DD213240A5 - PROCESS FOR REMOVING POLYSACCHARIDES - Google Patents

Abstract

The invention relates to a degradation process of particular cell wall polysaccharides of plants by means of a carbohydrase. The aim of the invention is to degrade vegetable protein products and to select the degradation products in great purity. The invention has for its object to provide a degradation process of polysaccharides via a carbohydrase. According to the invention, a SPS ase forming microorganism is grown on a fermentation medium whose main carbon source is SPS (soluble polysaccharide), which has been formed on the basis of vegetable crude protein, preferably defatted soybean meal, as starting material, and this SPS-ase preparation in an aqueous Medium is brought together with a substrate for the SPS ase preparation.

Description

The invention relates to a degradation process of polysaccharides by means of a novel enzyme.

The new enzyme is used in the food industry, for example in the total liquefaction of vegetables * in the juice and wine industry, in sugar production, beer production ^ for bakery production »in alcohol production and many other purposes»

Characteristic of the known technical solutions

A method for preparing a purified plant protein product (pvp) by enzymatic removal of the residue * without dissolving and reprecipitating the protein is described in Belgian Patent 882,769. However, the purity of the pvp obtainable by the known method is not satisfactory and therefore should be improved. In the examples, a purity of the pvp of about SS % is indicated. Even if it is possible to obtain a pvp with a purity of about 90 % according to the known method, this is only possible using certain pretreated starting substances. "It would be favorable, if possible, to have a purity of the pvp of approximately or more than 90 % with a much broader range of starting materials _# in particular from hulled and defatted soybean meal »

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The invention is based on the surprising discovery that a certain part of the residue decomposition product * as it occurs during the above-mentioned enzyraatischen treatment, d. h "a water-soluble high molecular weight carbohydrate partially attaches to the vegetable protein (binds), as will be explained later. This naturally leads to a low purity of the protein. It has also been shown 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 enable the degradation of a high molecular weight carbohydrate to produce a pvp of improved purity.

Explanation of the essence of the invention

The invention is based on the object * to provide a method for the degradation of polysaccharides.

The principle of the invention can be described as follows. Referring to Fig. 1, only products which are present as undissolved solids are indicated while all supernatant liquids (and solutes therein) have been omitted. A lot of soy flour was divided into two equal parts, Part I and Part II (column a in Fig. 1). Part I was administered proteolytically at a pH of about 8 with the aid of ALCALASE (a

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proteolytic enzyme that is formed is degraded by 8, licheniformis) and then further washed 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 FIG. 1). In this way, a pure residue (referred to as residue I) was isolated (column b in FIG. 1), part II of the soybean meal was not treated.

The residue in part II is referred to as residue II (column b in FIG. 1). 3 Residue I as well as part II were degraded with the aid of a commercially available pectinase (see columns b and c in FIG. 1).

Surprisingly, it has been found "that the undissolved portion of the residue I is substantially lower than the undissolved portion of the residue II on the basis of the nitrogen and dry matter balance" see Fig. 1 "where the shaded areas in column c the insoluble non- Protein substances in the above-mentioned step correspond to "When, moreover, the supernatant of pectinase-treated residue I is brought together with a soybean-red suspension at pH 4.5, a polysaccharide in the supernatant fluid is bound to the soy protein. This polysaccharide is bound to the supernatant of residue I, which is a part of the residual degradation product and which is clearly soluble in water in the absence of soy protein but "at or about the isoelectric point of the soy protein, if any" _t is called SPS (^ Soluble! polysaccharides) (see Fig. I) * The SPS

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has a Molekulargewichtsverteliung between 5 and 4.9 10 χ χ 10 · The formation of isolated SPS is apparent from the above in Fig, 2 scheme, which also includes a part of the specified in Fig, 1 procedure. Thus, the problem is to find an enzyme capable of degrading the SPS in such a way that the SPS degradation products do not attach to soy protein or bind to soy protein to a much lesser extent than SPS.

Although in the description specifically soy protein is mentioned, the invention is not limited to soy protein, but includes all kinds of vegetable proteins, see, "B", the proteins given in Belgian Patent 882,769, page ₉ I,

It has now been found, according to the invention, that by studying its ability to degrade soybean SPS, it is possible to select microorganisms capable of forming a compound which has enzymatic activity and is capable of degrading soybean SPS, and which will be discussed briefly below referred to as SPS-Ase ·

The invention therefore works primarily with a SPS ase, a Carbohydrase (glycosidase) in a suitable form, which is able to degrade soybean SPS under appropriate conditions to products that attach to protein in aqueous medium to a lesser extent than the soybean -SPS had accumulated before degradation to the same protein under appropriate conditions.

It has also been shown that this SPS ase, dis capable

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is able to degrade soybean SPS "polysaccharides that are similar to SPS and come from plants (vegetables and fruits), degrade more completely than common pectinases and common cellulases"

As used above, "in an appropriate form" is to be understood that he z, B _# a SPS-ase-containing agent excludes _e containing toxic substances or that such a small SPS-ase activity, in that more than 10% SPS-ase containing agent must be applied, based on the weight of the SPS in dera substrate in a reaction * 24 h at 50 ⁰ C at the pH optimum of the relevant SPS ase is carried out »to a reduction of PLC of practical importance to reach"

The purity of the resulting vegetable protein is improved compared with the purity of vegetable protein "that is obtained by the known from BE-PS 882 769 to known methods by complete or partial separation of the PLC of the resulting vegetable protein, since this vegetable protein product with PLC was contaminated.

It is currently unknown whether the particular SPSase described below obtains its enzymatic activity from a single enzyme or from an enzyme complex comprising at least two enzymes. "Some studies seem to indicate that there are at least two enzymes responsible for the degradation effect of the SPS." one of these enzymes can cause only a small degradation of the SPS, whereupon one or more enzymes are able

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to achieve ongoing degradation of the PLC. However, the invention should not be limited by this hypothesis »

The invention preferably relates to the use of a SPS ase which is capable of degrading soybean SPS in an aqueous medium to products which accumulate less in the aqueous medium to vegetable protein than the soybean SPS before degradation. Preferably, the SPS ase is able to degrade soy-SPS in an aqueous medium having a pH that does not deviate more than 1 * 5 from 4 * 5. The SPS ase leads to degradation products which accumulate after completion of degradation preferably less than 50%, in particular less than 20 % * based on the soybean SPS before degradation of vegetable protein in an aqueous medium. The SPS ase preferably leads to a positive SPS ase test * if it is determined qualitatively and quantitatively according to the method explained in more detail later »The SPS ase is preferably formed with the aid of a microorganism belonging to the genus Aspergillus, preferably to the species Aspergillus niger, heard. Conveniently, the SPSase is derived from enzymes that can be formed using Asp # aculeatus CBS <101 * 43 * The same SPSase can be formed using Asp »japonicus IFO 4408. It has been shown that Asp · Aculeatus C8S 101.43 also forms very effective remanases, cellulases, pectinases and hemiceilulases »It has also been shown» that not every strain belonging to the species Aspergillus aculeatus or Arpergillus japonicus forms an SPS ase which is useful for the invention As indicated later in the description (Section 5), the Asp * japonicus ATCC 20 236 strain is not shown to produce such levels of SPSase

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which can be detected by the enzymatic determination of SPS-ase _} as will be described later. The SPS ase is preferably immunoelectrophoretically identical to the SPS ase, which can be formed from Asp aculeatus CBS 101 * 43 and identifiable by immunoelectrophoresis overlaying methods (see sections 6 and 7),

When a SPS seed is microbiologically formed, it is formed into a mixture with various accompanying substances, especially other enzymes. If desired, the SPS seed in question can be purified, for example , by chromatographic separation techniques, as described in more detail below (Section 8).

In Agr, Biol. Chem. 40 (1), 87-92, 1976, it is described that a strain of Aspjaponicus, ATCC 20236, forms an enzyme complex capable of undergoing a specific degradation of an acidic polysaccharide in soy sauce, known as APS is called, a part of which is referred to as APS-I. This acidic polysaccharide is not identical to SPS, as discussed later in Section 3. "Thus, the HPLG gel filtration chromatograms of SPS and APS are distinctly different, and moreover, the gel filtration chromatograms of APS prepared by commercial pectinase _# Pectolyase * has been degraded and significantly different from SPS, which has been treated with their usual pectinase »pectolyase * Moreover, it is not clear from the article * that the acidic polysaccharide is bound to the soy protein and that the degraded acidic polysaccharide is not the soy protein or to a significantly lower

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In addition, it has been shown that this strain does not form an SPS ase in quantities determined by the enzymatic determination of SPSase as described in more detail below can be. This led to a prejudice against the use of any strain of Asp. Japonicus as a SPS ase maker. But surprisingly, it has been found according to the invention that some strains of Asp. Japonicus are SPS ase generators. The invention also relates to the isolated SPS based on raw vegetable protein as the starting material _#

In a preferred isolated SPS, the vegetable crude protein is defatted soybean meal. The preparation of the isolated SPS has been described above with reference to FIG.

In the method according to the invention, the strain Asp. Aculeatus CBS 101.43 or Asp. Japonicus IFO 4408 is preferably cultivated in a nutrient medium. It is further preferred that the cultivation is carried out as Submerskultur at a pH in the range of 3 to 7 _#, preferably from 4 to 6 at a temperature in the range of 20 to 40, preferably from 25 to 35 ⁰ C, wherein the nutrient medium Contains carbon and nitrogen sources as well as inorganic salts. The soybean meal is preferably treated before 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

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if preferred * a solution of pectin is added aseptically to the fermentation broth during the cultivation *

It has been found that Asp aculeatus CBS 101.43 also forms very effective residue solubilizing enzymes, cellulases, pectinases and hemicellulases in addition to the SPSase, and that the enzyme complex formed using Asp. Aculeatus CBS 101.43 is extremely well suited Thus, the complex formed by Asp »aculeatus CBS 101.43 can be applied 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 are also applied (as the dehydrating agent d · h. means for the degradation of polysaccharides and thus the release of the bound water in the polymeric structure of the polysaccharides (in the processing of vegetables). the invention further relates to the use of an SPS-ase or a method of Degradation of polysaccharides »v preferably plant cell wall polysaccharides »with the aid of a carbohydrase, wherein an SPS ase-containing agent according to the invention is brought together in an aqueous medium with a substrate for the SPS ase preparation.

Thus, according to the invention, "SPS ase preparations have valuable enzyme preparations for the partial or complete liquefaction or decomposition of various materials, preferably vegetable materials, for example, fruit and plant wastes containing protein, cellulose, hemicellulose (z. B "" glucans, xylan, galactans and araban), gums, pectin, lipids, inulin, polyphenols, starch and alginates

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For purposes related thereto, mention may be made of all the purposes in which commercial pectinases and cellulases are used today. Some examples are given later.

In the context of the extraction (isolation) procedure described in 2, B, in Example 2, it has been found that the SPS ase preparation is essentially proteinase-free due to the fact that the desired end product, d. Similarly, if other biological materials than proteins are extracted (isolated) from a biological raw material, the SPS ase preparation used should be substantially free of enzymes that degrade these other biological materials. Such modified SPS ase preparations may be formed by selective inactivation of the undesired enzyme, by 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 soy protein and related plant proteins from a biological raw material, the SPS ase preparation being substantially free of enzymes able to break down biological material «

Thus, according to the invention, it has been shown that the modified SPS-ase preparations (modified in the sense that they are substantially free of enzymes capable of *

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to degrade the biological material to be extracted or isolated) are valuable enzyme preparations for the extraction (isolation) of special biological materials such as starch, lipids, flavors, colors and juices from biological raw materials. Various examples are given later,

Erfindungsgeraäß it is preferred that one or more of the reaction products _(# if it desired end products or byproducts are not care) be treated simultaneously with or after the enzyme treatment. This will allow for a more flexible and adaptable mode of operation. This further treatment is preferably an alcoholic fermentation if one of the reaction products is a fermentable sugar. This gives a cheap process for producing alcohol.

In order to explain the essence of the invention in more detail, reference is made to the following sections 1 to 4, which respectively describe details in connection with the invention:

i · Production of PLC.

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

3 * Cleaning a PLC ase preparation »

4 * Dependence of the PLC ase activity on pH value and temperature as well as stability of the SPS ase *

63 376 12 - 12 Section 1 Production of SPS

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

Soy residue is the protein-free carbohydrate fraction (which may in practice contain small amounts of lignin and minerals) in defatted and ungested soybean meal * This carbohydrate fraction is insoluble in an aqueous medium at pH 4.5 and can be prepared in the following manner * Reference is made to flow chart 1 »

Defatted soy flour is in deionized water at 50 ⁰ C in a weight ratio of soy flour to water = 1: set suspended 5 in a tank with a pH-stat under temperature control, the pH value is 4n NaOH (I) to 8.0 * Oetzt is determined using Alkalase 0.6 L (a B. licheniformis proteolytic enzyme having an activity of 0.6 Anson units / g as determined by the Anson method described in Novo Enzyme Information IB No. 058 e -GB), a hydrolysis carried out under constant pH »wherein the ratio enzyme: substrate 4 % of the amount of protein in the soybean meal (II). After a hydrolysis time of 1 h, the slurry is centrifuged off (III) and washed (IV), these measures being repeated twice (V, VI, VII)

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Slurry obtained is hydrolyzed once more with Alkalse 0 _f 6 L for 1 h (VIII * IX), similar to the above. Then, the slurry is spun down (X) and washed twice (XI, XII, XII, XIV), and the slurry (6) obtained after the last washing step is spray-dried (XV). The thus-obtained spray-dried powder is the soy residue used as a raw material for Production of SPS serves »

SPS is the water-soluble polysaccharide fraction obtained by conventional treatment of the above-mentioned soy residue with pectinase. "The SPS is obtained in the following manner using the 14 reaction steps given below." Reference is made to Flow Sheet 2 ".

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

2 "The pH is adjusted to 4.50 with 6N NaOH,

3 »Pectinex 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 according to the prospectus" Determination of the pectinase units on Apple auice (MOU) "of 12 · 6, 1981). Further, CeI luclast 200 L is added in an amount of 20 g / kg of dry matter

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added (a commercial cellulases described in the prospectus Novo Enzyme, Information Sheet B 153-e-GB 1000 _# OuIi 1981).

4. The tank contents 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. The reaction mixture is allowed to stand for 30 minutes and the pH is then adjusted again to 4X5 with 6n HGl.

6. Centrifuge the reaction mixture and collect both the centrifugate (supernatant) and the residue (sludge).

7. The centrifugate 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 with a cutoff value of 30,000 using the following parameters ί

1 * ultrafiltration to a volcanic concentration of 6 *

2 »Diafiltration * until the percentage of dry matter in the permeate is O (O ° Brix)»

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3. Ultrafiltration to about 15% solids in the concentrate *

The temperature is 50 ⁰ C ₁ the pH 4 * 5 and the average pressure 3 bar ·

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

10 »The precipitate is collected with the help of a centrifuge *

11. The precipitate is washed twice with 50 % (vol / vol) ethanol in Η _Λ 0, corresponding to the volume of the centrifugate from stage 10, d. h, two centrifuging stages are carried out *

12 # The washed precipitate is redissolved in water having a volume equal to the volume of the ultrafiltered concentrate of Stage 9.

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

14. The clear filtrate containing pure SPS is lyophilized.

Flow chart I.

Defatted soy flour H ₀ O NaOH to pH »

hydrolysis

Alcalase 0.6L

(E / S »4 %) 4n NaOH

H ₂ O

Hydrolysis X In before being discharged pH-stat (pH = 8, T β 50 ⁰ C)

II

1, centrifugation 1 «washing stage III Centrifugate 1 > Waste slurry 1 IV

2 * centrifugation j V

centrifugate

slurry

waste

2 »washing stage

VI

3. centrifugation

VII

Centrifugate 3 waste

ι σ>

cn

(Continuation)

NaOH to pH 8

j slurry 3

New hydrolysis mixture

VIII

Alcalase 0.6L Hydrolysis 1 h before pH-stat the discharge (PH s 8.0, X Zem IX 4N NaOH ^ T = 50 ^° C) slurry XI irrifu 4 «Centrifugation 3 4 1 * VS / Aschstufe

waste

5 _# centrifugation

XII

Centrifugate 5

j Aufsch lahme 5 ^Abfa11

2 «washing stage

XIII

6 _# centrifugation

XIV

Centrifugate 6

Slurry 6

Abfair

spray drying

XV

j4 vj (Tt

Flow chart II

1000 g Pectinex Super cone. L 5 kg spray dried

Residue

g Celluclast 2 5O ₁ p

1

1

1 50 % C ₂ H ₅ OH

pectinase

Degradation with and cellulase

50 ⁰ Cj pH - 4.5j t u 24 h

1, 2 »3, 4, 5

centrifuge Transportation

slurry

Check filtration 38

Ultrafiltration, diafiltration, ultrafiltration (GR 6OP) (19-26 ⁰ C) 1

permeate

\ L

precipitation

Supernatant fluid

Centrifugation (Waste) Centrifugation

-"8th

10

Flow Chart II (continued)

2 x washing

precipitation

2 χ centrifugate

precipitation

1 Ho ⁰

redissolving

9 11

12

Check filtration

Protein slurry 13 (waste)

lyophilization

310 g lyophilized SPS

VD I

vj

cn

S] i »J

ro

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Section 2

Characterization of SPS and especially molecular weight distribution

Gel chromatography using an HPLC apparatus (Waters Pump Model 6000, Waters Data Module 730 and Waters Refractometer R 401) was used to determine the molecular weight distribution of the SPS, whose preparation was carried out as described in the description "(Fig. 4). using the same method and the molecular weight distribution of the determined by means of SPS-ase obtained degradation products of SPS · was (FIG. 5) Furthermore, using the same method, the annealing effect between soy protein and SPS (Figure _# 6) and the absence of the addition effect between soy protein and SPS mined using the agent of the invention (Figure 7), shown »

The calibration curve (the logarithm of the molecular weight versus Rf _$, where the Rl value for glucose is arbitrarily defined as 1 and the Rz value for a specific dextran is defined as the retention time for this dextran divided by the retention time for glucose is) was constructed using various standard dextrans of known molecular weight (T 4 »T 10, T 4 O _1, T 70, T 110, T 500). The R ^ for the maximum of each dextran peak was determined and the corresponding molecular weight calculated as V Ff * M, where M _{w is} the weight average molecular weight and M is the number average molecular weight. "Eluant for this chromatography method was 0.1m NaNO_. -Solution

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applied. The columns used for the chromatography were 60 cm PW 5000 and then 60 cm PW 3000. In this way, the relationship between the molecular weight and the Rr value for the above dextranes was established, see Fig. 3

On the basis of FIG. 4, it can be calculated that SPS has a molecular weight distribution corresponding to a value of

- 5 -

M of approximately 5.4 χ 10 and a value for M of approximately 4.2 x 10. It can also be seen from this figure 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 47.12 min (67 %) , corresponding to a molecular weight of around 4.9 χ 10 shows. This curve shows that there is a shoulder between these two peaks, with a retention time of 41.25 min (27 %) , corresponding to a molecular weight of 2.8 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, and that the remaining 45 % was reduced to one Polymer having three peaks with the following molecular weights: 5 × 10, 10 ⁴ and 4.4 × ³ , see FIG. 5

To show the attachment or binding effect between soy protein and SPS and the substantial reduction of the attachment effect between soy protein and SPS, which with

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The help of a PLC had been dismantled »the following tests were carried out:

3 % SPS in O _{1 of} acetate buffer at pH 4.5 was added to a slurry of soy isolate (Purina E 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 Figure 6 with Figure 4, it is apparent that the SPS is completely adsorbed to the soy isolate.

The same procedure as given in the previous paragraph was carried out with a 3% solution of SPS hydrolyzed with a SPS ase formed from CBS 101,43 (Fig. 7), a comparison between Fig. Figure 7 and Figure 5 shows that no compound in the hydrolyzed SPS having a molecular weight below about 10 was adsorbed to the soy isolate. The hydrolysis reduces the quantitative addition to about 10 to 15 %, based on the addition of SPS to soybean protein,

The NMR analysis of the PLC, the manufacture of which was carried out as described in the description, shows the following approximate composition of the SPS,

1 * c * -glacituronic acid in an amount of about 45 %, with about 40 % of the total amount of aer c < -galacturonic acid being present as methyl ester «

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2. Rhamnopyranose in an amount of about 20 % " galactopypranose" in an amount of about 15 % and 4 "β-xylopyranose in an amount of about 20 % *

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

Complete acidic hydrolysis of SPS (8 h in 1 η HpSO) and subsequent analysis by thin layer chromatography (TLC) showed that even smaller amounts of the monosaccharides fucose and arabinose were present in the hydrolyzed SPS »

HPLC analysis of SPS ₁ degraded by the SPS ase enzyme complex formed by CBS 101.43 showed a strong reduction in molecular weight. In agreement with this, the NMR spectrum of the degraded SPS indicated above showed that the major part of the ester groups had disappeared and that the content of xylose and galactose in the higher molecular weight material had also decreased. The NMR spectrum of the part of the SPS degradation product, which precipitates upon 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. »

63 376 12 - 24 Section 3 Cleaning a PLC ase preparation

Purification of the SPS ase preparation KRF 92 (see Example 1) was carried out by ion exchange. The buffer was 50 mM Tris (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 KRF 92 were dissolved in 450 ml of H ₂ O at 6 C and all other specified process steps between 6 and 10 C performed. 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 (ODp ₈₀ ) and the conductivity were measured on the eluate (see Fig. 12). Fraction 1 was the eluate which was not bound to the ion exchange material. Then, the column was washed with 2000 ml of buffer to obtain fraction 2. 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 exhibited SPS ase activity.

Fraction 1 was further purified by gel filtration, 1.5 g of fraction 1 was 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

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S-200th The flow rate 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. SPS is however reduced by the qualitative agar test if the factor G is mixed with a pectinase "It has been found that the factor G is able to cleave galactose, fucose and a part of the galacturonic acid of SPS but _t the main degradation product is correspondingly the HPLC analysis is still a high molecular weight product that is very similar to SPS,

Section 4

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

13 shows the pH-dependence of the activity of the SPS ase preparation KRF 68. From pH 2.7 to pH 3.5, 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 63 ".

Flg. 15 shows the temperature stability of the PLC-ase-zubeleltung KRF 68 »

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Working Example

For a more detailed explanation of the invention, reference is made to the following Examples 1 to 3, 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 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 Asp aculeatus and Asp # japonicus indicated here. This gave compositions containing SPS ase _# according to the SPS ase test given here. However, since relatively large amounts of SPS are required to carry out application experiments, similar pilot scale experiments have been carried out, see Example 1 below.

example 1

Production of SPS-ase in a pilot plant

A SPS ase was prepared by submerged fermentation of Aspergillus aculeatus CBS 101.43,

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

Peptone Difco 6 g

Aminolin Ortana 4 g

Glucose Ig

Yeast Extract Difco 3 g

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Meat Extract Difco 1.5 g

I <H PO ₄ Merck 20 g

Malt extract Evers 20 g

demineralized HO ad 1000 rnl

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 120 ⁰ C. The substrate is called E agar.

The strain was GBS 101.43 (37 ⁰ C) cultured on an E-agar slant * The spores from the slant were suspended in sterilized skim milk, and the suspension was lyophilized in glasses * The content of a vial of lyophilized contents were in the FERNBACH Given the piston. The flask was incubated at 30 ^° C. for 13 days *

A substrate with the following composition was prepared in a 500 1 seeding fermenter »

CaCO ₃ 1 2 kg glucose 7 2 kg Rofec (corn water festival materials) .3 "δ kg soy 1 # 2 kg

Tap water was added up to a total volume of about 240 1 * The pH was adjusted to about 5.5-CaCO before the addition of * The substrate was sterilized in the seed fermenter for 1 hour at 121 ⁰ C. The final volume

63 376 12 - 28 before inoculation was about 300 1 «

The spore suspension from the Fernbach flask was transferred to the seed fermentor. The conditions of the vaccine fermentation viaren:

Fermenter Type: Common aerated and stirred fermenter

with a ratio height; Diameter of about 2.3.

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 1 were transferred from the seed fermenter to the main fermenter »

In a 2 500 1 main fermentor, a substrate of the following composition was prepared:

Roasted soy flour 90 kg

KH ₂ PO ₄ 20 kg

Pluronic 150 ml

Tap water was added to a total volume of about 900 liters. The toasted soy meal was suspended in water "The pH was adjusted to 8.0 with NaOH and the temperature was raised to 50 ⁰ C" Then, about 925 Anson Units Alcalase 0 * 6 L was added to the suspension. The Geraisch was 4 h at 50 ⁰ C and pH 8 * 0

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(Na ₀ CO addition) without aeration under normal pressure (zero ato) and stirring at 100 rpm kept. Subsequently, the remaining components of the substrate were added and the pH was adjusted to about 6.0 with phosphoric acid. "The substrate was sterilized in the main fermenter for 1.5 h at 123 ° C. The final volume before inoculation was approximately 1080 1 *.

Then 150 1 seed culture were added »Ferraentation conditions were:

Fermenter type: Common aerated and stirred fermenter

with a ratio height: diameter of about 2.7

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:

Pectin genu ^x ^ 22 kg

Phosphoric acid"

conc. 6 kg

Pluronic 50 ml

'Genu pectin (citrus type NF)

63 376 12 - 30 -

Tap water was added to a total volume of about 325 liters. The substrate was sterilized in the dosing tank for 1 h at 121 ⁰ C. The final volume before the start of dosing was approximately 360. When this amount had run off, a second similar batch was prepared The total volume of pectin solution for fermentation was approximately 725 1.

After about 151 hours of fermentation, the fermentation was stopped. The approximately 1850 L of culture broth was cooled to approximately ⁵⁰ C and the enzymes recovered by the following procedure.

The culture broth was filtered using a vacuum drum filter (Dorr Oliver) coated with filter aid (Hy-flo-super-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 _IL- 25 % Hy-flo-sup_sr-ceL as a filter aid (referred to as Filtration I in the following table). The filtrate was precipitated with 561 g (NH ₄ ) ₂ SO ₄ / l at pH 5 * 5 and 4 % Hy-flo-super-cel diatomaceous earth was added as a filter aid. The precipitate and the filter aid were separated by filtration through a Rahraen filter »The filter cake was dissolved in water and insoluble constituents were filtered through a frame filter * The filtrate was (check) filtered» over a Seitz filter film (type supra 100) with 0, 25 % Hy-Flo super-cel filter aid (referred to as Filtration II in the table below). The filtrate was diafiltered through an ultrafiltration device »

63 376 12

After diafiltration, the liquid was concentrated to a solids content of 12.7 % (referred to as the solids content in the concentrate in the following table) *

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.

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

Four fermentation runs were carried out in the manner indicated below, changing the strain used for the fermentation, carrying out the optional base treatment and other parameters, as indicated in the following table.

Microorganism Base treatment X Code no. Concentration (%) of the filter aid at Aus precipitation Filtra tion II Solid content .ungen Yes No KRF 68 Filtra tion I 5 0.2 in concert ω ∈ C < CBS 101.43 X KRF 74 0.5 4 0.4 28 ATCC 20236 X KRF 83 2.0 5 0.25 7.5 IFO 4408 X KRF 92 1.0 4 0.25 12.4 χ) CBS 101.43 0.25 12.7

63 376 12 - 32 -

After filtration to reduce germs, the concentrate is concentrated at a ratio of 1: 2.3. A minor portion of the concentrated filtrate was spray dried and the remainder was freeze-dried.

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 being performed.

A, 100 g of SPS ase- preparation are dissolved in 1 1 of deionized water with stirring at 10 ⁰ C - 2 ° C. The pH is adjusted with NaOH to 9.1 »ßasenbehandlung This is carried out for 1 h» The pH is then adjusted with glacial acetic acid to 4.5 and dialyzed against the mixture of ice-cold deionized water to a conductivity of 3 mSi Then the product is frozen and lyophilized

S, 500 g of SPS ase preparation are dissolved in 4 l of deionized water with stirring at 10 ⁰ C 2 ⁰ C * The pH is adjusted to 9.1 with 4N 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 iyophilized *

C * 50 g of SPS ase preparation are dissolved in 400 ml of deionized water with stirring at 10 ⁰ C + _ 2 ⁰ C. The pH is adjusted to 9.1 with 4N NaOH. This base treatment is carried out for 1 h, then the pH is adjusted with ice

63 376 12

vinegar reduced to 5.7. The resulting material is lyophilized.

As a starting substance for the base treatment to clever SPS ase preparation Base treatment X Code no. i KRF 68 ABC KRF 68 BII KRF 68 X KRF 68 BIII KRF 92

The above-mentioned preparations are characterized in the following table by their Snzymaktivität according to the invention ».

63 376 12

Enzyme activity. Je g

KRF 68

KRF 63 BII. KRF 68 Bill KRF 74

SAE Plate test + 350 + 301 + 349 - 0 Quantitative test 737 507 481 142 SSU 2125 1560 1720 578 SRUM 120 67000 105 339 1630 CAP pH 3.2 8000 8044 9396 1320 ^C x 10300000 9000000 8800000 840000 PU 119400 72000 77700 4100 PGE - 78100 83700 76900 15130 UPTE 840 910 770 370 PEE 1600000 1100000 1000000 65000 VHCU

63 376 12

Enzyme activity

J ^e g Plate test KRF 83 KRF 92 KRF 92 BI SAE Quantitative test 168 476 430 SRU 120 683 626 757 SRUM pH 3.2 753 1640 1030 CAP 12800 5960 397 ^C x 8040 5700 3092 PU 7500000 8400000 7600000 PGE 64600 60000 68800 UPTE 327000 44000 62400 PEH 690 1000 790 VHCU 2200000 1100000 742000

63 376 12 - 36 Example 2 (application example )

This example describes the formation of a ρ.ν, ρ «from a hulled and defatted soybean meal" Sojamel 13 ". The solids content of this flour was 94 * 0 %, and the solids content (N χ 6.25) was .58.7 %. * The soy flour was spiked with the SPS ase preparation KRF 68 BII (Example 1) onto the treated the following VYeise:

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 to 4 _r 5 is set, 50 g of a solution containing 4 , 00 g of the aforementioned SPS-ase preparation were -zugegeben and then the reaction was stirred 240 minutes at 50 ⁰ C * the mixture was then placed in a laboratory centrifuge (Beckman Model 0-6B) 15 min χ at 3000 g centrifuged * the supernatant Liquid was weighed and analyzed for K and Kjeldahl's N and solid 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 performed twice. The solid phase was then lyophilized and weighed for Kjeldahl to N and for solids content. The results obtained in the experiment are shown in Table 2 * 1;

63 376 12 - 37 -

Table 2 _β 1 Results

Component Quantity N χ 6.25 Solid Yield Yield

g (%) substances on pro-solids

(%) tein (%) (95)

Soya flour 85.2 55.2 94.0 100 100

PLC ase-

Relation 4.00 75.6 - 6.4

1, centrifugate 666 1.50 5.04 21.2 42.0

ρ.ν, ρ. 44.5 87.5 95.7 82.7 53.2

Thus one obtained a pvp * with a protein purity, d # h, N χ 6.25, based on the solids, of 91.4 % and with a total yield of protein of 83 % *

Example 3 (application example )

This sex game was conducted to compare protein yields, nutritional quality and some functional properties of soy protein products made by the following three methods.

A «Typical isoelectric precipitation for the production of soy protein salad.

B _f Typical Isoelectric Washing Process for the Preparation of Soy Protein Concentrate Council

63 376 12 - 38 -

C isoelectric washing process according to the invention, comprising a residue-solubilizing enzyme for the formation of p.v.p »

In order to obtain a true comparison of the process (C) according to the invention with the usual processes for soy 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 temperatures and treatment times in all three cases were applied. Only the pH values were different due to the fundamental differences between the three attempts

A, Common isoelectric precipitate for the production of soy protein isolate

425.8 g of soy flour was extracted in 3574.2 g of tap water at 50 ⁰ C; the pH was adjusted to 8.0 with 20.1 g of 4N NaOH. After stirring for an hour, the slurry was centrifuged at 3000 g for 15 min using 41 L beakers in a laboratory centrifuge (Beckman Model 3-6B). The centrifugate I and the precipitate I were weighed. The precipitate I was again extracted with water to a total weight of 4000 g. The temperature was maintained at 50 ^° C. The pH was adjusted to 8 with 4N NaOH and the slurry was stirred for 1 h "The mixture was centrifuged and centrifugate II and precipitate II were weighed as above." Centrifuges I and II and precipitate II were sampled for Kjeldahl and analyzed for solids content

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I and II mixed and kept at 50 ⁰ C. The protein was then isoelectrically precipitated at pH 4.5 with the aid of 45 g of 6N HCl. After Istündigem stirring at 50 ⁰ C, the protein was gewonnen_ by 15 min centrifugation at 3000 g χ. The centrifugate III was weighed and analyzed for Kjeldahl to N and for solids content. The solid phase III was weighed and washed with water in an amount corresponding to the weight of the centrifugate I. Washing was carried out by stirring at 50 ^° C. for 1 h. The washed protein was recovered by centrifugation at 3000 g for 15 min. * Centrifuge IV was analyzed for K according to Kjeldahl and the solid content. The solid phase was dissolved in 1550 g of water of 50 ^° C. and the pH was adjusted to 6.5 with 17 g of 4N NaOH. The mixture was stirred for 1 h and the pH, if necessary, adjusted to 6.5 again. Finally, the product was subjected to freeze-drying and analyzed for Kjeldahl on N as well as on the solids content. The mass balance calculations are given in Table 3.1.

Table 3.1 - Calculation of the mass balance in the usual isoelectric

precipitation for production of protein Soy Protein Isolate yield 6.6 Yield 7.0 2.4 I 04 Operations and Amount of % (Nx6,25) Firmly on protein (%) 31.7 Solids (%) 35.2 • si fractions Fraction g 55.2 substances (SK) 100.0 100.0 Η · ro Extraction: soy flour 425.8 O 94.0 0 - 0 - water 3,574.2 O O .0 0 0.8 4n NaOH 20.1 5.9 16.0 100.9 100.4 1, centrifugation £ 4,020.1 4.4 10.0 58.8 54.1 Centrifugate I 3,141.0 - 6.9 - - Precipitation I 805.0 - Re-Extraktionj - - - Precipitation 1 805.0 O - 0 0 water 3,195.0 O 2 _# centrifugation 0.5 Centrifugate II 3,104.0 9.1 0.9 Precipitation II 820.0 17.2 Mix and acidify -. Centrifuges I + II 6,245.0 O - 6n HCl 45.0 21.3

Table 3.1 (continued)

Operations and fractions E III III Amount of fraction g Protein (° Nx6.25) Solids Yield of protein (%) Yield of solids 3 # centrifugation; Centrifugate precipitation III 6290.0 5650.0 308.0 0.3 7.2 26.8 Wash precipitate water IV IV 308.0 3141.0 O ι O O 0 4 _# centrifugation: centrifugate precipitation IV 3449.0 3113.0 291.0 0.4 0.15 • Mt. 0.5 mm Neutralization precipitate water 4 η NaOH 291.0 1550.0 17.0 O O O 16.0 0 0 0 0.7 Drying: powder 128.0 93.8 96.3 51.1 30, 8

63 376 12 - 42 -

B. Isoelectric Washing Process for the Production of Soy Protein Concentrate

425.6 g of soybean meal were washed in 3574 g of water at 50.degree. The pH was adjusted to 4.5 with 44.8 g of 6N HCl. Washing was carried out with stirring for 4 hours. The slurry was then centrifuged for 15 minutes in a laboratory centrifuge (Beckman Model 0-6B) at 3000 g by using 4 L-8 cups. Centrifuge I was weighed and assayed by Kjeldahl to N and analyzed for solids content. The solid phase I was weighed and washed again with water up to a total weight of 4000 g "The pH was readjusted _f 7 g to 4.5 with 6N HCl l, and the slurry stirred for 30 min at 50 C» The mass was The centrifugate II and the solids II were weighed as above. The solid phase II was resuspended in 1575 g of H ₂ O at 50 ^° C. and the pH was adjusted to 6.5 with 34.5 g of 4N NaOH. The mixture was stirred at 50 ° C. for 1 h and the pH was adjusted again to 6.5 as necessary. Finally, the protein product was freeze-dried, weighed and analyzed for H according to Kjeldahl and for the solids content. The quantitative 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 the protein fixed at yield% yield of fraction materials protein solids g (Nx6.25)%%%

To wash

Soy flour water 6n HCl

centrifugation:

Centrifugate I solids I

425 .8th 55.2 94 , 0 100 .0 100 , 0 3574 .0 0 0 0 0 44 .8th 0 21 3 0 2 A

4,044.6

3150.6 846.0

0.6

8.0

another wash 846.0 Solids I 3,154.0 To wash 1.7 6n HCl

0 0

0 0

25.2

0 0.1

2. Centrifugation: centrifugate II solids II Σ 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 0 0 1.4 Drying: powder 281.0 72.5 98.4 86.7 69.1

cn w

vj

cn

I *

ro

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C, isoelectric washing method using a residue-solubilizing enzyme to form p _e v »p,

425.8 g of soybean meal were washed in 3524.2 g of water at 50 C, the pH was adjusted to 4 * 5 with the aid of 43.7 g of 6N HCl. 24 g of the SPS-ase preparation KRF 68 BIII (Example 1) were dissolved in 26 g water and added to the wash mixture "The wash procedure was then 4 h performed" Then, while stirring the cleaning, _t carried out as described in B, wherein the amounts of 6N HCl, 4N NaOH and resuspending water are 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-solving enzyme for the preparation of ρ, ν, ρ.

Operations and fractions Amount of fraction g Protein (Nx6.25) ι _ Solids 1 21.3 Yield of protein Yield of solids I cn 04 scrubbers O ί Ui OJ Vj cn soy flour 425.8 55.2 O 94.0 100.0 100.0 ro water 3,540.2 O O O 0 6n HCl 43.7 O 21.3 O 2.3 SPS-ase: KRF 68 BIII 24.0 75.3 96.0 7.7 5.8 1 "centrifugation:) T 4,043.7 - f - - Centrifugate I 3,420.0 1.7 5 * 2 24.7 44.4 Solids I 620.0 - - Washing again Solids I 620.0 - - water 3,380.0 O 0 6n HCl 1.3 O 0.1

Table 3 »3 (continued)

Operations and fractions

Hange of the protein fixed '· yield of yield of fraction% materials: protein solids g (6.25 χ N) (%) (%) (%)

2 _# centrifugation: JT 4,001.3 0.2 0.6 j 0 2.9 5.1 I Centrifugate II 3,400.0 - 16.0 years! - - cn I Solids II 577.0 Neutralization: - - Solids II 577.0 O 0 0 water 1,700.0 0 0 1.0 4n NaOH 25.3

Dry; powder

211.0

87.3 86.9

2)

96.7 97.0

2)

78.2

51.1

Analysis of the Bioteknisk Institute, Holbergsvej 10, DK-6000 Kolding »Denmark

Analysis of Qvist's Laboratory, Marseiis Boulevard 169, DK-8000, Aarhus C, Denmark

63 376 12. - 47 Sewing 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 evaluation and the index of essential amino acids (EAAI) was calculated according to the FAO reference pattern of the 1957th

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 {A, O "C, S, is an abbreviation for American Oil Chemists' Society) method described. The results are given in Table 3 * 5 which also shows the yields and the protein to solids ratio of the three products.

Table 3 + 4 Composition of A ^m i ⁿ osäure and Nährbewertung of the three protein products A _1, B and C ι

amino acid

A »Soy protein isolate

B, soy protein isolate

g / 16 g N aas

g / 16 g N aas

t)

Nonessential

C * soy protein isolate (p _# v «p.)

g / 16 g n aas "

aspartic 12.4 - 11.3 - - 11 * 9 - serine 4.62 - 4.69 - - · 4.81 - glutamic acid 21.3 - 18.2 - 17.7 proline 6.07 - 5.19 - 4.76 - glycine 4.13 - 4.26 4.33 - alanine 3, .54 - 4.27 > i °° 4.55 - histidine 2.83 - 2.78 > 100 2.50 - arginine 8 * 09 - 7.57 > 100 7.04 Essential isoleucine 4.87 > 100 4.97 5.19 > 100 leucine 7.80 > 100 7.98 8.09 > 100 lysine 6.24 > 100 6.09 5.57 > 100

Table 3 «4 (continued)

2 38 3 4 41 5 6 42 7 I cn 1 5 «47 > ¹⁰⁰ years> 100 5.35 ^{> 100} *> 100 5.17 ^{> 100} *> 100 VD phenylalanine 3.38 56 > 100 ' 3.88 60 > 100 ^; 4.44 65, > 100 ' cn tyrosine 1.29 86 ⁶⁴ ' ⁵ * 56.4 1.32 90 ⁶⁶ '° * 60.2 1.44 91 ⁷² > ° \ 65.5 to cystine 1.08 49, 1 ^} 1.21 55.0 ' 1 * 31 59, 5 ^} methionine 3.10 > 100 3.60 > 100 3.97 7 100 threonine 1.06 75.7 1.37 97.9 1.32 94.3 tryptophan 4.90 7100 5.23 > 100 5.57 ₇ 100 valine % Total salary , 36 , 31 21 the essential amino acid , 4 % , 2 % 5 % Chem »rating , 7% , 2 % 3 % EAAI

aas = s Evaluation of the amino acid, based on the FAO reference pattern (1957)

63 376 12

- 50 -

Table 3 * 5 Method characteristics and trypsin inhibitor content of the three protein products A, B and C.

A. Soy Protein B. Soy Protein C, Soy Protein Isolate Concentrate Isolate (p, v, p,)

procedural Prote rens- in in character- the teristi- Firmly ka stof fen Prote in-off prey

97.4 %

· 7! 7 O /

90.0 %

51.1 %

86 J ^{Q /} -

/ Q

78.2 %

Trypsin inhibitor TU I / g PTOdu kt 34 000 21 000 19 000 TUI / g protein 36 250 28 970 21 810

Functional properties

The index of nitrogen solubility (NSI) was determined in a single dispersion of protein 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 4000 minutes at 4000 χ g centrifuged and the supernatant solution examined for nitrogen. The nitrogen solubility was calculated as (soluble N% / total N %) ♦ The results of this calculation for the three products are given in Table 3.6.

The emulsifiability was determined three times on each product by a slightly modified Swift titration * 4.0 g

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(N χ 6.25) of the product were mixed in 250 ml of 0.5 M NaCl with a Sorval Qmnimixer at low speed, 50 ml of the suspension were added to a mixing glass and 50 ml of soybean oil was added. Subsequently, the gesarate mixture was weighed. The ül-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 before the "end product" was added, was determined by weighing »

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 means of the provisions of the emulsification of the three products are shown in Table 3.6.

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

Extension on impact = 100 ^v

where V means the final volume of the whipped product in ml »

63 376 12 - 52 -

V was measured by refilling the mixer glass with water. Double tests were carried out for each of the three samples. The mean values obtained are given in Table 3.6 «

Foam stability was measured as the ratio between the amount of foam remaining after 3 minutes of drainage and the original foam amount. 1 g of the product foamed by the above procedure was placed in a plastic cylinder (diameter 7 cm, height 9 cm) with a wire mesh given with a mesh size of 1 χ 1 mm. The cylinder was placed on a funnel on a glass cylinder and the weight (3) of the elapsed liquid in the glass cylinder was determined. The foam stability FS is defined by the equation *

FS = 100 %.

The results of this determination are given in Table 3.6 «

The gel strength in this specification is defined as the Srookfield viscosity measured by T-spindles in a Brookfield Helipath stand. The gels were prepared by heat treating 12% protein suspensions in 0.5 M NaCl. The heat treatment was done in closed doses with a diameter of 7.3 cm and a height of 5 cm placed in a water bath maintained at 80 and 100 ° C, each within 30 minutes * The cans were cooled and before being opened and

53 375 12 - 53 -

(the content) was measured, thermostatically adjusted to 20 ° C »The results of the measurements are given in Table 3.6.

Table 3 "6 Functional properties of the three protein products

Function A. Sojapro-B, Sojapro- C. Sojapro-

tein isotein isotein isolate lat lat (p.v.p,)

% NSI in 0.2 Ra NaCl % NSI in water 39.5 53.9 20.3 25.1 25.6 28.6 Emulsifying ability: ml ull / g (N x 6.25) 218 182 354 Impactability (95) 120 120 340 Foam stability (SS) 50 50 20

Gel strength (dPa "s)

80 ⁰ C (0.5m NaCl) 1.7 χ ΙΟ ³ 1.2 x 10 ⁴ 3.3 x 100 ⁰ χ C (0.5m NaCl) 2.0 10 ⁴ 4.0 χ χ 10 1.3 ⁴ X

Example 4 (Application Example )

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

63 376 12 - 54 -

prepared by Novo Industri A / S was treated with a base at low temperature in the following manner. The pH of a 10% Celluclast solution in water was adjusted to 9.2 with NaOH and the resulting solution was adjusted to 5 ° C cooled. After 1 h at this pH and temperature, the pH was again adjusted to 4.7 with 20% acetic acid. The solution was kept at 5 C overnight and then sterile filtered. The filtrate was freeze dried. 4 g of the freeze dried product together with the SPS ase preparation KRF 68 BIII (Example 1) added »The two enzymes were dissolved in 172 g of water before adding to the washing mixture» The determination of the mass balance is given in Table 4 * 1 «

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. B * KRF 92, see in Table immediately before Example 2.

Table 4.1

Calculation of the quantity balance in isoelectric washing, including the SPS-ase preparation and Cellulast vJV for the preparation of ρ, ν, ρ. ι

Operations and fractions Amount of fraction g Protein (%) (Nx6.25) Solids Yield of protein Yield of solids (%) I W To wash Ul I soy flour 425.8 55.2 94.0 100.0 100.0 σ> water 3,546.2 0 0 0 0 6n HCl 43.1 0 21.3 0 2.3 SPS-ase: KRF-68-B-III Celluclast 24.0 4.0 75.3 43.6 96 96 7.7 0.7 5.8 1.0 Centrifugate JT 4,043.1 - - - - - Centrifugate I 3,382.0 1.9 5.5 27.3 46.5 Solids I 661.0 to wash again? Solids I 661.0 - - water 3,339.0 0 0 0 0 6n HCl 0 0 0 0 0

Table 4.1 (continued)

Operations and fractions Amount of fraction g Protein U) (Nx6.25) Solids Yield of protein Yield of solids (%) Ul cn 2 # centrifugation: ~ centrifugate II solids II '4000.0 3414.0 582.0 0.2 0.7 2.9 6.0 Neutralization: Solids II Water 4 η NaOH 582.0 1691.0 25.3 0 0 0 16.0 0 0 0 1.0

Drying: powder

206 _t o

88.8 98.9

77.8

50.9

63 376 12 - 57 -

Example 5 {application example)

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

Table 5 »! In the production of p * v.p * obtained theoretical protein yield

fractions Quantity g Protein (Nx6 * 25) Yield of Pro example 3 C tein < Protein, Nx6,25) Protein 0 / / O Soy flour SPS-ase KRF-68 B-III 85.2 4,, 8 55.2 75.3 100 55.2 75.3 100 ! ♦ centrifugate 2 # centrifugate ρ, ν.ρ, 639 595 0.99 0.13 87, 2 ^a 7.7 1.7 0.2 87.1 7.7 13.5 1.6 92.6 ^b 24.7 2.9 80, l ^b

^a average of 87.5 (Bioteknisk Institute) and 86.9 (Qvist's Laboratory) ι corresponding to solids of 97.6 and 98.0 %,

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

63 376 12 - 58 - '

Example 6 ( Example of Use) Detection of protein binding by SPS

40 g (N χ 6.25) from a commercially available soy protein isolate (Purina 500 E from Ralston Purina) were dissolved in 680 g of water. The mixture was warmed to 50 C in a water bath and the pH was raised to 6 η HCl 4.5 set. 90 g of this mixture was placed in 5 χ 250 ml Erlenmeyer flasks and 10 g of aqueous solutions containing O, 0.2, 0.4, 0.8 and 1.6 g, respectively, of the SPS prepared as previously described added. The flasks were then stirred for 240 minutes. »

240 min with a magnetic stirrer in a water bath at 50.degree.

The slurries were then centrifuged at 3000 χ g for 15 min and Kjeldahl centrifuge I analyzed for N and solids. The solid phases were washed in room temperature water and then recentrifuged. This procedure 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 N and on the solids content by the Kjeldahl method * Based on the specified in Table 6.1 analysis were the protein and obtained the percentage of SPS that was bound to the protein indicated by means in connection with Table 6.2 Calculated formulas.

This example shows that the SPS is firmly attached to the protein.

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is bound, so that the ratio of protein: solids decreases with increasing content of SPS. An SPS content, corresponding to approximately 0.4 g in 10 g of water, added to 5 g of protein isolate, is the protein: SP3 ratio present in the soybean meal.

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 in low protein: SPS ratios.

Table 6 * 1 measurements according to example 6

Ratio protein / SPS Zent rifugat I % N dry precipitation 93.1 , Νχ6,25 % N % DM % Nx6.25 % DM α 97.3 ^J DM OO 13.2 / I 97.9 88.6 25 0, 068 0.62 13.4 82.5 98.1 86.1 12.5 0,045 0.49 13.0 83.8 97.9 83.0 6.25 0, 038 0.45 12.6 81.3 80.3 3.125 0.031 0.45 11.8 78.8 75.3 0.026 0.61 73.8

63 376 12 - 60 -

Table 6 »2 Protein recovery and percent binding of SPS

Ratio % protein recovery ¹ ' % binding of SPS' protein / SPS

oc 91.5 O

25 94.4 ^ 77

12.5 95.3 90

6.25 96.1 70

3.125 96.8 60

¹ ^ % protein recovery = £ Ί - J χ 100, where NC-1 = % N in centrifugate I.

2)% binding of SPS =

_r 5x (^ Protein Recovery q ) - SxC ^ Protein Reqq ") " j

L i% ^p / H) {% p / h) «, ^ y

, , ,. χ 100,

j / ~ 5 / amount to PLC _J7

(% ^p / M) Protein / solid ratio in dry precipitate and

(% P / H) co applies to the precipitate without addition of SPS

Example 7 (Application Example )

This example describes the preparation of one using the SPS ase preparation KRF 92 BI in an amount of 5 % _t based on the solids, The method of preparation

63 376 12

was exactly the same as Example 3c, except that all amounts were reduced by a factor of 5. The p.v.p. was analyzed as described in Example 2. The results obtained in this experiment are shown in Table 7 * 1.

Table 7 »! Results obtained in Example 7

Ingredients Amount (Nx 6.25) Solid Yield Yield

g (%) Substances in protein Festig (%) (%)

Soya flour 85.2 55.2 94.0 100 100

Enzyme preparation 4.0 71.2 - 6.1 -.

1. centrifugate 632 1 , 88 5.44 25 3 43 , 0 2 * centrifugate 673 0 »30 0.80 4 # 3 6 .7 p. V. P. 39.8 85 , 6 * 98, ^a l 71 , 9 48 *8th 84 98, l ^b

^a Analysis of the Bioteknisk Institute, Holbergsvej 10, DK-6000 Kolding

Analysis of the Qvist's Laboratory, Marseiis Boulevard 169 * DK-8000

Example 8 (application example )

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

63 376 12 - 62 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 C could be maintained 25 s 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 to make ρ _# ν * ρ according to the invention, but in this case the slurry was spray dried at an inlet temperature of 200 ° C and an outlet temperature of 90 ° C. 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).

Production of ρ, ν.ρ.

The preparation was carried out in the following manner:

70 g of solids of the steamed and dried soybean meal was suspended in 560 g of water and stirred at 50 ⁰ C and the pH was adjusted to 4 »50 with the aid of 6.5 ml 6 η HCL, 6 χ 90 g of this suspension were in 6 250 ml Erlenmeyer flask and stirred on a water bath of 50 ⁰ C using magnetic stirrers. To each

63 376 12 - .63 -

Flasks were charged with 10 g of a solution containing O, 0.025, 0.050, 0.10, 0.20, * 0.40 g of SPS Ase preparation KRF 68 BIII. The reaction mixtures were then stirred at 50 ° C for 240 min. The mixture was then centrifuged at 3000 g for 15 min.

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

A similar experiment was conducted with untreated soybean meal (Sojamel 13 from Aarhus Oliefabrik A / S) as starting material. In this case, the ratios were enzyme: substrate O ₁ , 1, 2, 3, 4 or, S %,

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 dissolved after the reaction 100 % . The following table shows the results obtained in both experiments,

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Table 3.1

- 64 -

Ratio of protein yield and protein of the solids of p.v.p., prepared from steamed or ro> hera soy flour.

Steamed soy flour Protein of solids O / D untreated soy flour Protein of the solids E / S % Protein solids % 76.5 Protein solids % 73.9 O 92.9 86, 6 90.7 - 0.25 90.1 88, 7 - - 0.50 3 89 * 89.7 - 86.2 1.0 88.1 91.7 87.1 88.1 2.0 86.6 - 85.7 89.5 3.0 - 92.2 84.3 90.9 4.0 84.7 - 82.6 91.1 8.0 - 75.2

In the context of 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 given in Flowsheet 3 ".

The substrate may be one or more carbohydrates in a starting material or it may be all of the starting material.

This substrate can be a chemical or physical

63 376 12 - 65 -

Pretreatment »as explained later exemplarily, z. B, an acid or alkali treatment, soaking, wetting and / _or cooking with or without steam.

The starting material may be soaked (macerated), chopped, 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 Effect, z * B, at different pressures, which are only a fraction of the maximum pressure specified for the particular homogenizer. Different additives may be added before or during the homogenization, as shown in flow diagram 3 by b. bp ... b _n specified.

The reaction process, including SPS ase preparation, is carried out under special conditions, e.g. B. of temperature, pressure, time, pH and enzyme amount. Also, recommendations are given for the reactor used (eg, for batch operations or piston flow reactors) and stirring, if necessary. A number of additives can be given for different starting materials, as by c, C ₂ ... C in flow scheme 3 indicated.

The separation processes can also be carried out with different effects. In many methods, the separation is omitted or facilitated, for. For example, if the

63 376 12 - 66 -

gangsraaterial completely liquid (liquefied) is. Different separators can be used (eg «3, centrifuges, filters» ultrafiltration devices, hydrocyclones, thickeners, sieves or simple decanters).

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 solid or liquid phases obtained can be further treated, concentrated, dried, extracted with solvent to remove certain components, such as fat or oil, fermented to form biomass, alcohol or other product (enzymes, antibiotics or other valuable constituents). ,

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

Flow chart III

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substratum

SPS-ase

preparation

Horaogenisierung

reaction

homogenization

separation

liquid horn

= 0-100 %)

(special reaction conditions)

S ep

= 0 - 100

solids

Further treatment (see text) or reuse as substrate

63 376 12 - 68 -

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 accompanying Table I various characteristics are summarized together with the flow chart 3 «

63 376 12

Set up of possible applications of SPS ase preparations

Type of PLC ase- VoIl- 3e 1 - - ^ ,. ₌ ^ __ preparation stän- Extraction of starch from sharks » SPS-ase prepara- ended train number, 2 Wheat and potatoes _t the in the we Verflüs A Extraction of lipids from plant sentimentally free liquefaction 3 materials is from one or or A Extraction of essential oils several disturbing ones similar. 4 from vegetable materials Enzymaictivitäten behand A Extraction of natural color lung Substance of vegetable matter A 5 lien Extractions of rubber from the 1 Guayul-8usch A Production of a milk substitute 2 Fabric for pets. 3a Production of saccharified starch 3 Ke containing raw materials Ba Complete liquefaction of 4 Pears and other fruits Ba Production of juice by treatment 5 development of fruits and vegetables Ba Treatment in the cohort with the Not Extraction or pressing of modi- Ba 6 Sugar cane or sugar beet fi 7 Production of soy milk ornamental- Treatment to increase the profit th Ba amount of soluble coffee PLC Ba , components 3se-

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Type of PLC ase- machining loading 1 Prevention and / or reduction of preparation tungs- "Veiling" in apple juice accessory Help train no. 2 Use for clarifying white pure bb Wine do- 3 Production of ISSPH or others gene bb vegetable protein hydrolysates 4 Mash enzyme in the brewery bb 5. Enzyme additive in the Bierfermenta tion and / or storage bb 6 Means for removing almonds bb cuticle Other 1 Degradation of different waste To whom bb products Not Droppings 2 Saccharification and simultaneous modi species bc fermentation fi 3 Degradation of cellulose ed bc 4 Use as a baking aid PLC 5 Improvement of the alcohol yield ase- bc and biomass yield in the accessory bc Fermentation of sulphite lye from pure bc papermaking obligations 6 Drainage of biological whitewash 7 Silage Help bc bc

Table I

Sub-lit, strat

additions

a.

1 Homogenization _{0 /} r \ Hom ^h

2 'homo-separation

genisie-

tion

liquid phase

further treatments

solid phase

United phase (no separation

Corn water SO _n

What- NaOH or HCl

- 50 10 - 30 20 - 50 (contains washing

Germs washing, oil extraction

process

A 2 corn germs NaOH or HCl O 0 100 - Pasteurization Concentration spray drying Ba 1 Soya-Vi / as-flour ser (degreased) HCl O 0 0 - Yeast for alcohol fermentation, distillation Ba 2 Sweet Was- Karker potatoes NaOH or HCl 0 Crystal stallion - Ba 5 Sugar beets What ser NaOH or HCl 10 - 100

Bc 1 soy-wasquark ser o.

Soy milk residues

HCl

10

Yeast for alcohol fermentation, distillation

63 376 12 - 72 -

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

The extraction of starch from maize "wheat, potatoes and other starchy plants is carried out in one or more stages: soaking, wet milling and separation." 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 * Oer water consumption can be reduced

3, The release of corn germs is facilitated without release of corn oil

4 * The protein can be obtained in higher purity, 5 »The extraction 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 which 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 with a SPS ase preparation of the above

63 376 12 - 73 -

be treated kind specified. Similarly, the extraction of olive oil in the aqueous phase can be improved if the enzyme used for the enzymatic treatment is a SPS-ase preparation of the type indicated above, see z * 3, Food Pharmaceutical and Sioengineering No. 172, Vol. 74 * Pp. 93-94 «The aqueous extraction of z» B * soybean oil, rapeseed oil and sunflower oil can also be improved in a similar way.

A 3 · Extraction of essential oils from plant materials

If plant materials that contain essential oils, are treated with an aqueous solution of an SPS-ase preparation "which is substantially free of enzyme activity _f which is capable of reducing the essential oils or change in other ways, the essential oils in will high yields at very low cost »

A 4 »Extraction of natural dyes from plant- based material

yFor vegetable materials containing colorants, eg red beets containing the red dye betanin or the dye in cranberries, treated with a SPS ase preparation substantially free of enzyme activities capable of degrading the dyes or otherwise, the dyes are recovered in high yields at low cost.

63 376 12 - 74 -

A 5 * Extraction of rubber from the Guayul Susch

Another example of a substrate for a SPS ase preparation that is substantially free of enzyme activities capable of degrading crude rubber is the cell wall material of roots and branches of the guayul bush.

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

By complete liquefaction of soybeans, sunflower seeds, cottonseed, faba beans or canned peas, a calfrail substitute can be made which is soluble in cold water at a pH-1 / vert of about 4.5. When starchy raw materials such as faba beans or peas are used, starch liquefaction by means of a <X -amylase must be carried out before, after or simultaneously with treatment with a SPSsese which finally liquefies the non-starch polysaccharides in the structural material of the cell walls. A detailed example using faba beans is given below * and with respect to soybeans reference is made 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 Faba bean meal were suspended in 35 l of water. 75 g of Termamyl 60 L and 18 g of CaCl _p were added. The suspension was heated with stirring in a steam jacketed kettle at 95 ⁰ C

63 376 12 - 75 -

* Oie heated suspension was then treated for 60 min 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 300 L were added, the starch fraction was essentially converted to disaccharide (maltose). The reaction mixture was then pasteurized for 2 minutes at 90 ° C. 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 substance) and the solution of the product remained stable for days without sedimentation »

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

Example 8a 1,2

Soy flour (Sojamel 13) s' _s 25 described in Example 3 at 150 ⁰ C is treated with a steam jet "That treated © soy flour was" applied spray-dried and described for further investigation "as follows.

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50 g of the treated with water vapor soy flour were mixed with 450 g of water and the pH value with 4.1 ml of 6N HCl to 4.5 »The mixture was then heated in a water bath at 45 ⁰ C and 0.250 g of the SPS-ase- Preparation KRF 68 was added to the heated mixture, which then reacted with stirring for 5 hours. The mixture was then heated to 80 ^° C. for 2 minutes to inactivate the enzyme. A 100 ml sample was centrifuged at room temperature for 15 minutes at 3000 × g. The supernatant was treated with an ion exchanger and the carbohydrate composition analyzed by HPLC supernatant was also analyzed for nitrogen content by the Kjeldahl method and the solid content and the nitrogen solubility index (NSI) and the Feststof flöslichkeitsindex calculated (DSI) ,. see results in Table Ba I »100 ml of the reaction mixture *, which had been cooled to 20 ° C., was 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 resulting 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 as above, after 1 and 2 days *

A reaction was carried out as above, but in this case 1.00 g of SPS ase feed was applied *

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The same types of analyzes and stability measurements as described in paragraph A were performed. The results are given in tables Ba I and 3a II «

From the chemical analysis of the supernatants it is clear that the values for NSI (%) and DSI (%) are higher in experiment B than in experiment A. However, the stability tests carried out 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 at a lower enzyme level »

From the measured by HPLC carbohydrate composition shows that essentially mono- and disaccharides have been formed · How are oligosaccharides, which are known to be responsible for diarrhea and flatulence "when administered calves in excessive quantities, only in small Quantities available.

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Table Ba I

Chemical properties of the supernatant fluid

encryption

XX

Ratio NSI, % ^Λ DSI,% ^ΛΛ HPLC results (co-enzyme (at pH (neutral pH at pH)

amount = 4.5) = 4.5) sugar)

substratum

% (Wt./wt.)

A E / S = O , 5 % 39 , 9 62.4 DP ₁ + DP ₂ : 79.7 "A DP ₃ 7.4 % DP ₄ 12.2 % ^DP ₄₊ 8.1 O / Λ) B E / S = 2 , 0 % 57 , 0 67.1 DP ₁ + DP ₂ j * 84.4 % ° ^P 3 : 6.1 % ^DP 4 : 3.7 of / Q ^DP 4 ₊ : 5.7 %

NSI - Nitrogen Solubility Index DSI = Solubility Solubility Index

Table Ba II Stability test of the reaction mixtures

Relationship search

stability test

without oil

With 0Γ

Amount of dispersion Dispersion Emulsion Emulsion Substrate 1st day 2nd day 1, day 2, day » % ( w / w)

A E / S = 0.5 % 80 % 63 % 100% 87 % B E / S = 2, O % 66% 35 % 85% 71%

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Ba 2. Preparation of raw materials containing saccharose starch

In the context of saccharification of cassava and sweet potatoes and other starchy vegetable materials, the addition of a SPS ase formulation will solve viscosity problems. When using a SPS ase preparation, it is possible to prepare starch suspensions with a solids content of 25 to 30 % , and after the saccharification, the mash can be fermented, whereby a cheap ethanol can be obtained *

Example Ba 2 «!

On the basis of fresh and roasted sweet potatoes (Japanese), a massecuite having a solids content of 24 % was prepared * The starch content of the sweet potatoes was about 70 % of the solids content. A pre-liquefaction using the bacterial Aroylase of Termamyl 60 L ^ at a dose of 0.5 kg / tonne of starch was carried out by heating the slurry to 90 C »The mash was then maintained for 30 min at 90 ⁰ C. The viscosity y \. of the reaction mixture was then measured by means of a Haake spindle at 90 ^° C. *

Subsequently, the Reaktionsgeraiseheh was cooled to 55 ⁰ C and the pH with 2n H ₂ SO. set to .5.0 »Then, saccharification was initiated by adding the glucoarasease SAN 150 (tradename) at a dose of 1.75 L / t starch.» The saccharin mixture was then divided into three parts A, B

63 376 12

and C, which were treated with enzyme, as noted below, before enzyme viscosity was measured.

A: This part was for comparison. The viscosity η ₂ was measured, see Table Ba III.

B: Celluclast's Tricoderma viride cellulase was added at a dose of 1 kg / t solids of sweet potatoes. The viscosity was measured, see Table Ba III.

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

Reaction viscosity Viscosity

at 90 ^° C. at 55 ^° C.

pre-liquefied h ₁ = 770 mPa.s Ί? ^{= 2i9} ° mPa.s sweet potatoes

A - * l ₂ = 2190 mPa.s

B - ^ 3 ^{= 1970 mPa} * ^s

η ₄ = 950 mPa «s

From this table it can be seen that the viscosity of the reaction mixture can be substantially reduced by the SPS ase in a small amount, compared to the

Celluclast KrJ and SAN 150.

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- 81 Ba 3. 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 process can be applied to other similar fruits, eg, apples *

Example Ba 3 »!

Fresh apples were coarsely ground using a Bucher central mill. The apple mash was then pasteurized in a tank with heating jacket 5 min at 90 ⁰ C and then cooled to room temperature »verraaischten apples were then ground to a Fryma Mill Korundstein * to the mash was smooth · The mash was then again for 10 minutes at 80 ⁰ C pasteurized and cooled to 50 ° C »

Enzyme reactions were performed Oetzt 30 min at 50 ⁰ C with a Con-traves Rheomat 15, wherein stirring and viscosity measurements were carried out (in the context of a percent reading on the rheometer at speed 13) at the same time. After the enzyme reaction was complete, 100 g of P _r were withdrawn overhead and centrifuged in a calibrated tube for 15 min. At 3000 g. _# Here, the percentage of juice and the percentage of precipitates or solids were measured. The pH and the percentage of refractometer solids as ° Brix were also measured * Table Ba IV shows a comparison between the

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Effect of SPS-ase, the combination of Celiuclast and SPS-ase and the combination of Celluclast and Pectinex »The SPS-ase preparation KRF 68 was applied

Table Ba IV Results of the total liquefaction experiments

with apple mash at 50 ^° C., 30 min

PLC ase CelluclastCf) Pectinex C 200 1 3x g / hl 0 9 final ViS centrifugation 41 Saf t g / hi mash g / hl mash 0 kosity % Juice % solids 19 pH 3rix O 0 0 100 59 21 3.8 9.7 25 0 0 19 81 17 3.5 10.5 50 0 15 79 17 3.5 10.7 50 50 200 4.8 83 17 3.5 10.8 2000 3.8 83 18 3.1 12.5 0 50 9.5 83 3.2 12.9 0 50 4.σ 82 3.1 13.2

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 treating various fruits. Berries and vegetables, e.g. 8, carrots, peas, tomatoes »apples, pears, black currants , beans and cabbage. In this case, a better yield of juice and a better extraction of color and Geschraacksstoffen is achieved »compared with the commercial pectinase and

63 376 12 - 83 -

Cellulase preparation _e Example Sa 4 * 1

Reference is made to Example 3a 3, in which the SPS ase preparation has been compared with the commercially available cellulase and pectinase products Celluclast® 200 L and Pectinex® 3x *. The table shows that the yield of Juice can be easily improved if as little as 50 g / hl of SPS-Ase are used, compared to 2000 g / hl of Pectinex ^, both together with 50 g / hl of Celluclast ^. Also, the viscosity was 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 shown that it is possible to improve the yield in relation to simple extraction process when the PLC-ase preparation is applied before the treatment of sugar cane or sugar beet and / or during aer 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.

63 376 12 - 34 Example Ba 5 * 1

10 kg 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-IIO). "Process water was added during the milling process.

300 g portions of the pulp was now 18 h at 45 ⁰ C with enzyme treated using the amounts indicated in Table Ba V, the dry enzyme product (KRF 68) was added to the pulp, which was agitated during the first hour with a rod. 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 made on reaction mixtures and on the supernatants , Based on these results, the percentage was calculated dissolved solids, corrections for the content of soluble solids of the enzyme product were carried out in all calculations.

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

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Table Ba V Results in the enzymatic liquefaction of beet pulp

No. of Adhesives No., Enzyme amount to solids E / S %

End measurements

reaction

supernatant liquid

pH

(Full scale)

% Solids

% Solids

dissolved solids

α '/ ο

1 O 5.5 4.18 0.0 0.0 2 0.35 3.6 3.85 2.58 66.9 3 0.56 3.5 3.81 2.56 66.2 4 1.02 3.5 3.86 2.73 70.4 5 1.58 3.3 3.17 2.34 73.4 6 3.10 3.4 3.23 2.49 76.4 7 7.52 3.4 2.66 2.18 80.5

Reaction conditions:

H = 300 g

S = 4.18% solids

E / S as indicated above

pH not adjusted

T = 45 ^° C.

t =

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Table Ba VI HPLC values

Experiment No. 2 3 43.6 31.9 4 Sugar-Art % neutral sugar 4.6 4.8 (Neutral) 20.4 23.7 25.3 5.0 5.9 - High molecular weight (DP4 +) 26.4 32.2 27.8 disaccharides not measured • 7.3 glucose 33.2 galactose Fructose / arabinose galacturonic

All sugars resulting from Table Va VI above could be fermented to alcohol or used for other purposes.

Sa 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 produced by soaking soybeans in boiling water, grinding the soaked beans and extracting with water and then separating the insoluble residue, ζ * B, from proteins and polysaccharides. To improve the yield of soymilk, these insoluble residues can

63 376 12

be liquefied by reaction with the SPS ase * Example Ba 6.1

The soymilk method is illustrated by the following series of enzyme reactions, with protein solubility index (PSI, %) and solubility 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:

Full fat soy flour

(Dansk Sojakagefabrik A / S) Amount of the reaction mixture: 220 g Amount of substrate: 20 g Temperature: 50 ⁰ C pH: 4.5 (6n HCl) Reaction time: Series AIh Reaction time: Row B 0,5 -6h Enzyme: PLC-ase (KRF 68) Amount of enzyme: Row A: E / S ratio

(W / w) O - 8.0 row B: E / S ratio (w / w) 1.0

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

63 376 12 - 88 -

8a 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. For example, Z, B, spent coffee residues (coffee grounds) or green beans can be treated enzymatically with favorable results »

Table Ba VII

string

Calculation of the quantity balance in connection with the enzymatic soymilk process

Reaction rate of enzyme

reaction

supernatant liquid solubility indices

time h E / S% % Protein % Solids % Protein % Solids PSI % DSI % CU 1.0 O 3.65 8.70 1.87 5.72 49.6 63.7 KQ • I ₁ O 0.5 3.68 8.74 2.41 6.28 63.7 70.0 I A 1.0 1.0 3.71 8.78 2.48 6.43 65.1 71.4 1.0 2.0 3.78 8.86 2.98 7.18 77.5 79.5 1.0 4.0 3.92 9.03 3.36 7.69 84.6 83.9 1.0 8.0 4.19 9.37 3.76 8.08 88.5 85.0 cn 0.5 1.0 3.64 8.78 2.39 6.41 64.0 71.1 W C / l 1.0 1.0 3.64 8.78 2.56 6.60 68.7 73.4 Nj cn B 2.0 1.0 3.63 8.78 2.79, 6.91 75.2 77.1 to 4.0 1.0 3.63 8.78 3,1O ₁ 7.29 84.0 81.7 6, ο 1.0 3.63 8.78 3.39 7.69 92.3 86.5 I

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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 should be clear and which have been previously treated with usual pectinase and cellulase preparations »to prevent the formation of haze," fog "may occur. It has been found that the SPS ase preparations are well suited for the degradation of such veils consisting mainly of araban bound to proteins.

Example Bb 1

Pear juice concentrate, prepared by enzymatic liquefaction of residues from the preparation of canned pears using Celluclast® and Pectinex®, became cloudy on standing. The "veil" was isolated and hydrolyzed with 0.01% H-SO 2 for 24 hours analyzed by HPLC. The chromatogram showed arabinose and small amounts of oligosaccharides.

By incubation of 0.5 % (wt / vol) of this carbohydrate in 1 mmol acetate buffer, 3 h at pH 4.5 and 40 ⁰ C with SPS ase (KRF 68 and KRF 92 1: 1) with an Enzyrakonzentration 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 added 2 h

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at 40 ⁰ C with an enzyme amount of 0.15% (Gevv./Vol ") of the SPS-ase or with 1% (wt./vol.) mentioned above, a commercial product" which is called Clarex ^ treated. It was found that the SPS ase was able to reduce the relative HPLC peak area of an araban-like veil by 86 % while the corresponding reduction with Clarex (in a much higher amount than the SPS ase preparation) was only 78 %. scam,

Bb 2. Application as a clarifying agent for white wine

It has been shown that white wines "which show a" very undesirable turbidity, can be effectively clarified with the aid of SPS ase. It has been shown 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 described in US Pat. No. 4,100,024 or in Process Biochemistry, 8d, 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.

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Bb 4, mash enzyme for the brewing industry

In the production of beer, carbohydrates of the starting materials z, B. the S-glucans malt and barley affect the viscosity and filterability of the extract. "The addition of SPS ase during mashing reduces the viscosity of the extract and improves the filterability and the yield of extract »In addition, the addition of SPS-ase during the mashing process improves the fermentability of the extract and the nitrogen content in the extract»

Example Bb 4 »!

In the laboratory, 50 g milled grains were "consisting of 50% malt and 50% barley with 275 g water (15% solids content) according to the following mashing diagram mashed: 52 C (60 min) / 63 ⁰ C (60 min) / 76 ⁰ C (30 min)"

To demonstrate the effect of SPS ase, four experiments were carried out »see the table below where the enzymes were added during the mashing process (pH of the mash 5 _# 5 to 6 _t O)»

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enzyme

no cereflo

PLC-ase (KRF 68)

Activity of B-glucanase / g 0 200 BGU 1630 FBG 1630 FBG Amount of enzyme per kg grains 0 1 * 5 G 0.05 G 0 * 18 G

Total amount of enzyme activity units 0 per kg of grains

300 BGU

80 FBG 300 FBG

Filtration rate 120 ml 135 ml of the extract after 10 min

160 ml 170 ml

Viscosity of the 1.52 extract 10 balls (25 ° C)

mPa.s

1 * 36 snPa.s 1.36 mPa.s 1.30

mPa.s

3GU = β-glucanase units, determined by the analytical method AF 70/4-GB. available from NOVQ Industri A / S.

FBG = fungal β-glucanase units, determined by the analytical method AF 70.1 / 2-GB ₄ available from NOVO Industri A / S,

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

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Cereflo is a bacterial ß-Glucanasezubereitung, as described in the information sheet B 214b GS-1500 July 1931, available from NOVO Industri A / S,

Example Sb 4.2

In the laboratory, 50 g of ground grains consisting of 40% malt and 60% barley with 150 g water (25% solids) were mashed according to the following scheme: 45 C (60 min) / 63 ⁰ C (90 m × n) / 75 ⁰ C (15 minutes).

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

enzyme

none

SPS-Ase (KRF 68) + Ceremix as before

rprom · ν ν attempting

Leremxx _train ESpecify

Activity of β-glucanase / g

Enzyme amount per kg grains

Total amount of enzyme activity units per kg of grains

Filtration speed of the extract after 30 min

Extract * ° Balling

Viscosity of the extract 10 ° Balling (25 ⁰ C)

48 ml

200 BGU

1.65 g 330 BGU 98 ml

18 "6 19.0 1 72 1.37 Wed. mPa.s

1630 FBG 0.033 g

50 FBG

111 ml

19.5 1.27

63 376 12 - 95 -

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

Ceremix is a bacterial S-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 to reduce the content of β-glucans and thereby improve the filterability and stability against fogging of beer. SPS-ase also has an effect on proteins that are responsible for the turbidity that occurs during cooling *

8b 6. Means for easier removal of almond skins

During the mechanical removal of almonds after blanching almonds _f , a certain proportion 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

63 376 12 - 96 -

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 from Z, B, apples, pears or citrus fruits. It has been shown "that a SPS-asa preparation is capable of completely liquefying these carbohydrate-containing waste products and forming fermentable sugars" which can be used as starting material for ethanol fermentation,

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

10 lbs. Of whole soybeans were ground simultaneously with 70 l of boiling water in a Fryma type ΜΣ 110 grinder. The milled slurry was then held 15 min above 85 ⁰ C "to inactivate the natural enzymes beans which produce the known soybean flavor. 5 liters of this soybean slurry were 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 %) . 6N HCl was slowly added and worked into the residue with a spatula until a pH meter showed 4.50 when the electrode

  63 376 12 - 97 was immersed directly in the mass,

In a 500-ml beaker Giss enzyme reactions were performed on 2 χ 200 g of the composition with two different amounts of SPS-ase (KRF 68) carried out at 50 ⁰ C, namely E / S = 0.5% _t based on the solids, and E / S = 3 _t 0% _s based on the solids. The dry enzyme was added to the wet. The mixture was stirred with a spatula for the first 1 to 2 hours, and then the amount was liquefied so far that it was possible to stir successfully with a magnetic stirrer. The total reaction time was 21 h. During the Reactxor /. the osmolality measured with an osmometer. The results in Table Bc show the course of the reaction.

At the end of the experiment, the mixture was centrifuged at 3000 g for 15 min. On the surface of the supernatant liquid, an oil layer appeared * whose volume was determined. The soil layer was a loose mud layer. The supernatant, including the DI, was removed with a pipette. The oil was added to the clear aqueous phase by homogenization and a sample was withdrawn to determine the solids content. 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. Mach recovery of the oil, the dissolved residue can be used in various ways, eg. 8, for fermentation to valuable compounds or for concentration and drying and subsequent use as a feed or food product or after further purification for the production of valuable products.

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- SS -

Table 3c I Results obtained in the liquefaction of soymilk and soya quark

Reaction conditions and results

Trial A

Trial B

Amount of the residue t "min 200 g G C min 200 g ) Δ Os molarity mOsm 8 - 10 % Amount of PLC-Ase (KRF-68) 0 0.20 O 1.20 c O 93.5 % temperature IO 50 ° 10 50 ⁰ C 86 pH 40 95 4.50 Λ Osmolality mOsm 25 45 95 4.50 215 319 reaction time 250 21 h O 250 21 h 436 Results ranged with the osmometer during the reaction 1260 Osmo lali it mosmos 26 1260 Osmo lali it mosmos 593 287 104 214 282 863 313 347 368 O Osmolality is d * corrected value for the osmolality of the mixture at t = 0 391 501 620 497 601 718 634 % 875 t = o 907 1145 Reaction mixture solids 20.3 % 20.7 % supernatant liquid Q / speed: solids 18.0 % 19.4 % Upper liquid: oil content 8-10 Calculation % Soluble solids 88.6

53 376 12 - 99 Sc 2. Saccharification and simultaneous fermentation

Carbohydrate-containing plant materials, e.g. B, tubers, such as Jerusalem artichokes, potatoes, sweet potatoes. "Cassawa or pulp of such tubers, d. h, 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.

Example 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 SPSase and inulinase and four different pretreatments of Jerusalem artichokes.

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

Inulinase: The inulinase was produced by fermentation of Asp. Ficuum (CBS 55 565). The inulinase activity was determined as indicated on S, 99 of Research Disclosure No. 21,234 (December 1981) pp. 456-458.

Fermentation in the laboratory: 150 g portions of the pretreated mash (described later) were fermented as an antifoam agent after addition of 4.5 g baker's yeast and 1 ml of a 4% solution of Pluronic »The fermentation flasks were with COp traps containing 98 % sulfuric acid» provided and

63 376 12 - 100 -

the fermentation was monitored by measuring the weight loss due to released CO ₂ . The flask contents were stirred during the fermentation carried out at 30 ° C. Three flasks were used for each parameter investigated.

In Table Bc II, the weight loss due to release of CO _{2 is} converted to ethanol », assuming that 1 liberated CO 1 mol C ₇ H ₁ -QH, ie 1 g CO

~ g C ₂ H ₅ OH corresponds. Pretreatment of artichokes:

Treatment A: 14.1 kg of artichokes (22.8 % solids content) were cooked for 20 minutes at 140 ⁰ C and 4 to 5 bar (Henze). The weight after cooking was 19.0 kg (^ 16.9 % solids content). The fermentation was carried out directly on the mash.

Treatment B: Washed and sliced artichokes were mixed with water (1: 1) in a Waring blender. The mash was then treated for 1 h at 85 ^° C. and pH 4.5.

Treatment C: Same as B but without pH adjustment.

Treatment D: Same as S, but without heat treatment and pH adjustment.

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

Table Bc II

Fermentation results related to simultaneous fermentation and enzymatic saccharification of Oerusalera artichokes

Preventive action Add inulinase to 1 g of solid PLC-ase E / S % Loss after 42 Fermenta at CO (g) - 44 Π tion 0 / / O produced ethanol in relation to solids A 1 * 5 1.5 0 0.27 7.65 + 8.07 pounds 0.05 0 3.08 31.5 33.2 1.5 0 4 * 85 + _ 0 * 03 29, 7 3 1.5 0.40 5.41 +, 0.03 33.1 1 * 5 0 5 * 70 i 0.05 34.8 1.5 0.10 5.97 + _ 0.01 36.5 C 1.5 0.20 6 _f 13 +, 0.06 37.5 1 * 5 0.30 6.13 ± 0.00 37.5 1 * 5 0.40 6.18 ζ 0 _Λ 05 37.8 1.5 0 £ 5.77 0.02 35.3 1.5 0, 10 5, S9 +. O ₁ OO 36.0 1 * 5 0.20 6.04 ± 0.11 36.9 D 1.5 0.30 6.01 +, 0 * 01 36.7 1 * 5 0.40 6.02 ± 0.03 36.8 0 0.40 ' ⁵ * ^4S ± 0.02 33.5

63 376 12 - 102 Sc 3 » Degradation of cellulose

It has been found that cellulosic materials such as straw, 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) was treated with the aid of a common cellulase Celluclast® 200 produced by Trichoderma reesei and the SPS-ase preparation KRF 68.

Example 3c 3 «!

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

Table Bc III

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

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Bc 4 « Application as baking aid

It has been found that SPS ase preparations are excellently suited as baking aids. Thus, when a SPS ase preparation is added to the dry flour prior to making a dough, it is possible to have a bread of better quality in terms of volume _t preserving crumbs and flavor «It is thus 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 « Improving the yield of alcohol and biomass

during the fermentation of.Sulphitlauge of the

papermaking

It has been found 'that the yield can be improved to ethanol when Sulphitlauge before it is used as a carbohydrate source for the fermentation of ethanol is treated by the paper-making with an SPS-ase preparation, Sulphitlauge of paper making can also be used to prepare of biomass * z. B. 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.

63 376 12

Sc 6. Drainage of biological sludge products

During the usual water extraction of many biological materials from plant raw materials, large volumes of insoluble residue, consisting of large amounts of swollen polysaccharides, are produced. This is the case, for example, when soymilk, soya quark or soy isolate are produced by water extraction from 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 consequently obtains 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 offers the possibility of producing a cheaper dry animal feed or bulking agent for animal feed,

3c 7, silage aid

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.

63 376 12 - 105 -

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

Fig. Refers to no.

represents

general part of the description binding effect between SPS and soy protein

general part of the description Flowchart describing the production of SPS

Section 2 calibration curve for HPLC gel filtration chromatography

Section 2 HPLC gel filtration chromatogram of SPS

Section 2 HPLC Gel Filtration Chromatography of SPS Ase degraded SPS

Section 2 and 3

HPLC gel filtration chromatogram of supernatant from SPS incubated with soy protein

Section 2 HPLC gel filtration chromatogram of supernatant fluid from degraded SPS incubated with soy protein

63 376 12

Fig,

No.

refers to

represents

Section 3

HPLC-gel-filtration chromatogram of APS ₁ degraded by pectolyase

Section 3

HPL CG el-ration s-chromatogram of APS _t degraded by SPS-ase "

Section 3

HPLC gel filtration chromatogram of SPS treated with pectolyase

11-

Section 7

Immunoelectrophoretic peaks including a SPS ase peak identified by overexposure technique

12 Section 8 Ion exchange chromatogram of a SPS ase 13 Section 8 pH-dependence of the activity of a PLC-ase 14 Section 9 Teraturature 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 (11)

1 .. Method of decomposing polysaccharides, preferably cell wall polysaccharides of plants by means of a Garbohydrase, characterized in that a 3P3-ase forming microorganism, which is selectable in that the su examining microorganism is sought on a fermentation medium, the main carbon source SPS, the 3a3is of vegetable crude red wine, preferably defatted soybean meal, as starting material, whereupon a sample of the fermentation medium is analyzed for SPS-ase and the microorganism in question is considered to be a SPS-ase forming microorganism, if the analysis is based on SPS is positive in a medium, after which the 3PS-ase preparation prepared therefrom is brought together in an aqueous medium with a substrate for the SPS ase preparation. 2. The method according to item 1, characterized in that the SPS-ase is able to degrade SoJa-SPS in an aqueous medium su degradation products that attach themselves to vegetable protein in the aqueous medium to a lesser extent than the SoJa-SPS ago associated with degradation to the same plant protein in the aqueous medium. 3. The method according to item 1 or 2j, characterized in that the SPS-ase able to degrade SoJa-SPS in an aqueous medium having a pH value which does not deviate more than 1.5 from 4.5 to degradation products which attach themselves to soy protein in the aqueous medium to a lesser extent than the SoJa SPS would have attached to the soy protein in the aqueous medium prior to degradation. 4. Method according to item 1 to 3y characterized by 63 376 12 - 108 - that the decomposition products of soybean SPS after complete degradation attach themselves to vegetable protein to the extent of less than 50 %, in particular less than 20 % , when the soybean SPS had accumulated in the aqueous medium before degradation to the vegetable protein , 5. Method according to items 1 to 4 ,. characterized in that the SPS-ase gives a positive SPS-ase-test _t if it is examined according to the qualitative and quantitative SPS-ase-3estimmungsverfahren. 6 »method according to item 1 to 5», characterized in that the SPS ase has been formed with the aid of a microorganism * belonging to the genus Aspergillus, preferably to the species Aspergillus niger. 7. Method according to items 1 to 6 *, characterized in that the SPS ase is derived from the enzyme which can be formed by means of Asp aculeatus CBS 101,43. 8 "method according to item 1 to 7 _t characterized in that the SPS ase is immunoelectrophoretically identical to the SPS ase» which can be formed using Asp, aculeatus CBS 101.43 and identifiable by means of an immunoelectrophoretic Oberlagerungsverfahrens. 9. A process for the degradation of polysaccharides according to item 1, characterized in that the degradation is carried out by isolation or extraction of a biological component other than soy protein and related plant 63 376 12 - 109 - from a biological raw material, the SPS ase preparation being substantially free of any enzyme capable of degrading the biological material ♦ 10, process for the degradation of polysaccharides according to item 9 or Ί * characterized in that one or more reaction ProductsI), regardless of whether they are _t desired end-products or waste products "are further treated simultaneously with or after the Enzyrabehandlung, 11 «Process for the degradation of polysaccharides according to item 10, characterized in that the further treatment is an alcoholic fermentation» if one of the reaction products is a fermentable sugar »