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627-634, 1996 0 1997 Canadian Institute of Food Science and Technology
Published by Elsevier Science Ltd Printed in Great Britain 0963-9969/96$15.00+O.OO
Polymerization of soy protein digests by microbial transglutaminase for improvement of the functional properties
El Fadil E. Babiker, M. A. S. Khan, Naotoshi Matsudomi 81 Akio Kate*
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753, Japan
The protease- and acid-treated soy proteins were polymerized by microbial transglutaminase (TGase) in order to improve their functional properties. Although the protease digests and acid hydrolysates were considerably insoluble, the soy protein digests or hydrolysates polymerized by TGase were soluble, despite being composed of higher molecular weight fractions ((11.8-99.4)x 106) compared to that of the native soy protein (0.48x 106). The surface hydrophobicity of the polymerized proteins was greatly decreased, compared to that of the protease digests and acid hydrolysates, suggesting that the exposed hydrophobic residues of the polymerized peptides were buried inside the polymerized molecules. The emulsifying properties of the polymerized soy proteins were greatly improved compared to those of the untreated, protease- or acid-treated proteins. The foaming properties of the polymerized soy proteins were also improved. The bitterness of the protease digests and acid hydrolysates of soy proteins was diminished by the polymerization with TGase treatment. 0 1997 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd
Keywords: microbial polymerization.
INTRODUCTION The modifications of proteins by enzymatic and chemical reagents have been extensively studied and have been shown to be very powerful tools for improving the functional properties of these macromolecules. The effect of the chemical and enzymatic deamidation of food proteins on the functional properties of proteins has recently been of great interest in the food industry (Matsudomi et al., 1985a,b; Shih, 1991). A number of molecular parameters such as mass, conformation, flexibility, net charge, and hydrophobicity of proteins as well as interactions with other food components have already been shown to play an important part in both their emulsifying and foaming properties (Nakai and Voutsinas, 1983; Kato et al., 1985). Soybean and wheat proteins are usually rich in glutamine and asparagine. *To whom correspondence should be addressed.
These glutamine and asparagine residues can be enzymatically converted into glutamic and aspartic acid, respectively, and the resulting deamidated protein has a lower isoelectric point. Thus, its solubility increases in many mildly acidic food systems (Finley, 1975). It has been reported that deamidation levels as low as 2-6% could enhance the functional properties of proteins (Matsudomi et al., 1985a). Wu et al. (1976) found a significant improvement in the functional properties of gluten by mild acid hydrolysis. Kato et al. (1989) reported that proteolytic deamidation of gluten by chymotrypsin at alkaline pH was effective for the improvement of the functional properties and also (Kato et al., 1991~) reported that pronase digestion is the most promising way to further effectively solubilize gluten. Although functionality of proteins has generally been improved by solubility, contradictory results were reported with respect to emulsifying properties (Aoki et al., 1981; McWatters and Holmes, 1979). Studies on
1987) showed that the foaming properties were increased with increase in the molecular weight of the protein.0) containing 0.0) were reacted with TGase (0. MO).2% dipotassium hydrogen phosphate. chymotrypsin (52 units/mg). A.5 M NaCl.. (St Louis. Therefore. the supernatant was acidified to pH 4. 0.. After incubation. Kato et al. Preparation of proteasedigested APP A freeze-dried sample (4 g) of soy protein was suspended in 400 ml of 0. The mixture was incubated at 37°C for 6 h.05% sodium azide. The supernatant dialyzed against 0.. 1994. 1980. containing 10 mM 2-mercaptoethanol(2-ME) at 20°C.5% glucose.1 units/mg).. in addition to a problem of bitterness due to the presence of peptides enriched with hydrophobic amino acid residues (Arai et al. 1995.1 M phosphate buffer (pH 7.1 M phosphate buffer (pH 7.. Unless otherwise stated. The chymotrypsin and papain treatments were similar to the pronase one except that for the papain treatment.2% dipotassium hydrogen phosphate and 0.05 M Tris-HCl (pH 8. After centrifugation.1 M phosphate buffer (pH 7. MATERIALS AND METHODS Materials Enzymes Proteases (pronase E (4. S.). 100 ml of 0.05% Adekanol and then cultured for 3 days.05% sodium azide and 30 mg pepsin. Kato ovomucin (Kato et al. papain (14 units/mg) and pepsin (2345 units/ mg)) were purchased from the Sigma Chemical Co. Sergo et al. . The latter was used for transglutaminase treatment. 1991b.1 M HCl containing 0. Preparation of microbial transglutaminase Microbial transglutaminase was purified from the culture medium of Streptoverticillium cinnamoneum sub sp.0) for 24 h at 4°C.0). A. 0. The mixture was incubated at 55°C for 60 min. N. 1970). Pepsin digestion was carried out by dispersing 5 g soy protein in 300 ml of 0.05 M phosphate buffer (pH 6. However. The digested mixture was centrifuged (8000x g rpm) to precipitate a small amount of undigested protein. 1985) and ovalbumin (Kato and Takagi. 1989). pronase was inactivated by heating at 100°C for 3 min. 1989. The microorganism was inoculated in 200 ml of 0. M.. 1995) and is known to catalyze the transfer reaction between an amide group in a protein-bound glutamine and an e-amino group in a protein-bound lysine sidechain..1 M phosphate buffer (pH 7. The mixture was incubated at 37°C for 6 h.1% MgS04. This approach may be promising for the utilization of unutilized proteins and improvement in their functional properties. Sakamoto et al. The digested mixture was centrifuged (8000x g rpm) to precipitate a small amount of undigested protein. The enzyme was inactivated by heating at 100°C for 3 min. The treated samples were dialyzed against distilled water and then freeze-dried.0) composed of 2. Transglutaminase treatment The protease digests or acid hydrolysate (10 mg/ml) which dialyzed against 0.1%) (Kato ef al. the supernatant was dialyzed against distilled water or 0. The enzyme was inactivated by N-ethylmaleimide (0. Nio and Motoki. the clear supernatant was dialyzed against distilled water for 24 h at 4°C and then freeze-dried. Sakamoto et al.5) was applied to a column of amberlite CG-50 and then the adsorbed fraction was eluted with 0. 2.1% MgS04 for 48 h at 30°C.0.1 M phosphate buffer (pH 7. cinnamoneum IF012852 (Ando et al. an attempt was made to polymerize soy protein peptides through microbial transglutaminase treatment. the pH was adjusted to 7. resulting in poor functional properties.0) for 24 h at 4°C. and then 40 mg of pronase was added.0% polypeptone.0% lustergen.2% polypeptone.628 E.. polymerization of protein peptides should be considered in order to overcome this problem (Tanimoto et al. 0. 1991). the supernatant was dialyzed against distilled water or 0. resulting in cross-links between the protein molecules. the protease digestion causes a decrease in the molecular weight. The precipitated protein was dissolved in water at 4°C and the pH adjusted to 8.. Babiker. Matsudomi. pH 8.0) was used for transglutaminase treatment. Acid hydrolysis To 5 g of freeze-dried sample of APP. 0. The peak of TGase was collected and dialyzed against deionized water. The treated mixture was centrifuged (8000x g rpm) to precipitate a small amount of unhydrolysed protein. all reagents used in this study were of reagent grade.2 mg/ml). 1991b). Acid-precipitated soy protein preparation Acid-precipitated soy protein (APP) was prepared by the method of Iwabuchi and Yamauchi (1987). the supernatant was dialyzed against distilled water or 0. E..5) containing 0. Khan.1 ml.8 with 2 N HCl and then centrifuged. After centrifugation (8000x g rpm). A sample of defatted meal (100 g) was extracted once with 2 1 0. Therefore. The adsorbed sample was eluted with a gradient of 0 to 1 M NaCl. Transglutaminase as a polymerizer has been extensively studied (Folk. The culture medium (pH 6. The fraction having high activity of TGase was collected and then adsorbed to Blue Sepharose CL-6B (Pharmacia Co. 1983. The culture medium was added to 20 1 of fresh medium (pH 7. Nonaka et al. 0.03 M Tris-HCl buffer. 0.2% yeast extract and 0.05 N HCl was added and then the mixture was incubated at 100°C for 60 min.
Immediately the turbidity was measured at 500 nm. pH 7.05% sodium azide and 0.5 ml/min. at a flow rate of 0. where K is a constant depending on the instrumental and experimental conditions and is determined by using standard protein. Each sample solution (0. The emulsifying activity was determined from the absorbance immediately measured after the emulsion formation.1% cis-parinaric acid solution in ethanol containing butylated hydroxy toluene (BHT) as an antioxidant was added to 2 ml protein solution in 0..05 M NaOH slightly adjusted with 0.05 M acetate buffer.2%) of protease digests and acid hydrolysate with and without TGase treatment were used for the determination of solubility at various pH values (pH 2-3. 0. the gel sheets were stained with 0.05 M carbonate buffer and pH 12. in a glass filter (G-4) at a constant flow rate (90 cm3/min). The emulsion stability was estimated by measuring the halftime of the initial turbidity of the emulsion. NY 11716) for 10 s. 0. containing 0. Gel filtration by high-performance liquid chromatography (HPLC) Gel filtration was done by HPLC in a TSK Gel G-3000SW column (Tosoh Co. 0. Japan.05 M citrate buffer. SDS-polyacrylamide gel electropboresis SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Laemmli (1970) with 15% acrylamide separating gel and 5% acrylamide stacking gel containing 0. 0.1 M phosphate buffer.01 M phosphate buffer. The samples were dissolved in the buffer and then shaken with a vortex mixer (Scientific Industries.002% sodium dodecyl sulfate. using the same buffer as eluent.05 M phosphate buffer. 1983). (outpuths.2%) in 0. pH 7. Electrophoresis was carried out at a current of 10 mA for 1 h. Measurement of foaming properties The foaming properties were determined using the conductivity method (Kato et al. 20 mA for 2 h in electrophoretic Tris-glycine buffer containing 0. The chromatogram was depicted by monitoring the effluent at 280 nm. West Germany) at 12 000 rpm for 1 min at 20°C. 0. Samples (20 ~1. were shaken together and homogenized in an Ultra Turrax (Hansen & Co. containing 0.5 ml/min.Polymerization of soy protein digests 629 Measurement of solubility Freeze-dried samples (0. Elution from columns by using the same buffer was monitored with a low-angle laser light scattering photometer (LS-16) and then with a precision differential refractometer (RI-32). 4-8202. 0. respectively. Determination of surface hydrophohicity The surface hydrophobicity of proteins was determined by the method of Kato and Nakai (1980).. The parinaric acid-protein conjugate was excited at 325 nm and the relative fluorescence intensity at 420 nm was measured in an Aminco-Bowman spectrophotofluorometer (No.8 containing 1% SDS.0. (1) The average molecular weight was determined by eqn 2 (Takagi and Hizukuri. (output)nr. The molecular weight was estimated from the ratio of the output of a lowangle laser light scattering photometer.75x60 cm*). MW = K(output).1 M phosphate buffer. 1984). The foaming power was expressed as the maximum conductivity during aeration.9. thereafter. pH 4-5.1% SDS) was put into the column at a flow rate of 0.75x30 cm). 10 ul of 0.2% in 200 mM Na-phosphate buffer. After electrophoresis. Electric conductivity of foams was measured when air was introduced into 5 ml of a 0. To prepare emulsions.2% protein solution in 0. by eqn 1 (Takagi and Hizukuri.2%) were prepared in a Tris-glycine buffer at pH 8. 1984) where (area)Ls and (area)ni are the total areas in the elution peak of the LS photometer and the refractometer.O ml of corn oil and 3.1% SDS.. 0. Molecular weight determination The molecular weight of the native protein and that of the digests and hydrolysates polymers was determined according to the low-angle laser light scattering method of Kato and Takagi (1987).. 0..1% sodium dodecyl sulfate solution.2% Coomassie brilliant blue-R250 and destained with 10% acetic acid containing 20% methanol for 18 h to remove any traces of the staining solution. pH 6. The absorbance of the diluted emulsion was then determined at 500 nm.. to that of a refractometer. pH 68. The foam stability was indicated as the time for the disappearance of the foams (absence of conductivity).0 ml of protein solution (0. Bohemia. pH 9-l 1. consisting of a TSK gel G4000SW column (Toyo Soda Co./(output). A 50 pl sample of emulsion was taken from the bottom of the container at different times and diluted with 5 ml of 0.. containing 0. MW = K(area)&(area)nt Measurement of emulsifying properties The emulsifying properties of freeze-dried samples were determined by the method of Pearce and Kinsella (1978). pH 7. American Instrument (2) . pH 7.4. Tokyo.1% SDS. The prepared solutions (0.1% sodium dodecyl sulphate) were applied to a high-performance gel chromatography system.2% in 50 mM sodium phosphate buffer. 1.05 M HCl). Native ovalbumin was used as a molecular weight standard. Inc.
1994. Initial screening and selection of panelists were based on participant interest. A. HCl hydrolysates + TGase. 1980. 7. pepsin digests + TGase. pronase digests + TGase.). Each sample was divided into six parts. Water for rinsing between samples was provided. resulting in cross-links between the protein molecules (Folk.48. respectively. papain digests + TGase. TGase-treated samples Effect of TGase treatment on solubility of soy protein digests and hydrolysate The effect of TGase treatment on the solubility of soy protein digests was studied (Table 1). Kato et al. as shown in Fig. SDS-PAGE analysis was carried out. N. 10 and 12) or the boundary between the stacking and separating gels (Lane 4). RESULTS AND DISCUSSION Changes in the molecular mass by enzymatic modification of soy protein To determine whether the molecular mass of the digests and hydrolysate of soy protein was changed by enzymatic treatment or not. 1983. fluorescence intensity/% protein. Kato were composed of high molecular weight bands compared to the digests and hydrolysate. The average molecular weights of untreated soy protein. 1990). 2. Thus. The turbidity of soy protein was 0.20. which were served in random order to the panelists. protease Fig. 6. The results suggesting the formation of highly polymerized peptides as a result of the cross-links between the digested peptides by TGase treatment. USA). taste acuity and ability to understand test procedure (Meilgaard et al. the turbidity of TGase-treated samples was greatly decreased in the range of 0. Khan.9. The samples were tested at 24°C by a six-member panel selected from a pool of students and staff members of our department. On the other hand. 99. .. pepsin and acid hydrolysates polymers were 0. chymotrypsin digests. 5. The elution pattern showed that TGase-treated samples gave peaks with a larger area and with a shorter retention time than that of the protease digests and acid hydrolysate. 3. Sergo et al. HCl hydrolysates. SDS-PAGE patterns of acid-precipitated soy protein (APP) digested by protease or acid treatments followed by transglutaminase (TGase) treatment. S. The results indicated that TGase catalyzed the transfer reaction between an amide group in a protein-bound glutamine and an e-amino group in a protein-bound lysine side-chain. The slope (S. Matsudomi. Arrows indicate the boundary between the stacking (upper) and separating (lower) gels. chymotrypsin digests + TGase. pepsin digests. 11. Maryland. Molecular marker. pronase digests. It should be emphasized that the bands of giant molecules were observed in the top of the stacking gel (Lanes 6. chymotrypsin. 1991). Nio and Motoki..630 E. To further estimate the molecular size of the polymerized samples.4. gel filtration by high-performance liquid chromatography (HPLC) of protease digests and acid hydrolysate before and after TGase treatment was performed (Fig. 2). Scores obtained were indicated as means f standard deviation. 1. A. Sample solutions of protease digests and acid hydrolysate before and after TGase treatment were dialyzed against distilled water and the concentration was adjusted to OS%. 1986). The SDS-PAGE patterns also showed that the polymerized materials had broad molecular weight distribution. 4. Babiker. The average molecular weight of untreated soy protein and that of the digests and hydrolysates polymers was calculated by low-angle laser light scattering method (Kato and Takagi. E. Sakamoto et al. To elucidate the molecular mechanism of this interesting phenomenon.8 and 16. Company.0x lo6 Da. papain. Panelists evaluated the samples for bitterness in a randomized block design. 1. The bitterness score was expressed as the quinine sulfate equivalent (Tanimoto et al. 10.. 1.01 to 0. 98. The score was estimated on a nine-point scale from 1 (10p4%) to 9 (10-30h). Sensory evaluation of bitterness Freeze-dried samples of protease digests and acid hydrolysate with and without TGase treatment were used in this test. protease digestion and acid hydrolysis cause the decrease in solubility of soy protein. 1995). The solution ( 10e4 to 10-30h)was used as a control to which the bitterness of the samples was quantitatively estimated. 1991b. These broad molecular distributions may be attributed to the fact that transglutaminase has a stringent sequence specificity requirement for the acceptor site but can recognize a wide variety of alkylamines as donors (Yan and Wold.56 and that of the digests varied from 0. 1995.. 8..65 to 2. 8. when the digests and hydrolysate were polymerized by TGase. All the panelists had threshold values for quinine sulfate at about 10p4%. On the other hand. n = 6.. was calculated from the fluorescence intensity vs. protein concentration plot. 1987).. M. 11. APP. Nonaka et al.02. The results agree with those obtained for gluten (Matsudomi et al. the hydrophobicities of soy protein. 1984). papain digests. 1989. 12. and then the turbidity was measured at 500 nm. they converted to a transparent solution. 9.
pepsin (b).001) (f 0. 3 a b ___--__J _---- _.Polymerization of soy protein digests 631 digests and acid hydrolysate with and without TGase treatment were calculated from the fluorescence intensity vs. After polymerization with TGase.07 and that of the digests increased in the range of 24.003) (f 0..002) (It 0.70 (zt 0. protein concentration plot (Table 2).-) transglutaminase (TGase) treatment.39) 1.003) (*0.----------_.21) 1... the values were greatly decreased in the range of 1. pronase (c) or 0.04) (+0. while the TGase-treated proteins.80) b Values are means ( f SD).13) 1. Solubility at different pH levels of acid-precipitated soy protein (APP) (.05 N HCl (d).. the shifts of maximum insolubility at pH 6 to lower pH 4 were observed in TGase-treated digests and hydrolysates.36) & TGase + 3.63. protease digests and acid hydrolysate with and without TGase treatment were also measured (Fig. Effect of TGase treatment on the solubility of protease digests and acid hydrolysate of soy protein in distilled water Samples Turbidity (ODs..24 (zk0.98) (f 0. n = 3.__ --_A- 0 2 I 4 I 6 I 8 10 12 I TITTTT 2 4 6 8 10 12 14 Retention time(min) Fig. It is probable that TGase can deamidate the glutamine residues without cross-linkage between the amide group in glutamine and the E-amino group in lysine residues (Motoki et al. .20 2. In addition.07 25. except at pH 4. 2.01 0. pronase digest (b).-).0 to 37. Table 1.18 0.02) TGase + 0.56 1so 2..12) (hO. acid hydrolysate (c).08) ( zt 0. The results showed that the protease. The results indicated that the exposed hydrophobic residues of the protease digests and acid hydrolysate were buried inside the polymerized molecules. (- PH Fig.88) ( f 1.63 (f 1. Therefore.63.03) ( f 0. The pH dependence of the solubilities of soy protein.80 24. it seems likely that the deamidation of the digests and hydrolysates may occur by TGase treatment.87) (f 2.02 0.01 0.00 25. chymotrypsin digest (a). 3).20) (f 3.02 0.02) (i 0. This suggests that the hydrophobic residues were exposed to the surface of the peptide molecules. n = 3.65 0. with (-----) and without (.85 37.60 (*O.-) and without ) TGase treatment.17 to 3. Values are means of duplicate samples. Effect of TGase treatment on surface hydrophobicity (SO) of protease digests and acid hydrolysate of soy protein Samples TGase APP Pronase digest Chymotrypsin digest Papain digest Pepsin digest Acid hydrolysate 17. Therefore. 3).40 ( f 0.-) and APP treated with chymotrypsin (a).__.76) ( f 1. pepsin digest (d) and papain digest (e) with (.01 (zk 0.63 ( f 1. were completely soluble over a wide range of pH (Fig.17 (&0.and acid-treated soy protein were considerably insoluble at wide ranges of acidic pH vahtes.00 25.001) Values are means (i SD). Elution patterns by HPLC of APP (. 1986). Table 2.) TGase APP Pronase digest Chymotrypsin digest Papain digest Pepsin digest Acid hydrolysate 0. The hydrophobicity of the native soy protein was 17.. 3..Ol) 2.
which is estimated by the turbidity of emulsion measured immediately after emulsion formation. the emulsifying properties were improved by TGase treatment for all digests and acid hydrolysate.05 N HCl (d) with () and without (. The bitterness scores of the digests and hydrolysate were found to be in the range of the effect of polymerization on the solubility of digests and hydrolysate may be small and is mainly due to deamidation. the effect of TGase treatment was investigated.05 N HCl (d) with (glutaminase treatment. 1985). A. A. Effect of TGase treatment on surface properties of soy protein digests and hydrolysate The surface properties such as the emulsifying and foaming properties of soy protein peptides are poor (Aoki et al. . The emulsion stability (the half-time of the initial turbidity) of soy protein was 1.. 5.2 a b 0.5 to 9. 19856) caused an increase in the emulsifying properties due to the increase in the negative charges which result from the hydrolysis of the amide 1.0. while that of the digests and hydrolysate increased in the range of 200 to 240 and that of TGase-treated samples further increased in the range of 220 to 300 uu/cm.5 to 6.. Values are means of duplicate samples. pronase ) and without (. The results obtained showed that TGase treatment of soy protein peptides was very effective in the improvement of the emulsifying properties.54 to 0.0 min.-) with chymotrypsin (a).. Fig. pepsin (b). pepsin (b). E. while that of the digests and hydrolysate increased in the range of 5. the emulsifying properties of soy protein did not improve like that of gluten and ovalbumin. reflecting the importance of protein association or polymerization as a structural factor governing the foaming properties (Kato et al. The emulsifying activity of soy protein. .-) with chymotrypsin (a).8 e E' 0.. Khan. Protease treatment of gluten (Matsudomi et al. 1981).) transglutaminase treatment. The improvement in the solubility due to TGase treatments of the peptides digests at various pH values came mainly from the decrease in the surface hydrophobicity of the peptide molecules and the increase in the electrostatic repulsion as a result of partial deamidation of glutamine and asparagine.5 to 10. 4. and that of the TGase-treated samples increased in the range 2. pronase (c) or 0. Babiker.) trans(c) or 0. S.5 to 2. while that of the digests and hydrolysate was in the range of 0.63 to 0. the foaming properties of soy protein peptides were improved by TGase treatment. and that of TGase-treated samples further increased in the range of 6. . The foam stability (the time for the disappearance of the foam) of soy protein was 3. was 0. .8 I 1 1 I I I d % % I 0. The foaming properties of untreated soy protein were low. Effect of TGase treatment on the bitterness of soy protein digests and hydrolysate It is well known that protease digests are bitter due to the exposed hydrophobic peptides (Arai et al. M. 4.4 ( I I I I I I I I I I I I 0 2 4 6 810 0 Time (min) 2 4 6 810 012345 012345 Time (min) Fig. . 1986) and mild acid treatments of ovalbumin (Matsudomi et al. 5). N. Foaming properties of acid precipitated soy protein (APP) (. Matsudomi.632 E.82. Kato groups in glutamine and asparagine.64.74. As shown in Fig. while that of the digests and hydrolysate was in the range of 1. Emulsifying properties of acid-precipitated soy protein (APP) (. In order to improve these surface properties.. The bitterness scores of sample solutions of the digests and hydrolysate with and without TGase treatment are summarized in Table 3.0 min. The foaming properties of the soy protein were also improved by TGase treatment (Fig. Thus. Values are means of duplicate samples. The foaming power (maximum conductivity during aeration) of the untreated soy protein was 180 uu/cm... 1970). and that of the TGase-treated samples was increased in the range of 0.0 min.5 min. However.0 min.
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H. and Fujimaki. Kato. J. Kobayashi. Motoki. S. J. 3501-3504. F. Finley. Kato. Nakai. M.. 31. K.07 ( f 0.Polymerization of soy protein digests 633 Table 3. A. K. Food Chem. Deamidation and functional properties of food proteins by the treatment with immobilized chymotrypsin at alkaline pH.21 (*0. T.. Rev..04 to 1. and Yamauchi. P. 774776. A. Strength of protein gels prepared with microbial transglutaminase as related to reaction conditions..25) ( f 0. Food... 1027-1031. 49. Crosslinking between different food proteins by transglutaminase. H. Agric. Biophys. and Takagi. CRC Press. Tanaka.60 (f 0. Chem. Chem.. The results showed that the bitterness disappeared after polymerization of protease digests and acid hydrolysate. Biol.. and Motoki. T. The protease or acid treatments result in the bitter hydrophobic peptides (Arai et al.. 2619-2623. 2613-2617. Y. Matsudomi. 13-20.8. K. K. (1978). 1.11) Values are means ( f SD). M. H. 1192-1195.. N. 46. Chem. A. Matsudomi. 50(12). N. 1991). N. A.. J. as predicted by the decrease in surface hydrophobicity (Table 2). Shimokawa. (1970). Food Sci. (1983)..21. M. J. Food.13 (kO. E.. Polymerization of deamidated peptide fragments obtained with the mild acid hydrolysis of ovalbumin.31) ( f 0. Ovomucin-food protein conjugates prepared through the transglutaminase reaction. Chem. S. 48. 58-63. Biol. and Kinsella.13 7. 55. (1985).. M.. Effect of lipophilization of soy protein on its emulsion stabilizing properties. Kato. Matsudomi... (1987). Biochim..42) TGase + 1. and Holmes. M . 53. (1983).. Takahashi.13 to 7.36) (kO. J. S. 3025-3030. 1053-1056. and Kobayashi. although soy protein was solubilized by proteases and acid treatments.80 4. Seguro. K. S. 54. Bitterness scores of protease digests and acid bydrolysate of soy protein witb and without TGase treatment Samples Butterness score (quinine sulfate equivalent. Meilgaard. N. Food Sci. Matsudomi. 517-531. 561-566. Chem. K. K.91) (kO. This interesting phenomenon can be accounted for as follows. Chem. Okiyama. Agric. and Motoki. a considerable amount of hydrophobic peptides exist which produce problems. K. Functional properties of deamidated gluten obtained by treating with chymotrypsin at alkaline pH.04 (Zt 0. (1986).. and Motoki. Kurosawa. such as insolubility and bitterness. Folk. A. Functional and structural properties of ovomucin. p. Acta. 59. M. Nonaka. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. After polymerization Kato. Arai. A. N. Sci. 2. N. Nonaka. K. Chem.. U.. with TGase. Umeda. Agric. K. The polymerization of the bitter peptides by TGase seems to cause a decrease in the exposed hydrophobic peptides. Biochem. and Kitagawa. 1970). (1991).. Kato. 49. and Kobayashi. 6. J. V. and Kobayashi. Civille. Kato.02) 1. 624. 716723. (1975). J.. M. That is. J. McWatters. 62-65. 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