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NZ202876A - Enzymes for degradation of carbohydrates - Google Patents

Enzymes for degradation of carbohydrates

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Publication number
NZ202876A
NZ202876A NZ202876A NZ20287682A NZ202876A NZ 202876 A NZ202876 A NZ 202876A NZ 202876 A NZ202876 A NZ 202876A NZ 20287682 A NZ20287682 A NZ 20287682A NZ 202876 A NZ202876 A NZ 202876A
Authority
NZ
New Zealand
Prior art keywords
sps
ase
soy
protein
decomposition
Prior art date
Application number
NZ202876A
Inventor
J L Adler-Nissen
Georg Wilhelm Jensen
Steen Riisgaard
Henrik Guertler
Hans Aage Sejr Olsen
Martin Schuelein
Original Assignee
Novo Industri As
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Industri As filed Critical Novo Industri As
Publication of NZ202876A publication Critical patent/NZ202876A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F5/00—Coffee; Coffee substitutes; Preparations thereof
    • A23F5/16—Removing unwanted substances
    • A23F5/163—Removing unwanted substances using enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A23L11/65—Soy drinks
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
    • C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
    • C12H1/003—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages by a biochemical process
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Nutrition Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Botany (AREA)
  • Agronomy & Crop Science (AREA)
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  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

2 0287 Priority Date(s): <3?./ A- w DECI98Z, Patents Form No.5 NEW ZEALAND patents act 1953 COMPLETE SPECIFICATION "Enzyme degradation carbohydrates, process for producing the enzyme and materials used in and obtained by such processes" WE, NOVO INDUSTRI A/S, a Danish company of Novo Alle, 28 8 0 Bagvaerd, Denmark, hereby declare the invention, for which -P/we pray that patent may be granted to w®/us, and the method by which it is to be performed, to be particularly described in and by the following statement / D - 202876 SPS-ase 2390 o o -■/ v-//* O 1985 Improvements in and relating to an enzyme for decomposition of a high molecular carbohydrate, the isolated high molecular carbohydrate, a method for selection of a microorganism producing such enzyme and a method for production of such enzyme.
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 C -#V\J CUIOLMA- 0vv 3 described in BE patent No. 882.769^. 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 around or above 90% with a much broader spectrum of starting materials, especially dehulled and defatted soy meal.
The invention is based upon the surprising discovery that a certain part of the remanence decomposition product, as it appears during the enzymatic treatment indicated abo've, i.e. a water soluble, high molecular carbohydrate, attaches itself to part of the vegetable protein, as will be explained later in detail. This, of course, results in a lower purity of the protein. Also, it has been found that this high molecular carbohydrate has 'an ability to attach itself to proteins of animal origin. on i /i' 2 02876 Thus, an object of the invention is to provide an enzyme ; for decomposition of the above indicated high molecular carbohydrate, which will open up the possibility for production of a pvp with improved purity, and a method for production of such an enzyme.
The basis for the invention can be described in the following manner, reference being made to 'fig.' 1, in which only material existing as undissolved solids is. indicated, whereas all supernatants are left out. A charge of soy meal was divided in two equal parts, part I and part II (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 A/S, 2880 Bag-svaerd, Denmark), and then further washed at around pH 8 in order to eliminate the entire amount of protein, and the remanence was separatee fron the supernatant and washed (vide parE" I, column a and h, fig. 1). In this way a pure rerenence (designated remanence I) was "isolated (ooluran b in fig. 1). Part H of tte say meal was not treated? for the sake of brevity the remanence in "paxt II is designated remsnence H (colurnn b in fir. 1). Now, both remanence I 'and part II are Gecorrposed by means of a ccsrrrercial pectinase, e.g. EDCTIMEX- (a" pectolytic enzyme produced by Schweizeri^che Ferment A/G, BasleSwitzerland) (vide co-lunn 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, an the basis of nitrogen and dry matter mass balances, vide fig. 1, where the hatched areas in column c correspond to insoluble non-pro-tein 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 .1, 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) , vide fig. 1. The SPS has a molecular weight distribution ( between 5 x 1,06 and 4.9 x 104.aiie production of isolated. SPS appears frcm the flow, sheet shown on fig. 2, which also, enccirpasses scne of the processes depicted in fig. 1. Thus, the problem is to find an enzyme 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 specifically refers to soy protein, the invention is not restricted to soy protein, but encompasses all kinds of vegetable proteins, vide e.g. the proteins listed in BE patent No. 882.769, page 1. r c O 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 enzymatic activity effectively decomposing soy sps, in the following for the sake of brevity designated an SPS-ase.
Thus according to the invention there is provided in the first aspect a composition comprising an effective amount of an SPS-ase, capable of decomposing soy SPS as hereinbefore defined in aqueous medium into decomposition products which attach themselves to pr&tein in an aqueous medium to a lesser extent than the soy sps prior to decomposition would have attached itself to the same.protein under-corresponding conditions.
Furthermore it has been found that this SPS-ase capable of degrading soy SPS is able to degrade polysaccharides similar to SPS anrt originating from vegetables and fruits more completely than commercial pectinases and eonsaerci&l cellulases.
. The above indicated expression "in a usable form" is intended to exclude from the invention e.g. an SPS-ase containing preparation which contains toxic'substances or which exhibits such low SPS-ase activity, which necessita-o"^®s the use of more than 10% SPS-ase containing preparation, i&lated to the weight of SPS in the substrate, in a reaction - for 24 hours and at 50°C, at the pii optimum of the SPS-ase 1/ .
JJjjr question in order to obtain a deocnpositicn of SPS of any practical ^^^^Mportance. ■ i-» < T 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 not want to be restricted by such hypothesis or similar hypotheses.
A preferred embodiment of the SPS-ase according to the invention, is characterized by the fact that the SPS-ase is capable of decomposing soy SPS in an agueous medium into decomposition products which attach themselves to vegetable protein in the aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached itself to the same vegetable protein in the aqueous medium.
A preferred embodiment of the SPS-ase according to'the invention is characterized by the fact that the SPS-ase is capable of decomposing soy SPS in an aqueous medium with a pH value not deviating more than 1.5 from 4.5 into decomposition products which attach themselves to soy protein in the aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached itself to the soy protein in the agueous medium.
A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the 2 028 76 decomposition products of soy SPS after completed degradation attach themselves to the vegetable protein to an extent of less than 50%, particularly less than 20% than the soy SPS prior to decomposition would have attached itself to the vegetable protein in the agueous medium.
A preferred embodiment of the £P£-ase according to the invention is characterized by.the fact that the EPS-ase exhibits a positive SPS-ase test, when examined according to the qualitative and quantitative SPS-ase determination method described in this specification.
A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that the SPS-ase was produced by means of a microorganism belonging to the genus Aspergillus, preferably belonging to the Aspergillus niger- group.
A preferred embodiment of the SPS-ase according-to the invention is characterised by the fact that the SPS-ase 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 remanases, cellulases, pectinases, and hemicellulases. Furthermore, it has been found than not each and every strain belonging to the species Asp, aculeatus or Asp, japonicus generates an SPS-ase needed for the invention. Thus, as it'appears later in this specification"'(section 5), it has been demonstrated that the strain Asp, japonicus ATCC 20236 does not produce such amounts of an SPS-ase which can be detected by means of the enzymatic determination of SPS-ase described in this specification.
A preferred embodiment of the SPS-ase according to the invention is characterized by the fact that 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 immunoelectrophoretic over- 202876 lay technique, vide section 6 and 7.
When an SPS-ase is produced microbially, it is formed in admixture with several accompanying substances, particularly other enzymes. If desired, the SPS-ase in question can be purified, e.g. by means of chromatographic separation methods, as will appear later in this specification (section 8).
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 3i Thus, the EPLC gel filtration chromatograms of SPS and APS are clearly different, and furthermore, the gel filtration • chronatograms of APS. depomposed by means of the comramercial pectinase pectolyase and of SPS tjreated with the commercial pectinase pectolyase axe 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 extent than the undecomposed acidic 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 created 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 2 028 7 6 A preferred embodiment of the isolated EPS according to the invention is characterized by the fact that the vegetable raw protein is defatted soy meal. The production of this isolated SPS is described in the foregoing in relation to figure 2.
The invention comprises in its third aspect a method for selection of an SPS-ase producing microorganism for production of the SPS-ase according to the invention/ wherein the microorganism to be tested is grown on a fermentation medium, the main carbon source of which is SPS," whereafter & sample of the . < fermentation medium is analyzed for SPS-ase and the microorganism in question is selected as an SPS-ase1producing microorganism, if the analysis for SPS-ase is positive.
The invention comprises in its fourth aspect a method for production of SPS-ase, wherein a strain selectable according to the above method of selection of an SPS-ase producing microorganism is cultivated in a nutrient medium. The cultivation can be carried out as a submerged fermentation or as a surface fermentation.
A preferred embodiment of the method according to the invention is characterized by the fact that the strain Asp, aculeatus CBS 101.43 or Asp, japonicus IFO 4408 is cultivated in a nutrient medium.
A preferred embodiment of the method according to the invention is characterized by the fact that cultivation is carried out as a submerged cultivation at a pH in the range of from 3 to 7, preferably from 4 to 6, at a temperature in the range of from 20 to 40°C-, preferably from 25 to 35°C, and whereby the nijtrient medium contains carbon and nitrogen sources and inorganic salts.
A preferred embodiment of the method according to the invention is characterized by the fact that the nutrient medium contains toasted soy meal. . 2 028 76 A preferred embodiment of the method according to the invention is characterized by the fact that the soy meal is treated with a proteolytic enzyme before the use as a component of the substrate, preferably the proteolytic enzyme produced microbially by means of Bacillus licheniformis.
A preferred embodiment of the method according to the invention is characterized by the fact that a solution of pectin is added aseptically to the fermentation broth during the cultivation.
It has been found that Asp, aculeatus CBS 101.43 also produces very potent remanence solubilizing enzymes, cellulases, pectinases, and hemicellulases besides the SPS-ase, and that the enzyme complex produced by means of Asp, aculeatus CBS 101.43 is excellently suited as an agent for use in cell wall disintegration of vegetable materials.
Thus, the enzyme complex producible from Asp, aculeatus CBS 101.4 3 can be used in the food processing industry for treatment of fruit and vegetable mashes and for clarifying and viscosity reducing purposes in the processing of juice and wine with excellent results; also, it can be used as a dewatering agent (i.e. an'agent for decomposition of polysaccharides and hence for liberation of the water bound within the polymeric structure of the polysaccharides) in the processing of vegetables.
Thus, in its fifth aspect, the invention comprises-a use of an SPS-ase or a method for decomposition of polysaccharides, preferably plant cell wall polysaccharides, by means of a carbohydrase wherein an SPS-ase preparation according to the invention"in an aqueous medium is contacted with a substrate for said SPS-ase preparation. 2 028 7 6 Thus, according to the invention it has been found that SPS-ase preparations are valuable enzyme preparations for partial or total liquefaction or decomposition of several materials, preferably vegetable materials, e.g. fruits, and plant wastes, containing protein, cellulose, hemicellulose, (e.g. glucans, xylan, galactans, and araban) gums, pectin, lipids, inulin, polyphenols, starch, and alginates, and for purposes related thereto, vide the table shown later in this specification. As examples of such related purposes may be mentioned all purposes for which commercial pectinases and cellulases are used today. Several examples will be given later in this specification.
In relation to the extraction (isolation) process described in for instance example 2, it is noted that the SPS-ase preparation is essentially proteinase free, due to the fact that the wanted end product, i.e. the protein, otherwise would be degraded. Likewise, if it is wanted to extract (isolate) other biological materials than protein from a raw biological material, the SPS-ase preparation used should be essentially free of any enzyme degrading this other biological material. Such modified SPS-ase preparations can be produced by selective inactivation of the undesired enzyme, by fractionation, or by other methods known per se.
Thus, a preferred embodiment of the use according to the invention is characterized by the fact that the decomposition is accompanied by the isolation or extraction of a biological material other than soy protein and related vegetable proteins from a raw biological material whereby the SPS-ase preparation is essentially free of any enzyme which is able to degrade said biological material.
Thus, according to the invention it has been found that the modified SPS-ase preparations (modified in the sense that they are essentially free of enzymes capable of degrading the biological material to be extracted or isolated) are valuable enzyme preparations for extraction (isolation) of specified biological materials, e.g. starch, lipids, flavours, colours, and juices, from raw biological materials. Several examples will be given later in this specification. 2 028 A preferred embodiment of the use according to the invention is characterized by the fact that one or more of the reaction products (no matter whether they are wanted end products or waste products) are treated further simultaneously with or after the enzyme treatment. Hereby a more flexible and adaptable use is provided.
A preferred embodiment of the use according to the invention is characterized by the fact that the further treatment is an alcoholic fermentation in case one of the reaction products is a fermentable sugar. Hereby a simple and cheap method for production of alcohol is provided. 2 02876 .In.order to clarify the nature of the invention, reference is made to the following sections 1-10, all describing details related.to the invention: 1; • Production, of SPS. 2. Characterization of SPS, especially molecular weight distribution thereof. .3. Documentation for the fact that SPS and APS are different•compounds. 4i -Screening for SPS-ase producing microorganisms.
. . Characterization-, of some SPS-ase forming micro-organisms » 6. .General description of overlay technique associated ■ with.-Immunoelectrophoresis. 7. ..Immunoelectropboretic characterization of SPS-ase • vith polyspecific antibody and overlay. 8. Purification of an SPS-ase preparation. 9. pH-activity dependency, temperature activity dependency, and stability of an SPS-ase.
. Enzymatic activity determinations. 2028?S 0tk SECTION 1.
PRODUCTION OF SPS.
As previously mentioned the starting material for productio 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) I, in ctefatted and aehulled 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..01iefahrik A/.$) ...is suspended in deionized water of 50°C in a weight proportion soy meal:water = 1:5 in a tank with pK-stat and temperature control. pH is.adjusted to 8.0 with 4N NaOH (I). Nov; a pH-stat hydrolysis is performed with ALCALASE 0.6 L (a proteolytic enzyme on the basis of B-. . licheniformls. 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 (XI, 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 1 remanence with pectinase. The SPS is produced in the following mimner by means of the below indicated 14 reaction steps, 2-028 76 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 pE value is adjusted to 4.50 with 6N NaOH. 3. Pectinex Super conc. h is added in an amount of 200 g/kg dry matter (a commercial pectinase from Schweizerische Ferment AG, Basle Switzerland with a pectinase activity of 750,000 MX , as determined according to the leaflet "Determination of the Pectinase units on Apple Juice (MOU)" of 12.6.1981, obtainable from Schweizerische Ferment AG, Basel, Switzerland), and also Celluclast 200 L is added in an amount of 20 g/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 Bagsvasrd, 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 HC1. ■ 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 2 02876 ■once more ultrafiltered on a membrane with a cut-off value of 30,000 (DDE GR 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 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.
The precipitate is collected by means of a centrifuge.
The precipitate is w'ashed twice with 50% v/v ethanol in B2O, 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. . 11.
• W ii FLOW SHEET NO. 1, Defatted! soy meal H-,0 NaOH to pH *= 8 2 028 7 6 Hydrolysis mixture .Alcalase 0.6 L (E/S =4%) 4N NaOH Hydrolysis 1 hour before discharge pH-stat (pH = 8, T = 50 C) II 1st centrifugation ■ "B20 1st wash III Centrifucafcg 3 > Waste / Sludge 1 IV 2nd centrifugation V.
Centrifugate 2- ^ Waste H„0 2^—3 2nd wash Sludge 2 VI 3rd centrifugation NaOH to pH =2'6^New hydrolysis mixture Alcalase 0.6 L.
VII Centrifugate 3 ^Waste Sludge 3 VIII 4 4N NaOH Hydrolysis for 1 hour before discharge. pH-stat (pH = 8.0, T = 50 C) IX _S^o . 4th centrifugation X - V ' Sludge 4 1st wash XI V f 5t.h centrifugation XII - V / Sludge 5 2nd wash XIII V 6th centrifugation XIV - \ Sludge 6 / |Spray-drying XV Pprtrifngatp & yWaste Centrifuoate 5 v, Waste Centrifugate 6 ^ waste - 17 -PLOW SHEET NO. 2. 2-028 7€ 310 g lyophilized SPS 2 028 76 . SECTION 2.
CHARACTERIZATION OF EPS, ESPECIALLY MOLECULAR WEIGHT DISTRIBUTION 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 of the 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 R^-value 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 dextrans with 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 R^-value for the maximum of each dextran peak has been found, and the corresponding molecular weight has been calculated as Vm • M , whereby M is the average value of the molecular w ji u w weight according to weight and ia is the average value of the moleciiLar weight ac'cor ding to number. As an eluent for this chromatographic procedure 0.1 M NaNO^ 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 R^ for the above indicated dextrans •. has been established, vide figure 3. 2 028 7 6 On the basis of fig. 4 it can be calculated that SPS has.a molecular weight distribution which gives rise to a value of M of around c _ j W .4 x 10 and a value of M of 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 10^ and retention time 47.12 minutes (67%) corresponding to a molecular weight of around 4.9 x 10 . 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 205.
After decomposition of SPS with SPS-ase the hydrolysis mixture was irerribrane filtered, and the filtrate was chrcmatographed. It was four that around'55% of SPS is decomposed to mono-, di- and trisaccha-rides, and that the remaining 45% are decomposed to a polymer 4 with'three peaks with the following molecular weights: 5 x 10 , and 4.4 x 10^, vide figure 5.
In order 'to 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 rase. the following experiments .have been performed. .3%. SPS in 0.10 M acetate buffer at pH.4.5 is added to. ja. 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 50°C.
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 it appears that the SPS is -completely adsorbed to the soy isolate.
The same procedure as indicated in the previous paragraph, is p.erformed with a 3% SPS solution hydrolyzed with 202876 an SPS-ase produced by means of CBS 101.4 3 (fig. 7 ). A comparison between fig. 7 and fig. 5 shows that no compound in the hydrolyzed SPS with molecular weioht below around 4 is adsorbed to soy isolate. 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 approximate composition of the SPS: 1) £K-galacturonic acid in an amount of. approximately 45%» whereby approximately 40% of the total amount of #-galac- turonic acid is present as the methyl ester. / , 2) rhamnopyranose in an amount of approximately 20%, 3) galactopyranose in an amount of approximately 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 H^SO^) and subsequent TLC analysis revealed that also minor amounts of the monosaccharides fucose and arabinose were present -in the hydrolyzed SPS. 2 028 7 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 ga lactose in the higher moleciiLar weight material has decreased. Hie 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. 28 / SECTION 3 DOCUMENTATION FOR THE FACT THAT SPS AND APS ARE DIFFERENT COMPOUNDS.
APS was prepared as indicated in Agr.Biol.Chem., Vol. 36, No. 4, p. 544 - 550 (1972).
Now, this_ polysaccharide ana EPS were hydrolyzed with different enzymes, whereafter the decomposition mixture was gel chromatographed on HPLC equipment, as indicated in .section 2, ""Characterization of EPS, 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 63 or 30 mg Pectolyase. KRF 68 is an SPS-ase preparation,the preparation of which is described in exanple 1. Ihe results appear fron the following table.
Polysaccharide Enzyme HPLC gel chroma togram - Polvsaccharide Decomposed Not decomposed APS Pectolyase fig. 8 X • APS KRF 68 fig. 9 X SPS Pectolyase fig- ic X SPS KRF 68 fig. 5 X 2 028 76 SECTION 4.
SCREENING FOR SPS-ASE PRODUCING MICROORGANISMS.
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 NO^ , NH^*, urea, free amino acids, proteins or another nitrogen containing compounds, and y£iich 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. VThen the growth has taken place in a suitable period of time, of the order of magnitude of 1-7 d2ys, depending_upon the growth rate'of the microorganism in question, a sample of the fermentation broth is analyzed for SPS-ase according to the enzymatic SPS-ase determination described in this specification, or according to any other SPS-ase determinetioi tailored to other specific uses of the SPS-ase than the use as a cargxxnent' o an agent for decomposition of soy remanence.
In order to achieve a more sensitive method for the determination of enzymatic activity the temperature could be lowered to 4 0°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 foundjboth belonging to the genus of Aspergillus and to other genera.
SECTION 5 302873 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 Our identifying designation Official identifying designation First deposition year Yes NO Asp. japonicus Asp. aculeatus X X A 805 CBS 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 Culture 197 8 Centraalbureau voor Schiinmel-cultures, Baarn, The Netherlands.
Institute for Fermentation Osaka, List of Culture, 1972, 5th Edition, 17-85 Fuso-homnachi 2-chome, Yodogawa-ku, Osaka 532, Japan.
The American Type Culture Collection Catalogue of Strains I, 14th edition 1980, 12301 Parklawn Drive, Rockville, Maryland 20852.
All the strains in the above indicated table correspond closely to the taxonomic description of the species Asp. japonicus and Asp. aculeatus appearing in The genus Aspergillus of Raper and Fennell, 1965 (vide especially pages 327 - 330). (Available * * uf N\ <>\ on request). • § a < 1 These strains are deposited with: the German collection of 2T Ul/J . ^ Micro-organisms under Nos: DSM 2344, 2345 and 2346. 2 028 7€ SECTION 6.
GENERAL DESCRIPTION OF OVERLAY TECHNIQUE ASSOCIATED WITH IMMUNOELECTROPHORESIS.
A method designated the top-agar. overlay technique has been developed by the applicant for 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 enzyme-antibody binding, or otherwise stated that the active enzyme site is not identical to the site of the enzyme-antibody binding. The enzyme-antibody complexes precipitate as distinct arcs in the gel during the.electroforesis. The gel plate is covered with soluble SFS 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. 2 028 76 SECTION 7.
IMMUNOELECTROPHOKETIC CHARACTERIZATION OF SPS-ASE WITH POLY-SPECIFIC ANTIBODY AND 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. Axel-sen, et al., nA Manual of Quantitative Immunoelectrophoresis", 6' printing 1977. Reference is made to fig. 11 which shows the arcs corresponding to the different proteins produced by the microorganism-. By means of. the previously described top-agar overlay -technique it is found that the hatched area corresponds to SPSrase.
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. 2 028 7S section 8. purification of an 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 HCl. 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.
I 15 g of the SPS-ase preparation KRF 92 was dissolved in 4 50 ml H^O at 6°C, and all the following indicated operations were carried out between 6°C and 10°C. 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. OD^gQ and the conductivity was measured 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 0-500 mM NaCl gradient is established, giving rise to fractions 3-9. 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 activity.
Fraction 1 was further purified by gel filtration. 1,5 g of fraction 1 was dissolved in 10 ml 50 mM sodium acetate with pH 4.5 (500 mM KC1) . The column is 2.5x100 cm 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, calibrated 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 galac- turonic acid from SPS, but the main decomposition^roSic^? to the HPLC analysis is still a high molecular product very much like SPS. section 9. pH-ACTIVITY ' DEPENDENCY, TEflPERATURE ACTIVITY DEPENDENCY AND STABILITY OF AN SPS-ASE.
Fig. 13 shows the pH-activity dependency of the SPS-ase preparation KRF 68. Fran 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. 0s* SECTION 10 2 02876 ENZYMATIC ACTIVITY DETERMINATIONS The below indicated table is a survey of the different enzymatic activity determinations pertaining to the invention.
Definition of activity units and description of enzymatic activity determination Enzyme Kind of activity, short designation Publicly available Described later in this specification Reference SPS-ase SPS-ase X Remanence solubilizing SRU X 1 SCRUM-120 X Protease HUT X Cellulase cx x 2 Pectinase PU X 3 PGE X 4 UPTE X PEE X 6 Hemicellulase J VHCU X 7 The references indicated in the last column of the above table are detailed in the below indicated table. 2 028 76 Reference can be obtained from NOVO INDUSTRI A/S, Novo Alle, noon Schweizerische Ferment AG, Basel , Switzerland A libra-2T Reference NO Identification of reference /OOU xioj SVe£rd, Denmark 1 Analyseforskrift AF 154/4 of 1931-12-01 X 2 Analytical Biochemistry 84, 522 - 532 (1978) X Analytical method AF 149/6-GB of 1981-05-25 X 3 Determination of Pectinase Activity with Citrus Pectin (PU) of 23.3.1976 ■ X • 4 Viskosimetrische Poly-galacturonase-Bestim-mung (PGE) of 10.11.77 X Bestimmung der Pectin-trans elimin as e (UPTE/ g) of 24.Sept.1975 ■ X 1 6 Determination of the Pectinesterase activity (undated) with initials WJA/GVJ • X 1 1 i i i 7 Analytical method AF 156/1-GB X In relation to the.cellulase activity determination it can be notefi that the analysis was carried out as indicated in AF 14 9/6-GB and that the principles' of the detennination is explained in Analytical Biochemistry. 2 028 76 SECTION 10 a." 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 test.
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 jil of a~l% solution of the/eftzy™e Pr^e^ 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 " 2 028 7 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.
T' The purpose_ of' this test is the determination of enzymatic activities,:which are capable of decomposing SPS to.such an.extent that, the decomposition products exhibit a strongly reduced or no adsorption or bindinq -affinity to sqy 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.
ThelSPS-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, avail able., from NOVO INDUSTRI A/S, 2880 Bagsvaerd).
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 • >•3. ■ - 33 - Shaking water bath thermostatted VJhirlimixer stirrer Centrifuge Ice water bath 202876 at 50°c & The^reagents^comprise; Buffer: 0.6 M acetic acid in demineralized water (a) 1.0 M NaOK (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-ethahol. & 1 SPS-ase activity unit (SftE 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 ^jimol 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.
BV3 2 028 76 SECTION 10 b".
ENZYMATIC DETERMINATION OF REMANENCE SOLUBILIZING 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 insolxible part is filtered off. The amount of dissolved polysaccharides is determined spectro-photometrically after acid hydrolysis'according to AF 169/1 / available from Novo Industri A/S, 2880 Bagsvajrd.
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 pE 4.5 .24 g citric acid 1-hydrate (Merck Art 244) 8.12 g disodium hydrogen phosphate 2-hydrate (Merck Art 6580] Ad 1 1 demineralized 1^0 pB 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. - 35 2 028 76 Standard Conditions Temperature 50°C pH 4.5 Reaction time, sample 120 minutes - - blank . 5 - Unit Definition One soy remanence solubilizing unit (EKUM) 120 (M for manual) is the amount of enzyme which, under the given reaction conditions per minute, liberates solubilized polysaccharides equivalent to one micro-mole of galactose.
SECTION '10 c. 2 028 ENZYMATIC DETERMINATION OF PROTEOLYTIC ACTIVITY. HUT MEASUREMENT Method for the determination of proteinase 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%K 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 HC1.
After another 10 minutes of stirring, the pH is adjusted to pH 3.2 with IN 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 4 0 minutes. k 2 028 76 ■' For the blank, the hemoglobin substrate is JDrouglW 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 4 0°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 Noi 0, and placed in a spectrophotometer. The sample is re£d 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 spectrophotomete ' 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 HC1. This absorbancy value is 0.0084.. The reaction should take place at 40°C., pH 3.2, and in 30 minutes.
Sample-Blank Vol. in ml HUT =- 0.0084 reaction time in min.
Sample-Blank 11 . . - HUT «= 0.0084 X 30 * ( J 43.65 . , (S-B) x 43.65 /g enzyme - g.enzyme in 1 ml An investigation of the pH-stability dependency -of ^the protease in KRF 6B performed by means of the HUT analysis with pH values from 2.'o to 8.0 showed that the stability of the sp above nW B.O wag very gmall . vjde fSo_ 16. 202876 In order to illustrate the invention reference is made to the following examples 1-8, where for example 1 illustrates the production of SPS-ase, and where examples 2-8 illustrate the application of SPS-ase, and where examples 2-8 illustrate the application of SPS-ase, with a soy based raw material in order to produce a purified vegetable protein. Other applications of SPS-ase are indicated in the section between example 8 and the survey of the figures.
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.
Example 1 Production of an SPS-ase in pilot plant scale.
An SPS-ase was prepared by submerged fermentation of Aspergillus aculeatus CBS 101.43.
An agar substrate with the following composition was prepared in a Fernbach flask: Pepton Difco 6 g Aminolin Ortana 4 g Glucose 1 g Yeast extract Difco 3 g Meat extract Difco 1.5 g KH2P04 Merck 20 g Malt extract Evers 20 g Ion exchanged H2O ad 1000 ml 2 028 76 j?H was adjusted to between 5.30 and 5.35. Then 40 g of Agar Difco was added, and the mixture was autoclaved for 20 min. at 120°C (the substrate is named E-agar).
The strain CBS 101.43 was cultivated on an E-agar . slant (37°C). The spores from the slant were suspended in sterilized skim-milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the Fernbach flask. The flask was then incubated for 13 days at 30°C.
* A substrate with the following composition was prepared in a 500 liter seed fermenter: CaCO^ 1.2*kg Glucose 7.2 kg Rofec (corn steep liguor dry . matter) 3.6 kg Soy bean oil 1.2 kg • m " Tap water was added to a total volvmie of around 240 liters, pH was adjusted to around 5.5 before addition of CaCO^. The substrate was sterilized in the seed fermenter for 1 hour at 121°C.
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: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
Agitation: 300 rpm (two turbine impellers) Aeration: .l- 300 normal liter air per minute Temperature: 30 - 31°C Pressure: 0.5 ato •• Time: Around 28 hours Around 28 hours after inoculation 150 liters was transferred from the seed fermenter to the main fermenter. 2 02876 A substrate with the following composition was prepared in a 2500 liter main fermenter: Toasted soy meal 90 kg KH-PO. 20 kg 2 4 Pluronic 150 ml Tap water was added to a total volume of around 900 liters. 1 toasted soy meal was suspended in' water. pK was adjusted to 8.'0 With NaOH, and the temperature was raised to 50°C. There-after around 925 Anson units cf ALCALASE 0.6 L was added to the suspension. The mixture Was held for 4 hours at 50°C and pH = 8.0 (Na^CC^ addition) with ho 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 1% hours at 123°C.
Final volume before inoculation was around 1080 liter.
Then 150 liter of seed culture was added.
. Fermentation conditions, were:.
. Fermenter type: Conventional aerated and agitated fermenter -With a height/diameter ratio of around 2.7.
Agitation? 250. ;rpm (.two turbine impellers) Aeration: .1-200 -.normal :liter air per minute.
Temperature: 30°C Pressure: . QiS.ato Time: Around 151 hours.
From 24 fermentation hours to around 116 fermentation hours pectin solution was added' aseptically to the nain fernenter at a constant rate of around 8 liters per hour. The pectin solution Vith the following composition was prepared in a 500 liter dosing tank: y) Pectin genu 22 kg ■phosphbric "acid, conc. •* 6 kg Pluronic^1 "50 ml x^Genu pectin (citrus type NF from The Copenhagen pectin factory Ltd.) 2 028 7 6 Tap water was added to a total volume of around 325 liters. The substrate was sterilized in the dosing tank for 1 hour at 121°C.
Final volume before start of dosage was around 360 liters. Vlhen this portion ran out, another similar portion was made. Tbtal volune of pectin solution for one fermentation was around 725 liters.
After around 151 fermentation hours the fermentation process was stepped. The around 1850 liters 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 an a vacuum drum filter (Dorr Oliver), which was precoated with Ey-flo-super-cel diatcrnaceous 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 Seitz filter sheet • (type supra 100) with 0.25% Hy-flp-super-cel as a filter aid (in the following table referred to as filtration I). The filtrate was precipitated with 561 g of (NH^SO^/l at a pH of 5.5, and 4% By-flo-super-cel diatcrnaceous 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, ard unsoluble parts are separated by filtration on a frame filter. The filtrate is check filtered an a Seitz filter sheet (type supra '100) with 0.25% By-flo-super-cel as a filter aid (in the following table referred to as filtration H). The filtrate is diafiltered on an ultrafiltration apparatus. After diafil-txatian the liquid is concentrated to a dry matter content of 12.7% (in the following table referred to as dry matter ccntent in concentrate).
A facultative base treatment for partial raroval 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.
Naw the liquid is check filtered and filtered for the purpose of germ reduction and the filtrate is freeze-dried an a freeze-drying equipment frcen Stokes.
Four fermentations -were carried out in the manner 7 /L # O indicated below, whereby the strain used for the fer- mentation the use of the facultative base treatment and other parameters were varied, as indicated in the following table.
Preparation code Concentration (%) of filter aid in connection with Dry matter content in concentrate Ease treatment filtration I the precipitation filtration n Remarks Microorganism used not used CBS 101.43 X KRF 68 0.5 0.2 28 ATCC 20236 X KRF 74 2.0 4 0.4 7.5 IFO 4-508 X KRF 83 1.0 0.25 12.4 x) CBS 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 rsnaining 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 liter of deii nized 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 4.5 with glacial acetic acid, and it is dialyzed against ice cold, deianized water to a conductivity of 3'mSi. Then freezing and lyophilization are carried out.
B. 500 g SPS-ase preparation are dissolved in 4 liter of deianized 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 the adjusted to 5.0 with glacial acetic acid. The obtained material is lyophilized. 50 g SPS-ase preparation are dissolved in 400 ml of deicaiz stirring at 10°C - 2°C. pH is adjusted to 9.1 with C. ed water with 4 N NaOH. This base treatment is carried out for 1 hour. Then pB is reduced to 5.7 with glacial acetic acid. The obtained material is lyophilized.
SPS-ase preparation used as starting material for base treatment Base treatment used Preparation code A B C KRF 68 X KRF 68 BII KRF 68 X KRF 68 Bill 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.
Enzym activ per g e ity KRF 68 KRF 68 BII KRF 68 Bill KRF 74 KRF 83 KRF 92 KRF 92 BI SAE Plate test + + + - + + + Quantitative test 350 301 349 0 168 476 430 SRU . 737 507 481 142 683 626 757 SRUMi2o 2125 1560 1720 578 753 1640 1030 HUT pH 3.2 67000 105 339 1630 128U0 5960 397 Cx 8000 8044 9396 1320 8040 5700 3092 PU 10300000 9000000 8800000 • 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 2 028 76 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 BII (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 HC1. 50 g of a solution containing 4.00'g of said SPS-ase preparation was added, and the reac- o tion mixture was then agitated for 24 0 minutes at 50 C. The mixture was then centrifuged in a laboratory centrifuge (Beckman Model J-6B) for 15 minutes at 3000 x g. The supernatant was 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 Ovist'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 Component Mass S N x 6.25 % Dry matter % Yield of protein, • % Yield of dry matter, % Soy flour 85.2 55.2 94.0 100% 100% SPS-ase preparation 4.00 75.6 . - 6.4% - 1. Centrifugate 666 1.50 .04 21.2% 42.0% p.v.p. 44.5 87.5 95.7 82.7% 53.2% f 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 guality 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 treatment times are the same in all three cases. Only the pH-values I o / were different due to the fundamental differences between the three experiments.
A. The traditional isoelectric precipitation for produc tion of soy protein isolate. 425.8 g of soymeal (Sojamel 13 produced by Aarhus Oliefabrik A/S) were extracted in 3574.2 g of tap water at 50°C. pH was adjusted to 8.0 with 20.1 g of 4 N NaOH. After stirring for 1 hour the slurry was centrifuged at 3000 x g for 15 minutes using four one liter beakers in a laboratory 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 I and 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 HC1. After stirring for 1 hour at 50°C the protein was recovered by centrifugation 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 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 TV 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. 2 028 76 Table 3.1 Mass balance calculations of the traditional iso-. electric precipitation for production of soy protein isolate.
' Mass of Yield of Yield of Operations and fractions fraction Protein Dry mat protein/ dry mat g % (N x 6.25) ter % % ter, % Extraction: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3574.2 0 0 0 0 • 4 N NaOH .1 0 16.0 0 0.8 1. Centrifugation: X 4020.1 .9 .0 100.9 100.4 Centrifugate I 3141.0 4.4 6.9 58.8 54.1 Precipitate I 805.0 - - - - Re-extraction: Precipitate I 805.0 - - - - Water 3195.0 0 0 0 0 2. Centrifugation: Centrifugate II 3104.0 0.5 0.9 6.6 7.0 Precipitate II 820.0 9.1 17.2 31.7 .2 Mixing and acidifying: • Centrifugates I + II 6245.0 - - - — 6 N HC1 45.0 0 21.3 0 2.4 3. Centrifugation: H 6290.0 • Centrifugate HI 5650.0 0.3 1.9 1.2 26.6 Precipitate HI 308.0 - - - - Washing: Precipitate HI 308.0 — - — — Water 3141.0 0 0 0 0 4. Centrifuqation: JET 3449.0 Centrifugate IV 3113.0 0.04 0.15 0.5 1.2 Precipitate IV 291.0 - - - — Neutralization: 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 .8 2 028 76 •B. _ The isoelectric wash for production of soy protein concentrate 425.6 g of soy meal (Sojamel 13 produced by Aarhus Oliefabrik A/S) was washed in 3574 g of water at 50°C. pH was adjusted to 4.5 with 44.8 g of 6 N HC1. The washing was carried out for four hours by agitating. The slurry was then cehtrifuge'd at 3000 x g for 15 minutes in a laboratory centrifuge (Beckman Model J-6B) using four one liter beakers, mhe centrifugate I-was weighed and analysed for Kjeldahl N and dry matteri The solid, phase I was weighed and re-washed with water to a-total weight of 4 000 g. pH was re-adjusted to 4.5 with r.7 g "of "6 "N " HC1 -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 IT was resuspended in 1575 g of B^O at 50°C and pH was adjusted to 6.5 with 34.5 g of 4 H 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, weiglied, 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.
Mass of Yield of Yield of Operations and fractions: fraction Protein Dry mat protein, dry mat g % (N:x 6.25) ter % % ter, % Washing: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3574.0 0 0 0 0 6 N HC1 44.e 0 21.3 0 2.4 1. Centrifugation: 2 4044.6 — — — — Centrifugate I 3150.0 0.6 3.2 8.0 .2 Solids I 846.0 — — — - Re-washing: Solids I 846.0 — — — - Pfeter 3154.0 0 0 0 0 6 N HC1 1.7 0 21.3 0 0.1 2. Centrifugation: 2 4001.7 Centrifugate II 3130.0 0.1 0.4 1.3 3.2 Solids II 863.0 — — — - Neutralization: Solids U 863.0 - - - - Water 1575.D 0 0 0 0 4 N NaOH 34.5 0- 16.0 0 1.4 Drying: Powder 28110 72.5 98.4 86.7 69.1 C. • The isoelectric wash including a remanence solu- bilizing enzyme for production of p.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 50°C. pH was adjusted to 4.5 by use of 43.7 g of 6 N HCl. 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. Subsequently the purification was performed as described for B, the amounts of 6 N HCl, 4 N NaOH and water for resuspension being the only parameters with deviating values. The mass balance is shown in table 3.3. 2 028 7 Table 3.3 Mass balance calculations of the isoelectric wash .'including a remanence solubilizing enzyme for production of p.v.p.
Operations and fractions: Mass of fraction 9 Protein %{N x 6.25 Dry matter % Yield of protein, % Yield of dry mattery % Washing: Soy flour Water 6 N HCl SPS-ase.: KRF 68 BUI 425.8 3540.2 43.7 24.0 55.2 0 0 75.3 94.0 0 21.3 96.0 100.0 0 0 7.7 100.0 0 2.3 5.8 1. Centrifugation: £.
Centrifugate I Solids I 4043.7 3420.0 620.0 1.7 .2 . 24.7 44.4 Re-washing: Solids I water 6 N HCl 620.0 3380.0 1.3 0 0 0 21.3 0 0 0 0.1 2. Centrifugation: X Centrifugate H Solids n 4001.3 3400.0 577.0 0.2 0.6 2.9 .1 Neutral i r.atian: Solids U Water 4 N NaOH 577.0 1700.0 25.3 0- • 0 0 16.0 0 0 0 1.0 Drying: Powder 211.0 87.3-] 86.9 96"72) 97.0 78.2 51.1 1) Analysed at Bioteknisk Institut, Holbergsvej 10, DK-6000 Kolding, Denmark 2) Analysed at Ovist's Laboratorium, Marselis Boulevard 169, DK-8000, Aarhus C, Denmark ^ - 53 - 2 028/6 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 - 75 (A.O.C.S. is 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. 2 023 76 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/16 g N aas1* g/16 g N aas1 * g/16 g N aas1 ^ 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 - .19 • - 4.76 - Glycine 4.13 - 4.26 - 4.33 - Alanine 3.54 - 4.27 - 4.55 - Histidine 2.83 - 2.78 - 2.50 - Arginine 8.09 - 7.57 - 7.04 — Essential; Isoleucine 4.87 > 100 4.97 >100 .19 >100 .
Leucine' 7.80 >100 7.98 >100 8.09 >100 Lysine 6.24 >100 6.09 >100 .57 >100 Phenylalanine Tyrosine" .47 3.3B >100") >100 ) >100 .35 3.88 >100) f>100 >100) .17 4.44 >100*) f>100 >100 > Cystine Methionine 1.29 1.08 64.5 49.1. 56.4 1.32 1.21 66.0] \ 602 55.OJ 1.44 1.31 72.01 7 65.5 59.5 J Threonine 3.10 >100 3.60 >100 3.97 >100 Tryptophan 1.06 75.7 1.37 97.9 1.32 94.3 Valine 4.90 >100 .23 >100 .57 >100 % total content of essential 38.36 41. 31 42. 21 amino acids Chemical score 56.4% 60. 2% 65.
% EAAI 86.7% 90. 2% 91.3% ^ aas = amino acid score based on the FAO reference pattern (1957) 2 028 76 Table 3.5 Process characteristics and trypsin inhibitor content of the three protein products A, B, and C.
• '• A. Soy protein isolate B. Soy protein concentrate C. Soy protein isolate (p.v.p.) Process characteristics Protein of dry matter 97.4 % 73.7 % 90.0 % Protein yield 51.1 % 86.7 % 78.2 % Trypsin inhibitors TUI/g product 34,000 21,000 19,000 TUI/g protein 36,250 28,970 21,810 2 028 76 Functional properties (NET) 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 magnetic stirrer the suspension was centrifuged at 4000 x g for 30 minutes, and the supernatant was analysed for.nitrogen. The nitrogen solubility was calculated as (soluble N%/total N%). The results of this evaluation on the three products are shown in table 3.6.
ErnulsifYinc_capacitv 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 NaCl 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 amount 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.
WES determined in a 3% protein solution at pH = 6.5. 250 ml of the agueous* dispersion of the protein samples were whipped at speed III for 4 minutes in a Hobart mixer (model N-50) mounted with a wire whip. The whipping expansion was calculated according to the formula Whipping expansion - x 100%, 2 02876 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. 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 eguation -FS = A " B x 100% A The results of the determination is shown in table ' 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 NaCl. 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 80°C and 100°C each for 30 minutes. The cans were cooled and therroo- statted to 20°C before they were opened and measured. The results of the measurements are shown in table 3.6. 2028/6 Table 3.6 Functional properties of the three protein pro-' ducts A, B, and C.
Functionality A. Soy protein isolate B. Soy protein concentrate C. Soy protein isolate (p.v.p.) % MSI in 0.2 M NaCl % NSI in water 39.5 53.9 .3 25.1 .6 28.6 Emulsifying capacity: ml oil/g (N x 6.25) 218 182 354 Whipping expansion % 120 120 340 Foam stability % 50 50 Gel strength; [poise] 80°C (0.5 M NaCl) 100°C (0.5 M NaCl) 1.7 x103 2.0 x 104 1.2 x104 4.0 x104 3.3 x102 1.3 x1D4 . " " 2 028 76 Example 4 (application example) A p.v.p. was produced according to the procedure described in example 3 C except that the cellulase activity was partially derived from Trichoderma reseei. The commercial cellulase preparation -CELLUCLAST produced by Novo Indus tri 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 5°C. After 1 hour at this pH and this temperature the pH was re-adjusted to 4.1 with 20% acetic acid. This solution was kept at 5°C overnight and then sterile filtered. The filtrate was freeze dried. 4 g of the freeze dried product was added together with the SPS-ase preparation KRF 68 BUI (exanple 1). Ihe 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 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. -««.- 2 028 7 Table 4.1 Mass balance determinations of the iso-electric wash .including an SPS-ase preparation and CELLUCLAST for production of p.v.p.
Operations and Mass of Protein Dry matter Yield of Yield of fractions fraction % % protein dry matter gram (N x 6.25) % % Washing: Soy flour 425.8 55.2 94.0 100.0 100.0 Water 3546.2 0 .0 0 0 6 N HCl 43.1 0 21.3 . 0 2.3 SPS-ase: KKF-68-B-m 24.0 75.3 96 7.7 .8 CELLUCLAST 4.0 43.6 96 0.7 1.0 ■ Centrifugation: Z 4043.1 - - ■ - - Centrifugate I 3382.0 1.9 .5 27.3 46.5 Solids I 661.0 - - - - Re-washing: Solids I 661.0 - - - - Water 3339.0 0 0 0 0 6 N HCl 0 0 0 0 0 2nd centrifugation: <2* 4000.0 Centrifugate 11 3414.0 0.2 0.7 2.9 6.0 Solids II 582.0 - - - - Neutralization: Solids II 582.0 - - - - Water 1691.0 0 0 d 0 4 N NaOH .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 centrifugate 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.
Fractions Mass Protein (N x 6.25) * Yield of protein" % Example 3 C 9 Protein (N x 6.25) % Yield of protein,% Soy-flour 85.2 55.2 100 55.2 100 SPS-ase "KRF-68 B-HI 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. lb a Average of 87.5: (Bioteknisk Instit.ut) and 86.9 (Qvist's Labora-torium); dry matter is 97.6 and-98.0%, respectively. k; Calculated.as total mass of protein - protein lost in centri-fugates.; • j.:; - .• Example 6 (application example) Demons tration_of_the_protein_bindin2_of_SPS 40 grams of (Nx 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 50°C, and pH was adjusted to 4.50 with 6 N HCl. 90 g of this mixture was transferred 2 028 76 -to 5x 250 ml Erlerimeyer flasks, and 10 g of aqueous solutions containing respectively Og, 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 50°C 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 cm 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 e'x'ample 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. 2 028 76 Table 6.1 Measurements according to Example 6 Ratio Centrifugates I Dried precipitate Protein/SPS % N % DM % N % N x 6.
% DM % N x 6.25 DM oe> 12.5 6.25 3.125 0.068 0.045 0.03B 0.031 0.026 0.62 ' 0.49 0.45 0.45 0.61 13.2 13.4 13.0 12.6 11.8 82.5 . 83.8 81.3 78.8 73.8 93.1 97.3 97.9 98.1 97.9 88.6 86.1 83.0 80.3 75.3 Table 6.2 Protein recovery and % binding of SPS Ratio Protein/SPS % recovery of protein"* ^ % binding of SPS2^ o© 91.5 0 94.4 77 12.5 95.3 90 6.25 96.1 70. 3.125 96.8 60 % recovery of protein = [l - NC 1 * 6'25Jx 100, where • NC 1 = % N in centrifugate I ^ % binding of SPS = [ 5x (% recovery of protein) 5x (% recovery of protein) 1 L (% P/H) ~ (% P/H)^ J x 100, vdiere £ 5/ratio of SP&j (% P/H) is the protein/dry matter ratio in the dried precipitate, and ' (% 'P/H)^ is for the prftcipitate without addition of SPS. 2 028 7 6 Example 7 (application example) This example describes the production of a p.v.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, excej 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 Re suit is. obtained in Example 7 Component Mass g (N x 6.25) % Dry matter % '.Yield of protein, % Yield of dry matter, % Soy flour 85.2 55.2 94.0 100 100 Enzyme preparation 4. 0 71.2 - 6.1 - 1st centrifugate 632- • 1.88 .44 .3 43.0 2nd--cenir-i fiu gate 673 0.30 0.80 4.3 6.7 p. v.p.- 39.8 85.6®-84 .4 98.if 98.lb 71.9 48.8 a Analysed"at Bioteknisk Institut, Holbergsvej 10, DK-6000 Kolding b Analysed at Ovist's Laboratorium, Marselis Boulevard 169, DK-BOOO Aarhus C Example 8 (application example) ■ This example demonstrates the effect of pretreating the soy meal by jet cooking before the production of p.v.p.
Pretreatment" A slurry of soymeal in water consisting of 10 kg ■soymeal (f>ojamel 13 produced by Aarhus 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 150°C could be maintained for 25 seconds in a tubular pressurized reactor. Hereafter the pressure was released 2 028 7 6 in a flash chamber (a cyclone) and from here the slurry was sent through a plate heat, exchanger and cooled to about 50°C. 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 2 00°C and at an outlet temperature of 9 0°C. Ihe ©retreated product was found to have a dry matter content of 96.5% and a protein content of 56.9% (N x 6.25).
Production of p.v.p.
This production was carried out in the following way: 70 g of dry matter of the jet cooke'd 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 HCl. 6 x 90 g of this suspension was transferred to six 250 ml Erlenmeyer flasks and kept stirred on a 5D°C water bath by means of magnetic stirrers. To each flask were added 10 g of a solution containing respectively Og; 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 24 0 minutes at 50°C. Then a centrifugation at 3000 x g for 15 minutes was carried out.
The supernatant was 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 Oliefabrik A/S) as a starting material. In this case the enzyme substrate ratios were 0? -1%; 2%; 3%; 4%; and 8%.
Based on the protein content of the supernatants the percentage of 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. fjW Table 8.1 2 02B 7 6 Cooked soymeal Untreated soymeal E/S % Protein yield % Protein of dry matter. % Protein yield•% Protein of dry matter • % 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. 8 2..6 90.9 8.0 > — — 76.2. .91.1 Table. B. 1 .Protein yields and protein .dry.matter .ratio for p.v.p. .produced from-cooked or.raw soymeal... • .
In relation to either the extraction (isolation) processes for materials other than proteins or to the liquefaction processes and thereto related processes, reference is made to the general process scheme for applications as shown in flow sheet No. 3.
The substrate may be one or more carbohydrates present in a raw material, or it may be the entire raw material.
This substrate may be subjected to a pre-treatment of chemical or physical nature, as later exemplified, e.g. acid or alkaline treatment, soaking or steeping, and/or cooking with or without steam. i 2 028 7 6 The raw material may be macerated, chopped, wet milled, and/or homogenized (all these treatments being designated homogen-ization in flow sheet No. 3), with or without water additions, and other additives may be added during this step. The homogenization may be carried out with different efficiencies, e.g. different pressures being a fraction only of the maximum pressure given for the specific homogenizer used. Different additives may be added before or during homogenization, as symbolized by b]_, b2, ... bn in flow sheet No. 3.
The reaction process including the SPS-ase preparation is carred out under specified conditions, e.g. temperature, pressure, time, pH, and enzyme dosage; also recommendations regarding the reactor used (e.g. batch, plug-flow) and the stirring, if necessary, are relevant. A set of additives can be given for different raw materials, indicated as ci, C2 ... cn in flow sheet No. 3.
Also the separation processes may be carried out with different efficiencies. During many processes the separation is omitted or facilitated, e.g. when the raw material is totally liquefied. Different separation equipments may be used (e.g. centrifuges, filters, ultrafiltration equipment, hydrocyclones, thickeners, sieves or screens, or simple decanters).
The separation efficiency is defined as the proportion between the absolute sludge content in the solids phase and the absolute sludge content of the reaction mixture.
The liquid or the solid phases obtained may be further treated, e.g. concentrated, dried, or solvent extracted, to remove certain components like fat or oil, fermented for production of biomass, alcohol, or other products (enzymes, antiobiotics, or other valuable components).
Also the obtained products may be returned to repeated treatment through the process scheme.
Flow Sheet No. 3 n n. cl- n Substrate Pretreatments Homogeniz ation SPS-ase preparation — Horn 0 - 100%) (Reaction conditions to be specified) Homogenization (as above) Separation Sep 0 - 100%) Liquid Solids Further treatments (see text) or renewed usage as substrate 2 028 76 In the following some examples of applications of SPS-ase preparations are given, and a survey of these applications appears from the following list.
Also, in the adjoining Table I certain characteristics in relation to flow sheet No. 3 are listed.
List of exemplifying applications of SPS-ase preparations- 2 02 Kind of SPS-ase preparation SPS-ase preparation essentially free of one or more unwanted enzyme activities Reference No.
A 1 A 2 A J a: -it- Extraction of starch from corn, wheat and potatoes Extraction of lipids from plant material Extraction of etheric oils from plant materials Extraction of natural coloring agents from plant material Extraction of rubber from the guayul bush Un- mo- di- f ied SPS- ase pre- pa- ra- ti- ons Total Ba 1 lique Ba 2 faction or Ba 3 similar Ba 4 treat ment Ba 5 Ba 6 • Ba 7 Production of a milk substitute for domestic animals Production of saccharified starch containing raw materials Total liquefaction of pears and other fruits Production of juice by -treatment of fruits and vegetables Treatment in relation.to extraction.or pressing of sugar cane or sugar beet Production of soy milk Treatment in order to increase the recoverable amount of coffee solubles.
Pro- Bb 1 ces- .
Bb 2 sing Bb 3 aids Bb 4 Bb 5 Bb 6 Other Be 1 appli Be 2 cations Be 3 Be A Be 5 Be 6 Be 7 Prevention and/or decomposition of apple haze Use as a clarifying agent for white wine Production of ISSPH or other vegetable protein hydrolyzates Mashing enzyme in the brewing industry Enzymatic additive for use during beer fermentation and/or storage Almond skin release agent Decomposition of different waste materials Saccharification and simultaneous fermentation. Decomposition of cellulose Application as a baking aid Improvement of alcohol yield and yield of bio-mass during fermentation of sulfite liquor from paper production • Dewatering of biological sludge products Silage aid Table I Ref.
Substrate Additives 1' homogenization 1 Hom% 2' homogenization 1 Hom% Separation *lSep% Furtl ler treatments al bl C1 Liquid phase Solid phase Combined phase (no separation) A 1 corn water S02 water NaOH or HCl - 50 - 30 - 50 (Contains germ) washing, oil recovery Washing operations - A 2 corn germ — — NaOH or HCl 0 0 100 (Oil + water) purification (waste) - Ba 1 Soy-flour (defatted) water HCl 0 0 0 pasteurisation, concentration, spray drying Ba 2 sweet potatoes water Terma-myl NaOH or HCl : 0 ~ • yeast for alcohol fermentation, distillation Ba 5 sugar beets water " NaOH or HCl % ~ 100 crystallization (waste) !N1 Be 1 tofu or soy milk residue water HCl % 0 - ...... yeast for alcohol fermentation, dij^E tillation . , KJ GO Gs 2 028 /6 A 1. Extraction of starch from corn, wheat, and potatoes.
Extraction of starch from corn, wheat, potatoes, and other starch containing plants is carried out by one or more of the steps: steeping, wet milling, and separation. Application of an SPS-ase preparation with essentially no amylolytic activity will provide the following advantages, corn being used as an example: 1. The starch liberation will be facilitated during a shorter steeping period, 2. The water consumption can be reduced, 3. The liberation of corn germ would be facilitated without liberation of corn germ oil, 4. The protein can be obtained in a higher purity, . The recovery of corn steep water will be facilitated.
A 2. Extraction of lipids from plant material.
As the lipids in plant materials are trapped inside the cells and usually bound to proteins, lipids may be extracted in aqueous phase by treatment with an SPS-ase preparation which is essentially free from lipases. Thus, corn germ oil normally is isolated by hexane extraction of the dried corn germs. However, the drying operation is superfluous if the wet corn germs are treated with an SPS-ase preparation of the above indicated kind. Likewise, the extraction of olive oil in aqueous phase can be improved, if the enzyme used for the enzymatic treatment is an SPS-ase 2 028 preparation of the above indicated kind, vide e.g. Food, Pharmaceutical and Bioengineering, No. 172, vol. 74, p. 93 - 94. Also, aqueous extraction of e.g. soy oil, rape seed oil and sunflower oil may be improved in a similar manner.
A 3. Extraction of etheric oils from plant materials.
If vegetable materials containing etheric oils are extracted with an agueous solution of an SPS-ase preparation which is essentially free from enzymatic activity capable of decomposing or otherwise changing the etheric oils, the etheric oils will be recovered in high yields at a very low cost.
A 4. Extraction of natural colouring agents from plant material.
If vegetable materials containing colouring agents, e.g. beetroots containing the red colouring agent betanin or the colouring agent in cranberries, are treated with an SPS-ase preparation which is essentially free from enzyme activities capable of decomposing or otherwise changing the colouring agents, the colouring agents will be recovered in high yields at a very low cost.
A 5. Extraction of rubber from the guayul bush.
Another example of a substrate for an SPS-ase preparation which is essentially free from enzymatic activity capable of degrading native rubber is the cell wall material in roots and branches of the guayul bush.
Ba 1. Production of a milk substitute for domestic animals, preferably a calf milk substitute.
By a total liquefaction in aqueous medium of soy beans, sunflower seeds, cotton seeds, faba beans, or field peas a calf milk substitute which is soluble in cold water at a pH value of around 4.5 can be produced. Using the starch containing raw materials like faba beans or field peas a starch liquefaction by means of an alpha-amylase has to be accomplished before, after or simultaneously with a treatmei with an SPS-ase vdnch finally solubilizes the non starch polysaccharides present as the structural material in the cell walls. A detailed example is shown below using faba beans, and as regards soy beans reference is made to table I . The pre-treatment of said soy beans may preferably be a jet-cooking which improves the solubilization of the remanence.
Example Ba 1.1 kg of faba bean flour (Farine de Feves from GRANDES MI-NOTERIES A FEVES DE FRANCE, Paris) were suspended in 35 litres of water. 75 g of Termamyl 60 L and 18 g of CaC^ were added. The suspension was heated to 95°C using a steam jacketed vessel while stirred. The suspension was then treated at this temperature for 60 minutes. Hereafter pH was adjusted to pH = 4.5, and the product was cooled to 50°C. 300 g of the SPS-ase preparation KRF 68 was solubilized in 1 litre of water and added. The reaction was carried out for 4 40 minutes. 2 028 76 When 10 g of Fungamyl BOO L is included the starch fraction mainly will be converted to disaccharide (maltose). Hereafter the reaction mixture was pasteurized at 90°C for 2 minutes. An aliquot of the product was then freeze dried and used for stability tests. The sample was then solubilized at 10% dry matter, and the solution of the product could be kept stable without sedimentation for days.
Melted fat or oil may easily be emulsified in the product whereby a final composition very similar to cow milk may be obtained. An emulsion containing 3.5% oil (soy bean oil) could also be kept stable without sedimentation for days.
Example Ba 1.2 Soy flour (Sojamel 13) was jet cooked at 150°C for 25 seconds as described in example 8. The jet cooked soy flour was spray-dried and used for further studies described in the following. hL ■ ■ 50 g of the jet cooked soy flour was mixed with 450 g' of water, and pH was adjusted to 4.5 with 4,1 ml 6N HCl. The mixture was then heated to 45°C in a water bath, and 0.250 g of the SPS-ase preparation KRF-68 was added to the heated mixture which was then reacted for 5 hours with stirring. Thereafter the mixture was heat treated at 80°C for 2 minutes in order to inactivate the enzyme. A 100 ml sample was centrifuged at ambient temperature for 15 minutes at 3000 x g (g = gravity). The supernatant was ion-exchanged and analysed for carbohydrate composition by HPLC. Also the supernatant was analysed for Kjeldahl-N and dry matter and the nitrogen solubility index (NSI) and the dry matter solubility index (DSI) was calculated; vide results in table Bal. 100 ml of the reaction mixture cooled to 20°C was poured in a 100 ml graduated glass and kept at 4°C for two days. The dispersion stability (%) was measured by reading the volume of the 2 028 7 6 dispersions obtained (table Ball) after 1 and 2 days.
To 200 ml of the reaction mixture (at 20°C) was added 8 g of soy bean oil. An emulsion was made by blending for 2 minutes in a Waring Blender- The emulsion stability (%) was measured as above after 1 and 2 days.
Bj_ A reaction was carried out as above, however, in this case 1.00 g of the SPS-ase preparation was used. The same types of analyses and stability measurements as described in section A were performed. Results are shown in tables Bal and Ball.
It appears from the chemical analysis of the supernatants that the values for NSI (%) and DSI (%) obtained at the B experiment is higher than for the A experiment. However, the stability tests carried out on the reaction mixtures show a better value for the A samples. This probably is due to the higher peptide chain length of the proteins in the reaction mixture with the low enzyme dosage.
From the carbohydrate composition measured by HPLC it appears that mainly mono- and disaccharides are produced. Thus oligosaccharides known to be responsible for diarrhoea and flatulence when given to calves in too large amounts are present in small amounts only. " * ~77" 2 028 76 Table Bal. Chemical properties of the.supernatant.
Experiments Enzyme dosage in relation to substrate % w/w NSI, % * (at pH = • 4.5) DSI, % ** (at pH «= 4.5) HPLC results (neutral sugars composition) A E/S = 0.5% 3S.9 62.4 DP.. + DP, DP; ^ DP4 DP4+ 79.7% 7.4% 12.2% 8.1% B E/S = 2.0% 57.0 67.1 DP. + DP- DP3 DP4 DP4 + 84.4% 6.1% 3.7% 5.7% * NSI = Nitrogen Solubility Index ** DSI = Dry matter Solubility Index Table Ball. Stability tests of the reaction mixtures.
Experiments Enzyme dosage in relation to substrate % w/w Stability tests Without oil With oil Dispersion 1. day Dispersion 2. day Emulsion 1. day Emulsion 2. day A_ _ E/S = 0.5% 80% 63% 100% 87% B E/S = 2.0% 66% % 85% 71% 2 028 7-6 Ba 2. Production of saccharified starch containing raw materials.
In relation to the saccharification of cassava and sweet potatoes and other starch containing plant materials addition of an SPS-ase preparation is capable of solving viscosity problems. By using an SPS-ase preparation it is possible to produce starch suspensions with a dry matter content of 25-30%, and after sacchari-fication the mash may be fermented whereby a cheap ethanol may be obtained.
Example Ba 2.1 On the basis of fresh and grated sweet potatoes (Japanese) a mash with a dry matter content of 24% was produced. The starch content of sweet potatoes was found to be approximately 70% of the dry matter content hereof. A pre-liguefaction by ireans of the bacterial amylase of Termamyl® 60 L in a dosage of 0.5 kg/ton of starch was performed by heating the mash to 90°C. The mash was then held at 90°C for 30 minutes. The viscosity of the reaction mixture was then measured by means of a HAAKE spindle at 90°C.
Hereafter the reaction mixture was cooled to 55°C, and pH was adjusted to pH 5.0 with 2N H^SO^. A saccharif ication was then • initiated by addition of the gluco-amylase SAN 150 (trade mark from NOVO INDUSTRI A/S) in a dosage of 1.75 litre/ton of starch. The saccharification mixture was then divided into three parts, A, B, and C, which were enzyme treated for 15 minutes as shown below before measurement of viscosity: A: This part is the control. The viscosity/^ was measured# vide table Balll.
B: The Tricoderma viride cellulase of Celluclast^ 200 N was added in a dosage of 1 kg/ton of dry matter of sweet potatoes. The viscosity was measured, vide table BallI. i 2 028 7 C: The SPS-ase preparation of KRF-68 was added in a dosage of 0.25 kg/ton of dry matter and of sweet " potatoes. The viscosity/^ was measured, vide table Bal Table Balll; Viscosities Reaction mixture Viscosity at 90°C Viscosity at 55°C Pre-liguefied sweet potatoes A B C = 770 cp r^2 = 2190 cp /£2 = 2190 cp = 1970 cp /^4 = 950 cp Thus it can be seen that the viscosity of the reaction mixture could be effectively reduced with the SPS-ase in a low do sage compared to the Celluclast ® and SAN 150.
Ba 3. Total liquefaction of pears and other fruits.
If whole pears which are mechanically crushed are subsequently treated with an SPS-ase preparation, total liquefaction takes place, and a clear pear juice is produced after removal of minor amounts of solid matter. A similar method can be used in relation to other similar fruits, e.g. apples.
Example Ba 3.1 Fresh apples were coarsely milled by means of a Bucher Central mill. The apple mash was then pasteurized in a heat jacketed tank at 90°C for 5 minutes and then cooled to ainbient temperature. The pre-mashed apples were then milled on a Fryma mill with corund stone outfit until the mash was smooth to the touch. The mash was then re-pasteurized at 80°C for 10 minutes, and cooled to 50°C.
Enzyme reactions were now carried out at 50°C for 30 minutes with the Contraves Rheomat 15^ stirring and viscosity, measurement (in relation to a percentage reading on the Rheometer at speed 13) being carried out simultaneously. After completion of the enzyme reactions 100 g of samples were drawn out and centrifuged in a graduated tube at 3000 xg for 15 minutes. Hereby the percentage of juice and the percentage of deposit are measured. pH and the percentage of refractometer dry matter as ° Brix were also measured. Table BalV show! a comparison between the effect of SPS-ase, the combination of Cel-luclast and SPS-ase, and the combination of Celluclast and Pectinex. The SPS-ase preparation KRF-68 was used.
\ I Table BalV. Results of total liquefaction exp£ mash at 50°C for 30 minutes SPS-ase g/hl mash Celluclast^^ 200 1 (li") Pectinex^--' 3x g/hl mash Final viscosity Centrifugation Juice g/hl mash « % juice % deposit pH °Brix 0 0 0 100 59 41 3.8 9.7 0 0 19 81 19 3.5 .5 50 0 0 79 21 3.5 .7 50 50 0 4.6 3.8 83 83 17 17 3.5 3.1 .8 12.5 0 50 200 9.5 83 17 3.2 12.9 0 50 2000 4.0 82 16 3.1 13.2 Ba 4.
Production of juice by treatment of fruits and vegetables.
It has been found that SPS-ase preparations are well suited for production of juice by treatment of several fruits, berries and vegetables, e.g. carrots, peas, tomatoes, apples, pears, black currants, beans, and cabbage. Hereby an improved juice yield, and better extraction of colour and flavour components are achieved compare to commercially available pectinase and cellulase preparations.
Example Ba 4.1 Reference is made to example Ba 3.1 where the SPS-ase preparation has been corpared to the ccnmercially available conventional cellulase am pectinase products Celluclast ® 200 L and Pectinex 3x. It appears from the table that the juice yield may be slightly improved with as little as 50 g/hl of SPS-ase compared to 2000 g/hl of Pectinex ®, both in combination with 50 g/hl of Celluclast . Also the viscosity was slightly lower. Thus it seems that the SPS-ase is about 40 times more effective than Pectinex.
Ba 5. Treatment in relation to extraction or pressing of sugar -cane or sugar beet.
It has been found that it is possible to improve the yield related to simple extraction processes, if the SPS-ase preparation is used for treatment of sugar cane or sugar beet before and/or during the extraction or pressing thereof. Also, the remanence (the bagasse) can be treated with the SPS-ase preparation, whereby it is partially converted to fermentable sugars which may be used as a raw material for ethanol fermentation.
Example Ba 5.1 kg of sugar beet remanence (pulp) obtained from continuous countercurrent extraction in a DDS-diffuser at Nakskov Sugar Factory was milled twice in a Fryma mill (type MZ-110). Processing water was added during the milling operation. 300 g portions of the pulp were now enzyme treated at 45eC for 18 hours by means of the enzyme dosages shown in table Ba"\ The dry enzyme product (KRF-68) was added to the pulp which was stirred by a rod during the first hour. Thereafter the pulp was liquefied to such an extent' that magnet stirring could successively be performed for the remaining time. At the end of the reaction pH was measured (no pH-corrections were made during the—start of the reaction).and the reaction mixture was centrifuged until a .clear^ supernatant was obtained. Dry matter determinations were performed on the reaction mixtures and on the supernatants. Based on these results the percentages of solubi-lized dry matter were calculated. Corrections for the soluble dry matter of the enzyme product were made in all calculations.
' Supernatants Nos. 2, 3, and 4 were ionexchanged and analysed by HPLC for carbohydrate composition.
Table BaV. Results obtained by enzymatically liquefaction of beet pulp.
Experiment No Enzyme dosage in relation to dry mat-' ter, E/S % Final measurements Reaction mixture Supernatants pH (final) % dry matter % dry matter % solubi-lized dry matter 1 0 .5 4.18 0.0 0.0 2 0-35 3.6 3.85 2.58 66.9 3 0.56 3.5 3.81 2.56 66.2 4 1.02 3.5 3.86 2.73 70.4 * 1.58 3.3 3.17 2.34 73.4 6 3.10 3.4 3.23 2.49 76.4 7 - 7.52 3.4 2.66 2.18 1 80.5 Reaction conditions: M = 300 g S = 4.18% dry matter E/S as shown above pH not adjusted T = 45°C t = 18 hours 2 028 76 Table BaVI. HPLC data.
Sugar type (Neutral) Experiment No. 2 3 4 % of neutral sugars High molecular (DP4+) 43.6 31.9 .3 Disaccharides 4.6 4.8 - Glucose .4 23.7 27.8 Galactose .0 .9 7.3 Fructose/Arabinose 26.4 . 32.2 33.2 Galacturonic acid not measured All the sugars formed according to the above table BaVI could be fermented to alcohol or used for other purposes.
Ba 6. Production of soy milk.
Soy milk can easily be produced by total liquefaction of milled soy beans and subsequent homogenization of the resulting mixture. Soy milk is often produced, by soaking of soy beans in boiling water , milling of the soaked beans and water extraction followed by a separation of insoluble residues, e.g. pro- 86 2 0 7 teins and polysaccharides. In order to improve the yield of the soy milk these insoluble residues may be liquefied by reaction with the SPS-ase.
Example Ba 6.1 series of enzyme reactions, whereby calculations of the protein solubility index (PSI,%) and the dry matter solubility index (DSI, %) illustrate the yields obtained after separation at pH = 7 (vide table BaVI3). The enzyme reactions were carried out under the following conditions: The soy milk process is illustrated by the following Substrate: Full fat soy flour (Dansk Sojakagefabrik A/S) Mass of reaction mixture: 220 g Mass of substrate: Temperature: pH: Reaction time: g 50°C 4.5 (6 N HCl) Series A: 1 hour Series B: 0.5 - 6 hours SPS-ase (KRF-68) Series A: E/S-ratio (w/w): 0 - 8.0% Series B: E/S-ratio (w/w): 1.0% 1 hour ■ Enzyme: Enzyme dosage: of at After the reaction pH was adjusted to pB = 7 by means 4 N NaOH, and a separation was performed by centrifugation 3000 x g for 15 minutes.
Table BaVlI. Mass balance calculations in relation to the enzymatic soy milk process.
Reaction time hours Enzyme- Reaction,mixture Supernatant Solubility indices Series dosage E/S % % protein % dry matter * protein % dry matter PSI % DSI % 1.0 0 3.65 8.70 1.87 .72 49.6 63.7 1.0 0.5 3.68 8.74 2.41 G.2B 63.7 70.0 A 1.0 1.0 3.71 8.78 2.48 6.43 65.1 71.4 1.0 2.0 3.78 8.86 2.98 7.18 77.5 79.5 1.0 4.0 3.92 9.03. 3.36 7.69 84.6 83.9 1.0 8.0 4.19 9.37 3.76 8.0B 88.5 85.0 0.5 1.0 3.64 8.78 2.39 6.41 64.0 71.1 1.0 1.0 3.64 8.78 ■ 2.56 6.60 68.7 73.4 B 2.0 1.0 3.63 8.78 2.79 6.91 75.2 77.1 4.0 1.0 3.63 8.78 3.10 7.29 84.0 81.7 6.0 1.0 3.63 8.78 3.39 7.69 92.3 86.5 2 02876 Ba 7. Treatment in order to increase the recoverable amount of coffee solubles.
It has been found that treatment of coffee beans at different stages during the production of instant coffee results in an increased yield of coffee solubles. Thus, e.g. spent coffee grounds or green beans can be enzyme" treated with favourable results.
Bb 1. Prevention and/or decomposition of apple or pear haze.
After production of apple juice or pear juice and other fruit juices, which have to be clear, and which are previously treated with.. . conventional pectinase and cellulase preparations in order to prevent formation of turbidity, an apple haze or similar fruit hazes may appear. It has been found that the SPS-ase preparations are well suited for decomposition of such hazes, which mainly consist of araban bound t< proteins.
Example Bb 1 Pear juice concentrate produced by enzymatic liquefaction of pear cannery waste using Celluclast ® and Pectinex® was found to be cloudy on standing. The haze was isolated and hydrolyzed with 0.01 N H2S04 for 24 hours and analyzed by HPLC. The chromatogram showed arabinose and small amounts of oligosaccharides.
By incubation of 0.5% w/v of this carbohydrate in 1 mM acetate buffer at pH 4.5 for 3 hours at 40°C with SPS-ase (KRF-68 + KRF-92 1:1) with an enzyme concentration of 0.05% w/v it was found that 84% of the initial haze carbohydrate (dry matter) was converted to arabinose.
Also diluted pear concentrate (20°Brix) was treated for 2 hours at 40°C with an enzyme dosage of the above mentioned SPS-ase of 0.15% w/v or with a commercial product called Clarex ® in a dosage of 1% w/v. It was found that the SPS-ase was able to reduce the relative HPLC peak area of an araban-like haze with 86%, whereas the corresponding reduction with Clarex ® (used in a much higher dosage than the SPS-ase preparation) was only 78%. 2 028 76 Bb 2. Use as a clarifying agent for white wine. ** w It has been found that white wines exhibiting a highly undesired turbidity can be effectively clarified by means of SPS-ase. It has been shown that the cloudy material mainly consists of arabinogalactans which are bound to hydroxyprolin residues in a cell wall structural protein.
Bb 3. Production of ISSPH or other vegetable protein hydrolyzates.
Previously to the separation of the ISSPH (isoelectric soluble soy protein hydrolyzate) or other vegetable protein hydrolyzates from the sludge, as described in US patent No. 4,100,024 or in Process Biochemistry, vol. 14, No. 7 (1979), pages 6-8 and 10 - 11, the reaction mixture can be treated with an SPS-ase preparation. Hereby an easier separation is obtained.
Bb 4. Mashing enzyme in the brewing industry.
In production of beer carbohydrates of the raw materials, e.g. the beta-glucans of malt and barley, influence the viscosity and filterability of the wort. Addition of SPS-ase during mashing will reduce wort viscosity and improve filterability and extract yield. Furthermore addition of SPS-ase during mashing will increase the fermentability of the wort and the nitrogen content in the wort.
Example Bb 4.1 In the laboratory 50 g of milled grits consisting of 50% malt and 50% barley were mashed together with 275 g of water (15% dry matter) according to the following mashing diagram: 52° C (60 minutes)/63° C (60 minutes)/76° C (30 minutes).
In order to demonstrate the effect of the SPS-ase four tests were carried out, vide the below indicated table, whereby the enzymes were added during mashing in (pH of mash 5.5 - 6.0). 61 • 2 028 76 Enzyme None Cereflo SPS-ase (KRF 68) Activity of beta-gluca-nase/g 0 200 BGU 1630 FBG 163 0 FBG Dosage of enzyme per kg grits . 0 1.5 g 0.05 g 0.18 g Total dosage of enzyme activity units per kg grits 0 300 BGU 80 FBG 300 FBG Filtration rate of wort after 10 minutes 120 ml 135 ml 160 ml 170 ml Viscosity of wort 10° Balling (25° C) 1.52 CE 1.36 cP 1.36 cP 1.30 cP BGU is beta-glucanase units determined according to analytical method AF 70/4-GB obtainable from NOVO Industri A/S. " FBG is fungal beta-glucanase units determined according to analytical method AF 70.1/2-GB obtainable from NOVO Industri A/S.
The only difference between BGU and FBG is the pH, at which the enzyme determination is carried out: pH 7.5 for BGU and pH 5.0 for FBG.
Cereflo is a bacterial beta-glucanase preparation described in the information leaflet B 2l4b-GB 1500 July 1981 available from NOVO Industri A/S.
Example Bb4.2 In the laboratory 50 g of milled grits consisting of 40% malt and 60% barley were mashed together with 150 g of water (25% dry matter) according to the following mashing diagram: 45° C (60 minutes)/63° C (90 minutes)/75° C (15 minutes).
In order to demonstrate the effect of the SPS-ase three tests were carried out, vide the below indicated table, whereby the enzymes were added during mashing in (pH of mash 5.5 - 6.0). 2 028 76 Enzyme None Ceremix SPS-ase (KRF 68) + Ceremix added as in previous test Activity of beta-glucanase/g - 200 BGU 1630 FBG Dosage of enzyme per kg grits - 1.65 g 0.033 g Total dosage of enzyme activity units per kg grits 0 33 0 BGU 50 FBG Filtration rate of wort after 30 minutes 48 ml 98 ml 111 ml Extract, °Balling 18.6 19.0 19.5 Viscosity of wort 10° Balling (25° €) 1.72 cF 1.37 cP 1.27 cP The definition of BGU and FBG is as indicated in Example Bb^f.l. In the last column in the above table only the activity and dosage originating from SPS-ase is indicated.
Ceremix is a bacterial beta-glucanase preparation described in the information leaflet B 216 b-GB 1000 Feb. 1982-available from NOVO industri A/S.' Bb 5. Enzymatic additive for use during beer fermentation and/or storage.
SPS-ase can be added during fermentation of wort or storage of beer in order to reduce the content of beta-gluc'ans and thereby improve beer filtration and beer stability in regard to haze. SPS-sse will also exert an effect on proteins responsible for chill haze.
Bb 6. Almond skin release agent.
During the mechanical almond skin removal step following the blanching of almonds a certain percentage of the almond skins are not released. It has been found that enzyme treatment of the almonds results in a decrease of the above indicated percentage.
Be 1. Decomposition of different waste materials.
In relation to certain manufacturing process large amounts of carbohydrate containing waste materials are formed For instance this is the case in relation to production of soy isolate by water extraction and acid precipitation, soy milk and tofu (a special kind of Japanese cheese). Also in this connection waste pulp from e.g. apples, pears, or citrus fruits may be mentioned. It has been found that an SPS-ase preparation is able to liquefy this carbohydrate containing waste material completely and to produce fermentable sugars which can be used as a starting material for ethanol fermentation.
Example Be 1.1 By traditional production of soy milk or tofu soy beans are often soaked in boiling water, milled and extracted with hot water, whereafter a separation is carried out. The residue from this separation is the material used for this experiment. The liquid phase is the soy milk, which may be further treated to produce tofu. kg of whole soy beans obtained from Aarhus Oliefabrik A/S was milled simultaneously with 7 0 litres of boiling water in a Fryma mill type MZ 110. The milled ' slurry was then held above 85°C for 15 minutes in order to inactivate the natural bean enzymes which develop the well-known soy bean off-flavour. 5 litres of this soy bean slurry was then centrifuged in the laboratory for 15 minutes at 3000 x g (g = gravity). It was found by analysis that the remanence contained 20.45% and 20.06% dry matter (duplicate determinations, calculated average 20.26%). 6 N HCl was slowly added and worked into the remanence with a spatula until a pH-meter showed 4.50, when the electrode was introduced directly into the mass. 2 028 7 Enzyme reactions on 2 x 200 g of the mass with the two dosages of the SPS-ase (KRF-68) E/S = 0.5% in relation to dry matter and E/S = 3.0% in relation to dry matter were carried out in a 500 ml beaker at 50°C. The dry enzyme was added to the mass. During the first 1-2 hours the stirring was carried out with a spatula and hereafter the mass was liquified to such an extent that stirring with a magnet could be carried out successfully. The total reaction time was 21 hours. During the reaction the osmolality was measured with an osmometer (Advanced Digimatic 3DII from Advanced Instruments Inc.). The results in table Bel show the course of the reaction. At the end of the experiment the mixtures were centrifuged at 3000 x g for 15 minutes. A layer of oil appeared on the top of the supernatants and the volume thereof was determined. A loose layer of sludge appeared as a bottom-layer. The supernatant including the oil was removed with a pipette. The oil was combined with the clear water phase by homogenisation and a sample was drawn for dry matter determinations. The results shown in table Bel clearly demonstrate that this waste product can be liquefied by an enzymatic reaction and that crude oil can be produced as mentioned in section A4. After recovery of the oil the solubilized remanence may be used in different ways, e.g. for fermentation to valuable compounds or for concentration and drying and subsequent use as a fodder or food product, or after further purification for production of valuable products. 2 028 76 Table Bel. Results obtained during liquefaction of soy milk and tofu-sludge.
Reaction conditions and results Experiment A Experiment B Mass of residue 200 g 200 g Mass of SPS-ase (KRF-68) 0.20 g 1.20 g Temperature 50°C 50°C pH 4.50 •4.50 Reaction time 21 hours 21 hours Results measured on csmcrr.ster during the course of the t rain. osmolality mOsm & osmolality mOsm t min. osmolality mOsm & osmolality mOsm reaction. 0 287 0 0 282 0 313 26 368 86 4 Osmolality is - - - 497 215 the value corrected for the osmolality 40 215 391 m 104 m 45 2IS 601 Itt 319 III of the mixture at 1260 907 620 1260 1145 863 t = 0 Reaction mixture: Dry matter .3 % .-7% Supernatant: Dry matter 18.0% 19.4% Supernatant: Oil content 8 - 10% 8 - 10% Calculation %solubi-lized dry matter 88.6% 93.5% -96- 20287 Be 2. Saccharification and simultaneous fermentation.
Carbohydrate containing plant materials, e.g. tubers like Jerusalem artichokes, potatoes, sweet potatoes, cassawa, or pulp from such tubers, i.e. the material remaining after removal of the extracted components may be saccharified by treatment with an SPS-ase preparation, and simultaneously the formed fermentable saccharides may be fermented to ethanol.
Example Be 2.1 The production of ethanol by fermentation of the decomposed inulin containing Jerusalem artichokes was examined in laboratory scale by simultaneous saccharification with SPS-ase and inulinase and by four different pretreatments of the Jerusalem artichokes.
SPS-ase; The SPS-ase preparation KRF-6 8 was used.
Inulinase: The inulinase was produced by fermentation of Asp. ficuum (CBS 55 565) . The inulinase activity is determined as described in Research Disclosure No. 21234 (December 1981) p. 456 - 458.
Laboratory fermentation: 150 g portions of the pretreated mash (described later) were fermented after addition of 4.5 g of bakers yeast and 1 ml of a 4% solution of Pluronic as an antifoaming agent. The fermentation flasks are provided with CC>2 traps containing 98% sulphuric acid, and the fermentation is followed by measurement of the weight loss due to liberated C02« The content of the flasks is agitated during the fermentation carried out at 30°C. Three flasks were used for each parameter studied.
In table Bell the weight loss due to liberation of CO-j is converted to ethanol assuming that 1 mol liberated . . 46 CC>2 is equivalent to 1 mol C2H5OH, i.e. 1 g C02 ~ g C2H5OH Pretreatments of the artichokes: Treatment A: 14.1 kg artichokes (22.8% dry matter) was Henzecooked at 140°C and 4-5 atm. for 20 minutes. The weight after cooking was 19.0 kg (~ 16.9% dry matter). Fermentations were performed directly on the mash.
Treatment B: Washed and sliced artichokes were mixed with water (1:1) and blended in a Waring blender. The mash was then heat treated for one hour at 85°C and pH = 4.5.
Treatment C: As B, but pH was not adjusted.
Treatment D: As B, but no heat treatment and no pH adjustment Results: In table Bell the results show the effect of addition of SPS-ase to the pretreated mash on the ethanol yield. A significant improvement of the ethanol yield was obtained on all pretreated mashed when SPS-ase was added.
Table Bell Fermentation results in relation to simultaneous fermentation and enzyme saccharification of Jerusalem artichokes.
Pre- treat- ment Inulinase units added to 1 g of dry matter SPS-ase E/S % Loss of CO_ (g) 42 - 44 hours of mentation after fer- % ethanol produced in relation to dry matter 1.5 0 7.65 + 0.05 31.5 A 1.5 0.27 8.07 0.08 • 33.2 B 1.5 1.5 0 0.40 4.85 5.41 + + 0.03 0.03 29.7 33.1 1.5 0 .70 •+ 0.05 34.8 1.5 0.10 .97 + 0.01 36.5 C 1.5 0.20 6.13 0.06 37.5 1.5 0.30 6.13 + 0.00 37.5 1.5 0.40 • 6.18 + 0.05 37.8 "1.5 0 .77 + 0.02 - .3 1.5 0.10 .89 + 0.00 36.0 D 1.5 0.20 6.04 + 0.11 36.9 1.5 0.30 6.01 + 0.01 36.7 1.5 0.40 6.02 + 0.03 . 36.8 0 0.40 .48 + 0.02 33.5 Be 3. Decomposition of cellulose.
It has been found that cellulose containing materials like straw, e.g. wheat straw, saw dust, paper, and lignocellulose, may be hydrolyzed to a greater extent with an SPS-ase preparation than with conventional cellulases. This is illustrated by the following example in which a crystalline cellulose material (AVICEL) is treated by means of a conventional cellulase Celluclast 200 produced by Tricho-derma reesei and the SPS-ase preparation KRF 68# Example Be 3.1 Avicel was suspended in water (20% dry matter) ; pH was adjusted to 5, and the temperature was maintained at 50°C. After 24 hours reaction time the slurry was filtered, and the content of reducing sugar (mg glucose/g AVICEL) was measured. Using enzyme dosages of 5% and 20% of the cellulose content the following values were founc Table Belli Enzyme E/S % mg glucose / g AVICEL Celluclast 80 SPS-ase 200 Celluclast 100 SPS-ase 340 Be 4. Application as a baking aid. • It has been found that SPS-ase preparations are excellently suited as baking aids. Thus, when an SPS-ase preparation is added to the dry flour, before production of the dough, it is possible to obtain a bread with superior quality in regard to volume, crumb and taste. Thus it is possible to obtain a high quality bread with a low quality wheat flour if an SPS-ase preparation is used as an additive. 2 028 / 6 Be 5. Improvement of alcohol yield and yield of biomass during fermentation of sulphite liquor from paper production.
Also it is found that the yield of ethanol may be improved if paper'sulphite liquor is treated with an SPS-ase preparation before it is utilized as a carbohydrate source for fermentation of ethanol. Paper sulphite liquor may also be used for production of biomass, e-.g. single cell protein, by fermentation, and also in this case the yield of biomass has been improved when the sulphite liguor has been treated with an SPS-ase preparation previously. Also, decomposition due to the presence of the SPS-ase preparation and fermentation can be performed simultaneously.
/• Be 6. Dewatering of biological sludge products.
During traditional water extraction of many biological materials from plant raw materials, large volumes of insoluble residue consisting of great proportions of swelled polysaccharides are formed. This, for example, is the case when soy milk, tofu, or soy isolate is produced by water extraction of soy beans, defatted soy flour, or white flakes. The structural swelled polysaccharide material may then be treated to a slight extent with SPS-ase, whereby the network structure of the material is opened, and only slight amounts of carbohydrates are solubilized. Thereby the material is dewatered, and consequently a higher dry matter content is obtained in the sludge in comparison to a product obtained without the enzymatic treatment. Thus, the enzyme process exhibits the advantage of a considerably lower energy consumption for removal of water by drying, and it also opens up the possibility for production of a cheaper dry animal feed material or bulking agent for food applications.
Be 7. Silage aid.
It is known to add enzymatic silage aids to silage in order to increase the rate of the silage process and the dige-stability of the silage. It has been found that SPS-ase preparations are superior in compari-son to known enzymatic silage aids.
A survey of the figures, to which reference has been made already, is given 'below for the purpose of providing a better comprehensive view.
Fig. No.
Belongs to Describes 1 The general part of the specification Demonstration of binding effect between EPS and soy protein. • 2 The general part of the specification Flow sheet describing the production of SPS 3 Section 2 Calibration curve for KPLC gel filtration chromatography ■ 4 Section 2 HPLC gel filtration chromatogram of SPS Section 2 HPLC gel filtration chromatogram of SPS decomposed by SPS-ase 6 Sections 2 and 3 HPLC gel filtration chromatogram . of supernatant from SPS incubated with soy protein 7 Section 2 HPLC gel filtration chromatogram of supernatant from decomposed SPS incubated with soy protein B Section 3 HPLC gel filtration chromatogram of APS decomposed by Pectolyase 9 Section 3 . HPLC gel filtration chromatogram of APS decomposed by SPS-ase Section 3 HPLC gel filtration chromatogram of SPS treated with Pectolyase 7 Fig. No.
Belongs to Describes 11 Section 7 Immunoelectrophoretic peaks including an SPS-ase peak identified by overlay technigue 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 dependency of an SPS-ase Section 9 Temperature stability of an SPS-ase 16 Section 10 pH-stability of protease in an SPS-ase preparation

Claims (27)

1. - 103 - 202870 WHAT WE CLAIM IS:
2. A composition comprising an effective amount of an SPS-ase, capable of decomposing soy SPS as hereinbefore defined in aqueous medium into decomposition products which attach themselves to protein in an aqueous medium to a lesser extent than soy SPS prior to decomposition would have . attached itself to the same protein under corresponding conditions. ^ • A composition according to claim 1 wherein the SPS-ase is capable of decomposing soy SPS as hereinbefore defined in an aqueous medium into decomposition products which attach themselves to vegetable protein in the aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached itself to the same vegetable protein in the aqueous medium.
3.A composition according to claim 1 or 2, wherein the SPS-ase is capable of decomposing soy SPS as hereinbefore defined in an aqueous medium with a pH value not deviating more than 1.5 from 4.5 into decomposition products which attach themselves to soy protein in the aqueous medium to a lesser extent than the soy SPS prior to decomposition would have attached itself to the soy protein in the aqueous medium. - 104 - 2Q2S76
4. A composition according to any one of claims 1-3, wherein the decomposition products of soy SPS as hereinbefore defined after completed degradation attach themselves to the vegetable by weight protein to an extent of less than 50%/than the soy SPS prior to decomposition with SPS-ase would have attached itself to the vegetable protein in the aqueous medium.
5. A composition according to claim 4, wherein the decomposition products of soy SPS as hereinbefore defined after completed degradation attach themselves to the vegetable protein to an extent of less than 20% by weight than the soy SPS prior to decomposition with SPS-ase would have attached itself to the vegetable protein in the aqueous medium.
6. A composition according to any one of claims 1-5 wherein the SPS-ase exhibits a positive SPS-ase test, when examined according to the qualitative SPS-ase determination method.
7. A compo|^iggeGgjprising an effective amount of an SPS-ase according to/claims 1-6, wherein the SPS-ase was produced by means of a niicroorganism belonging to the genus Aspergillus.
8. A composition comprising an effective amount of an SPS-ase according to ■5$i§ ims°£-7, wherein the SPS-ase was produced by means of a microorganism belonging to the Aspergillus niger group.
9. A composition comprising an effective amount of an SPS-ase any one of according to/claims 1-8, wherein the SPS-ase is derived from the enzymes producible by means of Asp, aculeatus CBS 101.43. i - 105 - 202876 any one- of
10. ' A composition according to /Claims 1-9, 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 immunoelectrophoretic overlay technique.
11. A method suitable for selection of an SPS-ase producing microorganism, for production of the SPS-ase any one of according to/claims 1-10, wherein the micro-organism to be tested is grown on a fermentation medium, the main carbon source of which is an SPS as hereinbefore defined produced on the basis of vegetable raw protein as a raw material, whereafter a sample of the fermentation medium is analyzed for SPS-ase and the microorganism in question is selected as an SPS-ase producing microorganism, if the analysis for SPS-ase is positive.
12. A method for production of SPS-ase according to any one of /claims 1-10, wherein a strain selectable according to the method of selection according to claim 10 is cultivated in a nutrient medium.
13. A method according to claim 12., wherein the strain Asp, aculeatus CBS 101.43 or Asp, japonicus IFO 4408 is cultivated in a nutrient medium.
14. a method according to claim 12 or 13, wherein the cultivation is carried out as a submerged cultivation at a pH in the range from 3 to 7, at a temperature in the range of from 20 to 40®C and whereby the nutrient medium contains carbon and nitrogen sources and inorganic salts.. 202876 - 106 -
15. A method according to claim 14/ wherein the cultivation is carried out as a submerged cultivation at a pH in the range of 4-6, at the temperature in the range of 20 to 40°C and whereby the nutrient medium contains carbon and nitrogen sources and inorganic salts.
16. A method according to claim 14 or 15, wherein the cultivation is carried out as a submerged cultivation at a temperature in the range of 25 to 30°C and whereby the nutrient medium contains carbon and nitrogen sources and inorganic salts. any one of
17. A method according to/claims 12-16, wherein the nutrient medium contains soy meal.
18. A method according to claim 15, wherein the soy meal is treated with a proteolytic enzyme before using as a component of the substrate.
19. A method according to claim 18 wherein the soy meal is treated with the proteolytic enzyme produced microbially by means of Bacillus licheniformis. a'ny- one of
20. A method according to/claims 12-19, wherein a sterile solution of pectin is added aseptically to the fermentation broth during the cultivation.
21. A method suitable for decomposition of polysaccharides, wherein an SPS-ase composition according ^5iycfi?fm§^l-l0 in an aqueous medium is contacted with a substrate for said SPS-ase preparation.
22. A method for decomposition of polysaccharides | 27t1ARm6S! V, / i c5/ ( - 107 - 202876 according to claim 21 wherein the decomposition is accompanied by the isolation or extraction of a biological material other than soy protein and related vegetable proteins from a raw biological material, whereby the SPS-ase preparation is essentially free of any enzyme which is able to degrade said biological material.
23. A method for decomposition of polysaccharides according to claims 21 or 22, wherein one or more of the reaction products (no matter whether they are wanted end products or waste products) are treated further simultaneously with or after the enzyme treatment.
24. A method for decomposition of polysaccharides according to claim 23, wherein the further treatment is an alcoholic fermentation in case oneof the reaction products is a fermentable sugar.
25. A composition as claimed, in claim 1 substantially as herein particularly described.
26. A method for selection of an SPS-ase as claimed in claim n substantially as herein described with reference to any one of the examples.
27. A method for the decomposition of polysaccharides as claimed in claim 22 substantially as herein described with reference to any one of t" ^
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EP1067180A1 (en) * 1991-05-02 2001-01-10 Novo Nordisk A/S Aspergillus japonicus and Irpex lacteus rhamnogalacturonase and its use
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