NL1007158C2 - Enzymatic modification. - Google Patents

Description

ENZYMATIC MODIFICATION

The present invention relates to the enzymatic modification of various substrates.

The invention further relates to the products which can be manufactured by means of this chemical modification and their applications.

Proteins of vegetable and animal origin are used in the food and non-food industry. Animal proteins in particular have traditionally had various applications in a variety of foods and other fields. In principle, vegetable proteins are cheap and available in abundance, but have the drawback that they often do not have the functional properties desired for a particular application. Their use in said applications has therefore always been much more limited.

The functional properties of a protein are particularly related to their physicochemical and structural properties. For industrial application of proteins, it is often necessary to improve or change one or more such properties, such as dispersibility, color, foaming ability, adhesive strength, colloid protective properties, wettability, molecular size or molecular shape. This applies to both vegetable and animal proteins.

The chemical and enzymatic modification of proteins for different applications has been described in the literature. Such modifications are, for example, cross-linking of the individual protein molecules with each other and / or other molecules. The object of the invention is to provide a modification method which is improved over the known methods.

As will become apparent from the following description of the invention, the key step in said modification method is also suitable for initiating the polymerization of certain monomers and for the production of new dyes.

As a key step in said modification method (the invention provides an enzymatic oxidation process, __ * ^ λ c \ C \ / * 'i') \ 0 υ (1 "· ~ 2 comprising bringing together an oxidative enzyme, a hydrogen acceptor and a hydrogen donor in a reaction mixture and an oxidative reaction proceeding in the reaction mixture under the influence of the enzyme with at least the hydrogen acceptor and the hydrogen donor as substrate This enzymatic oxidation process is also referred to hereinafter as "key process" or "key step".

Depending on the desired application, substrates can be added and different types of oxidative enzymes, hydrogen donors and hydrogen acceptors can be selected.

The active bringing together of the components enzyme, hydrogen donor, hydrogen acceptor and optionally one or more other substrates to effect a modification of these substrates has not previously been described.

Suitable enzymes are, for example, peroxidases, in particular hydrogen peroxidase, polyphenol oxidase and lipoxygenase.

Hydrogen donors are typically phenols, phenolic acids or phenolic compounds, such as certain dyes, especially alizarin and its derivatives, antocyanins, flavonoids and the like.

Hydrogen acceptors are, for example, hydrogen peroxide and oxygen.

For example, if efforts are made to modify proteins, the reaction mixture further comprises one or more proteins as a substrate to be crosslinked and the hydrogen donor serves as a crosslinking agent for this. Particular preference is given to phenols, such as catechol, or phenolic acids, such as ferulic, caffeic, coumaric, vanillic, tyrosine, coniferyl, sinapyl, cinnamic, p-coumaryl, sinapic, p-hydroxybenzoic, gallic and flavonoids as crosslinking .

The substrate to be cross-linked is preferably formed by one or more naturally water-soluble 10071 π >>

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3 proteins and / or one or more water-solubilized proteins and / or mixtures thereof with one or more polysaccharides.

Naturally water-soluble proteins are, for example, ovalbumin, bovine serum albumin, cytochrome C, casein, collagen, gelatin, soy protein, proteins from vegetables or grains, in particular gluten. The proteins solubilized in water originate, for example, from potatoes. A suitable polysaccharide is starch. The (cross-linked) proteins or mixtures of proteins with polysaccharides modified in this way can have various applications. For example, it is possible to modify naturally foaming proteins, such as ovalbumin and bovine serum albumin, such that after shaking in an aqueous solution they lead to the formation of more and more stable foam than the unmodified versions of these proteins.

The gelling properties of some proteins can also be clearly improved by the modification. The 20 gels obtained by the modification can be used as an additive for desserts, as an extrusion aid in animal feeds and the like.

Protein cross-linking or cross-linking of mixtures of proteins with polysaccharides according to the invention can further lead to films which have better properties than films made from the corresponding uncrosslinked proteins or mixtures of proteins with polysaccharides. This will be further illustrated in the examples.

The type of product (gel, foam, film) ultimately formed depends, among other things, on the protein used and the conditions, enzyme amount and the like.

For foaming, proteins with favorable surfactant properties, such as ovalbumin or BSA, are preferably used, in combination with a relatively low concentration of the crosslinking agent (about 0.5-1% relative to the protein) and a relatively high 1. 0 0 7 1 5 3 4 concentration of hydrogen peroxide (about 1.5% relative to the protein).

For the manufacture of gels and films, casein-like proteins are used with a higher concentration of the crosslinking agent (about 1 to 5% relative to the protein) and lower concentrations of hydrogen peroxide (about 0.3-1.5% relative to the protein).

The products obtained by the method according to the invention are different from similar products, which are made from proteins and / or polysaccharides without crosslinking agents, due to the presence of crosslinking molecules in the final structure. They differ in both composition and properties. Such new products are therefore also part of the present invention.

The system of an oxidative enzyme, such as peroxidase, a hydrogen donor, such as a phenol, and a hydrogen acceptor, such as hydrogen peroxide, in combination with suitably selected proteins, such as casein, can be used as an additive in paints and glues, in starches and adhesives.

It has surprisingly been found that the key process according to the invention can also be used for the initiation of polymerization processes involving radicals. In that case, the hydrogen donor is converted into a radical by the oxidative enzyme, which subsequently serves as an initiator in the polymerization of monomers also present in the reaction mixture, in particular acrylates.

In another variant, the key process according to the invention can be used in the manufacture of new dye molecules, which are more stable and less sensitive to changes in pH. In that case, the hydrogen donor is an organic dye molecule, which is coupled by the oxidative enzymatic reaction to one or more other organic dye molecules of the same or a different kind. For example, the original organic dye molecule is alizarin or a derivative thereof, an antocyan or a flavonoid. Examples are alizarin sodium sulfonate, quercetin, myricetin, rutin.

The new dyes obtained in this way are also part of the present invention.

The present invention will be further elucidated on the basis of the accompanying examples, which are given by way of illustration only.

EXAMPLES EXAMPLE 1

Enzymatic modification of proteins 1.1. Modifying proteins to improve their foaming properties. Proteins with known foaming properties, such as bovine serum albumin (BSA) and ovalbumin, were treated with a system of peroxidase, hydrogen peroxide and a hydrogen donor to increase their foaming capacity and foaming stability.

A solution of 1% protein (10 mg / ml) was prepared in 0.1 M phosphate buffer pH 7. Of this solution, 10 ml was placed in a 50 ml measuring cylinder, and 0.056 mg of catechol (0.56 % relative to the protein) and 40 μΐ of a 0.00027% solution (0.001% relative to the protein) added and mixed gently with the protein solution. The enzymatic reaction was started by adding 50 μΐ 30% hydrogen peroxide (1.5% relative to the protein) to the reaction mixture. After fifteen minutes of incubation, the solution was shaken for 10 seconds. The volume of the liquid and of the foam formed were determined by direct reading. To determine foam stability, the heights of the foam and liquid, respectively, were measured over time at 30 minute intervals.

1007153 6

In all cases, foams of higher volumes and stability were obtained with the product of the invention. The best results were obtained with ovalbumin. When ovalbumin was incubated with peroxidase, hydrogen peroxide and catechol, the solution showed a darker color, but the volume of the foam formed was about six times larger than with ovalbumin alone. The foam obtained under these conditions was stable for at least a week.

Foam obtained with unmodified ovalbumin remained stable for only a few hours.

To obtain lighter color solutions and foams, ascorbic acid was added to the reaction mixture after the incubation with catechol. The foam obtained under these conditions had a very light white-yellowish color and a high volume. However, the stability of the foam decreased to days, but it was still much higher than the stability of the foam produced by the unmodified protein (Figure 1).

Foam obtained from cross-linked proteins, and characterized by high stability, can find application in the food industry for the production of whipped cream, mousses, soufflés and the like. 25 1.2. Modification of the proteins to improve gelling properties

Enzymatic cross-linking according to the invention can improve the gelation of biopolymers, such as proteins.

Incubation of 1% protein solution with 0.0136 mg or 0.00136 mg peroxidase (0.01-0.001% relative to the protein), 10 μΐ 30% hydrogen peroxide (0.3% relative to the protein) and 0, 62 mM phenol (1% over the protein) led to gelation after 5 minutes for bovine serum albumin or after 30 minutes for ovalbumin. The color of both solutions and gels varied from light yellow to dark brown depending on the type of crosslinking agent used. The use of a higher amount of protein for the cross-linking reaction resulted in the formation of gels with a higher firmness and improved properties. These gels can be used in various combinations for the preparation of desserts, as an additive in animal feed, etc.

1.3. Formation of protein films by enzymatic cross-linking. The biopolymers (ie proteins and polysaccharides) mentioned in the above examples were also used in the experiments below. All experiments were performed at room temperature (23-25 ° C).

Protein solutions were prepared in different ways, depending on the type of protein and the method of modification. A 10% solution of casein (1 g case-ine / 9 ml distilled water) is prepared by heating in a water bath at 60 ° C for 30 minutes.

Potato protein solutions were prepared in a different way, because potato protein is poorly soluble. In addition, lower potato protein concentrations were used to avoid high viscosity protein solutions. In a typical experiment, 0.6 g of potato protein is dissolved by adding 12 ml of 12% SDS. Demineralized water is added to this solution to a final volume of 16 ml, followed by 4 ml of ethanol. In other experiments, gluten or potato protein dispersions were used.

Starch solution was prepared by adding 9 ml of demineralized water and 1 g of starch, heating the mixture to 80 ° C until an opaque solution is obtained and cooling to room temperature. After cooling, the solution was used to use protein-starch mixtures for modification.

1.3.1. Casein Films i r. n 7 '* r o i u' J / i ü o 8

In the cross-linking experiments, 0.2 g of glycerol (1: 5, w / w, glycerol: protein) was added to the protein solution as an emulsifier, followed by addition of 1 ml of ethanol. Amounts ranging from 10-55 mg of a dissolved crosslinking agent (5% with respect to protein, w / w) and 10 µl of 30% -H2O2 (0.3% with respect to protein) were added to the solution under mix gently. Subsequently, five times 2.72 µg peroxidase (1: 100000, enzyme: protein) was added at 1 minute intervals. The reaction mixture was stirred gently for 15 minutes to 2 hours and the protein films then formed by pouring the solution onto plates with an internal diameter of 8.5 cm or on square plates of 12x12 cm. The films were then conditioned at 20 ° C and 60% humidity. The films are shown in Figure 2.

The compositions of the films, their water solubility, mechanical properties, water vapor and oxygen permeability of the films prepared in this way were measured. The results are shown in Table 1. To decrease the water vapor permeability of the protein films, experiments were performed in which the hydrophobicity of the protein was modified by adding chemical reagents (eg, glutaraldehyde or lipids) to the reaction mixture during the enzymatic crosslinking experiments. The properties of these films are also shown in Table 1.

The films obtained with enzymatically modified casein swelled, but did not dissolve in water compared to films of unmodified casein, which dissolved immediately upon addition of water. In addition, the films obtained with modified casein have lower oxygen permeability and improved mechanical properties compared to films obtained using unmodified casein.

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9 1.3.2. Casein starch films

Protein and starch films (as the polysaccharide used) were prepared in the same manner as above except that part of the protein was replaced with starch (protein: starch weight ratio ranging from 1: 1 to 1: 2, 1: 3, 1: 4). The films were cast, conditioned at 20 ° C and 60% humidity and analyzed to determine the properties (see Table 1). The films are shown in Figure 3.

The films obtained in this way have better properties than those made with unmodified casein or unmodified casein starch mixtures. The unmodified casein or casein / starch films could be easily dissolved by heating in water, while the films obtained from modified protein / starch were insoluble in water and also could not be dissolved by boiling in ether for 1 hour or by treatment with 10% SDS.

In addition, in the solution in which the enzymatically modified casein / starch films were suspended, the absence of starch was proven by the iodine test. However, starches were present in solutions in which films formed from unmodified starch and protein mixtures were suspended. This observation is an argument for cross-linking of starch with the protein through the oxidative peroxidase catalyzed reaction.

The films prepared from modified starch / protein mixtures had better mechanical properties than those obtained from unmodified starch / protein mixtures.

1.3.3. Gluten movies

By the modification of gluten by enzymatic crosslinking in the manner presented above, insoluble films with excellent mechanical properties could be obtained. The most interesting result was the formation of a very good film by treating 10 ml of gluten with only one phenol and hydrogen peroxide without adding peroxidase. The explanation is that gluten itself already contains peroxidase. The presence of this was detected experimentally and quantified with a modified version of the ABTS assay (Arnao et al. (1995) Analytical Biochemistry 18, 335-338). This gluten peroxidase catalyzes the crosslinking of gluten in the presence of the added substrates (phenol and hydrogen peroxide). This film had properties similar to those obtained from modified gluten due to the catalytic activity of added horseradish peroxidase.

The films prepared by the method of the present invention can find use in coatings for, for example, cheese or for fruit, which is used in yoghurt and puddings etc., for coating paper.

Number Ingredients E ^ Stress Strain WFP

__ [MPa] [MPa] (%) [g / msPa] 20 CHECKS: _ 1 casein 1773 ± 89 34 ± 2 9 ± 5 14 2 casein + glycerol 65 ± 11 3.1 ± 0.2 85 ± 8 16 -17 3 casein + glycerol + H202 63 ± 9 3.3 ± 0.3 85 ± 22 17-18 4 casein + glycerol + catechol 60 ± 6 2.8 ± 0.3 82 ± 9 16 + peroxidase 2 5 5 casein - (- glycerol + catechol 63 ± 8 2.9 ± 0.3 85 ± 14 18-19 __ + HA_____ 6 casein Ι-glycerol - (- starch 268 ± 69 6.0 ± 0.5 28 ± 10 14 __ + H2Q2_____ __PEROXIDASE + GLYCEROL + HA +: 7 casein-fcatechol (system 81 ± 4 4.5 ± 0.5 161 ± 18 15-17 according to the invention) 8 casein + starch 204 ± 18 5.5 ± 0.3 39 ± 14 16-19 30 9 casein + ferulic acid 491 ± 92 11 ± 2 33 ± 19 14-16 10 casein + vanillic acid 237 ± 36 8.6 ± 0.6 81 ± 11 13-14 11 casein + caffeic acid 442 ± 41 10 .9 ± 0.4 61 ± 14 15-16 ♦ *** / "Λ 1 Ou / 'υo 11 12 casein + catechol + extra 261 ± 47 7.9 ± 0.9 113 ± 23 14-15 peroxidase 13 casein + catechol at 65eC 431 ± 29 10.3 ± 0.5 110 ± 12 14-15 14 casein + ferulic acid at 622 ± 54 14.6 ± 0.6 66 ± 9 14-16 __6 £ C _____ 15 case ine + vanillic acid at 553 ± 43 13 ± 1 34 ± 17 13-14 65 ° C __ 5 16 casein + caffeic acid at 585 ± 66 13.6 ± 0.8 64 ± 23 15-16 __6 £ C _____ 17 casein + catechol 30 min .557 ± 141 13.1 ± 0.3 104 ± 16 14-15 reaction time 18 casein + catechol 2 hours re- 522 ± 28 12.9 ± 0.6 89 ± 25 14 action time 19 casein + catechol 5 hours re- 554 ± 32 13 ± 0.6 96 ± 14 14 action time 20 casein + tyrosine 626 ± 32 13.5 ± 0.8 37 ± 12 13-14 10 21 casein + catechol + glutar- 549 ± 25 13.0 ± 0.6 87 ± 17 15 aldehyde 22 casein + glutaraldehyde 825 ± 153 17.8 ± 0.1 47 ± 15 16 without enzyme 23 casein + catechol + stearic 467 ± 16 10.5 ± 0.9 11 ± 3 15-16 acid 24 casein + cinnamic acid 460 ± 16 11.3 ± 0.5 63 ± 10 16 25 casein + catechol + chi 505 ± 46 11.6 ± 0.6 50 ± 19 16-17 tosan 15 _ OTHER ENZYMES: 26 casein + catechol + polyfe- 463 ± 37 12.0 ± 1 85 ± 17 15 noloxidase 27 casein + tyrosine + polyfe- 851 ± 30 17.5 ± 0.5 17 ± 5 14-16 noloxidase 28 casein + linolenic acid + li-804 ± 74 17.8 ± 0.3 21 ± 11 14-15 poxygenase 29 casein + catechol + lino- 689 ± 26 15.9 ± 0.4 56 ± 27 15 lentic acid + lipoxygenase 20 f

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12 EXAMPLE 2

Combined chemical-enzymatic polymerization of organic monomers

In practice, the radical polymerization of organic monomers (eg acrylates, styrene) is carried out at temperatures of about 70-80 ° C in the presence of a chemical initiator (eg potassium peroxide sulfate or other radical precursor compounds).

In the method of the present invention, the polymerization is initiated at a temperature of 40 ° C by the enzymatic oxidative system ("keying process") of the invention consisting of peroxidase, a phenol and hydrogen peroxide. The phenol radicals formed by the chemical reaction are used as initiators for the radical polymerization of acrylates. The polymers obtained in this way have a higher molecular mass and better mechanical properties than those obtained by conventional chemical polymerization.

200 ml of water and 0.2 g of SDS are mixed in a reaction vessel. The solution is heated to 40 ° C and bubbled with nitrogen for 1 hour to remove any oxygen present. Then 12.5 ml of butyl-25 acrylate (0.078 mol), which had previously been washed with 10% NaOH solution, is added to remove the hydroquinone inhibitor. The reaction is initiated by adding 8.8 mg (8.74x105 mole) of catechol (dissolved in 5 ml of water), 0.34 ml of perhydrol (hydrogen peroxide) (10 mM in the reaction mixture) and 12 mg of horseradish peroxidase ( dissolved in 1 ml of water, corresponding to a ratio (w / w) of enzyme: monomer = 1: 10,000). The mixture is reacted at 40 ° C for one and a half hours while mixing. After completion, the polymer is separated and poured onto a Petri dish.

The polymer thus obtained has a molecular weight of about 700,000 D compared to a 1 0 n 7: c 13 control polymer obtained by chemical polymerization which has a molecular weight of 142,000 D.

EXAMPLE 3 5 Enzymatic Method for the Synthesis of Organic Dyes

Peroxidase catalyzes the oxidation of a variety of substrates in the presence of hydrogen peroxide (or methyl and ethyl hydrogen peroxides). A wide variety of organic compounds can serve as a substrate for peroxidase and thus play the role of hydrogen donor in radical reactions. This category includes phenols, aromatic amines and the like. Since alizarin, some of its derivatives, antocyanins (CORRECT ??) and flavonoids, carry a phenol unit in the molecule, they can be substrates for peroxidase. It is expected that the formation of a dimer as a result of the radical oxidation of each of these compounds will have a specific color and will be insensitive to variations in pH.

In this experiment, alizarin sodium sulfonate, quercetin, myricetin and rutin are used as substrates for peroxidase. In a spectrophotometric cuvette (d = 1 cm) with 3 ml 0.05 M phosphate buffer, pH 6.1, aliquots of the reagent solutions are added in the following order: 5 μΐ phenol solution and 40 μΐ hydrogen peroxide solution. The mixture is homogenized by mixing and the reaction is initiated by adding 10 μΐ peroxidase solution. The spectra are recorded in time for the spectral range 230-800 nm (UV-VIS spectrophotometer lambda 16, Perkin Elmer), relative to a reference containing only buffer.

The concentrations of the reagents in the reaction mixture were 5.6 μg / ml peroxidase, 2.6 mM hydrogen-35 peroxide, 7.18x10'4 M myricetin, or 1.12x10 * M alizarin sulfonate, or 8.82x10 ' 5 M quercetin, or 1.36x 10'4 M rutin. For each experiment, the spectrum of the phenol substrate solution is recorded at θ = 0 (Θ is 1 p.> W V ƒ r -V l. '* ^ U 14 time). The reaction endpoint is measured after approximately 24 hours.

The course of the enzymatic oxidation of different dyes is shown in Table 2.

5

Table 2

Experiment Substrate Product Reaction course color λ ™, color alizarin 260.8 red-yellow after 1 hour: yellow- very slow 333.6 260.8 brown reaction 518.4 332 420 516 after 24 hours: 260.8 420 myricitin 252.8 yellow 294 brown very fast 368 332.8 (light) reaction 10 quercitin 253.6 yellow after 1 hour: violet fast reaction 365.6 253.6 (after se-293.6 conden) 332.8 red- 484.0 brown (after after 24 hours: 1 hour ) 253.6 brown (after 293.6 24 hours) 332.8 484 rutin 255.2 yellow 242.4 brown rapid reaction 352 255.6 332.8

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Claims (19)

1. Enzymatic oxidation process comprising combining an oxidative enzyme, a hydrogen acceptor and a hydrogen donor in a reaction mixture and conducting an oxidative reaction in the reaction mixture under the influence of the enzyme with at least the hydrogen acceptor and the hydrogen donor as a substrate. 2. Process according to claim 1, wherein further a substrate to be crosslinked is present in the reaction mixture and the hydrogen donor serves as a crosslinking agent for this. Process according to claim 2, wherein the substrate to be cross-linked is formed by one or more naturally water-soluble proteins and / or one or more water-solubilized proteins and / or mixtures thereof with one or more polysaccharides. Process according to claim 3, wherein the naturally water soluble proteins are selected from ovalbumin, bovine serum albumin, cytochrome C, casein, collagen, gelatin, soy protein, proteins from vegetables or cereals, in particular gluten. The process of claim 3, wherein the water-solubilized proteins are from potatoes. Process according to claim 3, wherein the polysaccharide is starch. Process according to claim 1, in which the hydrogen donor is converted into a radical by the oxidative enzyme, which subsequently serves as an initiator in the polymerization of monorers, also in the reaction mixture, in particular acrylates. The process of claim 1, wherein the hydrogen donor is an organic dye molecule, which is coupled by the oxidative enzymatic reaction to one or more other organic dye molecules. Process according to claim 8, wherein the organic dye molecules are selected from alizarin, 1. 7 'kK I KJ Ό ί I-alizarin derivatives, antocyanins, flavonoids, in particular myricetin, quercetin, rutin. 10. Process according to any one of claims 1-9, wherein the hydrogen donor is selected from phenols, in particular catechol, phenolic acids, in particular ferulic acid, caffeic acid, coumaric acid, vanillic acid, tyrosine, coniferyl alcohol, sinapyl alcohol, cinnamic acid, p-coumaryl alcohol, sinapic acid, p-hydroxybenzoic acid, gallic acid and flavonoids, phenolic compounds or unsaturated fatty acids, especially linolenic acid. Process according to any one of claims 1-10, wherein the hydrogen acceptor is hydrogen peroxide and / or oxygen. A method for crosslinking one or more proteins or mixture of one or more proteins with one or more polysaccharides, comprising combining the proteins or mixture of proteins with polysaccharides with an oxidative enzyme, a hydrogen acceptor and a hydrogen donor in a reaction mixture and conducting an oxidative reaction in the reaction mixture under the influence of the enzyme, which leads to cross-linking of the proteins and / or the proteins with the polysaccharides. 13. Method according to claim 12, characterized in that the proteins are naturally water-soluble proteins, in particular ovalbumin, bovine serum malbumin, cytochrome C, casein, collagen, gelatin, soy protein, proteins from vegetables or grains especially gluten, or proteins solubilized in water, especially proteins derived from potatoes, the polysaccharide being starch where present, the hydrogen donor being selected from phenols, especially catechol, phenolic acids, in in particular ferulic acid, caffeic acid, coumaric acid, vanillic acid, tyrosine, coniferyl alcohol, sinapyl alcohol, cinnamic acid, p-coumaryl alcohol, sinapic acid, p-hydroxybenzoic acid, gallic acid and flavonoids, phenolic compounds or unsaturated fatty acids, in the, o r-7 1 h Pi io υ is special linolenic acid and where the hydrogen acceptor is hydrogen peroxide and / or oxygen. 14. A method of polymerizing acrylates, comprising bringing acrylic monomers into an reaction mixture with an oxidative enzyme, a hydrogen acceptor and a hydrogen donor and causing an oxidative reaction to proceed in the reaction mixture under the influence of the enzyme , to convert the hydrogen donor to a radical for initiating the polymerization of the monomers, and continuing the polymerization. 15. A method for the production of multimers of dye molecules, comprising combining in one reaction one or more phenolic dyes as hydrogen donor with an oxidative enzyme and a hydrogen acceptor and causing an oxidative to proceed in the reaction mixture under the influence of the enzyme reaction with the hydrogen donor as a substrate for making multimers of two or more dye molecules. 16. Stable foam, obtainable by carrying out the process according to claims 1-4, 10 or 11 or the method according to claim 12 with a protein already having foaming properties, such as ovalbumin or bovine serum albumin. Protein gel, obtainable by carrying out the process according to claims 1-4, 10 or 11 or the method according to claim 12 with a protein having naturally gelling properties, such as ovalbumin 30 or bovine serum albumin. Protein film obtainable by carrying out the process according to claims 1-4, 10 or 11 or the method according to claim 12 with one or more proteins, such as casein, or a mixture of one or more proteins and one or more polysaccharides such as starch. A dye molecule obtainable by carrying out the method according to claim 15. a "·; ar * i Ü Ü / ί J COOPERATION TREATY (PCT) REPORT ON THE NEW INTERNATIONAL TYPE ION IDENTIFICATION OF THE NATIONAL APPLICATION Characteristic of the applicant ol oe gemacnugoe j L / SF68 / MTC / 4 y --.— ...... .- ..... -. -, - ---- 1 NeoenanOse application no. Inoienngsoaïjm 1007158 29 September 1997 invoked priority oaojm Applicant (Name) ATO-DLO Date of reconciliation for a survey of intemal sewing type | By O u tnsanoe for intemonaai Impeach (ISA) to net I supply for an impurity of mtematronaaJ type affiliation nr SN 30063 NL I. CLASSIFICATION OF THE SUBJECT (in case of differences oe classifications. Specify all elassificaoe symbols) According to oe International aassilieaoe (IPC) Int.Cl.6: C 12 P 1/00, C 12 P 7/22, C 12 P 7/26, C 07 K 1/107, C 12 P 19/00, C 09 B 1/00, C 09 B 57/00, C 08 L 33/08, A 23 P 1/16 II. FIELDS OF TECHNIQUE EXAMINED _____ Minimum Documentation Inspected___ Classification System Classification Quotations_ ~ _ Int.Cl.6: C 12 P, C 07 K, C 09 B, C 08 L, A 23 P Unaerzocnte anoere oocumentaoe o oe the minimum Documentation insofar as similar documents in The investigated holes have been included. NO RESEARCH OPPORTUNITY FOR CERTAIN CONCLUSIONS (comments on top-up drawer): IV. ' y 1 LACK OF UNIT OF INVENTION (camerxincen cc supplement incspiafl) -z ”PCT.". S ^ - "2C ·" a - -S * 9 ^ ·