CA1198699A - Agent for decomposition of vegetable remanence, especially soy remanence, a method for production of a purified vegetable protein product, and a purified vegetable protein product - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The agent comprises an enzymatic activity which is able to decompose a high molecular carbohy-drater abbreviated SPS (soluble polysaccharide), and a remanence solubilizing activity together with no proteolytic activity or only a negligible amount of proteolytic activity. The method for production of purified vegetable protein comprises an almost quan-titative removal of the remanence from a raw vegetable protein by treatment thereof with the agent and separation of the solid phase containing the purified vegetable protein from the supernatant.

Description

A method for production of a purified vegetable pro-tein product (pvp) by enzymatic removal of the remanence, without dissolution and reprecipitation of the protein, is described in B~ patent No. 882.769. Also in this patent the importance of the fact that the proteolytic activity should be kep~ as low as pos-sible, is described. The purity of the pvp obtainable by the known method is not satisfactory and therefore open to ~mprove-ment. In the examples a purity of the p~p of about 85~ was de-monstrated. Even if it is possible to obtain a pvp of about 90%
purity according to the known method, this is only obtainable with certain pretreated starting materials, e.g. soy p~ctein concentrate. It would be desirable to be able to obtain a purity of the pvp of above 90% with a much broader spectrum of starting materials r especially dehulled and defatted soy meal.

The invention is kased upon the surprising discovery that a certain part of the remanence decomposition product, as it a~pears during the enzymatic trea~ment in~icated above, i.eO a water soluble, high molecular carbo-hydrate, attaches itself to part of the protein, as will be explained later in detail. This, of course r~sults in a lo-wer purity of the protein.

Thus, an object of the invention is to provide an agent for decomposition of veaetable remanence, in ~he Dresence of vegetable protein, whereby .he vegetable remanence especially ~s soy r~menence, which will result in a pvp with improved purity, and a method for production of a pvp.

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2 --In the accompanying drawings:
FIGURE 1 is a diagrammatic process flow sheet, ullustrating the background and context of the present invention;
FIGURE 2 is a diagrammatic flow sheet il.lustrating the production of SPS;
FIGURE 3 is a cal.ibration curve for HPLC gel filtration chromatography;
FI~U~E 4 is an HPLC gel filtration chroma~ogram or SPS;
FIGURE 5 is an HPLC gel filtration chromatogram of SPS
decornposed 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 filtration chromatogram of supernatant from decomposed SPS incubated with soy protein;
FIGURE 8 is an HPLC gel filtration chromatogram of APS
decomposeà by Pectolyase;
FIGURE 9 is an HPLC gel filtration chromatogram of APS
decomposed by SPS~ase;
FIGUR~ 10 is an HPLC gel filtration chromatogram of SPS
treated with Pectolyase;
FIGURE 11 is a graphical representation of imrnunoelectrophoretic peaks including an SPS-ase peak identified by overlay technique;
FI~URE 12 is an ion exchange chromatogram of an SPS-ase;
FIGURE 13 is a graphical representation of the pH
activity dependency o~ an SPS-ase;
FIGURE 1~ is a graphical representation of the tempera~ure activity dependency of an SPS-ase;
FIGURE 15 is a graphical representation of the temperature stability of an SPS-ase;
FIGURE 16 is a graphical representation of the pH
stability of protease in an SPS-ase preparation.

t ~ D~99 ( The basis for the invention can be described in the following manner, reference being made to fig 1 in which only materials existing as undissolved solids are i~dicated, whereas all supernatants are left out. A charge of soy meal was divided in two e~ual parts, part I and part II ~column a in fig. 1).
Part I was decomposed proteolytically at a pH value of about 8 ~y m~ of ALCALASE ~ a proteolytic enzyme produced by means of B. licheni~ormis and marketed by NOVO INDUSTRI A/S, 2880 Bag-svaerd, Denmark) and then further washèd at around pH 8 in or-der 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. l). In this way a ~ure ~emznence (designated C( remanence I~ was isolated (column b in fig. l). Part II of the soy meal was not treated; for the sake of brevity the remanence in part II is desisnated remanence II (column b in fig. l). Now, both remanence I and part II is decomposed by means of a commercial pectinase, e.g. PECTINEX ~ (a pectoly~ic enzyme produced by Schweizerische Ferment A/G, Basle, Switzerland)(vide column b and c in f ig. 1~ . Surprisingly it is foun~ th2t the undissol~ed pa~t of remanence I is much smaller - than the undissolved part of remanence II, on the basis of ni-trogen and dry matter mass balances, vide fig. 1, where the hatched areas in column c correspond to insoluble, non-protein materials in the above indicated stage. ~urthermore~ if the su-pernatant from the pectinase treated remanence I is brought to-gether with a soy protein suspension at pH 4.5, a polysaccharide in the supernatant is bound to the sov protein. This polysaccha-ride in ~he supernatant from remanence I, which is a part of ~he re-.
manence decomposition product, and which is clearly soluble in watex in the absence of soy protein, but bound ~o soy protein at or around the isoelectric point of soy protein, if soy pro-tein is presen., is designated SPS (Soluble Polysaccharide), ~ide fig. 1. The SPS has a molecular weight ~istribution be--tween 5 X106 and 4.9 x 104. The production of isolated SPS appears from the flow sheet sh~wn on fig. 2 which a`~ ~n~S~ ~ of the cesses depicted in fig. 1. Thus, the problem is to find an agent which is able to decompose the SPS in such a manner that the SPS decomposi~ioll products do not bind soy protein or do bind soy protein to a much lesser exten~ than SPS binds soy pro-tein. ^

Although the dis~losure mainly refers to decomposition of soy protein, the invention is not restricted to soy protein, but emcompasses all kinds of vege~able proteins, vide e.g. the pro~eins listed in BE patent No. 882,769, page 1.
Now, according to the invention it has been found that by screening for the ability to decompose soy SP5 it is possible to select microorganisms which are able to produce a compound which exhibits an anzymatic activity, which effectively decomposes soy SPS, in the following for the sake of brevity designated an SPS-ase.
In accordance herewitn it can be stated that an SPS-ase is a carbohydrase, which is capable of decomposing soy SPS under appropriate conditions into decomposition products which attach themselves to vegetable protein in an aqueous medium to a lesser extent that the soy SPS prior to decomposition would have attached itself to the same vegetable protein under corresponding conditions.
Thus, the invention in its first aspect comprises an agent for decomposition of vegetable remanenceO especially soy remanence, in the presence of vegetable protein, especially soy protein, suited for production of a pvp with a protein purity of around 30% with a vegetable protein~ which may be defatted or partially defatted, as a starting material, comprising an enzyme with remanence solubilizing ac~ivity, wherein the agent comprises an enzyme which is able to decompose soy SPS (SPS-ase) and wherein the agent is essentially free from proteolytic activity. The agent is essentially free from proteiolytic activity, when the proteolytic activity is equal to or less than the proteolytic activity, which will be accompanied by a total pro~ein loss in the finished pvp of not more than 30%, preferably not more than 15%, more preferably not more than 10%, when around 65~ of the remanence has been solubilized, on the basis of a nitro~en and dry matter mass balance.
It has been found that this SPS-ase capable of degrading soy SPS is able to degrade polysaccharides similar to SPS and orlginating from vegetables and fruits more completely than commercial pectinases and commercial cellulases.

8~FD~9 By total or partial elimination of the SPS from the final ve~etable protein the purity of the final vege-table protein necessarily is improved in comparison with the purity of the final vegetable protein obtainable accord-ing to the method known from BE patent No. ~2.769, as this known vegetable protein 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 sinyle enzyme or from an enzyme complex comprising at least two enz~mes. Some investigations seem to indicate that at least two enzymes ere responsible for the SPS-ase degrada-tion effect whereby one of these enzymes is capable of carry-ing out only a slight decomposi~ion 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 re-stricted by such hypothesis or similar hypotheses.

A preferred embodiment of the a~ent according to the invention is characterized by the fac, that .he SPS-ase was produced by means of a ~.icroorsanism belonging to the genus ~spergillus.

A preferred embodiment of the aaent according to the invention is characterized by the fact that the active component is derived from the enzymes producible by means of ~. aculeatus CBS 101.43. The same SPS-ase san be pro-duced by means of Asp. j~ponicus IFO 440~. It has been found that ~. aculeatus C~S 101.43 a~so produces very ~otent xema-nence solll~Jl;~;nr ~,;es, cellulases, ~;n~ ,' ana ~Pmicp~ es. Fur-thermore, it has been found that not each and every strain belonging to the species ~sp. aculeatus or AsP. japonicus generate an SPS-ase needed for the invention. Thus, as it appears from a later paragraph in ~his specific~tion, it has . .
~ em~nstrated ~hat the As~ 2n;~.~l~ AmC 20236.~ces-not Ero~uce such amounts o an SPS-ase which can be detected by means of the enzymatic determlnation of SPS-ase described in the ~ sDecif ication .
.

A preferred embodiment ol the agent according to the invention is characterized by the fact that the SPS-ase is immunoelectrophoretically identical to the SPS ase pro~ucible ~y means of AsP. aculeatus CBS
101.43 and identifiable by-means of the immunoelectro~
~horetical overlay technique, vide section 6 and 7.

'~ preferred embodiment o~ the agent according to the invention is characterized by the fact that the ratio between the proteolytic activity in HUT-units and the rëmanence solubilizing activity in SRUM-120-units is less than about 2:1,' preferably less than 1:1, more prefer2bly less than 0.25:1. It has been found that a clear correlation between the protease actlvity expressed in HUT units (to .
be defined'later) a't pH 3.2 (the pH acti~ity optimum of ~he protease) and t~e protein loss e~ists. This correlation , is~specific for the SPS-ase ~reparation producible by means of As~. aculeatus CBS 101.43. It is to be understood that this correlatlon may not'exist-in relation to another SPS-ase forming microorganism,-~thich-forms an SPS-ase which differs ~rom the SPS-ase producible by:means-of CBS 101 43, but ~ha~ a man skilled in-the art will be able to find a simi-lar correlation which will fulfill the requirement in regard to protein ioss stated in the main claim. The SRUM-120 activity (to be defined later) is a measure of conventional remanence solu~ilizing, cellulase, pectinase, and hemicellu-lase activities and other activities. The SRU~-120 units are measured at pH .4..5. It is to be understood that this pH is .chosen.bec.ause the decomposition of the remanence c~rr-e~ oa. at or~around the isoele~ic p~ o' ~he soy nr~tein,' and th~t a different enzyme activity method has to be used in'case the decomposition of the remanence is carried out at another p~-~alue. - :

' A preferred embodiment of the agent according to the-invention is characteri~ed by the fac~ that the agent contains ce~lu~ ase actlvity, and the cellulase activity is derived'partially or totally from Tric~oderma reseei. This cellulase is able to dissolve cryst~lline cellulose, and ( the agent gi~es rise to a pvp with high purity~

A prefexred embodLment of the agent according to the invention is characterized by the fact that the agent comprises cellulase activity (Cx), pectinase acti-vity (PU, PGE, UPTE, PEE) and hemicellulase activity (VHCU) In Agr.Biol.Chem. 40 (1), 87 - 92, 1976 i~ is described that a strain of As~. ja~onicus, ATCC 20236, produces an enzyme com~lex which is able to perform a partial ~egradztion of an acidic polysaccharide in soy sauce, named APS, a fraction of which is designated APS-I. This acidic polysaccharide is not identical to SPS, which will be shown later in this specification in more de~ail in section

3. Th~s, the HPLC gel filtration chromatograms of SPS and APS are clearly different, and furthermore, the gel filt~a-~o~ chromatograms of APS decomposed by means of the commerciai pe~t-inase-Pectolyase and of SPS treated ~ith the comme~cial pectinase Pectolyase are clearly different.
P~thermore, it does not aEpear frcm the article that the acidic ~bly~rh~ P is bound to the soy pr~tein and that the ~Y~osed acidic po-Iy~accXaride is not bound to the soy protein or is bound ~o ~e-soy protein to a much lesser degree than the undecom-posed acidi-c polysaccharide. Also, it has been demonstrated ~hat ~his strain does not form SPS-ase in such amounts, which ~an:bè de~ected by means o~ the enzymatic determination of SPS-ase d~scribed in this s~eci~ication. This creates a pre-judioe against any strain of As~. japonicus being a produ-cer o~ an SPS-ase, but surprisin~ly according to the inven-~ n it has been ~ound that some strains of AsP. japonicus -are ~roducers o~ an SPS-ase.
( .,:.

~'9~

he invzntion comprises in its second aspect a method for production of z puri:Eied vegetable protein product by remo~al of the remanence from a raw vegetable protein serving as starting material, wherein th~ startin~
material is treated with the agent according to the inven-tion in an aqueous medium a~ a pH value which does not differ more than 1.5 pH uni~s from the isoelectric point of the main part of the protein part of the starting mate-ria~, and at a temperature between about 20 and about 70C, until at least around 60~ of the remanence, on the basis of nitrogen and dry matter mass balance, preferably at least around 73% thereof, more preferably at least around ~0%
thereof, has been solubilized, followed by separation of the solid phase containing the purified ~regetable protein product from the suDernatant.

~ ~referred embodLment of the method according to the invention is characterized by the fact that the separation is carried o~t at a temperature betw~en room temperature and the freezin~ point of the supernatant.
Herehy a high protein yield is obtained.

A preferred embodiment of the method according to the invention is characterized by the ~act that the starting material is defatted or defatted and further partially pu-ified vegetable protein. This starting material is easily available.

A pxeferxed embodLment of the method according to the in~ention is characterized by the fact that the start-ing material is soy meal. This starting material is cheap and easily available.

A preferred embodiment o~ the method according to the invention is characterized by the fact that the starting material is heat treated soy meal, preferably jet cooked soy meal. Hereby a lower enz~me dosage can be used, and furtherm~re a higher yield can be obtained.

_ 9 _ A preferred embodiment of the method according to the invention is characterized by the fact that the starting material is able to pass a sieve with a mesh opening of around 2.5 mm. This ensures a reasonably short reaction period.

Only the dosage rate (in SAE units) for the S~S-ase acti~ity in treatment of soy meal can be provided. At least ab~ut 35 SPE units per l00 grams of jet co~ked soy meal, and 350 SA units per 100 grams oi uncooked soy meal. As a practic21 matter, the exact interrelation of enzyme ac,ivities needed to degrade S~S is not kno~m and, therefore, minimum dosages and proportions for pectinase cellulase and hemicellulase cannot be ~rovided. Rowever, a composite activity in treatme~t of soy meal in SR~.~ units can be provided, na~ely at le~st about 60~ SRUM-120 units per 100 grams for cooked sov meal, 600~ for unheated meal.
The exemplary values hereinafter provided are believed to be more than the minim~m dosages. Cut and try tests may be employed to establish optimum operating proportions and reacti~n time ~or the soy substrate to be converted into pvp, The invention comprises in its third aspect a purified vegetable protein product, produced by means of the meth~d according to the invention.

.

In order to clarify the nature of the invention, reference is made to the following sections 1 - 10, all describing details rei~ted to the invention:

1. Production of SPS.

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

3. Documenta~ion,for the fact that SPS and APS are different compounds~ ,

4. Screening for SPS-ase producing microorganisms.

5. Characterizatior of some SPS-ase forming micro-organi~ms.

6. General description of overlay technique associated with immunoelectrophoresis.

~,

7. Immunoelectxophoretic characterization of SPS-ase with polyspeciic antibo~y and overlay.

8. Purification o~ an SPS-ase ~reparation.

9. pH-activity dependency, temperature activity de~endency, and stability of an SPS-ase.

10. Enzymatic activity deter~inations.

:
~, '1 .SECTION l.

PRODUCTION OF SPS.

As previously mentioned the starting material for production of SPS may be sov remanence. Therefore, in the first place, the production of soy remanence is described.
Soy remanence is the protein free carbohydrate fraction (which in practice may contain minor amoun~s o~ nin an~ mineral~) ~n ~L~LLed and n~h~ Pd soy m~r which car~ohyarate frætion ~ ~oluble in an aqueous medium at pH 4.5, and it can be produced in the ol-lowing manner, reference also being made to flow sheet l.

Defatted soy meal (Soja~el.13 . from P.arhus..Oli~a~riX.
A/S) is suspended in deionized water of 50C in a wei~ht pro-portion soy meal:water = l:5 in a tank with p~-stat and temperature control. pH is adjusted to 8.0 ~ith 4N NaOH (I). No~ a pH-stat hy-drolysis is performed with ALCALASE 0.6 ~ (a proteolytic enzyme on the basis of B_ --. licheniformis with an àctivity o~ 0.6 Anson units/g, where~y the activity is determined according to the Anson method, as described in NOVO ENZY~E INFO~TION IB No. 058 e-GB), whereby the ratio enzyme/substrate equals 4~ of the amount of protein in the soy meal (II). After a hydrolysis of l hour the sludge is separated by centrifugation (III) and washing (IV) whereby this operation is perormed twice (V, VI, VII). The thus treated sludge ls hydrol~rzed once more for l hour with ALCALASE 0.6 ~ ~VIII, IX) similarly as indicated before~ Then the sludge is separated by cen-trifugation (X) and washed twice (XI, XII, XIII, XIVj, whereby the ~inal washed sludge (6) is spray-dried ~XV~. The thus produced spray-dried powder is the soy remane~ce serving as a raw material for the production of SPS.

SPS is thP water soluble polysaccharide fraction which i5 fonmed by conventional treatment of the above indicated soy r~manence with pectinase~ The S~S is produced in the following ma~ner by means of the below indica~ed 14 reaction steps .~2.

... . . . ..
reference also_beinq made to flow sheet 2.

1. The dry matter content in ~he above lndicated soy remanence is determined and the soy remanence is diluted with water to 2~ dry matter and kept in suspen-sion at 50~C in a tank with t~mperature control.

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

3. Pectinex Super conc. L is added in an amount o~
200 g~kg dry matter ~a commercial pectinase from Schweizerische Ferment AG, Basle Switzerland with a pec-tinase activity of 7S0,000 M3U , as determined according to the leaflet "Determination of ~he PectinasP units on Apple Juice (MOU)" of 12.6.1981, obt~in~hle from Schweizerische Ferment AG, Basel, Switzerland), ~nd also Celluclast 200 L is added in an amount of 20 g/kg dry mat~er (a commercial cellulase described in the leaflet NOVO enz~mes, information sheet B 153 e-GB
1000 July, 1381, obtainable from ~OVO INDUSTRI A/S, Novo Alle, 2880 Bagsv~rd, Denmark).

4. ~ The contents of the tank is kept at 50C durIng 24 hours with stirring.

5. . The enzymes are inactlvated by raising the pH value to 9.0 with 4N NaO~O The reaction mixture is kept for 30 minutes, and the p~-value is then re-adjusted to 4.5 with 6N HCl.

6. The reac~ion mixture ~s centrifugea, and both the centrifugate and the sludge are collected.

7A The centrifugate from step 6 is check filtered on a fil~er pr~ss t~he filter is washed with water before eheck filtration~.

8. The check f~ltrate is ultr~iltered, diafiltered and once more ultrafiltered on a membrane with a cut-off value of 30rO00 (DDS G~ 60-P from De Danske Sukkerfabrikker), whereby the following parameters are used:

1. Ultrafiltration corresponding to a volume concentration of 6.
2. Diafiltration until the percentage of dry matter in the permeate is 0 ~0 Brix).
3. Ultrafiltration to around 15% dry mat~er in the concentrate.

The temperature is 50C, p~ is 4.5 and the average pressure is 3 bar.

9. T~le ultrafiltered concentrate is cooled to 5C, and an equal volume of 96% ethanol is added.

10. The precipitate is collected by means of a c ntrifuge.

11. The precipitate is washed twice with 50~ v/v ethanol in H~0, corresponding to the volume of centrifugate from step 10, i.e. two centrifugations are performed.

12. The washed precipitate is redissolved in water with a volume which equals the volume of the ultrafiltered concentration from step 9~

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

14. The clear filtrate containing pure SPS is lyophilized.

~.

Defatted soy ~eal ~2 NaO~ to pH ~ 8 ¦Hydrolysls ixture ¦ I

Alcal~se 0.6 L ~ydrolysls 1 hour beforO discharge (E/S ~ 4 ~) Ip~-stat (pH ~ 8, T - 5~ C) 4N NaO~ ~ 1 ¦lst centrifugation ~ Centrlfuaate 1 ~ T~a5te Sludge 1 ~2 ~ 1st wash ¦IV ¦

¦ 2nd centrifugation ¦ V.~ Centrifuoate 2 ~?aste Sludge 2 ~2 ~ 2nd wash ¦VI¦

¦3rd ce~trifugation ¦VII ¦ Centrifuqate 3 ~'aste .~ Sludge 3 ~ O~ t ~ 2 ~ New hyarolysis mixt~re ! VIII , ¦ .
.atc~ se 0.6 L
ydx~lysis for.l hour before 4N-~ab~ 1echA~ge,.p~-.s~at ~p~ ~-8.0, -IX

¦4th centri~u~ation ¦ X ¦ ~ntrl~llpat~ aste Sludge 4 ~2 ~ 1st wash¦ X

¦5th centrlfugation ~ Centrlfuaate 5 )~aste ~ Sludge 5 : ~2 ~ 2nd wash¦XIII ¦
' .

¦~th centrifugation ¦XIV ¦ Centrifu5ate 6 ~ ~aste ~ Sludqe 6 ! SP~aY-drYing FLOW SHEET N0. 2.

1000 q Pectlnex Super conc. L 5 kg spray-dried remanence 100 q Celluclast 2 0 S 1 1 247 1 H Decomposltion wi~h pectinase 2 ~ anO cellulase 1, 2, 3, 4, 5 50 C; pH ~ 4.S; t - 24 hours ¦Centrifug~ion ¦ 38 kg 6 - ~Check flltrati~n ¦ 7 Ultralltrat~on~ diafiltra-18 1 H2O ~ tlon, ultraflltraOtlon 24~ 1 of ~er-(GR 60~1 (19 - 26 C) meate 8 5 1 ~0~ C2~ Precipltation ¦ Su~er~atAnt~9 5waste) ¦Centrifugatlon I (~ntrifl-qAt:~>10 1 P~ec~p~tate ~2 x washlng ¦ 2 x cent.rlfu~ate>t~
1 Preclpltate 2 1 H2O ~tlqsOlution ¦ 12 ., 1 .
Check iltration 1 Protein sludae 13 .(waste) >
yophilizatiorll l .1 310 g lyophilized SPS

SECTION 2.
CEARACTERIZA'I'ION OF SPS, ESPECIALLY MOLECULAK WEIGHT
DISTRIBUTION THE~EOF
By m~ans of gel chromatography on HPLC equipment ~Waters pump model 6000, Waters data module 730, and Waters refractometer K 4~1) the molecular weight distribution of the SPS, the production of which is carried out as indicated in this specificatlon, is determined (fig. 4). By means of the same method also the molecular weiyht disbribution of the decomposi~ion products of SPS by means of SPS-ase has been determined (fig~ 5). Furthermore, by means of the same method the binding effect between soy protein and SPS (fig.
6) and the absence of binding effect between soy protein and SPS clecomposed by means of the agent according to the invention (fig. 7) has been demons~rated.
The calibration curve (the logarithm of the molecular weight plotted against Rf, where the Rf-value for glucose is arbitrarily defined as 1 and the Rf-value for a specific dextran is defined as the retention time for this dextran divid~d by the retention time for glucose) has been established by means of several standard dextrans with known molecular weights (I' 4, T 10, T 40, T 70, T 110, ~r 500) from Pharmacia Fine Chemicals AB, Box 175, S-75104, Uppsala, Sweden. The Rf-value for the maximum of each dextran peak has been found, and the corresponding molecular weight has been calculated as ~ Mw . M , whereby M is the average value of the molecular weight according to weight and M~ is the average value of the molecular weight according to number. As an eluent for this chromatographic procedure 0.1 M NaNO3 has been used. The columns used in the chromatographic procedure are 60 cm PW 5000 followed by 60 cm PW 3000 from I'oyo Soda Manufacturing Co., Japan. In this manner the relationship between molecular weight and Rf for the above indicated dextrans has been established, vide figure 3.

', :

$~
- 17 ~

On the basis of fig. 4 it can be calculated that SPS has a molecular weight distribution which gives rise to a value of Mw of around 5.4 x 105 and a value of Mn f around 4.2 x 10 . Also, it appears from this figure that the chromatogram exhibits two distinct peaks at retention time 34.5 minutes (6%~ corresponding to a molecular weight of around 5 x 106 and retention time 47.12 minutes (67%) corresponding to a molecular weight of around 4.9 x 104.
Also, it appears from this curve that a shoulder exists between these two peaks at retention time 41.25 minutes (27~) corresponding to a molecular weight of 2.8 x 10 .
After decomposition of SPS with SPS-ase the hydrolysis mixture was membrane filtered, and the filtrate was chromatographed. It was found that around 55% of SPS is decomposed to mono-, di- and trisaccharides, and that the remaining 45~ are decomposed to a polymer with three peaks with the following molecular weights: 5 x 104, 104 and 4.4 x 10 , vide figure 5.
In order to d monstrate the binding effect between soy protein and SPS and the substantial reduction of binding effect between soy protein and SPS decomposed by means of an SPS-ase the following experiments have been performed.
3% SPS in 0.10 M acetate bu~fer at pH 4.5 is added to a slurry of soy isolate (Purina E 500) in order to genera e a suspension with a ratio isolate/SPS of 10:1~
This suspension is incubated for 18 hours on a shaking bath at 50C. After incubation the suspension is centrifuged, and the clear supernatant is analyzed on HPLC as previously described. Erom fig. 6 in comparison with fig. 4 it appears that the SPS is completely absorbed to the soy isolate.
~ he same procedure as indicated in the previous paragraph is performed with a 3~ SPS solution hydrolyzed with ;

a~ SPS-ase ~roduced by means of C3S 101.43 (fig.7 )-A comparison between ~ig. 7 and fig. 5 shows that no com-pound in the hyd~olyzed SPS with molecular weight below around is adsorbed to soy isola~e. The hydrolysis reduces the quantitative binding to around 10 - 15~ in relation to the binding of SPS to soy protein.

An NMR-analysis of the SPS, the production of which is carried out as indicated in this specification reveals the following approxLmate composition of the SPS:

1) ~-galacturonic acid in an ~mount of approxîmately 4S%, whereby approximately ao%of the total amount of ~-galac-turonic acid is present as the ~ethyl ester.

2) rhamnopyranose in an amount of appro~imately 20~, 3) galactopyrancse in an amount of approxLmately 15%, and 4) ~-xylopyranose in an amount of approximately 20%.

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

Complete acid hydrolysis of SPS (8 hours in 1 N H2SO4) and subsequent TLC analysis revealed that also minor amounts of ~he monosaccharides Lucose and arabinose were present in the hydrolyzed SPS.
. .

, . .

An HPLC analysis of the SPS decomposed by the SPS-ase enzyme complex formed by CBS 101.43 shows a po~terful reduction of molecular weight. In accordance therewith the N~-spectrum of the SPS decomposed as indicated above shows that the main ~2rt of the ester ~rou~s have disa~red and that also the C~l~lt o~ xylose and ga-lactose ~n the hi~h r molecular weight mater ~ has decreasea. The NMR-s~ru~
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-spectrum of the SPS, with the above- indicated modifications r concernina the ester groups and the content of xylose and galactose.

DOCUMENTATION ~OR THE FACT THAT SPS AND APS ~RE DIFFERENT
COMPOUNDS.

APS was prepared as indicated in Aar.Biol.Chem., Vol. 35, No. 4, p. 544 - 550 (1972).

Now, this polysacchariGe and cpc ~ere hydrolyze~ with different enz~mes, whereafter the decomposition mixture was ~ gel chromatographed on HPLC e~uipment, as indicated in sec-tion 2, "Charac~erization of SPS, espec'ially molecular ~eight distribution thereof".

In more detail, the hydrolyses were carried out by treatment of 25 ml solution of either 2~ APS or 2% SPS in O~1 M acetate buffer of pH 4.5 with 10 mg XRF 68 or 30 mg Pectolyase. K~ 6~ is an SPS-ase Dreoara,ion,the ~L~ ~ ~on of which is descr~ in ~Dle 1. The results appear from the ~ollo~ table.

Polysac- HPLC gel Polvsaccharide charide Enzyme chromatogrzm Decomposed Not decomposed APS Pecto~yase fig. 8 x ~PS ~RF 68 ~ig. 9 x SPS P~lyase fig. 1C x SPS KRF 6g fig. 5 x SCREENING ~OR SPS-ASE PRODI~CING MICROORG~NISMS.

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 liauid main substxate, in which the main carbon source is SPS (Dre~ared as indicated), in which the nitroge~ source is NO3 , NH4 , urea, free amino acids, proteins or another nitrogen containing co~pounds, which furthermore contains 2 mixture of necessary salts and vitamins, ?referably in the form of yeast extract. The composition of the main sub-strate depends ~on the microoraanism ~e~us, the principal issue being that the mair, substrate shculd be able to sup~ort growt~
~nd metabolism of the microor~anism. ~1hen the growth has taken place in a suitable period of time., of the order of magnitude of 1-? days, dependin~ unon the ~rowth rate of the microor~anism in question, 2 sample of the fermentation broth is analyzed for SPS-ase accordin~ to the enzymatic SPS-ase determination described in the spe.cification.
. . In order to achieve a more sensitive method for the de-~ termination of enzymatic activity the temperature could be lowered to 40C and the incubation tLme 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 CPS ase producins . microorg~nic~may be found3both belonains to the ~enus of Aspergillus and to other genera. .

., Dl l -q~

SECTION 5.

CHARACTERIZATION OF SOME SPS-ase FORMING MIC~OOR~ANISMS

According to the here indicated screening for SPS-ase producing microorganisms it has be~n found that the microorganisms listed in the upper part of the following table are SPS-ase producers. Also the table contains a strain belonging to the species Asp. japonicus which is not an SPS-ase producerO

SPS-ase Species Our identi- Official First procucer fying de- identi- Deposi~
Yes No Asp. Asp. signation fying de- on year japo- acule- signation nicus atus x x A 805 C~S 101.43;DSM 2344 1943 x x A 1443 IFO 4408;DSM~2346 1950 x x A 1384 ATCC 20236;DSM 2345- 1969 A short identification of the above indicated strains can be found in the following culture catalogs:

List of Cultures 1978 Centraalbureau voor Schimmel-cultures, Baarn, m e Netherlands.

Institute for Fermentation Osaka, List of Cultures, 1~72, 5th edition, 17-~S, Fuso-honmachi 2-chome, Yodo~awa-ku, Osa~a 532, Japan.

The Amnerican Type Culture Collection Catalogue of Strains I, 14th edition, 12301 Parklawn Drive, Rockville, ~aryland 20852.

All the strains in the above indicated table correspond closely to the taxonomic description o~ the species Asp.
japonicus an~ Asp. aculeatus appearing in The genus Aspergillus of Raper and ~ennell, 1~65 (vide especially pages 327 - 330).

.. .

.3~

SECTIO~ 6.

GEN~:RAL D~ SCRIl'TION O~ OVERLAY TECHNIQUE ASSOCIATED WIT~I
IMMUNO~LI :CTROPHORES I S .

A method designated the top-agar overlay technique has been developed by the applicant fox identification of individual components of an enzyme complex by crossed immunoelectroforesis with a polyspecific antibody against all enzyme components in the enzyme complex. The method is based on the fact that enzymes are still active after the specific en-zyme-antibody binding, or otherwise stated that the active enzyme site is not identical to the site of tne enzyme-antibody binding.
The enzyme~antibody complexes precipitate as distinct arcs in the gel during the ele~troforesis. The gel plate is ccvered with soluble SPS ~n a top-agar. After heating to 45C for 20 hours in an at~osphere with a relative humidity o~ 100% the arc which pos~esses SPS-ase activity will ap~ear as a clearin~ zone in the CPS cover after precipitation with a mixture of equal volume parts of ethanol and acetone when looked upon aaainst a black -background. Arcs which have no SPS-ase activity are left invi-sible. -3~9 SECTION 7.

IMMUNOELECTROPHOR~TIC CHARACTERIZATION O~ SPS-AS~ WITH POLY-SPECI~IC ANTIBODY ~ND OVERLAY~

Rab~its were immunized with the SPS-ase containing enzyme complex o~tained by fermentation of Aspergillus aculeatus CBS 101.43, as indicated in example 1 ~KRF 68) an~ the polyspec~ic antibody was recovered in a manner known per se. By means of this F polyspecific antibody a crossed immunoelectrophoresis of the enzyme complex obtained by fermentation of Asp. aculeatus CBS 101.43, as indicated in example 1 ~KRF 68) was p~ormed, as ~ec~ri ~ in N.H. Axel-sen et al., "A Manual of Quantitative Immunoelec'rophoresis", 6' printing 1977. Reference is made to fig.ll which shows the arcs corresponding to the different proteins produced by the microor-ganism. By means of the previously described top-agar ov2rla~
techni~ue it is lound that the hatched area corresponds to SPS-ase.

If the previously indicated hypothesis comprising the assumption that SPS ase consists of at least two enzymes is correct the hatched area is the area, in which all the enzymes responsible for the SPS~ase activity are pxesent. I~ these enzymes in other embodiments o r the invention should be sepa-rated by the immunoelectrophoresis in sucn a manner, that ~hey do not cover any mutual area, a part of the SPS-ase ac~ivity can still be identified by means of ~mmunoelectrophoresis with an overlay with both SPS and a comm~rcial pectinase.

al~

SECTION 8.

PURIFICATION OF AN SPS-ASE PREP~RATION
The purification of the SPS-ase preparation KRF 92 (vide example l) was Performed bY ion"exch~n~e. The buffer is 50 mM Tris ~tris-hydroxymethylaminometh2ne) which is adjusted to pH 7.0 with HCl. The column is X 5/30 from Pharmacia, Sweden. The ion exchange material i5 DEAE-trisacxyl from LKB, Bromma, Sweden r300 ml). The flow rate is 100 ml/hour.

t5 g of the SPS-ase preparation KRF 92 was dissolved in 450 ml H2O at 6C, and all the following indicated operations were carried out between 6C and 10C. pH was adjusted to 7.0,with 1 M
Tris. The column w2s eguilibra~ed with ~he buffer, and then the SPS-ase sample was introduced onto the column. OD280 and the conductivi-ty ~Jas measured on the eluate 5 reference being made to Fig. l~ Frac-tion 1 is ~he eluate which is not bou~ to thë ion ~change''m~terial- T~.e~ thQ CO-lumn is washed with 2000 ml buffer which . gives rise to ~raction ~. Now a 0 -500 mM NaCl gradient is established, giving rise to fractions 3-9. ~ll nine fractions were concentrated to 200 ml and dialyzed against water to a conductivity of 2 mSi by means of dialysis tHollow Fiber DP 2 from Amicon, Massachusetts, U.S.A.). Then the nine fractions were fxeeze dried. Only fractions 1 and 2 exhibited SPS-ase activity.

Fraction 1 was further purified by gel filtration. 1,5 g of fraction 1 wa~ dissolved in 10 ml 50 mM sodium acetate with pH
4.5 (500 mM KCl~. The column is 2.5 x 100 cm from LKB.' The gel fil-tration filling material i5 Sephacryl S-200 from Pharmacia! Sweden.
The flow rate is 30 ml/hour. The frac~ions containing materials with moleculax weigh.s be~ween 70,000 and 10~,000, ~alibrated with glo-,bular proteins, contained an enzyme complex designated factor Gwhich cannot deoompose SPS when tested according to the qualitative agar test; however, SPS is decomposed accordin~ to ~he qualitative agar test when mixing factor G with a pectinase. It has been found that actor ~- is able to split off galactose, ~se, and some galac-'1'1 . . . .

J u l turonic acid ~rom SPS, but the main decomposition product according to the HPLC analysis is still a high molecular product very much like SPS.

,.~F~aN 9.

AC~l lVl~L~ DEPENDENCY, TEHPERl!.TURE ,P.CTIVITY DEP~DENCY i~`dD
STABILITY CF A~ SPS-ASE, ~ig. 13 shows the pH-activity dependency o the SPS-ase ~ on ~RF 6S. Frcm ~Y 2,7 to p~ 3,5 a formic acid buffer system w2s use~, and from pH 3,7 to 5,5 an aoetate b~f~r syste~ was used.

Fig. 14 sho~s-the temperature activity depend~ncy of the SPS-ase preparation ~RE 68.

~ ig. 15 shows the temperature stability of the SPS-ase pxeparation KRF 68.

81~

SECTION 10.

EI~ZY~TIC ACTIVITY D_TERMINATIONS.

The below indicated table is a survey.of the dif~eren~ enzy-mz~ic ac~ivity determin2tions pertzinina to the invention.

Definition of 2c~ivity unit and descri~ion o' enzymatic ac~ivit~ determin2tion ~j I
Described Xind of ac- later in tivi~y, Publicly this short de- zvail- speci,i-Enzyme sisn2tion able cation ~ere~ence SPS-ase SPS-ase x ~ ~ n~n~e SRU x ii7in~ SRUM~120 -~` x Prote2se HUT X
Cellulase Cx x 2 PU x 3 PGE x 4 Pectinase UPTE x 5 PEE x HemiCel~ VHCU x 7 lulase The refe~ences indicated i~ the last ~column of the above table ~e detailed in the below indicated table.

~a ' Reference can be obtained from NOVO INDUS- Schwei-TRI A/S, zerische Novo Alle, ~exment ~880 Bag- AG, Ba-Reference Identification of sv2rd, sel, S~it- A libra-~o reference Denmark zerland ry ~nalyseforskrift 1 A~ 15~/4 of 1~ 12-~1 x .. , . , . . . . _ . . .
Analytical Biochemistry . 8g, 522 - 532 (197~) x Analytical method AF 1~9/6-~ of x 1~81-05~25 Determination of Pecti- .-nase Activity with Ci- .
trus Pectin (PU) of 3 23.~.19?6 x Viskosimetrische Poly-galactuxonase-Bestim-4 mung (PGE) of 10.11.77 x Besti~mung der Pectin~
. transeliminase~(UPTE/
- 5 g) of 24.Sept.1975 x Determination of t~e Pectinesterase acti-~ity (undated) with 6 initials ~JJA/G~ x Analytical method 7 AF 156/1-GB x In relation to the cellulase activity deter~;nation it can be noted that the analysis was carried out as indicated : in AF 149/~-GB and that the.~rinciples o~ the deter~ination is explained in Analytical Bio~hemistrv. - -. .

SECTION io a.

ENZY~TIC DETERMINATION OF SPS-ase.

The enzymatic determination of SPS-ase is carried out in two steps, i.e. a quzlitative agar plate test, and a quanti-tative SPS-ase activity determination based on measurement of the amount o~ total liber2ted sugars. If the qualitative agar plate test is negative, the SPS-ase activity is zero, reaardless of the value origin2tina 'rom the ~uantitative SPS-ase activity deter-mination. If the qualitztive 2gar plate test is positive, the SPS-ase activity is equal to the value originating from the quantita~ive SPS-ase activity determination.

I. Qualitative agar ~late test.
- ~ An SPS-asar ~late was prepared in the followin~
manner. A buffer (B~ is prepared by adjustin~ 0.3 M
acetic acid to a pH-value of 4.5 by means of l N NaO~.
1 g of SPS is dissolved in 20 ml of B. l 5 ~f agarose SB Litex) is mixed with 80 ml of B and heated to the boiling point with stirring. When t~e aaarose is dissolved - the SPS-solution is slowly added. The resulting 1% SPS-agarose solution is placed in 2 water bath o' 60C.
The plates are now cast by pourina 15 ml of the 1%
SPS-a~arose solution on a horizo~tal glass plate with dimensions 10 cm x 10 cm. Then 9 wells with a distance of 2.5 cm are punched out in the solldified layer of SPS-aaarose~ In each well a 10 ~1 of a 1% solution of the enzyme protein to b~ tested for~SPS-ase activity is lntroduced. The plate is incubated 'or l~ hours at 50C and with a relative hu~idity of 100%. No~ still . 1 D~'J

-- --r\lo~

undecomposed SPS is precipitated by a solution o equal volume parts of ethanol and acetone. The SPS-ase a~ar pl~te test i5 positive for a samDle Dlaced in a s~e-ci~ic well, if a clear annular zone appears around this well~ ' II. Quantitative SPS-ase activity determination test.
The purpose ol this test is the determination of enzymatic activities, which are capable of decomposing SPS to such an extent that the decomposition products , exhibit a s~r,ongly reduced or no adsorption or bindinq ,af~lnity to scy ~rotein. ExT~r;r~nts have sh~ that that.~xrt.
of the SPS decomposition products which are not preci~itated by a mixture of equal volumes of water and ethanol, do not have any adsor?tion or binding affinity to soy protein.

'''' The SPS-ase determination is based on a hydrolysis of SPS under standard conditions rollowed by a preci-pitation OL khat ~art o~ SPS, which is not hydrolyæed with ethanol. A~ter precipitation the content of ~~arbohydrate, which is not precipitated, is determined by ~uantitati~e analysis for total sugar (according to ~F 169/1, available from NOVO IND~STRI A/S, 2880 Ba~s~aerd).

The standard condi~ions are:
___~_______ .,______________ '~ Temperature: 50C
pH: 4.5 ~eaction time: control 210 minutes with substrate only, followed by 2 minutes with added enzyme - - . main value 212 minutes \

The esul~ment_com~rises:
Shaking water bath thermosta~ed at 50C
~irlimixer stirrer Centrifuge Ice water bath The reaq nts com~rise:
Buffer: 0.6 M acetic acid in demineralized water (a) 1.0 ~ NaO~ (b~
~ubstrate. The ~H value of 50 ml of a is adjusted . to 4.5 with b, then 4.0 g SPS are added, and after dissolution of the SPS the ~H is readjusted to 4.5, and the volume is adjusted to 100 ml with deionized water.
Stop rea-gent: Absolute ethanol.

1 SPS-ase activity unit SAE or SPSU is defined as SPS-ase ~activity which under the abo~e indicated standard conditions releases an amount of carbohydrate soluble i~ 50~ ethanol çquivalent to 1 ~mol galactose per minute.

~ en if the initial part of the enzyme standard curve is a straigh~ line, it has to be noted that it does not inters~ct the (000) point~

;, .~

I ~I V ~

,CF~Tc~l 10 b~ . -ENZYMATIC DETER~SINATION 0~ ANEN~E SOLUBILI3ING ACTIVITY EXPRESSED
AS SRUM 12 0 .
Principle In the method for determination of hydrolysis activity theinsoluble part of defatted, deproteinized~ and dehulled soy flour is hydrolyzed under standard conditions. The enz~e reaction is sto~ped with stop reagent and the insoluble part is filtered off. The amount of dissolved polysaccharides is determined soectro-photometrically after acid hydrolysis accordins to A~ 169~1, available from Novo Industri A/S~ 288~ Ba~svzrd.
. . .

~ 'arbohydrases with endo-'as well''as exo-activit,y are de texmine~ according to the method.

~ he substrzte pertaining to this enz~matic determination is identical to the remanence.substrate described for the SRU method.
, The substrate is dissolved as a 3~ solution in the below indicated . citrate buffer:
-0.1 N, citrate-phos~hate buffer pH 4.5 ., 5O24 g citric acid l-hydrate (Merck Art 244) 8.12 g disodium hydrogen phosphate 2-hydrate (Mexck Art 6580) Ad 1 1 demineralized H2O
pH 4.5 ~ 0~05 Stable for 14 days The stop rea~ent has the following composition:
100 ml 0.5 N NaOH
200 ml 96% ethanol To be kept in a refriserator until use.

- ~4 -Standard Conditions Temperature ........................ ~<. 50C
p~ ............. ~....._............... ,. 4.5 Reaction time, sample ........... 120 minutes - - blank ............................... 5 -Unit Definition ...
One soy r~n~nGe ~ hil~7ing unit (~I) 120 ~M for ~nual) is the amount of enzyme which, under the given reaction conditions per mi-nute, liberates solubilized ~olysaccharides equivalent to one mi~ro-mole of galactose.

"

. ~, ._ i' ~.~

3 ~ _ ,ECTION 10 c.

ENZYI~SATIC DE~ERMINATIo~ O~ ~ROTEOLYTIC ACTIVIT~!-~UT ~:ASUR:E:~ENT~e'chc)d for the determination of proteinase in an acid medium.

The method is based on the ~igestion of denatured hemoglo~in by the enzyme zt 40C., p~ 3.2, for ~0 ~inutes.
The undigested hemoglobin is precipitated with 14~ trichl~ro-acetic a~id (wt/v%).
. ~11 enzyme samples are prepared by dissolving them in 0.1 ~ acetate buf~ex, p~ 3.2.
The hemoglobin substrate is prepared using 5.0 g of ly~philized, bovine hemoglobin powder, preserve~ wit~ 1%
Thiomexsalate and 100 ml demineralized water which is stirred ~or 10 mi~utes, after which the pH is adjusced to pH
1.7 with 0.33 N ~Cl~ . -Af~ex another 10 minutes o~ stirring, the p~I is adjusted to p~ 3.2 with lN NaOH; The ~olume of th_s solution is increased to 200 ml with 0.2 ~ acetate buffer. This - ~ .
hemoglobin subs~rate must be refri~exated where it ~ill X2ep for S days.
The hemoglobin substrate is brought to ro~m .... .
temDerature. A~ ti~e zero, 5 ml of s~str~t~ is .~ to a test tube containing 1 ml of enz~ fter 5~2~.in~ Fo_ second, the tube is placed in a 40C. ~at~r bat~ for 30 mi2~utes. After exactly 30 mi~utes, 5 ml, 14~ trichloroacetic acid is added to the reaction tube, which i5 tl ~n ~sha)c~n and Srought ~o room tempera'cure i~or 4 0 s~inutes ..

3~ -For the blank, the hemoglobin substrate is brought to room temperature. At tirne zero, 5 ml of the substrate is added to a test tube containing l ml of enzyme. After shaking for l second, the tube is placed in a 40C. water bath for 5 minutes ~fter exactly 5 minutes, 5 ml of 14% trichloroacetic acid is added to the reaction tube, which is then shaken and brought to room temperature for 40 minutes.
After 40 minutes, the blanks and samples are shaken, filtered once or twice through Berzelius filter No~ 0, and placed in a spectrophotometer. The sample is read against the blank at 275 nm while adjusting the spectrophotometer against water.
Since the absorbance of tyrosine at 275 nm is a known factor, it is not necessary to make a tyrosine standard curve unless it is needed to check the Beckman spectrophotometer.

Calculations 1 HU'~' is the amount of enzyme which in l minute forms a hydrolysate equivalent in absorbancy at 275 nm to a solution of l.lO microyram/ml tyrosine in 0.006 N HCl. This absorbancy value is 0.0084. The reaction should take place at 40C.~ pH 3.2, and in 30 minutes.

Sample-Blank x Vol. in ml HUT = 0.0084 reaction time in min.
Sample-Blank x 1l = (S-B) x 43.65 EIUT = 0~0084 30 HUT/g enzyme = IS_B) x 43.65 g.enzyme in 1 ml An investigation of the p~-stability dependency o~
the protease in KRF 68 performed by means of the HUT analysis with pH values from 2.0 to B.0 showed that the stability of the protease above pH B.0 was very small, vide fig. 16.

. , r~ o\~o . . - 37 --. .. . . . .. ....

In order to illus.rate the invention reference is made to the ~ollowing examples 1 ~ 10 , where example 1 illustrates the production of SPS-ase, and where examples 2 - 10; illustrate the application of SPS-ase.
'~.
Several fermentations with the here indicated strains of Asp. aculeatus and Asp. japonicus were performed in laboratory scale. Hereby preparations were obtained which contained SPS-ase according to the hexe indicated SPS-ase tes~. Howe~er, as rather large amounts of SPS-ase are r~quired.in ~rder to run application tests, similar fermentations were run on ~ pilot plant sca7e, ~ide the following example 1.

Example 1 -Production of an SPS-ase in pilot plant scale.

. An SPS-ase was prepared by submerged fermenta~ion of Aspersillus aculea~us CBS 101~43.

An ayar substra~e with the following composi~ion was prepared in a Fernbach flask:
Pepton Di~co 6 g ~minolin Ortana : 4 g Glucose 1 g Yeast extract Difco 3 g Mea~ extrac~ DifcolvS o KH2po4 Merck ~0 g ¦ Malt ex~ract Evers 2~ g :~ Ion exchanged H2O ad1000 ml O~_ _ . p~ w~s adjusted to be~ 5.30 ~ 5.35. men 40 o of ~gar Difco W2S added, and the mixture was autoclaved for 20 min.
at 120~C (the substrate is nzmed E-agar).

The strain CBS 101.43 was cultivated on an E-agar slant (37C). The spores fxom the slant were suspended in sterilized skim-milk, and the suspension was lyophllized .
in vials. The contents of one lyophilized vial was trans-ferred to the Fernbach flask. The flask was then incubated for 13 days at 30C.

A su~strate with the following composition was prepared in a 500 liter seed fermenter:
g CaCO3 1.2 kg Glucose . 7.2 kg Rofec (corn steep liquor dry ma~ter) 3.6 kg Soy bean oil . 1.2 kg Tap water was added to ~ total volume of around 240 liters.
pH was adjusted to arou~d 5.5 before addition of CaC03. The su~c~
was st~.rilized in the seed fe~menter for 1 hour at 121C.
Final volume before inoculation was around 300 liters.

. The Fernbach flask spore suspension was transferred to ¢ the seed fermenter. Seed fermentation conditions were:

. Fermenter type: Conven~ional 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 mlnute ~emperature: 30 - 31~C
Pressure: 0~5 ato Time: Around 28 hours Around 2~ hours after inoculation 150 Liters was transferrea from the seed fermenter to the main fermenter.

.

r ~ ~, A subs~rate with the following composition was prepared in a 2500 liter main fermenter:

ed soy m~ 90 kg ~H2PO4 20 kg Pluronic~ 150 ml Tap water was added to a total volume of aro~d 900 lit~rs; The toasted soy meal was suspended in water. p~ was adjusted to .0 with NaOH, and the tem~erature was raised to 50~C. There-after ar~und 925 k~n units ~LCALASE~ 0.6 L~s add2d to the su~x~icn.
The mixture was held for 4 hours at 50C and pE = 8.0 (Na2CO3 additio~ with no aeration, zero ato and 100 rpm agit2tion.
Thereafter the r~m~ ng substrate components were adde~ and p'~ was.~djustedb~ ar~u~d 6.0 with phosphoric acid. The substrate was sterilized in the main fermenter for 1~ hours a~ 123~.
Final ~olume befcre inocula~ion was .æound 1080 li.~r.

Then 150 liter o~ seed culture was added.

Fermentation conditions were: .

~ermenter type: Conventional aerated and agitated fermenter with a height/diameter ~ ratio oX around 2.7.
Agitation: 250 rpm (two turbine impellers) Aera~ion: 1200 normal liter air per minute.
Temperatuxe: 30C
Pressure: 0.5 ato Time: Ar~und lSl hours.

~ rom 24 fermentation hours to æound 116 f~u~,~ ~on h~urs pectin solution was added asepticall~ '~ the m2in f~ ~ at a o~tant rate of a~ound 8 li~ers per hour.The pec~in solution with the follow-ing composi~ion was prepared in a 500 ~iter dosiny tank:

Pectin genu x) . 22 kg Phosphoric acid, conc. 6 kg Pluronic~ . 50 ml I x~
Genu pecti~ (citrus type NF ~rom The Copenhagen ¦ pectin factory ~td.) ;

i Tap wat~r was a~ded to a t~tal volume of arou~d 325 liters.
~he substrate W2S sterilized in the dosing tank for 1 h~ur at 121C.
~inal volu~e before start of dosage was around 360 li'uers. ~en this portion ran out, another s~m;l~r portio~ was m de. Tbtal volume of pectin s~ on for one fe~,~lLdLion was æ o~nd 725 liters.
hfter around 151 fermentation hours the fermentation process ~s sLo~d. ~ne axound 1850 liters of culture broth were c~oled to around 5C
and the enzymes were recovered ~cc~r~;ng t~ ~he following method.
The culture broth w~s drum filtered on a vacuum drum filter (Do~r Oliver), w~ich was pre~ated with Hy-flo su¢er-cel ~i~t~ P~us P ~
(f;lter aid)O The filtrate wzs ccncen-rated by evd~o~dLion t~ around 15% of the volu~e of the cultuL~e broth. m e conc~l~d~e was filtered on a Seitz ~ilter sheet (type supra 1003 with 0.25~ Hy-flo-super-cel æs a f;lter aid (in the following table referred to as filtration I). The filtrate was precipitated wi~h 561 g of (NH4)2SO4/1 at a pH of 5.5, and 4%
Hy-~lo-su~x~- cel di~t~rPous earth is added as a fil'Pr aid. The pre-cipitate and the filter aid are ~a1~Led by filtraL~n on a fram~ fil~erO
qhe filter cake is dissolved in water, and l~oluh1e parts axe s~aLd~ed ky ~iltration on a frame filter. ~ne filtrate is checX filtexed on a~
Ceitz filter sheet (t~pe su~ra l~O) with 0.25% Hy-flo-suDer-cel as a filter aid (in the follcwing table L~L~ll~d to as filtration II). The ~;ltrate is diafiltered on an ultrafiltration d~ald~S. AftPr diafil-tration the liquid is con~-LL~Led to a dry mattex ~J..~lL of 12.7~ (in ~he following table L~f~Ll~ to as dry ~a~ter c~l~lL in c~~ Lld~e).

A facultati~e base treatment for partial removal of the prote3se acti~ity c2n be c~rr;P~ out at th`Ls stage. In case the base LL~ r~lL is used it is carried out at a pH of 9.2 for 1 hour, where-after the pX value is adjusted to SØ

Now the li~uid is check filtered and filter~d for the pur-p~se of germ xeduction and the fi~trate is reeze-dried o~ a ~ree2e-dryin~ eqUipmQn~ from Stokes.

Four fenment~ ~e c~rr;~ out in the manner indicated below, whereby the strain used for the fer- .
mentation~ the use of the facultati.ve base treatment and other ~al~,~s ~re vaxied, as indicated in the followin~ table.

Concentration (%) m~tter of filter aid in con-connection with tent Pre- in par~ fil- thepre- oon-Base treatm~t tion tra- cipi- f;ltr2- cenr Re~
Mi~lo~Ly~sm us~d not used c~de tion I tation tion II trate mar~s CBS 101.43 x KR~ ~ 0.5 5 0.2 .~8 ~ICC 20236 x KRF 74 2.0 4 0.4 7.5 ~FO 4408 x XR~83 1.0 5 0.2~ 12.4 x) CBS 101.43 x KRF92 Ø25 4 0.25 12.7 ) After genm reducing filtration the filtrate is conc~,LLdLed ky ~vd~ tion in a ratio of 1:2.3. A minor part of the conc~lLLaLed filtrate was spray-dried, and ~he r~m~;n;~ part w~s freeze-dried.
In order to reduce the protease activity further, so~e of the above indicated preparations were treated as indicated below, where~y only one of the three al~ernatives A, B, a~ C
was used.

.
- A~ 100 ~ SPS-ase preparation are dissolved in 1 li~er of deio-ni2ed water wi~h stirring at 10C ~ 2C. pH is adjusted to 9ol with 4 N.NaOH. This base treatment is carried out for 1 hour~
The pH value is then adjusted to 4.5 with glacial acetic acid~
and it is dialyzed against ice cold, ~ ni7~ water tD a CJ~ ~vity of 3 mSi. Then freezing and lyophilization are carried out.

Bo 500 g SPS~ase prPparation are dissol~ed in 4 liter of ~Pi~i7.0~ water with st;rri~ at lOGC ~ 2&. p~ is adjusted to 9.1 with 4 ~ NaO~. This base txeatmen~ is carried out or 1 hour. The pH value i5 the adjusted to 5.0 with glacial ace-tic acid. The obtained material is lyophilized.

C. 50 g 5PS-asP preparation ~re dissoivedin 400 ml of ~ ni7-ed wa~er with stirring at 10C + ~CO pH is adjusted to 9.1 with 4 N NaO~. This base ~reatment is carried out for 1 hour. Then pH is reduced to 5.7 with glacial acetic acid. The obtained material is lyophillzed.
, i ..~

SPS-ase preparation Base treat-used as starting ment used Preparation material for base code treatment A B C
XRF 68 x KRF 68 BII
KRF 68 x KRF 68 BIII
KRF 92 x KRF 9~ BI

The above indicated preparations are characterized by their activities of the enzym.es relevant to the invention in the following table.

.

-~ t~

Enzyme activlty per g RRE 68 XRF 6~ BIIKRF 68 ~TIIKRF 74XRF 83 ~RF 92XR~ 92 BI
Pla~e te~t + ~ + +
S~ .
Quan~ta~ive test 350 3Q1 349 0 168 476 430 ~RU . 481 142 683 626 157 S~U~120 2125 1560 1720 578 753 1640 1030 HUT pH 3.2 67000 105 339 1630 128UU 5960 397 Cx - ~000 8044 9396 1320 8040 ~7~0 3092 PU ~03000~0 9000000 8800000 8~00007500000 8400000 7600000 PGE 119400 ;72000 77700 4100 64600 60000 68800 PEE . . 840 910 77U 370 690 1000 790 ~3 , . - 44 -6~

Example 2 (application example) This example describes the production of a p.v.p.
from a dehulled and de~atted soy flour, "sojamel 13'i~(commer-cially available from Aarhus Oliefabrik A/S). The dry matter con~ent of this flour was 94.0~ and the content of (N x 6.25) on a dry matter basis was 58.7%. The soy flour was treated with the SPS-ase preparations KRE 68 BII (example 1) in the following manner: .

85.2 g Ofthe soy flour were suspended and kept stirred at 50C in 664.8 g of water, and pH was adjusted to 4.5 by means of 7.5 ml of 6 N ~Cl. 50 g of a solution containing 4.oO y of said SPS-ase preparation was added, and the reac-tion mixture was ~hen agitate~ for 240 minutes at 50C.
The ~ixture was then centrifuged in a laboratoxy centrifuge (Beckman Model J-6Bl for 15 minutes at 3000 x g. The super-natant ~Jas weighed and analysed for Kjeldahl N and dry matter. .
The solid phase was then washed with a volume of water equiva-lent to ~he mass of supernatant obtained by the first cen-trifugation. This operation was perfo~med twice. The solid phase was then freeze-dried, weighed and analysed for Xj~ldahl N nd dry matter at Qvist's Laboratorium, Marselis Boulevard i6~, 8000 ~arhus ~, Denmark. This laboratory is state authorized for analyses of fodder and dairy products. The results obtained in the experiment a~pear from Table ~

~ ~r~ k Table 2.1 Results obtained Mass N x 6.25 Dry ma.t- Yield ~ Yield o' Component g % ter % prv~eL~ y ratter, 9~ %
Soy flour 85.2 55.2 94.0 100 SPS-ase prepa- .
ration 4.~ 75.6 - 6.4%
1. Centrifugate 66,6 1~ ~ 5.04 21.2% 42.0~
p.v.p. 44.5 87~5 95.7 ~2.7~ 53.2%

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

Example 3 (application example) This example was performed in order to compare the protein yields, the nutritional quality and some functional properties of soy protein products made by the following three pro~edures:
.. . .. .
A: The traditional isoelectric 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 ~ncluding a remanence solubilizing en~yme for p~o~uctio~ o~ p.v.p~

In order to generate a true comparison of the process according to the invention (C) with the conventional soy protein processes (~ and B) the same raw material has been used in all three cases. Also the experiments have been conducted in such a manner that corresponding temperatures and treat-me~t times are the same in all three cases. Only the pH~values .

were different due to the funda~e~tal differences between the ~hree 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 extracted in 357~.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 1~ minutes using four one liter ~eakers in a laboratory centrifuge (Beckman Model J~6B). The centrifugate I and the precipitate I were weighed. The precipitate I was re-extracted with water to a total weight of 4000 g. The temperature was kept at 50C, pH adjusted to 8 with 4 N NaOH and the slurry kept stirred for one hour. A cen.rifugation and weighing of centrifugate II and precipitate II were perlcrmed as above.
Samples were drawn from centrifuga~e I and II and precipitate II for Kjeldahl and dry matter determinations. Hereafter the centri~ugates I and II were mixed and held a~ 50C~ The pro-tein was then isoelectrically precipitated at pH 4.5 by means of 45 g of 6 N HCl. After stirring for 1 hour at 50C
the protein was recovered by centriugation at 3000 x g for

15 minutes. The centrifugate III was weighed and analysed for Kjeldahl-N and dry matter. The solid phase III was weighed and washed wi~h water in an amount corresponding ~o the weight of centrifugate I. The washing was carried out by stirring fox one hour at 50C. The washed protein was recovered by ce~tri-fu~ation at 3000 x g ~or 15 minutes. The centrifugate IV ~nd the solid phase IV were weighed. Centrifugate IV was analysed for Xjeldahl~N and dry matter.The solid phase was suspended in 1550 g of water at 50C and pH was ad~usted to 6.S with 17 a of 4 N NaOH. The mix~ure was kept stirred for one hour and re-adj~sted to pH = ~.5 if necessary. Finally the product was freeze dried, weighed, and analysed for Xjeldahl-N and dry ~atter. The mass balance calculations axe shown in table 3.1.

Table 3.1 Mass balance calculations of the traditio~al iso-electric precipitation for production of soy prot~in . isolate.

Mass of Yield of Yiel~ o CFerations and fxactions ~raction Prot~n Dry mat- proteLn, dry mat-g %~N x 6.25) ter % % ter,%
Extraction: S~y flour 425.8 55.2 94.0 100.0 100.0 W~ter3574~2 0 0 0 0 4 N NaCP. 20.1 0 16.0 0 0.8 i.Centrifugati~n: ~4020.1 5.9 10.0 100.9 100.4 Centrifu~ate I 3141.0 4.4 6.9 58.8 54.1 Precipitate I 805.0 Re~ . Ll d~; Liorl:
Precipitate I 805.0 - - -Water 3195.0 O Q 0 0 2. C~ri fugation:
Centrifuga~e II3104.0 0.5 0.9 6.6 7.0 Precipitate II 820.0 9.1 17.2 31.7 35.2 Mixing and acidi~ing:
Centrifu~ates I + Il6245.0 6 ~ HCl 45.0 0 21.3 0 2.a 3. C2ntrifu~ation: ~: 6290.0 Centrifugate III 5650 . 0 0 . 3 1. 9 7 . 2 26 . 8 Precipitate II:C 308 . O - - - -Wash~}g .
Preci2itate III 308. û - - - -~ater 3141. O O . 0 0 O
~, 4. c~ntrj Fugation: 3449.0 Cen~rifugat~ IV 3113.0 0.04 0.15 0.5 1.2 Precipitate IV 291.0 - - - -Pr~3cipita~e IV 291.~
~ater 1550. 0 ~ O û O
4 N NaO~I 17.0 0 16.0 0 0.7 ~yir2g: Pa~er 128.0 93.8 96.3 Sl.l. 3~.8 -B. The isoelectric wash for production of soy pxoteinconcentrate 425.6 g of soy meal (~ojamel 13 produced by ~arhus Oliefabrik A/S) was washed in 3574 g of water at 50C. pH
was adjusted to 4.5 with 44.8 g of 6 N HCl. ~he washing was carried out for four hours by asitating. The slurry was then centrifuged at 3000 x g for 15 minutes in a laboratory cen-trifuge (Beckman Modei J-6~) using four one liter beakers.
The centrifusate I ~las weighed and analysed for Kjeldahl N and dry matter. The solid phase I was weighed and re-washed with water to a total weight of 4000 g. pH was re-adjusted to 4.5 with 1.7 g of 6 N ~Cl and the slurry was kept stirred for 30 minutes at 50C. A centrifugation and weighi~y of centri-fugate II and solids II were performed as above. The solia phase XI was resuspended in 1575 g of H2O at 50C and pH
was adjusted to 6.5 with 34.5 g of 4 N NaOH. The mixtu~e was kept stirred a. 50C for one hour and re-adjusted to p~=6.5 if necessary. ~inally the protein product was freeze dried, weighed, and analysed for K~eldahl N and dry matt~r.
The mass balance is shown in table 3.2.

8~

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

Mass of Yield of Yield of OpDrations and fractions: fraction Pr~tein Dry maL- protein, dry mat-~ %(~:x6.253 ter % % ter,%

Soy flour425.8 55~2 94.0 100.0100.0 ~ater 3574.0 0 0 0 0 6 N HCl 44.8 0 21.3 0 2.4 l. Centrifugation: ~4044.~
Centrifugate I 3150.3 0.6 3.2 8.0 25.2 Solids I846.0 Re-~h;n~
Solids I846.0 - - - -Water 3154.0 0 .0 0 6 N HCl 1~7 0 21.3 0 0.1 2. Centrifugation ~4001.7 CentrifusateII 3130.0 0.1 0.4 1.3 3.2 Solids II863.0 ~eutralization:
Solids II~63.0 ~
~te~ 1575.0 O - O . 0 O
4 N NaC~34.~ G 16.0 .0 1.4 Drying: Pcwder 281.0 72.5 98.4 86.769.1 ., - 50~B~

C. me isoel~ic wash including a remanence solu-bilizing enz~me for production of p.~.p.

425 . 8 g of soy meal (So jamel 13 produced by Aarhus Oliefabrik A/S) was washed in 3524. 2 g of water at 50C. pH
was ad justed to 4.5 by use of 43.7 g of 6 N HCl . 24 g of the SPS-ase preparation KRF 68 BIII (example 1) were solubilized in.26 g of water and added to the washing mixture. The washing was then carried out for four hours by agitation. Subsequently the purification was performed as described ~or B, the amounts of 6 N ~Cl, 4 N NaOH and wa~er for resuspension being the only parameters with deviating values. The mass balance is shown in table 3.3.

~ ~ 8~?9 9 Table 3.3 Mass balance calculations of the isoelectric wash :including a remanence solubilizing enzyme for pro-duction of p.v.p.

Mass of Yield of Yield of ~ati~ns an~ ~ractions: fraction Protein Dry mat- PrO~J dry mat-g ~ x 6.25 ter % % te~
~Ja~L~g:
Soy flour425.8 55.2 g4.0 100.0 100~0 ~ater35~0.2 0 0 0 0 6 N HCl43.7 0 21.3 0 2.3 5PS-ase.:KR~ 68 BIII24.0 75.3 96.0 7.7 5.8 1. Centrifugation: ~4043.7 - . - - -Centr ~ gate I 3420.0 1.7 5.2 24.7 44.4 Solids I620.0 ~w2sh~:
So~ ~ 620.0 - -~ater3380.0 0 0 0 0 6 N HCl 1.3 0 21.3 0 0.1 2. Centri~uga~;o~. ~ 4001.3 Centrifuga.e II 3400.0 0.2 0.6 2.9 5.1 Solids II 577.0 Neutr~l; 7~tion S~lids II 577.0 ~'a~er ...1700:00 - 0 0 0 4 N NaOH 25.3 0 16.0 0 1.0 ing: Pcw~er 211.0 87.31)96.71) 78.2 51.1 86.9~97.o2) 1) Analysed at Bioteknisk Institut, Holbergs~ej 10, DK-6000 Kolding, Denmaxk 2) Analysed at QYist9 S ~aboratorium, Marselis ~oulevard 169, DK-~000, Aarhus C, Denmark I Nutritional Properties The amino acid compositions of the three protein products were determined, vidP 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 FAO
re~erence pattern from 1957.

The trypsin inhibitor content of the ~hree products was determined by means of the method described in A.O.C.S. Ten~tive th3d Ba 12 - 75 (A.O.C.S. is an abbreviation for pm~r;r~n Oil Chemists' Society).
The results zre shown in ~able 3.5, which also includes the yields and the proteinjdry matter ratio of the three products.

-Table 3.4 Am~no acid COmpOSLtiOn and nutritional evaluationof the three protein products A, B, and C~ .

~. Soy proteinB. 5Oy protein C. Soy protein Amino acid isolateconcentrate isolate (p.v.p.) g/16 gN aas ~ g/16 g N aas ) g/16 gN aas1) Non-essential:
___ _______ Aspartic acid 12.4 _ 11.3 _ 11,9 Serine 4.62 - 4.69 - 4.81 Glutamic acid 21.3 - 18.2 ~ 17.7 Proline 6.07 ~ 5.19 - 4.76 Glycine 4.13 ~ 4.26 - 4.33 ~lanine 3.54 - 4.27 - 4.55 Histidine 2.83 - 2.78 - 2.50 Axginine 8.09 - 7.57 - 7.C4 Essential:
Isoleucine . 4.37 ~ 100 4.g7 ~100 5.19 > 100 Leucine 7.80 > 100 7.98 ~100 8.09 ~100 ~sine 6.24 > 100 6.09 ~100 5O57 >100 Phenylalanine l>100 ~100 5.17 >10~}
Tyrosine- 3~38 >100~ 3488 ~100) 4.44 ~100 Cystine 1.29 64~5l56 1.32 66.0l 1.44 72.0 ~ethionine 1.08 49.1J 1.21 55.0J 1.31 59.5) Threonine 3.10 > 100 3.60 ~100 3.97 ~130 Tryptvphan lo 06 75.7 1137 97.9 1.32 94.3 Valine 4.90 ~100 5.23 ~100 5.57 >100 % total conte~t of essential 38~36 41O31 42:21 amino acids Chemical score 56.4% 63.2% 65~5%
EAAI 86~7~ 90~2% 91.3 1) aas ~ amino acid score based on the FAO reference pattern (1g57 , -. .. .. . ... . ... .. . _ . _ . ...

Table 3.5 Process characteristics and trypsin inhibitor . content of the three protein products A, B, and A. Soy protein B. Soy protein C. Soy protein isolate concentrate isolate (p.vOp.) Protein of 97,4 % 73 7 ~ 9 Process dry matter charac-teri5tiC5 Protein 51~1 ~ 86.7 % 78.2 %
- yield Trypsin inhibitors. 34 0OO 21,000 l9,000 TUI/g pxoduct TU~/g protein 36,250 28,970 21,810 n~tional Properties Nitro~en solubility ihdex ~NSI) was determined 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 s~irrer the suspension was centrifuged at 4000 x g for 30 minutes, and the supernatan~ was analysed for nitrogen.
The ~itxogen solubility was calculated as (soluble N%/total N%).
The results o~ this evaluation on the three products are shown in table 3.6.

Emulsifyin~ ca~acity was determined three times on _ ~ _ _ _ _ _ _ _ __ _ _ _ _ _ each product by a slightly modified Swift .itration. 4.0 g of (N x 6.25) of the product was blended in 250 ml of 0.5 ~M ~aCl with a Sorval Omnim~ xe ~at low speed. 50 ml of the suspension were ~ransferred to a glass blender jar and 50 ml of soy bean oil were added. ~ereafter the total mixture was weighed. The oil-water mixture was then homogenized at 10.000 rpm with the jar in an ice-bath. A supplementary amount of s~y bean oil was added at a rate of 0.3 ml per second until the emulsion collapses. The total amount of oil added before the "end point" wa$ found by weighing.

Emulsifying capacity was calculated as ml oil per gr~m protein (~ x 6.25). The density of the oil was taken as o . g g./~ ' The a~erage results of the determination of emul-sifying capaci~y on ~he ~hree products are shown in table 3.6.

Whi~in~ exPansion was determ;ned in a 3% protein s~lu-___ __ ___ ______ tion at pH = 6.5. 25Q ml of the aqueous dispersion of the pro-tein ~ ples were whipped at speed III for 4 minutes in a ~obar~ mixex (model N-50) mounted with a wire whip~ The whipping expansion was calculated according to the formula ~hipping expansion = V255~ x 100%, aole ~k5 whexe V = final whip volume in ml.

V was measur~ by refilling the mixer jar with water.
Duplicates were performed for each of the ~hree samples. The average results are shown in ta~le 3~6.

Foam stability was determined as the ratio between _____________ .
the amount of foam left after draining for 30 minutes and the ori~inal amount of foam. A gram of foam produced by the method above was introduced into a plastic cylinder (diameter 7 cm, hei~ht 9 cm~ having a wire net with a mesh size of 1 mm x 1 mm.
The cylinder was placed on a funnel on top of a glass cylinder and the weight (B) of drained li~uid in the glass cylinder is determined. The foam stability ~S is defined by the equation FS = A A B x 100%

The results of the determination is shown in table 3.6.

The ~el stren~th is in this specification defined as the Brookfield viscosity measured by means of T-spindles on a Brookfield Helipath stand. The gels were made by heat treatment of 12~ protein suspensions in 0.5 M NaCl. The heat treatment was performed in closed cans with a diameter of 7.3 cm and a height of ~.0 cm placed in a water bath maintained at 80C and 100C
each for 30 minutes. The cans were cooled and thermo-statted ~o 20C before they were opened and measuxed. The results o~ the measurements axe shown in table 3.6.

-;

Table 3.6 Functional properties of ~he three protein pro~
ducts A, B, and C.

. . A. Soy protein ~7 Soy protein C. Soy protein Functlonallty isolate concentrate isolate (p.v.p. ) % MSI in 0.2 ~ NaCl 39.5 20.3 25o6 % ~SI in water 53.9 25.1 28.6 Emulsifying capa-city: ml oil/g 218 ~82 354 (~ x 6.2S) Whipping expansion % 1~0 120 340 Foam stability % 50 50 20 Gel s~rength;~p~is~
80C (0.5 M NaCl)1.7 x 1031.2x 104 3.3 X102 100C (0.5 M NaCl)2.0 x104 4.0 x 10 1 3x104 Example 4 (applica,ion example) A p.v.p. was produced accordina to the procedure described in example 3 C except that the cellulase activity was partially derived from Trichoderma reseei. The commer-cial cellulase preparation CELLUCLA5T~ produced by Novo In-dustri A/S was treated ~lith a base at low temperature in ~he f~llowing manner. The pH value of a 10% CELLUCLAST solu-tion in water W2S adjusted to 9.~ with NaOH, and the thus resulting solution was cooled to 5C. After 1 hour at this pH and this tempexature the pH was re-adjusted to 4.7 with 20% acetic acid. This solution was kept at 5C overnight and then sterile filtered. The filtrate was freeze dried. 4 g of t the fre~ze drie~ pr~duct was added ~ether with the CDS-ase prenaration XRF 6 EIII (example 1). me tw~ en~ymes were solubilized in 172 g of water before addition to the washing mixture~ The mass balance determ nations of this example is shown in table 4.1.

The experiment demon-strates that 'hi~ p ~ ;~l~r SPS-ase preparation already contains an eflicient cellulase as addition of CELLUCLAST does not seem to effect the protein/
dry-matter`ratio. However, other SPS-ase preparations may contain less cellulase, e.g. KRF 92, vide the table immediately preceding-example 2.

~ ~rC~ k :
..

:

Table 4.1 Mass balance ~eterminations of the iso-electric wash including an SPS-ase preparation and C~LL~CLAST
for production of p.v.p.

Operations and Mass of Protein Dry matter Yield of Yield of fractions fraction % % protein dry matte~
gram (N x 6.25) % %
Washing:
S~y flour 425.8 55.2 9400 100.0 100.0 Water 3546.2 0 0 0 0 6 N ~Cl 43.1 0 21.3 0 2~3 SPS-ase: KR~-68-B-III X4.0 75.3 96 7.7 5.8 ~ . ..
CEL~VCLAST 400 43.6 .96 0.7 1.0 Centrifugation: ~ 4043.1 Centrifugate ~ 3382.9 1.9 5.5 27.3 46.5 Solids I 661.0 - - - -: Re-washing:
Solids I - 661.0 . - - -Water 3339.0 0 0 ~ 6 N ~Cl - O 0 ---- - -2nd centri~ugation: ~ 4000.0 Centrifugate II 3414.0 0.2 0.7 2.9 6.0 Solids II 582.0 -Neu~r~lization: ;
Solids II .582.0 . ~
Wa~er 1691.0 0 0 O O
4 N N~VH 25.3 0 16.0 0 1.0 . ~ . .. . .. .. .. . . . .
Dxying:
Powder - 206~0 88.8 98.9 77.8 5Q.9 .;

.., ! I .
~xample 5 (application example) A p.v.p. was produced according to the method described in example 3 C excep~ that all masses were scaled down with a factor of .
5, and that the reaction mixture was cooled to ~x~t 5~C prior to the cen-trifuga~io~. On the basis of the ~ytical results in relation to the cP~tr;fugates a theoretica~ yield OL precipitated protein is ob~ned, as s~ in table 5.1 Table 5.1 Theoretical pro~n yiPlds ob~ned in the production of p.v.p.

M Protein Yield of Example 3 C
Frac,ions ass ~N x 6.25~ protein . %protein,~
So~ flour 85.255.2 100 5S.2 100 S2S-ase K~F-68 B-III4,875.3 7O7 75.3 7.7 1st c~ntrifugate 6390.99 13.5 1.7 24.7 ~2nd centrif~gate 595 0.13 1.6 0.2 2.9 P-~ P- - 87.2a 92.6b 87.1 80.1b a A~erage of 87.5 (Bioteknisk Institut) and B6.9 (Qvistls Labora-torium); dry matter is 97.6 and 98.0%, respectively.
b Calculated as total mass of protein - protein lost in centri-fuga~es.

Example 6 (application example~

Demonstration of the ~rotein bindin~ of SPS

40 grams of (N x 6.25) ~rom a ~omm~rcial soy protein iso-late (Purina 500 E from Ralston Purina3 was dissolved in 680 g of water. The mixture was heated in a water bath to 50~C, and p~ was adjusted to ~.50 with 6 ~ HCl. gO g of this mixture was transferred Hereafter the slurries were centrifuged at 3000x G ~or 15 minutes, and the centrifugates I were analysed for K jeldahl-N
and dry mat~er. The solid phases were washed in water at room tem-perature and re-oentrifuged~ This procedure ~Jas repeated. Then the solids were dispersed in 50 ml of water, and pH was ad justed to 6.50 by drop-wise addition of 6 N NaOH. ~he neut~alized products were freeze dried and analysed for Kjeldahl-N and dry matter. Based on the analysis shown in Table 6.1, the protein recovery and the percentage of SPS which has b~en bound to the protein are calculated by means o~ the formulas shown in relation to Ta~le 6.2.

This example demonstrates-that the SPS is bound fir~ly to the protein so that the prote~n/dry matter ratio aecreases with in-creasing content of SPS. An SPS conient comparable to about 0.4 g in 10 g of water added to 5 g of protein isolate is ~he protei~/SPS
ratio present in the soy flour.

The ~ b~x~ng o SPS is a c~l~ttl~ted value. ~he % h;~in~ of SPS de-creases due to saturation of the protein with regard to SPS at the low proteintSPS ratios.

.:

- 6~ - .

Table 6.1 Measurements according to Example 6 Ratio Centrifugates I Dried precipitate Protein/SPS % N % DM 96 N % N x 6 0 25 % DM % ~ XD6M 5 oo 0.068 0.62 li.2 82;5 93;1 88.6 _ 2~ 0.045 0.~9 13.4 83.8 37.3 86.1 12.5 0.038 0.45 13.0 81.3 97.9 83.0 6.25 0.031 0~45 12.6 78.8 98.1 ~0.3 3.1250.026 0.61 11.8 73.8 97.g 75.3 Table 6.2 Protein recovery and % binding of SPS

Ratio % recovery of protein1) % binding of SPS2) Protein/SPS
oo 91.5 0 94.4 77 1~.5 9~.3 90 6.25 96.1 70.
3.. 1~5 9608 60 % xecovery of protein - [1 _ NC 1 6-25~ 100 NC 1 = ~ ~ in centrifuga~e I

2~ % binding of SPS =
r 5xl% r~x~ry of prot.ein) _ 5x(% r~ y of protein) ~% P/H) ~% P/H)~
x 10~, ~ere [S/ratio of sp~
~ P/H) is the prGtein/dry matter ratio in the dried preci-pitate, and ~ P~H~oo is ~or the pr~ci~itate wi~hout addition of SPS.

,"1 ta~

Example 7 (application example) This example describes the production of a p.~.p. using the SPS-ase preparation KRF 92 B-I in a dosage of 5% of the dry matter. The manner of production was exactly as in Example 3 C, except that all masses were scaled down with a factor of S. The p.v.p. was analysed as described in Example 2. The rPsults obtained in the experiment appPar from Table 7.l.

Table 7.1 Results obtained in Example 7 Mass (N x 6.25) Dry matter :Y.ield of Yield of dry Component g % % ~rotein, m~tter, Soy flour 85~2 55.2- 94.0 lO0 lO0 - Enzyme preparation 4.0 7l.2 - 6.l 1st centrifugate 632 l.88 5.44 25.3 43.0 2nd centrifuga~e 673 0~30. 0.80 4.3 6.7 p.v.p. 3g~ 85.6a . 98.la 71.. 9 48.
84 ~b 98 lb Analysed at Bioteknisk Institut, Holbergsvej lO, DX-6000 ~olding b Analysed at Qvist's Lahoratorium, Marselis Boulevard 169, DK-8000 Aarhus C
xample 8 (applicati~n example) This examp~e demonstrates ~he effect of pretreating the .soy meal by jet cooking before the production of p.v.p.

Pretreat~ent A slurry of soymeal in water consisting of lO kg soymeal ~soj~mel 13 produced by Aarhus Oliefabrik ~/S) per lO0 ~g was pumped through a steam-ejector (type Hydroheater B-300) and mixed with steam of ~ Bar in such amount and by such ~low that a final temperature o~ 150C could be maintained for 25 seconds in a tubulax pressurized reac~orO ~erea~ter the pressure was released , `'~1 ' ' , .

in a flash chamber (a cyclone) and from here the slurxy was 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 invention,but in this case the slurry W2S spray-dr.ied at an inlet temperature of 200C and at an outlet tempera-ture of 90C. Ihe pretreated E~ wzs found to have a dry m~tter ~ L~lL
of 96.5% and a pro~ein content of ~6.9~ (N x 6~25~o Pr~duction of p.v.P.

This production was carried out in the following way:

70 g ofdry matter of the jet cooked and dried soy flour was suspended and kept stirred at 50C in 560 g of water,and pH
was adjusted to 4.50 by means of 6~5 mlof 6N HCl. 6 x 90 g of this suspension was transferred to six 250 ml Erle~meyer flasks and kept stirxed on a 50C water bath by means of magnetic stirrers.
To each flask were added 10 g of a solution containing resp~ctively ~, 0c025 g; 0.050 g; 0.10 g; 0.20 g, and 0.40 g of the SPS-ase preparation K~F-68-B-III. The reaction mixtures were t~en agi~a~ed for 240 minutes at 50 C. Then a centrifugatio~ at 3000 x g for 15 minutes was carried out.

The supernatant was then analysed for ~jeldahl-~ and the solid phase was washed with water at equal volumes and centrifuged~ This procedure was performed twice. The solid phase was then freeze-dried and analysed.for Kjeldahl-N and dry mat~erO

A s~milar exper~ment was carried out wi~h an untreated soy meal (Sojamel 13 from Aarhus Oliefabrik A~S) as a starting material. In ~his case the enzyme suhstrate ra~ios were 0; 1~;
2%; 3%; 4%; and 8~.

Based on the pro~ein content of the supernatants the percentage of recovered protein can be calculated. The yield of protein is based on the assumption that the enzyme pxoduct is 100% solubilized ~.fter the reaction. The table below shows the results ob~a~ned by both experiments.

I

Table 8.1 Cooked soymeal Untreated soymeal E/S ~ Protein Protein of Protein Protein of yield % dry ma~ yield ~ ~ry mat~

- O 9~.9 76.5 gO.7 73.9 0.25 90.1 86 6 - -0.5~ 8~.3 88.7 - .
~- 1.0 88.1 ~9.7 87.1 86.~
~.0 85.6 91.7 ~5.7 ~8.1 3.0 - ' - 84.3 89.5 4.0 84.7 9~2 8~.6 . 90~9 76.2 91.1 Table ~.1 Protein yields and prot~in dry matter ratio for p.v.p.
.produced~from co`o~ed-or raw soymeal.~

Isolation of pro~ein from corn gluten Corn gluten is usually produced as a by-product in the corn starch manufacturing process. This raw corn gluten is not separated effectively from the fiber fraction, and this is one of the reasons why it is without any valuable functional properties. Also, differing degradable polysaccharides accompany raw corn gluten.
A series of SPS-ase treatments was run with an SPS~ase preparation produced according to example 1, except that an ultrafiltration was performed instead of the (NH4)2SO4 precipitation, whereby the isolated enzyme was base treated according to method A (this base treated SPS-ase preparation for the sake o~ brevity being referred to in the following as PPS
1305). PPS 1305 practically does not contain any proteolytic activity.
The following reaction conditions were used:
5ubstrate concentration: S = 10% dry matter (Staley Corn Gluten) pH = 4.50 T = 50C
t = 240 minutes Enzyme. PPS 1305 E/S = 0; 1; 2; 3; 4; 8%

The protein product was purified by centrifugation, washed twice, and finally freeze dried.

The results appear from the following table 9.

Table g Experiment Enzyme dose Protein Dissolved Protein No. E~S~ yield poIys~rh~ de purity % g (Nx6, 25/DM) %

~4 1 95 ~0 75.

. .. 876 3 95 52 ' 79 EXample 10 Isolation o~ protein from cotton seed meal, sunflower meal, or rape meaI.

The protein from cotton seed mezl,sunflowPr meal or rape meal can be isolated in the same manner zs the protei~ from soy bean meal and corn gluten. Isolation of protein from se~eral other pro-teinaceous vegetable raw materials can be performed i~ the same man-ner. Of course, the isolation and/or the separation should preferably be carried out at the isoeIectric point of the main part of the pro-tein part of the starting mat~rial, whereby the protein yield is .m~ ~; m ~1 ," . . . ' ,,: . . ,5 .. _, ~ . . .. . .

~ 68 -A survey of th~ ~igures, to which reference has been made already, is given below for the purpose of providi~g a better comprehensive view.

Fig. No. Belongs to Describes 1 The general part Demonstration of binding ef~ect of the specifica- between CPS and soy protein.
tion 2 The general part Flow sheet describing the pro-of the specifica- duction of SPS
tisn -3 Sec~ion ~ Calibration curve for Y.PLC
gel filtration chromatography 4 Section 2 HPLC gel ~iltration chromato-yram of SPS
Sectio~ 2 HPLC gel filtration chromato~ram of SPS decomposed by SPS-ase : 6 Sec~io~s 2 and 3 HPLC gel filtxation chromatogr~m . . of supernatant from SPS incubated with soy protein .
7 Section 2 ~PLC gel ~iltration chromatogram of supernatant from ~eco~posed SPS incubated with soy protein 8 Section 3 ~PLC gel iltra~ion chromatsgram of ~PS decom~osed by Pectolyase 9 Se tion 3 ~P~C gel filtration chromatogram of ~PS decomposed by SPS-ase Section 3 . ~PLC gel filtratio~ chromatogram of SPS treated with Pectolyase . ~

~ig. No. Belongs to Describes 11 Section 7 Immunoelectrophoretic peaks including an SPS-ase peak identified by overlay technique 12 Section 8 Ion exchan~e chromatogram of an CPS ase 13 Section 9 pH-actiYity dependency of an SPS-ase 14 Section 9 Temperature activity dependency o an SPS-ase Section 9 Temperature stability of an SPS-ase

16 Section 10 pH-stability of protease in an SPS-ase preparation -i ,(

Claims (20)

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

1. Agent for decomposition of vegetable remanence, in the presence of vegetable protein, suited for production of a p.v.p. with a protein purity of around 90% with a vegetable protein, which may be defatted or partially defatted, as a starting material, comprising an enzyme with remanence solubilizing activity wherein the agent comprises an enzyme which is able to decompose soy SPS (SPS-ase) and wherein the agent is essentially free from proteolytic activity.

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

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

4. Agent according to claim 1, wherein the active component is derived from the enzymes producible by means of Asp. aculeatus CBS 101.43.

5. Agent according to claim 1, wherein the SPS-ase is immunoelectrophoeretically idential to the SPS-ase producible by means of Asp. aculeatus CBS 101.43 and identifiable by means of the immunoelectrophoeretical overlay technique.

6. Agent according to claim 4 or 5, wherein the ratio between the proteolytic activity in HUT-units and the remanence solubilizing activity in SRUM-120-units is less than about 2:1.

7. Agent according to claim 1, claim 2 or claim 3, wherein the agent contains cellulase activity is derived partially or totally from Trichoderma reseei.

8. Agent according to claim 1, claim 2 or claim 3, wherein the agent contains cellulase activity (Cx), pectinase activity (PU, PGE, UPTE, PEE) and hemicellulase activity (VHCU).

9. Agent according to claim 4 or claim 5, wherein the ratio between the proteolytic activity in HUT-units and the remanence solubilizing activity in SRUM-120 units is less than about 1:1.

10. Agent according to claim 4 or claim 5, wherein the ratio between the proteolytic activity in HUT-units and the remanence solubilizing activity in SRUM-120-units is less than 0.25:1.

11. Method for production of a purified vegetable protein product by removal of the remanence from a raw vegetable protein serving as starting material, wherein the starting material is treated with the agent according to claim 1 in an aqueous medium at a pH value which does not differ more than 1.5 pH units from the isoelectric point of the main part of the protein part of the starting material, and at a temperature between about 20 and about 70°C, until at least around 60% of the remanence, on the basis of nitrogen and dry matter mass balance has been solubilized, followed by separation of the solid phase containing the purified vegetable protein product from the supernatant.

12. Method according to claim 11, wherein the separation is carried out at a temperature between room temperature and the freezing point of the supernatant.

13. Method according to claim 11, wherein the starting material is defatted or defatted and further partially purified vegetable protein.

14. Method according to claim 11, claim 12 or claim 13, wherein the starting material is soy meal.

15. Method according to claim 11, claim 12 or claim 13, wherein the starting material is heat treated soy meal.

16. Method according to claim 11, claim 12 or claim 13, wherein the starting material is jet cooked soy meal.

17. Method according to claim 12, wherein the treatment of the starting material with said agent is continued until at least about 70% of the remanence, on the basis of nitrogen and dry matter mass balance, has been solubilized.

18. Method according to claim 11 or claim 12, wherein the starting material is treated with said agent until at least around 60% of the remanence, on the basis of nitrogen and dry matter mass balance, has been solubilized.

19. Method according to claim 17, wherein the starting material is soy meal.

20. Method according to claim 11, claim 12 or claim 13, wherein the starting material is able to pass a sieve with a mesh opening of around 2.5 mm.