Synergistic Antimicrobial Effect of Sodium Chloride. essential oil components

Transcription

1 Agric. Biol. Chew., 46 (1), 159~165, Synergistic Antimicrobial Effect of Sodium Chloride and Essential Oil Components Nobuyuki Kurita and Shigeru Koike* Research Institute for Chemobiodynamics, Chiba University, Inohana, Chiba-shi, Chiba 280, Japan *Chiba Prefectural College of Dietitian, 111 Hanawa-cho, Chiba-shi, Chiba 280, Japan Received June 18, 1981 The antimicrobial effect of a variety of essential oil components was examined in the presence of various concentrations of NaCl, using air-borne microorganisms and purely cultured fungi. Even at a NaCl concentration of 15%, various kinds of microorganisms grew in 7 to 10 days of incubation at 27 C. All the essential oil components examined, at a concentration of as high as 1 mm,allowed the growth of various microorganisms within a few days of incubation at 27 C when the NaCl concentration of culture media was less than 3%. However, in the presence of 7 to 10% NaCl, cinnamaldehyde, perillaldehyde, citral (a,/?- unsaturated aliphatic aldehydes), citronellol, perillalcohol and geraniol (primary alcohols) all exhibited a potent antimicrobial effect at a concentration of less than 1 mm.cuminaldehyde and eugenol were also potent in the respect. L-Menthol at 1 mmwas only modest, but at 2mMwas potent in this effect. Citronellal, D-carvone, vanillin, and linalool were only modestly effective, and 1,8-cineole, anethole, and safrole were almost ineffective even at a concentration of 2mM. Hydrocarbons (a-pinene, /?-pinene, camphene, /?-myrcene, /?-caryophyllene, and />-cymene) even at a concentration of 2 mmwere all ineffective under the same condition. These results suggest that certain essential oil components are applicable to effectively preserve foods containing more than 7% There have been manyreports on the antimicrobial effect of essential oils and their components.1 ~14) However, from the practical point of view, these substances by themselves appear only moderately effective or not effective at all to inhibit various kinds of microorganisms from growing. In our preliminary experiments, all the essential oil components examined (e.g., cinnamaldehyde, perillaldehyde, cuminaldehyde, menthol, citronellol and perillalcohol), at a concentration of as high as 1mM or more, individually allowed the considerable growth of various kinds of air-borne microorganisms when using the usual culture media which contain less than 3% Recently we13) found that the fresh leaf of a kind of perilla (called "Aochirimen-shiso" in Japanese), or perillaldehyde, a major component ofperilla essential oil, exhibits a potent antimicrobial effect in the presence of 10% NaCl, while NaCl alone allowed considerable growth of various microorganisms, especially fungi, even at a concentration of 15%or more. Thereafter we found that fresh leaves of Japanese pepper, laurel and a kind of cherry also exhibit individually a potent antimicrobial effect in certain foods containing 7 to 10% Since each of these plants contain a relatively large widely used in amount of essential Japan as a spice, oil, and are it was predicted that ponents, in a variety addition of essential to perillaldehyde, oil com- may also exhibit a potent synergistic effect with antimicrobial In of the present study, various kinds of the antimicrobial effect essential oil components was examined in the presence of 5 to 10% The results possibility described that certain below will suggest the plants high in essential

2 160 N. Kurita and S. Koike oil content are applicable, in place of synthetic preservatives, to effectively preserve some foods which contain 7 to 10% or more of MATERIALS AND METHODS Chemicals. Perillalcohol and /?-caryophyllene were kindly supplied by Mr. Takashi Yasuda of Takasago Perfumery Co., Ltd. The other essential oil components here employed were from commercial sources. Assays of the antimicrobial effect. The growth-inhibitory effect on purely cultured fungi was assayed on 2% glucose Sabouraud agar slants at 27 C. All the fungi employed are listed in Table I. Ten to fifteen day-old culture of each fungus on 2%glucose Sabouraud agar slants at 27 C was used as an inoculum onto control and test media. The duration of inhibition of the fungal growth on a test mediumwas taken as a relative measure of the antifungal activity. Whether fungal growth occurs or not was determined macroscopically. The growth-inhibitory effect on air-borne microorganisms was assayed by an agar plate technique. After the agar plate medium was exposed to air for one hour, the petri dish containing the medium was sealed, and the contaminating microorganisms were incubated at 27 C for up to 20 days. The antimicrobial activity of each compoundwas estimated based on the numberand size of resulting microbial colonies. Each essential oil component to be tested for antimicrobial activity was dissolved in ethyl ether for sterilization, then added to the sterile culture mediumat specified concentrations. The small amount of ethyl ether added with each compound tested did not affect the growth of any microorganisms employed. Spices. Fresh leaves of four kinds of spices were examined for antimicrobial activity. These spices were a kind of perilla (Perilla frutescens Britton var. crispa Decaisne /. viridi-crispa Makino), laurel {Larrus nobilis L.), Japanese pepper (Zanthoxylum piperitum Da), and a kind of cherry {Prunus donarium Sieb.). RESULTS AND DISCUSSION The synergistic antifugal effect of essential oil components and NaCl As shown in Table I, NaCl, at a concentration of 10%, inhibited the growth of all the fungi employed for only less than a few days, and even at 20%its antifungal effect was not very potent on most of the fungi. All the essential oil components examined, at a concentration slightly effective of 1 or 2mM,were also only or virtually inactive in this respect. In general, the growth-inhibitory effect of a compoundis considerably variable depending upon the species or strain of the microorganism. The fungi employed here all appeared relatively resistant to the growth-inhibitory action of any of these substances. However, in the presence of 7% NaCl, the antifungal effects of cinnamaldehyde, perillaldehyde, cuminaldehyde, citronellol and eugenol were potent (Table II). The antifungal effects of citral, geraniol and perillalcohol were also potent under the same condition (data are not shown). In contrast, D-carvone, at a concentration of l mm, and citronellal, vanillin, linalool and D-limonene, even at 2 mm,were all very weak in this synergistic antifungal effect with L-Menthol was modestly effective at 1mM, but fairly potent at 2mMin this respect. In addition to limonene, several other hydrocarbons (a-pinene, /?-pinene, camphene, /?- mycene, /?-caryophyllene and /7-cymene) were also very weak in this effect (data are not shown). The synergistic growth-inhibitory effect of essential oil components and NaCl on air-borne microorganisms The degree ofmicrobial contamination from air is considerably variable depending upon a number of factors, such as the temperature and humidity of the atmosphere, the extent and direction of wind currents, the density of human and animal population, and so on. However, the growth-inhibitory effect of a substance on microorganisms contaminating from air would be, in a practical sense, one of reasonable measures of food-preserving ability. Thus the growth-inhibitory effect of essential oil components on such microorganisms was examined in the presence of various concentrations of NaCl, using 2% glucose Sabouraud agar plates. Brain heart infusion agar was also employed. Both of the agar media gave essentially the same results.

3 Table I. Antifungal Effect of NaCl and Components of Essential Oils ffl 9 I On

4 On Table II. Antifungal Effect of Essential Oil Components in the Presence of 7%NaCl

5 Antimicrobial Effect of Essential Oil Components 163 ClNNAMALDEHYDE (mm) EUGENOL (mm) G 7 10 Fig. 1. Synergistic Growth-inhibitory Effect ofnacl and Cinnamaldehyde or Eugenol on Microorganisms Contaminated by Air. Incubation was made at 27 C for 20 days. (Basal medium: 2% glucose Sabouraud agar.) As shown in Fig. 1, on the agar plate containing both 1 nim cinnamaldehyde and 10% NaCl, any microbial colony did not develop for more than 20 days after contamination, while either of them alone allowed considerable growth of a number of the contaminating air microorganisms within a week after contamination. On the agar plate containing both 0.5mM cinnamaldehyde and 7% NaCl, or containing both 1mMcinnamaldehyde and 5% NaCl, a few colonies developed, but the synergistic antimicrobial effect of cinnamaldehyde and NaCl was still fairly potent. Where the NaCl concentration was 3% or lower, cinnamaldehyde, even at 1mM, could not inhibit the growth of various air microorganisms effectively. One mmof eugenol alone, as well as 10% NaCl alone, allowed considerable growth of various air microorganisms. However,on the agar plate containing both 1 mmeugenol and 10% NaCl, any microbial colony did not develop for more than 20 days after contamination. Judging by the degree of the microbial growth at lower concentrations of eugenol and/or NaCl, the synergistic antimicrobial effect of eugenol appeared somewhat weaker than that of cinnamaldehyde (Fig. 1). Perillaldehyde, citral, cuminaldehyde, perillalcohol, geraniol and citronellol were nearly as potent as eugenol in this synergistic antimicrobial effect with The mechanism of the synergistic antimicrobial action of NaCl and either of these essential oil components yet remains to be clarified. In contrast to these components, D-carvone and citronellal, even at a concentration of 1 mm, were only modestly effective in the synergistic" antimicrobial effect with 10% The synergistic effect of 1 mml-menthol and 10% NaCi was, if any, very weak, but that of 2mML-menthol and 10%NaCl was potent. Linalool was ineffective at a concentration of 1mM, but synergistic slightly effective at antimicrobial effect 2mMin with the 10% Hydrocarbons from essential oils (dlimonene, a-pinene, /?-pinene, camphene, /?- caryophyllene, js-myrcene and />-cymene), at a concentration of as high as 2mM,all failed to exhibit any observable synergistic antimicrobial effect even with 10% Anethole,

6 164 N. Kurita and S. Koike Fig. 2. Food-preserving Effect of Leaves of Perilla, Japanese Pepper, Laurel and Cherry. Food: "Miso" containing no additive (A), containing the leaf of perilla (B), Japanese pepper (C), laurel (D) or cherry (E). Chopped fresh leaf of each plant was separately added to the miso at a ratio of 1/50 (w/w) (see text for details). 1,8-cineole and safrole, at a concentration of as high as 2niM, were also all ineffective in this respect. From the results of the growth-inhibitory effect on air-borne microorganisms and the purely cultured fungi, it is apparent that cinnamaldehyde, perillaldehyde, citral, citronellol, geraniol, perillalcohol, cuminaldehyde and eugenol are potent in the synergistic antimicrobial effect with 7% or more NaCl, whereas any of each by itself is only moderate in its antimicrobial activity. These results suggest that essential oils which contain one or more of these compounds as a major component should be effective in inhibiting the growth of various kinds of microorganisms under the presence of 7% or more The applicability of certain plants to preserve foods of high NaCl content The results described in the preceding sections strongly suggest that certain plants relatively high in essential oil content, such as spices, would exhibit a potent antimicrobial effect in foods which contain 7 to 10%or more Thus, from the practical point of view, we examinedthe antimicrobial effect of several plants widely used in Japan as spices, using a Japanese food, "miso" (a bean paste). Chopped fresh leaves of laurel, Japanese pepper, cherry or perilla were added, at a ratio of 1/50 (w/w), to a home-made miso containing 10% NaCl but without synthetic preservatives. The miso was kept at 27 C with its container sealed. On the miso to which no leaf of the spices was added, a number of fungal colonies developed and spread within a month after storing it at 27 C. In contrast, on the miso which contained the leaves of either of the spices, no microbial colony developed over a period of one year (Fig. 2). These results suggest that certain plants high in essential oil content would be applicable to preserve some foods which contain 10% or more On the other hand, the leaves of these spices failed to exhibit a potent antimicrobial effect in foods of low NaCl content, such as boiled rice and a miso containing only 5% Further studies on the food-preserving effect of various kinds of plants are now in progress in our laboratories. Acknowledgments. Wewish to thank Mr. T. Yasuda for his generous supply of perillalcohol and /?-caryophyllene. Weare also grateful to Mr. Haruo Takahashi of the Chiba Prefectural Institute of Hygiene for his generous supply of the identified fungi employed here. This work was supported in part by a Grant-in-Aid for Scientific Research to N. Kurita from the Ministry of Education, Science and Culture of Japan. REFERENCES 1) K. Okazaki and S. Oshima, /. Pharmaceut. Soc. Jpn., 72, 558, 564, 1131 (1952). 2) K. Okazaki and S. Oshima, J. Pharmaceut. Soc. Jpn., 73, 344, 690 (1953). 3) K. Okazaki and T. Kawaguchi, J. Pharmaceut. Soc. Jpn., 72, 561 (1952). 4) K. Okazaki and A. Honma, J. Pharmaceut. Soc. Jpn., 74, 174 (1954). 5) J. C. Maruzzella and P. A. Henry, J. Am. Pharm. Assoc. Sci. Ed., 47, 294, 471 (1958). 6) D. I. Murdock and W. E. Allen, Food Technol, 14, 441 (1960). 7) L. B. Bullerman, /. FoodSci., 39, 1163 (1974).

7 Antimicrobial Effect of Essential Oil Components 165 S. Miyao, /. FoodHyg. Soc. Jpn., 16, 412 (1975). L. R. Beuchat, /. FoodScL, 41, 899 (1976). L. B. Bullerman, F. Y. Lieu and S. A. Sair, /. Food ScL, 42, 1107 (1977). S. Morozumi, Jpn. J. Med. Mycol, 19, 172 (1978). N. Kurita, M. Miyaji, R. Kurane, Y. Takahara and K. Ichimura, Agric. Biol. Chem., 43, 2365 (1979). 13) N. Kurita and S. Koike, Nippon Nogeikagaku Kaishi, 55, 43 (1981). 14) N. Kurita, M. Miyaji, R. Kurane and Y. Takahara, Agric. Biol. Chem., 45, 945 (1981).