Effect of salts on the thermal properties of pectin solution on freezing and thawing

Food Hydrocolloids Vol.5 no.4 pp.393-405, 1991

Effect of salts on the thermal properties of pectin solution on freezing and thawing S.Sawayama and A.Kawabata Faculty of Agriculture, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156, Japan Abstract. The effect of salts on the thermal properties of pectin solutions on freezing and thawing was examined by means of differential scanning calorimetry (DSC). When NaCl or KCl was added to a I % pectin solution, two peaks were observed in both the endothermic and exothermic DSC curves, while a single peak was observed after the addition of CaCl, or MgClz. The presence of two peaks in the former case indicates that the existence of non-freezing water, freezing bound water and free water could be distinguished. Differences between the pectin samples were small, but the effect of added salts was markedly greater.

Introduction Pectins are being widely used in food processing as gelling agents and for other purposes to improve the dispersion, emulsification and stability of foods. With the recently increasing demand for treating and preserving food in low temperature ranges, changes in the food quality by freezing and thawing have been drawing attention. Studies on the behavior of water in macromolecular polymer gels such as agarose gels (1-3), carrageenan gels (4-6) and starch gels (7-10) by differential scanning calorimetry (DSC) have been reported. However, very little is actually known about the thermal properties of pectins by DSC. Light-scattering measurements have been conduced to examine the aggregation induced by the pH value of various pectins with different functional groups (11). In this study the effect of salts on the thermal properties of pectins on freezing and thawing was examined, using the same pectin samples as those reported in the previous studies (11). Materials and methods Pectin samples

Pectin-NF was provided by Sunkist Growers Inc., sodium polygalacturonic acid (PGA) was supplied by Sigma Chemical Co., and two low-methoxyl pectins were also tested. These last two samples were ammonia-demethylated pectin (ALM-pectin) from Sunkist Growers Inc. and acid-demethylated pectin (CLMpectin) from Copenhagen Pectin Factory Inc. Analysis

Pectin content was estimated by determining the anhydrogalacturonic acid (PGA) content by the m-hydroxydiphenyl method (12). Methoxyl content was 393

S.Sawayama and A.Kawabata

determined by measuring the methanol liberated from the pectin by saponification, following the method of Wood and Siddiqui (13). Determination of the amide content was carried out according to the procedure of Black and Smit (14). The weight-average molecular weight (M w ) and radius of gyration (R G ) were examined by the one-concentration method from light-scattering measurements (15,16). The solvent used for the light-scattering method was a 0.1 mol/dm' NaCI solution, and the measuring temperature was 20°C.

Differential scanning calorimetric (DSC) measurement The DSC measurement was carried out with a Sensitive DSC-lO from Seiko Instruments & Electronics Ltd , alpha alumina being used as the reference material. The concentration of each pectin solution was fixed at 1% (dry matter basis), and NaC!, KCl, CaClz or MgClz was added to the pectin solution in varied amounts of 1, 5 and 10% on a w/v basis (concentrations are shown in the figures and tables). These solutions were prepared by soaking a predetermined amount of pectin in a given amount of water for 30 min, dissolving the swollen pectin by heating at 95 to 100°C, and then adding the necessary volume of a salt solution. Each salt-added pectin solution (10 ± 0.1 mg) was sealed into a 15 ILl silver pan. The temperature was lowered by liquid nitrogen at a cooling rate of 2°C/min from ambient to -60°C, after which this temperature was maintained for 10 min and then raised at a heating rate of 2°C/min. In this manner, the exothermic and endothermic peaks could be measured, and the heat of transition, and the contents of non-freezing water (W nf), freezing bound water (W fb) and free water (W f ) were calculated from the areas of these peaks. The amount of frozen water was calculated from the endothermic peak of thawing of frozen pure water measured by means of DSC, and this calculated amount was subtracted from the total water content in the system to give the amount of W nf- In the case of the presence of two peaks during the thawing process, the peak at the lower temperature was regarded as Wfb (17) . The value for pure water of 332.9 mJ/mg was used as the latent heat of thawing for ice . Results and discussion

Mole cular characteristics of the pectins Table I summarizes the numerical results from chemical analysis and lightscattering measurements of the pectin samples in 0.1 rnol/dnr' aqueous NaC!. The values for M; in this table may be considered to refer to single pectin chains , since it was previously found (18) that the molecular weight dependence of R G and intrinsic viscosity was consistent with the behavior usually observed for single randomly coiled chains.

Freezing and thawing beha vior of the pectin-NF solution Freezing and thawing DSC curves of various 1% pectin solutions are shown in 394

Properties of pectin solutions

Table I. Molecular characteristics of pectins studied

Pectin-NF' Al.Mcpectin'' eLM-pectin e PGA f

33 36 22 15

590 670 780 540

Methoxyl content (%)

Amide content (%)

12.08 6.10 6.18

o

o

5.50

o

o

M; and R G were examined by the one-concentration method for light-scattering measurement. • Weight-average molecular weight. bZ-average radius of gyration. "Pectin national formula. d Ammonia-demethylated pectin. e Acid-demethylated pectin. fSodium polygalacturonic acid.

Figure 1. When the temperature of these solutions was lowered to -60°C at a rate of 2°C/min and then raised again at the same rate, a single peak was found at -6SC in the freezing curve of pectin-NF, at -10.0°C in the curve of ALMpectin as one low-methoxyl pectin, at -WAoC in CLM-pectin as the other lowmethoxyl pectin and at -11.2°C in PGA, showing only slight differences between the tested samples. In the case of the thawing curves, each sample showed a large endothermic peak from the melting ice crystals within the range 3.7-4.2°C. It seems that such differences, although slight in degree, originated from differences in the molecular weight, molecular size and functional groups. It is known that the addition of a small amount of a divalent metal salt to a pectin solution causes association of the pectin molecules and enhances gelation (16,19) and, depending on the conditions, imparts various characteristics to the pectin gels (20,21). Monovalent metal salts are frequently used during the processing and cooking of food. In consequence, it is important to understand changes in the thermal properties of a pectin solution by the addition of salts. Freezing and thawing DSC curves of NaCl-added 1% pectin-NF solutions are shown in Figure 2. As the concentration of NaCI was increased, the first exothermic peak in the freezing curve shifted to a lower temperature from -10.9 to -14.9 and further to -16.1°C, while the second peak shifted to a higher temperature from -40.2 to -39.0 and further to -38.7°C. A similar tendency was found in the endothermic peak: the first peak in the thawing curve shifted to a slightly higher temperature from -22.1 to -20.0 and then to -19.1°C, with a slightly enlarged peak area. The second peak shifted to a lower temperature from 2.4 to -1.0 and further to -6.0°C. Two peaks were observed in both cases, and the difference between the lower and higher peak temperatures became less as the concentration of NaCI was increased. The appearance of two peaks indicates that the presence of at least three kinds of water can be distinguished from one another: that is, Wn f , Wfb and Wf • The total water (W t ) content in the system is represented by the following formula:

395

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The amount of frozen water was calculated from the thawing peak of ice measured by means of DSC, and this calculated amount was subtracted from the total water content in the system to give the amount of non-freezing water. The enthalpy was calculated from the area of the high-temperature peak to obtain the amount of bound water and free water. Freezing and thawing DSC curves of KCl-added 1% pectin-NF solutions are shown in Figure 3. As the concentration of KCI was increased, the first

396

Properties of pectin solutions

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Fig. 2. Freezing and thawing DSC curves after adding NaCl to the 1% pectin-NF solutions. Concentration of NaCI : A, 1% (0.171 mol/dm "); B, 5% (0.856 mol/drn"); C, 10% (1.711 mol/dm"). °Tp lo .oTp 2 '

exothermic peak in the freezing curve shifted to a lower temperature from -9.9 to -11.2 and further to 13.9°C, while changes in the second peak were not significant (-17.1 , -16.4 and -17.1°C, in that order) . Regarding the thawing peaks , the first peak shifted to a higher temperature as the concentration of KCI was increased (-10.7, -9.2 and -8.0°C, in that order), while the second peak shifted to a lower temperature from 3.0 to -0.4 and further to -3.6°C, thus showing a tendency for the two peaks to approach each other as the concentration of KCI was increased. Freezing and thawing DSC curves of CaClr added and MgClz-added 1% pectin-NF solutions are shown in Figures 4 and 5 respectively. In the case of either salt , only a single exothermic peak and a single endothermic peak were observed , with no trace of the second peaks, as was the case with Na + and K+. There was a tendency for the exothermic and endothermic peaks to shift to a slightly lower temperature with increasing salt concentration, the ionic radius and hydration phenomenon probably being concerned in such results. 397

S.Sawayama and A.Kawabata

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Fig. 3. Freezing and thawing DSC curves after adding KCl to the 1% pectin-NF solutions. Concentration of KCI: A , 1% (0.134 rnol/drrr'}; B , 5% (0.671 rnol/drrr'); C, 10% (1.341 mol/drrr'). °Tp t> . oTp2 '

Table 2 shows properties of water obtained from freezing and thawing DSC curves of salt-added 1% pectin-NF solutions. The size of the ionic radius is in the order of K+ > Na+ > Ca2+ > Mg'", with the hydration entropy being in the same order (22). In each case of the added salt, the proportion of free water showed a tendency to decrease with increasing salt concentration. Especially, in the case of the thawing DSC curve by adding KCl or NaCl, two peaks were observed; the lower temperature peak seems to have originated from the freezing bound water, and the other higher temperature peak from free water. When the concentration of each of these two salts was 1%, the proportion of freezing bound water was low (NaCl , 2.8%; KCl, 4.7%), while the proportion was high when the salt concentration was 10% (NaCl, 41.6% ; KCI, 65.3%). In the case of adding CaCh or MgCh , the presence of freezing bound water was not observed and, instead , non-freezing water was observed in a high proportion.

398

Properties of pectin solutions

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Such a result indicate s that , because a pectin molecule (especially one having free carboxyl groups) is likely to combin e with divalent metal ions, water was incorporated into the pectin network and, as a result, the proportion of nonfreezing water increased. Peak temperature by fr eezing and thawing the salt-added pectin solution

Peak temperatures of the freezing and thawing DSC curves for pectin-NF and ALM-pectin solutions are shown in Figure 6(a), and those for CLM-pectin and PGA solutions in Figure 6(b). When NaCI was used , two peaks were obser ved in the freezing and thawing DSC curve of the pectin solutions. The lower temperature peak in the freezing curves was slightly lower for the two LM-pectins (ALM and CLM) , but with a tendency to increase slightly with increasing salt concentration , although within

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Free water (%)

14.4 12.3 11.6

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Salt (%)

(mol c.)

Non-freezing water (gig) (%)

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1 (0.171) 5 (0.856) 10 (1.711)

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1 (0.134) 5 (0.671) 10 (1.341)

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400

Properties of pectin solutions

(a)

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a range close to -40°C. The higher temperature peak was roughly within the range -10 to -20°C. There was a trend for the lower- and higher-temperature peaks to become closer as the salt concentration increased. The lowertemperature peak in the thawing curves was --20°C in each case. The higher temp erature was at 4 to 5°C for the addition of 1% NaCI, and tended to shift to a lower temperature (-5 to - 6°C) as the salt concentration was increased. Similar to the case of NaCl , two peak s were observed in the freezing and thawing DSC curves for all of the KCl-added pectin solutions. However, the 401

S.Sawayama and A.Kawabata (b)

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lower-temperature peak in the freezing DSC curves was higher (-15 to -20°C) than in the case of NaCI, and the higher-temperature peaks were mostly -lOoC. The lower-temperature peak in the thawing curves was also higher (~-100C) than in the case of NaC!, With the addition of CaCl z and MgCl z, as shown in Figure 6(a and b), no significant difference was observed between these salts in terms of the DSC peak temperatures of each pectin solution, Endothermic enthalpy by thawing the salt-added pectin solutions Data for the endothermic enthalpy on thawing the four frozen salt-added 1% 402

Properties of pectin solutions

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Fig. 7. Rel ationship between the endothermic enthalpy by thawing and the concentration of various salts. • NaCI, • .6 H in P2 , 0 KCI, 0 .6H in PI ' '" CaCI2 , .6 MgCI2 • wI, free water; w2, freezing bound water.

pectin solutions are summarized in Figure 7. Since two endothermic peaks were observed in each case for the addition of NaCI and KCI, WI (free water ) and W 2 (freezing bound water) in the figure are shown as the endothermic enthalpy calculated from the thawing peaks of free water and freezing bound water respectively. Although differences among the pectin samples were not clear because of their low concentration compared to the concentration of salts, CLMpectin showed a slightly higher endothermic enthalpy in general. Since CLMpectin has the largest radius of gyration compared to its molecular weight as 403

S.Sawayama and A.Kawabata

shown in Table I, such a result supports the idea that the network structure of molecules in this pectin is loose . The endothermic enthalpy of PGA was slightly smaller than that of the other pectin samples, probably because of its lowest molecular weight among the tested samples. Non-freezing water by freezing the salt-added pectin solutions

The amount of non-freezing water by freezing the four salt-added 1% pectin solutions is shown in Figure 8. The proportion of non-freezing water by freezing each 1% pectin solution without salt to the whole water content of the system is indicated by a double circle. This ratio of non-freezing water was remarkably small (2-6%), although such water could be regarded as being strongly coordinated to pectins. Among the tested salts, monovalent NaCl and KCl showed smaller effects, even with the decreasing tendency for non-freezing water with increasing concentration of K+ in the case of pectin-NF and ALMand CLM-pectins. In the case ofthe divalent salts, CaCh and MgClz, the amount of non-freezing water increased as the salt concentration increased. Especially, 60% or more non-freezing water content was observed in all the tested pectin solutions after the addition of 10% MgCIz Such a high proportion of nonfreezing water by the addition of divalent metal salts indicates that these metal ions are coordinated into the network structure of the pectin molecules, thus making freezing of the water difficult. If conformation of pectin does not change, the carrier that binds water does not change either. However, such change is conceivable because conformation of the pectin changed with the existence of divalent ions. In particular, when the concentrations of Caz+ and Mgz+ were increased, it is conceivable that the values of Wnf increased, because the system as a whole becomes more difficult to freeze to the bound water molecule when it coordinates into the network structure of the pectin molecules. A

B

D

C

70

'i

P

':'60 til

;50 til

~

40

til til

.:;: 30

c ~ 20 10

@ 5

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5

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Concentration of salt (%)

Fig. 8. Amount of non-freezing water by freezing the four salt-added pectin solutions . A, pectin-NF; B, ALM-pectin; C, CLM-pectin; D, PGA . 0 NaCl, • KCI, c: CaCI2 , . . MgCI2 , e non-added pectin.

404

Properties of pectin solutions

Conclusion The th ermal properties of pectin solutions by freezing and thawing were examined. When freezing and thawing DSC curves of 1% pectin solutions were constructed, a single peak was found at -6.5°C in the freezing curve for pectinNF , at -lO.O°C in the curve for ALM-pectin, at -lO.4°C for CLM-pectin and at -l1.ZoC for PGA, while a large endothermic peak by the melting of ice crystals was observed within the range 3.7-4.ZOC in the case of the thawing curves for the pectin solutions. When NaCI or KCl was added to the 1 % pectin solutions, two peaks were observed in each of the freezing and thawing curves , while each curve showed a single peak after the addition of CaCl2 or MgCI 2 • Since the appearance of two peaks in the freezing and thawing curves by the addition of NaCI or KCl indicated that the presence of at least three kinds of water could be distinguished , the exothermic enthalpy was calculated from the thawing peak to obtain the proportions of non-freezing water, freezing bound water and free water. Differences between the pectin samples were small but the effect of added salts was markedly greater. Acknowledgement This study was supported in part by a grant-in-aid from the Salt Science Foundation. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Watase,M . and Nishinari,K. (1986) Food Hydrocoll., 1,25-36. Watase,M. and Nishinari,K. (1987) Makrornol. Chem . , 188, 1177-1186. Watase,M . , Nishinari ,K. and Hat akc yama,T. (1988) Food Hydrocoll. , 2, 427-438. Wata se ,M . and Nishinari,K. (1985) Nippon Shokuhin Kogyo Gakkaishi , 32, 630-638. Wata se,M. and Nishinari ,K. (1987) Makr ornol. Chem ., 188,2213-2221. Watase ,M ., Nishinari ,K. , Williams,P.A . and Phillips,G .O. (1990) Food Hydrocoll. , 4, 227-237. Roulet ,Ph . , Raemy,A . and Wuersch ,P. (1987) Food Hydrocoll., 1,575-578. Biliaderis,C.G ., Maurice ,T .T. and Vose,J .R . (1980) s. Food Sci. , 45, 1169-1674 . Biliaderis,C.G ., Page ,C.M ., Sladc ,L. and Sirell,R.R. ( 1985) Carbohyd r. Polym. , 5, 367-389 . Ish ida ,N. , Kobayashi,T . and Kainuma,K . (1988) Nippon Shokuhin Kogyo Gakkaishi, 35, 98-

104. Sawayama,S., Kawabala ,A .. Nak ahara,H . and Kamala,T. (1988) Food Hydroco//., 2, 31-37. Blumenkrantz,N. and Asboe -Han sen.G , (1973) Anal. Biochern. , 54, 484-489. Wood,P.J. and Siddiqui,I.R. (1971) Anal. Biochem., 39, 418-428. Black,S .A. and Smit,G.J.B. (1972) l . Food Sci., 37, 726-729. Kamata,T. and Nakahara,H. (1973) J, Coli. Interface Sci., 43, 89-96. Kawabala,A., Sawayama,S. and Kamata,T . (1979) Nippon Nogeika gaku Kaishi, 53, 61-67. Halakeyama,T., Nakamura,K. and Hatakeyarna.H. (1988) Thermochem . Acta, 123, 153-161. Kawabala ,A. and Sawayama,S. (1977) Nippon Nogeikagaku Kaishi , 15, 15-20. Kawabata,A., Sawayama,S ., Nakahara,H. and Kamata,T. (1981) Agric. Bioi. Chem . , 45, 965973. 20. Kawabata,A. and Sawayama,S. (1975) Eiyo to Shokuryo, 28, 17-24. 21. Kawabata.A,, Sawayama,S., Nagashima ,N . and Uchimura,Y. (1981) Kaseigakukaishi, 32, 73911. 12. 13. 14. 15. 16. 17. 18. 19.

744. 22. Ar akawa,K ., Tokiwano,K ., Ohtomo.N. and Uedaira,H . (1979) Bull . Chern. Soc. Jpn, 52,24832488.

Received on December 10, 1m; accepted on March 13, 1991

405