Properties of Alkyl Hydroxycinnamates and Effects on Pseudomonas fluorescenst

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1983, p /83/1218-5$2./ Copyright 1983, American Society for Microbiology Vol. 45, No. 1 Properties of Alkyl Hydroxycinnamates and Effects on Pseudomonas fluorescenst J. D. BARANOWSKI AND C. W. NAGEL* Department of Food Science and Technology, Washington State University, Pullman, Washington Received 7 June 1982/Accepted 13 September 1982 Alkyl esters of six hydroxycinnamic acids, shown to be active antimicrobial agents when tested against Pseudomonas fluorescens, were further investigated for their effects against this organism. There was no statistically significant adaptation by this organism to either of the methyl esters of caffeic, p-coumaric, cinnamic, or p-hydroxybenzoic acids. Mixtures of these compounds taken two at a time gave at least additive effects, with some mixtures showing synergism. Preliminary work was also performed to determine the mode of inhibitory action for these compounds. The inhibition of oxygen utilization by the methyl esters correlated well with growth inhibition. Short-term lethality studies showed that none of the alkyl esters or methyl or propyl paraben produced any bacteriocidal effects. Oil-water partition coefficients were determined for these compounds and were shown to have no correlation with growth inhibitions. These all point to a specific mode of action, based in part on cellular energy depletion, rather than the nonspecific membrane-disrupting effects of other phenolic antimicrobial agents. Phenolic antimicrobial agents, including benzoic acid and the parabens, have had a long history in food usage. Closely related to benzoic acid are the naturally occurring cinnamic acid derivatives. Although generally recognized for their antimicrobial activities, very little work has been done on their range of activity or on their mode of action. Depending on the test system used, certain of the hydroxycinnamic acids show considerable antimicrobial activity. Caffeic acid has been found to be active against potato fungii (23), ferulic and p-coumaric acids are known to be active against Saccharomyces cerevisiae (3), and chlorogenic acid (3-caffeoyl quinate) has been found to be active against both gram-positive and gram-negative bacteria (14). Conversely, other researchers using the same compounds have reported finding no antifungal or antibacterial activity (1, 18). These variable results are probably due to the differences in experimental procedures. It can be concluded that under certain conditions these cinnamic acid derivatives do possess a range of antimicrobial activities. One reason for the discrepancies noted above is the differences in ph of the media employed. The hydroxycinnamates are ionizable compounds, and their activity will likely vary with ph, as does benzoic acid (7). An obvious way to t Scientific paper no. 625 of the College of Agriculture Research Center, Washington State University, Pullman, WA negate these effects is through the synthesis of non-ionizable derivatives, the simplest being the alkyl esters. Certain of these alkyl esters do exist naturally, although their concentrations are usually low (4, 15, 2). Ethyl p-methoxycinnamate has been isolated from a tumeric-type plant and has been shown to inhibit a wide range of fungi at 1 to 5 ppm (,xg/ml) (15). Baranowski and Nagel (J. Food Sci., in press) synthesized the methyl esters of six hydroxycinnamic acids as well as the propyl and ethyl esters of caffeic acid for antimicrobial screening. They found that the caffeoate esters, methyl p-coumarate, and methyl ferulate possessed antibacterial activity toward Pseudomonasfluorescens, a gramnegative bacterium associated with refrigerated meat spoilage. The most active of these derivatives was, in fact, found to be more inhibitory than either methyl or propyl paraben on a weight basis. These authors have suggested the use of these alkyl hydroxycinnamates (to which they have given the acronym alkacins) in food products. This paper attempts to identify the mode of activity of the alkacins against P. fluorescens as well as some of the general properties of these compounds which may influence their effectiveness as antimicrobial agents. MATERIALS AND METHODS A culture of P. fluorescens ATCC was obtained from the culture collection of the Department of Food Science and Technology, Washington State Uni- 218

2 VOL. 45, 1983 ALKACIN EFFECTS ON PSEUDOMONAS SPP. 219 versity. This culture was maintained on Trypticase soy (Sigma Chemical Co., St. Louis, Mo.) were prepared agar slants, prepared by the addition of 1.5% agar to in distilled 95% ethanol at the same concentration, and Trypticase soy broth (TSB; BBL Microbiology Systems, Cockeysville, Md.) adjusted to ph 7. with 1 N milliliters of this stock solution was transferred to a their partition coefficients were also determined. Five HCl before autoclaving. The synthesis of the alkyl test tube containing 5 ml of soy oil (Sona Food esters has been described elsewhere (Baranowski and Products, Co., Los Alamitos, Calif.), sealed, inverted Nagel, in press). Stock solutions of these compounds 1 times, and shaken for 5 s. After being allowed to were prepared at 12,5 ppm in distilled 95% ethanol. separate, the aqueous layer was removed by pipette A sample of a 1:1, dilution (in phosphate buffer, and centrifuged at 4, x g for 3 min. The absorbances of this sample (Aaqueous) and of the stock solu- ph 7. [2]) of a 2-h seed culture grown from the stock slant in TSB at 2 C was used as the inoculum for tion (Astock), after any necessary dilutions with.1 M growth studies. Generally this was diluted 1:5 by the phosphate buffer, were determined at 28 nm. The oilbuffer partition coefficient was defined as: (Astock - test medium to provide ca. 1, cells per ml. All growth results are discussed in terms of relative effectiveness (RE) (Baranowski and Nagel, in press), i.e., Oxygen usage analysis. For oxygen usage measure- Aaqueous)IAaqueousthe ratio of the time for the treated culture to reach a ments, a Spectroplus-D (MSE Scientific Instruments, turbidity of 7 Klett units (an arbitrary point in the Crawly, England) oxygen apparatus was used, coupled to an Omniscribe recorder (Houston Instruments, exponential phase of growth; Klett-Sumerson photoelectric colorimeter with a red filter; Klett Manufacturing Co., New York, N.Y.) to the time for the in TSB (ph 7.) at 2 C was diluted 1:1 with phos- Austin, Tex.). A 2-h culture of P. fluorescens grown control cultures to reach the same turbidity. Turbidity phate buffer (ph 7.). Four milliliters of this was added measurements were made at approximately 12-h intervals. density of 4 x 17 bacteria per ml. Early experiments to 46 ml of TSB (ph 7.), resulting in a population Effect of alkacins on growth and survival. The shortterm lethality of P. fluorescens was investigated under available oxygen in about 1 min. The culture was indicated that this population density would use all conditions identical to that of the growth inhibition kept on ice throughout any waiting period. At 2 min assays previously performed (Baranowski and Nagel, before the measurements, a sample of the culture was in press). Duplicate samples of TSB containing 25 placed at room temperature in a screw-cap test tube ppm of the inhibitor were inoculated and manually and agitated with a magnetic stir bar. Two milliliters of shaken at 1-min intervals for 3 min. Dilutions were this suspension was transferred to the oxygen chamber, the stirrer was started, and the readout was then pour plated with Trypticase soy agar, and counts were made after 48 h of incubation at 2 C. allowed to stabilize (5 to 1 min). The gain was then To determine whether any adaptation or crossadaptation to the alkacins occurred, the effects of plunger was inserted into the cell, and the recorder adjusted to read full scale on the equipment. The pregrowth in media containing 125 ppm of an inhibitor was started. Basal oxygen usage was measured for 1 were investigated. In this experiment, only the methyl min. Then 8,ul of stock antimicrobial agent (providing 5 ppm) was then added by means of a syringe, esters of caffeic, p-coumaric, cinnamic, and p-hydroxybenzoic acids were used, as these were the only and the experiment was allowed to continue until a methyl esters with any appreciable growth-inhibiting new rate was stabilized (elapsed time of ca. 1 min). activity (Baranowski and Nagel, in press). For each of Each compound (the methyl esters of p-hydroxybenzoic, p-coumaric, caffeic, ferulic, and sinapic acids) the four test compounds, 49 ml of TSB containing 125 ppm of the compound and 2% distilled 95% ethanol and ethanol control was run in triplicate. Data are was inoculated and incubated at 2 C with constant presented as percentages of control respiration, i.e., agitation until the turbidity level of the original 2-h (KexplKcontrol) x 1, where: Kexp = Kexp treated/kexp seed culture was reached. These were then diluted in pretreatment' and Kcontrol = Kcontrol with ethanol/kcontrol the same manner as the original inoculum. A control before ethanolfor each of these inocula was included for the comiputation of RE. Each of four test compounds was then with phosphate buffer (ph 7.) 1 min after the addi- In an identical experiment, the cells were diluted evaluated for its antimicrobial activity at 125 ppm tion of the compound and plated with Trypticase soy against each of the four pregrowth inocula. These agar. Counts were made after a 48-h incubation at 2 C antimicrobial activities were assayed in duplicate, and to determine whether any cell mortality was occurring the entire experiment was repeated once. Data are under the oxygen assay conditions, which would influence oxygen consumption rates. reported as the averages of the two duplicates. The additive or synergistic effect of the combination of two alkacins at 125 ppm each was also investigated. RESULTS AND DISCUSSION These experiments were carried out for combinations of methyl esters only and performed with the system Unlike the effects of other phenolic antimicrobial compounds such as BHA (9), no mortality outlined earlier for the general assay. All statistical analyses were performed with the Student t-test (22). of Pseudomonasfluorescens was found after 3 Oil-phosphate buffer partition coefficients. Stock min of exposure to 25 ppm of the alkacins or antimicrobial agent solutions were diluted 1:5 with the parabens in TSB. The parabens also have.1 M potassium phosphate (ph 7.) to simulate the TSB system. This resulted in a solution containing 25 been observed to cause no mortality when tested ppm of the alkacins and 2% distilled ethanol. For against Serratia marcescens (13). Other studies comparative purposes, butylated hydroxyanisole have employed phosphate buffer systems for (BHA) and TBHQ (both from Eastman Chemical Co., short-term mortality studies (1, 16), however, Kingston, Tenn.), and methyl and propyl paraben as the growth inhibition studies were performed

3 22 BARANOWSKI AND NAGEL -J z CL) I-t z 4 r. co 1r 5F 1. r RELATIVE EFFECTIVENESS FIG. 1. Effects of methyl alkacins and methyl paraben on P. fluorescens. Correlation of growth inhibition with inhibition of respiration. in TSB, mortality was also studied in this medium Ṫhe inhibition of oxygen usage correlated well with the inhibition of growth (Baranowski and Nagel, in press) (Fig. 1). This is consistent with a physiological action based on the depletion of ATP, rather than through a genetic inhibition of some type (12). Again, as shown with plate counts, no mortality was observed under the oxygen analysis conditions. Also, no correlation of the soy oil-phosphate buffer (ph 7.) system to the growth inhibition (r =.8) at 25 ppm was found. These results point to a specific action of the inhibitors, rather than the nonspecific membrane action of various other phenolic antimicrobial agents such as BHA and phenol (9, 17, 21). The parabens are known to inhibit both membrane transport of amino acids and electron transport (11, 12). The data herein are consistent with those results. Table 1 presents the results of the adaptation studies. Although there was a decrease in the effectiveness of methyl caffeoate and methyl p- coumarate after growth of the organism on all of the inhibitors, these results demonstrate a trend and were not found to be significantly different 2. APPL. ENVIRON. MICROBIOL. from the inhibitions found without the pregrowth treatments. The only statistically determined decrease in activity was shown by methyl paraben after pregrowth in methyl cinnamate (P =.5). The lack of adaptation by a microorganism to an inhibitor is an obvious necessity for its use in a food product and has been previously noted for the parabens (19). P. fluorescens was able to adapt to BHA through a similar pregrowth treatment, and did become somewhat resistant to its effects (9). Quite commonly, mixtures of antimicrobial compounds are used in a product so as to circumvent such problems as solubility or taste of any individual compound. This practice is common for the parabens where a 2:1 or 3:1 mixture of the methyl and propyl ester is used. This allows an additive effect of the antimicrobial activities and avoids off tastes brought about by a high concentration of any one paraben (taste effects for these compounds are not additive [6]). The action of mixtures of compounds is commonly additive, as with the parabens, although occasionally synergistic actions are found. Mixtures of BHA and sorbic acid tested against both P. fluorescens and Pseudomonas fragi were found to have additive results (9). The effects of a mixture of two alkacins, each at 125 ppm against P. fluorescens (Table 2), are seen to be at least an additive effect. If both were active in the same manner, it would seem reasonable that the activity of a mixture would be compared with the average activity of the two compounds at twice the concentration used in the study. Conversely, if they acted in a different manner, the activity of the mixture should be the additive activities of the two compounds at the concentration tested. With either comparison method, all mixtures gave at least additive results, and certain of these compounds were found to have a slightly synergistic activity. Although this is a favorable property, only one mixture, methyl p-coumarate-methyl caffeoate, had an activity greater than methyl caffeoate (the most active methyl ester) at 25 ppm alone (RE = 2.84; Baranowski and Nagel, in press), and this difference was not significant. These additive and synergistic effects would become important with respect to flavor if these alkacins were to be used in food products. TABLE 1. Adaptation of P. fluorescens to 125 ppm alkacins and methyl parabena RE (pregrowth in 125 ppm) Antimicrobial agent Noneb Methyl Methyl p- Methyl Methyl caffeoate coumarate paraben cinnamate Methyl caffeoate 1.71 (.3) 1.48 (.8) 1.57 (.2) 1.62 (.16) 1.68 (.9) Methyl p-coumarate 1.72 (.18) 1.36 (.14) 1.4 (.18) 1.49 (.13) 1.49 (.13) Methyl paraben 1.31 (.3) 1.2 (.3) 1.24 (.2) 1.27 (.4) 1.2 (.2) Methyl cinnamate 1.28 (.1) 1.18 (.9) 1.25 (.16) 1.16 (.5) 1.22 (.7) a Data are presented in terms of RE; see text for explanation. Standard deviations are within parentheses. b Data from Baranowski and Nagel (in press).

4 VOL. 45, 1983 TABLE 2. ALKACIN EFFECTS ON PSEUDOMONAS SPP. 221 Effect of 125 ppm each of methyl hydroxycinnamate mixtures on P. fluorescensa Mixture RE Additive comparisons lb 2c p-coumarate-caffeoate 2.89 (.4) 2.48 (.17) 2.43 (.18) p-coumarate-ferulate 2.4 (.5) 1.66 (.11)d 1.74 (.18) p-coumarate-sinapate 1.8 (.13) 1.59 (.11) 1.74 (.18) p-coumarate-p-methoxycinnamate 1.93 (.5) 1.56 (.11)d 1.73 (.18) p-coumarate-3,4-dimethoxycinnamate 1.94 (.1) 1.57 (.11)d 1.75 (.18) Ferulate-caffeoate 2.38 (.53) 2.2 (.13) 1.73 (.4) Ferulate-sinapate 1.1 (.4) 1.13 (.6) 1.4 (.3) Ferulate-p-methoxycinnamate 1.35 (.2) 1.11 (.4) 1.3 (.4)d Ferulate-3,4-dimethoxycinnamate 1.32 (.1) 1.11 (.4)d 1.5 (.4)d Caffeoate-sinapate 1.9 (.12) 1.95 (.13) 1.73 (.3) Caffeoate-p-methoxycinnamate 1.91 (.24) 1.93 (.11) 1.72 (.4) Caffeoate-3,4-dimethoxycinnamate 1.99 (.13) 1.93 (.13) 1.74 (.4)d Sinapate-p-methoxycinnamate 1.3 (.3) 1.4 (.5) 1.3 (.3) Sinapate-3,4-dimethoxycinnamate 1.1 (.) 1.4 (.5) 1.5 (.2)d p-methoxy-3,4-dimethoxycinnamate 1.2 (.) 1.2 (.2) 1.4 (.) a Additive comparisons 1 and 2 are based on data from Baranowski and Nagel (in press). Standard deviations are given within parentheses. b Additive comparison 1 = [RE1 (25 ppm) + RE2 (25 ppm)]/2. c Additive comparison 2 = RE1 (125 ppm) + RE2 (125 ppm) d Statistically lower activity than the mixture at P =.5 level. BHA has been shown to be an active antimicrobial agent in model systems against a wide range of bacteria and fungi (5, 9, 1, 16). Problems arise, though, when this additive is used in a food product for this purpose, presumedly due to its very large oil-water partition coefficient. This causes the majority of the compound to partition into the lipid phase of the product, effectively removing it from any antimicrobial capabilities. The active alkacins (the methyl esters of caffeic, p-coumaric, and ferulic acids and ethyl and propyl caffeoate; Baranowski and TABLE 3. Soy oil-.1 M phosphate buffer (ph 7.) partition coefficients (D) for phenolic preservatives Compound D REa Methyl caffeoate Ethyl caffeoate Propyl caffeoate Methyl p-coumarate Methyl ferulate Methyl sinapate Methyl p-methoxycinnamate Methyl 3,4-dimethoxycinnamate Methyl paraben Propyl paraben TBHQ 1.79 BHA 4.37 a Data from Baranowski and Nagel (in press) at 25 ppm. Nagel, in press) have oil-water partition coefficients ranging from 1.33 to (Table 3), much less than the 4.37 of BHA or 3.43 of propyl paraben. In fact, methyl caffeoate has a significantly smaller partition coefficient (1.33) than methyl paraben (3.43), pointing to the compatability of the alkacins for their use in food systems. Overall, based on their effects on P. fluorescens, certain alkacins show promise for further antimicrobial use based on (i) their non-selfadaptive nature, (ii) their favorable oil-buffer partition coefficients (which are comparable to methyl and propyl paraben), and (iii) their additive or possibly synergistic antimicrobial effects. Further research is under way in the testing of these compounds against other microorganisms in broth cultures and in food systems. LITERATURE CITED 1. Alberghina, A., A. Benni, and M. G. Fantino Effects of temperature and polyphenols on Fusaria growth. Phytopathol Z. 96: American Public Health Association Standard methods for the examination of dairy products, 14th ed., p. 56. American Public Health Association, Washington, D.C. 3. Baranowski, J. D., P. M. Davidson, C. W. Nagel, and A. L. Branen Inhibition of Saccharomyces cerevisiae by naturally occurring hydroxycinnamates. J. Food Sci. 45: Bhattacharya, J., and D. K. Chaudhuri Isolation and characterization of a crystalline antithiamine factor from mustard seed, Brassica juncea. Biochim. Biophys. Acta 343: Chang, H. G., and A. L. Branen Antimicrobial

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