Chemical, Physical and Microbiological Indexes to the Surface Deterioration of Melamine Resin. (Received April 10, 1987)
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1 Chemical, Physical and Microbiological Indexes to the Surface Deterioration of Melamine Resin (Received April 10, 1987) Takiko INOUE, Hajimu ISHIWATA and Kunitoshi YOSHIHIRA (National Institute of Hygienic Sciences: , Kamiyoga, Setagaya-ku, Tokyo, Japan) The surface deterioration of melamine resin caused by repeated treatment with 4% acetic acid at 95C for 30min was determined by chemical, physical and microbiological methods, and the relation between the deterioration of the resin and the number of repetitions of the treatment was determined. The molar concentration ratio (y) of formaldehyde to melamine released into 4% acetic acid decreased with increase in the number of repetitions (x) of the heat treatment, according to the formula in y= in x over 5 repetitions of the treatment. Methylene blue absorption (y) increased accoring to the formula In y= in x between the second and the sixth repetitions of the treatment. Surface roughness (y) increased between the seventh and the 20th repetitions according to the formula lny= nx The retention of water on the surface of the resin increased between the seventh and the 20th repetitions according to the formula lny= In x Escherichia toll (y) washed out into rinsings after the inoculation of the microorganisms on the surface of the resin increased between the fifth and the 20th repetitions of the treatment according to the formula In y= In x These indexes are considered to be most useful in combination with evaluation by exterior observation. Key words: deterioration index; melamine resin; molar ratio of formaldehyde to melamine; methylene blue absorption; surface roughness; Escherichia toll Introduction The long-term use of plastic products induces deterioration caused by light, heat, air and physical impact. Although many methods have been authorized for the determination of the durability of plastic products, few indexes are known for the deterioration of plastic tableware in the field of food hygienics. Baba et al.2) reported deterioration indexes in broad terms such as dirt, flaws, cracks and tarnish, by exterior observation, but statistical evaluation of these results obtained by observation seems difficult. To solve this problem, they developed a methylene blue absorption method* for the determination of the deterioration of tableware. We previously reported that there is a relation *Baba, T., Hosokawa, M., Yamada, A.: The 50th Meeting of the Food Hygienic Abstract p. 29 (1985). Society of Japan, between the ratio of the molar concentration of formaldehyde to melamine (FJM ratio) in the migration solution obtained from cups made of melamine resin, using 4% acetic acid and a repetition of the migration test.3) In the present research, we evaluated the effect of heat treatment at 95 for 30min in the presence of Ono acetic acid on the acceleration of surface deterioration of melamine resin, using chemical, physical and microbiological methods. Experimental 1. Materials and reagents As a sample of melamine resin, cups made of that material obtained from a wholesaler of plastic tableware in Tokyo were used. The cups were 69mm in upper and 50mm in bottom inner diameter, and 85mm in depth. They were rinsed with tap water and distilled water before use. Polyoxyethylene (20) sorbitan monolaurate
2 was of chemical analysis grade (Wako Pure Chemical Industries Ltd., Osaka). All other reagents used were JIS special grade. Melamine, 99% pure, and the hexamethylenetetramine used as a standard substance for the determination of formaldehyde, 99.5% pure, were obtained from Wako Pure Chemical Industries, Ltd., Osaka. Nutrient broth and desoxycholate agar were obtained from Eiken Chemical Co. Ltd., Tokyo. 2. Apparatus A Yanaco Model L-2000 high performance liquid chromatograph (Yanagimoto Mfg. Co. Ltd., Kyoto) equipped with an ODS-A column (250x2.5mm i.d.), a Model 215 spectrometer set at 235nm, and an integrator (Hewlett-Packard 3390A, Pennsylvania, U.S.A.) was used. A Shimadzu UV-240 spectrophotometer (Shimadzu corporation, Kyoto), an SB-35 water bath (Tokyo Rikakikai Co. Ltd., Tokyo) and a surface roughness measuring instrument, Surfcom 5A, (Tokyo Seimitsu Co. Ltd., Tokyo) set at a scanning speed of 0.3mm/sec, and a chart speed of 30mm/sec were used. 3. Test microorganisms Escherichia coli ATCC (E. coli) was used as a test microorganisms to determine surface contamination. 4. Acceleration of the deterioration of melamine resin A 220ml portion of 4% acetic acid preheated to 95C was poured into each cup. The solvent surface was 5mm below the upper edge of the cups. The cups were covered with watch glasses, then allowed to stand at 95C for 30min. After the treatment, the solution was transferred to a flask. When the treatment was repeated using the same cup, the cup was rinsed with a small portion of distilled water, then filled with fresh 4% acetic acid, and the treatment was repeated under the same conditions as above. Cups were handled as aseptically as possible. Three cups were used in each test. 5. Inoculation of E, coli on the surface of the resin E. coli cultured at 37C for 18hr in nutrient broth was diluted 10 times with sterilized saline. Two hundred mililiters of the diluted solution containing 1.05x10cells/ml were poured into the cups and allowed to stand for 5min at room temperature, 25C. 6. Determination 1) Melamine A high performance liquid chromatographic method4) was used. Conditions for the determination of melamine were as follows: mobile phase, 0.1M phosphate buffer at ph3.0; and a flow rate of 0.78ml/min. 2) Formaldehyde Formaldehyde in the migration solution was determined by an acetylacetone method.b) 3) Methylene blue absorption ability The method reported by Baba et al.* was improved. The cups were filled with 220ml of 0.2% methylene blue in a 10% ethanol solution and allowed to stand for 5min, then the methylene blue solution was discarded. The cup was rinsed with tap water for 30sec to remove excess methylene blue, then the methylene blue absorbed on the inner surface was desorbed with 220ml of 4% acetic acid heated to 95C for 30min. After cooling, the optical density of the solution was measured at 665nm. The solution was concentrated to 5ml, if necessary. 4) Surface roughness Cups were cut into strips vertically, and the surface roughness was measured. The roughness is given as the ratio of the actual length of undulating line (cm) to a definite length of reference line (cm) on the chart of the surface roughness (Fig. 3). 5) Moisture retained on the inner surface of cups Cups were filled with water. The water was discarded and the cups were suspended upside down for 10sec to remove excess water from the surface, then the water retained on the inner surface was weighed. 6) Number of E. coli remaining on the surface of the resin A partially modified rinse solution methode6) was used to determine the E. coli remaining on the surface of the cups. An outline of the method follows. After the E. coli suspension was discarded, 20ml of sterilized saline was poured into the cup and the inside was rinsed by rotating the cup slowly for 1min. The rinsings were discarded, and the same process was repeated. Then, 20ml of the sterilized saline containing 1% polyoxyethylene (20) sorbitan mono-
3 laurate was poured in and the inside was rinsed again in the same way for 1min. The rinse solution was diluted with saline, and 1ml of the diluted solution was poured into a plate and mixed with 10ml of desoxycholate agar. The plate was incubated at 37C for 2 days and the resulting colonies were counted. The results are shown as the number of cells in 1ml of the rinse solution. Results 1. Relation between the F/M ratio and deterioration Release of melamine from the melamine resin at the first treatment was pg/ml of the migration solution, and that at the seventh repetition of the treatment showed the maximum concentration, pg/mi. After the seventh repetition, the release of melamine decreased, the concentration at the 20th repetition being pg/ml. The maximum concentration of formaldehyde also occurred at the seventh repetition, but the change in the concentration of formaldehyde was not as large as that of melamine. The F/M ratio (y) decreased biexponentially between the first and fifth treatments according to the following formula; Fig. 1. Relation between the F/M ratio and the deterioration of melamine resin Deterioration of melamine resin was accelerated by the repetition of treatment with 4% acetic acid at 95C for 30min. The ratio of the molar concentration of formaldehyde to melamine released into 4% acetic acid is shown as the F/M ratio. Fig. 2. Relation between the absorption of methylene blue and the number of treatment repetitions The absorbed methylene blue is shown as optical density of the desorption solution. In y= inx , r= , where x=the number of repetitions of the heat treatment and r=the correlation coefficient. After the sixth treatment, the F/M ratio was maintained at , ranging from 1.57 to 1.67 until the 20th treatment (Fig. 1). 2. Relation between the absorption of methylene blue and deterioration The absorption of methylene blue on the inner surface of the resin increased between the second and sixth repetitions according to the formula in y= In x , r=0.9864, where y=the optical density of the desorption solution, x=the number of repetitions of the treatment and r=the correlation coefficient. The absorption of methylene blue on the surface of the resin before the treatment was After the seventh repetition the optical density was maintained at a constant , with a range from to (Fig. 2). 3. Relation between the surface roughness and deterioration Little increase in surface roughness was observed until the fifth repetition of the treatment, but the roughness increased after the seventh repetition, in contrast to the results of the former two experiments. The surface rough-
4 A B C D E F Fig. 3. Change of patterns of surface roughness of melamine resin with repetition of the heat treatment A: before treatment; B, C, D and E: after 5, 10, 15 and 20 treatments, respectively; F: straight reference line for the ratios shown in Fig. 4 Fig. 5. Relation between the retention of water on the surface of the resin and the number of treatment repetitions Fig. 4. Relation between the surface roughness and the number of treatment repetitions The surface roughness is shown as the ratio of the actual length of the undulating line relative to a straight reference line drawn across the same width of the graph (Fig. 3). ness (y) after the seventh repetition increased according to the formula In y= nl x , r=0.9805, where x=the number of repetitions of the treatments and r=the correlation coefficient. Changes in the patterns of the surface and its roughness are shown in Fig. 3 and Fig. 4, respectively. Fig. 6. Relation between the bacterial contamination and the number of treatment repetitions An E. coli suspension (220ml) containing 1.05x10a cells/ml was poured into each cup and allowed to stand for 5min. The cup was rinsed twice with 20ml of saline, then the E. coil remaining on the surface was rinsed out with 20ml of saline containing 1% polyoxyethylene (20) sorbitan monolaurate.
5 4. Relation between the retention of water on the surface and deterioration The retention of water on the inner surface of the resin before the third repetition of the treatment was less than 44mg per cup, but increased to more than 200mg after the fifth repetition (Fig. 5). A biexponential linear relation, In y= In x , r=0.9464, occurred between the seventh and 20th repetitions, where y=the weight (mg) of water retained on the inner surface of cups, x=the number of repetitions of the treatment and r=the correlation coefficient. 5. Relation between bacterial contamination and deterioration The relation between the number of E. coli in the rinse solution and the deterioration is shown in Fig. 6. The bacterial contamination (y) increased between the fifth and 20th repetitions according to the formula In y= nl x , r=0.7983, where x=the number of repetitions of the treatment and r=the correlation coefficient. Before the third repetition of the treatment, the concentration of E. coli was less than 1x103/ml of the rinse solution. Discussion It has been reported that the repeated heat treatment of melamine resin at 95C for 30min in the presence of 4% acetic acid causes a biexponential decrease in the F/M ratio in the migration solution.3) This change in the F/M ratio was reconfirmed in the present study. This relationship can be used as an index of surface deterioration of the resin. In the first stage, before the fifth repetition of the treatment, excess of both hydrolyzed formaldehyde and melamine is released.7) However, in the second stage, after the seventh repetition, the melamine resin is hydrolyzed by the 4% acetic acid into its constitutent elements, melamine and formaldehyde, as indicated by the constant F/M ratio, When cups were heated at 95C for 30min with water,3) the F/M ratio decreased according to the equation In y= In x The particular point at which the F/M ratio reaches 1.60 is considered to be the 183rd repetition of the treatment. Like the F/M ratio, absorption of methylene blue increased biexponentially until the sixth repetition of the treatment, and remained stationary after the seventh repetition. Thus, the methylene blue absorption is closely related to the decrease in the F/M ratio. The surface roughness increased, also biexponentially, but only after the seventh repetition of the treatment. This indicates that there is an essential difference between the chemical and physical indexes of surface deterioration of the resin. The retention of water and bacterial contamination increased, also biexponentially, after the fifth or the seventh repetition of the treatment. Although the number of E. coli in the rinsings was presumably affected by the increased retention of water on the surface of the resin, other effects were also involved, since the number of E. coli in the rinsings obtained from the 20th repetition of the treatment was two orders of magnitude higher than that obtained from the non-treated resin, but the retention of water on the surface increased by only one order of magnitude between the non-treated resin and that treated 20 times. Thus, the retention of E. coli on the surface of the resin is apparently attributable to some direct interaction between the cell surface and the resin, as in the absorption of methylene blue. The absorption of methylene blue by the resin treated 20 times was also two orders of magnitude higher than it was by the non-treated resin. The indexes obtained in the present paper could be classified into two types, one being biexponentially linear before the fifth or sixth treatment and the other also being biexponentially linear but only after the seventh repetition. The surface of melamine resin may deteriorate in two steps, as reflected by the F/M ratio and the methylene blue absorption for the first stage and by the surface roughness, the contamination with E, coli and the retention of water for the second stage. These 5 indexes of the surface deterioration of the resin are all closely related to food hygiene; i.e., the release of components from the plastic, the absorbability of food component, and the adherence and retention of microorganisms and particles of food and water. These are considered to be most useful in combination with evaluation by the exterior observation methods reported by Baba et al.2),8)
6 Acknowledgement The authors are grateful to Dr. T. Maruyama, Tokyo Metropolitan Research Laboratory of Public Health, for the gift of the test microorganisms and to Dr. K. Tsuji, National Institute of Hygienic Sciences, for his helpful advice on determining surface roughness. References 1) Japanese Standard Association: JIS Handbook No. 11 "Plastics", (1983). 2) Baba, T., Hosokawa, M., Yamada, A.: J. Food Hyg. Soc. Japan 27, (1956). 3) Ishiwata, H., Inoue, T., Tanimura, A.: Food Addit. Contam. 3, (1986). 4) Inoue, T., Ishiwata; H., Yoshihira, K., Tanimura, A.: J. Chromatogr. 346, (1985). 5) Pharmaceutical Society of Japan: "Standard Methods of Analysis for Hygienic Chemists" p. 81, (1980), Kanehara Shuppan, Tokyo. 6) Favero, M., Gavis, D. A., Vesley, D: Environmental Monitoring Procedures, p , in "Compendium of Methods for the Microbiological Examination of Foods" 2nd Ed. ed. by Speck, M. L., (1984), American Public Health Association, Washington, D. C., U.S.A. 7) Katan, L. L, ed.: Food, Cosmetics and Drug Packaging 9, No. 1, p. 4-5 (1986). 8) Baba, T., Hosokawa, M., Yamada, A.: J. Food Hyg. Soc. Japan 28, (1987)
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