GB2116977A - An 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

An 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 Download PDF

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GB2116977A
GB2116977A GB08236263A GB8236263A GB2116977A GB 2116977 A GB2116977 A GB 2116977A GB 08236263 A GB08236263 A GB 08236263A GB 8236263 A GB8236263 A GB 8236263A GB 2116977 A GB2116977 A GB 2116977A
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sps
protein
ase
remanence
soy
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Jens Lorenz Adler-Nissen
Georg Wilhelm Jensen
Steen Riisgaard
Henrik Guertler
Hans Aage Sejr Olsen
Martin Schuelein
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Novo Nordisk AS
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Novo Industri AS
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Abstract

The agent comprises an enzymatic activity which is able to decompose a high molecular carbohydrate, 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 quantitative 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

SPECIFICATION Improvements in and relating to an agent for decomposition of vegetable remanence, especially soy remanence, a method for production of a purified vegetable protein product, and the purified vegetable protein product A method for production of a purified vegetable protein product (pvp) by enzymatic removal of the remanence, without dissolution and reprecipitation of the protein, is described in BE patent No. 882.769.
Also in this patent the importance of the fact that the proteolytic activity should be kept as low as possible, is described. The purity of the pvp obtainable by the known method is not satisfactory and therefore open to improvement. In the examples a purity of the pvp of about 85% was demonstrated. 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 protein 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, especially dehulled and defatted soy meat.
The invention is based upon the surprising discovery that a certain part of the remanence decompose tion product, as it appears during the enzymatic treatment indicated above, i.e. a water soluble, high molecular carbohydrate, attaches itself to part of the protein, as will be explained later in detail. This, of course results in a lower purity of the protein.
Thus, an object of the invention is to provide an agent for decomposition of vegetable remanence, in the presence of vegetable protein, whereby the vegetable remanence especially is soy remenence, which will result in a pvp with improved purity, and a method for production of a pvp.
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 indicated, whereas all supernatants are left out.
A charge of soy meal was divided in two equal parts, part land part II (column a in fig. 1). Part I was decomposed proteolytically at a pH value of about 8 by means of ALCALASE a proteolytic enzyme produced by means of B. licheniformis and marketed by NOVO INDUSTRI AIS, 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 bin fig. 1).Now, both remanence I and part II is decomposed by means of a commercial pectinase, e.g. PECTINEXs (a pectolytic enzyme produced by Schweizerische Ferment AIG, 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, where 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 brought together with a soy protein suspension at pH 4.5, a polysaccharide in the supernatant is bound to the soy protein.This polysaccharide in the supernatant from 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 bound to soy protein at or around the isoelectric point of soy protein, if soy protein is present, is designated SPS (Soluble Polysaccharide), videfig. 1.
The SPS has a molecular weight distribution between 5x106 and 4.9x 104. The production of isolated SPS appears from the flow sheet shown on fig.
2 which also encompasses some of the processes 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 decomposition products do not bind soy protein or do bind soy protein to a much lesser extent than SPS binds soy protein.
Although the disclosure mainly refers to decomposition of soy protein, the invention is not restricted to soy protein, but emcompasses 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 to decompose soy SPS 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 herewith 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 priorto 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 remanence, especially soy remanence, in the presence of vegetable protein, especially soy protein, suited for production of a pvp 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.
The agent is essentially free from proteiolytic activ The drawing(s) originally filed were informal and the print here reproduced is taken from a later filed formal copy.
This print takes account of replacement documents later filed to enable the application to comply with the formal requirements of the Patents Rules 1982.
ity, when the proteolytic activity is equal to or less than the proteolytic activity, which will be accompa nied by a total protein 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 nitrogen 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 originating from vegetables and fruits more completely than commercial pectinases and commercial cellulases.
By total or partial elimination of the SPS from the final vegetable protein the purity of the final vegetable protein necessarily is improved in comparison with the purity of the final vegetable protein obtainable according to the method known from BE patent No.882.769, 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 single enzyme or from an enzyme complex comprising at least two enzymes. Some investigations seem to indicate that at least two enzymes are responsible for the SPS-ase degradation effect whereby one 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 notwantto be restricted by such hypothesis or similar hypotheses.
A preferred embodiment of the agent according to the invention is characterized by the fact that the SPS-ase was produced by means of a microorganism belonging to the genus Aspergillus.
A preferred embodiment of the agent according to the invention is characterized by the fact that the active component is derived from the enzymes producible by means of Asp. aculeatus CBS 101.43.
The same SPS-ase can be produced by means of Asp. japonicus IFO 4408. It has been found that Asp.
aculeatus CBS 101.43 also produces very potent remanence solubilizing enzymes, cellulases, pectinases, and hemicellulases. Furthermore, it has been found that not each and every strain belonging to the species Asp. aculeatus or Asp. japonicus generate an SPS-ase needed for the invention. Thus, as it appears from a later paragraph in this specification, it has been demonstrated that the Asp. japonicus ATOC 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 the specification.
A preferred embodiment of the agent according to the invention is characterized by the fact that the SPS-as is immunoelectrophoretically identical to the SPS-ase producible by means of Asp. aculeatus CBS 101.43 and identifiable by means of the immunoelectrophoretical overlay technique, vide section 6 and 7.
A preferred embodiment of the agent according to the invention is characterized by the fact that the ratio between the proteolytic activity in HUT-units and the remanence solubilizing activity in SRUM 120 - units is less than about 2:1, preferably less than 1 :1, more preferably less than 0.25:1. It has been found that a clear correlation between the protease activity expressed in HUT units (to be defined later) at pH 3.2 (the pH activity optimum of the protease) and the protein loss exists. This correlation is specificforthe SPS-ase preparation producible by means of Asp. aculeatus CBS 101.43.It is to be understood that this correlation may not exist in relation to another SPS-ase forming microorganism, which forms an SPS-ase which differs from the SPS-ase producible by means of CBS 101.43, but that a man skilled in the art will be able to final similar correlation which will fulfill the requirement in regard to protein loss stated in the main claim.
The SRUM-120 activity (to be defined later) is a measure of conventional remanence solubilizing, cellulase, pectinase, and hemicellulase activities and other activities. The SRUM-120 units are measured at pH 4.5. It is to be understood that this pH is chosen because the decomposition of the remanence is carried out at or around the isoelectric pH of the soy protein, and that a different enzyme activity method has to be used in case the decomposition of the remanence is carried out at another pH-value.
A preferred embodiment af the agent according to the invention is characterized by the fact that the agent contains cellulase activity, and the cellulase activity is derived partially or totally from Trichoderma reseei. This cellulase is able to dissolve crystalline cellulose, and the agent gives rise to a pvp with high purity.
A preferred embodiment of the agent according to the invention is characterized by the fact that the agent comprises cellulase activity (Cx), pectinase activity (PU, PGE, UPTE, PEE) and hemicellulase activity (VHCU) In Agr. Biol. Chem. 40 (1), 87-92, 1976 it is described that a strain of Asp. japonicus, ATCC 20236, produces an enzyme complex which is able to perform a partial degradation 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 detail in section 3.Thus, the HPLC gel filtration chromatograms of SPS and APS are clearly different, and furthermore, the gel filtration chromatograms of APS decomposed by means of the commercial pectinase Pectolyase and of SPS treated with the commercial pectinase Pectolyase are clearly different. Furthermore, it does not appear from the article that the acidic polysaccharide is bound to the soy protein and that the decomposed acidic polysaccharide is not bound to the soy protein or is bound to the soy protein to a much lesser degree than the undecomposed aciddic polysaccharide. Also, it has been demonstrated that this strain does not form SPS-ase in such amounts, which can be detected by means of the enzymatic determination of SPS-ase described in this specification. This creates a prejudice against any strain of Asp.
japonicus being a producer of an SPS-ase, but surprisingly according to the invention it has been found that some strains of Asp. japonicus are producers of an SPS-ase.
The invention comprises in its second aspect a method for production of a purified vegetable pro tein 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 the invention 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, preferably at least around 70% thereof, more preferably at least around 80% thereof, has been solubilized, followed by separation of the solid phase containing the purified vegetable protein product from the supernatant.
A preferred embodiment of the method according to the invention is characterized by the fact that the separation is carried out at a temperature between room temperature and the freezing point of the supernatant. Hereby a high protein yield is obtained.
A preferred embodiment of the method according to the invention is characterized by the fact that the starting material is defatted or defatted and further partially purified vegetable protein. This starting material is easily available.
A preferred embodiment of the method according to the invention is characterized by the fact that the starting material is soy meal. This starting material is cheap and easily available.
A preferred embodiment of 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 enzyme dosage can be used, and furthermore a higher yield can be obtained.
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 SPS-ase activity in treatment of soy meal can be provided. At least about 35SAE units per 100 grams of jet cooked soy meal, and 350 SAE units per 100 grams of uncooked soy meal. As a practical matter, the exact interrelation of enzyme activities needed to degrade SPS is not known and, therefore, minimum dosages and proportions for pectinase ceiluiase and hemicellulase cannot be provided. However, a composite activity in treatment of soy meal in SRUM units can be provided, namely at least about 60, SRUM-120 units per 100 grams for cooked soy meal, 600, for unheated meal. The exemplary values hereinbefore provided are believed to be more than the minimum dosages. Cut and try tests may be employed to establish optimum operating proportions and reaction time for 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 method according to the invention.
In order to clarify the nature of the invention, reference is made to the following sections 1 to 10, all describing details related to the invention: 1. Production of SPS.
2. Characterization of SPS, especially molecular weight distribution thereof.
3. Documenation for the fact that SPS and APS are different compounds.
4. Screening for SPS-ase producing microorganisms.
5. Characterization of some SPS-ase forming microorganisms.
6. General description of overlay technique associated with immunoelectrophoresis.
7. Immunoelectrophoretic characterization of SPS-ase with polyspecific antibody and overlay.
8. Purification of an SPS-ase preparation.
9. pH-activity dependency, temperature activity dependency, and stability of an SPS-ase.
10. Enzymatic activity determinations.
SECTION 1 PRODUCTION OFSPS As previously mentioned the starting material for production of SPS may be soy 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 amounts of lignin and minerals) in defatted and dehulled soy meal, which carbohydrate fraction is insoluble in an aqueous medium at pH 4.5, and it can be produced in the following manner, reference also being made to flow sheet 1.
Defatted soy meal (Sojamel 13 from Aarhus Oliefabrik A/S) is suspended in deionized water of 50"C in a weight proportion soy meal: water water =1:5 in a tank with pH-stat and temeperature control. pH is adjusted to 8.0 with 4N NaOH (I). Now a pH-stat hydrolysis is performed with ALCALASE 0.6 L (a proteolytic enzyme on the basis of B.
licheniformis with an activity of 0.6 Anson units/g, whereby the activity is determined according to the Anson method, as described in NOVO ENZYME INFORMATION IB No.058 e-GB), whereby the ratio enzyme/substrate equals 4% of the amount of protein in the soy meal (II). After a hydrolysis of 1 hour the sludge is separated by centrifugation (III) and washing (IV) whereby this operation is performed twice (V, VI, VII). The thus treated sludge is hydrolyzed once more for 1 hour with ALCALASE 0.6 L (VIII, IX) similarly as indicated before. Then the sludge is separated by centrifugation (X) and washed twice (Xl, XII, XIII, XIV), whereby the final washed sludge (6) is spray-dried (XV). The thus produced spray-dried powder is the soy remanence serving as a raw material for the production of SPS.
SPS is the water soluble polysaccharide fraction which is formed by conventional treatment of the above indicated soy remanence with pectinase. The SPS is produced in the following manner by means of the below indicated 14 reaction steps, reference also being made to flow sheet 2.
1. The dry matter content in the above indicated soy remanence is determined and the soy remanence is diluted with water to 2% dry matter and kept in suspension at 50"C in a tank with temperature control.
2. The pH value is adjusted to 4.50 with 6N NaOH.
3. Pectinex Super conc. L is added in an amount of 200g/kg dry matter (a commercial pectinase from Schweizerische Ferment AG, Basle, Switzerland with a pectinase activity of 750,000 MOU, as determined according to the leaflet. "Determination of the Pectinase units on Apple Juice (MOU)" of 12.6.1981, obtainable from Schweizerische Ferment AG, Basle, Switzerland), and also Celluclast 200 L is added in an amount of 20g/kg dry matter (a commercial cellulase described in the leaflet NOVO enzymes, information sheet B 153 e-GB 1000 July, 1981, obtainable from NOVO INDUSTRI A/S, Novo Alle, 2880 Bagsvaerd, Denmark).
4. The contents of the tank is kept at 50"C during 24 hours with stirring.
5. The enzymes are inactivated by raising the pH value to 9.0 with 4N NaOH. The reaction mixture is kept for 30 minutes, and the pH value is then re-adjusted to 4.5 with 6N HCI.
6. 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 filter is washed with water before check filtration).
8. The check filtrate is ultrafiltered, diafiltered and once more ultrafiltered on a membrane with a cut-off value of 30,000 (DDS GR 60-P from 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 matter in the concentrate.
The temperature is 50"C, pH is 4.5 and the average pressure is 3 bar.
9. The ultrafiltered concentrate is cooled to 5"C, and an equal volume of 96% ethanol is added.
10. The precipitate is collected by means of a centrifuge.
11. The precipitate is washed twice with 50% v/v ethanol in H2O, 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 concentrate 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.
FLOW SHEET NO. 1
Defatted soy meal H20 NaCH to pH = 8 í Hydrolysis mixture I I I Alcalase 0.6 L | Hydrolysis 1 hour before discharge II (elm = 4% | pH-stat (pH = 8, T=500C F = 50 C 4N NaOH 9 | 1st centrifugation | Centrifugate 1 Waste }lud0e 1 h2O > Ilet wash TV 1 I Znd centrifugation I V I Centrifugate 2 Waste tiludge 2 H2H I 2nd wasp | V Zndwash / VII j, 3rd 3rd centrifugation I VII I Centrifugate 5 Waste trudge 3 20 New hydrolysis mixture 1 VIII NaOn to pH = 8 t Alcalase 0.6 L Hydrolysis for 1 hour before 4N NaOH > discharge. pH-stat (pH = 8.0, IX T=500C) I FLOW SHEET NO. 1 - continued
| centrifugation IL1 X | Centrifugate 4 slua9e Waste Sludge 4 2e I 1st wash XI | 5th centrifugation ( XII | Centrifuqate 5 > Waste Sludge 5 24 1 2nd wasp | II Zndwash)XIIII | centrifugation I XIV I Centrifugate 6 Waste Sludge 6 Spray-drying | FLOW SHEET NO. 2 1000g Pectinex Super conc. L 5kg 5 ra spray-dried remanence e2F I necoeposition with pectinase 247 1 H20 > l and cellulase 1, 2, 3, 4, 5 500C; PS = 4.5; t = 24 hours Lcentriiugation 0 Sludge > 6 fffiitration 38kg Ultra-filtration, diafiltration, 18 1 2 ultrafiltration (GR 60P) permeate 8 > (19 to 260C) P 5 1 50% C2H5OH | Precipitation Supernatant 9 (waste) Centrifugation Centrifugate 10 Precipitate | 2 x washing | 2 x centrifugate > 11 } Precipitate 2 1 H20 > | Redissolution | 12 Check filtration 1 Protein sludge 13 (waste) I Lyophilization I 310g lyophilized SPS SECTION 2 CHARACTERIZATION OF SPS, ESPECIALLY MOLECULAR WEIGHTDISTRIBUTION THEREOF By means of gel chromatography on HPLC equipment (Waters pump model 6000, Waters data module 730, and Waters refractometer 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 weight distribution ofthe decomposition 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 decomposed by means of the agent according to the invention (Fig. 7) has been demonstrated.
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-vaiue for a specific dextran is defined as the retention time for this dextran divided by the retention time for glucose) has been established by means of several standard dextranswith known molecular weights (T 4, T 10, T 40, T 70, T 110, T 500) from Pharmacia Fine Chemicals AB, Box 175, S-75104, Uppsala, Sweden.
The Rf-vaiue for the maximum of each dextran peak has been found, and the corresponding molecular weight has been calculated as
whereby Mw is the average value of the molecular weight according to weight and ln 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 Toyo 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.
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 of around 4.2 x 104. 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 106and 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 105.
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 103, vide Figure 5.
In orderto demonstrate 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 buffer at pH 4.5 is added to a slurry of soy isolate (Purina E 500) in order to generate a suspension with a ratio isolate/SPS of 10:1. This suspension is incubated for 18 hours on a shaking bath at 505C. After incubation the suspension is centrifuged, and the clear supernatant is analyzed on HPLC as previously described. From Fig.
6 in comparison with Fig. 4 is appears that the SPS is completely adsorbed to the soy isolate.
The same procedure as indicated in the previous paragraph is performed with a 3% SPS solution hydrolyzed with an SPS-ase produced by means of CBS 101.43 (Fig. 7). A comparison between Fig. 7 and Fig. 5 shows that no compound in the hydrolyzed SPS with molecular weight below around 104 iS adsorbed to soy isolate. The hydrolysis reduces the quantitative binding to around 10 to 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 approximate composition of the SPS: 1) oc-galacturonic acid in an amount of approximately 45%, whereby approximately 40% of the total amount of a-galacturonic acid is present as the methyl ester.
2) rhamnopyranose in an amount of approximate ly 20%, 3) galactopyranose in an amount of approximately 15%, and 4) ,8-xylopyranose in an amount of approximately 20%.
The constituents seem to be present in a structure comprising a rhamnogiacturonic 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 the monosaccharides fucose 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 powerful reduction of molecular weight. In accordance therewith the NMR-spectrum of the SPS decomposed as indicated above shows that the main part of the ester groups have disappeared and that also the content of xylose and galactose in the higher molecular weight material has decreased.
The NMR-spectrum 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, concerning the ester groups and the content of xylose and galactose.
SECTION 3 DOCUMENTATION FOR THE FACT THATSPSAND APS ARE DIFFERENT COMPOUNDS APS was prepared as indicated in Agr. Biol. Chem., Vol.36, No.4, p. to 550 (1972).
Now, this polysaccharide and SPS were hydrolyzed with different enzymes, whereafter the decomposition mixture was gel chromatographed on HPLC equipment, as indicated in section 2, "Characterization of SPS, especially molecular weight distribution thereof".
In more detail, the hydrolyses were carried out by treatment of 25 ml solution of either 2% APS or 2% SPS in 0.1 M acetate buffer of pH 4.5 with 10 mg KRF 68 or 30 mg Pectolyase. KRF 68 is an SPS-ase preparation, the preparation of which is described in example 1. The results appear from the following table.
Polysaccharide )olysacc- HPLC gel Not aride Enzyme chromatogram Decomposed decomposed APS Pectolyase Fig. 8 X APS KRF 68 Fig. 9 X SPS Pectolyase Fig. 10 X SPS KRF 68 Fig. 5 x SECTION 4 SCREENING FOR SPS-ASE PRODUCING MICROOR GA 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 liquid main substrate, in which the main carbon source is SPS (prepared as indicated), in which the nitrogen source is NO3-,NH4+, urea, free amino acids, proteins or another nitorgen 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 to 7 days, depending upon the growth rate of the microorganism in question, a sample of the fermentation broth is analyzed forSPS-ase according to the enzymatic SPS-ase determination described in this specification.
In order to achieve a more sensitive method for the determination of enzymatic activity the temperature could be lowered to 40"C 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 producing microorganisms may be found, both belonging to the genus of Aspergillus and to other genera.
SECTION 5 CHARACTERIZATION OF SOME SPS-ASE FORMING MICROORGANISMS According to the here indicated screening for SPS-ase producing microorganisms it has been 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 producer.
SPS-ase producer Species Official Yes No Asp. Asp. Our ident- identi- First X japo- acule- ifying de- fying de- deposit nicks atus signation signation ion yea X X A 805 CBS 101.43; 1943 X OSN 2344 X - X A 1443 IFO 4408; 1950 X DSM 2346 X Z . A 1384 ATCC 20236; 1969 X OSW 2345 A short identification of the above indicated strains can be found in the following culture catalogues: List of Cultures 1978 Centraalbu reati voor Sch im melcultures, Baarn, The Netherlands.
Institute for Fermentation Osaka, List of Cultures, 1972,5th Edition, 17-85, Fuso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan.
The American Type Culture Collection Catalogue of Strains 1,14th Edition 1980,12301 Parklawn Drive, Rockville, Maryland 20852.
All the strains in the above indicated table correspond closely to thetaxonomic description of the species Asp. japonicus and Asp. aculeatus appearing in The genus Aspergillus of Raper and Fennell, 1965 (vide especially pages 327 to 330).
SECTION 6 GENERAL DESCRIPTION OF OVERLAY TECHNIQUE ASSOCIATED WITH IMMUNOELECTROPHORESIS A method designated the top-agar overlay techni que has been developed by the applicant for identification of individual components of an enzyme complex by crossed immunoelectrophoresis with a polyspecific antibody against all enzyme compo nents in the enzyme complex. The method is based on the fact that enzymes are still active after the specific enzyme-antibody binding, or otherwise stated that the active enzyme site is not identical to the site of the enzyme-antibody binding. The en zyme-antibody complexes precipitate as distinct arcs in the gel during the electrophoresis. The gel plate is covered with soluble SPS in a top-agar.After heating to 45"C for 20 hours in an atmosphere with a relative humidity of 100% the arc which possesses SPS-ase activity will appear as a clearing zone in the SPS cover after precipitation with a mixture of equal volume parts of ethanol and acetone when looked upon against a black background. Arcs which have no SPS-ase activity are left invisible.
SECTION 7 IMMUNOELECTROPHORETIC CHARACTERIZATION OF SPS-ASE WITH POL YSPECIFIC ANTIBOD YAND OVERLAY Rabbits were immunized with the SPS-ase containing enzyme complex obtained by fermentation of Aspergillus aculeatus CBS 101.43, as indicated in example 1 (KRF 68) and the polyspecific 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 (KRF 68) was performed, as described in N.H. Axelsen et al., "A Manual of Quantitative Immunoelectrophoresis", 6' printing 11977. Reference is made to Fig. 11 which shows the arcs corresponding to the different proteins pro duced by the microorganism.By means of the previously described top-agar overlay technique it is found 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 present. If these enzymes in other embodiments of the invention should be separated by the immunoelectrophoresis in such a manner, that they do not cover any mutual area, a part of the SPS-ase activity can still be identified by means of immunoelectrophoresis with an overlay with both SPS and a commercial pectinase.
SECTION 8 PURIFICATION OFAN SPS-ASE PREPARATION The purification of the SPS-ase preparation KRF 92 (vide example 1) was performed by ion exchange.
The buffer is 50 mM Tris (tris - hydroxymethylaminomethane) which is adjusted to pH 7.0 with HCI.
The column is K 5/30 from Pharmacia, 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 KRF 92 was dissolved in 450 ml of H20 at 6"C, and all the following indicated operations were carried out between 6"C and 105C. pH was adjusted to 7.0 with 1 M Tris. The column was equilibrated with the buffer, and then the SPS-ase sample was introduced onto the column. OD280 and the conductivity was mea sured on the eluate, reference being made to Fig. 12.
Fraction 1 is the eluate which is not bound to the ion exchange material. Then the column is washed with 2000 ml buffer which gives rise to fraction 2. Now a 0 to 500 mM NaCI gradient is established, giving rise to fractions 3 to 9. All nine fractions were concentrated to 200 ml and dialyzed against water to a conductivity of 2 mSi by means of dialysis (Hollow Fiber DP 2 from Amicon, Massachusetts, U.S.A.).
Then the nine fractions were freeze-dried. Only fractions 1 and 2 exhibited SPS-ase activitv.
Fraction 1 was further purified by gel filtration. 1.5 g of fraction 1 was dissolved in 10 ml 50mM sodium acetate with pH 4.5 (500 mM KCI). The column is 2.5x 100cm from LKB. The gel filtration filling material is Sephacryl S-200 from Pharmacia, Sweden. The flow rate is 30 ml/hour. The fractions containing materials with molecular weights between 70,000 and 100,000, caiibrated with globular proteins, contained an enzyme complex designated factor G which cannot decompose SPS when tested according to the qualitative agar test; however, SPS is decomposed according to the qualitative agar test when mixing factor G with a pectinase.It has been found that factor G is able to split off galactose, fucose, and some galacturonic acid from SPS, but the main decomposition product according to the HPLC analysis is still a high molecular product very much like SPS.
SECTION 9 pH-ACTIVITY DEPENDENCY, TEMPERATURE ACTIV ITYDEPENDENCYAND STABILITY OFAN SPS-ASE Fig. 13 shows the pH-activity dependency of the SPS-ase preparation KRF 68. From pH 2,7 to pH 3,5 a formic acid buffer system was used, and from pH 3,7 to 5,5 an acetate buffer system was used.
Fig. 14 shows the temperature activity dependency of the SPS-ase preparation KRF 68.
Fig. 15 shows the temperature stability of the SPS-ase preparation KRF 68.
SECTION 10 ENZYME TIC ACTIVITY DETERMINATIONS The below indicated table is a survey of the different enzymatic activity determinations pertaining to the invention.
Definition of activty unit and description of enzymatic activity determination Described Kind of act- later in ivity short Publicly this spec Enzyme designation available ification Reference SPS-ase SPS-ase X Remanence SRU X 1 solubil- SRU.'4-120 X izing Protease HUT X Cellulase Cx X 2 ~ PU ------ X 3 PGE X 4 Pectinase UPTE X 5 PEE X 6 Hemicell- VHCU X 7 ulase The reference indicated in the last column of the above table are detailed in the below indicated table.
Reference can be obtained from NOVO INDUS- Schweiz TRI A/S, erische Novo lile, Ferment 2880 AG, Bagsvaerd, Basle. Reference Identification of Denmark Hasle, No. reference erland rary 1 Analyseforskrift X AP 154/4 of 1981-12-01 Analytical Bio chemistry 84, X 2 522 - 532 (1978) Analytical me thod AF 149/6-Ge X of 1981-05-25 3 Determination X of Pectinase Activity with Ci trus Pectin (PU) of 23.3.1976 4 Viskosimetrische X Polygalacturonase Restimmung (pre) of 10.fl.77 Reference can be obtained from NOVO INDUS- Schweiz- TRI A/S, erische Novo Alle, Ferment 2880 AG, Bagsvaerd, Basle, Reference Identification of Denmark Switz- A lib No. reference erland rary 5 Bestimmung der X Pectintranselimin ase (UPTE/g) of 24.Sept.1975 6 Determination of X the Pectinesterase activity (undated) with initials WJA/GW 7 Analytical method X AF 156/1-Ge In relation to the cellulose activity determination it can be noted that the analysis was carried out as indicated in AF 149/6-GB and that the principles of the determination is explained in Analytical Biochemistry.
SECTION 10a ENZYMATIC DETERMINATION OF SPS-ASE The enzymatic determination of SPS-ase is carried out in two steps, i.e. a qualitative agar plate test, and a quantitative SPS-ase activity determination based on measurement of the amount of total liberated sugars. If the qualitative agar plate test is negative, the SPS-ase activity is zero, regardless of the value originating from the quantitative SPS-ase activity determination. If the qualitative agar plate test is positive, the SPS-ase activity is equal to the value originating from the quantitative SPS-ase activity determination.
I. Qualitative agar plate base.
An SPS-agar plate was prepared in the following manner. A buffer (B) is prepared by adjusting 0.3 M acetic acid to a pH-value of 4.5 by means of 1 N NaOH. 1 g of SPS is dissolved in 20 ml of B. 1 g of agarose (HSB Litex) is mixed with 80 ml of B and heated to the boiling point with stirring. When the agarose is dissolved the SPS-solution is slowly added. The resulting 1% SPS-agarose solution is placed in a water bath of 60"C. The plates are now cast by pouring 15 ml of the 1% SPS-agarose solution on a horizontal glass plate with dimensions 10 cm x 10 cm. Then 9 wells with a distance of 2.5 cm are punched out in the solidified layer of SPS-agarose. In each well a 10 p of a 1% solution of the enzyme protein to be tested for SPS-ase activity is introduced.The plate is incubated for 18 hours at 50"C and with a relative humidity of 100%. Now still undecomposed SPS is precipitated by a solution of equal volume parts of ethanol and acetone. The SPS-ase agar plate test is positive for a sample placed in a specific well, if a clear annular zone appears around this well.
II. Quantitative SPS-ase activity determination test.
The purpose of this test is the determination of enzymatic activities, which are capable of decom posing SPS to such an extent that the decomposition products exhibit a strongly reduced or no adsorption or binding affinity to soy protein. Experiments have shown that that part of the SPS decomposition products which are not precipitated by a mixture of equal volumes of water and ethanol, do not have any adsorption or binding affinity to soy protein.
The SPS-ase determination is based on a hydrolysis of SPS under standard conditions followed by a precipitation of that part of SPS, which is not hydrolyzed with ethanol. After precipitation the content of carbohydrate, which is not precipitated, is determined by quantitative analysis for total sugar (according to AF 169/1, available from NOVO INDUS TRI A/S, 2880 Bagsvaerd).
The standard conditions are: Temperature 50"C pH: 4.5 Reaction time: control 210 minutes with substrate only, followed by 2 minutes with added enzyme: main value 212 minutes.
The equipment comprises: Shaking water bath thermostated at 50"C Whirlimixer stirrer Centrifuge Ice water bath The reagents comprise: Buffer: 0.6 M acetic acid in demineralized water (a) 1.0 M NaOH (b) Substrate: The pH 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 pH is readjusted to 4.5, and the volume is adjusted to 100 ml with deionized water.
Stop reagent: Absolute 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 minute.
Even if the initial part of the enzyme standard curve is a straight line, it has to be noted that it does not intersect the (0.0) point.
SECTION lOb ENZYMATIC DETERMINATION OFREMANENCE SOL UBILIZING ACTIVITY EXPRESSED AS SRUM 120 Principle In the method for determination of hydrolysis activity the insoluble part of defatted, deproteinized, and dehulled soy flour is hydrolyzed under standard conditions. The enzyme reaction is stopped with stop reagent and the insoluble part is filtered off. The amount of dissolved polysaccharides is determined spectrophotometrically after acid hydrolysis according to AF 169/1, available from Novo Industri A/S, 2880 Bagsvaerd.
Carbohydrases with endo- as well as exo-activity are determined according to the method.
The substrate pertaining to this enzymatic 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-phosphate buffer pH 4.5 5.24 g citric acid 1-hydrate (Merck Art 244) 8.12 g disodium hydrogen phosphate 2-hydrate (Merck Art 6580) Ad 11 demineralized H2O pH 4.5 + 0.05 Stable for 14 days The stop reagent has the following composition: 100 ml 0.5 N NaOH 200 ml 96% ethanol To be kept in a refrigerator until use.
Standard conditions Temperature 50"C pH 4.5 Reaction time, sample 120 minutes blank 5 minutes.
Unit Definition One soy remanence solubilizing unit (SRUM) 120 (M for manual) is the amount of enzyme which, under the given reaction conditions per minute, liberates solubilized polysaccharides equivalent to one micromole of galactose.
SECTION lOc ENZYMATIC DETERMINATION OFPROTEOLYTIC ACTIVITY HUT MEASUREMENT Method for the determination of preteinase in an acid medium.
The method is based on the digestion of denatured hemoglobin by the enzyme at 40 C, pH 3.2, for 30 minutes. The 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 HCI.
After another 10 minutes of stirring, the pH is adjusted to pH 3.2 with 1 N NaOH. The volume of this solution is increased to 200 ml with 0.2 M acetate buffer. This hemoglobin substrate must be 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. After shaking for 1 second, the tube is placed in a 40"C 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.
For the blank, the hemoglobin substrate is brought to room temperature. At time zero, 5 ml of the substrate is added to a test tube containing 1 ml of enzyme. After shaking for 1 second, the tube is placed in a 40"C water bath for 5 minutes. After 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 HUT is the amount of enzyme which in 1 minute forms a hydrolysate equivalent in absorbancy at 275 nm to a solution of 1.10 microgram/ml tyrosine in 0.006 N HCI. This absorbancy value is 0.0084. The reaction should take place at 40eC, pH 3.2, and in 30 minutes.
Sample-Blank x Vol. in ml HUT = 0.0084 reaction time in min.
Sample-Blank x 11 = (S-B) x 43.65 HUT = 0.0084 30 (S-B) x 43.65 HUT/g enzyme = g.enzyme in 1 ml An investigation of the pH-stability dependency of the protease in KRF 68 performed by means of the HUT analysis with pH values from 2.0 to 8.0 showed that the stability of the protease above pH 8.0 was very small, vide Fig. 16.
In orderto illustrate the invention reference is made to the following examples 1 to 8, where example 1 illustrates the production of SPS-ase, and where examples 2 to 8 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 here indicated SPS-ase test. However, as rather large amounts of SPS-ase are required in order to run application tests, similar fermentations were run on a pilot plant scale, vide the following Example 1 Production of an SPS-ase in pilot plant scale.
An SPS-ase was prepared by submerged frementation of Aspergillus aculeatus CBS 101.43.
An agar substrate with the following composition was prepared in a Fernbach flask: Pepton Difco 6g Aminolin Ortana 49 Glucose 1g Yeast extract Difco 89 Meat extract Difco 1.5 g KH2PO4Merck 20 g Malt extract Evers 20 g lon exchanged H2O ad 1000 ml pH was adjusted to between 5.30 and 5.35. Then 40 g of Agar Difco was added, and the mixture was autoclaved for 20 minutes at 120"C (the substrate is named E-agar).
The strain CBS 101.43 was cultivated on an E-agar slant (375C). The spores from the slant were suspended in sterilized skim-milk, and the suspension was lyophilized in vials. The contents of one lyophil ized vial was transferred to the Fernbach flask. The flask was then incubated for 13 days at300C.
A substrate with the following composition was prepared in a 500 litre seed fermenter: CaCO3 1.2kg Glucose 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 litres. 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 litres.
The Fern batch flask spore suspension was transferred to the seed fermenter. Seed fermentation conditions were: Fermenter type: Conventional aerated and agitated fermenter with a heightldiameter ratio of around 2.3.
Agitation: 300 rpm (two turbine impellers) Aeration: 300 normal litre air per minute Temperature: 30 to 31"C Pressure: 0.5 ato Time: Around 28 hours Around 28 hours after inoculation 150 litres was transferred from the seed fermenter to the main fermenter.
A substrate with the following composition was prepared in a 2500 litre main fermenter: Toasted soy meal 90 kg KH2PO4 20 kg Pluronic (registered Trade Mark) 150 ml Tap water was added to a total volume of around 900 litres. The toasted soy meal was suspended in water. pH was adjusted to 8.0 with NaOH, and the temperature was raised to 50"C. Thereafter around 925 Anson units of ALCALASE (registered Trade Mark) 0.6 Lwas added to the suspension. The mixture was held for 4 hours at 50"C and pH = 8.0 (Na2CO3 addition) with no aeration, zero ato and 100 rpm agitation. Thereafter the remaining substrate components were added and pH was adjusted to around 6.0 with phosphoric acid.The substrate was sterilized in the main fermenter for 11/2 hours at 123eC. Final volume before inoculation was around 1080 litres.
Then 150 litres of seed culture was added.
Fermentation conditions were: Fermentertype: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
Agitation: 250 rpm (two turbine impellers) Aeration: 1200 normal litre air per minute Temperature: 30"C Pressure: 0.5 ato Time: Around 151 hours From 24fermentation hours to around 116fermentation hours pectin solution was added aseptically to the main fermenter at a constant rate of around 8 litres per hour. The pectin solution with the following composition was prepared in a 500 litre dosing tank: Pectin genuX) 22 kg Phosphoric acid, conc. 6 kg Pluronic (registered Trade Mark) 50 ml x) Genu pectin (citrus type NF from The Copenhagen pectin factory Ltd.) Tap water was added to a total volume of around 325 litres. The substrate was sterilized in the dosing tank for 1 hour at 1215C. Final volume before start of dosage was around 360 litres.When this portion ran out, another similar portion was made. Total volume of pectin solution for one fermentation was around 725 litres.
After around 151 fermentation hoursthefer- mentation process was stopped. The around 1850 litres of culture broth were cooled to around 5"C and the enzymes were recovered according to the following method.
The culture broth was drum filtered on a vacuum drum filter (Dorr Oliver), which was precoated with Hy-flo-supercel diatomaceous earth (filter aid). The filtrate was concentrated by evaporation to around 15% of the volume of the culture broth. The concentrate was filtered on a Seitzfilter sheet (type supra 100) with 0.25% Hy-flo-super-cel as a filter aid (in the following table referred to as filtration 1). The filtrate was precipitated with 561 g of (NH4)2SO4/l at a pH of 5.5, and 4% Hy-flo-super-cel diatomaceous earth is added as a filter aid. The precipitate and the filter aid are separated by filtration on a frame filter.
The filter cake is dissolved in water, and unsoluble parts are separated by filtration on a frame filter. The filtrate is check filtered on a Seitz filter sheet (type supra 100) with 0.25% Hy-flo-super-cel as a filter aid (in the following table referred to as filtration II). The filtrate is diafiltered on an ultra-filtration apparatus.
After diafiltration the liquid is concentrated to a dry matter content of 12.7% (in the following table referred to as dry matter content in concentrate).
A facultative base treatment for partial removal of the protease activity can be carried out at this stage.
In case the base treatment is used it is carried out at a pH of 9.2 for 1 hour, whereafter the pH value is adjusted to 5.0.
Now the liquid is check filtered and filtered for the purpose of germ reduction and the filtrate is freezedried on a freeze-drying equipment from Stokes.
Four fermentations were carried out in the manner indicated below, whereby the strain used for the fermentation, the use of the facultative base treatment and other parameters were varied, as indicated in the following table.
Dry Concentration mat (z) of filter ter aid in connect- con ion with tent Pre- fil- the fil- in Base para- tra- pre- tra- con Elicro- treatment tion tion cipi- tion cen organism used not used code I tation II trate Remarks CBS 101.43 X 0 KRF 68 0.5 5 0.2 28 FCC 20236 X KBF 74 2.0 4 0.4 7.5 IFO 4408 X KRF 83 L 0 5 0.25 12.4 x) CNS 101.43 X KRF 92 0.25 4 0.25 12.7 x) After germ reducing filtration the filtrate is concentrated by evaporation in a ratio of 1:2.3. A minor part of the concentrated filtrate was spray-dried, and the remaining part was freeze-dried.
In order to reduce the protease activity further, some of the above indicated preparations were treated as indicated below, whereby only one of the three alternatives A, B, and C was used.
A. 100 g SPS-ase preparation are dissolved in 1 litre of deionized water with stirring at 100C+2"C. pH is adjusted to 9.1 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, deionized water to a conductivity of 3 mSi. Then freezing and lyophilization are carried out.
B. 500 g SPS-ase preparation are dissolved in 4 litre of deionized water with stirring at 10 C+2 C. pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The pH value is then adjusted to 5.0 with glacial acetic acid. The obtained material is lyophilized.
C. 50 g SPS-ase preparation are dissolved in 400 ml of deionized water with stirring at 100Cf20C. pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. Then pH is reduced to 5.7 with glacial acetic acid. The obtained material is lyophilized.
SPS-ase preparation used Base treatment as starting material for used base treatment A B C Preparation code KRF 68 X i XRF 68 BII KRF 68 X XHF 68 BIII KRF 92 ~ X KRF 92 BI The above indicated preparations are characterized by their activities of the enzymes relevant to the invention in the following table.
Enzyme activity per g KRF 68 KRF 68 BII KRF 68 BIll KRF 74 KRF 83 KRF 92 KRF 92 BI Plate test + + + - + + + S's Quantitative test 350 301 349 0 168 476 430 SRU 737 i 507 481 142 683 626 757 120 2125 1560 1720 578 753 1640 1030 HUT pH 3.2 67000 105 339 1630 12800 5960 397 C 8000 8044 9396 1320 8040 5700 3092 PU 10300000 9000000 8800000 1 840000 7500000 8400000 7600000 PGE 119400 72000 77700 4100 64600 60000 68800 UPTE 78100 83700 76900 15130 327000 44000 62400 PEE 840 910 770 370 690 1000 790 VHCU : 1600000 1100000 1000000 65000 2200000 1100000 742000 Example 2 (application example) This example describes the production of a p.v.p.
from a dehulled and defatted soy flour, "Sojamel 13" (commercially available from Aarhus Oliefabrik A/S).
The dry matter content of this flour was 94.0% and the content of (N x 6.25) on a dry matter basis was 58.7%. The soy flour was treated with the SPS-ase preparations KRF 68 Bll (Example 1) in the following manner: 85.2 g of the soy flour were suspended and kept stirred at 50"C in 664.8 g of water, and pH was adjusted to 4.5 by means of 7.5 ml of 6 N HCI. 50 g of a solution containing 4.00 g of said SPS-ase preparation was added, and the reaction mixture was then agitated for 240 minutes at 505C. The mixture was then centrifuged in a laboratory centrifuge (Beckman Model J-6B) for 15 minutes at 3000 x g. The supernatantwas weighed and analysed for Kjeldahl N and dry matter.The solid phase was then washed with a volume of water equivalent to the mass of supernatant obtained by the first centrifugation. This operation was performed twice. The solid phase was then freeze-dried, weighed and analysed for Kjeldahl N and dry matter at Qvist's Laboratorium, Marselis Boulevard 169, 8000 Aarhus C, Denmark. This laboratory is state authorized for analyses of fodder and dairy products.The results obtained in the experiment appear from Table 2.1: Table 2.1 Results obtained
yield of yield Mass N x 6.25 Dry mat- protein of dry Component g % ter % % matter % Soy flour 85.2 55.2 94.0 100% 100% SPS-ase prepara tion 4.00 75.6 - 6.4% 1. Centri fugate 666 1.50 5.04 21.2% 42.0$ p.v.p. 1 44.5 87.5 95.7 82.75 53.2% Thus, a p.v.p. was obtained with a protein purity, i.e. Nx 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 procedures: 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 including a remanence solubilizing enzyme for production of p.v.p.
In order to generate a true comparison of the process according to the invention (C) with the conventional soy protein processes (A 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 treatmenu times are the same in all three cases. Only the pH-values were different due to the fundamental differences between the three experiments.
A. The traditional iso electric precipitation for production of soy protein isolate 425.8 g of soy meal (Sojamel 13 produced by Aarhus OliefabrikA/S) were extracted in 3574.2 g of tap water at 500C. 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 litre beakers in a laboratory centrifuge (Beckman Model J-6B). The centrifugate I and the precipitate I were weighed. The precipitate I was re-extracted with water to a total weight of 4000 g. The temperature was 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 precipitate II for Kjeldahl and dry matter determinations. Hereafter the centrifugates land II were mixed and held at 50"C. The protein was then isoelectrically precipitated at pH 4.5 by means of 45 g of 6 N HCI. After stirring for 1 hour at 505C the protein was recovered by centrifugation at 3000 x g for 15 minutes. The centrifugate Ill was weighed and analysed for Kjeldahl-N and dry matter. The solid phase lil was weighed and washed with water in an amount corresponding to the weight of centrifugate I. The washing was carried out by stirring for one hour at 50"C. The washed protein was recovered by centrifugation at 3000 x g for 15 minutes. The centrifugate IV and the solid phase IV were weighed. Centrifugate IV was analysed for Kjeldahl-N and dry matter.The solid phase was suspended in 1550 g of water at 50"C and pH was adjusted to 6.5 with 17 g of 4 N NaOH. The mixture was kept stirred for one hour and re-adjusted to pH=6.5 if necessary. Finally the product was freeze-dried, weighed and analysed for Kjeldahl-N and dry matter. The mass balance calculations are shown in Table 3.1.
Table 3.1 Mass balance calculations of the traditional isoelectric precipitation for production of soy protein isolate.
yield Mass of Dry yield of dry operations and fraction Protein mat- of pro- matter fractions g 8 (N x 6.25) ter % tein * 8 Extraction: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3574.2 0 0 0 0 4 N NaOH 20.1 0 16.0 0 0.8 1. Centrifug ation: # 4020.1 5.9 10.0 100.9 100.4 Centrifugate I 3141.0 4.4 6.9 58. 8 54.1 Pracipitate I 805.0 Re-extraction: Precipitate 2 805.0 Water 3195.0 0 0 0 0 2. Centrifug ation: CentrifugateII 3104.0 0.5 0.9 6.6 7.0 Precipitate II 820.0 9.1 17.2 31.7 35.2 Mixing and acidifying: Centrifugates I oF II 6245.0 - - - 6 N HC1 45.0 / 0 21.3 0 2.4 3. Centrifug ation: t 6290.0 Centrifugate III 5650.0 0.3 1.9 7.2 26.8 Precipitate III 308.0 - - - Washing: Precipitate III 308.0 - - - Water 3141.0 0 0 0 0 4. Centrifug ation: # 3449.0 Centrifugate IN 3113.0 0.04 0.15 0.5 1.2 Precipitate IV 291.0 - - - - Table 3.1 (continued)
Mass of Dry Yield of dry Operations and fraction ffi Protein mat- of pro- matter fractiong g % (N x 6.25) ter % tein % % Neutraliza tion:: Precipitate IV 291.0 - - - - Water 1550.0 0 0 0 0 4 N NaOH 17.0 0 16.0 0 0.7 Drying: Powder 128.0 93.8 96.3 51.1 30.8 B. The isolectric wash for production of protein concentrate 425.6 g of soy meal (Sojamel 13 produced by Aarhus Oliefabrik A/S) was washed in 35749 of water at 505C. pH was adjusted to 4.5 with 44.8 g of 6 N HCI.
The washing was carried outforfour hours by agitating. The slurry was then centrifuged at 3000 x g for 15 minutes in a laboratory centrifuge (Beckman Model J-6B) using four one litre beakers. The centrifugate I was weighed and anaylsed 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 HCI and the slurry was kept stirred for 30 minutes at 50"C. A centrifugation and weighing of centrifugate II and solids II were performed as above. The solid phase II was resuspended in 1575 g of H2O at 50"C and pH was adjusted to 6.5 with 34.5 g of 4 N NaOH. The mixture was kept stirred at 50"C for one hour and re-adjusted to pH = 6.5 if necessary.Finally the protein product was freeze-dried, weighed, and analysed for Kjeldahl N and dry matter. The mass balance is shown in Table 3.2.
Table 3.2 Mass balance calculations of the isoelectric wash for production of soy protein concentrate.
yield Nass of Dry yield of dry Operations and fraction Protein mat- of pro- matter fractions g % (1 x 6.25) ter % tein % $ Washing: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3574.0 0 0 0 0 6 N HC1 44.8 0 21.3 0 2.4 1. Centrifug ation: Z 4044.6 - - - - Centrifugate 1 3150.0 0.6 3.2 8.0 25.2 Solids I 846.0 - - - - Re-washing: Solids I 846.0 - - - - Water 3154.0 0 0 0 0 6 N NC1 1.7 0 21.3 0 0.1 2. Centrifug ation: Z 4001.7 Centrifugate II 3130.0 0.1 0.4 1.3 3.2 Solids II 863.0 - - - -
Yield Sass of Dry yield of dry Operations and fraction Protein mat- of pro- matter fractions 9 8 (N x 6.25) ter % tein % % Heutraliza- tion:: Solids II 863.0 - - - - Water 1575.0 0 0 0 0 4 N NaOH 34.5 0 16.0 0 1.4 Drying: Powder 281.0 72.5 98.4 86.7 60.1 C. The isoelectric wash including a remanence solubilizing enzyme for production ofp.v.p.
425.8 g of soy meal (Sojamel 13 produced by Aarhus Oliefabrik A/S) was washed in 3524.2 g of water at 505C. pH was adjusted to 4.5 by use of 43.7 g of 6 N HCI. 24 g of the SPS-ase preparation KRF 68 BIll (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. Subse quentlythe purification was performed as described for B, the amounts of 6 N HCI, 4 N NaOH and water for resuspension being the only parameters with deviating values. The mass balance is shown in Table 3.3.
Table 3.3 Mass balance calculations of the isoelectric wash including a remanence solubilizing enzyme for production of p.v.p.
yield Mass of 0 Dry yield of dry Operations and fraction Protein mat- of pro- matter fractions g % (N x 6.25) ter % tein % % Washing: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3540.2 0 0 0 0 6 N HC1 43.7 0 21.3 0 2.3 SPS-ase: KRF 68 BIII 24.0 75.3 96.0 7.7 -5.8 1. Centrifug ation: Z 4043.7 - - - - Centrifugate I 3420.0 1.7 5.2 24.7 44.4 Solids I 620.0 - - - - Re-washing: Solids I 620.0 - - - - Water 3380.0 0 0 0 0 6 N HC1 1.3 0 21.3 0 0.1 2. Centrifug ation: Z 4001.3 Centri fugate I I 3400.0 0.2 0.6 2.9 5.1 Solids II 577.0 - - - - Table 3.3 (continued)
yield Mass of Dry yield of dry Operations and fraction Protein mat- of pro- matter fractions g % (N x 6.25) ter % tein % 8 Neutraliza tion:: Solids II 577.0 - - - - Water 1700.0 0 0 0 0 4 N NaOH 25.3 0 16.0 0 1.0 Drying: Powder 211.0 87.31) 96,71) 78.2 51.1 86.92) 97.02) 1) Analysed at Bioteknisk Institut, Holbergsvej 10, DK-6000 Kolding, Denmark 2) Analysed at Qvist's Laboratorium, Marselis Boulevard 169, DK-8000, Aarhus C, Denmark.
Nutritional Properties The amino acid compositions of the three protein products were determined, vide Table 3.4. The total content of essential amino acids, the chemical score and the essential amino acid index (EAAI) is calculated using the FAO reference pattern from 1957.
The trypsin inhibitor content of the three products was determined by means of the method described in A.O.C.S. Tentative Method Ba 12 to 75 (A.O.C.S. in an abbreviation for American Oil Chemists' Society).
The results are shown in Table 3.5, which also includes the yields and the protein/dry matter ratio of the three products.
Table 3.4 Amino acid composition and nutritional evaluation of the three protein products A, B, and C.
A. Soy protein B. Soy protein C. Soy protein amino acid isolate concentrate isolate (p.v.p) g/16g N alas 1) gXl6g N asps 1) g/16g N aas 1) On-essent vial:: partial acid 12.4 - 11.3 - 11.9 Serine 4.62 - 4.69 - 4.81 lutamic acid 21.3 - 18.2 - 17.7 reline 6.07 - 5.19 - 4.76 Glycine 4.13 - 4.26 - 4.33 canine 3.54 - 4,27 - 4.55 istidine 2.83 - 2.78 - 2.50 rginine 8.09 - 7.57 - 7.04 Essential:: tsoleucine 4.87 > 100 4.97 > 100 5.19 > 100 Leucine 7.80 > 100 7.98 > 100 8.09 > 100 ;sine 6.24 > 100 6.09 > 100 5.57 > 100 Phenylala nine 5.47 > 100) 5.35 > 100) 5.17 > 100) > 100 3.38 > 100 > 100 Fyrosine 3.38 > 10 3.88 > 10 4.44 > 10 Cystine 1.29 64.5 1.32 66.0) 1.44 72.
56.4 60.2 65.5 Nethionine 1.08 49.1 1.21 55. SS.@ 1.31 59.5 Threonine 3.10 > 100 3.60 > 100 3.97 > 100 Tryptophan 1.06 | 75.7 : 1.37 0 97.9 1.32 94.3 Valine 4.90 > 100 5.23 > 100 5.57 > 100 z total content of essential amino acids 38.36 41.31 42.21 Chemical score 56.4% 60.2% 65.5% EAAI 86.7% 90.2% 91.3% Table 3.5 Process characteristics and trypsin inhibitor content of the three protein products A, B, and C.
A. Soy protein B. Soy protein C. Soy protein isolate concentrate isolate (p. v.p) Protein Process of dry 97.4% 73.7% 90.0% charact- matter eristics Protein 51.1% 86.7% 78.2% yield Trypsin inhibit ors TUI/g prod- 34,000 21,000 19,000 uct TUI/g protein 36,250 28,970 21,810 Functional Properties Nitrogen solubility index (NSI) was determined in a 1% protein dispersion at pH = 7.0 in 0.2 M NaCI and in distilled water respectively.After stirring for 45 minutes with a magnetic stirrer the suspension was centrifuged at 4000 x g for 30 minutes, and the supernatant was analysed for nitrogen. The nitrogen solubilitywas calculated as (soluble N%/total N%).
The results of this evaluation on the three products are shown in Table 3.6.
Emulsifying capacity was determined three times on each product by a slightly modified Swift titration. 4.0 g of (N x 6.25) of the product was blended in 250 ml of 0.5 M NaCI with a Sorval Omnimixer 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 weighed. The oil-water mixture was then homogenized at 10,000 rpm with the jar in an ice-bath. A supplementary amount of soy bean oil was added at a rate of 0.3 ml per second until the emulsion collapses. The total of oil added before 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 taken as 0.9 g/ml.
The average results of the determination of emulsifying capacity on the three products are shown in Table 3.6.
Whipping expansion was determined in a 3% protein solution at pH = 6.5. 250 ml of the aqueous dispersion of the protein samples were whipped at speed Ill for 4 minutes in a Hobart mixer (model N-50) mounted with a wire whip. The whipping expansion was calculated according to the formula V-250 Whipping expansion = w x 100%, where V = final whip volume in ml.
V was measured by refilling the mixer jar with water. Duplicates were performed for each of the three samples. The average results are shown in Table 3.6.
Foam stability was determined as the ratio between the amount of foam left after draining for 30 minutes and the original amount of foam. A gram of foam produced by the method above was introduced into a plastic cylinder (diameter 7 cm, height 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 liquid in the glass cylinder is determined. The foam stability FS is defined by the equation - A A x 100% The results of the determination is shown in Table 3.6.
The gel strength 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 NaCI. The heat treatment was performed in closed cans with a diameter of 7.3 cm and a height of 5.0 cm placed in a water bath maintained at 805C and 1 005C each for 30 minutes.
The cans were cooled and thermostatted to 20 C before they were opened and measured. The results of the measurements are shown in Table 3.6.
Table 3.6 Functional properties of the three protein products A, B, and C.
A. Soy protein B. Soy protein C. Soy protein Functionality I isolate concentrate isolate (p.v.p) % NSI in 0.2 M NaCl 39.5 20.3 25.6 % NSI in water 53.9 25.1 28.6 Emulsifying capacity: ml 218 182 354 oil/g (Nx6.25) Whipping expan sion % 120 120 340 Foam stability 8 50 50 20 Gel strength;; poise3 80 C (0.5 M NaCl) 1.7 x 103 1.2 x 10 3.3 x 102 1000C (0.5 H NaCl) 2.0 x 104 4.0 x 104 1.3 x 104 Example 4 (application example) A p.v.p. was produced according to the procedure described in Example 3 C. except that the celulase activity was partially derived from Trichoderma reseei. The commercial cellulase preparation CEL LUCLAST produced by Novo Industri A/S was treated with a base at low temperature in the following manner.The pH value of a 10% CELLUCLAST solution in water was adjusted to 9.2 with NaOH, and the thus resulting solution was cooled to 55C. After 1 hour at this pH and this temperature the pH was re-adjusted to 4.7 with 20% acetic acid. This solution was kept at 5 C overnight and then sterile filtered.
The filtrate was freeze-dried. 49 of the freeze-dried product was added togetherwith the SPS-ase preparation KRF 68 BIll (Example 1). The two enzymes were solubilized in 172 g of water before addition to the washing mixture. The mass balance determinations of this example is shown in Table 4.1.
The experiment demonstrates that this particular SPS-ase preparation already contains an efficient cellulase as addition of CELLUCLAST does not seen 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.
Table 4.1 Mass balance determinations of the isoelectric wash including an SPS-ase preparation and CELLUCLAST(registered Trade Mark) for production of p.v.p.
yield Mass of Dry yield of dry Operations and fraction Protein mat- of pro- matter fractions g % (N x 6.25) ter % tein t Washing: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3546.2 0 0 0 0 6 N HC1 43.1 0 21.3 0 2.3 SPS-ase: KRF-68-B-III 24.0 75.3 96 7.7 5.8 CELLUCLAST 4.0 43.6 96 0.7 1.0 Centrifoga- tion: I 4043.1 - - - - Centrifugate I 3382.0 1.9 5.5 27.3 46.5 Solids 1 661.0 - - - - Re-washing: Solids I 661.0 - - - - Water 3339.0 0 0 0 0 6 N HC1 0 0 0 0 0 2nd centrifug ation: Z 4000.0 Centrifugate II 3414.0 0.2 0.7 2.8 6.0 Solids II 582.0 - - - - Neutralization Solids II 582.0 - - - - Water 1691.0 0 0 0 0 4 N NaOH 25.3 0 16.0 0 1.0 Drying: Powder 206.0 88.8 98.9 77.8 50.9 Example 5 (application example) A p.v.p. was produced according to the method described in Example 3 C. except that all masses were scaled down with a factor of 5, and that the reaction mixture was cooled to about 5 C prior to the centrifugation.On the basis of the analytical results in relation to the centrifugates a theoretical yield of precipitated protein is obtained, as shown in Table 5.1 Table 5.1 Theoretical protein yields obtained in the production of p.v.p.
Example 3 C.
Protein yield Protein yield Mass (N x 6.25) of pro- (N x 6.25) of pro Fractions g 8 tein % B tein % Soy flour 85.2 55.2 100 55.2 100 SPS-ase KRF-68 B-III 4.8 75.3 7.7 75.3 7.7 1st centrifugate 639 0.99 13.5 1.7 24.7 2nd centrifugate 595 0.13 1.6 0.2 2.9 p.v.p. - 87.2a 92.6b 87.1 80.1b a Average of 87.5 (Bioteknisk Institut) and 86.9 (Qvist's Laboratorium); dry matter is 97.6 and 98.0%, respectively.
b Calculated as total mass of protein - protein lost in centrifugates.
Example 6 (application example) Demonstration of the protein binding ofSPS 40 grams of (N x 6.25) from a commercial soy protein isolate (Purina 500 E from Ralston Purina) was dissolved in 680 g of water. The mixture was heated in a water bath to 505C, and pH was adjusted to 4.50 with 6 N HCI. 90 g of this mixture was transferred to 5 x 250 ml Erlenmeyerflasks, and 10 g of aqueous solutions containing 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 was added.
The flasks were then held under stirring with a magnet in a water bath at 505C for 240 minutes.
Hereafter 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 temperature and re-centrifuged. This procedure was repeated. Then the solids were dispersed in 50 ml of water, and pH was adjusted to 6.50 by drop-wise addition of 6 N NaOH.
The neutralized 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 been bound to the protein are calculated by means of the formulas shown in relation to Table 6.2.
This example demonstrates that the SPS is bound firmly to the protein so that the protein/dry matter ratio decreases with increasing content of SPS. An SPS content comparable to about 0.4 g in 10 g of water added to 5 g of protein isolate is the protein/ SPS ratio present in the soy flour.
The % binding of SPS is a calculated value. The % binding of SPS decreases due to saturation of the protein with regard to SPS at the low protein/SPS ratios.
Table 6.1 Measurements according to Example 6
Centrife ates I Dried recipitate Ratio x 6.25 Protein/SPS % H 8 OW t N % N x 6.25 % DM OH 0.068 0.62 13.2 82.5 93.1 88.6 25 0.045 0.49 13.4 83.8 97.3 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 80.3 3.125 0.026 0.61 11.8 73.8 97.9 75.3 Table 6.2 Protein recovery and % binding of SPS
Ratio Protein/SPS % recovery of proteinl) % binding of sPs2) 91.5 0 25 94.4 77 12.5 95.3 90 6.25 96.1 70 3.125 96.8 60
1) 8 recovery of protein = 1 - HC 1 x 6.22 x 100, where NC 1 = % N in centrifugate I 2) 8 binding of SPS =
5 x (% recovery of protein) - 5 x (% recovery of proteins 1% P/H) (% P/H) x 100, where ES/ratio of SPSu (8 P/H) is the protein/dry matter ratio in the dried precipitate, and (8 P/H) is for the precipitate without addition of SPS.
Example 7(application example) This example describes the production of a p.v.p.
using the SPS-ase preparation KRF 92 B-l 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 5. The p.v.p. was analysed as described in Example 2. The results obtained in the experiment appear from Table 7.1.
Table 7.1 Results obtained in Example 7
yield Yield Mass (N x 6.25) Dry mat- of pro- of dry Component g 8 ter % tein % matter Soy flour 85.2 55.2 94.0 100 100 Enzyme preparation 4.0 71.2 - 6.1 1st centrifugate 632 1.88 5.44 25.3 43.0 2nd centrifugate 673 0.30 0.80 4.3 6.7 p.v.p. 39.8 85.6a 98.1a 71.9 48.8 84.4b 98.1b a Analysed at Bioteknisk Institut, Holbergsvej 10, DK-6000 Kolding b Analysed at Qvist's Laboratorium, Marselis Boule vard 169, DK-8000 Aarhus C.
Example 8 (application example) This example demonstrates the effect of pretreat ing the soy meal by jet cooking before the production of p.v.p.
Pretreatment A slurry of soy meal in water consisting of 10 kg soy meal (Sojamel 13 produced byAarhus Oliefabrik A/S) per 100 kg was pumped through a steam -ejector (type Hydroheater B-300) and mixed with steam of 8 Bar in such amount and by such flow that a final temperature of 1505C could be maintained for 25 seconds in a tubular pressurized reactor.Hereafterthe pressure was released in a flash chamber (a cycione) and from here the slurry was sent through a plate heat exchanger and cooled to about 505C. The cooled slurry could be used directly for production of p.v.p. according to the invention, but in this case the slurry was spray-dried at an inlet temperature of 2005C and at an outlet temperature of 90"C. The pretreated product was found to have a dry matter content of 96.5% and a protein content of 56.9% (N x 6.25).
Production ofp.v.p.
This production was carried out in the following way: 70 g of dry matter of the jet cooked and dried soy flour was suspended and kept stirred at 50"C in 560 g of water, and pH was adjusted to 4.50 by means of 6.5 ml of 6N HCI. 6 x 90 g of this suspension was transferred to six 250 ml Erlenmeyer flasks and kept stirred on a 50"C water bath by means of magnetic stirrers. To each flask were added 10 g of a solution containing respectively 0 g; 0.025 g; 0.050 g; 0.10 g; 0.20 g, and 0.40 g of the SPS-ase preparation KRF-68-B-III. The reaction mixtures were then agitated for 240 minutes at 505C. Then a centrifugation at 3000 x g for 15 minutes was carried out.
The supernatantwas then analysed for Kjeldahl-N 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 matter.
A similar experiment was carried out with an untreated soy meal (Sojamel 13 from Aarhus OliefabrikA/S) as a starting material. In this case the enzyme substrate ratios were 0; 1 %; 2%; 3%; 4% and8%.
Based on the protein content of the supernatants the percentage of recovered protein can be calculated. The yield of protein is based on the assumption that the enzyme product is 100% solubilized after the reaction. The table below shows the results obtained by both experiments.
Table 8.1
Cooked soy meal Untreated soy meal Protein Protein of Protein Protein of E/S % yield % dry matter z yield z dry matter s 0 92.9 76.5 90.7 73.9 0.25 90.1 86.6 - - 0.50 89.3 88.7 - - 1.0 88.1 89.7 87.1 86.2 2.0 86.6 91.7 85.7 88.1 3.0 - - 84.3 89.5 4.0 84.7 92.2 82.6 90.9 80 - - 76.2 91.1 Table 8.1 Protein yields and protein dry matter ratio for p.v.p. produced from cooked or raw soy meal.
EXAMPLE 9 Isolation of protein 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 (N H4)2SO4 precipitation, whereby the isolated enzyme was base treated according to method A (this base treated SPS-ase preparation for the sake of 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: Substrate concentration: S=10% dry matter (Staley Corn Gluten) pH=4.50 T= 50"C 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.
Experiment Enzyme dose Protein ) Dissolved Protein No. E/S% yield polymacchtridc purity (Hx6,25/DM)% 873 0 96 0 64 874 1 95 40 75 875 2 95 50 78 B76 3 95 52 79 877 4 95 63 83 Example 10 Isolation of protein from cotton seed meal, sunflower meal, or rape meal.
The protein from cotton seed meal, sunflower meal or rape meal can be isolated in the same manner as the protein from soy bean meal and corn gluten. Isolation of protein from several other proteinaceous vegetable raw materials can be performed in the same manner. Of course, the isolation and/or the separation should preferably be carried out at the isoelectric point of the main part of the protein part of the starting material, whereby the protein yield is maximal.
A survey of the figures, to which reference has been made already, is given belowforthe purpose of providing a better comprehensive view.
Fig.No. Belongs to Describes 1 The general part Demonstration of binding effec of the specification between SPS and soy protein.
2 The general part Flow sheet describing the of the specification production of EPS 3 Section 2 Calibration curve for HPLC gel filtration chromatography 4 Section 2 NPLC gel filtration chromato gram of SPS section 2 HPLC gel filtration chromatogr of SPS decomposed by SPS-ase
ig.No. Belong to I Describes 6 Section 2 and 3 HPLC gel filtration chromato gram of supernatant from SPS incubated with soy protein 7 Section 2 HPLC gel filtration chromato gram of supernatant from decomposed SPS incubated with soy protein 8 Section 3 HPLC gel filtration chromato gram of APS decomposed by Pectolyase 9 Section 3 HPLC gel filtration chromato gram of APS decomposed by SPS-ase 10 Section 3 HPLC gel filtration chromato gram of BPS treated with Pectolyase 11 Section 7 Immunoelectrophoretic peaks including an SPS-ase peak identified by overlay technique 12 Section 8 Ion exchange chromatogram of an SPS-ase 13 Section 9 pH-activity dependency of an SPS-ase 14 Section 9 Temperature activity depend ency of an SPS-ase 15 Section 9 Temperature stability of an SPS-ase 16 Section 10 pH-stability of protease in an SPS-ase preparation

Claims (14)

1. Agent for decomposition of vegetable remanence, especially soy remanence, in the presence of vegetable protein, especially soy 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, preferably belonging to the Aspergillus niger group.
3. Agent according to Claim 1-2, wherein the active component is derived from the enzymes producible by means of Asp. aculeatus CBS 101.43.
4. Agent according to Claim 1-3, wherein the SPS-ase is immunoelectrophoretically identical to the SPS-ase producible by means of Asp. aculeatus CBS 101.43 and identifiable by means of the immunoelectrophoretical overlay technique.
5. Agent according to Claim 3 or 4, 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, preferably less than 1:1, more preferably less than 0.25:1.
6. Agent according to Claim 1-5, wherein the agent contains cellulase activity, and the cellulase activity is derived partially or totally from Trichoderma reseei.
7. Agent according to Claim 1-6, wherein the agent comprises cellulase activity (Cx), pectinase activity (PU, PGE, UPTE, PEE) and hemicellulase activity (VHCU).
8. 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 1-7 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 ofthe starting material, and at a temperature between about 20 and about 705C, until at least around 60% of the remanence, on the basis of nitrogen and dry matter mass balance, preferably at least around 70% thereof, more preferably at least around 80% thereof, has been solubilized, followed by separation of the solid phase containing the purified vegetable protein product from the super natant.
9. Method according to Claim 8, wherein the separation is carried out at a temperature between room temperature and the freezing point of the supernatant.
10. Method according to Claim 8 or 9, wherein the starting material is defatted or defatted and further partially purified vegetable protein.
11. Method according to Claim 8-10, wherein the starting material is soy meal.
12. Method according to Claim 8-11, wherein the starting material is heat treated soy meal, preferably jet cooked soy meal.
13. Method according to Claim 8-12, wherein the starting material is able to pass a sieve with a mesh opening of around 2.5 mm.
14. Purified vegetable protein product, produced by means of the method according to Claim 8-13.
GB08236263A 1981-12-22 1982-12-21 An 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 Expired GB2116977B (en)

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GB2225326A (en) * 1988-10-27 1990-05-30 Atomic Energy Authority Uk Recovery of substances comprising enzymatic degradation of polysaccharides
WO1992013945A1 (en) * 1991-02-06 1992-08-20 Novo Nordisk A/S Sg(b)-1,4-galactanase and a dna sequence

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GB1161419A (en) * 1965-12-13 1969-08-13 Hiram Walker & Sons Inc Starch Hydrolyzing Enzyme System for use in Grain Alcohol Fermentation
GB1227211A (en) * 1968-05-30 1971-04-07
GB1421448A (en) * 1972-05-05 1976-01-21 Gates Rubber Co Process for preparing alpha amylase
GB1446965A (en) * 1974-02-14 1976-08-18 Agricultural Vegetable Prod Preparation of food products
GB1485502A (en) * 1974-06-05 1977-09-14 Aarhus Oliefabrik As Process for removal of water-soluble carbohydrates in the production of plant protein products
GB1489145A (en) * 1975-04-28 1977-10-19 Bio Research Center Co Enzymatic hydrolysis of cellulose
GB2016476A (en) * 1977-11-29 1979-09-26 Walker & Sons Inc Hiram High potency glucamylase and alpha amylase enzyme system by cultivation of aspergillus niger
WO1980001080A1 (en) * 1978-11-20 1980-05-29 Us Commerce Cellulase-producing microorganism

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US4119733A (en) * 1977-05-13 1978-10-10 Massachusetts Institute Of Technology Method of making soybean beverages
US4200694A (en) * 1977-10-08 1980-04-29 Kikkoman Shoyu Co., Ltd. Novel pectin esterase, process for its production, and process for producing demethoxylated pectin by the use of said pectin esterase
JPS54163848A (en) * 1978-06-14 1979-12-26 Toyo Seikan Kaisha Ltd Juice making method
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GB1161419A (en) * 1965-12-13 1969-08-13 Hiram Walker & Sons Inc Starch Hydrolyzing Enzyme System for use in Grain Alcohol Fermentation
GB1227211A (en) * 1968-05-30 1971-04-07
GB1421448A (en) * 1972-05-05 1976-01-21 Gates Rubber Co Process for preparing alpha amylase
GB1446965A (en) * 1974-02-14 1976-08-18 Agricultural Vegetable Prod Preparation of food products
GB1485502A (en) * 1974-06-05 1977-09-14 Aarhus Oliefabrik As Process for removal of water-soluble carbohydrates in the production of plant protein products
GB1489145A (en) * 1975-04-28 1977-10-19 Bio Research Center Co Enzymatic hydrolysis of cellulose
GB2016476A (en) * 1977-11-29 1979-09-26 Walker & Sons Inc Hiram High potency glucamylase and alpha amylase enzyme system by cultivation of aspergillus niger
WO1980001080A1 (en) * 1978-11-20 1980-05-29 Us Commerce Cellulase-producing microorganism

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2225326A (en) * 1988-10-27 1990-05-30 Atomic Energy Authority Uk Recovery of substances comprising enzymatic degradation of polysaccharides
WO1992013945A1 (en) * 1991-02-06 1992-08-20 Novo Nordisk A/S Sg(b)-1,4-galactanase and a dna sequence
US5474922A (en) * 1991-02-06 1995-12-12 Novo Nordisk A/S β-1,4-galactanase and a DNA sequence

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CA1198699A (en) 1985-12-31
ES8402352A1 (en) 1984-01-16
NL8204925A (en) 1983-07-18
AR245955A1 (en) 1994-03-30
MX7203E (en) 1987-12-24
ES518412A0 (en) 1984-01-16
FR2518571B1 (en) 1987-02-13
GB2116977B (en) 1985-07-03
FR2518571A1 (en) 1983-06-24

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