CA1198700A - Enzyme for decomposition of a high molecular carbohydrate, the isolated high molecular carbohydrate, a method for selection of a microorganism producing such enzyme and a method for production of such enzyme - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

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

Description

.

SPS-ase 2390 ~ V~i~l~ in and rela~ing to an enz~me for decomposition of a high molecular arbohydrate, the iso~
lated high molecular carbohydrate, a method fox selec~ion of a microorganism producing such enzyme and a method for pro-duction of such enzyme.

A method for production of a purified vegetable protein product (p~p) by enzymatic removal OI the remanence, without dissolution and reprecipitation o~ the protein, is described in BE patent No. 882.769. The purity of the pvp obt2i~able by the known methoa is not satisfactory and there-fore open to improvement. In the examples a purity; of the pvp of about 85~ was demonstrated. Even if it is possible to obt~in a pvp ~f a~out 90% purity according to the Xno~m method; this is only obt~inahle with certain pretreated starting materials, e.~. soy protein concentrateO It would be d~sirable to be able to o~tain a purity of the pvp of around or above 90~ with a much broader spectrum of starti~g materials, especially dehulled and defatte~ soy meal.

The invention is based upon th~ surprising dis-co~ery that a certain part of the remanence decomposition product, as it appears during the enzymatic treatment indicated above, i.e. a water soluble, high mole~
cular carbohydrate, attaches itself to part of the v ~e~able protein, as will be explained later i~ detail. ~his, of course, results i~ a lower purity o~ the protein. Also, it h~s ~een lound that this hi~h ~olecular carbohydra~e has ~n ability to atkach itself to proteins of ~; m~l origin.
~i Thus, an object of the i.nvention is to provide an enzyme for decomposition of the above indicated high molecula~ carbohydrAte, which will open up the possibilit~
for produc~ion of a pvp with improved purity, and a method for production of such an enzyme.
In the accompanying drawings:
FIGUR~ a diagrammatic process flow sheet, ullustrating the background and context of the present invention;
FIGURE 2 is a diagrammatic flow sheet illus~rating the production of SPS;
FIGURE 3 is a calibration curve for HPLC gel filtration chromatography;
FIGURE 4 is an HPLC gel filtration chromatogram of SPS;
FIGURE 5 is an HPLC gel filtration chromatogram of SPS decomposed by SPS-ase;
FIGURE 6 is an HPLC gel filtration chromatogram of supernatant from SPS incubated with soy protein;
FIGURE 7 is an HPLC gel fi1tration chromatogram of supernatant from decomposed SPS incubated with soy protein;
FIGURE 8 is an HPLC gel filtration chromatogram of APS decomposed by Pectolyase;
FIGURE 9 is an HPLC gel filtration chromatogram of APS decomposed by SPS-ase, FIGURE 10 is an HPLC gel filtration chromatogram of SPS treated with Pectolyase;
FIGURE 11 is a graphical represe~tation of immunoelectrophoret.ic peaks including an SPS~-ase peak identifiea by overlay technique;
FIGURE 12 is an ion exchange chromatogram of an SPS-ase;
FIGURE 13 is a graphical representation of ~he pH
activity dependency of an SPS-ase;
FIGURE 14 is a graphical representation of the tempera~ure activity dependency of an SPS-ase;

- 3a -FIGURE 15 is a graphical representation of the temperature stability of an SPS-ase;
FIGUR~ 16 is a graphical representation of the pH
stability of protease in an SPS-ase preparation.
The basis Eor the invention can be described in the following manner, reference being made to fiy. 1, in which only material e~isting as undissolved solids is indicated, whereas all supernatants are left out. A charge of soy meal was divided in two equal parts, part I and part II lcolumn a in fiy. 1).
Part I was decomposed proteolytically at a pH ~alue of about 8 by means of ALCALASE* (a proteolytic enzyme produced by means of ~. licheniformis and marketed by NOVO INDUSTRI A/S, 2880 Bagsvaerd, Denmark), and then further washed at around pH 8 in order to eliminate the entire amount of protein, and the remanence was separated from the supernatant and washed (vide part I, column a and b, fig. 1). In this way a pure remanence (designated remanence I) was isolated (column b in fig. 1).
Part II of the soy meal was not treated; for the sake of brevity the remanence in part II is designated remanence II (column b in fig. 1). Now, both remanence I and part II are decomposed by means of a commercial pectinase, e.g. PECTINEX* (a pectolytic enzyme produced by Schweizerische Ferment A/G, Basle, Switzerland) (vide column b and c in fig. 1). Surprisingly it is found that the undissolved part of remanence I is much smaller than the undissolved part of remanence II, on the basis of nitrogen and dry matter mass balances, vide fig. 1, ~here the hatched areas in column c correspond to insoluble non-protein materials in the above indicated stage. Furthermore, if the supernatant from the pectinase treated remanence I is brough~
together with a soy protein suspension at pH 4.5, a polysaccharide in the supernatant i6 bound to the soy protein.
This polysaccharide in the supernatant ~rom remanence I, which is a part of the remanence decomposition product and which is clearly soluble in water in the absence of soy protein~ but *Trade Mark 3~7~

bound to soy protein at or around the isoelectric point of soy protein, i~ soy protein is present, is designated SPS (Soluble Polysaccharide), _ide figO 1. The SPS has a molecular ~eight distribution between 5 x 10~ and 4.9 x 10~. The production of isolated SPS appears from the flow sheet shown on fig. ~, which also encompasses some of the processes depicted in fiy.
1. Thus, the problem is to find an enzyme which is able to decompose the 5PS in such a manner that the SPS decomposition products do not bind soy pro-tein or do bind soy protein to a much lesser extent than SPS binds soy protein.
Although the disclosure specifically refers to soy protein, the invention is not restricted to soy protein, but encompasses all kinds of vegetable proteins, vide e.g. the proteins listed in BE patent No. 882.769, page 1.
Now, according to the invention it has been found that by screening for the ability of decompose soy SPS it is possible to select microorganism which are able to produce a compound which exhibits an enzymatic activity effectively decomposing soy SPS, in the following for the sake of brevity designated an SPS-ase.
Thus the invention in its first aspect comprises an SPS-ase, a carbohydrase in a usable form and capable of decomposing soy SPS under appropriate conditions into decomposition products which attach themselves to protein in an a~ueous medium to a lesser extent than the soy SPS pxior to decomposition would have attached itself to the same protein under corresponding conditions.
Furthermore it has been found that ~his SPS-ase capable of degrading soy SPS is able to degrade polysaccharides similar to SPS and originating from vegetables and fruits more completely than commercial pectinases and commercial cellulases.

- 4a -The above indica~ed expression "in a usable form" is intended to exclude rom the invention e.g. an 5PS-ase containing preparation which contains ~oxic substances or which exhibits such low SPS-ase activity, which necessitates the use of more than 10% SPS-ase containing preparationl related to the weight of SPS in the substrate, in a reaction conducted for 24 hours and at 50 C, at the pH optimum of the SPS-ase in question, in order to obtain a decomposition of SPS of any practical importance.

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By to~al or partial elimination of the SPS from the final ~egetable protein the purity of the final vegetable pro-tein necessarily is improved in comparison with the purity of the final vegetable protein obtain2ble accord~ng to the method .nown from BE patent No. ~82.769, as this known vege,able pro-tein product was contaminated with SPS.

At present it is not known if the particular SPS-ase described in the following derives its enzymatic activity from a single enzyme or from an enzyme complex comprising at least two enzymes. Some investigations seem to indicate that at least two enzymes zre responsible for the SPS-ase degradation effect, O whereby o~e of these enzymes is capable of carrying out only a slight decomposition of SPS, whereafter one or more enzymes are able to perform a more extensive degradation of the SPS. The applicant, however, does not want to be restricted by such hypo-thesis or similar hypotheses.
A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS~ase is capable of decompsing soy SPS in an aqueous medium into dacomposition products which attach themselves to vegetable protein in the aqueous medium to a lesser extent than the soy SPS prior to decomp~sition would have attached itself to the same vegetable protein in the aqueous medium.

A preferred em~odiment o, the SPS-ase according to the invention is characterized by the fact that the SPS-ase is capable of decomposing soy SPS in an aqueous medium with a p~ value not deviating more than 1.5 fro~ 4.5 into decom-position roducts which attach themselves to soy protein in the aqueous medium to a lesser extent than the soy SPS
prior to decomposition would ha~e attached itself to the soy protein in the aqueous medium.

A preferred embodiment o the SPS-ase according to the invention is characterized by the fact that the 7~

decomposition products of soy SPS after completed degradation attach theMselves to the ve~etable protein to an extent of less than 50%, particularly le~s than 20~ than the soy SPS prio~ to decomposition would have attached itself to the veyetable protein in the aqueous medium.
A ~ e~bodiment of the CP~-ase acco*ding to the invention is characterized by.the fact that the SPC-ase eY.-hibits a positive SPS-ase test, when examined according to the qualitati~e and ~uantitative SPS-ase determination method describQd in this specification.

A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS-ase was produced by means of a microoraanism belonging to the senus Aspergillus, preferablv belonging to the As-pergill.us niaer group.
A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS-ase is derived from the enzymes Droducible by means of Asp. aculeatus CBS 101.43. The same SPS-ase can be produced by mezns of ~. japonicus IFO 4408. It has been foun~ that As~. aculeatus CBS 101.43 also produces very potent remanases, cellul.~ses, pectinases, and hemicellulases.
Furthermore, it has been found than not each and every str.ain belo~aing to the species As~ aculeatl~s or As~
nicus generates an SPS-ase needed for the invention. Thus~
as it a~pears later in this s~ecification-(section 5), it has been demonstrated that the strain As~. ja~onicus ATCC
20236 does not produce such amounts of an SPS ase which can be detected by means of the enzymatic determination of SPS-ase described .in this specification.

~ preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS-ase is ~mmunoelectrophoretically identical to the SPS-ase producible by means of ~. aculeatus CBS 101.43 and identifiable by means of the immunoelectrophoretic ovex-lay technique, vide section 6 and 7 When an SPS-ase is produced microbially, it is forrned in admixture with several accompanying substances, particularly other enzy~es. If desired, the SPS-ase in ouestion can be purified, e.g. by ~eans of chromatographic separation methods, as will appear later in this specifi-cation (section B).

In Agr.Biol.Chem 40 (1), 87 - 92, 1976 it is de~
scribed that a strain or ~. japonicus, ATCC 20236, pr~duces an enzyme complex which is able to perform a nartial de-gradation of an acidic polysaccharide in soy sauce, n~med APS, a fraction of which is designated A~S-I. ~his acidic polysaccharide is not identical to SPS, ~Ihich will be shown later in .his specifi~ation in more detail in sec-tion 3, Thus, the ~P~C gel filt.ation chroma~ogr~ms of SPS and APS are clearly di ferent, and furthermore, the gel filtration d~ ~k~ s o APS decomposed by means of the commmercial pectinase Pectolyase and of SPS
~reated with the comm~rcial peckinase Pectolyase are elearly differen~. ~urthe-rmore, it does not appear from the article that the acidic polysaccharide is bound to the soy protein and that the de-omposed acidic polysaccharide is not bound to the soy protein or is bound to the soy protein to a much lesser extent than the undecomposed acidic poly-saeeharide. Also, it has been demonstrated that this strain does not form SPS-ase in such amounts which ean be detected by means of the enzymatic determination of ~PS-ase deseribed in this speeification. This ereated a prejudice aaainst any strain of ASD . japonicuS being a producer of an SPS-ase, but surprisingly according ~o the invention it has been found that some strains of Asp. ja~onieus are producers of an SPS-ase.

The invention comprises in its second aspect the isolated SPS, pro~uced on the basis of ~egetable raw protein as a starting material.

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A preferred embodiment of the isolated CPS accor~in~
to the invention is ch2racterized by the fact that the vegetable raw protein is defatted soy meal. The production of this isolated SPS is described in the foregoing i~ rela-tion to figure 2.

The invention comprises in its third aspect a method for selection of an SPS-ase producing microorga-nism for production o the SPS-~se according to the in-vention, wherein the microorganism to be tested is grown on a fermentation medium, the main carbon source of which is SPS, whereafter a sam?le of the fermentation medium is analyzed for SPS-ase and the micro-organism in question is selected as an SPS-ase producing microorganism, if the analysis for SPS-ase is positive.

The invention comprises in its fourth aspect a method for production of SPS-ase, wherein a strain se-lectable according to the above method of selection of an SPS-ase producing microorganism is cultivated in a nutrient medium. The cultivation can be carried out as a submerged fermentation or as a surface fermentation~

A preferred embodiment of the method according to the inventioIl is characterized by the fact that the strain AsP. aculeatus C3S 101.43 or Asp. japonicus IFO
4408 is culti~ated in a nutrient medium.

A preferred ~mbodiment of the method according to the invention is characterized by ~he fact that culti-vation is carried out as a submerged cultivation at a p~
in the xange of from 3 ~o 7, preferably from 4 to 5, at a temperature in the range of from 20 to 40C, preferably from 25 to 35C, and whereby the nutrient medium contains car~on and nitrogen sources and inorganic salts.

A preferxed embodiment of the method accordîng to the invention is charac~crized by the fact tha~ the nutrient medium contains toasted soy meal.

~ 9 -A preferred embodiment o~ ~he method according to the invention is characterized by the fact that the soy meal is treated with a proteolytic enzyme before the use as a component of the substrate, preferably the pxoteo-lytic enzyme produced microbially by means of Bacillus licheniformis.

A preferred embodiment of the method according to the in~ention is characterized by the fact that a solution of pectin is a~ded aseptic~Lly to the r~ L~tion broth during the cultivation.

It has been found that Asp. aculeatus CBS 101~43 also produces very potent remanence solubilizing enzymes, cellulases, pectinases, and hemicellulases besides the SPS-ase, and that the enzyme complex produced by means of . aculeatus CBS 101.43 is excellently suited 2S an agent for use in cell wall disintegration of veset2ble materials.
Thus, the enzyme complex producible rom ~. aculeatus CBS 101.43 can be used in the ood processing industr~
for treatment of fruit and vegetable mashes and for clari-~ying and viscosity xeducing purposes in the processing of juice and wine with excellent results; also, it can be used as a dewatering agent ~i. e. an aoent for decom~o-sition of polysaccharides and hence for liberation o, the water bound within the polymeric structure of the polysaccha-xides) in the processing of vegetables.
;

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- 9a -Thus, in its fifth aspect, the invention comprises use of an SPS-ase or a metnod for decomposition of polysaccharides, preferably plant cell wall polysaccharides, by means of a carbohydrase wherein an SPS-ase preparation according to the invention in an aqueous medium is contacted with a substrate for said SPS-ase preparation, except Eor the decompositions already described in Danish patent application No. 5691/81.

I\ I o~Jo ~ 7q 3 ~ - ~

- 9b -Thus, according to the invention it has been found that SPS-ase preparations are valuable ~nzyme preparations for partial or total lique~action or decomposition of several materials, pre-ferably vegetable materials, e.g. fruits, and plant ~Jastes, cont~ng protein , cellulose, hemicellulose, (e.g. glucans, xylan, galactans, and araban) ~m~, p~in, lipids, in~lm, polyphenols, starch, and ~ginates, a~d for purposes related thereto, ~ide tne table shoT~n later in this s~ecification. As examples of such related purposes may be mentioned zll purposes for which commercial pectinases and cellulases are used today. Several examples will be given later in this specification.
In relation to the extraction (isolation) process described in for instance example 2 it is noted that the SPS-ase preparation is essentially proteinase free, due to the fact that the wanted end product, i.e. the protein, otherwise would be degraded. Likewise, if it is wanted to extract (isolate) other biological materials than protein from a raw biological material, the SPS-ase preparation used should be essentially free of any enzyme degrading this other biological material.
Such modified SPS-ase preparations can be produced by selective inactivation of the undesired enzyme, by fractionation, or by other methods known per se.
Thus, a preferred embodiment of the use according to the invention is characterized by the fact that the decomposition is accompanied by the isolation or e~traction of a biological material other than soy protein and related vegetable ;proteins from a raw biological material whereby the SPS-ase preparation is essentially free of any enzyme which is able to degrade said biological material.
Thus, according to the in~ention it has been f~und that ~he modified SPS-ase prepara~ions -(modified in the sense that they are essentially free of enzymes capable of degrading the bioloyical material to be extracted or isola~ed3 are valuable enzyme prepara-tions for extractîon tisolation) of specified biological materials, e.g. starch, lipids, 1avours, colours, and juices, from raw biolo-gical materials. Several examples will be ~i~en later in this speci-fic'~tion~

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, . , -- gc --A preferred embodiment of the use accordinq to the invention is characterized by the fact that one or more of the reaction products (no matter whether they are wanted end products or waste products) are treated further simultaneously with or after t~e enzyme treatment. Hereby a more fleY~ible and adaptable use is provided.

A preferred embodiment of the use according to the invention is characterized by the fact that the further treatment i5 an alcoholic fermentation in case one of the reaction products is a fermentable sugar. ~ereby a simple and cheap method for production of alcohol is provided.

In order to clarify the nature of the invention, reference is made to the following sections 1 - LO~ all describing details related to the invention~

1. Production of SPS.

2. Characterization of SPS, especially molecular weight distribution thereof.

3. Documentation for the fact that SPS and APS are liffexent compounds.

-Screeni~g ~or S~S-ase producir.g microorganisms.

5, C~aracte~zatiPn:p~ some S~S-ase forming micro-org~ n i sm c ~

. General descriptio~ of overlay technique associated with immunoelectrcphoresis.

7. Immunoelectrop~oretic characterization of SPS-ase ~ith polyspecific antibo~y and overlay.

8. Purification of an SPS-ase reparation~

9. pH-activity dependency, t~mperature activity depenaency, and stability of an SPS-ase.

10. Enzymatic ac ivity deter~inations.

SECTION 1. . .

PRt:)DVCTION O~? SPS.

As previously mentioned the starting ma~erial ~or productio~
o~ SPS may be soy remanence. Therefore, in the flrst place, the production o~ soy remar~ence is described.
Soy remanence is the protein free c~rbohydrate fraction (which in Dxactice may contain mi~or amoun~s o lignin an~ minerals~
~n ~Pf~tt~ ~nd ~ Soy m~31, which car~ohycrate fr~ion is ;n~31lhl~
in an aqueous medium at p~ 4.5, and it can pe produced in the fol-lowing manner, reference also being made to flow sheet 1.

Defatted soy meal (Soja~el I3 from ~.arhus.Olie~ri~
~/$~ is suspended in deionized water of 50C in a weight pro-portion soy meal:water = 1:5 in a tank with p~-stat and tem~eratuxe control. p~ is adjusted to 8.0 with 4N NaOH (I). Now a p~-stat h~-drolysis is per~ormed with ~LCALASE 0.6 L (a pro~eolytic enz~e on the basis of B licheniformis wi~h an activity of 0.~ Anson lmits/g, where~y the activity is determined according to the Anson method, as described in NOVO ENZY~ FORMATION IB No. 058 e-GB), whereby the ratio enzyme/subs~ra~e equals 4% of the amount of protein in t~e soy meal (I~). After a hydrolysis of 1 hour the sludge is separated b~ centrifugation (III) and washing (IV) wh2reby this operatio;~ is performed twice (V, VI, VII). The thus treated sludge is hydrolyzed once more for 1 hour with ALCALASE 0~6 ~ ~VIII~ IX) similarly as indicated before. Then the sludge is separated by cen~
trifugation (X) and washed twic~ (X~, XII, XIII, XIV), whereby the final washed sludge (6) is spray-dried ~XV)~ The thus produced spray-dried powder is thP soy remanence serving-as a raw materi21 for the production of SPS.

SPS is the watex solu~le polysaccharide fraction which is formed by conventional treatment of the above indica~.d soy remanence with pectinase. The SPS is produced in the following manner by means o~ the below indicated 14 reac~ion steps~
~ o 2 - lZ -reference also bein~ made to flow sheet 2.

The dry matter content in the above indicated soy remane~ce is determined and the soy remanence is diluted with water to 2~ dry matter and kept in suspen-sion at 50C in a tank with temperature control.

The pB value is adjusted to 4.50 with 6N NaOH.

Pectinex Super conc. L is added in an amou~t of 200 g/kg dry matter (a commercial pectinase from Schweizerische Ferment AG, Bzsle Switzerland with a pec-tinase activity of 750,000 M~U , as determired according to the leaflet "Dete_mination of the Pectinase uni~s on Apple Juice (MOU)'i of 12.5.1981, obtai~able from Schweizerische Ferment AG, Basel, Swi~zerland), and al50 Celluclast 200 L~is zdded i~ an amount o' 20 g/kg dry matter (2 commercial cellulase described in the leaflet NOVO enz~mes, in'ormation sheet B 153 e-GB
1000 July, 19~1, obtainable from NOVO INDUSTRI A/S, No~o Alle, 2880 Bagsvzrd, Denmark).

The contents of the tank is ke~t at 50C during 24 ho~rs with stirring.

5. The enzymes are inactivated by raising the pH ~alue to 9.0 with 4N NaOHO The reaction mixture is kept for 30 minutes, and the pH-value is ~hen re-adjusted to 4.5 with 6M HCl.

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

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

8. The check filtrate is ul~rafiltered~ dia~iltered and ~L`I r~al~ k v~
once more ultrafiltered on 2 membrane with a cut-off value of 30,000 (DDC GR 6G-P fro~ De Dans~e 'ukkerfa~rik~er), whereby the following parameters are used:
.... .

1. ~ltrafiltrztion corxesponding to a ~olume concentra-tion of 6.
2. Diafiltration until the percentage of dry matter in the permeate is 0 (~ 0 Brix).
3. Ultrafiltration ko around 15% dry matter in the concentrate.

The tem~erature is 50C, p~ is 4..5 and the a~erage pres-sure is 3 bar.
-3. The ultrafiltexed concentrate is cooled to 5 C, and an equal volume of 96~ ethanol is added.

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

11. The precipitate is washed twice with 50% v/v ethanol in H~O, corresponding to the volume of centrifugate from step ~ , i.e. two centrifugations are perLormec.

12. The washed ~recipitate is redissolved in water with a volume which equals the volume of the ultrafilte_ed concentrate from step 9.

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

14. . The clear filtrate ~ont~; n; ng pure SPS is lyophilized.

7~
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Defatted soy meal H2O NaOH 'o pH ~ 8 . ¦Hydrolvsis mixture ¦ I

Alcalase 0.6 L ~ydrolysls 1 hour befor& discharge (E/S = ~ p~-stat (pn = 8, T = 50 C) 4M NaO~ 1 ¦lst centrifugation ~ ¦ C~htrlfuate ] > ~-~aste ~l Slud9e 1 ~2 ~ 1st wash ¦ rv ¦ -¦ 2nd centrifugation ¦ V.¦ Cent~ifuaate 2 ) ~aste - ~ Sludoe 2 n2 ~ 2nd wash ¦VI¦

¦3rd centrifugz-ion ¦VII ¦ Centrifuqate 3 ~ ~aste ¦ Sludge 3 . ~-NaO~ to ~H _26;~New hydrolysis ~lxtu-e l V~
Alcalase 0.~ L
~ Hydrolysis _or 1 hour before 4N NaO~ ; dischaOge. p~-stat (p~ - 8.0, IX

¦4th centrlf~gation ¦ X ¦ ~nt~1 f!7a9 P ~ ~aste Sludge 4 ~2 ~ 1st wzsh¦ XI
¦Sth centrifugation ¦XII ¦ Cent~ifu~ate S )~;zste ~ Sludse 5 B2O ~ ~nd wash¦XIII ¦

¦6th cen_ri.usati~n ¦XIV ¦ Ce~t-i-u~ate 6 ~ ~aste ¦ Sludoe 6 Spray-dryin~ j XV

~ 15 -7~7~

1000 g Pectlnex Super conc. L 5 kg spray-drled remanence 100 ~ Celluclast 2'0 S" 1 1 247 1 H 0 Deco~position witn pectinase 2 ~ an~ cellulase 1, 2 3 4, 5 50VC; pH ~ 4.5; t ~ 24 hours ¦Centrlfugation ¦ Slut~e ~
~, 38 kg 6 rCheck filtration ¦ 7 Ultrafiltration, diafiltr2-18 1 R20 _ion, ultrafiltraOtion 241 1 of ger-tGR 60P) (19 - 26 C) meate B
~1, 5 1 50i C2H~0~ ~Precipitation ¦ Supern~nt>g (waste) entrifugatio~ nt~
¦ Precipitate ~2 x washlng ¦ 2 x centrtfu~ate)l 1 Precipltate 2 1.~20 ~ R~sso~ution ¦ 12 ¦ChecX flltration ¦ P~otein sludae > 13 1 (waste) ¦Lyophilization 310 g lyoph~lized SPS

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~CTI ON ~ .

C~ARACT~RIZATION OF SPS, E:SPECIALLY MOLECULAR 1~7EI~T DISTRI-BUTION THEREOF.

By means of gel chromatography on X~LC equipment (Waters pump model 6000, Waters data module 730, and Waters refraotomete~ R 401) the molecular weight distribution of the SPS, the production of which is carried out as indicated in this specification, is determined (fig. 4). By means of the same method also the molecular weisht distribution of the decomposition ~roducts of SPS by means of ~PS-ase has been determined (fig. 5). Furthexmore, by means of the same method the bindina effect between soy protein 2nd SPS (fig.6 ) and the absence of binding effect between soy protein and SPS
decomposed by means of the agent according to the invention (fig. 7) has been demonstrated.
The calibration curve (tl~e logarithm of the ~olecular weight plotted against Rf, where the R~-value for glucose is arbi.trarily defined as 1 and the Rf-value for a specific dextran is de~ined as the retention time for this dextran divided by the retention time fo~ ~lucose)has been established by means of seve-ral standard dextrans with known molecular weiahts (T 4, T 10, T 40 T 70, T 110, T 500) from Pharmacia ~ine ~hemicals AB, Box 175, 5-75104, Uppsala, Sweden. The Rf~value ~or the mzximum o~ each dextran peak has been ~ound, and the corresponding molecular weight has been calcu-lated as ~Mw M~, whexeby Mw is the average value of the molecularweight æoording to weigh. and~n is the dV~ld~l~ value of the m~l~ar wei~ht ~c~r-ding to number. As an eluent for this chromatographic procedure O.1 M ~aN03 has been used. The columns used in the chromatog.raphic procedure are 60 cm PW ~000 followed by 60 cm P~ 3000 ~rom Toyo Soda Manufacturing Co., Japan. ~n this manner the relatlonship between molecular weight and R~ for ~he abo~e indic~ted de~tr~ns : has been established, ide figure 3.

On the ~2sis of fig. 4 it can ke c~ ted that SPS has,a m~leculzr weiqht distrikution which ~L~es rise to a vzlue of ~w -of aro~d 5.4 x 1~ and a value of Mn of around 4.2 ~ 10 . Also, it appears from this figure that the chromatogrzrn e~.hibits t~"o distinct peaks at retention time 39.5 minutes (6%) corres~ond-ing to a molecular weight of around 5 x 10 and retention time 47.12 minutes (67%) correspondins to a molecular weight of around 4.9 x 104. Also, it appears from this curve 'chat a sh~ulder exists between these two pezks at retention time 41.25 minutes (27%) corresponding to z molecular weight of 2.8-x 10 ' ' ' ~ fter decomposition of sPC with SPS-ase the hydrolysis mixture wzs~ L~le ~iltered, and the filtr,te was ~ raphed. It wr~ fo~nd that zround'55% of spc is decomposed to mono-, di- and trisaccha-rides, and ~at the remaining -45% are decomposed to a polymer with three peaks with the following moleculzr wei~hts: 5 x 104, lo'4 znd 4.4 x 103, vide figure 5.

In orde'r to demonst.ate the bindina effeot between soy protein an~ SPS and the substantial reduction o' binding effec~ ~.e~w.een,.soy protein and SPS decomposed by means of an:sps-ase: the following experimen's ~ve b~en ~erf,o,~ned.

3% ,S,PS ln 0.10 M acetate buffer at pH.4.5 is added t~,a,slurry, o,f, soy isolate (Purina E 500 ) in order to aene-rate ,~ suspension with a ratio isolate/SPS of 10:1. This sus-~ension is incubated for 18 hours on a shakin~ bath at 50C.
. ~ftex incubation the suspension is centrifuged, and the clear supernatant is analyzed on HPLC as previously described. ~rom fig. 6 in comparison with fig. 4 it appears ~hat the SPS is completely adsorbed to the soy isolate.

The same procedure as indicated in ~he prev.ious para~ra.p,h. is p,erfor~ned with a 3% SPS solution hydrolyzed with 7~P~

an SPS-ase produced by means of CBS 101.43 (fig.7 ).
A comparison between fig. 7 and fiy. 5 shows that no com-pound in the hydrolyzed SPS with molecular weiaht ~elol,l around is adsorbed to soy isolate. The hydrolysis reduces the quantitative bindi~g to around 10 - 15~ in relation to the binding of SPS to soy proteln.

An N~R-analysis of the SPS, the production of which is carried ou~ as indicated in this specification reveals the following zppro~imate composition of the SPS:

1) ~-galacturonic acid in an amount of.approximately 45%, whereby ap~roximately ~o% of the total a~ount of ~-galac-turonic acid is present as the methyl ester.

2) rhamnopyranose in an amount of approximately 20%, 3) galactopyranose in an amount of approximztels~ 15%, and ' 4) ~-xylopyranose in an amount of approximately 20~.

The constituents seem to be present in a structure comprising a rhamnogalac'uronic backbone and side chains of xylose and galactose.

Complete acid hydrolysis of SPS (8 hours in 1 N X2SO4) an~ subsequent TLC an21ysis revealed that also minor amounts of ~he monosaccharides fucose and arabinose were present in the hydrolyzed SPS.

., -- 19 --An HPLC analysis of the SPS decomposed by the SPS-ase enzyme comple~ formed by CBS 101.43 shows a powerful reduction of molecul2r weight. In accordance therewith the N~R-spectrwn of the SPS decomposed as indicated above shows that the main ~rt of the ester yroups have disap~eared and that aLso the co~tent of ~ylose ~d ga-lac~ose in the hiaher m~lecular~e,ight material h2s decre~sed. The N.~ ~nxn of the part of the SPS decomposition product which precipitates by addition of one,volume of ethanol to one volume of SPS
decomposition product is similar to the NMR-spectrurn of the SPS, with the above indicated modifications, concernina the ester groups and the content of xylose and galactose.
(0 '~.

j. - 20 -~ '7 SECT I O~ 3 DOCUMENTATION FOR THE FACT THAT SPS AND APS ARE DIFE'ERENT
COMPOUNDS .

APS was prepared 2s indicated in Agr.Biol.Chem., Vol. 36, No. 4, p. 544 - 550 (1972).

Now, this polysac~ha-ide and cpc ~:ere h~drolyzed with different enzymes, wherea ter the decomposition mixture was gel chromatographed on ~PLC equipment, as indicated in sec-tion 2, "Characterization of SPS, especially ~.olecular weight distribution thereof".

In more detail, the hydrolyses were carried out by treatment of 25 ml solution o~ either 2% APS or 2~ SPS in O.1 M acetate buffer of pH 4.5 with 10 mg KRF 6~ ox 30 m~ Pectolyase. KR~ 68 is an SPS-2se preparatio~,the ~ kld~iOn OI W~iG~ i5 described in exa~le 1. ~he r~ts Z~D~P~I frcm the follcwina table.

Polysac- HPLC gel ~ Polvsaccharide charide Enzyme chromatogr2m Decomposed `I~Ot decomposed APS Pectolyase fig. 8 x APS ~RF 68 fig. 9 x 5PS Pec~olyase fig. 1C x SPS K~F 68 fig. 5 x SECTION 4. ~ ~87~

SCREENING FOR SPS-ASE PRODUCING MICROOE~A~lISMS.

The microorganism to be tested is incubated on an agar slant substrate with a composition which enables growth of the microorganism. After initial growth on the agar slant substrate the microorganism is transferred to a liquid main substrate, in which the main carbon source is SPS (prepared as indiGated), in which the nitrogen source is NO3 , NH4 , urea, free amino acids, proteins or another nitrogen containing compounds, and which furthermore contains a mixture of necessary salts and vitamins, preferably in the form of yeast extract. The composition of the main substrate depends upon the microorganism genus, the principal issue being that the main substrate should be able to support growth and metabolism of the microorganism.
When the growth has taken place in a suitable period of time, of the order of magnitude of 1-7 days, depending upon the growth rate of the microorganism in question, a sample of the fermentation broth is analyzed for SPS-ase according to the enzymatic SPS-ase determination described in this specification, or according to any other SPS-ase determination tailored to other specific uses of the SPS~ase than the use as a component of an agent for decomposition of soy remanence.
In order to achieve a more sensitive method for the determination of enzymatic activity the temperature could be lowered to 40C and the incubation time could be raised to 20 hours during the determination of SPS-ase activity, whereby antibiotics should be added to the substrate in order to avoid infection.
By following this test method other SPS-ase produciny microorganisms may be found, both belonging to the genus of Aspergillus and to other genera.

,;

., _.
SECTION 5.

~HARACTERIZATION OF SOME SPS-ase FORMI~G MICROORGANIS~S

According to the here indicated screening for SPS-ase producing microorganisms it hzs been found that the microorganisms listed in the upper part of the following table are SPS-ase pxoducers. Also the table contains a strain belonging ~o the species Asp. japonicus which is not an SPS-ase producer.

SPS ase Species Our identi- Official ~posit Yes ~o As~. Asp fying de- identi- on .japo- acuie- signation fying de- . ye~
nicus atus signation x . x ~ 805 CBS 101.43; DSM 2344 1343 x x A 1443 IFO 4408~ ~M 2346.195C
x x A 1384 ATCC 20236; ~M 2345 1969 ~ short identification of the above indicated strains can be found in the Xollowing culture catalogs:

hist of Cultures 1978 Centraalbureau voor SchLmmel-cultures, Baarn, The Netherlands.

Institute for Fermentation Osaka, List of Cultures, 1~72, 5th edition, 17-85, Fuso-honmachi 2-chome, Yodoyawa-ku, Osaka 532, Japan.

The Amnerican Type Culture Collection Cataloyue of Strains I, 14th edition, 12301 Parklawn Drive, Rockville, Maryland 20852.

All the strains in the above indicated table correspond closely to the taxonomic description o~ the species Asp.
japonicus and Asp. aculeatus appearing in The genus Aspergillus of Raper and Fennell, 1965 (vide especially pages 327 - 330).

SECTION 6.

GENERAL DESCRIPTION OF OVERLAY T~CHNIQUE ASSOCIATED WI'T~
IMMU~OELECTROPHORESIS~

A metrhod designated the top-agar overlay technique has be~n developed by the applicant for identification of individual components of an enzyme comDlex by crossed Lmmunoelectroforesis with a polyspeciic antibody against all en~mP components in the enzyme complex. The method is based on the fact that enzymes are still active after the specific en-zyme-antibody bindin~, or otherwise stated that the active enzyme s~te is not identical to the site of the enzyme-anti~ody binding.
The enzyme-antibody comple~es precipitate as distinct arcs in the gel durin~ the electroforesis. The gel plate is covered with soluble S~S in a top-agzr. ~fter heating to 45C for 20 hours in an at~osphere wi',h a relative-h,~midity, o' 100% the arc which possesses SPS-'ase activitV will a~ear as a clearin~ zone in ' '' the SPS cover a,ter precipitation with a mixture of equal volume paxts of ethanol and acetone when looked upon a~ainst a black back~round. Arcs which have no SPS-ase activity are left invi-sible.

31.~

SECT ION 7 .

IMMUlNOELECTROPHORETIC CHARACTERIZATION O~ SPS-P~SE WITH POLV-SPECIFIC ~NTIBODY AND OVERLAY.

Rabb.its were ;mmlln;zed with the SPS-ase containing enzyme complex o~ained by fermentation of Aspergillus aculeatus CBS 101.43, as indicated in example 1 (KRF 68) -and the poly~if~
antibody was recovered in a manner known per se. By means of this polyspecific antibody a crossed immunoelectrophoresis of the enzyme complex obtained by fermentation of Asp. aculeatus CBS 101.43; as indicated in example 1 (RRF 68) was p~rfonm~1, as described in N.H. Axel-sen e, al., "A Manual of Quantitative Immunoelectrophoresis", 6' ~rinting 1977. ReferPnce is made to fig.ll which shows the arcs ~orresponding to the dif~erent proteins produced by the microor-ganism_ B~ means o' the previously descri~ed top-agar overlay t~hni que it is found that the hatched area corresponds to SPS-ase.

If the previously indicated hypothesis comprisin~ the assum~tion that SPS-ase consists of at least two enzymes is c~rrect the hatched area is the area, in which all the enzymes responsible for the SPS-ase activity are present. Ic these enzymes in other embodiments o~ the invention should be sepa-rated by the immunoelectrophoresis in such a manner, that they do not cover any mutual area, a part of the SPS-ase acti~ity ca~ still be identified by means of immunoelectrophoresis with an overlay with both SPS and a commercial pectinase.

~3 SECTION 8 .

PURIF':I:CATION O~ A~l SPS-ASE PREPARATION
The purification of the SPS-ase preparation K~F 92 (vide example 1) w25 performed by ion exch~n~e. The buffer ,.
is 50 mM Tris (tris-hydro~ymethylaminomethane) which is adjusted to pH 7.0 wi~h ~Cl. The column is K 5/30 from Ph2rmacia, Sweden. The ion exchange material is DEAE trisacryl from LKB, Bromma, Sweden ~300 ml). The flow rate is 100 ml/hour.

15 g of the SPS-ase preparation ~RF 92 was dissolved in 450 ml H20 at 6C, and all ~he following indicated opera~ions were carried out between 6C and 10C. pH was adjusted to 7.0 with 1 M
Tris. The column was eauilibrated with the bufer, and then the SPS-ase sample was ~introduced onto ~he column. OD280 and the conducti~l-ty was measured on the eluate, reference being made to Fig. 12. Frac-tion 1 is the eluate whlch is not bound to the.ion,P~rh~n~e~m~teri?l. ~nen the co-lumn is washed with 200Q ml buffer which gives rise to fxaction 2. Now a 0 - SQQ m~ NaCl gradient is established, giving ' rise to fractions 3-9. All nine fractions were concentrated to ~00 ml and' dialyzed against water to a condllctivity of 2 mSi by mean5 G' dialysis (Hollow Fiber D~ 2 from Amicon, Massachusetts, V.S.A.). Then the nine fractions were freeze dried. Only fractions 1 and 2 exhibited SPS-ase activity.

~ raction 1 was further purified by gel filtration. 1,5 g of fraction 1 was dissolved .in 10 ml 50 mM s~dium acetate with pH

4.5 (500 ~ KCl). The column is 2.5 x 100 cm from L~B. The gel fil-tration filling material is Sephacryl S~200 from Pharmaci2, Sweden.
The flow rate is 30 ml/hour. The fxacti.ons containing materials with molecular weiyhts between 70,000 and 10~,000, calibrated with glo-bular pro~eins, contained an enzyme complex designated factor G
which cannot decompose SPS when tested according to the ~ualitative agar test; however, SPS is decomposed according to the qualitati~e agar test when mixing factor G with a pectinase. It has been found that f actor C- is a~le to split off galactose, fucose, and some galac-turonic acid from SPS, but the mair. decomposition product according to the HPLC analysis is still a high molecular product vexy much li~e SPS.

~ICN 9.

ACTIVITY DÉPENDENCY, TE~IP~::RP.~URE PCTIVITY DEPE~DENC~ I~ND
STABILITY OF ~I SPS ASE.

~ ig. 13 shows the p~-activity dependency of the S~S-ase pre~tion K~'68. ~o~ p~ 2,7 to p~ 3,5 a formic acid buffer system was used, and fr~m p~ 3,7 to 5,5 2n aoeta~e buffer system was used.

Fig; 14 shows the te~.perature activity dependcncy of the SPS ase preparation KRF 63.

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

.. . ... . . .. ...

a~

~9~370~3 SECTION 10.

ENZYI~TIC ACTIVII'Y D~TERMl:NATIONS.

The below indicated table is a survey.of the di~ferent enzy-matic ac,ivity determinations pertaini~a to the invention.

Definition of ac~ivity unit and descriDtion o' enzymatic activit~r determination ~j ' ' . I
Described Kind of ac later in tivity, Publicly this short de- avail- specifi-Enzyme si~nation able cation ~e~erence SPS-ase SPS-ase x n~e SRU x scl~bi- SRUM-120 Protease HUT X
Cellulase Cx x 2 PU x 3 PGE x 4 Pectinase UPTE x 5 P~ x . 6 lulaCel V~CU x 7 The references indicated in the last ~column of the above table are d~tailed in the below ind~cated tableO

Dl-~

~913~

Reference can ~e obtaincd from NOVO I~D~S- Schwei-~RI A/S, zerische Novo Allé, Fer~;ent 2880 Bag- AG, Ba-Re~erence Identification of sv2rd, sel, S~lit- A libra-No reference Denmark zerland ry Analyseforskri~t 1 AF 154/4 of lsal-].~-ol X
Analytical Biochemistry . 84, 5~2 - 532 (197 Analytical method AF 149/6-GB of x 19~ 5-25 Determi~ation of Pecti-nase Activity with Ci- .
trus Pectin (PU) of 3 23~3.1976 x Viskosimetrische Poly-galacturonase-Bestim-4 mung (PGE) OI 10.11.77 x Bestimmung der Pectin-trznseliminase (UPTE/
g) of 24.Sept.1975 x Determination of t~e Pe~tinesterase acti-vity (undated3 with 6 initials ~JA/G~ x ~' Analytical method ( 7 ~F 156/1-GB x In relation to the cellulase activity determination it can be noted that the anali~sis was carried out as indicated in AF 149/6-GB and that ~he principles of the determination is explained in Analytical Biochemistry.

!. _ ~9~

SECTION 10 a.

ENZYMATIC DETERMINA~ION OF SPS ase.

The enzymatic detexmination of SPS-ase is carried out in two steps, i.e. a qualitative aga_ plzte tes~, and a quanti-tative SPS-ase activity determination based on measurement o the amount of total li~erated sugars. If the qualitative agar plate test is negati~e, .he SPS-ase activity is zero, regardless of ~he vallle originatin~ from the quantitative SPS-ase activity deter-mination. If the quzlitative agar plate test is positive, the SPS-~se activity is e~uzl to the v21u2 originating from the quantitative SPS-ase activity determination.

I. -Qualitative agar ~lzte test.
An SPS-agaT plate was prepared in the followina, manner . A buffer (B) is prepared by adjustin~ 0.3 M
acetic acid to a pH-value of 4.5 by means of 1 ~ NaOH.
1 g of SPS is dissolved in 20 ml of ~. 1 S of agarose (HSB Litex) is mixed with 80 ml of ~ and heated to the boiling point with stirring. ~hen the a~arose is dissolved the SPS-solution is slowly added. The resulting 1~ SPS-agarose solution is placed in a water bath o~ 60C.
The plates are now cast by pourin~ 15 ml of the 1%
SPS-aaarose solution on a horizontal glass plate with ~1m~cions 10 cm x 10 cm. Then 9 wells with a distance of 2 ;5 cm are pu~ched out in the solidified layer of SP5~aaarose. In each well a 10 ~1 of a~l% 5o~ on of the enz~ne protein ~o be tested 'ox SPS-ase activity is lntxoduced. The plate is incubated ~or 18 hours at 50C and with a relative humudity o' 100~. Now still ~8~

undecomposed SPS is precipita'ed by a solution of e~ual volume parts of ethanol and acetone. The SPS-ase agar plate test is positive for a sam~le ~laced in a s~e-cific well, if a clear annular zone a~pears around this well.

II. Qua~titative SPS-ase activity determination test.
~- The puxpose o, this test is the determination of enz~matic.activities,:which are capable of decomposing SPS ~o.such an,extent ~hat.~he decomposition products exhibit a strongly reduce~ or no adsorption or bindinq affi~it~tD s~i protein. E~rPrir~nts have sh~ that that Part ,of the,SPS decomposition products which are not preci~itated by.a mixture of equal volumes Oc water -a~d:ethanol, do not have an~ adsorption or bindina a~finlty to soy protein.

.. ~he:SPS-ase de!erminati~n is based on a hydroiysis of SPS.under.standard.condi.tions followed'by a pr~ci-~i~ation of that ~art,of.SPS,,.which is not hydrolyzed with.ethanol~.After precipitation the content of ~carbohydrate, which is not precipitated, is determined b~,auan~itative.,analysis ~or ~otal sugar (according to.AF..169~1, available..from NOVO INDUSTRI A/S, 2880 Ba~svaerd).

The standard conditions are:
Temperature~ 50C
pH: .4.~
Reaction time: control 210 minutes with substrate only, ~ollow~d by 2 minu-~es with added en~yme - - '-main value:21~ minutes 31.1 The e~ui~ment com~rises-_____ __ _______ ______ Shaking water bath the~nosta~ed at 50C
~irlimixer stirrer Centrifuge Ice w~ter ~ath ~he rea~ents com~rise:
Buffer: 0.6 ~ acetic acid in demineralized ~Ja~er (z) 1.0 M NaO~ (b) Substrate: The DH value of 50 ml of a is adjusted to 4.5 with b, then 4.0 g S~S are added, and after dissolution of the SPS the ~H is readjusted to 4.5, and the volume is adjusted to 100 ml with deionized water.
Stop rea-gent: AbsolutQ ethanol;

1 SPS-ase activity unit (SAE or SPSU) is defined as the SPS-ase activity which under the above indicated standard conditions releases an amount of carbohydrate soluble in 50%
ethanol equivalent to 1 umol galactose per minu~e.

Even if ~he initial part of the enzyme standard curve is a straigh~ line, it has to be noted that it does not intersect the (0.0) point.

I

7~1 c~TT~N 10 b;

ENZYMATIC DETERMIN~TION oP ~E'~ANENCE SOLUBILIZING ACTIVITY EY.PRESS~D
~S SRUM 120.
Principle In the method for determination of hydrolysis activity the insoluble part of defatted, deproteinized, and dehulled soy flour is hydrolyzed under st2Ildaxd con~itions. The enz~e reaction i5 sto~ped with stvp reagent and the insoluble par. is filtered off. The amount of dissolved p~lysaccharides is detennined spectro-photometrically after acid hydrolysis accordins to AF 169/1, available from Novo Industri A/S, 288~ Bassvzrd.

Carbohydrases ~ith endo- as ~ell as exo-activity are de-~rmi ~ed according to the meihodO

- ThP substrate pertaining to this enzymatic deter~i~ation is identical to the re~anence~substrate described ~or the SRU method.
The substrate is dissolved 2S a 3~ solution in the below indicated citrate buf.er:
0.1 N; citrate-phosphate buff~r pH 4.5

5.24 g citric acid 1-hydrate (Mexck Art 244) 8.12 g disodium hydro~en phosphate 2-hydrate (~lerck Art 5580) ~d 1 1 demineralized H20 p~ 4.5 ~ 0O05 Stable for 14 days The stop rPa~ent has the following composition:
100 ml 0.5 N NaO~
200 ml 96~ ethanol To be kept in a refrigerator ~ntil US20 D:-~

8~
Standard Conditions .
Temperature ...................... ~ 50C
p~ .................................. 4.5 P~eac~ion tirne, sample ..... 120 minutes - - blank ........................... 5 -Unit Defin~tion ..
' ~ne so~ reranence s.~ h;l;7;ng unit (9~5) 120 (M for r2nual) is the amount of enzyme which, under the given reaction conditions per mi-nute, liberates solubilized ~olysaccharides equivalent to one micro-mole of galactose.

~s~

11987~
.

SECT ION 10 C .

ENZ~MATIC DETERMINATION OF PROTEOLYTIC ACTIVITY.

HUT MEASUREMENT
Method for the determination of proteinase in an acid rnedium~

The method is based on the digestion of denatured hemoglobin by the enzyme at 40C, pH 3.2, for 30 minutes. I'he undigested hemoglobin is precipitated with 14~ trichloroacetic acid (wt/v%).
All enzyme samples are prepared by dissolving them in 0.1 M acetate buffer, pH 3.2.
The hemoglobin substrate is prepared using 5.0 g of lyophilized, bovine hemoglobin powder, preserved with 1%
Thiomersalate and 100 ml demineralized water which is stirred for 10 minutes, after which the pH is adjusted to pH 1.7 with 0~33 N ~Cl.
After another 10 minutes of stirring, the pH is adjusted to pH 3.2 with lN naOH. The volume of this solution is increased to 200 ml with 0.2 M acetate buffer. This hemoglobin substrate must be refrigerated where it will keep for 5 days.
The hemoglobin substrate is brought to room temperature. At time zero, 5 ml of substrate is added to a test tube containing 1 ml of enzyme. Af~er shaking for l second, the tube is placed in a 40C water bath for 30 minutes. After exactly 30 minutes, 5 ml, 14~ trichloroacetic acid is added to the reaction tube, which is then shaken and brought to room temperature for 40 minutes.

'` ,:

~ 7~ 36 ~
~ or the blan~, the hemoglobin subs~rzte is brough~
to room temperature. At time zero, 5 ml of the substrate is added to a test tube containiny 1 ml of enzyme. After sha~ing for 1 second, the tube is placed in a 40C. water bath for 5 minutes. After exactly S minutes, 5 ml of 14~
trichloroacetic ~cid is added to the reaction tube, which is then shaken and brousht to room temperzture for 40 minutes.
After 40 minutes, the blan~.s 2nd samples are shaken, filtered once or twice through Berzelius filter No. 0, and placed in 2 spectropho~ometer. The sample is read against the blank a~ 275 nm while 2djusting the spectro-photometer against water.
Since the absorbance of tyrosine a- 275 nm is 2 ~nown factor, it is not necessary to ma~e a tyrosine ~t~nda_a curve unless it is needed to check ~he Bec~m2n spectro~hotometer Calculations 1 HUT is the zmount of e~zyme which in 1 minu~e forms a hydrolysate equivzlent in absor~ancy at 275 nm to a solution of 1.10 microsr~m/ml tyrosine in 0.006 N HCl. This zbsorbancy Ya1ue is 0.0084. The reaction should take place at 40C., pH 3~2, and in 30 minutes.

Sam~le-Blank Vol. in ml ~UT = 0O0084 x reaction time in min.

-SamDle-Blank 11 ~UT = 0.0084 x 34 - (S-B) x 43.65 ~T/g enzy~e = ~S-B) x 43.65 g.enzyme in 1 ml - An inves.igation of the pH-stability dependency of the prote2se in K~ 68 performed by means of the ~ analysis with p~ values from 2.0 to 8.0 showed that the stability of the p~otease above pH 8~0 wzs very small, vide fig. 16.
, ~L98~QO

In order to illustrate the invention reference is made to the following examples 1 - 8, where example 1 illustrates the production of SPS-ase, and where examples 2 - 8 illustrate the application of SPS-ase with a soy based raw material in order to produce a purified vegetable protein. Other applications of SPS-ase are ndicated in the section between example 8 and the survey of the figures.
Several fermentations with the here indicated strains of Asp. aculeatus and Asp. jaDonicus were performed in laboratory scale. Hereby preparations were obtained which contained SPS-ase according to the here indicated SPS-ase tes'. However, as rather large amounts of SPS-ase are r-e~uired in order to run application tests, similar ~exmentations were run on a pilot plant scale, vide the following example 1.

Example 1 Production of an SPS ase in pilot plant scale.
.... .
P~ SPS-ase was prepared by submerged fermentation of Aspergillus aculeatus CBS 101.43.

An agar substrate with the following composition was prepared in a Fernbach flask:
Pepton Difco 6 g Aminolin Ortana 4 g ' Glucose 1 g Yeast extract Difco3 g Meat extract Difco1~5 ~X2PO4 Merck 20 g Malt extract Evers20 g Ion exchanged ~O ad1000 ml ~3L1987~ ~

pH was adjusted to between 5430 and 5.35. ~en 40 g of Agar Difco was added, and the mixture was autoclavecl ~or 2C) rnin.
at 120C (the sllbstrate is named E-agar) .
The strain CBS 101.43 was cultivated on an E-agar slant (3 7C) . The spores from the slant were suspended in sterilized skim-milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transf erred to the Fernbach flask. 'rhe flask was then incubated for 13 days at 3 0C .
A substrate with the following cornposition was prepared in a 500 liter seed fermenter:

CaCO3 1.2 kg Glucos e 7 . 2 kg Rofec (corn steep liquor dry matter) 3 . 6 kg Soy bean oil 1.2 kg Tap water was added to a total volume of around 240 liters. pH was adjusted to around 5.5 before addition of CaCO3. The substrate was sterilized in the seed fermenter for 1 hour at 121 C. Final volume before inoculation was around 300 liter s.
The Fernbach flask spore suspension was transf erred to the seed fermenter. Seed fermentation conditions were:

Ferrnenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
Agitation: 300 rpm (two turbine impellers) Aeration: 300 normal liter air per minute Temperature: - 30 - 31C
Pressure: 0 . 5 ato Time: Around 28 hours Around 28 hours after inoculation 150 liters was transferred from the seed fermenter to the main fermenter.
;

~:~L9~37~
A substrate with the following com~osition was p.ep,ared in a 2500 liter main fermenter:

Tbasted soy meal 90 kg , XH2 4 20 ky ' ~luronic~ 150 ml Tap water was added to a' total volume of' ar~ 900 lit~rs. ~ne ~casted soy meal ~7as s~spended in water. pH was adjusted to 8.'0 wit~ NaO~, and the tem~erature was raised to 50C. There-a~ter ar~und 925k~tn unitst~ALCALASE~ 0.6 L was added to the sl.~p~nsit~n.
The mixture ~as held for 4 hou~s at ~0C and p~ = 8.0 (Na2CO3 '~ 'addition)Wit~ no aeration, zero ato and 100 rpm agitation.
Thereafter the remaining substrate components were added and p~ was.~djusted,~tanound 6.0 ~ h pho~t~r;c acid. Thrr~ substrate was sterilized' in the main fermenter for l~ hours at 123C.
Final voIu~e befor~ inoculation was around 1080 liter.

Then 150 liter of seed culture W25 added.

Fe,rmentation conditions,were;,, ,~,erme,nt,er ty~e:, Conventioral aerated and ayitated , ~ermenter ~'ith a height/diameter `
- ratio o~ art3und 2.7.
C~ .~gitation. 2$0 rpm (two turbine impellers) ( '~tæ ation: ,1200 normal liter air per minuteO
Tt~mperature: 30 C
~ressure: , -Q.S,ato , Time: hr~und l51 hoNrs.

~rom 24 fermentation hours to ~ 116 fPt~ la~ion hours ... . .
pectin soiu~ion was added'aseptically tCt ~h main ~ at a o~k~lL
rate of around 8 liters per hou~.The pectin solution with the follow-lng composition was prepared in a 500 liter dosing tank:

Pec in genu x3 22 X~
P~osphoric-a~id, conc. 6 kg Pluroni ~ 50 ml ~)Genu pectin (citrus type NF from The Copenhagen pectin ~actoxy ~td~3 - ~o -~i9~
Tap ~ater w~s added to a ~otal volume of around 325 liters.
lhe substrate ~s stPril i7~A in the dosing tank for 1 hour at 121C.
Final volume before start of dosage was around 360 liters. ~lhen this portion ran out, a~other s;m;l~r portion ~Jas ~ade. Tbtal volumP of ~l~c~in solu',ion for one f~l"~,Ld~ion was around 725 liters.
After around 151 fe~mPntation hours the fenm~ntation process ~s sto~ped. Ihe around 1850 liters of culture broth were cooled to arcund 5C
and the en_ymes were rec~vered according to the following methcd.
The culture broth ~as drum filtered on a va~uum drun filter (~orr Oliver), which was precoated ~7ith Hy-flo-super-cel dia'.omaceous earth (filter aid). m e filtrate was co~c~iLldLed by eva~oratiDn to around 15% of the vol~me of the culture bro,hO The crmcentra.e was filtered on a Seitz filter sheet (type supra 100) with 0.25~ Hy-flo-super-cel zs a filter aid (in the following table referre2 t~ zs filtration I). The filtrate w2s precipitated with 561 g of ~N~4)25O4/1 at a p~ of 5.5, and 4~
Hy~flo-suser-cel diabo~aceouLc ea,-~h is ad2ed as a filter aid. The pre-cipitaue and the filter aid are sepera.ed ky fiitra~ion ~n a frz~e filter.
m e filter cake is 2issolved in water, and unsoluble parts dre S~dld~ed ~y filtration on a fr~e filter. ~ne riltrate is che~k liltered on a ~eitz filter sheet (type supra 100) wi~h 0.25% By-flo-super-cel as a f;lter aid (in the foll~ing ~hle referred to zs filtration II). Ihe ~iltrate is diafiltered on an ultrafiltrztion aF~aratus, ~fter diafil-L,ation the liquid is conc~-Ll~L~d to a 2ry mztter co~ ,L of 12,7% (in the following table referred to ac dry m2tter content in conc~ L~Le).

, A fac~ltative bYce treatment for partial remo~zl of ~he pr~tease activity can be ~rri~ out at this stage. In ~se the base tr~tm~t is used it i carried out at a pH of 9,2 for 1 hour, whÆre-after the p~ value is a~ Le~ to 5Ø

~ bw the liquid is check filter~d and filtered for the pur-pose of yenm reducti~n and the filtr2~e is freeze-drie~ on ~ freeze-dkying ~ lir~t fro~ Stokes.

Four fe~-t~ ns were c~rr;e~ ou~ in the man~er I

7~
indicated below, whereby the strain used for the fer-mentation~ the use of the facultative base treatment ard other F~ldll~eL~ ~re v2ried, as indicated in the foll~in~ table.

Con~l~dLion (~ er of f~ter aid in con-connectior.wlth tent Pre- in B2se treat~ent Ption tra- cipi- filtra- c_n- ~
Microorgani~l used not u~ed ccde tion I tatior. tion II trate marks CBS 101.43 x KgF ~ 0.5 ~ 0.2 28 AICC ~0236 x XRF74 2.0 4 0.4 7.5 IFO 440~ x ~ ~3 1.0 5 0.25 12.4 x) ~BS 101.43 x KRF92 0.25 4 0.25 12.7 x3 After germ reducing filtrati~n he filtrate is c~c~nll~ed by evapor2 ion in a ratio of 1:2.3. A munor p2rt of the c~llc~l~dt~d l;ltrate was spray-dried, and the re~ ~ng part wzs freeze-dried.
In order to reduce the protease activity further, some of the above indicated preparations ~ere treated as indicated below, whereby only one of the three alternatives A, B, and C
was used.

A 100 g SPS ase prepzration are dissolved In 1 liter of deiD-nized water wlth stirring at 10C ~ 2C. p~ is adjusted to 9.1 with 4 N NaOH. ~his base treatment is carried out for 1 hour.
The pH value is then adjusted to 4.5 with glacial acetic cid, and it is dialyzed against ice cold, ~P;~n~7el water to a ~onducti~ity of 3 mSi~ Then ~reezing and lyophilization are carried out.

B. 500 g SPS-ase preparation are dissolved in 4 liter of ~ n;7~ wa~er wi~h st;rr;~ at lo& ~ 2C~ pH ~s ~dj~s~d to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The p~ value is the adjusted to S.0 with glacial ace-tic acid. The obtained material is lyophilized.

C. 50 g SPS-ase pre,paration are dissolvedin 400 ml of ~P;0~;7-ed water with stirring at 10C ~ 2C. p~ is adjusted to 9.1 with 4 N NaO~. This base treatment is carried ou~ for 1 hour. ~hen pH is reduced to a.7 with glacial acetic acid. The obtained material is lyophilized~

SPS-ase preparation Base treat-used as starting ment used Preparation material ror base code treatment A B C
KRF 68 x KRF 68 BII
KRF 68 x KR~ 68 BIII
KRF 92 x KRF 92 BI

The above indicated preparations are characterized by ~heir activities of the enzymes relevant to the invention in the following table.

.

i:

Enzyme ~c~ y per g XRF 68~RF 68 BIIXRF 68 BIII KRF 74 K~ 83~RF 92KR~ 92 BI
Plate test ~ + ~ - + ~ +
SAE
Qu~ntitative test 350 301 349 0 168 476 430 HUT pH 3.2 67000 105 339 1630 128~0 5960 397 Cx 8000 8044 g396 1320 8040 37~0 3092 PU 10300000 9000000 8800000 ~40000 750000Q 84000007600000 UPTE 78100 83700 76900 15i30 327000 44000 62400 PEE . 840 910 77~ 370 690 1000 7yo VI~CU 1600000 1100000 1000000 65000 2200000 1100000 742000 . . .

~ 44 -37(~

~xaJnple 2 (application example) _ This example describes the production of a p.v.p.
from a dehulled and defatted soy flour, "Sojamel 131' (commer-cially available from Aarhus Olie abrik A~S). The dry matter content of this flour was 94.0% and the content of ~N x 6.25 o~ a dry matter basis was 58.7%. The soy flour was treated with the SPS-ase preparations KRF 68 BII (example 1) in the following manner: .

85~2g ofthe soy flour were suspended and kept stirred at 50C in 664.8 g of water, and p~ was adjusted to 4.5 by means of 7.5 ml of 6 N HCl. 50 g of a solution con~in;ng 4.oO g of said SPS-ase preparation W25 added, and the -eac-tion mixture was then agitated for 240 minutes at 50C.
~=mixture was then centrifuged in a laboratory centrifuge ~Beckman Model J-6B) for 15 minutes at 3000 x g. The super-natant ~25 weighed and analysed for Kjeldahl N and drv inatter. ' The solid phase was then washed with a vol~ne of water equiv2-lent to the mass of supernatant obtained by the first cen trlfugationO This operation was perfonmed twice. The solid phase was then freeze-dried, weighed and analys~d for ~jeldahl N and dxy matter at Qvistls Laboratori~n, Marselis Boulevard 16g, 8000 Aarhus C, De~nark. This laboratory is _ _ .
state authorized for analyses of fodder and dairy products. The results obtained in the exper~nent appear from Table a~

.9~
Table 2.1 Results obt2ined Mass N x 6.25 Dry mat- Yieldo~ Yield of Component g % ter ~prv~ei~ y r~t~r, ~5 Soy flour 85.2 55.2 94.0 100% 100%
SPS-ase prepa-ration 4.~ 75~6 . - 6.4%
1. Centrifugate 666 1~53 5.0421.2% 42.0~
p.v.p. 44.5 87.5 ~5.7 82.7% 53.2%

Thus,.a p.v.p. was obtained wi.h a protein purity, i.e.
N x 6.25 on dry matter basis, of 91.4%, and with a total yield of ~rotein of 83%.

Example 3 (applicatio~ ex~mple~

This example was performed in order to compare the protein yields, the nutritional quality and some functional pxoperties of soy protein products made by the following three proce-dures: .

A: The traditional isoelectxic precipitation for production of soy protein isolate.
.
B: The traditional isoelectric wash for production of soy protein concentrate C: The isoelectric wash according to the invention including a remanence solubilizing enzyme for prod~ction of p.v.p.

In order ~o generate a true comparison of the process according 'o the i~vention (C) with the conventional soy protein processes (A and B) t~e same raw material has been used in all thre~ cases~ Also ~he experiments have been conducted ` ~n such a manner that corresponding temperatures and treat-ment times are the same in all three cases.. Onlv the pH-values - 46 ~
~9~

were different due to the fund~mental di~erences between the three experiments.

A. The traditional isoelectric precipitation for produc-tion of soy protein isolate.

425.8 g o~ soymeal (Sojamel 13 produced by Aarhus Oliefabrik A/S) were extra~ted in 3574.2 g of tap water at 50 C. pH was adjusted to 8.0 with 20.1 g of 4 N NaOH. After stirring for 1 hour the slurry was centrifuged at 3000 x g for 15 minutes using four one liter beakers in a laboratory eentrifuge (Beckman Model J-6B). The ~entrifugate I and the precipitate I were weighed. The precipitate I was re-extracted with water to a total weight of 4000 g. The temperature WZ5 kept at 50~C, pH adjusted to 8 with 4 N NaOH and the slurry kept stirred for one hour. A centrifugation and weighing of centrifugate II and precipitate II were performed as above.
Samples were drawn from centrifugate I and II and preci~itate II for Kjeldahl and dry matter determinations. Hereafter the centrifugates I and II were mixed ~nd held at 50C. The pro-tein was then isoelectrically precipitated at pH 4.S by means of 45 g of 6 N HCl. After stirring for 1 hour at 50C
the protein was recovered by centrifugation at 3000 x g for 15 minutes. The centrifugate III was weighed and analysed for Xjeldahl-N and dry matter. The solid phase III was weighed ~nd washed with water in an amount corresponding to the weight of centri~ugate I. The washing.was carried out by stirring for one hour at 50C. The washed pro~ein W2S recovered by ce~tri-fugation at 3000 x g for 15 minutes. The centrifugate IV and the solid phase IV were weighed. Centrifugate IV was analysed for Kjeldahl-N and dry matter~he solid phase was suspended in 15S0 g of water at 50C and pH W25 adjusted to 6.5 with 17 a o~ 4 N NaOH. The mixture was kept stirred for one hour a~d re-adjusted to pH = 6.5 if necessary. Finally the product was freeze dried, weighed, and anzlysed for Kjeldahl-N and d~y matter. The mass balance calculations are shown in table 3~

o able 3.1 Mass balance calculations of the traditional iso-electric precipitation for production of soy protein isolate.

Mass of Yield of Yiel~ o' O2erations 2n ractions fraction Protein Dry mat- protein, dry ~at~
g %(N x 6.25~ ter ~ ~ ter,~
EXtraction: Soy flour 425.8 55.2 94.0 100.0 100.0 ~2ter 3574.2 0 0 0 0 .4 N NaC~I 20.1 0 16.0 0 0.8 1. Centrifuga'ion: ~ 4020.1 5.9 10.0 100.9 100.4 Centri~ug2te I3141.0 4.4 6.9 58.8 54.1 PreciPitate I 805.0 - - - -Re-extra tion:
Precipitate I .805.0 - . - - -h'ater 3195.0 0 0 0 0 2. Centrifugation:
Cen',ri~ugate II3104.0 0.5 0.9 6.6 7~0 Precipitate II 820.0 9.1 17.2 31.7 35.2 Mixing and acidifying: .
C2ntrifu~ates I ~ II 6245.0

6 N HCl 45.0 0 21.3 0 2.4 3. Centrifu~ation: ~6290.0 Cen'r; ~ te III 5650.0 O.3 1.9 7.2 26.8 Precipitate III 308.0 Washing:
Precipita~e III 308.0 - - - -Wlater 3141.0 0 0 0 0 9. Cen~ri~ugation: 3449.0 :Centri~uga~e IV 3113.0 0.04 0.15 0.5 1.2 Precipitate IV 291.0 r~;7~tion:
Px~cipita~e IV 291.0 - - -Water 1550.0 ' O O O
4 N NaO~ 17.0 0 16.0 0 0.7 ~rying: Pcw~er 128.0 93.~ 96.3 51.1 30.8 - 48 - ~ ~ ~8~

B~ The isoelectric wash for production of soy protein concen~rate ~ 425.6 g of soy meal (~ojamel 13 produced by Aarhus Oliefabrik A/S) was washed in 3574 g of water at 50C~ p~
... ~ .. ~, .. ....
was adjusted to 4.5 with 44.8 g of 6 N HCl. The washing was carxied out for four hours by agitating. The slurry was then centrifuyea at 3000 x g 'or 15 minutes in a laboratory cen-trifuge (Beckman Model J-6B) using four one liter beakers.
The ce~trif~gate I was weighed and analysed for Kjeldahl N and dr~~matter; The solid phase I was weighed and -e-washed with water to a-total weight of 4000 ~. pH was re-adjusted to 4.5 r.7 g of-6 ~ HCl.and the slurry was kept stirred for 30 ~inutes at 50C. A centrifuyation and weighing of centri-fllqA~P II ~n~ solids II were performed as above. The solid pha~e II W25 resuspended in 1575 g of H~O at 50C and pH
~as adjusted to 6~5 with 3~.5 g of 4 N NaOH. The mixture was ke~t stirred at 50C for one hour and re-adjusted to pH-6.5 if:nëcessary. Finally the protein product was ~reeze ~rle~,~we~ghed, ~n~ analysed for Kjeldahl N and dry matter.
The mass-balanGe -is shown in table 3O2~

- 49 - ~ ~ 987~

Table 3.2 ~lass balance calculations of the isoelectric wash for production of soy protein concentrate.

Mbss of Yield of Yield of O?erations and fractions: fraction ~rotem Dry mat- protein, dry mat~
g %(Nx6.~) ter ~ % ter,%
Washing:
Soy flour425.8 55.294.0 100.0100.0 Water3574.
6 N HCl44~B 0 21.3 0 2.4 1. Centrifugation: ~ 4044.6 Centrifugate I 3150.0 0.6 3.2 8.0 25.2 Svlids I846.0 - - - -Re-washing:
S~lids I846.0 - - - -~ater3154.0 0 0 0 0 6 N HCl1.7 0 21.3 0 0.1 2. Centrifugation: ~ 4001.7 Centri~ gateII 3130.0 0.1 0.4 1.3 3.2 Solids II863.0 - - - -Neutralization.
Solids IIB63.0 Water 1575.0 0 O 0 O
4 N NaO~34.5 0 16.0 0 1.4 Drymg: ~o~er 281.072.5 98.4 86.7 69.1 ~' . ~
., - 50 - ~ 7~

The isoelectric wash including a remanence solu-bilizing enzyme for production of p.v.p.

425.8 g Gf SOy meal (Sojamel 13 produced by Aarhus Oliefabrik A/S) was washed in 3524.2 g of water at 50C. pH
was adjusted to 4.5 by use of 43.7 g of 6 N HC1. 24 g of the SPS-ase preparation KRF 68 BIII (e~æmple 1) were solubilized in 26 g of water and added to the washing mixture. The washing was then carried out for four hours by agitaticn. Subsequently the purification was performed as described for B, the amounts of 6 N HCl, 4 N NaOH and water for resuspension being the only parameters with deviating values. The mass balance is shown in table 3.3.

- 51 ~ ~1 g ~i7 ~ ~

Table 3.3 Mass balance calculations of the isoelectric wash :including a remanence solubilizing enzyme for pro-duction of p.v.p.

M2s5 of Yield of Yield o Op~rations zn~ fractio~s: fraction Protein Dry mzt- pro~ein, ~-y mat-g %(~1 x 6.25, ~er ~ % te~
~I2shing:
Soy flour425.8 55.2 94.0 100.0 100.0 Water3540~2 0 0 0 0 6 N HCl43.7 0 21.3 0 2.3 SPS-ase.:KRr 68 BIII24.0 75.3 96.0 7.7 5.8 1. Centrifugation: ~ 4043.7 - - - -~entrifu~ate I 3420.0 1.7 502 24.7 44.4 Solids I620.0 - - - -Solids I620.0 - - ~ -h'ater3380.0 0 0 0 0 6 N HCl1.3 0 21.3 0 0.1 2. Centrifugation: ~ 4001.3 C~n~r;fugateII 3400.0 0.2 0.6 2.9 5.1 So~ids Il 577.0 Neutralization~
Solids II 577,0 - ~ _ _ Water 1700:0 0 O 0 O
4 N NaO~ 25.3 0 16.0 0 1.0 ~hying: PcwdRr 211.0 87.31) 96.71~73~2 5101.
86.9 97.~
1) Analysed at Bioteknisk Institut, Holberysvej 10, DK-6000 Kolding, De~maxk 2) Analysed at Qvist's Laboratorium, Marselis :E3Oulevard 169, DK-8000, Aarhus C/ Denmark ~1 987(~
Nutritional Properties The amino acid com?ositions of the three protein products were determined, vlde table 3.4. The total content of essential amino acids, the chemical score and the essen-tial amino acid index (EAAI) is calculated using the ~AO
reference pzttern from 1957.

The trypsin inhi~itor content of the three products was determined by means of the method described in A.O.C.S. Tenka'Live ~e-th~d Ba 12 75 (A.O.C.S. is an a~bre~iation for Pm~ric~n Oil Che~is,s' Scciety).The results zre shown in table 3.5, which also includes the yields and the pro~ein/dry matter ratio of the ~hree products.

~,

7~)~
Table 3.4 Amino acid composition and nutritional evaluation of the three protein products A, B, and C.

A. Soy pro~ein B. Soy protein C. Soy protein Amino acid isolate concentrate isolate (p.v.p.) g/16 g N aas1~ g/16 g Naas ) g/16 gN aaS1) Non-essential~
Aspartic acid 12.4 - 11.3 - 11.9 Serine 4~62 - 4.69 - 4.8l 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 - 4.55 Histidine 2.83 - 2.78 - 2.50 Arginine 8.09 - 7O57 - 7.04 Essential:
Isoleucine 4.87 ~ lO0 4.g7 ~ lO0 5.19 ~ lO0 Leucine 7.80 ~100 7.98 >100 B.09 ~100 Lysine 6.24 >100 6.09 ~100 5.57 > 109 Phenylalanine - 5.47 ~lO01 5.35 ~lO0) 5.17 >lO0¦
~'100 ~100 ~iOO
Tyrosine 3.38 >loOJ 3.88~100 4.44 >100 Cystine 1.29 64.5~ 1.3266.0l 1.44 72~0 Me~hionine 1~08 49.1l 1.2155.0J 1.31 59.5J
Threonine 3.10 ~100 3.60~100 3.97 ~100 Tryptophan 1.06 . 75.7 1.37 97.9 1.32 94.3 - -Valine ~.90 --~100 -----5.23~ 100- - - -5.57 ~100 total contPnt of essential 3g.36 41.31 42.21 amino acids Chemical score 56. 4% 63.2~ 65.5~
EAAI B6. ~% 90.2% 91~3%

1) aas = amino acid score based on ~he F~0 reference pattern (1957l

8'7(~

Table 3.5 Process chzracteristics 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.) Protein of 97 4 % 73.7 % 90 0 %
Process dry matter cha:rac-teriStics Pr~tein 51.1 % 86.7 % 78.2 %
.~ yield Trypsin inhibitors 34 0OO 21,00C l9,000 TUI/g product TUI/g protein 36,250 28,970 21,810 .

~a9~37(~(~
~nctional Properties Nitroqen solubility ihdex (NSr) was dete~nined in a _____ ____________ ______ 1% protein dispersion at pH = 7.0 in 0.2 M NaCl and in destilled water respectively. After stirring for 45 minutes with a mag-netic stirxer the suspension was centrifuged at 4000 Y. g for 30 minutes, and the supernatant was anal~sed for nitrogen.
The nitrogen solubility was calculated as (soluble N%/total M~j.
~he results of this evaluation on the three products are shown in table 3.6.

Emulsifyin~ capaci,y was de~ermine~ three times on each product by a slightly modified Swift titration. ~.0 g of (N x 6.25) of the product ~a~s blended in 250 ml of 0.5 M NaCl with a Sor~al Omn; m; xer at low speed. 50 ml of the suspension were transferred to a glass blender jar and 50 ml of soy bean oil were added. Hereafter the total mixture was wei~hed. The oil-water mixture was ,hen homogenized at 10.000 rpm with the jar in an ice-bath. ~ supplementary amount of soy bean oil was added at a rate of 0.3 ml per second until the emulsion collapses. The total amount o~ oil added be~ore the "end point" was found by weighing.

Emulsifying capacity was calculated as ml oil per gram protein (N x 6.25). The density of the oil was ta~en as O . 9 g/ml.

The average results of the determination of emul-sifying capacity on the three products are shown in table 3.6.

Whl~inq ex~ansion W2S determined in a 3~ protein solu-tion at pH - 6.5. 250 ml of the aqueous` dispersion of the pro-tein samples were whipped at speed III for 4 minutes in a Hobart mixer (model ~-50) mounted wi~h a wire whip. The whipping expansion was calculated according to the formula ~ hipping expansion _ VzS0 x 100~, k ~137(~ .
where V - final whip volume in ml.

V was measured by refilliny the mixer jar with w~ter.
Duplicates were performed for each of the three samples. The average results are shown in table 3.6.

Foam s~bilit~ was determined as the ratio between the amount of foam left after draining for 30 minutes and the oriqinal amount of foam. A gram of foam produced by the method above was introduced into a pl~stic cylinder (diameter 7 cm, heiaht 9 cm) having a wire net with a mesh size ~f l mm x l mm.
The cylinder was placed on a funnel on top of a glass cylinder and the weight (B) of drained liquid in the glass cylinder is determined. The foam stability FS is defined by the equation -FS c A ~ ~ x lO0~

The reslllts of the determination is shown in table T~e gel strenqth is in this s~ecification defined as the Broo~ield viscosity measured by means of T-spindles on a Brookfield Helipath stand. The gels were made by heat treat~ent of 12% protein suspensions in 0.5 M NaCl. The heat treatment was performed in closed cans with a diameter ~f 7 .-3 cm and a height of 5.0 sm placed in a water bath main~ained at 80C and 100C
each for 30 minutes. The cans were cooled and thermo-sta~ted to ~0C before ~hey were opened and measured. The results of the measurements are shown in table 3.6~

71~C~
T~ble 3.6 Functional properties of the three protein pro-ducts A, B, and C.

Functionality A~ Soy protein B. Soy protein C. Soy protein isolate concentrate isolate (p.v.p.) ~SI in 0.2 M NaCl 39.5 20.3 25.6 NSI in water53.9 25.1 28.6 ~mulsifying capa-city: ml oil/g 21B 182 354 (N x 6.25) Whipping expansion % 120 120 340 Fo~n stability ~ 50 50 20 Gel strength;rpoise3 80C (0.5 M NaCl) 1.7 x 101.2 x 104 3.3 x 102 100C ~0.5 M NaCl) 2.0 x 1044.0 x 10 1.3 x 104 ~ ' ~.~

~ 37(~
Example 4 (application example) A p.v.p. was produced accordiny to the ~rocedure described i~ exzmple 3 C except that the cellulase acti~ity was partially derived from Trichoderma reseei. The commer-cial cellulase preparation CELLUCLAST ~produced by Novo In-dustri A/S was treated with a bzse at low temperature in the following manner. The pH value of a 10% CELLUCLAST solu-tion in water was adjusted to 9.2 wit~ NaO~, znd the thus resulting solution was cooled to 5C. After 1 hour zt this pH and this temperature the pH wzs re-adjusted to 4.7 with 20% acetic acid. This solution was kept at 5C overnight and then sterile filtered. The filtrate was free~e dried. 4 g of the freeze dried proluct wzs added together with the CDS-ase pLe~cLion K~- 68 BIII (~xam~le 1). The tw~ enz~es were solubilized in 17~ g of water before addition to the washing mixture. The mass baiance determinations of this example is shown in iable 4.1.
....

The experiment demonstrates that this p~;~ll~r SPS-ase prepara~ion already contains an ef,icient cellulase as addition of CELLUCLAST does not seem to effect the proteinj . dry ma4ter ratio. However, other SPS-ase pre?arations may contain less cellulase, eOg. RRF 92, vide the table immediatel~
preceding~example 2.
I

~~ Tf ac~ ~C,~k _ 59 ~ 7~

Table 4.1 Mass balance determinations ~-t~ iso-electric wash including an SPS-ase preparation and CELLUCLAST
for production of p.v.p.

Mass of Protein Dry matter Yield of Yield o~
Operatiorls and fraction % % protein d~y mztter fractions gram (N x 6.25) % %
Washing:
Soy flour 425.8 5S.2 94.0 100.0 109.0 Wzter 3546.2 0 0 0 0 6 ~ HCl 43.1 0 21,3 0 2.3 SPS-ase: I~R~-68-B-III 24.0 75.3 96 7.7 5.8 CELLUCL~ST 4.0 43.6 96 Q.7 1.0 Centrifugation: ~4043.1 - - -Centrifugate I3382.0 1.9 5.5 27.3 46.5 Solids I 661.0 ~ - -Re-washing:
Solids I 661.0 - - -Water 3339.0 0 0 0 0 6 N HCl 0 0 0 0 ~nd centrifugation: ~ 4Q00.0 Centrifugate II3414.0 0O2 0.7 2.9 6.0 Svlids II 582.0 - - -~eutralization: .
Solids II 582.0 - - ~ ~
Wa~er 1691.0 0 0 0 0 4 W ~aO~ 2503 0 16.0 0 100 Drying:
Powder 206.0 88.8 98.9 77.8 50.9 , -7~
Example 5 (application example) A p.v.p. was produced according to the method descrihed in example 3 C except that all masses were scale~ down with a factor of 5, and that the reaction mixture was cooled to ah~ut 5GC prior to the cen-trifuga~ion. On the basis of the analytical results in relation to ~he centrifugat~s a theoretical yield of pre~ipitated protein is obtained, as sh~n in table 5.1.

~ble 5;1 l~eoretic~l pro~in yields ob~ned in the p~ction of ~.v.p.
~1 M Protein Yield of ~xample 3 C
Fractions ass tN x 6.25) protein Proteln Yleld of ~ ~ ~ (N x 6.25)protein,%
Soy flour 85.2 55.2 100 55.2 100 SPS ase ~ 68 B-III4.8 75.3 7.7 75.3 7.7 ~ . .
1st centrifugate6390.99 13.5 i.7 24.7 2nd centrifugate5950~13 106 0 2 2.9 p.v.p. ~8702a 92,6b 87.1 80,1b a Ayerage of 87.~ (Bioteknisk Institut) and 86.9 (Qvist's Labora-' torium); dry ~atter is g7~6 and-98.0~, respectively.
~a~çula$ed as total mass of pro~ein - protein lost in centri-fugates. -Example 6 (application example) Demonstration of the ~rotein bindin~ of SPS
_____________________ ________ _____ 40 grams of (N x 6.25) from a ~ommercial soy protein iso-Late (Purina 500 E from Ralston Purina) was dissolved in 680 g of water. The ~ixture wzs heated in a water bath to 50C, and p~ was adjusted to 4~50 with 6 ~ HCl. 90 g of this tnix~ure was transferred 1~87~
to 5x 250 ml Erlenmeyer flasks, and 10 g of aqueous solutions con~
taining respectively 0 g, 0.2 g, 0.4 g, 0.8 g, and 1.6 g of the SPS produced as described previously in this specification Wa5 added. The flasXs were then held under stirring with a magnet in a water bath at 50C for 240 minutes.

Hereaft'er the slurries were centrifuged at 3000 x G for 15 minutes, and the centrifugates I were analysed for Kjeldahl-N
and dry matter. The solid phases were washed in water at room tem-perat~re and re-centrifuged. This procedure was repeated. Then the soIids were dispersed in 50 ml of water, and pH was adjusted to 6.50 by drop-wise addition of 6 N NaO~. The neutralized products ~re f-reeze ~r-ed and analysed for Kjeldahl-N and dry matter. Based on th,e analysis shown in Table 6.1, the protein recovery and the percentage of SPS which has been bound to the protein are czlculated ~y mean~ the ~m~las shown in relation to Table 6.2.

'Th'is eXample demonstrates that the SPS is bound firmly to the protein, s,o, that the protein/dry matter ratio decreases with in-creasin~, c,ont,e~t of SPS. An S~S content comparable to about 0.4 g i~ 10 g Oc water added to 5 g of protein isolate is the proteinjSPS
ratio present in the soy flour.

he:% ~in~ing of SPS is a c~ lated valu~. Ihe ~ h;n~;n~ of SPS de-crea,ses due to saturation of the protein with xegard to SPS at the low, pxotein/SPS ratios.

7~(~
Table 6.1 Measureme~ts according to Example 6 Ratio Centrifugates I Dried precipitate Protein/SPS % N % DM % N % N x 6~25 % DM ~ N x 6-25 co 0.068 0.6213.282.5 93 1 88.6 0.045 0.4913.4. 83.8 97.3 86.1 12.5 0.038 0.4513.0~1.3 97.9 83.0 6.25 0.031 0.4512.678.8 98.1 80.3 3.125 0.026 0.6111.873.8 97.9 75.3 Table 6.2 Protein recovery and ~ binding of SPS

~atio ~ recovery of protein1) % binding of SPS2) Protein/SPS
o~ 91.5 94.4 77 12.5 95.3 90 6.25 g6.1 70.
3.125 96.8 60 ~ reCovery of protein = [~ _ NC 1 x 6.25] 100 h NC 1 = ~ N in centrifugate I

2) % binding o~ SPS ~
r 5x(% xecovery of protein7 5x(~ reo~y of protein7]
L 1% P/~ P~
~ x 100, where [5/ra~io o SP~]
t% P/H) is the proteinjdry matter ratio in the dried preci-pitate, and (~ P/H~o is ! or thP preci~ltate without addition of SPS.

Example 7 (application example) This exa~ple describes the production o~ a p.v.p. u~ing the SPS-ase preparation XRF 92 B-I in a dosage of 5% of the d-y matter. The manner of production was exactly as in Example 3 C, e~.cept that all masses ~ere scaled down ~7ith a factor of 5. The p.v.p. ~Jas analysed as described in Example 2. The results obtained in the experiment appear from Table 7.1.
-Table Z~1 Results ob~ained in Example 7 A~l Mass (N x 6 25) Dry matter :Y.ield of Yield of dry Component g % ~ rotein, mHtter, Soy flour 85.2 55.2 94.0100 100 - Enz~me preparation 4.0 71.2 - 6 1 1st centrifugate 632 1.885.44 25.3 43.0 2nd-~n~ f~g2~ 3 0.30 0.804.3 6.7 p.v.p; 39.8 85.6a 98.la71.9 48.8 84.4b 98.1b Analysed at Bioteknisk Institut, Holbergs~ej 10, DK-6000 Kolding Analysed at Qvist's Laboratorium, Maxselis Boule~ard 169, DK-~000 Aarhus C
Ex~nple 8 (application example) This example demonstrates the effect o pretreating the soy meal by jet cooking before the production of p.v.p.

Pretreat~ent-A slurry ~f soymeal in water consisti~g of 15 kg soymeal (Sojamel 13 produced by A2rhus Oliefabrik ~/S~ per 100 ~g ~was pumped through a steam-ejec~or (type Hydrohea~er B-300~ and mixed with steam of 8 Bar in such amount and by such flo~7 that a inal temperature of 150C could be maintained for 25 seconds in I a tubular pressuxized reactor. ~lereafter the pressure was released J ~ lC~ f~ .

~L98~

in a flash chamber (a cyclone) and from here the slurry ~Jas sent ~hrough a plate heat~exchanger and cooled to about 50C. The cooled slurry could be used directly for production of p.v.p.
according to the inve~tion,but in this case ~he sluxry w~s spray-dried at an inlet temperature of 200C and at an outlet tempera-ture of 90~C. ~ne ~retreatea prcduct was found to have a dry matter c~ntent of 96.5% and a protein content of 56.9% (N x 6.25)~

Production of p.v.p.

This production was carried out in ~he following way:

70 g ofdry matter of the jet cooked and dried soy flour was suspended and ~ept stirred at 50C in 56G g of water,and p~
was adjusted to 4.50 by means of 6.5 mlof 6N ~1. 6 x 90 g of this suspension was transferred to six 250 ml Erlenmeyer flzsks and kept stirred on a 50C water bath by means of magnetic stirrers.
To each flask were added 10 g of a solution containing res~ecti~ely G ~, 0.025 g; 0:050 g; 0.10 g; 0.20 g, and 0.40 g of the SPS-ase pxeparation K~F-68-B-III. The reaction mixtures were khen agitated for 240 minutes at 50C. Then a centrifugation at 3000 x g for 15 minutes was carried out.
' " .
The su~ernatant was then analysed for Kjeldahl N and ~he solid phase was washed with water at equal volumes and centriEuged. This procedure was performed twice. The solid phase was then freeze-dried and analysed for Kjeldahl~N and dry matter.

A similar experiment was carried out with an untreated 50y meal (Sojamel 13 from Aarhus Oliefabrik A~S) as a starting material. In this case the enzyme substrate ratios were 0; 1~;
2%; 3~; 4%; and 8~.

Based on the protein content of the supernatants the percentage of rec~vered protein can be calculated. The yield of protein is based on the assumption that ~he enzyme produc~ is l10~% solubilized after the reac~ion. The ~ahle below shows the Iresults obtained by bo~h experiments.
I

~87~3~
Tzble 8.1 Cooked soymeal ~ Untreated soymeal ~/S % Protei~ Protein of Protein Protein of yield ~ ~ ~atter % yield ~ dryr~tter 0 92.9 76.5 90.7 73.9 0.25 90.1 86.6 - -0~50 89.3 88.7 1.0 ~ 9.7 87.1 ~6.2 2.0 ~6.6 91.7 85.7 88.~
3.0 - - 84.3 8g.5 .0 84.7 92.2. 82.6 90.9 8.0 - - 76.2 91.1 Table ~.l.Pro~ein yields and protein dry matter ratio for p.v.p.
produced frQ~ cooke~ or raw soymeal.

`:;

7~

In relation to either the extraction (isolation) process for materials other t~lan proteins or to the liquefactisn processes and thereto related processes, re~erence is made to the general process scheme for applications as shown in flow sheet No. 3.
The su~strate may be one or more carbohydrates present in a raw material, or it may be the entire raw material.
This su~strate may be subjected to a pre-treatment of chemical or physical nature, as later exemplified, e.g. acid or alkaline treatment, soaking or steeping, and/or cooking with or without steamO
The raw material may be macerated, chopped, ~et milled, and/or homogeniæed (all these treatments being designated homogenization in flow sheet No. 3), with or without water additions, and other additives may be added during this step.
The homogenization may be carried out with different efficiencies, e.g. different pressures being a fraction only of the maximum pressure given for the specific homogeniæer used.
Different additives may be added before or during homogenization, as symbolized by 51~ b2, ...brl in fLow sheet No. 3.
The reaction process including the SPS-ase preparation is carried out under specified conditions, e.g. temperature, pressure, time, pH, and enzyrne dosage; also recommendations regarding the reactor used (e.g. batch, plug-flow) and the stirring, if necessary, are relevant. A set of additives can be given for different raw materials, indicated as cl, c2,....cn in flow sheet No. 3.
Also the separation processes may be carried out wi~h different efficiencies. Vuring many processes the separation is omitted or facilitated, e.g. when the raw material is totally liquefied. Different separation equipments may be used ~e.g.
centrifuges, filters, ultrafiltration equipment, hydrocyclones, thickeners, sieves or screens, or simple decanters).

v~ ~9~37~

The separation efficiency is defined as the proportion be-~ween the ~bsolute sludge content in the solids phase and the abso-lute sludqe content of the reaction mixture.

The liquid or the solid phases obtained may be further treated, e.g. concentrated, dried, or solvent extracted, to remove cextain components like fat or oil, fermented for production o bio-mass/ alcohol, or other products (enzymes, antibiotics., ~r other valuable components).

Also the obtained products may be returned to repeated ' I treatment through the process scheme.

( ~" ~

8~

FLOW SHEET NO. 3 ¦Substrate ,a2 . >¦ PretreatmentS

b ~
~b2 ~ Homogeni2ation ~ Hom 0 - 100%) bn~
SPS-ase c~ prepara~ion ~ ~
.C2 ~ Reaction ~Reaction conditions to be cn specified) ¦ Homogenization (as above) ¦

¦ Separation ¦ ~ S~p = ~ 100%) Liquid Solids Further treatments (see text) or renewed usage as substrate 7(~

In the following some examples of applications of SPS-ase preparations are given, and a survey oE these applications appears from the following list.

Also in the adjoining Ta~le I certain charac~eristics in relation to flow sheet No. 3 are listed.

- 70 ~

~198~

Lise of exemplifying applicationS of SPS-ase pre?arations , .

Kind of Refe-sPS-ase pre- rence p ra~ion No.
SPS-zse pre~ A 1 Extraction of starch from corn, wheat and poea~oesparation es- ~ 2 Extraction of lipids from p~ant material senti~lly A -~ Extraction of etheric oils-from plant m~teri ls free of one A: ': Extraction of natural coloring agents from pl~nt or ~ore un- materiAl wanted en-zyme hCtiVi- A ; Extrac~ion of rubber from the guayul bush tL~s ., ~otal sa 1 Production of a ~ilk substitute for domestic animals, lique- Ba 2 Production of saccharified starch containing raw faction materials 'or Ba 3 ~otal liquefaction of pears ~nd other fruits Un- si~ilar Ba 4 Production of juice by trertment of f_uits and mo- ~reat- ~egetables --ai- ment Ba 5 Treat~ent in relztlon.to extraction,,or press$ng fied of sugir cane o~ sugar bee~
SPS- Bn 6 Production of soy mil~
psre_ Ba 7 ~reatment in order to increase the recoverable amoune of coffee solubles ra-ti- Pro- Bb 1 Prevention and/or d~ ion o~ appl2 haze ons ces- Bb 2 Use as a clari~ying aqent for whlte wine sinq Bb 3 Production of ISSP~ or other vegetable protei~
aids hydrolyzates Bb 4 Mashing enzyme in the brewing indus~ry Bb S Enzymatic additive for use during beer fermentation and/or storage Bb 6 Almond skin release agent oeher sc 1 Decomposition of different waste materials appli- Bc 2 Saccharification and simultaneQus fermentation cations Bc 3 Decomposition of cellulose Bc 4 Application as a baking aid Bc 5 Improvement of alcohol yield and yleld of bio-mass during fermentaeion of sulfite llquor from paper production Bc 6 De~ater~ng of biological sludge products Bc 7 Silage aid ~ ~ c~
o c~
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o o ~
o o 0 o r~ J ~
C
- I r ~ ~ h O ~ I O O
a ~ ~ ~ ~ J
L D '. >~ ~) ~ J a~
~, ~ O n ~ -~ Cl ~ r ( j n ~ ~ rd ~ 4~ ~
C~ ^
Cl J _~
-I r~ ~ r I ~
r-/ O
' C) >~ + ~ ~ ~ ~ ~
D ~1 ~-1 n a O h C ~1 ~ N
O .) ~ 3 P~
.

~ ~P O
I O C4 n o O
o o o O
o l~ ~p o ~`

~ .
:~ ~P O
O O
O I O O l ~ ~-1 ' O 1 .
~ ' O

O ~ ~ O ~ ~ C) O ~I O ~I
~~d O t~ O t.t :~ a~ t-l O 1~] h t) Z ~ Z ~ Z O ~ Z 0 Cq I I
R
~:t ~ V V 1) .V
D
~( ~ O I ~ ~ 3 :~1 3 t)~ .
Q~ , ~ ~a ~" 3 ~ w Sl h h ~ O 11) 1~ (D ~ a3 t11 ID ~ l Cl ~D
,~ _1 0 0 ~D O r~l ro 1~ 3 0 0 ~ O O ~ (D :~
U ~ ~cq .~ ~J Cl .4 ~J Cl E~ h r~
ec m m m m 87~

A 1 Extraction of s~arch from corn, wheat, and potato~s.

Extraction of starch from corn, wheat, potatoes, and other starch conta~ng plants is rArr;ed out by one or more of the steps: steeDing, we~m;il;n~- and ~eparation. P~plication of an SPS-ase PreParati~n with essentially no amylolytic activity will proYide the following advantages , corn being used as.an example:
: 1. The starch liberation will be ~acilitated during . a shoxter steeping period, 2. The water consumption can be reduced, .
3. The liberation of corn germ would be facilitated without liberation of corn germ oil, 4. The protein can be obtained in a higher purity/

5. The recovery of corn steep water will be facili-tated.

,\lr,~/o ~ '7~

A ~. Extraction of lipids from plant material.

As the lipids in plant materials are trapped inside the cells and usually bound to proteins, lipids may be extxacted in aqueous phase by treatment with an SPS-ase preparation ~7hich is essentially free from lipases. Thus, corn germ oil normally is isolated by hexane extraction of the dried corn germs. However, the drying operation is superfluous if the wet corn germs are treated with an SPS-ase prepaxation ~f the above indicated kind. Li~ewise, the extraction of olive oil in aqueous phase can be i~lproved, if the enz~me used for the enzymatic treatment is an SPS-ase prepara-tion of the abo~e indicated kind, vide e.g. Food, Pharmaceutical and Bioengineering, No. 172, vol. 74, p. 93 - 94. Also, aqueous ex-traction of e~g. soy oil, rape seed oil and sunflower oil may be improved in a similar manner.

A 3. Extraction of etheric oils from plant materials.

~f vegetable materials containing etheric oils are ~a~ted _ with ~n a~u~m1~ solution of an SPS-ase preparation which is essentially free from enzymatic activity capable of decomposing or otherwise changing he etheric oils, the etheric oiis will be recovered in high yields at a very low cost.

A 4- Extraction of natural colouring agents from plant material.

If vegetable materials containing colouxing agents, e.g.
beetroots containing the red colouring agent bet~nin ox the colour-ing agent in cranberries, are treated with an SPS-ase preparation whlch is essential~y free from enzyme activities capable ~f decompos-.ing ox otherwise changing the colouring agents, the colouring agentswill be recovered in high yields at a very low cost~

N O ~/O ~ ~B~

A s. Extraction of rubber from the guayul bush.

Another ~xample of a substrate for an SPS-ase preparation which is essentially free from enzymatic activity capable of de-grading native rubber is the cell wall material in roots and ~r~nch-es of the guayul bush.

a 1. Production of a milk substitute for domestic animals, pre'erably a calf milk substitute.
By a total liquefaction in aqueous medium of soy beans, sunflower seeds, cotton seeds, faba beans, or field peas a calf milk substitute which is soluble in cold water at a pH value of arsund 4.5 can be produced. Using the starch containing raw mate-rials like faba beans or field peas a starch liquefa~tion by mezns of an alpha-amyl~se has to be A~cnmp1;s~ed before, after or s~taneously ~th a ~P~t with an .SæS-ase which finally solubilizes the non s~arch polysaccharides present 2S the structural material in the cell walls. A detailed example is shown below using fab2 beans, and as xegards soy beans !-reference is made to table I. The pre-tre~tment of said soy beans ma~ preferably be a jet-cooking which improves the solubilization of the rP~nence.

Example Ba 1.1 15 kg of faba bean flour (Farine de Feves from GRANDES MI-NOTERIES A F~VES DE PRANCE, Paris ) were suspended in 35 litres of water. 75 g of Termamyl 60 L an~ 18 g of CaCl2 were added. The su-spension was heated to 95C using a steam jacketed vessel while stirred. The suspension was then treated at ~his temperature or 60 ~inutes. Hereafter pH was adjusted to pH - 4.5, and the product was cooled to 50C. 300 g of ~he SPS-ase preparation KR~ 68 was solubilized in l litre of water and added. The reaction was carried out for 440 minutes.

Sl.~

87(~

~en 10 g of Fungamyl 800 L is included the starch fraction mainly will be converted to disaccharide (maltose). He~eater the reaction mixture was pasteurized at gOC for 2 minutes. An ali~uot of the product was then free~e dried and used for stability tests. The sample was then solubilized at 10~ dry matter, and the solution of the product could be kept stable without sedimentation for days.

~ elted ~at or oil may easily be emulsified in the product whereby a final com~osition very similar to cow milk may be obtain-ed. An emulsion containing 3.5% oil (soy bean oil) could also be kept stable without sedimentation for days.

Soy flour (Sojamel 13) was jet cooked at l50C for 25 seconds as aescribed in example i. The jet cooked soy flour was spray-dried and used for further studles described in the following.

A:
50 g o~ the jet cooked soy flour was mixed with 450 g of water, and pH was adjusted to 4.5 with 4.1 ml 6N HCl. The mixture was then heated to 45C in a water bath, and 0.250 g of the SPS-ase preparation KRF-68 was added to the heated mixture which was then reacted for 5 hours with stirring.
Thereafter the mixture was heat treated at 80C for 2 minutes in order to inactivate the enzyme. A lO0 ml sample was centrifuged at ambient temperature for 15 minutes at 3000 x g (g = gravity). The supernatant was ion-exchanged and analyzed for carbohydrate composition by HPLC. Also the supernatant was analyzed for Kjeldahl-N and dry matter and the nitrogen solubility index (NSI) and the dry rnatter solubility index (DSI) was calculated; vide results in table Ba I lO0 ml of the reaction mix~ure cooled to 20C was poured in a lO0 ml graduatea glass and kept at 4C for two days. The dispersion stability (%) was mea~ured by reading the volume of the r~ lovo 119871~o dispersions obtained ~tahle BaII) after 1 and 2 days.

To 200 ml of the reaction mixture (at 20C) was added 8 g of soy bean oil. An emulsion was made by blending for 2 mi-nutes in a Waring ~lender. The emulsion stability ~%) was measured as above after 1 and 2 days.

-A reaction was czrried out as above, however, in thiscase 1.00 g of the SPS-ase preparation was used. The same types of analyses and stabili~y measurements as ~escribed in section A
were performed. Results are shown in tables BaI and BaII.

It appears from ~he chemical analysis of the supernatants that the values for NSI (~) and DSI (%) obtained at the B expe-riment is higher than for the A experiment. However, the stabi-lity tests carried o~t on the reaction mixtures show a better value for the A samples. This probably is due to the higher pep-tide chain length of the proteins in the reaction mixture with the low enzyme dosage.

From the carbohydrate composition measured by HPLC it appears that mainly mono- and disaccharides are produced. Thus oligosaccha-rides known to be responsible ~or diarrhoea and flatulence ~hen given to calves in too large amounts are present in small amounts only.

DtJ

N O\/L~ 37 ! ' I
Table B~ Chemical properties of the supernatant.

En~yme dosage in ~xperi- relation NSI, % * D5I, % ** HPLC results ments to sub- (at pH = - (at pH = (neutral sugars strate 4.5) 4.5) cornposition) % w/w DPl + DP2 79-7%
A E/S = o 5% 39 9 62.4 DP3 12 2~
DP4+ : 8.1-%

DPl D 2 B E/S = 2.0% 67.1 DDp3 6 1%
DP4+ : 5.7 * NSI = Nitrogen Solubility Index ** DSI = Dry matter Solubility Index Table BaIIstability tests of the reaction mixtures.
Enz~yme Stability test dosage in Without oi_ With oil Experi~ relation ments to sub- Disperslon Disperslon Emulsion Emulsion strate 1. day 2. day lo day 2. day % w/w A E/S = 0.5~ 80% 63g 100% 87%
_ _ _ B E/S = 2.0% 66% 35~ 85% 71%

Dl.~

r\ o~o ~ 7LP~

Ba 2. Production of saccharified starch containing ra~ mat~rials.

In relation to the saccharification of cassava and s"eet potatoes and other starch containing plant materials addition of an SPS-ase preparation is capable of solving viscosit~ problems. By using an SPS-ase prep~ration it is possible to produce starch su spensions with a dry matter content of 25-30%, and after sacchari-fication the mash may be fermented whereby a cheap et~anol may be obtained.

Example Ba 2.1 On the basis of fresh and grated sweet potatoes (Japanese) a mash with 2 dry matter content of 24~ was produced. The starch content of sweet potatoes was fo~md to be approximately 70% of the dry matter conten~ hereof. A pre-1iquefaction by ~eans OL the b ~ OEial amylase of Termamyl ~ 60 L in a dosage of 0.5 Xg/ton of starch was performed by heating the mash to 90C. The mash was then held at 90C for 30 minutes. The viscosity ~1 of the reaction mixture was then measured by means of a H~KE spindle at 90C.

~ ereafter the reaction mixture was cooled to 55C, and pH
was adjusted to pH 5.0 with 2N H2SO4. A sacchzrification was then initiated by additlon of the gluco-amylase SAN 150 ~trade mark from NOVO INDVSTRI A/S) in a dosage of 1.75 litre/ton of starch The saccharification mixture was then divided into three parts, A, B, and C, which were enzyme treated for 15 minutes as shown below before measurement of viscosity:

A: This part is the control. The viscosity ~ was measured, vide table B Q 111 B: The Tricoderma viride cellulase of Celluclast 200 N was added in a dosage of 1 kg/ton of dry matter of sweet potatoes. The viscosity ~3 was measured, vide table . B ~l~l a - J

~lg~37~

C: The SpS-ase preparation of KXF-68 was added in a dosage o~ 0.25 kg/ton of dry ~natter o~ sweet potatoes. The viscosity ~ 4 was measure~, v le table Ba III

Table ~a III Viscosities Viscosity Viscosity Reaction mix'ure at 90C at 55C

Pre-liquefied = 770 cp ~ = 2190 cp sweet potatoes ~1 . 2 A ~ ~2 = 2190 cp B ~ ~3 = 1970 cp C _ ~4 = 950 cp ,~

Thus it can be seen that the viscosity of the reaction mixture could be effectively reduced with the SPS-ase in a low do- -sage compared to the Celluclast ~ and SAN 150.

r~IOVC~ 8~

Ba 3. Total liquefaction of pears and other fruits.

If whole pears which are mechanically crushed are subse-quently treated with an SPS-ase preparation, total liquefaction takes place, and a clear pear juice is yroduced after rernoval of minor amounts of solid matter. A similar method can b~ used in re-lation to other similar fruits, e.g. apples.

Example Ba 3.1 Fresh apples were c02rsely milled by means of a Bucher Cen-tral mill. The apple mash wzs then pasteuri~ed in 2 heat jacketed tank at 90C for 5 minutes and then cooled to ambient temperature.
The pre-mashed apples were then milled on a Fryma mill with corund stone outfit until the mash W25 smooth to the touch. The mash W2S
then re-pzsteurized at 80C for 10 minutes, and cooled to 50C.

Enzyme react~ons were now carried out at 50C for 30 mi-,nutes with the Contra~es Rhéomat 15, stirring and viscosity measure-ment (in relation to a percentage reading on the Rheometer at speed 13) being carried out simultaneously. After completion of the enzyme reactions 100 g of samples were drawn out and centrifuged in a gra-duated tube at 3000 x g for 15 minutes. ~ereby the percentage of juice and the percentage o r deposit are measured. pH and the perc~ntaqe of refractometer dry matter 2s Brix were also measured. Table Ba IV sh~ws a comparison between the effect of SPS-ase, the combination of Cel-luclast and SPS-ase, and the combination of Celluclast and Pectinex~
The SPS-ase preparation KRF-68 was used~

a t3 r~vo ~987~0 T~ rV Results of total liquefaction experiments with apDle mash at 50C for 30 minutes SPS-,~se Celluclzst ~ viscosity Centrifugatio~ Juice g~hl ~ash g/hl mzsh g/hl mash ~ juice ~ de~osit p~ Brix.

0 0 0 100 59 ~1 3.8 9.7 ~5 0 0 19 81 19 3.5 10.5 S0 0 0 15 79 21 3~5 10.7 43 8 ~3833 17 3 1 12 5 0 50 200 9.5 83 17 3.2 12.9 0 ~0 2000 4.0 82 18 3.1 13.2 ~1~

r~lovo ~a 4. Production of juice by treatment of fruits and vet3etables~

It has been found that SPS-ase preparations are well suited for production of juice by treatment of several fruits, berries and vegetables, e.g. carrots, peas, tomatoes, apples, pears, black cur-rants, beans, and cabbage. Hereby an improved ~uice yield, and be~-ter extraction of colour and flavour components are achieved compared to commerci211y available pectinase and cellulace preparations.

Example Ba 4.1 Reference is made to example Ba 3.1 where the SPS-ase pre-p2ration hzs bP~ com~ared to ~he c~",~ially available conventional c~ 7l~Se andp~i~se pro~s Celluclast ~ 200 L and Pectinex ~ 3x. It appears from the table that the juice yield may be slightly improved wi'h as little as 50 g/hl OL SPS-ase compared to 2000 g/hl of Pectinex ~ , both in combination with 50 g~hl of Celluclast ~ . Also the viscosity was slightly lower. ~hus it seems that the SPS~ase is about 40 times more effective than Pectinex.

a 5. Treatment in relation to extraction or pressing of sugar czne or sugar beet.

It has been found that it is possible to improve the yield related to simple extraction processes, if the SPS-ase preparation is used for treatment of sugar cane or sugar beet before and/or dur-ing the extraction or pressing thexeofO ~lso, the remanence (the bagasse) can be treated with the SPS-ase preparation, whereby i~ is partially converted to fermentable sugars which may be used as a raw material for ethanol fermentation.

D~-~

~ vo ~9~

Example Ba ~.1 10 kg of sugar beet remanence (pulp~ obtained from con-tinuous countercurrent extraction in a DDS-di~fuser at Nakskov Sugar Factory was milled twice in a Pryma mill (type MZ-llO).
Processing water was added during the milling operation.

300 g portions of the pulp were now enzyme treated at 450C fox 18 hours by means of the enzyme dosages shown in table Ba V
. The dry enzyme product (KRF 68) was added to the pulp which was stirred by a rod during the first hour. Thereafter the pulp was liquefied to such an extent' that magnet stirring could suc-cessively be performed for the remaining time. At the end of the reaction pH was measured (no pH-corrections were made during the_star* of the reaction) and the reaction mixture was centri-fuged until a clear supernatant was obtained. Dry matter deter-~i~ations were performed on the reaction mixtures and on the supernatants. Based on these results the percentages of solubi-lized dry matter were calculated. Corrections for the soluble dry matter of the enzyme product were made in all calculations.

Sup~tants ~os. 2, 3, and ~ were ionexchanged and ~ysed by HPLC for carbohydrate composition.

7(~
o\~o Table Ba V Results obtained by enzymatically liquefaction of beet pulp .

Final measurements Experi- ~nzyme Reaction mixture SUpernatdntS
NOnt in r~ela- pH ('i- ~ dry ~ dry ~ solubi-tion tonal)matter matter lized dry dry mat- matter ter, E/S %
5.5 4.18 0_0 0.0 3.85 2.58 66.9 0,563.5 3.81 2.56 56.2 1.023.5 3.86 2.73 70.4 1.583.3 3.17 2.34 73.4 6 3.103.4 3.23 2.49 76.4 -7.52 3.4 2.66 2.1R 80.5 eaction conditions: M = 300 g S = 4.18% dry matter E/S as sho~n above p~ not adjusted T ~ 45~C
t = 18 hours r~ovo ~ 9~37(~

Table Ba VI. HPLC da-ta.

E~periment No.
Sugar type 2 ¦ 3 ¦ 4 (Neutral) g of neutral sugars (DP4+) 43.6 31.9 25.3 Disaccharides 4.6 4.8 Glucose 20.4 23.7 27.8 Galactose . 5.0 5.9 7.3 Fructose/Arabinose 26.4 , 32.2 33.2 Galacturonic acid not measured i~
All the sugars formed according to the abo~e table Ba VI
could be fermented ~o alcohol or used for other purposes~

Ba 6. Production of soy milk.

Soy milk can easily be produced by total liquefaction of milled soy beans and subsequent homogenization of the resul-ting mixture Soy milk is often produced by soaking of soy beans in boiling water , milling of the soa~ed beans and water extrac-tion foliowed by a separation of insoluble residuesf e.g. pro-o ~87~

teins and polysacchzrides. In order to improve the yield of the soy milk these insoluble residues may be liquefied by reac-tion with the SPS-ase.

Example Ba 6.1 The soy milk process is illustrated by the following series of enzyme reactions, whereby calculations of the protein solubility index (PSI,%) and the dry matter solubility index (DSI, %) illustrate t~e yields obtained after separation at pH = 7 (vide Table Ba VII)The enzyme reactions were carried out under the following conditions:

Substrate: Full fa, soy flour (Dansk Sojakagefzbrik A/S~
Mass of reaction mixture: 220 g Mass of substrate: 20 g Temperature: 50C
p~: 4,5 (6 N ~Cl) Reaction time: Series A: 1 hour - - Series B: 0,5 - 6 hours Enzyme: SPS-ase (KRF-68) Enzyme dosage: Sexies A: E/S-ratio (w/w):
O - ~,0~
Series B: E/S-ratio (w/w):
- 1.0%

After the reaction pH was adjusted to pH = 7 by means of 4 N NaOH, and a separation was performed by centrifugation a~ 3000 x g for 15 minutes.

31~

Ta~le Ea VII ~ass balance calculations in relation to the enzymatic soy mi~k process. J
O
Reaction Enzyme- Reaction,mixture Supernatant Solubility indice~
SerieS hours dojsage ~ protein ~ dry matter % prote~n ~ dry matter PSI ~ DSI ~

1.0 0 3.65 8.70 1.87 5.72 49.6 63.7 1.0 0.5 3.~8 8.74 2.41 6.2a 63.7 70.0 1.0 1.0 3.71 ~.78 2.4~ 6.43 65.1 71.4 1.0 2.0 3.78 ~.~6 2.98 7.1~ 77.5 79.5 1.0 4.0 3.92 9.03 3.36 7.69 84.6 i33.9 1.0 8.0 4.19 9.37 3.76 8.08 88.5 B5.0 ~
0.5 1.0 3.6~ 8.7~ 2039 6.41 64.0 71.1 ~b 1.0 1.0 3.64 8.78 2.56 ~.60 68.7 73.4 2.0 1.0 3.63 8.78 2.79 6.91 75.2 77.1 4.0 1.0 3.63 8.78 3.10 7.29 84.0 81.7 g 6.0 1.0 3.63 B.78 3.39 7.69 92.3 B6.5 87~
I\IC:~VO

a 7. Treatment in oraer to increzse the recoverable am~ur:t of co.fee solubles.

It has been found that treatment of coffee beans at different s,ases during tne production of instant coffee results in an increased yield of coffee solubles. Thus, e.g. spent coffee grouncs or qreen beans can be enzyme- treated with favourable results.

~ 1~

r~vo ~9~17(~

Bb l. Prevention and/or decomposition of apple or pear haze.

After production of apple juice or pear juice and other fruit juices, which have to be clefar, and which a~e ~reviouslv trea~d ~7ith ., conventional ~e and cellul~se Dre~aratio~s in order to ~revent f~rma-tion of turbidity, an apple haze or similar fruit hazes may appear.
It has been found that the SPS-ase preparations are well suited for decomposition of such hazes, which mainly consist o. araban bound to proteins.
i Example Bb 1 Pear juice concentrate produced by enzymatic liquefaction of pear cannery waste using Celluclast ~ and Pectinex ~ was found to be cloudy on standing. ~he haze w~s isolated and hydrolyzed with 0.01 N H2SO4 for 24 hours and analyzed by HPLC. The chromatogram showed arabinose and small amounts of oligosaccharides.

By incubation of 0.5~ s/v of this carbohydrate in l mM
acetate buffer at pH 4.5 for 3 hours at 40C with SPS-ase (K~F-~8 ~ DRF-92 l:l) with an enzyme concentration of 0.05~ w/v it was found that 84% of the initial haze carbohydrate (dry matter~ was converted to arabinose.

Also diluted pear concentrate (20Brix) was treated for 2 hours at 40C with an enzyme dosage of the above mentioned SPS-ase of 0~15% w/v or with a commercial product called Clzrex ~ in a do-sage of 1~ w/v. It was found tha~ the SPS-ase was able to reduce the relative HPLC peaX area of an araban-like haze with 86~, whereas ~he corresponding reduction with Clarex ~ (used in a much higher dosage than the SPS-ase preparation) was only 78%.

o~., c ~ 7~

Bb ~. Use 25 a clarifying agent for ~hite ~ine.
It has been found that white ~ines e~hibiting a highly undesired turbidity can be eCfectively clarified b~ means of SPS-ase. It has been sho~ that the cloudy mate-rial mainly consists of arabinogalactans wh-ch ~re ~ound to hydro~yprolin residues in a cell wall structural protein.
Bb 3. Production of ISSPH or othe~ vegetable protein hydrolyzates.

Previously to the separation of the ISSPH (isoelectric soluble soy pro.ein hydrolyzate) or other veget2ble p~otein hydroly-zates from the sludge as described in ~S patent No. 4 100,024 or in Process Bioche~istry vol. 14 No. 7 (1979), pages 6 - 8 and 10 - 11 the re2ction mixture can be treated with an SPS-ase preparation.
Hereby an e2sier sep2ra.ion is obtained.

:~9~7~
~ 'O~fO

Bb ~. Mashins enzyme in ~he brewing industry.

In production of beer car'oohydra.es of the raw rnaterials, e.g. the beta-gluczns of malt and barley, influence the viscosity an~A fil'erability of the wort. Addition of S~S-ase duriny mashing wil,l reduce wort v~scosity and im~rove filterability and e~tract yield~ Furthermore addition of SPS-ase during mashing will in-crease the fermentability of the wort and the nitrogen content in the wort.

Example 3b 4.1 In the laboratory 50 g of milled grits consisting of 50% malt and 50% barley were mashed together with 275 g of water (15% dry matter) according to the following mashing diagram: 52 C (60 minutes)/63 C (60-minutes)/76- C (30 minutes).

In order to demonstrate the effect of the SPS-ase four tests were carried out, vide the below indicated table, where~y the enzymes were added during mashing in (pH of mash 5.5 - 6.0).

~.., ~ll9B7(~0 ~30\/o Enzyme NoneCereflo SPS-ase (KRF 68) Activity of o 200 BGU 1630 FBG 1630 FsG
beta-gluca-nase/g Dosage of 0 1 5 g 0.05 g 0.18 g enzyme per kg grits Total dosage 0 300 BGU 80 FBG 300 F~G
of enzyme ac-tivity units per kg grits Filtration 120 ml 135 ml160 ml 170 ml rate of wort after 10 minutes Viscosity of 1.52 c~ 1.36 cP 1.36 cP 1.30 cP
wort 10 Bal-ling (25 C) BGU is beta-glucanase units determined according to analytical method AF 70/4-GB obtainable from NOVO Industri A/S.

FBG is fungal beta-glucanase units determined according to analytical method AF 70.1/2-GB obtainable from NOVO Industri A/S.

The only difference between BG~ and F~G is the pH,.
at which the enzyme determination is carried out: pH 7.5 for BG~ and pH 5.0 for FBG.

C Cereflo is a bacterial beta-glucanase preparation described in the information leaflet B 214b-GB 1500 July 1981 available from ~OVO Industri A/S.

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

In order to demonstrate the effect of the SPS-ase thr~e tests were carried out, vide the below indicated table, wher~by the enzymes were added during mashing in (p~ of mash 5.5 - 6.0).

iovo ~l 37{~

SPS-ase (KRF 68) ~
Enzyme None Ceremix Ceremix added as inprevicus tr~-,t Activity Oc beta- - 200 BG~ 1630 FBG
glucanase/g Dosage of enzyme - 1.65 g 0.033 g per kg grits Total dosage of 0 330 BGU 50 FBG
enzyme activity units per ky grits Filtra~ion rate of 48 ml g8 ml 111 ml wort a:Eter 30 minutes ~xtract, ~Balling 18.6 19.0 19.5 Viscoslty of wort 1.72 c~ 1.37 cP 1.27 cP10 Balling ~25 C) The definition of BGU and FBG is as indicated in Example Bb 4.1. In the last column in the above table only the activity and dosage originating from SPS-ase is indicated.

Ceremix is a bacterial beta-glucanase preparation described in the informa~ion leaflet B 21~ b-GB 1000 Feb. 1982 zvailable fro~ NOVO Industri A/S;

~ 5. Enzymatic additive for use during beer fermen~ation anc/or storage.

SPS-ase can be added during fermentation of wort or storage of beer in order to ~educe the content Or beta-gl-~cans and thereby improve beer filtration anci beer stabili~y in xegard to haze. SPS-ase ~-i'l also exert an effect on pro~eins respon-sible xor chill haze.

Bb 6. Almonci s~i,n release agent.

~ uring the mechanical almond skin removal step following the blanching o' almonds a ce.tain percen.age of the almond skins are not released. It has been found that enz~me trea~ment of the alm~nds results in a decrease of the above indicated percentage.

~L~g~37(~

Bcl. Decomposition of different waste rnaterials.

In relation to certain manufacturing processes larye amounts of carbohydrate containiny waste mdterials are formed. For instance this is the case in relation to production of soy isolate by water extraction and acid precipitation, soy milk and tofu (a special kind of Japanese cheese). Also in this connection waste pulp from e.y. apples, pears, or citrus fruits may ~e ment~onea. It has been found tnat an SPS-ase preparation is able to liquefy this carbohydrate containing waste material completely and to produce fermen~able sugars which can be used as a starting material for ethanol fermentation.

7~3~
~vo I

Example Bc 1.1 ~ y traditional production of soy milk or tofu soy beans are often soaked in boiling water, milled and extracted with hot water, whereafter a separation is carried out. The residue from this separation is the material used ~or this experiment. The liquid phase is the soy milk, which may be ~urther treated to produce tofu.

10 kg of whole soy beans obtained from Aarhus Oliefabrik A/S was milled simultaneously with 70 litres of boiling water in a Fryma mill type MZ 110. The milled slurry was then held above 85C for 15 minutes in order to ina~vate the natural bean enzymes which develop the well-known soy bean off-flavour. 5 litres of this soy bean slurry was then centrifuged in the laboratory for 15 minutes at 3000 x g (g = gravity). It was found by analysis that the remanence contained 20,45~ and 20.06% dry matter (dupli-cate determinations, calculated average 20.26%).-6 N HCl was slowly added and worked into the r'emanence with a spatu-la until a pH-meter showed 4.50, when the electrode was introduced directly into the mass.

Enzyme reactions on 2 x 200 g of the mass with the two dosages of the SPS-ase (KRF-68) E/S = 0.5% in rela-tion to dIy matter and E/S = 3.0% in relation to dry matter were carried out in a 500 ml beaker at 50C. The dry enzyme was added to the mass. During the first 1 - ~ hours the stirrîng was carried out with a spatula and hereafter the mass was liquified to -such an extent that stirring with a magnet could be carried out successfully. The total reac-tion time was 21 hours. During the reaction the osmolality was measu-ed with an osmometer (Advanced Digimatic 3DII
from Advanced Instruments Inc.). The results in table Bc I

~, ~

f\10~0 ~9~37~3~

( show the course of the reaction. At the end of the experi-ment the miY~tures were centrifuged at 3000 x g for 15 minu-tes. A layer of oil appeared on the top of the supernatants and the volume thereof was determi.ned. A loose layer of sludge appeared as a bottom-layer. The supernatant including the oil ~as removed with a pipette. The oil was combined with the clear water phase by homogenisation and a sample was drawn for Zry matter determinations. The results shown in table Bc I clearly demonstrate, that this ~aste product can be llquefled by an enzyntatic reaction and that crude oil can be produced as mentioned in section A4. After reco-very of the oil the solubilized remanence mày be used in different ways, e.g. for fermentation to valuable compounds or for concentration and drying and subscquent use as a fodder or food product, or after further purification for production of valuable products.
( o Takle BcIResults obtained during liquefaction of soy milk and tofu-sludge.

Reaction conditions and results ~xperiment A Experiment B
Mass of residue 200 g 200 -g Mass of SPS-ase (KRF-68) 0.20 g 1.20 g Temperature 50C 50C
pH 4.50 4.50 Reaction time 21 hours 21 hours osmo- a OS- osmo- ~ os-Results measured on t lality mola- t lality molali-^s~cmete~ ~uring mln- mOsm lity mln- mOsm ty . mOsm mOs~
the course of the reaction. 0 287 0 0 282 0 ~ Osmolality is - - - 25 497 215 the value corrected 40 391 104 45 601 315 for the osmolality 2~0 ~ 7 2~ 8~58 ~36 of the mixture at1260 907620 1260 1145 863 t = 0 Reaction mixture:
Dry matter 20.3 ~ 20.7%
Supernatant:
Dry matter 18.0% 19.4 Supernatant:
Oil content 8 - 10% 8 ~ 10%
Calculation %solubi-lized dry matter88.6% 93.5%

0l ~

1~987~

Bc2. Saccharification and sirnultaneous fermentation.

Carbohydrate containing plant rnaterials, e.y.
tubers llke Jerusalern artichokes, potatoes, sweet potatoes, cassawa, or pulp froM such tubers, i.e. the material remaining after removal of the extracted cornponents may be saccharified by treatment with an SPS-ase preparation, and sirnultaneously the formed fermentable saccharides may be fermented to ethanol.

,, .

r~Jo\fo ~g87~

The production of ethanol by fermentation of the decomposed inulin contailling Jerusale~n artichokes was e~amined in laboratory scale by simultaneo~s saccharification with SPS-ase and inulinase and by four different pretreatments of the Jerusalem artichokes.

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

Inulinase: The inulinase was produced by fermentation of ~sp. ficuum ~C3S 55 565). The inulinase activity is determined as described in Research Disclosure No. 212~4 (December 1981) p. 456 - 458.

Laboratory fermentation: 150 g portions o. the pretreated mash (described later) were fermented after addition of 4.5 g of bakers yeast and 1 ml of a 4% solution of ~luronic as an antifoaming agent. The fermentation flasks are provided with CO2 traps containing 98~ sulphuric acid, and the fer-mentation is followed by measurement of the weight l~ss due to liberated CO2 . The content of the flasks is agitated during the ferment~tion carried out at 30C. Three flasks were used for each parameter studied.

In table Bc II the weight loss due to libera-tion of CO2 is converted to ethanol assuming that 1 mol liberated CO~ is equivalent to 1 mol C2~5OH, i.e~
1 g CO2 ~v 44 g C2~50 Pretreatments of the artichokes~
~eatment A: 14.1 kg artichokes (22.8% dry matter) was Henze-cooked at 140C and 4 - 5 atm. for 20 minutes.
The weight after cooking ~Jas 19~0 ~g (~J 16.9 ~1~

~o\/o ~L~9870~

dry matter)~ Fermentations were performed di-rectly on the mash.

Treatment B: Washed and sliced artichokes were mi~.ed with water (1:1) and blended in a Waring blender.
The ~ash was then heat treated for one hour at 85C and pH = 4 5.

Treatment C: As B, but pH was not adjusted.

Treatment D: As B, but no heat treatment and no pH adjustment.

Results~ In table BcII the results show the effect of addition of SPS-ase to the pretreated mash on the ethanol yield. A
significant ~mprovement of the ethanol yield was obtained on all pretreated mashed when SPS-ase was added.

( Dl-3 7(~)~
N 0~/0 Table ~c IIFermentation results in relation 'co simultaneous fermentation and enzyme saccharification of Jerusa-lem artichokes.

Inuli~ase units ad-ded to Loss of C02 (g) zfter % ethanol pro-Pre- 1 g of 42 - 44 hours of fer- duced in rela-treat- dry mat- SPS-ase mentation tion to dry ment ter E/S % matter 1.5 0 7.65 + 0.05 31.5 1.5 0~27 8.07 + 0.08 . 33.2 B 1.~ 0 4.85 + 0.03 29.7 0.40 5.41 + 0.03 1.5 0 5.70 -~ 0.05 1.5 0.10 5.97 + 0.01 C 1.5 0.20 6.13 ~ 0.06 37.~
1.5 0.30 6.13 + 0.00 37.5 1.5 0.40 6.18 + 0.05 37.8 -1.5 0 5.77 + 0.02 35.3 1.5 0.10 5,89 + 0.00 36.0 D 1.5 0.20 6.04 ~ 0.11 36.9 1.5 0.30 6.01 + 0.01 - 36.7 1.5 0.40 6.02 ~ 0.03 3~.8 0-40 5.4~ + 0.02 33,5- ~

~1~

7V~
r~ ~ ovO

Bc 3. Decomposition of oellulose.

It has been found that cellulose containing materials like straw, e.g. wheat straw, saw dust, paper, and liynocellulose, rnay be hydrolyzed to a greater extent with an SPS-ase preparation thar with conventional cellulases. This is illustrated by th~ following eY.ample in which a crystalline cellulose material (AVI OE L~ is treated by means of a conventio~al cellulzse Celluclast~ 200 proouced by Tricho-~ma reesei and ~e.5PS-ase prepzration ~ 680 Example Bc 3.1 Avicel was suspended in water (20~ dry matter); pH W2S ad-justed to 5, and the ~emperature was maintained at 50C. After 24 hours reaction ti~e the slurry was filtered, and the content of re-ducing sugar (mg glucose/g AVICEL) was measured. Using enzyme dosages of 5% znd 20~ of the cellulose content the following values ~ere foun~:
Table Bc III

Enz~me E/S ~ mg glucose /g AVI OE L

Celluclast 5 80 SPS-ase 5 - 200 Celluclast 20 100 SPS-ase 2~ 340 Bc 4. Applicati.on as a baking aid.

It has been found th2t SPS-ase preparations are excellent-ly suited as baking aids. Thus, when an S~S-ase preparation is added to the dry flour, before production o~ the dough, it is possible to obtain a bread with superior quality in regard to volume, crumb and taste. Thus it is possible to obtain a high quali~y bread with a low /~-quality wheat flour i~ an SPS--ase preparation is used as an additive. ~

c 5. Improvemen. of alcohol yield and yield of biomass during fermentation of sulphite liquor from paper pro~uction.

Also it is found that the yield of ethanol may be improved if paper sulphite liquor is treated with an SPS-ase preparation be-fo~e it is utilized as a carbohydrate source for fermentation of ethanol. Pzper sulphite liguor may also be used for production of biom2ss, e:g. sirsle cell protein, by fermentation, and also in th~s case the yield of biomass has been improved when the sulphite li~uor has been treated with an SPS-ase preparation previously. Also, decom-position due to the presence of the SPS-ase preparation and fermenta-tion can be performed simultaneously.

Bc 6. Dewatering of biological sludge products.

During traditjonal water extraction of many bi~logical materials from planL raw materials,large volumes of insoluble residue consisting o~ great ~roportions of swelled polysaccharides are formed.
This, for e~ample, is the case when soy milk, tofu,or soy isolate is produced by water extraction of soy beans, defatted soy flour, or white flakes. The structural swelled polysacchaxide material may then be treated to a slight extent wi~h SPS-ase, whereby the network structure of the material is opened, and only slight amounts of car-bohydrates are solubilized. Thereby the material is dewatered, and consequently a higher dry matter content is obtained in the sludge in comparison ~o a product obtained wi~hou~ the enzymatic treatment Thus, the enzyme process exhibits the advantage of a considera~ly lower energy consumption for removal of water by drying, and it also opens up the possibility for produc~ion of a cheaper dry animal feed material or bulking agent for food applications.

~c 7. Silage aid.

It is known ~o add enzymatic silage aids to silage in order to increase the rate of the silage process and the dige-stability of ~he silage. It has been found that SPS ase prepa~
rations are superior in coT.parison to known enzy.m~tic silage aids.

o~-~

87(~(~

A survey of the figures, to which re~erence has been made already, is given below for the p~rpose of pxoviding a better comprehensive viewO

Fig. No. Belongs to Describes 1 ~he general part Demonstration of binding effect of the specif iC2- between SPS and soy protein.
tion 2 The general part Flow sheet describing the pro-of the specifica- duction of SPS
tion 3 Section 2 Calibration curve for ~PLC
gel filtration chromatography . 4 Section 2 ~PLC gel filtra'tion chromato-gram of SPS
S Section ~ HPLC gel filtration chromatosram of SPS decomposed by SPS-asP
6 Sections 2 and 3 HPLC gel filtration chromatogram . of supernatant from SPS incubated with soy protein .
7 Section 2 HPLC gel filtration chromatogram of supernatant from decomposed SPS incubated with soy ~rotein Section 3 HPLC ~el filtration chromatogr2m of APS decom~osed by Pectolyase

9 Section 3 .HPLC gel filtration chromatogram of APS decom~osed by SPS-ase Section 3 ~PLC gel Çiltration chromatogram of SPS treated with Pectolyase 91.a - ~987G~

Fig. N~. Belongs to Describes Section 7 Immunoelectrophoretic peaks includin~ an SPS-ase peak identified by ovexlay techni~ue 12 Section 8 Ion exchange chromatograM of an SPS~ase 13 Section 9 pH-activity dependency of an SPS-ase 14 Section 9 Tem~erature activity dependency of an SPS~ase Sectio~ 9 Temperature stability of an SPS-ase 16 Section 10 pH-stability of protease in an SPS-ase preparation ~' , --

Claims (22)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An SPS-ase, a carbohydrase in a usable form and capable of decomposing soy SPS under appropriate conditions into decomposition products which attach themselves to protein in an aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached itself to the same protein under corresponding conditions.

2. An SPS-ase according to claim 1 wherein the SPS-ase is capable of decomposing soy SPS in an aqueous medium into decomposition products which attach themselves to vegetable protein in the aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached itself to the same vegetable protein in the aqueous medium.

3. An SPS-ase according to claim 1, wherein the SPS-ase is capable of decomposing soy SPS in an aqueous medium with a pH
value not deviating more than 1.5 from 4.5 into decomposition products which attach themselves to soy protein in the aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached inself to the soy protein in the aqueous medium.

4. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the decomposition products of soy SPS after completed degradation attach themselves to the vegetable protein to an extent of less than 50% than the soy SPS prior to decomposition would have attached itself to the vegetable protein in the aqueous medium.

5. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the decomposition products of soy SPS after completed degradation attach themselves to the vegetable protein to an extent of less than 20% than the soy SPS prior to decomposition would have attached itself to the vegetable protein in the aqueous medium.

6. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the SPS-ase exhibits a positive SPS-ase test, when examined according to the qualitative and quantitative SPS-ase determination method.

7. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the SPS-ase was produced by means of a microorganism belonging to the genus Aspergillus.

8. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the SPS-ase was produced by means of a microorganism belonging to the Aspergillus niger group.

9. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the SPS-ase is derived from the enzymes producible by means of Asp. aculeatus CBS 101.43.

10. An SPS-ase according to claim 1, claim 2 or claim 3, wherein the SPS-ase is immunoelectrophoretically identical to the SPS-ase producible by means of AsP. aculeatus CBS 101.43 and indentifiable by means of the immunoeletrophoretically overlay technique.

11. Method of selection of an SPS-ase producing microorganism for production of the SPS-ase as defined in claim 1, wherein the microorganism to be tested is grown on a fermentation medium, the main carbon source of which is SPS
produced on the basis of vegetable raw protein as a raw material, whereafter a sample of the fermentation medium is analyzed for SPS-ase and the microorganism in question is selected as an SPS-ase producing microorganism, if the analysis for SPS-ase is positive.

12. Method of selection of an SPS-ase producing microorganism for production of the SPS-ase as defined in claim 1, wherein the microorganism to be tested is grown on a fermentation medium, the main carbon source of which is SPS

produced on the basis of vegetable raw protein as a raw material, whereafter a sample of the fermentation medium is analyzed for SPS-ase and the microorganism in question is selected as an SPS-ase producing microorganism, if the analysis for SPS-ase is positive and is cultivated in a nutrient medium.

13. Method according to claim 12, wherein the strain Asp.
aculeatus CBS 101.43 or AsP. japonicus IFO 4408 is cultivated in a nutrient medium.

14. Method according to claim 12, wherein the cultivation is carried out as a submerged cultivation at a pH in the range of from 3 to 7, at a temperature in the range of from 20 to 40°C, and whereby the nutrient medium contains carbon and nitrogen sources and inorganic salts.

15. Method according to claim 14 wherein the pH is in the range of 4 to 6 and the temperature is in the range of 25 to 35°C.

16. Method according to claim 12, wherein the nutrient medium contains soy meal.

17. Method according to claim 16, wherein the soy meal is treated with a proteolytic enzyme before the use as a component of the substrate.

18. Method according to claim 17 wherein said proteolytic enzyme is the proteolytic enzyme produced microbially by means of Bucillus licheniformis.

19. Method according to claim 12, claim 14 or claim 17, wherein a sterile solution of pectin is added aseptically to the fermentation broth during the cultivation.

20. Method for decomposition of polysaccharides, by means of a carbohydrase, which comprises treatment with an SPS-ase preparation as defined in claim 1 in an aqueous medium is contacted with a substrate for said SPS-ase preparation.

21. Method for decomposition of polysaccharides according to claim 20, wherein the decomposition is accompanied by the isolation or extraction of a biological material other than soy protein and related vegetable proteins from a raw biological material, whereby the SPS-ase preparation is essentially free of any enzyme which is able to degrade said biological material.

22. Method for decomposition of polysaccharides according to claim 20, wherein one reaction products is a fermentable sugar which is further treated by alcoholic fermentation.