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oil emulsifier
Food Hydrocolloids Vol.7 no.4 pp.327-335, 1993
Pepsin-solubilized elastin as a water/oil emulsifier Makoto Hattori and Koji Takahashi
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu City, 183 Tokyo, Japan Abstract. Elastin is a major protein component of elastic tissues and is thought to be a valuable dairy resource. The emulsifying properties of pepsin-solubilized elastin (PSE) were investigated so as to find the possibility of PSE utilization as a novel emulsifier. The emulsifying properties of PSE were evaluated in the light of the emulsion stability of PSE-oleic acid emulsion. PSE could stabilize either for the oillwater (O/W) type and waterloil (W/O) type emulsion. PSE was unique in that it was especially effective for stabilizing the W/O type emulsion. From the effect of environmental factors on the stability of a W/O type emulsion, it was clarified that the best emulsification could be achieved when W/O type emulsification was performed at 25°C at pH 8 without salt with a 1% PSE solution. PSE was thought to be valuable as a novel W/O emulsifier.
Introduction Elastin is a major protein component of elastic tissues like the arterial wall, ligament and skin (1,2). The amino acid composition of elastin is unique in that elastin contains a lot of nonpolar amino acids like Gly, Ala, Val and Pro (>80%) (3). Elastin also has specific cross-linkages of desmosine, isodesmosine (4,5), lysino-norleucine and merodesmosine (6,7). Consequently, elastin is highly insoluble in diluted acid, diluted alkali, hot water and various denaturants, leading to difficulty of structural analysis and utilization. Various chemical solubilization processes such as hot oxalic acid treatment (8) and alkali extraction with or without alcohol (9,10,11) have already been studied. However since these methods are very severe it is difficult to regulate cleavage of the peptide chain, resulting in the production of peptide fragments that are widely distributed in sequence and molecular weight. In comparison with these methods, solubilization by protease digestion could leave amino acid sequences peculiar to insoluble elastin (IE), owing to the substrate specificity of protease. It can be expected that the solubilized peptides would be rich in nonpolar amino acids and have a good affinity to oil. In this study we carried out a pepsin digestion of IE and investigated the emulsifying ability of pepsin-solubilized elastin (PSE) as a water/oil (W/G) emulsifier. Materials and methods
Preparation of pepsin-solubilized elastin IE was prepared from minced bovine ligamentum nuchae by extracting with a 10% sodium chloride solution and acetone-ether, before autoclaving seven times at 2 kg/ern? for 1 h according to the method of Partridge et al. (8). IE was recovered by lyophilization and then pulverized by cooling with dry ice-acetone. 327
M.Hattori and K. Takahashi
IE was solubilized by pepsin digestion using 20 g of IE suspended in 1000 ml of 0.5 mol/dm" acetic acid. To the suspension, 200 mg of pepsin (E.C. 3.4.23.1 , Sigma, St. Louis, USA , 3200 U/mg) was added, and the reaction mixture was incubated for 30 h at 25°C with gentle stirring. The reaction was stopped by adjusting to above pH 12.0 with a 30% NaOH solution. The reaction mixture was neutralized with 1 rnol/drrr' HCI and centrifuged at 12 000 g for 30 min at 25°C. The resulting supernatants was dialyzed against distilled water and lyophilized. Lyophilized material was designated as pepsin-solubilized elastin (PSE).
Determination of the amino acid composition PSE was hydrolyzed in 6 mol/dnr' HCI at 110°C for 20 h in vacuo. An amino acid analysis of the hydrolysate was carried out on an automatic amino acid analyzer (model 835, Hitachi, Tokyo , Japan) . To determine the desmosine and isodesmosine content, PSE was hydrolyzed in 6 mol/dm ' HCI at 110°C for 48 h in vacuo, and hydrolysate was analyzed by ion-pair chromatography. A TSK gel ODS-80 T M column (4.6<1> x 150 mm, Tosoh, Tokyo, Japan) was equilibrated with 0.09 mol/drrr' methane sulfonic acid (pH 2.0) containing 5.4 mmol/drrr' sodium heptanesulfonate and 10% (v/v) acetonitrile. The hydrolysate was applied to the column and eluted at a flow rate of 1.0 ml/min at 25°C. The absorbance was monitored at 275 nm.
Size-exclusion chromatography (SEC) The molecular weight of PSE was measured by SEC. A TSK gel G3000SW column (7.8<1> x 300 mm, Tosoh) was equilibrated with a 0.07 mol/dm' phosphate buffer containing 0.3 mol/dnr' NaCl at pH 7.0. The PSE sample (100 I-1g/50 1-11) was applied to the column and eluted at a flow rate of 1.0 ml/min. The absorbance was monitored at 280 nm.
Emulsification of oleic acid with the PSE solution PSE was dissolved in a 0.05 mol/dm? acetate buffer at pH 7.0 to give a concentration of 1%. A measured amount of oleic acid and this PSE solution were homogenized at 25°C by a Polytron PTA-7 (Kinematica, Switzerland) for 5 min at full speed. The reason why oleic acid was employed as oil phase instead of triacyl glycerol or alkanes was to acquire an insight into the emulsifying properties of PSE in simple and homogenous systems.
Distribution of PSE from the water phase to oil phase Emulsification was carried out by homogenizing 1 ml of the 1% PSE solution and 0.1, 0.3 or 0.5 ml of oleic acid. After this emulsification , the emulsion was centrifuged twice at 18 000 g for 30 min at room temperature. The PSE concentration in the water phase was measured by the micro-biuret method (12). The decrease of PSE in the water phase is assumed to be due to that distributed to the oil phase. 328
Emulsifying properties of a pepsin-solubilized elastin
E vaLuation of the emuLsion stability (ES)
ES was evaluated according to the method of Yamano et aL. with some modifications (13 ,14) . The oil/water (O/W) type or W/O type emulsion was sampled to a hematocrit capillary tube from the bottom or top layer respectively, after standing for 0, 3, 6 or 12 h , and then centrifuged at 12 000 g for 5 min in a Kub ota (Tokyo, Japan) Hem atocrit KH-1200M centrifuge. Th e change in oil separation or water sep aration was used as an ES index. In case of an O/W type emulsion , as the emulsion becomes un stable the emulsion in the bottom layer rele ases oil to the top layer. In case of a W/O type emulsion , as the emulsion becomes unstable the emulsion in the top layer releases water to the bottom layer. T ype of emulsion was determined by dispersibility when the emulsion was dropped in distilled water. Results and discussion StructuraL features of PSE
IE obtained from bovine ligamentum nuchae was completely solubilized by pepsin digestion at 25°C for 30 h. The yield of PSE from IE was -52%, which is higher than that of the extraction with 0.25 mol/drrr' oxalic acid at 100°C for 1 h (7% yield) or with 1 N potassium hydride/80% eth yl alcohol for 4 h (35% yield). The amino acid composition of PSE shown in Table 1 is similar to that of the IE preparation and native elastin from bovine ligamentum nuchae reported by Table I. A mino acid composition of"PSE I A mino acid
4-Hy p A sp Thr Ser Glu Pr o
Gly Al a Cys V al Met
IIe Leu Tyr Phe H yl Lys His Arg D es Ide
IE
PSE
8 6
9
180 312 205 6 129 1 22 53 7 29
12 10 12 19 107 345 199 4 129 1 25 60 10 34
4
2
3 1 6 2.4 2 2. 12
4
8 8 14
1
5 3.22 2.3 2
Bovine ligamentum nuchae ela stin" 8.1 5.8 9.3 8.7 15.4 115.5 328.1 227.0
o
131.6
o
23.9 59.4 5.9 29.3 0.5 3.3 0.5 5.8 5.4 10.1
Residues per 1000 amin o acid residu es. g. 3 Field et al. (3). I
2 mo l/105
329
M.Hattori and K.Takahashi
Field et at. (3), suggesting that PSE has a peculiar amino acid sequence rich in hydrophobic residues. PSE contained a similar amount of desmosine and isodesmosine to that in IE. The SEC pattern of PSE indicates a relatively homogenous molecular weight with a major peak of about 10 000 as shown in Figure 1. Distribution of PSE from the water phase to oil phase
The distribution of PSE from the water phase to oil phase is shown in Table II. The amount of PSE distributed to the oil phase increased as the oil phase volume increased. However the PSE concentration in the oil phase in each case was 7-8 mg/ml. Under the condition of >0.3 ml of oleic acid per 1.0 ml of PSE solution, the PSE concentration in the oil and water phases became almost equal. Therefore though PSE concentration in oil phase in Table II may contain PSE adsorbed at the surface of oil droplets as well as PSE solubilized in oil phase, it is thought that PSE was easily distributed from the water phase to the oil phase due to its amphiphilic property. This strongly suggests that PSE has good emulsifying ability.
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Retentiontime (min) Fig.!. HPLC pattern for PSE. HPLC conditions: column, TSK gel G3000SWX L (7.8<1> x 300 mrn, Tosoh); sample, 100 /log/50 p.l; mobile phase, 0.07 mol/dm? phosphate buffer containing 0.3 mol/drrr' NaCi (pH 7.0); flow rate, 1.0 ml/min; detection, absorbance at 280 nm. Table II. Distribution of PSE from the water phase to oil phase Volume of oil Volume of water PSE distributed to Concentration of PSE Concentratio of phase (m!) phase (ml) oil phase (mg) in oil phase (mg/m!) PSE in water phase (mg/ml) 0.1 0.3 0.5
330
1.0 1.0 1.0
0.7 2.3 3.3
7.0 7.7 6.6
9.3 7.7 6.7
Emulsifying properties of a pepsin-solubilized elastin
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Time{hr) Fig. 2. Stability of PSE-oleic acid emulsion at various mixture ratios. The oleic acid conce ntratio n was (a) 20% , (b) 30% , (c) 40% , (d) 50% , (e) 60% , (f) 70% and (g) 80% . Emul sion stability was evaluate d by the centrifugation method . In a-f the emulsion was sampled from the wate r phase . In g, the emulsion was sampled from the oil phase . • , PSE ; 0 , BSA; A , control (withou t emulsifying agent ).
331
M.Hattori and K.Takahashi
Emulsifying ability of PSE Various amounts of oleic acid were emulsified with the 1% PSE solution at pH 7.0 in order to compare the emulsifying ability of PSE with that of bovine serum albumin (BSA). Figures 2a-f indicate that BSA was good for the O/W type emulsion with a mixture ratio (v/v) of oleic acid up to 0.7. Using the PSE solution, the quantity of separated oleic acid was greater than that with BSA at any selected time. Therefore, it can be said that PSE had greater emulsifying ability for the O/W emulsion than BSA. Non-polar amino acids content in PSE is 95.9% (Table I), while that in BSA is 66.2% (15). Hence, high hydrophobicity of PSE is thought to lead to strong emulsifying ability. However at a mixture ratio 0.8, BSA lost most of its emulsifying ability because of the phase inversion between the ratio of 0.7 and 0.8 (Figure 2g). On the other hand, the emulsifying ability of PSE remained substantially unchanged. Therefore, it was concluded that PSE was capable of stabilizing not only the O/W type emulsion but also the W/O type emulsion. Since most proteineous emulsifiers are effective with the O/W type emulsion PSE is thought to be valuable as a novel W10 type emulsifier. In this experiment, droplet-size distribution of the emulsion just after emulsification was clarified by a microscopic observation as follows. As for O/W emulsion, 97% of droplets were proved to be <10 urn and the rest were 1040 u.m in the emulsion containing PSE, which was similar to the droplet-size distribution of the control emulsion without emulsifying agent. As for W/O emulsions, droplet-size distribution of the emulsion containing PSE was similar to that of an O/W emulsion containing PSE, while the control emulsion without PSE was unstable and heterogenous (mixture of O/W type and W/O type). Droplet-size distribution of the control W/O emulsion could not be measured. Stability of the W/O emulsion with PSE The effects of several environmental factors on the stability of a W/O emulsion composed of 80% of oleic acid and 20% of a PSE solution were studied.
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Fig. 3. Effect of temperature on emulsion stability. The oleic acid concentration was 80% . • , 15°C; 0, 25°C; ~, 35°C; _,45°C.
332
Emulsifying properties of a pepsin-solubilized elastin
The effect of emulsifying temperature was investigated in the range 15-40°C (Figure 3). The emulsion prepared at 25°C was more stable than that at 15°C, but increasing temperature further resulted in the stability of emulsion being decreased due to the aggregation of PSE chains. Thus, the emulsion was most stable when the emulsifying temperature was 25°C. The effect of PSE concentration was investigated by varying the concentration in the range 0-2.0% (Figure 4). When the PSE concentration was 1.0% the emulsion was most stable. A PSE concentration <1% appeared to be inadequate for stabilizing an emulsion. When the PSE concentration was> 1%, it is thought that an increase in the interaction between PSE molecules resulted in the decreased emulsion stability. The effect of pH on the emulsion stability was investigated by varying pH in the range 6-9 (Figure 5). When the pH value was 7 or 8, the emulsion was the
--
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e, 0%;
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e, pH 6; 0, pH 333
M.Hattori and K.Takahashi
most stable. Since the isoelectric point of PSE has been estimated to be pH 4.7 by isoelectric focusing (data not shown), PSE might take an expanded conformation as the pH value increased. The conformation of PSE at pH 7 or 8 is believed to be suitable for interacting with oleic acid, and this could be confirmed by an additional experiment such as circular dichroism measurement. The effect of ionic strength was investigated with NaCI and CaClz (Figures 6 and 7). In the presence of only 0.05 mol/dnr' NaCI or CaCl z the emulsion completely lost its stability. The effect of PSE on the emulsion stability was strongly weakened by shielding the electric charge in PSE.
Concluding remarks PSE was prepared by pepsin-treating IE from bovine ligamentum nuchae. PSE was rich in nonpolar amino acids, suggesting that PSE had a strong amphiphilic
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Fig. 6. Effect of NaCI concentration on the emulsion stability. The oleic acid concentration was 80%. "',0.05 mol/dm'; ., 0.1 mol dm",
e, 0 mol/dm"; 0, 0.01 mol/dm";
25
o '--_.....__......__..... o 2 4 6 Time (hr) Fig. 7. Effect of CaCl z concentration on the emulsion stability. The oleic acid concentration was 80%. e, 0 mol/drrr'; 0, 0.01 mol/dm'; "',0.05 mol/drrr'; .,0.1 mol/dm",
334
Emul sifying properties of a pepsin-solubilized elastin
property. The emulsifying properti es of PSE wer e evaluated from the sta bility of a PSE- oleic acid emulsion . PSE stabilized both the O/W type and W/O type emulsion . PSE was unique in that it was especially effecti ve for stabilizing the W/O type emulsion. When the PSE solution/oleic acid ratio was 2/8, i.e . W/O type emuls ion , the best emulsification was achieved at pH 8 and 25°C without salt, using a 1% PSE solution. PSE was thought to be valuable as a novel emulsifier. References 1. Serafini-Fracassini,A. , Field,J .M., Rodger ,G.W. and Spina,M. (1975) Biochim . Biophys. Acta, 386, 80-86. 2. Spina ,M. , Garbin, G ., Field,J .M. and Serafini-Fracassini,A. (1975) Biochim . Biophys. Acta , 400,162-166. 3. Field,J .M . , Rodger,G.W ., Hun ter,J .c. , Sera fini-Fracassini.A . and Spina,M . (1978) Arch. Biochim . Biophys. , 191, 705-713. 4. Partridge ,S.M., Elsden ,D .F . and Thomas,J. (1963) Nature, 197, 1297-1298. 5. Thom as,J .M ., Elsden,D . and Partridge,S.M. (1963) Nature, 200, 651-652. 6. Starcher ,B.C., Partridge ,S.M. and Elsden ,D .F. (1967) Biochemistry, 6, 2425-2432. 7. Franzbl au ,C. , Sinex,F .M . and Fabris ,B. (1965) Biochem. Biophys. Res. Commun. , 21, 575-581. 8. Part ridge ,S.M. , Davis,H .F . and Adai r ,G .S. (1955) Biochern. J. , 61, 11-21. 9. Moschett o ,Y., Bach er ,M.D . , Pizieux,a. , Bouissou .H .; Pieraggi,M.T. and Julian,M . (1974) Paorodiarterialle/Arterial Wall, 2,116-175. ' 10. Lowry,a .H . , Gilligan,R . and Katersky,E .M. (1941) J. Bioi. Chern., 139, 795-804. 11. Lansing,A.! ., Rosenth al,T.B. , Alex,M. and Dempsey,E.W. (1952) A nat. Rec. , 114.555-575. 12. Itzhaki,R .F. and Gill,D .M. (1964) A nal. Biochern., 9, 401-410. 13. Vold,R .D . and Gro ot ,R .C. (1962) J. Phys. Chem ., 66 , 1969-1975. 14. Yamano, Y., Tsuru ,T ., Sugihara, S. and Miki,E . (1982) Nippon Shok uhin Kogyo Gakkaishi, 29, 137- 142. 15. Brown ,J .R. (1976) Fed. Proc. , 35, 2141- 2144.
335
Pepsin-solubilized elastin as a water/oil emulsifier Makoto Hattori and Koji Takahashi
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu City, 183 Tokyo, Japan Abstract. Elastin is a major protein component of elastic tissues and is thought to be a valuable dairy resource. The emulsifying properties of pepsin-solubilized elastin (PSE) were investigated so as to find the possibility of PSE utilization as a novel emulsifier. The emulsifying properties of PSE were evaluated in the light of the emulsion stability of PSE-oleic acid emulsion. PSE could stabilize either for the oillwater (O/W) type and waterloil (W/O) type emulsion. PSE was unique in that it was especially effective for stabilizing the W/O type emulsion. From the effect of environmental factors on the stability of a W/O type emulsion, it was clarified that the best emulsification could be achieved when W/O type emulsification was performed at 25°C at pH 8 without salt with a 1% PSE solution. PSE was thought to be valuable as a novel W/O emulsifier.
Introduction Elastin is a major protein component of elastic tissues like the arterial wall, ligament and skin (1,2). The amino acid composition of elastin is unique in that elastin contains a lot of nonpolar amino acids like Gly, Ala, Val and Pro (>80%) (3). Elastin also has specific cross-linkages of desmosine, isodesmosine (4,5), lysino-norleucine and merodesmosine (6,7). Consequently, elastin is highly insoluble in diluted acid, diluted alkali, hot water and various denaturants, leading to difficulty of structural analysis and utilization. Various chemical solubilization processes such as hot oxalic acid treatment (8) and alkali extraction with or without alcohol (9,10,11) have already been studied. However since these methods are very severe it is difficult to regulate cleavage of the peptide chain, resulting in the production of peptide fragments that are widely distributed in sequence and molecular weight. In comparison with these methods, solubilization by protease digestion could leave amino acid sequences peculiar to insoluble elastin (IE), owing to the substrate specificity of protease. It can be expected that the solubilized peptides would be rich in nonpolar amino acids and have a good affinity to oil. In this study we carried out a pepsin digestion of IE and investigated the emulsifying ability of pepsin-solubilized elastin (PSE) as a water/oil (W/G) emulsifier. Materials and methods
Preparation of pepsin-solubilized elastin IE was prepared from minced bovine ligamentum nuchae by extracting with a 10% sodium chloride solution and acetone-ether, before autoclaving seven times at 2 kg/ern? for 1 h according to the method of Partridge et al. (8). IE was recovered by lyophilization and then pulverized by cooling with dry ice-acetone. 327
M.Hattori and K. Takahashi
IE was solubilized by pepsin digestion using 20 g of IE suspended in 1000 ml of 0.5 mol/dm" acetic acid. To the suspension, 200 mg of pepsin (E.C. 3.4.23.1 , Sigma, St. Louis, USA , 3200 U/mg) was added, and the reaction mixture was incubated for 30 h at 25°C with gentle stirring. The reaction was stopped by adjusting to above pH 12.0 with a 30% NaOH solution. The reaction mixture was neutralized with 1 rnol/drrr' HCI and centrifuged at 12 000 g for 30 min at 25°C. The resulting supernatants was dialyzed against distilled water and lyophilized. Lyophilized material was designated as pepsin-solubilized elastin (PSE).
Determination of the amino acid composition PSE was hydrolyzed in 6 mol/dnr' HCI at 110°C for 20 h in vacuo. An amino acid analysis of the hydrolysate was carried out on an automatic amino acid analyzer (model 835, Hitachi, Tokyo , Japan) . To determine the desmosine and isodesmosine content, PSE was hydrolyzed in 6 mol/dm ' HCI at 110°C for 48 h in vacuo, and hydrolysate was analyzed by ion-pair chromatography. A TSK gel ODS-80 T M column (4.6<1> x 150 mm, Tosoh, Tokyo, Japan) was equilibrated with 0.09 mol/drrr' methane sulfonic acid (pH 2.0) containing 5.4 mmol/drrr' sodium heptanesulfonate and 10% (v/v) acetonitrile. The hydrolysate was applied to the column and eluted at a flow rate of 1.0 ml/min at 25°C. The absorbance was monitored at 275 nm.
Size-exclusion chromatography (SEC) The molecular weight of PSE was measured by SEC. A TSK gel G3000SW column (7.8<1> x 300 mm, Tosoh) was equilibrated with a 0.07 mol/dm' phosphate buffer containing 0.3 mol/dnr' NaCl at pH 7.0. The PSE sample (100 I-1g/50 1-11) was applied to the column and eluted at a flow rate of 1.0 ml/min. The absorbance was monitored at 280 nm.
Emulsification of oleic acid with the PSE solution PSE was dissolved in a 0.05 mol/dm? acetate buffer at pH 7.0 to give a concentration of 1%. A measured amount of oleic acid and this PSE solution were homogenized at 25°C by a Polytron PTA-7 (Kinematica, Switzerland) for 5 min at full speed. The reason why oleic acid was employed as oil phase instead of triacyl glycerol or alkanes was to acquire an insight into the emulsifying properties of PSE in simple and homogenous systems.
Distribution of PSE from the water phase to oil phase Emulsification was carried out by homogenizing 1 ml of the 1% PSE solution and 0.1, 0.3 or 0.5 ml of oleic acid. After this emulsification , the emulsion was centrifuged twice at 18 000 g for 30 min at room temperature. The PSE concentration in the water phase was measured by the micro-biuret method (12). The decrease of PSE in the water phase is assumed to be due to that distributed to the oil phase. 328
Emulsifying properties of a pepsin-solubilized elastin
E vaLuation of the emuLsion stability (ES)
ES was evaluated according to the method of Yamano et aL. with some modifications (13 ,14) . The oil/water (O/W) type or W/O type emulsion was sampled to a hematocrit capillary tube from the bottom or top layer respectively, after standing for 0, 3, 6 or 12 h , and then centrifuged at 12 000 g for 5 min in a Kub ota (Tokyo, Japan) Hem atocrit KH-1200M centrifuge. Th e change in oil separation or water sep aration was used as an ES index. In case of an O/W type emulsion , as the emulsion becomes un stable the emulsion in the bottom layer rele ases oil to the top layer. In case of a W/O type emulsion , as the emulsion becomes unstable the emulsion in the top layer releases water to the bottom layer. T ype of emulsion was determined by dispersibility when the emulsion was dropped in distilled water. Results and discussion StructuraL features of PSE
IE obtained from bovine ligamentum nuchae was completely solubilized by pepsin digestion at 25°C for 30 h. The yield of PSE from IE was -52%, which is higher than that of the extraction with 0.25 mol/drrr' oxalic acid at 100°C for 1 h (7% yield) or with 1 N potassium hydride/80% eth yl alcohol for 4 h (35% yield). The amino acid composition of PSE shown in Table 1 is similar to that of the IE preparation and native elastin from bovine ligamentum nuchae reported by Table I. A mino acid composition of"PSE I A mino acid
4-Hy p A sp Thr Ser Glu Pr o
Gly Al a Cys V al Met
IIe Leu Tyr Phe H yl Lys His Arg D es Ide
IE
PSE
8 6
9
180 312 205 6 129 1 22 53 7 29
12 10 12 19 107 345 199 4 129 1 25 60 10 34
4
2
3 1 6 2.4 2 2. 12
4
8 8 14
1
5 3.22 2.3 2
Bovine ligamentum nuchae ela stin" 8.1 5.8 9.3 8.7 15.4 115.5 328.1 227.0
o
131.6
o
23.9 59.4 5.9 29.3 0.5 3.3 0.5 5.8 5.4 10.1
Residues per 1000 amin o acid residu es. g. 3 Field et al. (3). I
2 mo l/105
329
M.Hattori and K.Takahashi
Field et at. (3), suggesting that PSE has a peculiar amino acid sequence rich in hydrophobic residues. PSE contained a similar amount of desmosine and isodesmosine to that in IE. The SEC pattern of PSE indicates a relatively homogenous molecular weight with a major peak of about 10 000 as shown in Figure 1. Distribution of PSE from the water phase to oil phase
The distribution of PSE from the water phase to oil phase is shown in Table II. The amount of PSE distributed to the oil phase increased as the oil phase volume increased. However the PSE concentration in the oil phase in each case was 7-8 mg/ml. Under the condition of >0.3 ml of oleic acid per 1.0 ml of PSE solution, the PSE concentration in the oil and water phases became almost equal. Therefore though PSE concentration in oil phase in Table II may contain PSE adsorbed at the surface of oil droplets as well as PSE solubilized in oil phase, it is thought that PSE was easily distributed from the water phase to the oil phase due to its amphiphilic property. This strongly suggests that PSE has good emulsifying ability.
,
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o
5
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Retentiontime (min) Fig.!. HPLC pattern for PSE. HPLC conditions: column, TSK gel G3000SWX L (7.8<1> x 300 mrn, Tosoh); sample, 100 /log/50 p.l; mobile phase, 0.07 mol/dm? phosphate buffer containing 0.3 mol/drrr' NaCi (pH 7.0); flow rate, 1.0 ml/min; detection, absorbance at 280 nm. Table II. Distribution of PSE from the water phase to oil phase Volume of oil Volume of water PSE distributed to Concentration of PSE Concentratio of phase (m!) phase (ml) oil phase (mg) in oil phase (mg/m!) PSE in water phase (mg/ml) 0.1 0.3 0.5
330
1.0 1.0 1.0
0.7 2.3 3.3
7.0 7.7 6.6
9.3 7.7 6.7
Emulsifying properties of a pepsin-solubilized elastin
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TIme(hr)
.....
20
.!!
15
'C
-a
10
f!
5
..
(g)
~ ~
~
GI
GI
(/)
0
0
2
4
6
Time{hr) Fig. 2. Stability of PSE-oleic acid emulsion at various mixture ratios. The oleic acid conce ntratio n was (a) 20% , (b) 30% , (c) 40% , (d) 50% , (e) 60% , (f) 70% and (g) 80% . Emul sion stability was evaluate d by the centrifugation method . In a-f the emulsion was sampled from the wate r phase . In g, the emulsion was sampled from the oil phase . • , PSE ; 0 , BSA; A , control (withou t emulsifying agent ).
331
M.Hattori and K.Takahashi
Emulsifying ability of PSE Various amounts of oleic acid were emulsified with the 1% PSE solution at pH 7.0 in order to compare the emulsifying ability of PSE with that of bovine serum albumin (BSA). Figures 2a-f indicate that BSA was good for the O/W type emulsion with a mixture ratio (v/v) of oleic acid up to 0.7. Using the PSE solution, the quantity of separated oleic acid was greater than that with BSA at any selected time. Therefore, it can be said that PSE had greater emulsifying ability for the O/W emulsion than BSA. Non-polar amino acids content in PSE is 95.9% (Table I), while that in BSA is 66.2% (15). Hence, high hydrophobicity of PSE is thought to lead to strong emulsifying ability. However at a mixture ratio 0.8, BSA lost most of its emulsifying ability because of the phase inversion between the ratio of 0.7 and 0.8 (Figure 2g). On the other hand, the emulsifying ability of PSE remained substantially unchanged. Therefore, it was concluded that PSE was capable of stabilizing not only the O/W type emulsion but also the W/O type emulsion. Since most proteineous emulsifiers are effective with the O/W type emulsion PSE is thought to be valuable as a novel W10 type emulsifier. In this experiment, droplet-size distribution of the emulsion just after emulsification was clarified by a microscopic observation as follows. As for O/W emulsion, 97% of droplets were proved to be <10 urn and the rest were 1040 u.m in the emulsion containing PSE, which was similar to the droplet-size distribution of the control emulsion without emulsifying agent. As for W/O emulsions, droplet-size distribution of the emulsion containing PSE was similar to that of an O/W emulsion containing PSE, while the control emulsion without PSE was unstable and heterogenous (mixture of O/W type and W/O type). Droplet-size distribution of the control W/O emulsion could not be measured. Stability of the W/O emulsion with PSE The effects of several environmental factors on the stability of a W/O emulsion composed of 80% of oleic acid and 20% of a PSE solution were studied.
--
20
?fl.
Jo.
~
15
~CD 10 1; ~ 5
c. CD
en
01.--.......- - - - - - - - ' 2 4 6 o Time (hr)
Fig. 3. Effect of temperature on emulsion stability. The oleic acid concentration was 80% . • , 15°C; 0, 25°C; ~, 35°C; _,45°C.
332
Emulsifying properties of a pepsin-solubilized elastin
The effect of emulsifying temperature was investigated in the range 15-40°C (Figure 3). The emulsion prepared at 25°C was more stable than that at 15°C, but increasing temperature further resulted in the stability of emulsion being decreased due to the aggregation of PSE chains. Thus, the emulsion was most stable when the emulsifying temperature was 25°C. The effect of PSE concentration was investigated by varying the concentration in the range 0-2.0% (Figure 4). When the PSE concentration was 1.0% the emulsion was most stable. A PSE concentration <1% appeared to be inadequate for stabilizing an emulsion. When the PSE concentration was> 1%, it is thought that an increase in the interaction between PSE molecules resulted in the decreased emulsion stability. The effect of pH on the emulsion stability was investigated by varying pH in the range 6-9 (Figure 5). When the pH value was 7 or 8, the emulsion was the
--
20
ffl.
o ------'------... o 2 4 6 Time (hr) Fig. 4. Effect of PSE concentration on the emulsion stability. The oleic acid concentration was 80%. 0, 0.5%; .A., 1.0%; _, 2.0%.
e, 0%;
--
20
?ft
0 - - - -......- ......."-------' 2 4 6 o Time (hr) Fig. 5. Effect of pH on the emulsion stability. The oleic acid concentration was 80%. 7; .A., pH 8; _, pH 9.
e, pH 6; 0, pH 333
M.Hattori and K.Takahashi
most stable. Since the isoelectric point of PSE has been estimated to be pH 4.7 by isoelectric focusing (data not shown), PSE might take an expanded conformation as the pH value increased. The conformation of PSE at pH 7 or 8 is believed to be suitable for interacting with oleic acid, and this could be confirmed by an additional experiment such as circular dichroism measurement. The effect of ionic strength was investigated with NaCI and CaClz (Figures 6 and 7). In the presence of only 0.05 mol/dnr' NaCI or CaCl z the emulsion completely lost its stability. The effect of PSE on the emulsion stability was strongly weakened by shielding the electric charge in PSE.
Concluding remarks PSE was prepared by pepsin-treating IE from bovine ligamentum nuchae. PSE was rich in nonpolar amino acids, suggesting that PSE had a strong amphiphilic
--
20
G:i
15
== "0
10
?fl
as Q)
as
lo.
co 5 c. Q)
en
0'---...&.--.......----' o 2 4 6 Time (hr)
Fig. 6. Effect of NaCI concentration on the emulsion stability. The oleic acid concentration was 80%. "',0.05 mol/dm'; ., 0.1 mol dm",
e, 0 mol/dm"; 0, 0.01 mol/dm";
25
o '--_.....__......__..... o 2 4 6 Time (hr) Fig. 7. Effect of CaCl z concentration on the emulsion stability. The oleic acid concentration was 80%. e, 0 mol/drrr'; 0, 0.01 mol/dm'; "',0.05 mol/drrr'; .,0.1 mol/dm",
334
Emul sifying properties of a pepsin-solubilized elastin
property. The emulsifying properti es of PSE wer e evaluated from the sta bility of a PSE- oleic acid emulsion . PSE stabilized both the O/W type and W/O type emulsion . PSE was unique in that it was especially effecti ve for stabilizing the W/O type emulsion. When the PSE solution/oleic acid ratio was 2/8, i.e . W/O type emuls ion , the best emulsification was achieved at pH 8 and 25°C without salt, using a 1% PSE solution. PSE was thought to be valuable as a novel emulsifier. References 1. Serafini-Fracassini,A. , Field,J .M., Rodger ,G.W. and Spina,M. (1975) Biochim . Biophys. Acta, 386, 80-86. 2. Spina ,M. , Garbin, G ., Field,J .M. and Serafini-Fracassini,A. (1975) Biochim . Biophys. Acta , 400,162-166. 3. Field,J .M . , Rodger,G.W ., Hun ter,J .c. , Sera fini-Fracassini.A . and Spina,M . (1978) Arch. Biochim . Biophys. , 191, 705-713. 4. Partridge ,S.M., Elsden ,D .F . and Thomas,J. (1963) Nature, 197, 1297-1298. 5. Thom as,J .M ., Elsden,D . and Partridge,S.M. (1963) Nature, 200, 651-652. 6. Starcher ,B.C., Partridge ,S.M. and Elsden ,D .F. (1967) Biochemistry, 6, 2425-2432. 7. Franzbl au ,C. , Sinex,F .M . and Fabris ,B. (1965) Biochem. Biophys. Res. Commun. , 21, 575-581. 8. Part ridge ,S.M. , Davis,H .F . and Adai r ,G .S. (1955) Biochern. J. , 61, 11-21. 9. Moschett o ,Y., Bach er ,M.D . , Pizieux,a. , Bouissou .H .; Pieraggi,M.T. and Julian,M . (1974) Paorodiarterialle/Arterial Wall, 2,116-175. ' 10. Lowry,a .H . , Gilligan,R . and Katersky,E .M. (1941) J. Bioi. Chern., 139, 795-804. 11. Lansing,A.! ., Rosenth al,T.B. , Alex,M. and Dempsey,E.W. (1952) A nat. Rec. , 114.555-575. 12. Itzhaki,R .F. and Gill,D .M. (1964) A nal. Biochern., 9, 401-410. 13. Vold,R .D . and Gro ot ,R .C. (1962) J. Phys. Chem ., 66 , 1969-1975. 14. Yamano, Y., Tsuru ,T ., Sugihara, S. and Miki,E . (1982) Nippon Shok uhin Kogyo Gakkaishi, 29, 137- 142. 15. Brown ,J .R. (1976) Fed. Proc. , 35, 2141- 2144.
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