Inhibition of Clostridium botulinum by Antioxidants, Phenols,

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1982, p /82/ $02.00/0 Vol. 43, No. 4 Inhibition of Clostridium botulinum by Antioxidants, Phenols, and Related s N. R. REDDY, M. D. PIERSON,* AND R. V. LECHOWICH Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia Received 28 July 1981/Accepted 14 December 1981 A total of 75 compounds, including antioxidants, preservatives, gallic acid and p-hydroxybenzoic acid esters, hydroquinones, hydroxyquinolines, phenol derivatives, and related compounds, were screened for their antibotulinal activity in prereduced Thiotone-yeast extract-glucose broth. The most effective inhibitors of Clostridium botulinum growth and toxin production were long-chain esters of p- hydroxybenzoic acid and gallic acid, antioxidants, and butylphenol derivatives. The antioxidant nordihydroguaiaretic acid at 100,ug/ml delayed the growth and toxin production for the entire incubation period (7 days). Other antioxidants, such as butylated hydroxytoluene, butylated hydroxyanisole, and tert-butylhydroquinone were also very effective (at 200 to 400,ug/ml) for the inhibition of C. botulinum growth and toxin production. Toxin was detected, although no detectable growth was found by daily absorbance measurements, in the prereduced medium containing 50 to 400,ug of 8-hydroxyquinoline, pentylphenol, tertpentylphenol, 3,5-ditert-butylphenol, 3,5-detert-butylcatechol, (2-hydroxydiphenyl)methane, or (4-hydroxydiphenyl)methane per ml. Chemicals such as derivatives of 5-nitrothiazoles (3), alkyl esters of p-hydroxybenzoic acid (2, 7), certain antioxidants (8), aliphatic alcohols and spice extracts (5), and aliphatic amines and long-chain aliphatic aminodiamides (6) have been evaluated for the inhibition of Clostridium botulinum growth. Robach and Pierson (7) reported that methyl and propyl esters of p-hydroxybenzoic acid at 1,200 and 200,ug/ml, respectively, inhibited the growth and toxin production of C. botulinum 10755A in a prereduced medium. In another study, Robach and Pierson (8) showed that the growth and toxin production of C. botulinum could be inhibited by using various concentrations of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate. Other than in these studies, antioxidants have not been studied extensively for the inhibition of C. botulinum growth and toxin production. Since several antioxidants are used as food additives, it was thought that these and related phenolic compounds might be potential antibotulinal additives in food products such as cured meats. With this objective in view, we screened 75 compounds (antioxidants, phenol derivatives, etc.) for their ability to inhibit the growth and toxin production of C. botulinum strains in a prereduced medium. MATERIALS AND METHODS Chemicals. Tables 1 to 3 list the chemicals selected for the study of the inhibition of C. botulinum growth and toxin production. All of the 75 compounds were purchased from Sigma Chemical Co., St. Louis, Mo., Aldrich Chemical Co., Milwaukee, Wis., and Pfaltz and Bauer, Inc., Stamford, Conn. The chemicals were used without further purification. Except for sodium benzoate (which was dissolved in sterile water), the chemicals were dissolved in 95% ethyl alcohol to a final concentration of 4.0 to 10.0% (wt/vol), filter sterilized (Millipore Corp., Bedford, Mass.; 0.22-,m filter), and stored in sterile glass screw-capped bottles at 4 C. Growth media. The basal prereduced growth medium consisted of 1.0% Thiotone (BBL Microbiology Systems, Cockeysville, Md.), 1.0% yeast extract (BBL), 0.1% glucose, 0.05% cysteine hydrochloride, and % resazurin (TYG medium). The TYG medium was prepared anaerobically by the methods of Holdeman et al. (4). The prereduced TYG medium was adjusted to ph 7.0 with 5 N sodium hydroxide or 5 N hydrochloric acid, dispensed into anaerobic culture tubes (18 by 150 mm; Beilco Glass, Inc., Vineland, N.J.), stoppered under oxygen-free nitrogen, and autoclaved for 15 min at 121 C in a tube press (Bellco Glass, Inc.). The tubes were allowed to cool to room temperature before the addition of chemicals. The chemicals were aseptically added to the desired concentrations (0, 50, 100, 200, 400, 800, and 1,000,ug/ml) while the tubes were sparged with anaerobe-grade carbon dioxide by the VPI Anaerobic Culture System (Bellco Glass, Inc.). Growth studies. Culture tubes containing 10 ml of prereduced TYG medium and the appropriate concentration of chemical were inoculated by the anaerobic methodology of Holdeman et al. (4). The inoculum consisted of a mixture of equal numbers of 10 strains 835

2 836 REDDY, PIERSON, AND LECHOWICH APPL. ENVIRON. MICROBIOL. TABLE 1. C. botulinum inhibition by p-hydroxybenzoic acid esters, gallic acid esters, and antioxidantsa b Inhibition" at concn (i,g/ml) of: ,000 Hydroxybenzoic acid esters o-hydroxybenzoic acid <1 <1 <1 <1 <1 1 Methyl p-hydroxybenzoate <1 <1 <1 <1 <1 1 Ethyl p-hydroxybenzoate <1 <1 <1 <1 5 7 Propyl p-hydroxybenzoate <1 < Butyl p-hydroxybenzoate <1 1 7 Gallic acid esters Ethyl gallate <1 <1 <1 <1 <1 1 Propyl gallate <1 <1 <1 <1 1 6 Butyl gallate <1 < Isopropyl gallate <1 <1 <1 <1 1 2 Isobutyl gallate <1 <1 <1 2 7 Isoamyl gallate < p-octyl gallate <1 <1 <1 7 p-dodecyl gallate <1 <1 7 Antioxidants BHA <1 4 7 BHT Nordihydroguaiaretic acid 3 7 tert-butylhydroquinone <1 < ',4',5'-Trihydrobutyrophenone <1 <1 <1 1 7 a Tubes containing TYG medium and inhibitor were inoculated with C. botulinum spores (2.5 x 102/ml) and incubated for up to 7 days at b 37 C. m-hydroxybenzoic acid, p-hydroxybenzoic acid, gallic acid, pyrogallic acid, methyl gallate, ethoxyquin, a- tocopherol, syringic acid, benzyl alcohol, hydroquinone, methylhydroquinone, 2,5-ditert-butylhydroquinone, and 4-hydroxyquinoline at 1,000,ug/ml did not inhibit the growth and toxin production of C. botulinum for up to 1 day. c Number of days that growth and toxin production were inhibited. of C. botulinum spores (33A, 36A, 62A, 77A, 10755A, 9B, 40B, 41B, 53B, and 67B). The spore crops were prepared according to the procedures described previously (L. A. Smoot and M. D. Pierson, J. Food Sci., in press). The spore inoculum was prepared by diluting the refrigerated mixed spore crop in a prereduced diluent of 0.1% peptone, 0.05% cysteine hydrochloride, and % resazurin (ph 7.0) to a concentration of approximately 2.5 x 104 spores per ml. The spore suspension was heat shocked for 10 min at 80 C, and 0.1 ml of this spore suspension was inoculated anaerobically into each tube. The inoculated tubes of TYG medium were incubated at 37 C. The initial concentration of the spore inoculum in the tubes was approximately 2.5 x 102 spores per ml. Uninoculated controls were also used at each concentration of chemical. The growth of the test organisms in the culture tubes was monitored by determining the absorbance of the tubes at 600 nm in a Bausch & Lomb Spectronic 20 spectrophotometer. Absorbance readings were taken at 24-h intervals for up to 7 days, and quadruplicate tubes were used at each concentration of chemical. Selected tubes with visible growth were checked for C. botulinum toxin. Toxin detection. The culture fluid in the selected tubes was centrifuged at 34,000 x g for 10 min at 4 C to remove cells and debris. The resulting supernatant liquid was divided into two parts. One part was boiled for 10 min to inactivate the toxin, and 0.5 ml of the boiled sample was injected intraperitoneally into each of two mice (ICR white male mice, 18 to 22 g). The unboiled supernatant fluid was injected intraperitoneally (0.5-ml portions) into each of three mice. Each chemical was tested for toxicity by intraperitoneal injection of 0.5 ml of uninoculated TYG broth containing the chemical. RESULTS AND DISCUSSION A total of 75 compounds, including antioxidants, preservatives, phenol derivatives, etc., were screened for the inhibition of C. botulinum growth and toxin production in prereduced TYG medium. The inhibition of C. botulinum by the compounds tested is documented in Tables 1 to 3. Of the 75 compounds screened, 24, at a concentration of 1,000,ug/g of growth medium, did not inhibit the growth and toxin production of C. botulinum for up to 1 day (Tables 1 and 2, footnote b). Seven compounds showed no C. botulinum inhibition at concentrations of up to 800,ug/ml, but did inhibit the growth and toxin production for up to 1 day at a concentration of 1,000,ug/ml.

3 VOL. 43, 1982 TABLE 2. C. botulinum inhibition by phenol derivativesa, b INHIBITION OF C. BOTULINUM 837 Inhibitionc at concn (>tg/ml) of: ,000 Phenol <1 <1 <1 <1 <1 1 2-Phenylphenol < Phenylphenol Isopropylphenol < Isopropylphenol < ,4,6-Trimethylphenol <1 <1 < Chloro-5-methylphenol <1 < Chloro-4,5-dimethylphenol Chloro-2-methylphenol < Chloro-3-methylphenol < Chloro-3,5-dimethylphenol tert-Butylphenol tert-Butylphenol < tert-Butylphenol < ,4-ditert-Butylphenol 4 7 2,6-ditert-Butylphenol tert-Butyl-4-methylphenol tert-Butyl-6-methylphenol Thymol tert-Butylcatechol <1 1 7 Cresol <1 <1 <1 <1 <1 1 m-cresol <1 <1 <1 <1 2 7 o-cresol <1 <1 < p-cresol <1 <1 <1 <1 1 5 Dehydroacetic acid <1 <1 <1 <1 <1 1 Sodium p-chlorobenzoate <1 <1 <1 <1 <1 1 a Conditions were as in Table 1. b Resorcinol, catechol, benzoic acid, sodium benzoate, 2,4,6-tritert-butylphenol, m-methoxyphenol, o- methoxyphenol, p-methoxyphenol, 2,6-dimethoxyphenol, m-aminophenol, and o-aminophenol at 1,000,ug/ml did not inhibit the growth and toxin production of C. botulinum for up to 1 day. c Number of days that growth and toxin production were inhibited. Of the classes of compounds tested, the most effective inhibitors were long-chain esters of gallic acid and p-hydroxybenzoic acid, antioxidants (Table 1), and butylphenol derivatives (Table 2). The antibotulinal activity was increased with an increase in the chain length of the ester for p-hydroxybenzoic acid (C1 to C4) and gallic acid (C1 to C12) esters (Table 1). Similar conclusions were also made by Dymicky and Huhtanen (2) and Robach and Pierson (7) for the esters of p-hydroxybenzoic acid. The maximum antibotulinal activity was observed with long-chain esters, such as the butyl esters of p- hydroxybenzoic acid (C4) and dodecyl gallate (C12), at concentrations of 200 plg/ml of prereduced medium. The short-chain esters (methyl and ethyl gallates and methyl ester of p-hydroxybenzoic acid) were not effective in inhibiting the growth and toxin production, even at high concentrations (800 to 1,000,ug/ml). Medium-chain esters (isobutyl gallate, isoamyl gallate, octyl gallate, and ethyl and propyl esters of p-hydroxybenzoic acid) were effective against the growth and toxin production only at higher concentrations (400 to 800,ug/ml). Octyl gallate and dodecyl gallate added to TYG medium at >400 and >200,ug/ml, respectively, inhibited the growth and toxin production for the entire incubation period, although they were not completely soluble in the prereduced medium. Compared with the findings of Dymicky and Huhtanen (2) and Robach and Pierson (7), we found slightly higher inhibitory levels for the entire incubation period (7 days) for the esters, except the butyl ester, of p-hydroxybenzoic acid. This could be due to the use of mixed A and B strains of C. botulinum in our study. Their observations were based on the growth of only one C. botulinum type A strain in each study. Among the antioxidants tested, BHA, BHT, nordihydroguaiaretic acid, and tert-butylhydroquinone appeared to be very effective at lower concentrations (200 to 400 jig/ml) for the inhibition of C. botulinum growth and toxin production (Table 1). Nordihydroguaiaretic acid at 100 jig/ml delayed the growth and toxin production for the entire incubation period (7 days). BHT appeared to be slightly more effective than BHA in inhibiting C. botulinum growth and toxin production. BHT at a concentration of 50,ug/g of

4 838 REDDY, PIERSON, AND LECHOWICH TABLE 3. s that inhibited detectable C. botulinum growth but allowed toxin productiona Inhibition' at concn (,ug/ml) of: Hydroxyquinoline ND Pentylphenol tert-pentylphenol ,5-ditert-Butylphenol ,5-ditert-Butylcatechol (2-Hydroxydiphenyl)methane (4-Hydroxydiphenyl)methane a Conditions were as in Table 1. b There was no detectable growth of C. botulinum in the presence of each compound listed. Samples were tested for toxin after 3 and 7 days of incubation at 37 C. Numbers represent the day at which toxin was detected; ND, none detected at day 7. medium retarded C. botulinum growth and toxin production for up to 2 days, whereas BHA at the same concentration did not inhibit the growth and toxin production for 1 day. In contrast, Robach and Pierson (8) found that BHA was more effective than BHT when used against single strains (A or B). They obtained significantly lower inhibitory levels for BHT and BHA than we did. This variation could also be related to our use of mixed A and B spores of C. botulinum. The antioxidants propyl gallate and 2',4',5'-trihydrobutyrophenone markedly delayed the growth and toxin production at somewhat higher concentrations (800 to 1,000 jxg/ml) (Table 1). Other antioxidants, such as a-tocopherol, methyl gallate, hydroquinone, methyl hydroquinone, and ethoxyquin, did not inhibit the growth and toxin production for up to 1 day, even at 1,000,ug/ml. These antioxidants appeared to have the least promise as possible inhibitors of C. botulinum. Some of the direct and indirect food additives (benzoic acid, sodium benzoate, dehydroacetic acid, o-hydroxybenzoic acid, and sodium p- chlorobenzoate), aminophenols, methoxyphenols, and cresol (Table 2) showed little or no inhibition at 1,000 jig/ml. A difference in the inhibitory levels was observed for m-, o-, and p- cresols. At 800,ugIml, o-cresol was more effective in the inhibition of the growth and toxin production than were the m- and p-cresols. At 1,000,ug/ml, m-, o-, and p-cresols delayed the growth and toxin production for 7, 6, and 5 days, respectively (Table 2). Thymol was an effective inhibitor of C. botulinum when added at 200,ug/ml (Table 2). Among the phenols listed in Table 2, isopropylphenols and butylphenol derivatives at low concentrations (c400,ug/ml) inhibited the growth and APPL. ENVIRON. MICROBIOL. toxin production of C. botulinum for the entire incubation period. Of the butylphenol derivatives, seven compounds, namely, 2-tert-butylphenol, 3-tert-butylphenol, 4-tert-butylphenol, 2,4-ditert-butylphenol, 2,6-ditert-butylphenol, 2- tert-butyl-4-methylphenol, and 2-tert-butyl-6- methylphenol, delayed the growth and toxin production for the entire incubation period at a concentration of 400,ug/ml or lower. The position of the butyl group on the phenol ring significantly changed the inhibitory levels of butylphenol derivatives. For example, 2,4-ditert-butylphenol was more effective at 100 jig/ml than was 2,6-ditert-butylphenol at the same concentration. Most of the butylphenols are toxic to humans, and their harmful effects need to be studied further before they are considered for application in foods. The addition of 1,000,ug of phenol per ml of medium delayed growth for 1 day, and heavy growth was observed on day 2. Toxin was not detected in this sample when the sample was tested by the mouse bioassay. In contrast to this observation, growth was not detected by daily absorbance measurements during the entire 7-day incubation period in the inoculated medium that contained various concentrations (50, 100, 200, and 400,ug/ml) of 8- hydroxyquinoline, pentylphenol, tert-pentylphenol, 3,5-ditert-butylphenol, 3,5-ditertbutylcatechol, (2-hydroxydiphenyl)methane, and (4-hydroxydiphenyl)methane; however, toxin was detected by the mouse bioassay. We screened these chemicals twice at concentration levels of 50, 100, 200, and 400 jig/ml for growth and toxin production. The results of the second screening are presented in Table 3. Samples were examined for toxin at 3 and 7 days of incubation. In the cases of 3,5-ditert-butylphenol and tert-pentylphenol (at 50 to 400,ug/g of medium), toxin was detected on day 3, even though no change in the absorbance was found. The addition of 8-hydroxyquinoline (at 50, 100, and 200,ug/ml), pentylphenol (at 400,xg/ml), 3,5- ditert-butylcatechol (at 200 and 400,g/ml), and the hydroxymethane derivatives (at 200 and 400,ug/ml) to the growth medium containing C. botulinum spores resulted in the production of toxin between days 3 and 7 of incubation. In all cases, no change in the absorbance at 600 nm was found when measured daily. This may be due in part to limited cell growth and subsequent cell lysis and release of the toxin into the medium. Costilow (1) suggested that it is possible for nonviable spores of C. botulinum to produce toxin in an appropriate medium. However, his research involved the use of a much higher level of spores than we used. From the results presented in this paper, it is clear that several antioxidants, long-chain esters of p-hydroxybenzoic acid and gallic acid, and a

5 VOL. 43, 1982 number of butylphenol derivatives are effective in inhibiting C. botulinum growth and toxin production. These compounds may prove to be useful in direct or indirect food applications. We are currently investigating several of these compounds for their antibotulinal activity in a meat system. Also, certain pharmaceutical, sterilization, and disinfection applications may be derived from these chemicals. ACKNOWLEDGMENT This study was supported by the U.S. Department of Agriculture under agreement no U administered by the Eastern Regional Research Center, Philadelphia, Pa., and the Virginia Polytechnic Institute and State University Agricultural Experiment Station. LITERATURE CITED 1. Costdlow, R. N. 1%2. Fermentative activities of control and radiation-"killed" spores of Clostridium botulinum. J. Bacteriol. 84: INHIBITION OF C. BOTULINUM Dymicky, M., and C. N. Huhtanen Inhibition of Clostridium botulinum by p-hydroxybenzoic acid n-alkyl esters. Antimicrob. Agents Chemother. 15: Dymicky, M., C. N. Huhtanen, and A. E. Wasserman Inhibition of Clostridium botulinum by 5-nitrothiazoles. Antimicrob. Agents Chemother. 12: Holdeman, L. V., E. P. Cato, and W. E. C. Moore Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University Anaerobe Laboratory, Blacksburg. 5. Huhtanen, C. N Inhibition of Clostridium botulinum by spice extracts and aliphatic alcohols. J. Food Protect. 43: Huhtanen, C. N., and T. J. Micich Inhibition of Clostridium botulinum by aliphatic amines and long chain aliphatic aminodiamides. J. Am. Oil Chem. Soc. 55: Robach, M. C., and M. D. Pierson Influence of p- hydroxybenzoic acid esters on the growth and toxin production of Clostridium botulinum 10755A. J. Food Sci. 43: Robach, M. C., and M. D. Pierson Inhibition of Clostridium botulinum types A and B by phenolic antioxidants. J. Food Protect. 42: Downloaded from on March 9, 2019 by guest