Properties of Soy Protein

Transcription

1 Agric. Biol. Chem., 46 (1), 91~96, Effect of Tryptic Digestion on Emulsifying Properties of Soy Protein Kazuo Ochiai, Yoshiro Kamata and Kazuo Shibasaki Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Sendai 980, Japan Received April 17, 1981 The effect of tryptic digestion on the solubility and emulsifying properties of heat-denatured and native soy protein (crude glycinin) was studied as a function of ph. Fractionation of the tryptic digests of heat-denatured soy protein was achieved by centrifugation and ultra-filtration. The, resulting precipitate (PPT), high molecular weight fraction (HMF) and low molecular weight fraction (LMF) were constituted mainly with polypeptide. They had molecular weights of approximately 30,000, 20,000 and less than 10,000, respectively. The isoelectric region of HMFand that of DNG(digest of native glycinin) shifted to the acidic side, and the solubility of PPT and LMFwas not influenced by ph. Emulsifying properties, such as emulsifying capacity (EC) and emulsion stability (ES) after 30min and 24hr, were measured. It was found that the emulsifying properties were improved by tryptic hydrolysis but the low molecular weight digests showed poorer emulsifying properties. Among these digests, digestion intermediates (HMF and DNG) showed the highest emulsifying properties. The behavior of EC and ES after 30 min of these digests was related to that of their solubility and these values showed the minimum in the isoelectric region. Conversely, the ES value after 24hr was maximumin this region. The use of soy protein products in the food industry is expanding greatly1} due in part to their relatively low cost and unique functional characteristics.2* The emulsifying properties of soy protein have been utilized as processing aids in the comminuted meat field.3) Recently, they have also begun to be used for coffee whitener, milk-type beverages, mayonnaise etc.4) For the use of soy protein as a food ingredient, it is necessary to improve its emulsifying property and several investigators have reported that it was improved by modification.5'6) Manystudies have been made on the 7S and US proteins, the main components of soy protein, and much information has been accumulated concerning their molecular structure and subunits of these proteins.7) Kamata and Shibasaki8) investigated the degradation process of glycinin during tryptic hydrolysis and found that native glycinin was split by trypsin Abbreviations: SDS, sodium dodecyl sulfate; 2-ME, 2-mercaptoethanol; TPCK, tosyl-l-phenylalanine chloromethyl ketone. in limited sites under a high ionic strength condition and, consequently, converted into an intermediate named glycinin-t. The present study is concerned with improvement of the emulsifying properties of glycinin (US globulin) by tryptic hydrolysis and the aim of this work is to investigate the relationship between the emulsifying property and the composition or conformation of the tryptic digests as a function of ph. MATERIALS Soybean seeds (var. Raiden) harvested at Iwanurna, Miyagi, Japan in 1978 was used throughout this work. Trypsin (twice crystallized) was obtained from Worthington Biochemical Corp. and the contaminated chymotrypsin activity was removed by the TPCKtreatment as described by Carpenter.9) Soybean trypsin inhibitor was obtained from Sigma Chemical Company. METHODS Preparation of crude glycinin. The crude glycinin was prepared from defatted soybean meal, stored at 5 C, ground with a coffee mill, screened through a 60-mesh

2 92 K. Ochiai, Y. Kamata and K. Shibasaki sieve, and defatted with hexane, according to the method of Thanh and Shibasaki.10) Preparation of the digests of heat-denatured and native crude glycinin. A digest of heat-denatured crude glycinin was prepared as follows: crude glycinin was dissolved in 0.01 m Tris-HCl buffer (ph 8.0) containing 0.5 m NaCl and 0.01% sodium azide. The sample was incubated with TPCK-trypsin (40 /zg/ml) at 30 C for 5 hr after heating in a boiling bath for 30 min. Soybean trypsin inhibitor (40 //g/ml) was added to terminate this reaction. A precipitate (PPT) formed during the digestion was separated by centrifugation (10,000 rpm, 20 C, 20 min) and dissolved in 0.53 n acetic acid. The supernatant obtained was separated into two fractions: a high molecular weight fraction (HMF) and a low molecular weight fraction (LMF), by using the ultra-filtration system Mini-Module M-3 (molecular weight cut off; 13,000). A digest of native crude glycinin (DNG)was obtained in the same manner as that for heat-denatured protein, but without heating. No precipitate was formed in the sample and this sample was dialyzed without centrifugation. Electrophoresis. An SDSgel electrophoresisn) was conducted with 0.1 m Tris-phosphate buffer (ph 6.8) for 6hr at 100V in a 10% acrylamide gel. The gel obtained was stained with Coomassie Blue G-250 and destaining was in 7.5% acetic acid-5% methanol-water. Determination of the protein solubility. After the ph and protein concentration (0.4%) were adjusted with 0. 1 n HC1 or NaOH and deionized water, the supernatant was separated by centrifugation (10,000 rpm, 20 C, 20 min) and the protein content measured by the Biuret method.12) The protein solubility was expressed as the percentage of protein content in the supernatant to the original protein content. Measurement of the emulsifying capacity (EC). The emulsifying capacity was measured according to the method of Swift et al.,13) using commercial salad oil (Ajinomoto Co. Inc., Tokyo). A 0.5% protein solution (2ml) was put into a vessel and oil added from a buret at an average rate of 0.5 ml/min while the protein mixture was blended at a constant speed by a homogenizer (Model HC, Nihon Seiki Co., Tokyo). Oil was added until a sudden drop in the electric current occurred due to phase inversion. That breakpoint was considered to be the emulsifying capacity of the protein solution. This emulsifying capacity was expressed as ml of the oil emulsified per g of protein. The test for measuring the emulsifying capacity was repeated three times. Measurement of the emulsion stability (ES). The method of Acton and Saffle14) was modified and applied to measure the emulsion stability. Oil and a 0.5% protein solution were homogenizedat a volume ratio of 35:65 (3.5ml : 6.5ml) and the emulsion obtained was immediately placed into a 14x 104mm test tube. After standing for 30 min at 37.5±2 C, 2.5ml of the emulsion was removed from the bottom and the moisture content measured. The emulsion stability after 30 min was calculated as follows: Emulsion stability after 30 min 100-M test30m 100 -M original xloo where Mtest30 m refers to the percentage of moisture after 30 min and Moriginal to the percentage of moisture in the original emulsion. Further, the emulsion stability after 24hr was measured. The residual emulsion (7.5ml) was allowed to stand for 24hr at the same temperature and then 2.5ml of the emulsion was similarly removed from the bottom. The emulsion stability after 24hr was calculated as follows: Emulsion stability after 24 hr 100-M test24h 100- M original xloo where Mtest24 h refers to the percentage of moisture after 24hr and Moriginal to the percentage of moisture in the residual emulsion (7.5ml). The test for measuring emulsion stability was repeated three times. RESULTS Figure 1 shows the SDS gel electrophoretic patterns obtained for several digests and unhydrolyzed crude glycinin. When native glycinin is incubated with trypsin under the high ionic strength condition, only a limited number of a b c d e f g h IST-3 ISM Fig. 1. SDS Gel Electrophoretic Patterns of Tryptic Digests. Unhydrolyzed glycinin (a), unhydrolyzed glycinin + 2-ME (b), PPT (c), PPT +2-ME (d), HMF (e), HMF +2-ME (f), DNG (g), DNG +2-ME (h).

3 Emulsifying Properties of Soy Tryptic Digests 93 the potentially sensitive peptide bonds are cleaved and the protein is converted into glycinin-t.8) The high molecular weight fraction (HMF)and the digest of native glycinin (DNG) showed a similar pattern in the high molecular weight (M.W.) region and the mobilities of the two high molecular weight bands were the same as those of the stable fragments of glycinin-t (IST-3; M.W. 32,500 and IST- 4; M.W. 29,000). In addition the HMF contained low molecular weight fragments. PPT consisted of polypeptides smaller than the basic subunit of glycinin and they were insoluble in 0.01 m Tris-HCl buffer (ph 8.0, 0.5 m NaCl, 0.01% sodium azide). The low molecular weight fraction (LMF) which was not shown in Fig. 1 did not exhibit distinct bands. It was considered that the LMFconsisted of small peptides (M.W.: below 10,000), too small to be detected under these analytical electrophoretic conditions. As shown in Fig. 2, the solubility of the crude glycinin, the HMFand DNGindicated minimum values at ph 5~6, 4~5 and 4~5, respectively, suggesting that the isoelectric regions of the HMFand DNGshifted to the acidic side by tryptic hydrolysis. The PPT was insoluble throughout the ph range (2 ~9) studied, and conversely the LMFwas soluble throughout this ph range except for a small decrease in solubility between ph 3 and 5. As is well known, the emulsifying capacity and emulsion stability of soy proteins generally decreased to the minimumlevel in their isoelectric region and indicate higher values in the more acidic or alkaline regions.15'16* Figure 3 shows the emulsifying capacity of several digests and crude glycinin at various ph (2 ~9). Just as with the unhydrolyzed glycinin, I. = '-å -'-'-'-'-'-å - a> o Fig. 3. Effect of ph on the Emulsifying Capacity (EC) of Tryptic Digests. The protein solution was titrated by salad oil and its critical point was determined by the increase in electrical resistance. The emulsifying capacity was expressed as ml of oil per g of protein at the critical point. O-O, unhydrolyzed glycinin; A-A, PPT; #-#, LMF; A-A, HMF; Q-D, DNG. PH 60 I..... r-, I ll 1 20 å à"->^ ^2^t^^#-# " p i t f f. r~* ph Fig. 2. Effect of ph on the Solubility oftryptic Digests. Protein solution was adjusted to ph 2~9 with 0.1 n HC1 or NaOH. Solubility was measured by the Biuret method12) and expressed as the percentage ratio of protein content in the supernatant to the original protein content after centrifugation. O-O, Unhydrolyzed glycinin; A-A, PPT; #-#, LMF; A-A, HMF; D-D, DNG PH Fig. 4. Effect of ph on the Emulsion Stability (ES) after 30 min of Tryptic Digests. Salad oil and the protein solution were homogenized at the volume ratio of 35:65. After standing for 30 min at constant temperature, 2.5 ml of the emulsion was removed from the bottom and the moisture content was determined by the method of Acton and Saffle.14) O-O, unhydrolyzed glycinin; A-A, PPT; #-#, LMF; A-A, HMF; D-D, DNG.

4 94 K. Ochiai, Y. Kamata and K. Shibasaki å -.,,.. å å. 0^-9- à" *-«æf à" à" ol r-r^-^; ; t; PH Fig. 5. Effect of ph on the Emulsion Stability (ES) after 24hr of Tryptic Digests. The emulsion was prepared by the same method as the ES after 30 min. After removing 2.5ml of emulsion after 30 min, the emulsion residue wasagain stood for 24hr at the same temperature. Then 2.5 ml of emulsion was removed by the same method and the emulsion stability after 24hr wascalculated. O-O, unhydrolyzed glycinin; A-A, PPT; #-0, LMF; A-A, HMF; D-D, DNG. the HMFand DNGindicated the lowest levels in their isoelectric regions, and increased at ph's above and below these regions. On the other hand, the emulsifying capacity of the PPT and LMF, which have no isoelectric region, was not influenced by ph and no change was observed throughout the ph range 2^9. Figure 4 shows the emulsion stability after 30 min of each protein at various ph's. The behavior of each protein in its emulsion stability after 30 min was similar to that of its emulsifying capacity. That is, crude glycinin, the HMFand DNGshowed the lowest levels in their isoelectric regions and in the behavior of the PPT and LMF,no change was observed throughout the ph range (2~9). Figure 5 shows the emulsion stability after 24hr of each protein at various ph's. The behavior of the crude glycinin, the HMFand DNGwas similar to that of either their emulsifying capacity or emulsion stability after 30 min, showing higher values in the more acidic or alkaline regions, except for their isoelectric regions. The maximumvalues, however, were observed in their isoelectric regions. The behavior of the PPT and LMFwas also similar to that of either their emulsifying capacity or emulsion stability after 30 min, showing no change at all values of ph studied. From the results of the emulsion stability after 30 min and 24hr, it was suggested that the rate of creaming or the flocculation of emulsion particles in the isoelectric region differs from that at other ph's. As shown in Figs. 3 and 4, a remarkable increase in the emulsifying capacity and emulsion stability of the HMF and DNG, which were hydrolyzed by trypsin, was observed, especially in the acidic or alkaline region, in comparison with those of the unhydrolyzed glycinin. Conversely, the emulsifying properties of the PPT and LMF, which were low molecular weight digests, showedlower values than for unhydrolyzed glycinin. It was found that the emulsifying properties were improved by tryptic hydrolysis but the low molecular weight digest showed a lower value. DISCUSSION The HMFshows higher emulsifying properties than the DNG.The digest of native glycinin (DNG) used in this study is the digestion intermediate named glycinin-t. It has been found that glycinin-t maintains the quaternary structure which is almost similar to that of glycinin, except for partial fragmentation, and the resulting fragments are combined by the disulfide bond, hydrogen bond, etc.ll) The pattern of the HMF,which was heated before digestion, was also similar to that of the DNG (glycinin-t) in the high molecular weight region on the SDS gel electrophoresis (Fig. 1). However, it is considered that the HMFdoes not maintain the samequaternary structure as the DNGbecause of heat-denaturation, and somefragments which are smaller than those of the DNGare formed by tryptic hydrolysis. To reveal the above consideration, it is required to investigate the structural change of heatdenatured glycinin during tryptic digestion. Kamata et al.18) investigated the tryptic digestibility of urea-denatured glycinin by using a ph-stat, several kinds of gel electrophoresis and column chromatography. It follows that

5 Emulsifying Properties of Soy Tryptic Digests 95 the glycinin-t-like protein was generated from urea-denatured glycinin by tryptic hydrolysis and the glycinin-t-like protein had more kinds of component whose quaternary structure is destroyed than glycinin-t which is generated from native glycinin. The quaternary structure of the HMF is also destroyed by heatdenaturation and tryptic digestion (data not shown), suggesting that the HMF had a quaternary structure similar to that of the glycinin-t-like protein which is generated from urea-denatured glycinin. The higher emulsifying properties of the HMFover the DNGmay be due to the destruction of the quaternary structure. The emulsifying properties are affected by the rate of reduction of inter facial tension by proteins and this rate is determined by three consecutive or concurrent processes: (1) the diffusion of whole protein molecules or aggregates to and attachment at the interface; (2) spreading or unfolding of already-adsorbed molecules on the interface; (3) molecular rearrangements and reconformations of adsorbed molecules. The results of the emulsifying properties are interpreted as follows: (1) the unhydrolyzed glycinin has a complex quaternary structure in bulk with a particle weight 363,00019) and the migrating unit of high particle weight, therefore, diffuses slowly to the oil-water interface and cannot spread or unfold on the interface; (2) though glycinin-t has a quaternary structure similar to that of glycinin except for partial fragmentation, its structure is unstable because its quaternary structure varies by a mild change in the experimental conditions, such as lowering of the ionic strength.200 The protein, therefore, diffuses and spreads to and on to the interface faster than glycinin; (3) the HMFdoes not maintain the same quaternary structure as glycinin-t and, therefore, spreads or unfolds more easily on the oil-water interface than glycinin-t. The decrease in emulsifying properties of the PPT and LMFmay be due to excessive fragmentation. Aoki et al.21} investigated the effect of partial hydrolysis on the emulsifying properties and also obtained similar results. "aterqcp *JLT r Creami ng Flocculation Coalescence V-^ Coalescence Fig. 6. A Schematic Representation of Emulsion Instability. The behavior of the emulsifying capacity and emulsion stability after 30 min shown in Figs. 3 and 4 is similar to the commontrend wherein the emulsifying properties of vegetable proteins, including soy protein, indicate the lowest value in their isoelectric region. However, the behavior of the emulsion stability after 24hr shown in Fig. 5 is reversed in the isoelectric region compared with that of the other emulsifying properties. It is considered that the rate of creaming or flocculation of the emulsion particles in the isoelectric region is faster than that at other ph's, but the emulsion particles in the isoelectric region are more stable than those at other ph levels after creaming of the particles (Fig. 6). At present, we are investigating the emulsifying properties from the standpoint of the colloidal stability of the emulsion, using spectroturbidimetry22) to reveal the above consideration. REFERENCES 1) W. J. Wolf, /. Am. OilChemists'Soc, 54, 112(1977). 2) M. D. Wilding, /. Am. Oil Chemists' Soc, 51, 128 (1974). 3) P. A. Inklaar and J. Fortuin, Food TechnoL, 23, 103 (1969). 4) A. K. Smith and S. J. Circle, "Soybeans: Chemistry and Technology," Vol. 1, ed. by A. K. Smith and S. J. Circle, Avi Publishing Co., Inc., Westport, Conn., 1972, p ) F. Yamauchi, H. Ono, Y. Kamata and K. Shibasaki, Agric. Biol. Chem., 43, 1309 (1979). 6) H. Aoki, O. Taneyama and M. Inami, /. Food Sci.,

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