Maillard-type protein-polysaccharide conjugates

G. Doxastakisand V. Kiosseoglou(Editors) Novel Macromolecules in Food Systems 9 2000 Elsevier Science B.V. All rights reserved.

385

M a i l l a r d - T y p e Protein-Polysaccharide Conjugates Akio Kato Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan

I. INTRODUCTION It has been proposed that protein-polysaccharide conjugates prepared by a naturally occurring reaction, without the use of any chemicals, are useful as new functional biopolymers having excellent emulsifying, antioxidant and antimicrobial properties for food applications [ 1-3]. It was first reported that a safe ovalbumin-dextran conjugate can be prepared by covalent binding between the e-amino groups on the protein and the reducing-end carbonyl group in the polysaccharide through a controlled Maillard reaction in the dry state without using any chemical reagents [1]. The reaction scheme is shown in Figure 1. As shown here, a limited number of polysaccharide molecules are attached to the protein. It seems likely that the limited number of saccharides attached to protein is due to the steric hindrance of the macromolecular polysaccharide. Only one or two polysaccharide molecules attach to folded proteins such as ovalbumin and lysozyme, while several polysaccharides attach to unfolded proteins such as casein. This limitation is suitable for designing the functional properties of proteins, because the functionality of proteins deteriorates if most of the lysyl residues are masked by the saccharide, as was observed in the conjugates of proteins with monosaccharides and oligosaccharides. In the case of casein, the protein-polysaccharide conjugation is completed within 24 hr. On the other hand, it takes a long time for the protein-polysaccharide conjugates to form in the case of folded or rigid proteins. Interestingly, the emulsifying properties of this conjugate were much better than those of commercial emulsifiers even under acidic pH or at high salt concentrations [4]. In addition, other protein conjugates with polysaccharides also showed a significant improvement in their functionality such as in their emulsifying performance and solubility, and also in their antioxidative and antimicrobial activity. For instance, insoluble wheat gluten was solubilized and its functional properties were improved by pronase treatment followed by conjugation with dextran [5]. Furthermore, Nakamura et al. [2] reported that the lysozyme-dextran conjugate exhibited bifunctional properties, that is, excellent emulsifying properties combined with antimicrobial activity against both Gram-positive and Gram-negative bacteria. Thus, various hybrid proteins with polysaccharides, such as dextran or galactomannan, often exhibited excellent functional properties. On the other hand, the conjugation of proteins with small carbohydrate molecules such as glucose or lactose, under controlled dry-heating, resulted in insoluble aggregates having poor surface properties [1]. Therefore, in order to improve the surface

386

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Figure 1. Scheme for the binding of polysaccharide with protein through Maillard reaction (A) and the binding mode (B)

properties of the proteins, the conjugation with a polysaccharide, not with an oligosaccharide, is desirable for industrial applications. However, it remains to be solved how long the attached polysaccharide chain has to be in order to result in an improvement of the functional properties of the protein and how many polysaccharide molecules should be attached to the protein molecule. This chapter describes the effect of the length and binding number of the carbohydrate chains on the functional properties (emulsifying properties and heat stability) of protein-polysaccharide conjugates, using lysozyme and soy protein as the model proteins. Galactomannan and xyloglucan, derived from guar gum and tamarind seed, respectively, are used as the model polysaccharides, because they are well characterized and, furthermore, are utilized as thickeners, binders and stabilizing agents in food applications. In addition to the improvement of the functional properties, such as emulsifying properties, heat stability, solubility etc., as will be mentioned in the later part of this chapter, the allergen structure of proteins can be masked by the attachment of polysaccharide. This finding suggests that the protein-polysaccharide conjugation is effective in lowering of allergenicity of food proteins.

2. METHODS OF PREPARATION 2.1. Materials

Galactomannan (GM, mannase hydrolysate of guar gum) was supplied by Taiyo Chemicals Co. Xyloglucan (XG, hydrolysate of tamarind gum, average MW of 1.4 kDa) was supplied from

387 Dainihon Pharmaceutical. Guar gum (galactomannan) is composed of a (1-4)-13-D-mannan backbone substituted with a side chain of ct-D-galactose linked (1-6) to mannan residues. In addition, tamarind seed (xyloglucan) contains a relatively similar polysaccharide chain framework composed of a (1-4)-13-D- glucan backbone substituted with a side chains of ct-D-xylose and 13-Dgalactosyl-(1-2)- ot-D-xylose linked (1-6) to glucose residues. Lysozyme was crystallized from fresh egg white at pH 10.0 in the presence of 5 % sodium chloride and recrystallized five times [6]. Soy protein was prepared by the method of Iwabuchi et al.[7]. Sephadex G-50 and CMToyopearl 650M were purchased from Pharmacia and Tosoh Co., respectively. Dialysis membranes with a molecular weight cut-off from 3.5 kDa to 12-14 kDa, were obtained either from Sanko Junyaku Co. (with 12-14 kDa) or from Spectrum Medical Industries (with 6-8 kDa and 3.5 kDa). All other chemicals were of analytical grade.

2.2. Preparation of galactomannan with various molecular sizes by dialysis. The galactomannan solution (5%, w/v) was dialyzed against deionized water at 4 ~ for 48 hr using dialysis tubing with a molecular weight cut-off (MWCO) of 12 - 14 kDa. The inner dialyzate was collected and lyophilized. The molecular weight (24 kD) was determined using a low angle laser light scattering technique combined with HPLC by the method of Takagi and Hizukuri [8]. The outer dialyzate was collected and lyophilized, then dissolved in distilled water, and further fractionated using a dialysis membrane with a MWCO of 6 -8 kDa. The outer and inner dialyzates were separately collected and lyophilized. The former was used as a galactomannan 6-12 kDa and the latter was further fractionated using a dialysis membrane with a MWCO of 3.5 kDa. The inner dialyzate was collected, lyophilized and used as a galactomannan 3.5-6 kDa. 2.3. Preparation of lysozyme conjugates with galactomannans and xyloglucan. The lysozyme and the galactomannans, prepared as described above, were dissolved in distilled water, each in a mole ratio of 1:4, and then lyophilized. Each powder mixture was incubated at 60 ~ for two weeks, in a desiccator containing a saturated KI solution (relative humidity of 65%). The lysozyme-xyloglucan conjugate was prepared in the mole ratio of 1:8 by a 24 hr incubation in a controlled dry state, as described above. The lysozyme-saccharide conjugates were separated from the mixture by gel filtration on a Sephadex G-50 and CM-Toyopearl 650M column, as described below. 2.4. Preparation of soy protein conjugates with galactomannans and xyloglucan. The conjugates of soy protein with saccharides were prepared by the same method, as the lysozyme-saccharide conjugates, except for the application of cation exchange chromatography on CM-Toyopearl 650M. 2.5. Gel filtration of lysozyme conjugates on a Sephadex G-50 column. Lysozyme-polysaccharide conjugates were separated by gel filtration on a Sephadex G-50 column (2 x 82 cm), equilibrated with 20 mM sodium phosphate buffer (pH 7.0), at a flow rate of 20 ml/hr. The protein content in each fraction was detected by measuring the absorbance at 280 nm, and the carbohydrate was determined by measuring the absorbance at 490 nm, after color development with the phenol-sulfuric acid reaction. The void volume fraction containing the lysozyme-polysaccharide conjugates was collected, dialyzed against deionized water at 4 oC for 48 hr and lyophilized.

388

2.6. Separation of conjugates by cation-exchange chromatography on CM-Toyopearl 650M column. For further purification, the lysozyme conjugates with saccharide were loaded on a CMToyopearl 650M column, equilibrated with 20 mM sodium phosphate buffer (pH7.0), at a flow rate of 30 ml/hr. Elutions were conducted stepwise with the same buffer, containing 0.1, 0.2 and 0.3M NaCI. The protein and carbohydrate contents were determined by the same methods described above. Each peak was collected, dialyzed against deionized water at 4 oC for 48 hr and lyophilized. 2.7. Measurement of emulsifying properties. The emulsifying properties were determined according to the modified method of Pearce and Kinsella [9]. To prepare an emulsion, 1.0 ml of corn oil and 3.0 ml of a sample solution (0.1%), in 1/15 M sodium phosphate buffer at pH 7.4, were homogenized with the aid of a Polytron PT 10-35 homogenizer (Kinematica Co. Switzerland) at 12,000 rpm for 1 min. One hundred microliters of the emulsion were obtained from the bottom of a test tube at different times (0, 1, 2, 3, 5 and 10 min) and diluted with 5 ml of a 0.1% sodium dodecyl sulfate solution. The turbidity of the diluted emulsion was then determined at 500 nm. The emulsifying activity was determined from the turbidity measured immediately after the emulsion formation. The emulsion stability was estimated by measuring the half-time of the turbidity measured immediately after the emulsion preparation. 2.8. Measurement of heat stability. The apparent heat stability was estimated by measuring the turbidity developed when a 0.074 % solution in protein of native lysozyrne and lysozyme-saccharide conjugate was heated from 50~ to 95 oC at a rate of I~ min in a 1/15 M sodium phosphate buffer (pH 7.4). Following heating to a given temperature, the sample was immediately put into a cuvette and the turbidity was measured at 500 nm. The temperature interval of the measurements was I~ near melting point (Tm).

3. PROPERTIES AND FUNCTIONALITY

3.1. Effect of the size of polysaccharide chain on the emulsifying properties of proteinpolysaccharide conjugates. The effect of the size of the polysaccharide chain on the emulsifying properties of the lysozyme-galactomannan conjugates is shown in Figure 2 [ 10]. The emulsifying properties of the lysozyme galactomannan conjugates increased in proportion to the length of the polysaccharide chains. The emulsion stability of the lysozyme-GM (3.5-6 kDa) conjugate was very low. This result suggests that the conjugation of protein with galactomannan, having a molecular size of more than 6-12 kDa, is at least essential for the improvement of the emulsifying properties. In order to further elucidate the effect of the oligosaccharide chain on the emulsifying properties of the protein-saccharide conjugate, xyloglucan was used in the experiment. The xyloglucan used was is a well-characterized oligosaccharide, composed of hepta-, octa- and nona-saccharides, at a weight ratio of 10.4, 33.3 and 53.2 %, respectively. The emulsifying properties of the lysozymexyloglucan conjugate were almost the same as those of the lysozyme-GM (3.5-6 kDa) conjugate. Thus, it was confirmed that the lysozyme-oligosaccharide conjugate formation does not result in an improvement of the emulsifying properties. A similar attempt was done using soy protein-

389 saccharide conjugates. As shown in Figure 3, the emulsifying activity and emulsion stability were improved in proportion to the molecular size of attached saccharides. The saccharide of more than Mw 10 kDa is effective in improving the protein emulsifying properties. A

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a 0V

1.6

1.2 A

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0.8 uJ A m

0.4 m

a mm

m Ix: =)

0

2

4

6

8

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TIME (rain) Figure 2. Effects of the molecular size of saccharide chains on the emulsifying properties of lysozyme-polysaccharide conjugates. /x, mixture of lysozyme with galactomannan (GM, 24 kDa); O, lysozyme-GM (24 kDa) conjugate; A, lysozyme-GM (6-12 kDa) conjugate; I , lysozyme-GM (3.5-6 kDa) conjugate; O, lysozyme-xyloglucan (1.4 kDa) conjugate. (Reprinted from ref. 10)

3.2. Heat stability of lysozyme-polysaccharide conjugates. The effect of the length of polysaccharide chains on the heat stability of the lysozymepolysaccharide conjugates is shown in Figure 4. The apparent heat stability of the native and conjugated lysozyme was examined at a pH of 7.4, by heating from 50 to 95 oC. The turbidity of the native lysozyme solution gradually increased with a transition point at 88 oC and reached a maximum of 1.6 (ODs00) at 95 ~ On the other hand, the lysozyme-galactomannan conjugates solution showed no sign of aggregation up to 95 oC, regardless of the molecular size of the attached galactomannan. This result suggests that the lysozyme is converted into a heat stable form by conjugation with a galactomannan molecule of several kDa. In order to elucidate the critical size of the saccharide chain that stabilizes the protein-saccharide conjugate, xyloglucan (1.4 kDa) was used in the experiment. As shown in Figure 4, the lysozyme-xyloglucan conjugate was not as heat-stable as the lysozyme-polysaccharide one, although it was more stable than the native lysozyme. Therefore, a saccharide of several kDa may be necessary for the lysozyme to be stabilized against heating. This suggests that the polysaccharide attached to lysozyme may stabilize the protein molecule according to the manner in which the unfolded protein, during heating, is sterically protected against aggregation by the polysaccharide.

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Figure 3. Relationships between emulsifying properties and molecular weight of saccharides in the conjugates with soy protein. 1, glucose; 2, lactose; 3, xyloglucan (Mwl,400); 4,galactomannan (Mw 3,500-6,000); 5, galactomannan (Mw 10,000); 6, galactomannan (Mw 20,000); 7, dextran (Mw 9,300); 8, dextran (Mw 19,600); 9, dextran (Mw 200,000-300,000); 10, xyloglucan (Mw 470,000).

3.3. Effect of the glycosylation site number of lysozyme-galactomannan conjugates.

In order to further elucidate the molecular mechanism of the improvement of protein emulsifying properties, following the Maillard-type polyglycosylation, conjugates having single and double glycosylation sites were separated by cation-exchange chromatography, on a CMToyopearl column. The conjugates were stepwise eluted with the same buffers containing 0.1, 0.2 and 0.3 M NaC1. Two peaks (peaks I and II) were eluted with 0.1 M NaCI concentration. These peaks are considered to be separated by the difference in the number of the positive charges of the lysozyme-GM conjugates and the size of the attached saccharide, because the Maillard-type protein-polysaccharide conjugates are formed between the free amino groups in the proteins and the reducing end carbonyl groups in the polysaccharides. Therefore, peaks I and II seem to elute in proportion to the number of attached polysaccharide chains through the amino groups in the lysozyme. Table 1 exhibits the number of free amino groups and the molar ratio of carbohydrate to protein in peaks I and II. Taking into account these data and the elution position on a cation-exchange chromatography, peak I should correspond to a two moles of galactomannanattached conjugate, and peak II to one mole of galactomannan-attached conjugate. The emulsifying properties of each peak are shown in Figure 5. The two polysaccharideattached lysozyme (peak I) showed better emulsifying properties than the one polysaccharideattached protein (peak II). We reported that the lysozyme molecule offers two reactive amino groups (N-terminal and 97-1ysines) for the Maillard reaction [11 ]. Therefore, it seems likely that

391

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HEATING TEMPERATURE (~

Figure 4 Heat stability of lysozyme-galactomannan and lysozyme-xyloglucan conjugates. O, native lysozyme; V, lysozyme-xyloglucan (1.4 kDa) conjugate; I--l, lysozyme-GM (24 kDa) conjugate; ~ , lysozyme-GM (6-12 kDa); A, lysozyme-GM (3.5-6 kDa). (Reprinted from ref. 10)

the protein corresponding to peak I is glycosylated both at the N-terminal and at the 97 position and that of peak II is glycosylated at the 97-position. It was reported that the lysine residue at the position 97 was preferentially involved in the formation of the lysozyme-galactomannan conjugate [10]. The two moles of polysaccharide-attached lysozyme showed better emulsifying properties than the one mole of polysaccharide-attached protein (Figure 5.). This suggests that the binding number of polysaccharide chain may affect the emulsifying properties of proteinpolysaccharide conjugate. Table 1 Residual free amino groups and carbohydrate content in peak I and peak II separated by CMToyopearl column chromatography of lysozyme-galactomannan conjugate.

lysozyme peak I peak II

number of free amino groups

carbohydrate content

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Figure 5. Emulsifying properties of peak I and II in lysozyme-galactomannan conjugate separated by a CM-Toyopearl column.O, native lysozymeD , Peak I; ~ , Peak II (Reprinted from ref. 10)

The length of the polysaccharide and the binding number to a protein greatly affect the functional properties of the Maillard-type lysozyme-polysaccharide conjugates. The emulsifying properties of the conjugates were greatly increased in proportion to the length of polysaccharide chains, while the heat stability of the conjugates was enhanced with the lower molecular size (several kDa) of the saccharide. However, lysozyme-oligosaccharide (1.4 kDa) conjugate formation does not result in an improvement of the emulsifying properties. This suggests that the dramatic improvement of lysozyme functional properties is brought about by the attachment of a polysaccharide chain but not an oligosaccharide one. The role of polysaccharide chain in the stabilization of emulsion of protein-polysaccharide conjugates is considered as follows" Proteins adsorb at the oil-water interface during emulsification and form a coherent viscoelastic layer. On the other hand, polysaccharides confer colloidal stability through the thickening of the bound water around the oil droplets. Thus, the oil droplets are stabilized through the bound water layer that prevents the coalescence of oil droplets. Therefore, the protein-polysaccharide conjugate could exhibit improved emulsifying properties. In addition to lysozyme-polysaccharide [2] and casein-polysaccharide conjugate [4], soy protein-polysaccharide conjugate formation [12] also exhibited a dramatic enhancement of the protein emulsifying properties. The heat stability of lysozyme was also dramatically increased by the conjugation with a polysaccharide but not with an oligosaccharide. This suggests that the attachment of the polysaccharide results in the formation of a stable protein structure. It seems likely that the protein denaturation process may be a reversible process in the case of the protein-polysaccharide conjugates, because of the inhibition of the unfolded protein-protein intermoleculer interaction, due to the attached polysaccharide. The attached polysaccharide may, in order words, function like a molecular chaperon that prevents the aggregation of unfolded protein molecules.

393 4. APPLICATIONS AND FUTURE PROSPECTS 4.1. Applications as multifunctional food additives. As previously reported, in addition to their excellent emulsifying and heat stability properties, the lysozyme-galactomanan conjugates were demonstrated to exhibit antimicrobial action against two Gram-positive and Gram-negative bacteria [2]. Thus, the improvement of various functional properties of the Maillard - type protein- polysaccharide conjugates has been reported from a viewpoint of the applications to new-type of food additives, medicines and cosmetics. In order to evaluate their potential in industrial applications, the emulsifying properties of a dried egg white (DEW)-polysaccharide conjugate were compared with those of commercial emulsifiers [13]. As shown in Figure 6, the DEW-polysaccharide conjugate showed much better emulsifying properties than the commercial emulsifiers (SE-11, sucrose-fatty acid ester and Q-18, glycerol-fattyacid ester). In addition, the emulsifying properties of the conjugate were not affected by acidic pH, and/or heating, conditions which are commonly encountered in industrial application. Since DEW contains a number of functional proteins such as lysozyme and ovalbulnin, the DEWpolysaccharide conjugate can be used as a multifunctional food additive. On the other hand, the use of galactomannan is desirable as a food ingredient, because it is not so expensive as dextran and is already utilized as a thickener, binder and stabilizing agent in food. The commercial mannase hydrolysate (galactomannan) of guar gum is contaminated with considerable amounts of small molecular carbohydrates, which result in the deterioration of its emulsifying properties. Therefore, the low-molecular weight contaminants of galactomannan should be removed prior to the preparation of the DEW-polysaccharide conjugate. 1.5 A

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Time (min) Figure 6. Comparison of the emulsifying properties between dried egg white-galactomannan conjugate and commercial emulsifiers. Q , dried egg white- galactomannan conjugate; &, commercial emulsifier (Q-18); El, commercial emulsifier (SE-11); O , untreated dried egg white-galactomannan mixture.

394 4.2. Masking of the allergen structure of proteins. Lysozyme is one of the better known allergens to allergy patients against egg white proteins. Soy protein is also a well known allergenic protein. The 34 kDa protein is a well identified allergenic protein in soy protein[ 12]. Most patients against soy protein are sensitive to the 34 kDa protein fraction. It has been reported that the allergenic protein of soy bean can easily react with a polysaccharide by Maillard reaction. Therefore, it was expected that the allergen structure might be masked by the attachment of a polysaccharide. The masking of the allergen structure of lysozyme and soy protein were investigated by using the solid phase ELISA method and monoclonal antibodies were prepared to measure the binding affinity with lysozyme and soy protein. As shown in Figure 7, the binding affinities of antibody with lysozyme or soy protein were greatly decreased by the polysaccharide attachment, using Maillard reaction in the dry state. This means that the masking of allergen structure of proteins occurs following proteinpolysaccharide conjugate formation. It has been reported that the protease digestion and transglutaminase treatment are partially effective in decreasing the allergenicity of food proteins. Therefore, the comparison of polysaccharide attachment with the protease digestion and transglutaminase treatment was done and it was found that the polysaccharide attachment is the best method to reduce the allergenicity of soy protein, as shown in Figure 7. These results were obtained from the in vitro experiments. The conclusion should be elucidated by the in vivo system. We are now trying to confirm that the polysaccharide conjugation is effective in lowering the allergenicity of these proteins in vivo.

(A)

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Figure 7. Assay of antibody titers by EI~ISA of glycosylated lysozymes (Panel A) and soy proteins (Panel B). Oligomannosyl and polymannosyl lysozymes were constructed by genetic modification using yeast expression system. Galactomannan conjugate indicates the Maillardtype one. Soy protein was treated with protease and transglutaminase (TG).

395

4.3. Attachment of polysaccharide to protein by genetic modification. In yeast cells, the proteins having an Asn-X-Thr/Ser sequence are N-glycosylated in the endoplasmic reticulum and the attached oligosaccharide chain can be elongated with further extention of a large polymannose chain in the Golgi apparatus. Therefore, it was attempted to construct the yeast expression plasmid carrying the mutant cDNA of hen egg white lysozyme, having N-glycosylation signal sequence (Asn-X-Thr/Ser) at the molecular surface. The expression plasmid vector was introduced into S. cerevisiae. As expected, a large amount of polymannosyl lysozyme was predominantly secreted in the yeast medium [14]. We were successful in constructing single polyannosyl lysozyme at the position 19 and 49, and double polymannosyl lysozyme at both 19 and 49 positions [15]. These polymannosyl lysozymes showed remarkable heat stability and excellent emulsifying properties as the Maillard-type lysozyme-polysaccharide conjugates [16]. Furthermore, the molecular mechanism of the functionality of polymannosyl lysozyme was elucidated in detail [ 17]. The oligomannasyl and polymannosyl lysozymes in Figure 7 were constructed by genetic engineering to elucidate the molecular mechanism of the lowering of allergen structure. Most proteins can be polymannosylated in the yeast expression system in a similar manner as lysozyme. The advantage of genetic modification is to enable to design the binding site and the length of saccharide as required. Therefore, this methodology will produce the novel functional products for the food and pharmacutical industry in future.

REFERENCES 1. A. Kato, Y. Sasaki, R. Furuta, and K. Kobayashi, Agric. Biol. Chem., 54 (1990) 107-112. 2. S. Nakamura, A. Kato and K. Kobayashi, J. Agric. Food Chem. 39 (1991) 647-650. 3. S, Nakamura, A, Kato and K. Kobayashi, J. Agric. Food Chem. 40 (1992) 2033-2037. 4. A. Kato, R. Mifuru, N. Matsudomi and K. Kobayashi, Biosci. Biotech. Biochem. 56 (1992) 567-571. 5. A. Kato, K. Shimokawa and K. Kobayashi, J. Agric. Food Chem. 39 (1991) 1053-1056. 6. G. Alderton and H.L. Fevolt, J. Biol. Chem. 164 (1946) 1-5. 7. S. Iwabuchi and F.Yamauchi, J. Agric. Food Chem., 35 (1987) 200-205. 8. T. Takagi and S. Hizukuri, J. Biochem. 95 (1984) 1459-1467. 9. K.M. Pearce and J.E. Kinsella, J. Agric. Food Chem. 26 (1978) 716-723. 10. Yu-Wei Shu, S. Nakamura and A. Kato, J. Agric. Food Chem., 44 (1996) 2544-2548. 11. S. Nakamura, K. Kobayashi, A. Kato, J. Agric. Food Chem. 42 (1994) 2688-22691. 12. E. E. Babiker, H. Azakami, N. Matsudomi, H.Iwata, T. Ogawa, N. Bando and A.Kato, J. Agric. Food Chem., 46 (1998) 866-871. 13. A. Kato, K. Minaki and K.Kobayashi, J. Agric. Food Chem. 41 (1993) 540-543. 14. S. Nakamura, H. Takasaki, K. Kobayashi and A. Kato, J. Biol. Chem., 268 (1993) 1270612712. 15. Yu-Wei Shu, S. Maki, S. Nakamura and A. Kato, J. Agric. Food Chem., 44 (1996) 25442548. 16. S. Nakamura, K. Kobayashi and A. Kato, FEBS Letters, 328 (1994) 259-262. 17. A.Kato, S.Nakamura, H.Takasaki and S. Maki: In "Macromolecular Interactions in Food Technology" ed. by N.Parris, A.Kato, L.Creamer and J.Pearce, ACS Symposium Series 650; American Chemical Society, pp.243-256 (1996)