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Change in antibacterial characteristics with doping amount of ZnO in MgO–ZnO solid solution
International Journal of Inorganic Materials 2 (2000) 451–454
Change in antibacterial characteristics with doping amount of ZnO in MgO–ZnO solid solution Osamu Yamamoto*, Jun Sawai, Tadashi Sasamoto Department of Applied Chemistry, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi-shi, 243 -0292 Japan Accepted 11 May 2000
Abstract Antibacterial activity for MgO–ZnO solid solution was studied by measuring the change in electrical conductivity with bacterial growth. MgO–ZnO solid solution powders were prepared by heating at 14008C for 3 h in air. A single phase with cubic type structure was obtained in the weight ratio range (MgO / ZnO) of 4.0 and 1.5, but the ratio of 0.67 resulted in a ZnO phase in addition to solid solution. After milling the solid solution powders by planetary ball mill, the average particle size and the specific surface area of these powders became 0.1 mm and 26 m 2 / g, respectively, which were used in the test of antibacterial activity. From the results of antibacterial tests, the activity increased with increasing the powder concentration in the medium. With increasing the doping amount of ZnO in MgO–ZnO solid solution, it was found to show a decrease in the antibacterial activity against Escherichia coli and Staphylococcus aureus. The pH value in physiological saline at the powder concentration of 2.5 mg / ml showed the alkali region above 10.0, and decreased with the increase of ZnO amount in solid solution. The decrease in antibacterial activity, therefore, was associated with the decrease of pH value in medium 2000 Elsevier Science Ltd. All rights reserved. Keywords: MgO; ZnO; Solid solution; Antibacterial activity
1. Introduction Microbial pollution and contamination that take place by microorganisms have produced various problems in industry and other vital fields, including medicine, such as degradation and infection, etc. In order to solve these problems, therefore, new pasteurization and antibacterial techniques have been demanded and studied [1–3]. Recently, the occurrence of antibacterial activity by using ceramic powders has been pointed out with much attention as a new technique that can substitute for conventional ones using organic agents. Ceramic powders of zinc oxide (ZnO), calcium oxide (CaO) and magnesium oxide (MgO) were found to show a marked antibacterial activity without the presence of light [4–7]. The use of these ceramics has the following advantages; mineral elements essential to the human body and strong antibacterial activity in small amount without the irradiation of light [8–10]. However, it is not yet clear what change in antibacterial activity is expected by the formation of solid solution among these powders, either reinforcement or *Corresponding author. Tel.: 181-46-291-3148; fax: 181-46-2428760.
cancellation in activity, because these three ceramics are known to form solid solutions easily. In the present work, MgO–ZnO solid solution powders were prepared with the various weight ratios (MgO / ZnO) at 14008C for 3 h in air. After preparing the slurries of the powders obtained, the change in antibacterial activity as a function of MgO–ZnO composition in the solid solution was studied by measuring the change in electrical conductivity with bacterial growth.
2. Experimental
2.1. Preparation of samples and test bacteria MgO and ZnO powders of reagent grade were used as starting materials. These powders were mixed with different weight ratio (MgO / ZnO) and then heated at 14008C for 3 h in air. The as-prepared powder samples of MgO– ZnO solid solution were milled by planetary ball mill. The sample code and the chemical composition of solid solution are listed in Table 1. In order to examine the formation of solid solution, X-ray diffraction measurement (XRD) was carried out and the specific surface area of
1466-6049 / 00 / $ – see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S1466-6049( 00 )00045-3
452
O. Yamamoto et al. / International Journal of Inorganic Materials 2 (2000) 451 – 454
Table 1 Sample code and chemical composition of solid solution powders used in this study Sample code
Mass ratio MgO:ZnO
SS-10 SS-82 SS-64 SS-46
1:0 8:2 6:4 4:6
powder samples was measured. The powder samples obtained were suspended with physiological saline in the concentration range from 1.6 to 100 mg / ml and then the slurries prepared were used in antibacterial tests. Staphylococcus aureus 9779 (S. aureus) and Escherichia coli 745 (E. coli) were used as test bacteria and stored at Tokyo Metropolitan Research Laboratory of Public Health. These bacteria were cultured in Brain Heat Infusion (BHI) at 37C for 24 h on a reciprocal shaker. The bacterial culture was suspended in a sterile physiological saline with a final concentration of approximately 10 3 CFU / ml (CFU: Colony Forming Unit).
2.2. Test of antibacterial activity Antibacterial activity of powder samples was judged by
Fig. 1. Schematic illustration of the apparatus used in antibacterial test.
measuring the change in electrical conductivity with bacterial growth. The apparatus for measuring the conductivity was Bactometer Microbial Monitoring System Model 64 (company: bioMerieux) as shown in Fig. 1. Preparation of bacteria into the wells of a module for the Bactometer was carried out as follows; adding the powder samples into the well containing Modified Plate Count Agar (MPCA) and then dispensing the bacterium suspension into the well. After setting the module in the Bactometer, the change in electrical conductivity was monitored during the incubation at 378C for 25 h in a dark place. The details of the procedures were reported in a previous paper [4]. In order to examine indirectly the pH values when the powder samples were added into the well, the samples were dispersed into physiological saline at a powder concentration of 2.5 mg / ml. After keeping the dispersed solutions for 24 h, the pH values of physiological saline were measured. 3. Results
3.1. Powder samples In Fig. 2, XRD patterns of the powder samples are shown. A single phase of solid solution with NaCl type structure was formed in SS-82 and SS-64 samples. For the SS-46 sample, however, ZnO with the hexagonal wurtzite
Fig. 2. XRD patterns of MgO–ZnO solid solution heated at 14008C for 3 h in air.
O. Yamamoto et al. / International Journal of Inorganic Materials 2 (2000) 451 – 454
type structure coexisted with the solid solution of the cubic phase with the NaCl type structure. This result agreed with the phase diagram [11]. A diffraction line of solid solution with the index of 200 shifted to high-angle side with increasing the amount of ZnO and the lattice constant of the cubic phase changed from 0.211 to 0.204 nm. This shift is considered to be due to the replacement of Mg 21 ions (ion radius: 0.065 nm) with larger Zn 21 ions (ion radius: 0.074 m). ZnO detected in SS-46 sample seems to be due to excess ZnO in the formation of solid solution. After milling the powder samples by planetary ball mill, it was found that the specific surface area and particle size was about 26 m 2 / g and 0.1 mm, respectively.
453
change occurs at the bacterial concentration of about 10 7 CFU / ml in the medium. Fig. 3(a) and (b) show the changes in electrical conductivity of SS-10 and SS-64, respectively, with the incubation time of S. aureus. In the figures, DT (Detection Time) shows the incubation time at which an electrical change can be detected. Hence, if the value of DT is delayed by adding the powder samples, it can be judged that the samples have the effect of an inhibition of the
3.2. Antibacterial activity of powder samples With the growth of bacteria, it is known that electrolytes such as organic and amino acids are produced with the digestion of proteins in the medium [12]. The electrical conductivity in such a growth medium, therefore, increases with an increase of the electrolytes produced, of which the
Fig. 3. The change in electrical conductivity with incubation time on S. aureus. (a) SS-10 and (b) SS-64 sample.
Fig. 4. Comparison of antibacterial activity against (a) S. aureus and (b) E. coli by adding powder samples.
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O. Yamamoto et al. / International Journal of Inorganic Materials 2 (2000) 451 – 454
bacterial growth. In the case of no addition of SS-10 (control), the DT value was approximately 15 h. By adding SS-10, however, the DT value increased with the increase of powder concentration and no DT value was detected in the powder concentration of 6.3 mg / ml (see Fig. 3(a)). The change of DT value of SS-64 was similar to that of SS-10 and no DT was observed in powder concentration of 12.5 mg / ml (see Fig. 3(b)). The results indicate an increase in the antibacterial activity against S. aureus by increasing the powder concentration in medium. Based on the change in electrical conductivity described above, the antibacterial activity of all powder samples prepared was examined on two bacteria, S. aureus and E. coli. Fig. 4(a) and (b) show the comparison of antibacterial activity of four samples on S. aureus and E. coli, respectively. The vertical axis, ‘DT / DT control ’ represents the ratio of the DT value at specified concentration of powder samples to that at no addition of powder samples (control). If the values of DT / DT control are changed with a steep rise at the lower powder concentration, it can be judged to show the stronger antibacterial activity. As shown in Fig. 4(a), with the increase of ZnO amount in solid solution, the pronounced change of the value is observed at high powder concentration, that is, the decrease of ZnO in solid solution results in an effective antibacterial activity on S. aureus. In E. coli (see Fig. 4(b)), the change of DT / DT control value occurs at a little higher powder concentration with the increase of ZnO amount in solid solution than in S. aureus. The change of antibacterial activity of powder samples on E. coli was similar to those on S. aureus. The pH value in physiological saline dispersing powder samples was examined, in order to know the pH value in medium. The value in saline at the powder concentration of 2.5 mg / ml showed pH510.7 in SS-10 sample, 10.5 in SS-82 sample, 10.4 in SS-64 sample and 10.1 in SS-46 sample.
4. Discussion The following four factors may affect the antibacterial activity of ceramic powders: (1) the cations eluted from powder, (2) active oxygen generated from powder, (3) the pH value, and (4) the mechanical destruction of cell membrane [4,6,9,13,14]. However, Yamamoto et al. [4,13] and Sawai et al. [14] reported that factors (1) and (4) had no effect on the activity. In the case of MgO powder, it has been reported that the pH value in physiological saline increases with the increase of powder concentration and antibacterial activity appears with increasing value of pH, i.e., under alkali above pH510 [15,16]. Therefore, it is essential to examine the pH value in physiological saline dispersed powder samples. In the results of the examination, the pH value in saline at the powder concentration of 2.5 mg / ml decreases with an increase in doping amount of
ZnO in MgO–ZnO solid solution. From this result, it is found that bacteria growth is inhibited with increasing the pH value in the medium. Sawai et al. [16] have even found the generation of super-oxide, O 2 2 , from the surface of MgO and considered to have an effective activity for the inhibition of bacterial growth. And also, it has been known that the super-oxide is stable under alkali region and then the diffuse distance of super-oxide increases with increasing pH value [17]. In the present work, therefore, super-oxide is expected to generate from the surface of MgO–ZnO solid solution. Based on the above discussion, the decrease in the antibacterial activity with increasing ZnO content in solid solution is anticipated to be due to the decrease of stability of O 2 2 generated from the surface of the solid-solution and the decrease of pH value in the medium. In conclusion, by measuring the change in electrical conductivity with bacterial growth, it was found that the antibacterial activity against Escherichia coli and Staphylococcus aureus decreased by increasing the doping amount of ZnO in MgO–ZnO solid solution.
Acknowledgements The authors thank Professor Michio Inagaki of Aichi Institute of Technology for his discussion and encouragement.
References [1] Kusaka T, Takagi Y. J Antibact Antifung Agents 1992;20:451. [2] Saito M. J Antibact Antifung Agents 1993;21:17. [3] Tsunoda Y, Egawa H, Yuge O. J Antibact Antifung Agents 1992;20:571. [4] Yamamoto O, Hotta M, Sawai J, Sasamoto T, Kojima H. J Ceram Soc Jpn 1998;106:1007. [5] Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1995;28:288. [6] Sawai J, Kawada E, Kanou F, Igarashi H, Hashimo A, Kokugan T, Shimizu M. J Chem Eng Jpn 1996;29:251. [7] Yamamoto T, Uchida M, Kurihara Y. J Antibact Antifung Agents 1991;19:425. [8] Kurihara Y. New Ceramics 1996;1996:39. [9] Yamamoto O, Sawai J, Hotta M, Kojima H, Sasamoto T. J Mater Sci Soc Jpn 1998;35:258. [10] Sawai J, Yamamoto O, Hotta M, Kojima H, Sasamoto T. J Chem Soc Jpn 1998;1998:633. [11] Segnit ER, Holland AE. J Am Ceram Soc 1965;43:412. [12] Oya A, Banse T, Ohashi F, Otani S. Appl Clay Sic 1991;6:135. [13] Yamamoto O, Sawai J, Ishimura N, Kojima H, Sasamoto T. J Ceram Soc Jpn 1999;107:853. [14] Sawai J, Kojima H, Igarashi H, Hashimoto A, Shoji S, Kokugan T, Shimizu M. J Ferment Bioeng 1998;86:521. [15] Ohkouchi S, Murata R, Ishihara Y, Maeda N, Moriyoshi Y. Inorg Mater 1996;3:111. [16] Sawai J, Kawada E, Kanou F, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1996;29:627. [17] Kobayashi K. Protein, Nucleic Acid and Enzyme 1988;33:2678.
Change in antibacterial characteristics with doping amount of ZnO in MgO–ZnO solid solution Osamu Yamamoto*, Jun Sawai, Tadashi Sasamoto Department of Applied Chemistry, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi-shi, 243 -0292 Japan Accepted 11 May 2000
Abstract Antibacterial activity for MgO–ZnO solid solution was studied by measuring the change in electrical conductivity with bacterial growth. MgO–ZnO solid solution powders were prepared by heating at 14008C for 3 h in air. A single phase with cubic type structure was obtained in the weight ratio range (MgO / ZnO) of 4.0 and 1.5, but the ratio of 0.67 resulted in a ZnO phase in addition to solid solution. After milling the solid solution powders by planetary ball mill, the average particle size and the specific surface area of these powders became 0.1 mm and 26 m 2 / g, respectively, which were used in the test of antibacterial activity. From the results of antibacterial tests, the activity increased with increasing the powder concentration in the medium. With increasing the doping amount of ZnO in MgO–ZnO solid solution, it was found to show a decrease in the antibacterial activity against Escherichia coli and Staphylococcus aureus. The pH value in physiological saline at the powder concentration of 2.5 mg / ml showed the alkali region above 10.0, and decreased with the increase of ZnO amount in solid solution. The decrease in antibacterial activity, therefore, was associated with the decrease of pH value in medium 2000 Elsevier Science Ltd. All rights reserved. Keywords: MgO; ZnO; Solid solution; Antibacterial activity
1. Introduction Microbial pollution and contamination that take place by microorganisms have produced various problems in industry and other vital fields, including medicine, such as degradation and infection, etc. In order to solve these problems, therefore, new pasteurization and antibacterial techniques have been demanded and studied [1–3]. Recently, the occurrence of antibacterial activity by using ceramic powders has been pointed out with much attention as a new technique that can substitute for conventional ones using organic agents. Ceramic powders of zinc oxide (ZnO), calcium oxide (CaO) and magnesium oxide (MgO) were found to show a marked antibacterial activity without the presence of light [4–7]. The use of these ceramics has the following advantages; mineral elements essential to the human body and strong antibacterial activity in small amount without the irradiation of light [8–10]. However, it is not yet clear what change in antibacterial activity is expected by the formation of solid solution among these powders, either reinforcement or *Corresponding author. Tel.: 181-46-291-3148; fax: 181-46-2428760.
cancellation in activity, because these three ceramics are known to form solid solutions easily. In the present work, MgO–ZnO solid solution powders were prepared with the various weight ratios (MgO / ZnO) at 14008C for 3 h in air. After preparing the slurries of the powders obtained, the change in antibacterial activity as a function of MgO–ZnO composition in the solid solution was studied by measuring the change in electrical conductivity with bacterial growth.
2. Experimental
2.1. Preparation of samples and test bacteria MgO and ZnO powders of reagent grade were used as starting materials. These powders were mixed with different weight ratio (MgO / ZnO) and then heated at 14008C for 3 h in air. The as-prepared powder samples of MgO– ZnO solid solution were milled by planetary ball mill. The sample code and the chemical composition of solid solution are listed in Table 1. In order to examine the formation of solid solution, X-ray diffraction measurement (XRD) was carried out and the specific surface area of
1466-6049 / 00 / $ – see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S1466-6049( 00 )00045-3
452
O. Yamamoto et al. / International Journal of Inorganic Materials 2 (2000) 451 – 454
Table 1 Sample code and chemical composition of solid solution powders used in this study Sample code
Mass ratio MgO:ZnO
SS-10 SS-82 SS-64 SS-46
1:0 8:2 6:4 4:6
powder samples was measured. The powder samples obtained were suspended with physiological saline in the concentration range from 1.6 to 100 mg / ml and then the slurries prepared were used in antibacterial tests. Staphylococcus aureus 9779 (S. aureus) and Escherichia coli 745 (E. coli) were used as test bacteria and stored at Tokyo Metropolitan Research Laboratory of Public Health. These bacteria were cultured in Brain Heat Infusion (BHI) at 37C for 24 h on a reciprocal shaker. The bacterial culture was suspended in a sterile physiological saline with a final concentration of approximately 10 3 CFU / ml (CFU: Colony Forming Unit).
2.2. Test of antibacterial activity Antibacterial activity of powder samples was judged by
Fig. 1. Schematic illustration of the apparatus used in antibacterial test.
measuring the change in electrical conductivity with bacterial growth. The apparatus for measuring the conductivity was Bactometer Microbial Monitoring System Model 64 (company: bioMerieux) as shown in Fig. 1. Preparation of bacteria into the wells of a module for the Bactometer was carried out as follows; adding the powder samples into the well containing Modified Plate Count Agar (MPCA) and then dispensing the bacterium suspension into the well. After setting the module in the Bactometer, the change in electrical conductivity was monitored during the incubation at 378C for 25 h in a dark place. The details of the procedures were reported in a previous paper [4]. In order to examine indirectly the pH values when the powder samples were added into the well, the samples were dispersed into physiological saline at a powder concentration of 2.5 mg / ml. After keeping the dispersed solutions for 24 h, the pH values of physiological saline were measured. 3. Results
3.1. Powder samples In Fig. 2, XRD patterns of the powder samples are shown. A single phase of solid solution with NaCl type structure was formed in SS-82 and SS-64 samples. For the SS-46 sample, however, ZnO with the hexagonal wurtzite
Fig. 2. XRD patterns of MgO–ZnO solid solution heated at 14008C for 3 h in air.
O. Yamamoto et al. / International Journal of Inorganic Materials 2 (2000) 451 – 454
type structure coexisted with the solid solution of the cubic phase with the NaCl type structure. This result agreed with the phase diagram [11]. A diffraction line of solid solution with the index of 200 shifted to high-angle side with increasing the amount of ZnO and the lattice constant of the cubic phase changed from 0.211 to 0.204 nm. This shift is considered to be due to the replacement of Mg 21 ions (ion radius: 0.065 nm) with larger Zn 21 ions (ion radius: 0.074 m). ZnO detected in SS-46 sample seems to be due to excess ZnO in the formation of solid solution. After milling the powder samples by planetary ball mill, it was found that the specific surface area and particle size was about 26 m 2 / g and 0.1 mm, respectively.
453
change occurs at the bacterial concentration of about 10 7 CFU / ml in the medium. Fig. 3(a) and (b) show the changes in electrical conductivity of SS-10 and SS-64, respectively, with the incubation time of S. aureus. In the figures, DT (Detection Time) shows the incubation time at which an electrical change can be detected. Hence, if the value of DT is delayed by adding the powder samples, it can be judged that the samples have the effect of an inhibition of the
3.2. Antibacterial activity of powder samples With the growth of bacteria, it is known that electrolytes such as organic and amino acids are produced with the digestion of proteins in the medium [12]. The electrical conductivity in such a growth medium, therefore, increases with an increase of the electrolytes produced, of which the
Fig. 3. The change in electrical conductivity with incubation time on S. aureus. (a) SS-10 and (b) SS-64 sample.
Fig. 4. Comparison of antibacterial activity against (a) S. aureus and (b) E. coli by adding powder samples.
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O. Yamamoto et al. / International Journal of Inorganic Materials 2 (2000) 451 – 454
bacterial growth. In the case of no addition of SS-10 (control), the DT value was approximately 15 h. By adding SS-10, however, the DT value increased with the increase of powder concentration and no DT value was detected in the powder concentration of 6.3 mg / ml (see Fig. 3(a)). The change of DT value of SS-64 was similar to that of SS-10 and no DT was observed in powder concentration of 12.5 mg / ml (see Fig. 3(b)). The results indicate an increase in the antibacterial activity against S. aureus by increasing the powder concentration in medium. Based on the change in electrical conductivity described above, the antibacterial activity of all powder samples prepared was examined on two bacteria, S. aureus and E. coli. Fig. 4(a) and (b) show the comparison of antibacterial activity of four samples on S. aureus and E. coli, respectively. The vertical axis, ‘DT / DT control ’ represents the ratio of the DT value at specified concentration of powder samples to that at no addition of powder samples (control). If the values of DT / DT control are changed with a steep rise at the lower powder concentration, it can be judged to show the stronger antibacterial activity. As shown in Fig. 4(a), with the increase of ZnO amount in solid solution, the pronounced change of the value is observed at high powder concentration, that is, the decrease of ZnO in solid solution results in an effective antibacterial activity on S. aureus. In E. coli (see Fig. 4(b)), the change of DT / DT control value occurs at a little higher powder concentration with the increase of ZnO amount in solid solution than in S. aureus. The change of antibacterial activity of powder samples on E. coli was similar to those on S. aureus. The pH value in physiological saline dispersing powder samples was examined, in order to know the pH value in medium. The value in saline at the powder concentration of 2.5 mg / ml showed pH510.7 in SS-10 sample, 10.5 in SS-82 sample, 10.4 in SS-64 sample and 10.1 in SS-46 sample.
4. Discussion The following four factors may affect the antibacterial activity of ceramic powders: (1) the cations eluted from powder, (2) active oxygen generated from powder, (3) the pH value, and (4) the mechanical destruction of cell membrane [4,6,9,13,14]. However, Yamamoto et al. [4,13] and Sawai et al. [14] reported that factors (1) and (4) had no effect on the activity. In the case of MgO powder, it has been reported that the pH value in physiological saline increases with the increase of powder concentration and antibacterial activity appears with increasing value of pH, i.e., under alkali above pH510 [15,16]. Therefore, it is essential to examine the pH value in physiological saline dispersed powder samples. In the results of the examination, the pH value in saline at the powder concentration of 2.5 mg / ml decreases with an increase in doping amount of
ZnO in MgO–ZnO solid solution. From this result, it is found that bacteria growth is inhibited with increasing the pH value in the medium. Sawai et al. [16] have even found the generation of super-oxide, O 2 2 , from the surface of MgO and considered to have an effective activity for the inhibition of bacterial growth. And also, it has been known that the super-oxide is stable under alkali region and then the diffuse distance of super-oxide increases with increasing pH value [17]. In the present work, therefore, super-oxide is expected to generate from the surface of MgO–ZnO solid solution. Based on the above discussion, the decrease in the antibacterial activity with increasing ZnO content in solid solution is anticipated to be due to the decrease of stability of O 2 2 generated from the surface of the solid-solution and the decrease of pH value in the medium. In conclusion, by measuring the change in electrical conductivity with bacterial growth, it was found that the antibacterial activity against Escherichia coli and Staphylococcus aureus decreased by increasing the doping amount of ZnO in MgO–ZnO solid solution.
Acknowledgements The authors thank Professor Michio Inagaki of Aichi Institute of Technology for his discussion and encouragement.
References [1] Kusaka T, Takagi Y. J Antibact Antifung Agents 1992;20:451. [2] Saito M. J Antibact Antifung Agents 1993;21:17. [3] Tsunoda Y, Egawa H, Yuge O. J Antibact Antifung Agents 1992;20:571. [4] Yamamoto O, Hotta M, Sawai J, Sasamoto T, Kojima H. J Ceram Soc Jpn 1998;106:1007. [5] Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1995;28:288. [6] Sawai J, Kawada E, Kanou F, Igarashi H, Hashimo A, Kokugan T, Shimizu M. J Chem Eng Jpn 1996;29:251. [7] Yamamoto T, Uchida M, Kurihara Y. J Antibact Antifung Agents 1991;19:425. [8] Kurihara Y. New Ceramics 1996;1996:39. [9] Yamamoto O, Sawai J, Hotta M, Kojima H, Sasamoto T. J Mater Sci Soc Jpn 1998;35:258. [10] Sawai J, Yamamoto O, Hotta M, Kojima H, Sasamoto T. J Chem Soc Jpn 1998;1998:633. [11] Segnit ER, Holland AE. J Am Ceram Soc 1965;43:412. [12] Oya A, Banse T, Ohashi F, Otani S. Appl Clay Sic 1991;6:135. [13] Yamamoto O, Sawai J, Ishimura N, Kojima H, Sasamoto T. J Ceram Soc Jpn 1999;107:853. [14] Sawai J, Kojima H, Igarashi H, Hashimoto A, Shoji S, Kokugan T, Shimizu M. J Ferment Bioeng 1998;86:521. [15] Ohkouchi S, Murata R, Ishihara Y, Maeda N, Moriyoshi Y. Inorg Mater 1996;3:111. [16] Sawai J, Kawada E, Kanou F, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1996;29:627. [17] Kobayashi K. Protein, Nucleic Acid and Enzyme 1988;33:2678.