 100 - Ba 5 Soy quark od. Sojanüh- Water - HCl 10% 0 He f e fi - alcohol fermentation ,; distillation Bc 1 water 10%

246 17 5 2

- 80 -

A 1. Extraction of starch from corn, wheat and potatoes.

The extraction of starch from corn, wheat, potatoes and other starchy plants is carried out in one or more stages: soaking, wet-milling and separating. The use of a SPS ase preparation with essentially no amylolytic activity leads to the following advantages, using corn as an example:

1. The starch release is facilitated within a shorter soak time,

2. Water consumption can be reduced

3 »The release of maize germs is facilitated without the release of maize germ oil,

4. The protein can be obtained in higher purity,

5. The recovery of corn water is facilitated. A 2. Extraction of lipids from plant material,

Since the lipids are entrapped in plant materials within the cells and usually bound to proteins, lipids can be extracted in the aqueous phase by treatment with a SPS ase preparation that is substantially free of lipases. Thus, corn oil is usually isolated by extraction of the dried corn germ with hexane. However, the drying process is superfluous if the wet maize germs are cultivated with a SPS ase

- 81 -

2Λ6 17 5 2

- 81 -

treatment of the type indicated above. Similarly, the extraction of olive oil in aqueous phase can be improved if the enzyme used for the enzymatic treatment is a SPS-ase preparation of the type indicated above, see e.g. Food, Pharmaceutical and Bioengineering, No. 172, Vol. 74, p. 93-94. Also, the aqueous extraction of e.g. Soybean oil, rapeseed oil and sunflower oil can be improved in a similar way.

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

When vegetable materials containing essential oils are treated with an aqueous solution of SPS ase preparation which is substantially free of enzyme activity capable of degrading or otherwise altering the essential oils, the extracted essential oils in high yields at very low cost.

A4. Extraction of natural dyes from plant material.

ν When vegetable materials containing dyes

e.g. Beets containing the red pigment betanin or the colorant being treated in cranberries with a SPS-ase preparation containing essentially

Since enzymes are capable of degrading or otherwise altering the dyes, the dyes are recovered in high yields at low cost.

- 82 -

2Λ6 V7 5 2

61 626 12 - 82 -

A 5 · Extraction of gum from the Guayul bush

Another example of a substrate for a SPS preparation which is substantially free of enzyme activity capable of degrading crude rubber is the cell wall material of roots and branches of the Guayul bush.

Ba 1 · Preparation of a milk substitute for pets, preferably substitutes for calf milk

Complete liquefaction of soybeans, sunflower seeds, cottonseed, pabobeans or pea-tubers can produce a calf milk substitute that is soluble in cold water at a pH of about 4.5. * Starch liquefaction must be used when using starchy raw materials such as pabobo beans or pea peas with the aid of a C-amylose before, after or simultaneously with a treatment with a SPS-ase, which finally liquefies the starch-starch polysaccharides in the structural material of the cell walls. A detailed example using pababeans is given below, and with reference to FIG the soybean is referred to Table I. The pretreatment of the soybeans may preferably be a steam jet cooking which improves the solubility of the residue.

Example Ba 1.1

15 kg of pabobo bean flour were used in 35 1

246175 2

- 83 -

Water suspended. 75 g of Termamyl 60 L and 18 g of CaCl ₂ were added. The suspension was heated to 95 ° C. with stirring in a jacketed steam kettle. Those suspension was then treated for 60 minutes at this temperature. Subsequently, the pH was adjusted to 4.5 and the product is cooled to 50 ⁰ C ,, 300 g of the SPS-ase - Preparation KRF 68 were dissolved in 1 1 of water and added. The reaction was carried out for 440 minutes. When 10 g of Fungamyl 800 L were added, the starch fraction was essentially converted to disaccharide (maltose). The reaction mixture was then pasteurized at 90 ^° C. for 2 minutes. A portion of the product was then freeze-dried and used for stability studies. The sample was then dissolved in an amount of 10 % solids (dry matter) and the solution of the product remained stable for days without sedimentation.

Molten fat or oil can be easily emulsified in the product, whereby a final product very similar to cow's milk can be obtained. An emulsion containing 3 g of 5 g of oil (soybean oil) could also be kept stable for days without sedimentation.

B e i s ρ i el Ba 1.2

Soy flour (Sojamel 13) was 25 s as described in Example 8, treated at 15O ⁰ C with a steam jet. The soymeal flour so treated was spray dried and used for further studies / as described below.

- 84 -

2 Λ 617 5 2

- 84 -

A:

50 g of the steamed 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 in a water bath at 45 ⁰ C and 0.250 g of the SPS-ase preparation KRF added to the heated mixture 68, which reacted then 5 h with stirring. Then, the mixture was heated at 80 ^° C. for 2 minutes to inactivate the enzyme. A 100 ml sample was centrifuged at room temperature for 15 min at 3000 g. The supernatant was treated with an ion exchanger and also the carbohydrate composition analyzed by HPLC. The supernatant was also analyzed for Kjeldahl nitrogen content and the solids content and nitrogen solubility index (NSI) and solids solubility index (DSI) were calculated, see results in Table Ba I. 100 ml of the reaction mixture cooled to 20 ^° C. placed in a calibrated 100 ml glass and kept at 4 ° C for 2 days. The dispersion stability (%) was measured by reading the volume of the obtained dispersions (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 in a Waring Blender for 2 minutes. The emulsion stability {%) was measured after 1 and 2 days as above.

A reaction was carried out as above, but in this case 1.00 g of the SPS ase preparation was obtained

24 617 5--2

- 85 -

applied. The same types of analyzes and stability measurements as described in paragraph A were performed. The results are given in tables Ba I and Ba II.

From the chemical analysis of the supernatant fluids, it is seen that the values for NSI {%) and DSI (%) are higher in Experiment B than in Experiment A. However, the stability tests performed on the reaction mixtures show a better value for samples A. This may be due to the greater length of the peptide chain of the proteins in the reaction mixture with a smaller amount of enzyme.

It is apparent from the carbohydrate composition measured by HPLC that mainly mono- and disaccharides have been formed. Thus, oligosaccharides _f known to be responsible for diarrhea and flatulence, when administered to calves in excessive amounts, are present only in minor amounts.

- 86 -

24617 5 2

6 -

Table Ba I Chemical properties of the supernatant

Search Ratio of enzyme amount to substrate% (Gsw./Gew) N.SI,% '* (at pH =. ·· 4,5). DSI,% ** (at pH = '4.5) HPLC results (composition of neutral sugars) A E / S = 0 _r 5% .39j 9 62.4 DP ₁ + DP ₀ : 79.7% DP * ^z : 714% DP ^: 12.2% DP ^ ₊ : 8 ₇ 1% B E / S = 2 _; 0% 57.0 67.1 DP ₁ + DP ₉ : 84.4% DP ^ ^: 6 1% DP ^: 3 7% ^DP 4 ₊ ^{: 5} ) ^7%

* NSI = nitrogen solubility index ** DSI = Solubility Index

Table Ba II Stability tests of the reaction mixtures

Tries Ratio of enzyme amount to substrate% (w / w) Stability tests Dispersion 2nd day • with oil Emulsion 2nd day A E / S = 0.5% without 01 .. 63% Emulsion 1st day 87% B E / S = 2 _; 0% Dispersion 1st day 35% 100% 71% 80% 85% 66%

24617 5 2 ^6162612

- 87 -

Ba 2 · Production of saccharified starch-containing raw materials

In connection with the saccharification of cassava and sweet potatoes and other starch-containing plant materials, the addition of a SPS-ase preparation leads to the solution of viscosity problems. · When using a SPS ase preparation, it is possible to use starch suspensions with a pesticide content of 25 to 30 %. and after saccharification, the mash can be fermented, whereby a cheap ethanol can be obtained.

B e is ρ ie 1 Ba 2 * 1

On the basis of fresh and roasted sweet potatoes (Japanese), a mash was made with a pesticide content of 24 % * The starch content of the sweet potatoes was about 70 % of the pesticide content. A pre-liquefaction using the bacterial amylase, Termamyl 60 L ^ at a dose of 0.5 kg / tonne of starch was carried out by heating the mash to 90 ⁰ C. The slurry was then held for 30 minutes at 90 ⁰ C · The viscosity \ ^ The reaction mixture was then measured at 90 ^° C. using a Haake spindle.

Then the reaction mixture was cooled to 55 ⁰ C and the pH with 2N HpSO- 5 "is set to 0 · Then, a saccharification was initiated by addition of the glucoamylase SAN 150 (trade name) at a dose of 1.75 l / t starch · The saccharification mixture was then divided into three parts A, B and C, which were enzyme treated for 15 minutes, as noted below, before the viscosity was measured.

2Λ6 17 5 2

-88 -

A: This part was for comparison. The viscosity '.'Ti ρ was measured, see Table Ba III.

B: Celluclast®> 200 N Tricoderma viride cellulase was added at a dose of 1 kg / t of sweet potato solids. The viscosity <ΎΙ -ζ was measured, see table Ba III.

C: The SPS ase preparation KRF 68 was added in an amount of 0.25 kg / t of sweet potato solids. The viscosity Ί \ ^ was measured, see Table Ba III.

reaction viscosity viscosity 55 ° C mPa. s mixture at 90 ^° C. at. 2190 mPa. s pre-liquefied sweet potatoes ¹ ^ ₁ = 770 mPa.s 2190 mPa. s '.. V A - 1970 mPa. s B - 73 = 950 C -

From this table, it can be seen that the viscosity of the reaction mixture can be significantly reduced by the SPS ase in a small amount compared to the Celluclast® and SAN 150.

- 89 -

24617

- 89 -

Ba 3β Total liquefaction of pears and other fruits. '/. ·. \. '. '.'

When whole pears that have been mechanically crushed are subsequently treated with a SPS ase formulation, complete liquefaction takes place and, after removal of smaller amounts of solids, a clear pear juice is obtained. A similar procedure can be applied to other similar fruits, eg ₀ apples.

10 Be is piVl Ba 3.1

Fresh apples were coarsely ground using a Bucher central mill. The apple mash was then pasteurized in a boiler with a heating mantle for 5 min at 90 ⁰ C and then cooled to room temperature. The mashed apples were then ground on a Fryma mill with corundum until the mash was smooth. The mash was

_{o o}

then pasteurized again at 80 C for 10 min and at 50 C

cooled. '

Now enzyme reactions were performed for 30 min at 50 ⁰ C with a Contraves Rheomat-15, wherein stirring and, viscosity measurements were carried out (in the context of a percent Abie solution on the rheometer at speed 13) at the same time. After complete enzyme reaction 100 g samples were withdrawn and centrifuged in a calibrated tube for 15 min at 3000 χ g. Here, the percentage of juice and the percentage of precipitates or solids were measured. The pH and percentage of refractometer solids as ° Brix were also measured. Table Ba IV shows a comparison between the effect of SPS-ase, the combination of Celluclast and SPS-ase and the combination of

- 90 -

246 175 2

- 90 -

Celluclast and Pectinex. The SPS-ase- ^ preparation KRF 68 was used.

Table Ba I'v results of the overall liquefaction tests with apple mash at 50 ⁰ C, 30 min

SPS-ase g / hl Celluclast ^ 200 1 g / hl mash Pectinex ^ - '3x g / hl Final viscosity (*) centrifugation % Firmly- ·. matter juice Brix 0 25 50 50 0 0 0 0 ο 50 50 50 0 0 ο ο 200 2000 100 · 19 15 4.8 3.8 9-5 4.0 % Juice 41 19 21 .17 17 17 18 pH > 1 ⁷ , 10 ^ 5; .io _V 7 10 8 12 _f 5 12 ^ 59 81 79 83 83 83 82 3.8 ³ J ^{5 3} J ^{5 3} J ⁵ V ³ ) ^{2 3} J ¹

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

It has been found that SPS ase preparations are well suited for the production of juice by treatment

24617 5 2

- 91 -

of various fruits, berries and vegetables, e.g.

Currants Carrots, peas, tomatoes, apples, pears, black beans and cabbage. In this case, a better yield of juice and a better extraction of colorants and flavors is achieved, compared with the commercial Pectinase and Cellulase-- preparation.

Example Ba A. 1

Reference is made, for example, to Ba 3 <> 1, in which the SPS ase preparation has been compared with the commercially available cellulase and pectinase products Celluclast ^ 200 L and Pectinex® 3x. From the table it can be seen that the yield of juice can be improved slightly using as little as 50 g / hl of SPS-ase compared to 2000 g / hl of Pectinex, both

together with 50 g / h Celluclast ^ H The viscosity was also slightly lower. So it seems that the SPS ase is about 40 times more effective than Pectinex.

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

It has been found that it is possible to improve the yield associated with simple extraction processes when the SPS ase formulation is used to treat sugar cane or sugar beet before and / or during extraction or pressing.

Also, the residue (bagasse) can be treated with the SPS-ase preparation, whereby it is partially converted into fermentable sugars, which can be used as starting material for ethanol fermentation.

- 92 -

24 617.5 2

- 92 -

B e i pi el Ba 5.1

Ten kilograms of sugar beet (pulp) residue obtained by continuous countercurrent extraction in a DDS diffuser at Nakskov Sugar Factory were ground twice in a Fryma mill (type MZ-110). During the milling process process water was added.

300 g portions of the pulp were now treated with enzyme at 45 ° C. for 18 h using the amounts indicated in Table Ba V. The dry enzyme product (KRF 68) was added to the pulp which was stirred with a rod for the first hour. As a result, the pulp liquefied so much that the rest of the time was successfully stirred magnetically. At the end of the reaction, the pH was measured (no pH correction was made during the beginning of the reaction) and the reaction mixture was centrifuged until a clear supernatant liquid was obtained. The determination of the solids content was carried out on reaction mixtures and on the supernatants. Based on these results, the percentage of dissolved solids was calculated. Corrections for the soluble solids content of the enzyme product were made in all calculations.

The supernatant fluids 2, 3 and 4 were treated by ion exchange and analyzed for carbohydrate composition by HPCL.

Λ 6 17 5

- 93 -

Table Ba V Results in the enzymatic liquefaction of beet pulp

Test no. Ratio of the amount of enzyme to Solids E / s'% End measurements. % Solids supernatant liquid % dissolved solids , 1 0 reaction 4 ₅ 18 % Solids 0.0 2 0.35 pH (final value) 3.85 0 ^ 0 66 _; 9 3 0 _y 56 3.81 2 j 58 66 _; 2 4 -. ¹ ^ ⁰² 3.6 3.86 2.56 70.4 5 I ₁ 58 : ³ J ^{5 3} I ¹⁷ 2 _; 73 .. ⁷³ ) ⁴ 6 3 y 10. '   ν , ' ³ I ²³ 2.34 76 _; 4 "7 /. -7y52 ^: , ³ I ³ , 2.6 6 2.49 80.5 "-.:"'...' ³ I ⁴ 2,18 ' ^: ' ³ T ⁴ .-'

Reaction conditions: M = 3 00 g

S = .4.18% solids E / S as indicated above pH not adjusted T = 45 ⁰ C t = 18

246 17

Table Ba VI HPLC values

Sugar Type (Neutral) Experiment No. 3 4 43.6 23.7 25.3 not measured , High molecular weight (DP4 +); . 2 % neutral sugars; 4.6 - disaccharides 20.4 ³² I ^2. " 27 ^ 8 Glucose · : ⁵ I ^{0 7} J ³ galactose ^'26 I ⁴ - 33.2 Fructose / Arabinοse galacturonic

All sugars produced according to the above table Ba VI could be fermented to alcohol or used for other purposes.

- 95 -

2Λ6 17 5 2

95 -

Ba 6. Production of soymilk »

Soy milk can be easily prepared by fully liquefying ground soybeans and then homogenizing the resulting mixture. Soy milk is often made by soaking soybeans in boiling water, grinding the soaked beans and extracting with water and then separating the insoluble residue, e.g. of proteins and polysaccharides. In order to improve the yield of soymilk, these insoluble residues can be liquefied by reaction with the SPS ase.

B e i s pi e 1 Ba 6.1

The soymilk method is illustrated by the following series of enzyme reactions, with protein solubility index (PSI, %) and solubility index (DSI, %) calculations showing yields obtained after separation at pH 7 (see Table Ba VII). The enzyme reactions were carried out under the following conditions:

substrate:

Amount of the reaction mixture:

Amount of substrate:

Temperature:

pH: reaction time:

Reaction time:

Enzyme:

Amount of enzyme:

Full-fat soybean meal (Dansk soybean factory A / S) 220 g 20 g 50 ° C

4.5 (6n HCl) Row A 1h Series B 0.5 -6h PLC Ase (KRF 68) Row A: E / S Ratio (w / w) 0-8.0 Row B: E / S ratio (w / w) 1.0

- 96 -

24617 5 2

-96 -

After the reaction, the pH was adjusted to 7 with 4N NaOH, and the separation was carried out by centrifuging at 3000 g for 15 minutes.

Ba 7. Treatment to increase the recoverable amount of soluble coffee ingredients.

It has been found that the treatment of coffee beans at different stages during the production of instant coffee leads to an increased yield of soluble coffee constituents. Thus, e.g. Consumed coffee residues (coffee grounds) or green beans are treated enzymatically with favorable results.

-97 -

Table Ba VII Calculation · of the quantity balance in connection with the enzymatic

Soymilk method. -: ''

string Reaction time h Enzyme amount E / S% reaction % Solids supernatant liquid % Solids Solubility indices DSI% Λ 1.0 1.0 1.0 1.0 1.0 1.0 0 0.5 1.0 2 _; 0 4.0 8.0. % Protein 8,70 8,74 8,78 8,86 9 j 03. 9.37 % Protein 5.72 6.28 6.43 7 j 18 7.69 8.08 PSI% 63.7 70.0 71.4 79.5 83 ₇ 9 85.0 B 0.5 1.0 2.0 4.0 6 _y 0 l _; 0 1.0 ¹ T ⁰ -.- 1.0 1.0 3,65 3,68 3,71 3,78 3 ^ 92 4,19 8.78 8.78 8.78 8.78 8.78 1.87 2.41 2.48 2.98 3.36 3.76 6,41 6,60 6 ₁ 91 7,29 7,69 49.6 63.7 65.1 I 77.5. .84.j6 88.5 71.1 73 _T 4 77.1 81.7 86.5 3.64 3 _? 64 3.63 3.63 3.63 2.39 2.56 2.79 3.10 3.39 64.0 68.7 75.2 84.0 92 _; 3

246 17 5 2

- 98 -

Bb 1. Prevention and / or removal of the "veil" in apple or pear juice.

After the production of apple juice or pear juice and other fruit juices, which are to be clear and which have been previously treated with conventional pectinase and cellulase preparations to avoid the formation of cloudiness, "throat eggs" may appear -ase preparations are well suited to degrade such veils which consist mainly of araban bound to proteins.

Example Bb 1

Pear juice concentrate prepared by enzymatic liquefaction of residues from the preparation of pear preserves using Celluclast® and Pectinex® H became cloudy on standing. The "veil" was isolated and hydrolyzed with 0.01 η H ₂ SO _{4 for} 24 h and analyzed by HPLC. The chromatogram showed arabinose and small amounts of oligosaccharides.

By incubation of 0.5 % (w / v) of this carbohydrate in 1 mmol of acetate buffer, 3 h at pH 4.5 and 40 ⁰ C with SPS ase (KRF 68 and KRF 92 1: 1) with an enzyme concentration of 0.05 % (w / v) showed that 84 % of the original fog-forming carbohydrates (solids) were converted to arabinose.

Also a diluted pear concentrate (20 ° Brix) was 2 h at 40 ⁰ C with an enzyme amount of 0.15% (Gew./ vol.) Of the above-mentioned SPS-ase or with 1% (Gew./ Vol.) Of a commercial product which is called Clarex

246175 1

- 99 -

is being treated. It turned out that the SPS-; relative

It has been shown that the level of VHPLC peak of an araban-like veil was reduced by 86 % while the corresponding reduction with Clarex (in a much higher amount than the SPS-ase preparation) was only 78 % .

Bb 2. Application as a clarifying agent for white wine «

It has been found that whites which show a very undesirable turbidity can be effectively clarified by means of SPS ase. It has been found that the turbidity-forming material consists mainly of arabinogalactans bound to hydroxyproline residues in a cell wall structural protein.

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

Prior to separation of the ISSPH (isoelectrically soluble soy protein hydrolyzate) or other vegetable protein hydrolysates from the slurry as disclosed in U.S. Patent No. 4,100,024 or in Process Biochemistry, Vol. 14, No. 7 (1979), pp. 6-8 and 10 -11 described, the reaction mixture can be treated with a SPS-ase preparation. This achieves easier separation.

Bb 4. Mash enzyme for the brewing industry.

In the production of beer, carbohydrates of the starting materials, e.g. the ß-glucans of malt and barley, the viscosity and filterability of the extract. The addition of SPS ase during mashing reduces the viscosity of the extract and improves

- 100 -

246 17 5 2

- 100 -

the filterability and the yield of extract. Moreover, the addition of SPS-ase during the mashing process leads to an improvement in the fermentability of the extract and the nitrogen content in the extract.

B e i s ρ i e 1 Bb 4.1

In the laboratory, 50 g of ground grains consisting of 50 % malt and 50 % barley were milled with 275 g of water (15 % solids) according to the following mash diagram: 52 ° C (60 min) / 63 ° C (60 min) / 76 ° C (30 min).

To demonstrate the effect of SPS ase, four experiments were performed, see the table below, where the enzymes were added during the mashing process (pH of the mash 5.5 to 6.0).

enzyme none CerefIp PLC-ase (KRF 68) 1630 FBG 1630 FBG Activity of the β-glucanase / g 0. 200 BGU OjOS g 0.18 g Amount of enzyme per kg grains .0 1.5 g 80 FBG 300 FBG Total amount of enzyme activity units per kg of grains. 0. 300 BGU 160 ml 170 ml Filtration speed of the extract after 10 min 120 ml 135 ml 1.3 6 raPa.s 1.30 mPa.s Viscosity of the extract is ⁰ Baling (25 ° C) 1.52 'mPa.s 1.36 mPa. s

 - 101 -

246175 2

- 101 -

BGU = β-glucanase units, determined by the analytical method AF 70/4-GB

FBG '.. = fungal β-glucanase units, determined by the analytical method AF 70.1 / 2-GB

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

Cereflo is a bacterial β-glucanase preparation as described in the information sheet B 214b-GB 1500 July 1981,

B e i sp e e 1 Bb 4.2

In the laboratory, 50 g of ground grains consisting of 40 % malt and 60 % barley were mixed with 150 g of water (25 % solids) according to the following scheme; 45 ° C (60 min -) / 63 ⁰ C (90 min) / 75 ⁰ C (15 min).

In order to demonstrate the effect of SPS ase, three experiments were carried out, see the table below, where the enzymes were added during mashing (pH of the mash 5.5 to 6.0).

- 102 -

2 * 6 175 2

- 102 -

enzyme none Ceremix SPS-ase (KRF Ceremix, as receded added 68) + in the preliminary test Activity of the β-glucanase / g -; 200 BGU 1630 FBG Enzyme amount per kg grains - 1.65 g 0.033 g Total amount of enzymatic activity units per kg grains. 0 330 BGU 50 FBG Filtration speed of the extract after 30 min 4 8 ml 98 ml 111 ml Extract, ^o Balling 18.6 19.0 19 , 5 Viscosity of the extract 10 ° Balling (25 ° C) 1.72. mPa. έ 1.37 mPa. s 1; 27

The definition of BGU and FBG is given as in Example Bb 4 »1. The last column of the above table shows only the amount and activity of SPS-ase.

Ceremix is a bacterial β-glucanase preparation as described in Bulletin B 216b-GB 1000 Feb. 1982 available from NOVO Industri A / S.

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

SPS ase may be added during the fermentation of extract or during the storage of beer in order to reduce the content of β-glucans and thereby improve the filterability and stability against fogging.

- 103 -

ι / j

to improve education of beer. SPS-ase also has an effect on proteins responsible for turbidity occurring during cooling. .

Bb 6. Means for easier removal of almond skins.

During the mechanical removal of almonds after blanching almonds, a certain percentage of almonds are not removed. It has been found that enzyme treatment of the almonds leads to a reduction of the mentioned percentage.

Bc 1. Decomposition of different waste products.

In connection with certain manufacturing processes, large quantities of carbohydrate-containing waste products are formed. For example, This is the case in connection with the production of soy isolate by water extraction and acid precipitation, soy milk and soybean curd (a special type of Japanese cheese). Also in this context, waste pulp of e.g. Apples, pears or citrus fruits are mentioned. It has been found that a SPS ase preparation is capable of completely liquefying these carbohydrate-containing waste products and forming fermentable sugars which can be used as a starting material for ethanol fermentation.

25 Example Bc 1.1

In the usual production of soymilk or soybean curd, soybeans are often soaked in boiling water, ground and extracted with hot water, whereupon a separation is carried out. The residue

- 104 -

246175 2 -.04. ^6162612

of this separation is the material used for this experiment. The liquid phase is the soybean milk, which can be further used to make soybean curd.

10 kg of whole soybeans were ground together with 70 1 of boiling water in a Pryma-mill type MZ 110 · The milled slurry was then held for 15 min above 85 ⁰ C, to inactivate the natural beans enzymes that generate the known soybean flavor x 5 1 of this Soybean slurry was then centrifuged in the laboratory for 15 minutes at 3000 g. The analysis showed that the residue contained 20.45 % and 20.06 % solids (duplicate determinations, calculated mean 20.26 %) . Ss was added slowly to 6N HCl and incorporated into the residue with a spatula until a pH meter showed 4.50 when the electrode was immersed directly in the mass

In a 500 ml Beeher glass at 50 ⁰ C enzyme reactions were carried out on 2 χ 200 g of the mass with two different amounts of SPS ase (KRF 68), namely E / S = 0.5%, based on the pesticides and e / S "3.0%, based on the solids · the dry enzyme was added to the mass · During the first 1 to 2 hours was stirred with a spatula, and then the amount was so far liquefied that successfully stirred with a magnetic stirrer The total reaction time was 21 h. During the reaction, the osmolality was measured with an osmometer. The results in Table Bc I show the progress of the reaction. At the end of the experiment, the mixture became active for 15 min

617

- 105 -

Centrifuged 3000 xg. On the surface of the supernatant liquid, an oil layer, the volume of which was determined, appeared as a bottom layer, a supernatant sludge layer was removed, the supernatant liquid including the oil was removed with a pipette, the oil was combined with the clear aqueous phase by homogenization, and a The results given in Table Bc I clearly show that this waste product can be liquefied by enzymatic reaction and that crude oil can be obtained as indicated in Section A 4. After recovery of the oil, the residue that has dissolved can be recovered be used in various ways, for example for fermentation to valuable compounds or for concentration and drying and subsequent use as feed or food product or after further purification to produce valuable products.

-106 -

24617 5 2

- 106 -

Table Bc I Results obtained in the liquefaction of soymilk and soya quark.

Reaction conditions and results Trial A min Osmo lalität mOsm Δ os molarity mOsm 20.3% 18 _T 0% 88.6% 8-10% Trial B Osmo lalität mOsm Δ Os molali ity MoSra 20.7% 19.4%. 8-10% 93 _; 5% Amount of. Residue Amount of PSA (KRF-68) Temperature. ph ..; , Reaction s sit 200 g Mo g 50 ⁰ C 4 ^ 50 21 h 0 10 40 2? 8 1260 287 313 3 91 ISI 907 0 26 104 m 620 200 g 1) 20 g 50 ° C Aj 50 21 h. 2 82 368; 497 601 1145 0 86 215 319 863 Results Measured with the Osmometer During the Reaction Δ Osmolality is the corrected value for osmolality of the mixture at t = 0 t = 0 t.; - 'min Reaction mixture Solids supernatant liquid: solids supernatant: Oil content calculation% Soluble solids o 10 25 45 2? a 1260

24617 5 2

- 107 -

Bc 2. Saccharification and simultaneous fermentation.

Carbohydrate-containing plant materials, e.g. Tubers such as Jerusalem artichokes, potatoes, sweet potatoes, cassawa or pulp of such tubers, i. The material remaining after removal of the extracted components can be saccharified by treatment with a SPS ase preparation and at the same time the resulting fermentable saccharides are fermented to ethanol. ·. · '' ·. , '. ; ; , ·. ' '.

10 B ic ρ i el Bc 2.1

The production of ethanol by fermentation of degraded inulin-containing Jerusalem artichokes was investigated on a laboratory scale by simultaneous saccharification with SPS-ase and inulinase and four different pretreatments of Jerusalem artichokes.

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

Inulinasei The inulinase was formed by fermentation of Asp. Ficuum (CBS 55 565). Inulinase activity was determined as indicated on page 99 of Research Disclosure Item 21234 (December 1981) pp. 456-458.

Fermentation in the laboratory: 150 g portions of the pretreated mash (described later) were fermented after addition of 4.5 g of baker's yeast and 1 ml of a 4% solution of Pluronic as an antifoam agent. The fermentation flasks were fitted with CO ₀ traps containing 98% sulfuric acid and the fermentation was monitored by measuring the weight loss due to released CO ₂ . The contents of the piston

- 108 -.

2-4.6 17 5 2 -

108 -

was stirred during the fermentation carried out at 30 ° C. Three pistons were used for each parameter studied.

In Table Bc II, the weight loss due to release of CO _{2 is} converted to ethanol, assuming that 1 mole of CO ₂ liberated contains 1 mole of C ₂ HcOH, d. 1 g CO ₂ > ^ ^ C ₂ H ₅ OH corresponds.

Pretreatment of artichokes:

Treatment A: 14.1 kg of artichokes (22.8 % solids) were stirred at 140 ^° C. for 20 minutes and 4 to

bar cooked (Henze). The weight after cooking was 19.0 kg '(-ry 16.9 % solids content). The fermentation was carried out directly on the mash.

Treatment Bs Washed and sliced

Artichokes were mixed with water (1: 1) in a Waring blender. The

Mash was then treated 1 4.5 »

h at 85C and pH

Treatment C: Same as B but without pH adjustment.

Treatment Dj Like B, but without heat treatment and without pH adjustment «

Results: In Table Bc II, the results show the effect on the ethanol yield of adding SPS ase to the pretreated mash. A significant improvement in ethanol yield was achieved on all pre-treated mashes when SPS asese was added.

_ -IAQ _

246 175 2

- 109 -

Table Bc II Fermentation Results Related to the Simultaneous Fermentation and Enzymatic Saccharification of Jerusalem Artichokes,

Preventive action Add inulinase to 1 g of solid PLC-ase E / S% Loss of _{G0 (g) after 42-44} h fermentation % ethanol produced relative to solids A ¹ A 0 0 ^ 27 7.65 + 0 ^ 05 8.07 + 0.08 - 31.5 33.2 B 0 0 ·, 4 0 4,85 + 0 _Λ 03 5,41 + 0,03 29.7 33.1 C h ^{5 1} T ⁵ ! .5 ¹ I ⁵ 0 0.10 0 ^ 20 0.30 0.40 5.70 + 0.05 5.97 + 0.01 • 6.13 + 0.06 6.13 + 0.006.18 + 0.05 34.8 36.5 · 37 _T 5 37.5 37.8 D " ¹ I ⁵ * ¹ T ⁵ 1.5 ¹ I ⁵ o 0 0.10 0 _r 20 0.30 0.40 0.40 5.77 + 0.02 5.89 + 0.00 6 ^ 04 + O ₁ Il 6.01 + 0.01 6.02 + 0 _T 03 5 ₇ 48 + 0.02 35.3 36.0 36.9 36 _r 7 36.8 33.5

246 17 5 2

- 110 -

Bc 3. Degradation of cellulose.

It has been found that cellulosic materials, such as straw, e.g. Wheat straw, sawdust, paper and lignocellulose can be hydrolyzed to a greater extent with a SPS ase preparation than with conventional cellulases. This is demonstrated by the following example in which a crystalline cellulosic material (AVICEL) has been treated with the aid of a standard cellulase Celluclast ™ 200 produced by Trichoderma reesei and the SPS ase preparation KRF 68.

Example Bc 3.1

Avicel was suspended in water (20 % solids), the pH adjusted to 5, and the temperature maintained at 50 ^° C. After 24 hours of reaction, the slurry was filtered and the content of reducing sugar (mg glucose / g Avicel) was measured. When using enzyme amounts of 5 and 20 % , the following values for the cellulose content were found:

Table Bc III

enzyme E / S% mg glucose / g AVICEL Celluclast SPS-ase 5 5 80 200 Celluclast SPS-ase 20 20 100 340

- 111 -

24617 5 2

- 111-

Bc 4. Application as baking aid.

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

Bc 5 «Improvement of the yield of alcohol and of biomass during the fermentation of sulphite liquor from paper production.

It has also been found that the yield of ethanol can be improved by treating papermaking sulphite liquor with a SPS ase preparation before it is used as a source of carbohydrate for the fermentation of ethanol. Sulphite liquor from papermaking can also be used to produce biomass, e.g. Single cell protein by fermentation and also in this case, the yield of biomass is improved when the sulphite liquor has been previously treated with a SPS-ase preparation. Also, the degradation due to the presence of the SPS ase preparation and the fermentation can be carried out simultaneously.

Bc 6. Drainage of biological sludge products.

During the usual water extraction of many biological materials from plant raw materials, large volumes of insoluble residue are formed.

- 112 -

24 61.7 5 2 -H ₂ -

standing from large quantities of swollen polysaccharides. This is the case, for example, when soymilk, soybean curd or soy isolate are 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 SPSsese, which opens the mesh structure of the material and dissolves only small amounts of carbohydrates. This dehydrates the material and results in a higher solids content in the sludge compared to a product obtained without enzyme treatment. Thus, the enzyme process provides the advantage of significantly lower energy consumption for removing water by drying, and also opens up the possibility of producing a cheaper dry animal feed or bulking agent for animal feed.

Bc 7. Silage aids.

It is known to add enzymes as silage adjuvants to improve the speed of the ensiling process and the fermentation of the silage. It has been found that SPS ase preparations are better in comparison with known enzymatic silage aids.

113-

24617

- L13 -

Overview of the figures to which reference has previously been made.

Fig. No. Refers to represents 1 general part of the description Binding effect between SPS and soy protein 2 general part of the description Flow chart describing the production of SPS 3 Section 2 Calibration curve for HPLC gel filtration chromatography 4 Section 2 HPLC gel filtration chcomotogram from SPS 5 Section 2 HPLC-Ge1-Filtration-Chromato- log of PLC-aseeded PLC 6 Section 2 and 3 Supernatant liquid HPLC gel chromatography chromatogram of SPS. incubated with soy protein 7 t Section 2 HPLC gel filtration chromatogram of supernatant from degraded SPS incubated with soy protein 8th Section 3 HPLC-gelation chromatogram of APS degraded by pectolyase 9 Section 3 HPLC gel filtration chromatogram of APS _# degraded "by SPS ase 10 Section 3 HPLC gel filtration chromatography of SPS, treated with pectolyase

24617

- 114 -

Fig.Nr. Refers to represents \ 11 Section 7 Immunoelectrophoretic peaks, including a SPS peak, identified by overlay technique 12 Section 8 Ion exchange chromatogram of a SPS ase 13 Section 8 pH-dependence of the activity of a SPS-ase 14 Section 9 Temperature dependence of the activity of a SPS ase : 15 Section 9 Temperature stability of a PLC ase 16. Section 10 pH stability of protease in a SPS ase preparation

Claims (6)

invention claim 1 A process for producing SPS ase, a carbohydrase in useful form and capable, under suitable conditions, of degrading soybean SPS into products which attach to protein in an aqueous medium to a lesser extent than the soybean SPS before degradation to the same protein under appropriate conditions, characterized in that a strain is grown in a nutrient medium whose main carbon source is SPS, which has been formed on the basis of vegetable raw, preferably defatted soybean meal, as starting material, followed by Sample of the fermentation medium on SPS-ase is examined and the microorganism in question is considered SPS-ase forming microorganism, if the analysis on SPS-ase is positive · 2. The method according to item 1, characterized in that the strain Asp. Aculeatus CBS 101 * 43 or Asp. Japonicus IFO 4408 is grown in a nutrient medium. Method according to item 1 or 2, characterized in that the cultivation is carried out as a submerged culture at a pH in the range from 3 to 7 ", preferably from 4 to 6, at a temperature in the range from 20 to 40 ° C, preferably from 25 to 35 ⁰ C, and wherein the nutrient medium contains carbon and nitrogen sources and inorganic salts. 4. The method according to item 1 to 3t, characterized in that the nutrient medium contains soybean flour. 31 AUG. 1983 * 1 _A 61 626 12 246 175 2 Method according to item 4, characterized in that the soya flour has been treated before use as part of the substrate with a proteolytic enzyme, preferably the proteolytic enzyme which has been formed microbiologically with the aid of Bacillus licheniformis. Method according to items 1 to 5 »characterized in that a sterile solution of pectin is added aseptically to the fermentation broth during the cultivation» For this SLSeiten drawings