An antimicrobial and antifungal agent derived from montmorillonite

An antimicrobial and antifungal agent derived from montmorillonite

Applied Clay Science, 6 (1991) 135-142 135 Elsevier Science Publishers B.V., Amsterdam An antimicrobial and antifungal agent derived from montmoril...

790KB Sizes 1 Downloads 10 Views

Applied Clay Science, 6 (1991) 135-142

135

Elsevier Science Publishers B.V., Amsterdam

An antimicrobial and antifungal agent derived from montmorillonite Asao Oya, Takanobu Banse, Fumihiko Ohashi and Sugio Otani Faculty of Engineering, Gunma University, Kiryu, Gunma 376, Japan (Received April 26, 1990; accepted after revision February l, 1991 )

ABSTRACT Oya, A., Banse, T., Ohashi, F. and Otani, S., 1991. An antimicrobial and antifungal agent derived from montmorillonite. Appl. Clay Sci., 6: 135-142. Montmorillonite sol was passed through a column charged with the cation exchange resin supported by Ag + to convert into Ag+-exchanged montmorillonite sol. The thin film and fine particles were prepared from the resulting Ag +-exchanged montmorillonite sol of which the Ag content was about l 0 wt%. These specimens exhibited strong antimicrobial activity to both Staphylococcus aureus and Escherichia coil, and antifungal activity to Aspergillus niger. Fine silver particles were deposited more abundantly with increasing the heating temperature to 800 ° C, leading to a lowering of antimicrobial activity.

INTRODUCTION

Change of our life style asks for a new type of antimicrobial and antifungal agent instead of a conventional organic agent with a low durability and a loose fixing on a substrate. Recently a novel antimicrobial agent, an Ag+-ex changed zeolite, was developed and commercialized in Japan (Hagiwara and Nohara, 1985 ). On the other hand, the present authors have been attempting to develop new functional materials by using the ion exchange reaction of clay mineral (Oya et al., 1987; Oya et al., 1988 ) and found independently that the Ag+-exchanged montmoriUonite (abbreviation in this paper: Ag-Mont) shows very strong antimicrobial and antifungal activities (Oya et al., 1990). Fundamental mechanisms of such activities are not revealed yet, but this agent seems to be quite promising in view of its simple preparation procedure and being a durable inorganic material. The purpose of this paper is to describe the preparation procedure, structure, antimicrobial and antifungal activities of Ag-Mont. 0169-1317/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.

136

A. 0YA ETAL.

EXPERIMENTAL

Raw materials and preparation procedure of Ag-Mont Raw montmorillonite from Aterasawa, Yamagata, Japan, was kindly supplied by Kunimine Ind. Co. Fractions smaller than 2/zm (cation exchange capacity: 107 meq/100g) were collected by the sedimentation technique. Its chemical formula is M(A13.4oFe3+o.o9FeE+o.o6Mgo.47) (Si7.65A1o.35)O2o(OH )4 • nH2o, where M is an alkali metal. The cation exchange resin is Amberlite IR118 produced in Organo Co. Inc. in Tokyo. The other agents used are all reagent grade. The resin charged in a column (40 cm length and 3 cm in thickness) was first converted into the H+-exchanged one by passing a 2N HC1 solution and furthermore into the Ag+-exchanged one by 0.5N AgNO3 solution. The AgMont sol was prepared by passing the raw montmorillonite sol (about 1 wt.%) through this column, followed by centrifugal concentration to about 3.5 wt.% sol. A part of the Ag-Mont sol was subjected to spray-drying to make fine particles. Another part of the Ag-Mont sol was mixed with a prefixed amount of the raw montmorillonite sol to prepare the Ag-Mont containing various Ag contents, followed by preparation of films according to the method reported elsewhere ( 0 y a et al., 1986 ). The specimens thus prepared were heat-treated to prefixed temperatures below 800 °C under air, nitrogen or carbon monoxide. Unless otherwise noted, however, the specimen was heated at a prefixed temperature for 1 hr under nitrogen. In this paper, the specimens are labelled by appending the heattreatment temperature ( X 10-2°C) after Ag-Mont e.g., Ag-Mont-0 and AgMont-4 are the Ag+-exchanged montmorillonites before heating and after heating to 400 °C for 1 h under nitrogen, respectively.

Measurements The Ag content in a specimen was measured by using the atomic absorption method after decomposing Ag-Mont by the HF/H2SO4 mixed acid. Ag-Mont particles and the fracture surface of the Ag-Mont film were observed by a scanning electron microscopy (SEM). The Ag-Mont particles before and after heating were observed with transmission electron microscopy (TEM) in order to study the deposition state of silver particles. The film was attached on a glass plate for X-ray diffraction analysis using C u K a radiation. Antimicrobial and antifungal tests were carried out as follows. A culture medium melted was poured in a Petri dish and solidified. Then the medium containing bacteria or mold was layered over it. The Ag-Mont specimen was put on it and then incubated. Incubation conditions were 1 day at 37°C for the bacteria and 5 days at 28 °C for the mold, respectively. Antimicrobial and

137

ANTIMICROBIAL AND -FUNGAL AGENT DERIVED FROM MONTMORILLONITE

antifungal activities were evaluated by the transparent halo around the AgMont specimen after incubation (so-called halo test), that is, when a agent has antimicrobial or antifungal activity, the halo is formed along the pedphcry of the agent. In this work, a conventional and Waksman ager culture media were used for antimicrobial and antifungal tests, respectively. The composition of the conventional agar culture medium is an extract of meat (5 g), peptone ( l0 g), NaC1 (5 g), agar powder ( 15 g) and water ( 1000 ml). The pH of this medium was controlled between 6.8 and 7.2. The composition of the Waksman ager culture is grape sugar (10 g), peptone (5 g), K H 2 P O 4 ( 1 g), MgSO4"7H20 (0.5 g), agar powder ( 15 g) and water ( 1000 ml) with a pH of 5-6. Two bacteria (Staphylococcus aureus ATCC 6538p, Escherichia coil IFO 3301 ) and two molds (Aspergillus niger FERM S-1 and Penicillium citrium FERM S-5 ) were used in this work. RESULTS AND DISCUSSION

SEM observation Figure 1 show SEM photographs of the Ag-Mont particles before and after heating to 800 ° C. The particles with less than several 10 gm are irregular in shape. A particle had a petal-like structure under a higher magnification. Such structure remained substantially unchanged even after heating to 800 oC. Figure 2 shows a SEM photograph of the fracture surface of the film before heat-

Before healing

Before heating

800"C

Fig. 1. SEM photographsof Ag-Montpanicles before and after heatingto 800°C.

138

A.0YAET AL

Fig. 2. SEM photograph of the fracture surface of Ag-Mont film before heating. t ~

Ag-M-8

L

Ag-M-6

I~

~

Ag-M-5

~

J,

Ag-M-O

~.

A

M~':'r" : ":t ~-_Ji~ 10 20

30

o:Ag o

?

40 50 2e (CuK~)

60

70

80

Fig. 3. Change of X-ray diffraction profiles of Ag-Mont film with heating temperature.

ANTIMICROBIAL AND -FUNGAL AGENT DERIVED FROM MONTMORILLONITE

139

o

lO' ='m Fig. 4. TEM photographs of Ag-Mont panicles before and after heating to 600 ° and 800°C. ing. The film was about 10 g m in thickness and showed a preferred orientation o f the flaky Ag-Mont particles. The film was very flexible.

X-ray diffraction analysis Figure 3 shows the change o f X-ray diffraction profiles o f the Ag-Mont film

140

A. 0YA ET AL.

with heat-treatment. There was no substantial change in X-ray diffraction profiles of the specimens heated below 300 ° C. After heating to 600 ° C, some peaks appeared at 2.342 A, 2.031 A, 1.440 A and 1.229 A and strengthened by heating to higher temperature. These are all assigned to silver (ASTM File No. 4-783 ). The Ag-Mont somewhat remained even after heating to 800°C. TEM observation TEM photographs of the Ag-Mont particles before and after heating are shown in Fig. 4. Fine black spots on the photographs seemed to be silver particles through the comparison with X-ray diffraction data. The silver particles were not necessarily deposited homogeneously throughout the specimen. In the Ag-Mont before heating (Ag-Mont-0), fine silver particles can be seen rarely together with the coagulated large silver particles (see arrow). Heating to 600°C resulted in the denser deposition of the fine silver particles (AgMont-6 ). After heating to 800 ° C, the silver particles increased remarkably in both distribution density and size (Ag-Mont-8). Antimicrobial and antifungal tests In some cases it was difficult to show the real situation after the halo test on the photograph. The results of the halo test described below, therefore, were mainly based on the observation by the naked eye. At first antimicrobial activities of the Ag-Mont films with various amounts of Ag content were examined. MontmoriUonite without Ag (reference sample) showed no halo for both bacteria, which means no antimicrobial activity of the raw montmorillonite. The Ag-Mont containing 10.3 wt.% of Ag resulted in a very d e a r halo around the specimen. A thin halo was also observed in the case of the AgMont containing 2.8 wt.% of Ag in both cases. Antimicrobial activity of the Ag-Mont lowered remarkably after heating to 750 ° C, that is, the Ag-Mont samples containing l 0.3 and 4.4 wt.% of Ag, after heating to 750 ° C, showed a slight activity and no activity against Escherichia coli IFO 3301, respectively. According to TEM photographs and XRD analysis, it is clear that the size of the silver particles increases with the heat treatment. It is reasonable to conclude, therefore, that the antimicrobial activity fo Ag-Mont depends on the availability ofAg + or of very fine silver particles not showing an easily detectable XRD pattern. The Ag-Mont with 10.3 wt.% of Ag was heated at 500 and 600°C under various pressures and subjected to antimicrobial tests using Escherichia coil IFO 3301. All specimens exhibited a clear halo along the sample periphery, showing an antimicrobial activity regardless of the heating pressure as shown in Fig. 5. Figure 6 shows antifungal activities of the Ag-Mont with 10.3 wt.% of Ag,

ANTIMICROBIALAND-FUNGALAGENTDERIVEDFROMMONTMORILLONITE

] 41

Fig. 5. Antimicrobial activity of Ag-Mont films (Ag content: 10.3 wt.%), after heating to 500 ° C under various atmospheres, against Escherichia coli IFO 3301.

Penicittium citrinum FERM 5-5

AspergitLus niger FERM $-1

Fig. 6. Antifungal activity ofAg-Mont (Ag content: 10.3 wt.%) after heating to 500 ° and 600°C.

142

A. OYA ET AL.

after heating to 500 ° and 600°C, against Aspergillus niger F E R M S-l and Penicillium citrium F E R M S-5. The specimen was active to the former mold alone. As stated above, Ag-Mont shows a very strong antimicrobial activity and antifungal activity against Aspergillus niger. This specimen can be prepared quite easily and is favorable to make films because of its thin flaky shape. In addition, different from a conventional organic agent, this is an inorganic and thermostable agent. This agent seems to be so promising. For practical applications, however, some problems remain to be solved, e.g., how to suppress reduction of Ag + into silver particles on standing and how to suppress releasing of Ag + into water. Further details will be reported later. ACKNOWLEDGEMENTS The authors wish to thank Kunimine Ind. Co. Ltd. for supplying raw montmorillonite and Daiwa Chemical Ind. Co. for antimicrobial and artifungal tests.

REFERENCES Hagiwara, Z. and Nohara, S., 1985. An antimicrobial agent using zeolite as a support and its preparation procedure. Japanese Patent Publication 1985-181002. Oya, A., Omata, Y., Kizu, K. and Otani, S., 1986. SEM study on the fractured surfaces of films and block prepared from montmorillonite and its complex with a-naphthylamine. J. Mater. Sci., 21: 1219-1226. Oya, A., Kikuchi, H. and Otani, S., 1987. Characteristics of mouldings made from the saponite/ acriflavine complex after heating under nitrogen. J. Mater. Sci., 22:1045-1050. Oya, A., Matsunaga, K., Satou, A., Mita, H. and Otani, S., 1988. Changes of tensile strength and electrical resistivity of taeniolite/acriflavine complex film by carbonization. J. Mater. Sci. Lett., 7: 985-986. Oya, A., Takeda, Y. and Iino, M., 1990. An antimicrobial silicate having a film-forming ability. Japanese Patent Publication 1990-19308.