polymerization of soy protein digests by microbial transglutaminase for improvement of the...

7/27/2019 Polymerization of Soy Protein Digests by Microbial Transglutaminase for Improvement of the Functional Properties

1/8

ELSEVIER

Food Research nternat ion al,Vol. 29, No. 7, pp. 627-634, 19960 1997Canadian Institute of Food Science and TechnologyPublished by Elsevier Science LtdPrinted in Great Britain

PII: SO963-9969(96)00069-S 0963-9969/96$15.00+O.OO

Polymerization of soy protein digests by microbialtransglutaminase for improvement of thefunctional propertiesEl Fadil E. Babiker, M. A. S. Khan, Naotoshi Matsudomi 81 Akio Kate*

Department of Bi ological Chemistry, Yamaguchi Uni versit y, Yamaguchi 753, Japan

The protease- and acid-treated soy proteins were polymerized by microbialtransglutaminase (TGase) in order to improve their functional properties.Although the protease digests and acid hydrolysates were considerably insoluble, thesoy protein digests or hydrolysates polymerized by TGase were soluble, despitebeing 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 hydro-phobicity of the polymerized proteins was greatly decreased, compared to that ofthe protease digests and acid hydrolysates, suggesting that the exposed hydro-phobic residues of the polymerized peptides were buried inside the polymerizedmolecules. The emulsifying properties of the polymerized soy proteins weregreatly improved compared to those of the untreated, protease- or acid-treatedproteins. The foaming properties of the polymerized soy proteins were alsoimproved. The bitterness of the protease digests and acid hydrolysates of soyproteins was diminished by the polymerization with TGase treatment. 0 1997Canadian Institute of Food Science and Technology. Published by ElsevierScience LtdKeywords: microbial transglutaminase, soy protein, protease, hydrolysis,polymerization.

INTRODUCTIONThe modifications of proteins by enzymatic and chemi-cal reagents have been extensively studied and havebeen shown to be very powerful tools for improving thefunctional properties of these macromolecules. Theeffect of the chemical and enzymatic deamidation offood proteins on the functional properties of proteinshas recently been of great interest in the food industry(Matsudomi et al., 1985a,b; Shih, 1991). A number ofmolecular parameters such as mass, conformation,flexibility, net charge, and hydrophobicity of proteins aswell as interactions with other food components havealready been shown to play an important part in boththeir emulsifying and foaming properties (Nakai andVoutsinas, 1983; Kato et al., 1985). Soybean and wheatproteins are usually rich in glutamine and asparagine.*To whom correspondence should be addressed.

These glutamine and asparagine residues can beenzymatically converted into glutamic and aspartic acid,respectively, and the resulting deamidated protein has alower isoelectric point. Thus, its solubility increases inmany mildly acidic food systems (Finley, 1975). It hasbeen 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 asignificant improvement in the functional properties ofgluten by mild acid hydrolysis. Kato et al . (1989)reported that proteolytic deamidation of gluten bychymotrypsin at alkaline pH was effective for theimprovement of the functional properties and also(Kato et al., 1991~) reported that pronase digestion isthe most promising way to further effectively solubilizegluten. Although functionality of proteins has generallybeen improved by solubility, contradictory results werereported with respect to emulsifying properties (Aoki etal., 1981; McWatters and Holmes, 1979). Studies on

627


2/8

628 E. E. Babiker, M . A. S. Khan, N. Matsudomi, A. Katoovomucin (Kato et a l ., 1985) and ovalbumin (Kato andTakagi, 1987) showed that the foaming properties wereincreased with increase in the molecular weight of theprotein. However, the protease digestion causes adecrease in the molecular weight, resulting in poorfunctional properties, in addition to a problem ofbitterness due to the presence of peptides enriched withhydrophobic amino acid residues (Arai et al., 1970).Therefore, polymerization of protein peptides should beconsidered in order to overcome this problem (Tani-moto et al., 1991). Transglutaminase as a polymerizerhas been extensively studied (Folk, 1980; Nio andMotoki, 1983; Nonaka et al ., 1989; Kato et al ., 1991b;Sakamoto et al., 1994; Sakamoto et al., 1995; Sergo etal., 1995) and is known to catalyze the transfer reactionbetween an amide group in a protein-bound glutamineand an e-amino group in a protein-bound lysine side-chain, resulting in cross-links between the proteinmolecules. Therefore, an attempt was made topolymerize soy protein peptides through microbialtransglutaminase treatment. This approach may bepromising for the utilization of unutilized proteins andimprovement in their functional properties.

MATERIALS AND METHODSMaterialsEnzymesProteases (pronase E (4.1 units/mg), chymotrypsin (52units/mg), papain (14 units/mg) and pepsin (2345 units/mg)) were purchased from the Sigma Chemical Co. (StLouis, MO). Unless otherwise stated, all reagents usedin this study were of reagent grade.Preparation of microbial transglutaminaseMicrobial transglutaminase was purified from theculture medium of Streptoverticil l ium cinnamoneum subsp. cinnamoneum IF012852 (Ando et al., 1989). Themicroorganism was inoculated in 200 ml of 0.2% poly-peptone, 0.5% glucose, 0.2% dipotassium hydrogenphosphate and 0.1% MgS04 for 48 h at 30C. Theculture medium was added to 20 1 of fresh medium (pH7.0) composed of 2.0% polypeptone, 2.0% lustergen,0.2% dipotassium hydrogen phosphate, 0.1% MgS04,0.2% yeast extract and 0.05% Adekanol and thencultured for 3 days. The culture medium (pH 6.5) wasapplied to a column of amberlite CG-50 and then theadsorbed fraction was eluted with 0.05 M phosphatebuffer (pH 6.5) containing 0.5 M NaCl. The fractionhaving high activity of TGase was collected and thenadsorbed to Blue Sepharose CL-6B (Pharmacia Co.).The adsorbed sample was eluted with a gradient of 0to 1 M NaCl. The peak of TGase was collected anddialyzed against deionized water.

Acid-precipitated soy protein preparationAcid-precipitated soy protein (APP) was prepared bythe method of Iwabuchi and Yamauchi (1987). A sam-ple of defatted meal (100 g) was extracted once with2 1 0.03 M Tris-HCl buffer, pH 8, containing 10 mM2-mercaptoethanol(2-ME) at 20C. After centrifugation,the supernatant was acidified to pH 4.8 with 2 N HCland then centrifuged. The precipitated protein wasdissolved in water at 4C and the pH adjusted to 8.After centrifugation (8000x g rpm), the clear super-natant was dialyzed against distilled water for 24 h at4C and then freeze-dried.Preparation of proteasedigested APPA freeze-dried sample (4 g) of soy protein was suspen-ded in 400 ml of 0.05 M Tris-HCl (pH 8.0) containing0.05% sodium azide, and then 40 mg of pronase wasadded. The mixture was incubated at 37C for 6 h.After incubation, pronase was inactivated by heating at100C for 3 min. The digested mixture was centrifuged(8000x g rpm) to precipitate a small amount of undi-gested protein, the supernatant was dialyzed againstdistilled water or 0.1 M phosphate buffer (pH 7.0) for24 h at 4C. The chymotrypsin and papain treatmentswere similar to the pronase one except that for thepapain treatment, the pH was adjusted to 7.0. Pepsindigestion was carried out by dispersing 5 g soy proteinin 300 ml of 0.1 M HCl containing 0.05% sodium azideand 30 mg pepsin. The mixture was incubated at 37Cfor 6 h. The enzyme was inactivated by heating at100C for 3 min. The digested mixture was centrifuged(8000x g rpm) to precipitate a small amount of undi-gested protein, the supernatant was dialyzed againstdistilled water or 0.1 M phosphate buffer (pH 7.0) for 24 hat 4C. The supernatant dialyzed against 0.1 M phosphatebuffer (pH 7.0) was used for transglutaminase treatment.Acid hydrolysisTo 5 g of freeze-dried sample of APP, 100 ml of 0.05 NHCl was added and then the mixture was incubated at100C for 60 min. The treated mixture was centrifuged(8000x g rpm) to precipitate a small amount of unhydro-lysed protein, the supernatant was dialyzed againstdistilled water or 0.1 M phosphate buffer (pH 7.0). Thelatter was used for transglutaminase treatment.Transglutaminase treatmentThe protease digests or acid hydrolysate (10 mg/ml)which dialyzed against 0.1 M phosphate buffer (pH 7.0)were reacted with TGase (0.2 mg/ml). The mixture wasincubated at 55C for 60 min. The enzyme was inac-tivated by N-ethylmaleimide (0.1 ml; 0.1%) (Kato efal., 1991b). The treated samples were dialyzed againstdistilled water and then freeze-dried.


3/8

Polymeri zati on of soy protein digests 629

Measurement of solubilityFreeze-dried samples (0.2%) of protease digests andacid hydrolysate with and without TGase treatmentwere used for the determination of solubility at variouspH values (pH 2-3, 0.05 M citrate buffer; pH 4-5,0.05 M acetate buffer; pH 68, 0.05 M phosphatebuffer; pH 9-l 1, 0.05 M carbonate buffer and pH 12,0.05 M NaOH slightly adjusted with 0.05 M HCl). Thesamples were dissolved in the buffer and then shakenwith a vortex mixer (Scientific Industries, Inc., Bohemia,NY 11716) for 10 s. Immediately the turbidity wasmeasured at 500 nm.

that of a refractometer, (output)nr, by eqn 1 (Takagiand Hizukuri, 1984), where K is a constant dependingon the instrumental and experimental conditions and isdetermined by using standard protein. Native ovalbu-min was used as a molecular weight standard.

MW = K(output),,/(output),, (1)The average molecular weight was determined by eqn 2(Takagi and Hizukuri, 1984) where (area)Ls and(area)ni are the total areas in the elution peak of the LSphotometer and the refractometer, respectively.

MW = K(area)&(area)nt

SDS-polyacrylamide gel electropboresis Measurement of emulsifying propertiesSDS-polyacrylamide gel electrophoresis (SDS-PAGE)was performed using the method of Laemmli (1970)with 15% acrylamide separating gel and 5% acrylamidestacking gel containing 0.1% SDS. Samples (20 ~1,0.2%) were prepared in a Tris-glycine buffer at pH 8.8containing 1% SDS. Electrophoresis was carried out ata current of 10 mA for 1 h, thereafter, 20 mA for 2 h inelectrophoretic Tris-glycine buffer containing 0.1%SDS. After electrophoresis, the gel sheets were stainedwith 0.2% Coomassie brilliant blue-R250 and destainedwith 10% acetic acid containing 20% methanol for 18 hto remove any traces of the staining solution.Gel filtration by high-performance liquidchromatography (HPLC)

The emulsifying properties of freeze-dried samples weredetermined by the method of Pearce and Kinsella(1978). To prepare emulsions, 1 O ml of corn oil and3.0 ml of protein solution (0.2%) in 0.1 M phosphatebuffer, pH 7.0, were shaken together and homogenizedin an Ultra Turrax (Hansen & Co., West Germany) at12 000 rpm for 1 min at 20C. A 50 pl sample of emul-sion was taken from the bottom of the container at dif-ferent times and diluted with 5 ml of 0.1% sodiumdodecyl sulfate solution. The absorbance of the dilutedemulsion was then determined at 500 nm. The emulsify-ing activity was determined from the absorbance imme-diately measured after the emulsion formation. Theemulsion stability was estimated by measuring the half-time of the initial turbidity of the emulsion.

Gel filtration was done by HPLC in a TSK GelG-3000SW column (Tosoh Co., Tokyo, Japan,0.75x30 cm). Each sample solution (0.2% in 200 mMNa-phosphate buffer, pH 6.9, containing 0.1% SDS)was put into the column at a flow rate of 0.5 ml/min,using the same buffer as eluent. The chromatogram wasdepicted by monitoring the effluent at 280 nm.

Measurement of foaming properties

Molecular weight determinationThe molecular weight of the native protein and that ofthe digests and hydrolysates polymers was determinedaccording to the low-angle laser light scattering methodof Kato and Takagi (1987). The prepared solutions(0.2% in 50 mM sodium phosphate buffer, pH 7, con-taining 0.05% sodium azide and 0.1% sodium dodecylsulphate) were applied to a high-performance gel chro-matography system, consisting of a TSK gel G4000SWcolumn (Toyo Soda Co.; 0.75x60 cm*), at a flow rate of0.5 ml/min. Elution from columns by using the samebuffer was monitored with a low-angle laser light scat-tering photometer (LS-16) and then with a precisiondifferential refractometer (RI-32). The molecular weightwas estimated from the ratio of the output of a low-angle laser light scattering photometer, (outpuths, to

The foaming properties were determined using theconductivity method (Kato et al., 1983). Electricconductivity of foams was measured when air wasintroduced into 5 ml of a 0.2% protein solution in 0.1 Mphosphate buffer, pH 7, in a glass filter (G-4) ata constant flow rate (90 cm3/min). The foaming powerwas expressed as the maximum conductivity duringaeration. The foam stability was indicated as the timefor the disappearance of the foams (absence ofconductivity).Determination of surface hydrophohicityThe surface hydrophobicity of proteins was determinedby the method of Kato and Nakai (1980). 10 ul of 0.1%cis-parinaric acid solution in ethanol containingbutylated hydroxy toluene (BHT) as an antioxidant wasadded to 2 ml protein solution in 0.01 M phosphatebuffer, pH 7.4, containing 0.002% sodium dodecylsulfate. The parinaric acid-protein conjugate was excitedat 325 nm and the relative fluorescence intensity at420 nm was measured in an Aminco-Bowman spectro-photofluorometer (No. 4-8202, American Instrument

(2)


4/8

630 E. E. Babiker, M. A. S. Khan, N. Matsudomi. A. KatoCompany, Maryland, USA). The slope (S,), fluores-cence intensity/% protein, was calculated from thefluorescence intensity vs. protein concentration plot.Sensory evaluation of bitternessFreeze-dried samples of protease digests and acidhydrolysate with and without TGase treatment wereused in this test. Each sample was divided into six parts,which were served in random order to the panelists. Thesamples were tested at 24C by a six-member panelselected from a pool of students and staff members ofour department. Initial screening and selection ofpanelists were based on participant interest, taste acuityand ability to understand test procedure (Meilgaard etal., 1990). The bitterness score was expressed as thequinine sulfate equivalent (Tanimoto et al., 1991). Allthe panelists had threshold values for quinine sulfate atabout 10p4%. The solution ( 10e4 to 10-30h)was used asa control to which the bitterness of the samples wasquantitatively estimated. Water for rinsing betweensamples was provided. Panelists evaluated the samplesfor bitterness in a randomized block design. The scorewas estimated on a nine-point scale from 1 (10p4%) to 9(10-30h). Scores obtained were indicated as meansf standard deviation, n = 6.

RESULTS AND DISCUSSIONChanges in the molecular mass by enzymaticmodification of soy proteinTo determine whether the molecular mass of the digestsand hydrolysate of soy protein was changed byenzymatic treatment or not, SDS-PAGE analysis wascarried out, as shown in Fig. 1. TGase-treated samples

Fig. 1. SDS-PAGE patterns of acid-precipitated soy protein(APP) digested by protease or acid treatments followed bytransglutaminase (TGase) treatment. 1, Molecular marker; 2,APP; 3, pronase digests, 4, pronase digests + TGase; 5, chymo-trypsin digests; 6, chymotrypsin digests + TGase; 7, papaindigests; 8, papain digests + TGase; 9, pepsin digests; 10, pepsindigests + TGase; 11, HCl hydrolysates; 12, HCl hydrolysates+ TGase. Arrows indicate the boundary between the stacking(upper) and separating (lower) gels.

were composed of high molecular weight bands com-pared to the digests and hydrolysate. It should beemphasized that the bands of giant molecules wereobserved in the top of the stacking gel (Lanes 6, 8, 10and 12) or the boundary between the stacking andseparating gels (Lane 4). The SDS-PAGE patterns alsoshowed that the polymerized materials had broadmolecular weight distribution. These broad moleculardistributions may be attributed to the fact that trans-glutaminase has a stringent sequence specificity require-ment for the acceptor site but can recognize a widevariety of alkylamines as donors (Yan and Wold, 1984).The results indicated that TGase catalyzed the transferreaction between an amide group in a protein-boundglutamine and an e-amino group in a protein-boundlysine side-chain, resulting in cross-links between theprotein molecules (Folk, 1980; Nio and Motoki, 1983;Nonaka et al., 1989; Kato et al., 1991b; Sakamoto et al.,1994, 1995; Sergo et al., 1995). To further estimate themolecular size of the polymerized samples, gel filtrationby high-performance liquid chromatography (HPLC) ofprotease digests and acid hydrolysate before and afterTGase treatment was performed (Fig. 2). The elutionpattern showed that TGase-treated samples gave peakswith a larger area and with a shorter retention time thanthat of the protease digests and acid hydrolysate. Theaverage molecular weight of untreated soy proteinand that of the digests and hydrolysates polymers wascalculated by low-angle laser light scattering method(Kato and Takagi, 1987). The average molecularweights of untreated soy protein, chymotrypsin, papain,pepsin and acid hydrolysates polymers were 0.48, 99.4,98.9, 11.8 and 16.0x lo6 Da, respectively. The resultssuggesting the formation of highly polymerized peptidesas a result of the cross-links between the digestedpeptides by TGase treatment.Effect of TGase treatment on solubility of soy proteindigests and hydrolysateThe effect of TGase treatment on the solubility of soyprotein digests was studied (Table 1). Sample solutionsof protease digests and acid hydrolysate before andafter TGase treatment were dialyzed against distilledwater and the concentration was adjusted to OS%, andthen the turbidity was measured at 500 nm. The turbidityof soy protein was 0.56 and that of the digests variedfrom 0.65 to 2.20. On the other hand, the turbidity ofTGase-treated samples was greatly decreased in therange of 0.01 to 0.02. Thus, protease digestion and acidhydrolysis cause the decrease in solubility of soyprotein. The results agree with those obtained for gluten(Matsudomi et al., 1986). On the other hand, when thedigests and hydrolysate were polymerized by TGase,they converted to a transparent solution. To elucidatethe molecular mechanism of this interesting phe-nomenon, the hydrophobicities of soy protein, protease


5/8

Polymeri zati on of soy protein digests 631

digests and acid hydrolysate with and without TGasetreatment were calculated from the fluorescence inten-sity vs. protein concentration plot (Table 2). Thehydrophobicity of the native soy protein was 17.07 andthat of the digests increased in the range of 24.0 to37.63. This suggests that the hydrophobic residues wereexposed to the surface of the peptide molecules. Afterpolymerization with TGase, the values were greatlydecreased in the range of 1.17 to 3.63. The resultsindicated that the exposed hydrophobic residues of theprotease digests and acid hydrolysate were buried insidethe polymerized molecules. The pH dependence of thesolubilities of soy protein, protease digests and acidhydrolysate with and without TGase treatment werealso measured (Fig. 3). The results showed that theprotease- and acid-treated soy protein were consid-erably insoluble at wide ranges of acidic pH vahtes,while the TGase-treated proteins, except at pH 4, werecompletely soluble over a wide range of pH (Fig. 3). Inaddition, the shifts of maximum insolubility at pH 6 tolower pH 4 were observed in TGase-treated digests and

b

___--__J _---- -_A_.----------_.__.__

Retention time(min)Fig. 2. Elution patterns by HPLC of APP (- - -), chymo-trypsin digest (a), pronase digest (b), acid hydrolysate (c),pepsin digest (d) and papain digest (e) with (- - -) and without

(- ) TGase treatment.

hydrolysates. Therefore, it seems likely that the deami-dation of the digests and hydrolysates may occur byTGase treatment. It is probable that TGase can deami-date the glutamine residues without cross-linkagebetween the amide group in glutamine and the E-aminogroup in lysine residues (Motoki et al., 1986). Therefore,Table 1. Effect of TGase treatment on the solubility of protease

digests and acid hydrolysate of soy protein in distilled waterSamples Turbidity (ODs,,,,)

TGase - TGase +APP 0.56 (zt 0.02)Pronase digest 1so (i 0.04) 0.02 (zk 0.003)Chymotrypsin digest 2.20 (+0.12) 0.01 (f 0.001)Papain digest 2.18 (hO.08) 0.01 (f 0.002)Pepsin digest 0.65 ( zt 0.03) 0.02 (It 0.003)Acid hydrolysate 0.70 ( f 0.02) 0.01 (*0.001)Values are means (i SD), n = 3.Table 2. Effect of TGase treatment on surface hydrophobicity(SO) of protease digests and acid hydrolysate of soy proteinSamples &

TGase - TGase +APP 17.07 ( f 1.20)Pronase digest 25.80 (f 3.87) 3.63 (f 1.39)Chymotrypsin digest 24.00 (f 2.88) 1.17 (&0.21)Papain digest 25.00 ( f 1.98) 1.24 (zk0.13)Pepsin digest 25.85 (f 0.76) 1.60 (*O.Ol)Acid hydrolysate 37.63 ( f 1.36) 2.40 ( f 0.80)Values are means ( f SD), n = 3.

3 a b

I I I I TITTTT0 2 4 6 8 10 12 2 4 6 8 10 12PH

14

Fig. 3. Solubility at different pH levels of acid-precipitated soyprotein (APP) (- - -) and APP treated with chymotrypsin(a), pepsin (b), pronase (c) or 0.05 N HCl (d), with (-----)and without (- - -) transglutaminase (TGase) treatment.Values are means of duplicate samples.


6/8

632 E. E. Babiker, M. A. S. Khan, N. Matsudomi, A. Katothe effect of polymerization on the solubility of digestsand hydrolysate may be small and is mainly due todeamidation. The improvement in the solubility due toTGase treatments of the peptides digests at various pHvalues came mainly from the decrease in the surfacehydrophobicity of the peptide molecules and theincrease in the electrostatic repulsion as a result ofpartial deamidation of glutamine and asparagine.Effect of TGase treatment on surface properties of soyprotein digests and hydrolysateThe surface properties such as the emulsifying andfoaming properties of soy protein peptides are poor(Aoki et al., 1981). In order to improve these surfaceproperties, the effect of TGase treatment was investi-gated. As shown in Fig. 4, the emulsifying propertieswere improved by TGase treatment for all digestsand acid hydrolysate. The emulsifying activity of soyprotein, which is estimated by the turbidity of emulsionmeasured immediately after emulsion formation, was0.64, while that of the digests and hydrolysate was in therange of 0.54 to 0.74, and that of the TGase-treatedsamples was increased in the range of 0.63 to 0.82. Theemulsion stability (the half-time of the initial turbidity)of soy protein was 1.5 min, while that of the digests andhydrolysate was in the range of 1.5 to 2.0 min, and thatof the TGase-treated samples increased in the range 2.5to 9.0 min. Protease treatment of gluten (Matsudomi etal., 1986) and mild acid treatments of ovalbumin (Mat-sudomi et al., 19856) caused an increase in the emulsi-fying properties due to the increase in the negativecharges which result from the hydrolysis of the amide

1.2

0.8

%%I 0.8eE'

0.4

(

a

I I I I I I0 2 4 6 810

b

I 1 1 I I Id

I I I I I I0 2 4 6 810Time (min)

Fig. 4. Emulsifying properties of acid-precipitated soy protein(APP) (- - -) with chymotrypsin (a), pepsin (b), pronase(c) or 0.05 N HCl (d) with (- ) and without (. . .) trans-glutaminase treatment. Values are means of duplicate samples.

groups in glutamine and asparagine. However, theemulsifying properties of soy protein did not improvelike that of gluten and ovalbumin. The results obtainedshowed that TGase treatment of soy protein peptideswas very effective in the improvement of the emulsifyingproperties. The foaming properties of the soy proteinwere also improved by TGase treatment (Fig. 5). Thefoaming properties of untreated soy protein were low.The foaming power (maximum conductivity duringaeration) of the untreated soy protein was 180 uu/cm,while that of the digests and hydrolysate increased in therange of 200 to 240 and that of TGase-treated samplesfurther increased in the range of 220 to 300 uu/cm. Thefoam stability (the time for the disappearance of thefoam) of soy protein was 3.0 min, while that of thedigests and hydrolysate increased in the range of 5.5 to6.0, and that of TGase-treated samples further increasedin the range of 6.5 to 10.0 min. Thus, the foamingproperties of soy protein peptides were improved byTGase treatment, reflecting the importance of proteinassociation or polymerization as a structural factorgoverning the foaming properties (Kato et al., 1985).Effect of TGase treatment on the bitterness of soyprotein digests and hydrolysateIt is well known that protease digests are bitter due tothe exposed hydrophobic peptides (Arai et al., 1970).The bitterness scores of sample solutions of the digestsand hydrolysate with and without TGase treatment aresummarized in Table 3. The bitterness scores of thedigests and hydrolysate were found to be in the range of

012345 012345Time (min)

Fig. 5. Foaming properties of acid precipitated soy protein(APP) (- - -) with chymotrypsin (a), pepsin (b), pronase (c)or 0.05 N HCl (d) with (- ) and without (. . .) transglu-taminase treatment. Values are means of duplicate samples.


7/8

Poly meri zati on of soy protein digests 633

Table 3. Bitterness scores of protease digests and acid bydro-lysate of soy protein witb and without TGase treatment

Samples Butterness score(quinine sulfate equivalent, x 10e3%)TGase -

Chymotrypsin digest 6.50 (f 0.25)Papain digest 6.40 ( f 0.36)Pepsin digest 7.80 (kO.91)Pronase digest 4.13 (kO.31)Acid hydrolysate 7.60 ( f 0.42)Values are means ( f SD), n = 6.

TGase +1.04 (Zt 0.02)1.12 (*0.13)1.21 (*0.07)1.07 ( f 0.05)1.13 (kO.11)

4.13 to 7.8. After polymerization with TGase, the scoreswere greatly decreased in the range of 1.04 to 1.21. Theresults showed that the bitterness disappeared afterpolymerization of protease digests and acid hydrolysate.This interesting phenomenon can be accounted for asfollows. The protease or acid treatments result in thebitter hydrophobic peptides (Arai et al., 1970). Thepolymerization of the bitter peptides by TGase seems tocause a decrease in the exposed hydrophobic peptides,as predicted by the decrease in surface hydrophobicity(Table 2). That is, TGase treatment causes debitteringby shielding of the bitter peptides in the interior of themolecules during polymerization (Tanimoto et al., 1991).In conclusion, although soy protein was solubilizedby proteases and acid treatments, a considerableamount of hydrophobic peptides exist which produceproblems, such as insolubility and bitterness, forindustrial applications. Therefore, polymerization ofsoy protein digests and hydrolysate should be carriedout to avoid such problems. In addition to the bitternessreduction, we found that the polymerization withTGase also improved the solubility and surfacefunctional properties of soy protein peptides.

REFERENCESAndo, H., Adashi, M., Umeda, K., Matsuura, A., Nonaka,M., Uchio, R., Tanaka, H. and Motoki, M. (1989).Purification and characteristics of a novel transglutaminasederived from microorganisms. Agric. Biol. Chem. 53,

2613-2617.Arai, S., Noguchi, M., Kurosawa, S., Kato, H. and Fujimaki,M. (1970). Applying proteolytic enzymes on soybean. 6.Deodorization effect of aspergillopeptidase A and debit-tering effect of aspergillus acid carboxypeptidase. J. FoodSci. 35, 392-395.Aoki, H., Taneyama, O., Orimo, N. and Kitagawa, I. (1981).Effect of lipophilization of soy protein on its emulsionstabilizing properties. J. Food. Sci . 46, 1192-1195.Finley, J. W. (1975). Deamidated gluten: a potential fortifierfor fruit juices. J. Food Sci. 40, 1283-1285.Folk, J. E. (1980). Transglutaminase. Ann. Rev. Biochem. 49,517-531.Iwabuchi, S. and Yamauchi, F. (1987). Determination ofglycinin and /3-conglycinin in soy proteins by immunologicalmethods. J. Agr i c. Food Chem. 35,20& 205.

Kato, A., Lee, Y. and Kobayashi, K. (1989). Deamidationand functional properties of food proteins by the treatmentwith immobilized chymotrypsin at alkaline pH. Immo-bilized chymotrypsin at alkaline pH. J. Food Sci. 54,1345-l 347.Kato, A. and Nakai, S. (1980). Hydrophobicity determined bya fluorescence probe method and its correlation with surfaceproperties of proteins. Biochim. Biophys. Acta. 624, 13-20.Kato, A., Oda, S., Yamanaka, Y., Matsudomi, N. andKobayashi, K. (1985). Functional and structural propertiesof ovomucin. Agric. Biol. Chem. 49, 3501-3504.Kato, A. and Takagi, T. (1987). Estimation of the molecularweight distribution of heat-induced ovalbumin aggregatesby the low-angle laser light scattering technique combinedwith high-performance gel chromatography. J. Agri c. Food.Chem. 35, 633637.Kato, A., Shimokawa, K. and Kobayashi, K. (1991).Improvement of the functional properties of insolublegluten by pronase digestion followed by dextran conjuga-tion. J. Agric. Food Chem. 39, 1053-1056.Kato, A., Wada, T., Kobayashi, K., Seguro, K. and Motoki,M . (1991). Ovomucin-food protein conjugates preparedthrough the transglutaminase reaction. Agric. Biol. Chem.55, 1027-1031.Kato, A., Takahashi, A., Matsudomi, N. and Kobayashi, K.(1983). Determination of foaming properties of proteinsby conductivity measurement. J. Food Sci. 48, 62-65.McWatters, K. H. and Holmes, M. R. (1979). Influence ofmoist heat on solubility and emulsification properties of soyand peanut flours. J. Food. Sci . 44, 774776.Laemmli, U. K. (1970). Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature 227,68& 685.Matsudomi, N., Tanaka, T., Kato, A. and Kobayashi, K.(1986). Functional properties of deamidated gluten obtainedby treating with chymotrypsin at alkaline pH. Agric. Biol.Chem. 50, 1989-1994.

Matsudomi, N., Sasaki, T., Kato, A. and Kobayashi, K.(1985). Conformational changes and functional propertiesof acid-modified soy protein. Agric. Biol. Chem. 49,1251-1256.Matsudomi, N., Sasaki, T., Tanaka, A., Kobayashi, K. andKato, A. (1985). Polymerization of deamidated peptidefragments obtained with the mild acid hydrolysis of oval-bumin. J. A gri c. Food. Chem. 33, 738-742.Meilgaard, M., Civille, G. V. and Carr, B. T. (1990). InSensory Evaluation Technique. CRC Press, Boca Raton, FL,Vol. 1, p. 125, Vol. 2, p. 159.Motoki, M., Seguro, K., Nio, N. and Takinami, K. (1986).Glutamine-specific deamidation of crsi-casein by transgluta-minase. Agri c. Bi ol. Chem. 50(12), 3025-3030.Nakai, S. and Voutsinas, L. P. (1983). A simple turbidimetricmethod for determining the fat binding capacity of proteins.J. Agric. Food. Chem. 31, 58-63.Nio, N. and Motoki, M. (1983). Crosslinking between differentfood proteins by transglutaminase. J. Food Sci . 48, 561-566.Nonaka, M., Tanaka, H., Okiyama, A., Motoki, M., Ando,H., Umeda, K. and Matsura, A. (1989). Polymerization ofseveral proteins by Ca*+-independent transglutaminasederived from microorganisms. Agric. Biol. Chem. 53,2619-2623.Pearce, K. N. and Kinsella, J. E. (1978). Emulsifying proper-ties of proteins: evaluation of a turbidimetric technique. J.Agric. Food Chem. 26, 716723.Sakamoto, H., Kumazawa, Y. and Motoki, M. (1994).Strength of protein gels prepared with microbial trans-glutaminase as related to reaction conditions. J. Food Sci .59, 866-871.


8/8

634 E. E. Babiker, M. A. S. Khan, N. Matsudomi, A. KatoSakamoto, H., Kumazawa, Y., Toiguchi, S., Serguro, K.,Soeda, T. and Motoki, M. (1995). Gel strength enhance-ment by addition of microbial transglutaminase duringonshore surimi manufacture. J. Food Sci. 60, 3OG304.Sergo, K., Kumazawa, Y., Ohtsuka, T., Toiguchi, S. andMotoki, M. (1995). Microbial transglutaminase and E(Y-glutamyl)lysine cross-links effects on elastic properties ofkamaboko gels. J. Food Sci. 60,305-311.Shih, F. F. (1991). Effect of anions on the deamidation of soyprotein. J. Food Sci . 56, 425-454.Tanimoto, S., Tanabe, S., Watanabe, M. and Arai, S. (1991).Enzymatic modification of zein to produce a non-bitterpeptide fraction with a very high Fischer ratio for patients withhepatic encephalopathy. Agri c. Biol . Chem. 55, 1119-l 123.

Takagi, T. and Hizukuri, S. (1984). Molecular weight andrelated properties of lily amylose determined by monitoringof elution from TSK-GEL PW high performance gel chroma-tography column by the low-angle laser light scatteringtechnique and precision differential refractometry. J. Bi ochem.951459-1467.Wu, C. H., Nakai, S. and Powrie, W. P. (1976). Preparationand properties of acid solubilized gluten. J. Agr i c. FoodChem. 24, 504-510.Yan, S. B. and Wold, F. (1984). Neoglycoproteins: in v i t rointroduction of glycosyl units at glutamines in @-caseinusing transglutaminase. Bi ochemi str y 23, 3759-3765.

(Received 14 June 1996; accepted 22 October 1996)

polymerization of soy protein digests by microbial transglutaminase for improvement of the...

Documents