Effect of Phenolic Acids and Esters on Respiration

Transcription

1 APPLIED MICROBIOLOGY, May, 196 Copyright 196 American Society for Microbiology Vol. 13, No. 3 Printed in U.S.A. Effect of Phenolic Acids and Esters on Respiration and Reproduction of Bacteria in Urine' ELIZABETH S. KEITH AND JOHN J. POWERS Department of Food Science, University of Georgia, Athens, Georgia lieceived for publication 8 October 1964 ABSTRACT KEITH, ELIZABETH S. (University of Georgia, Athens), AND JOHN J. POWERS. Effect of phenolic acids and esters on respiration and reproduction of bacteria in urine. Appl. Microbiol. 13: Vanillic, syringic, gallic, and protocatechuic acids, methyl-p-hydroxybenzoate, and propyl-p-hydroxybenzoate generally inhibited respiration in vitro of Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, and Aerobacter aerogenes in human urine. In the absence of any other available carbon source, certain of the phenolic compounds were utilized. Reproduction was generally suppressed in urine buffered to ph 7,.6, 4., and 4.. The phenolic compounds were used in the range of.11 to,mole/ml. Masquelier and Jensen (193a, b) reported that malvidin, one of the anthocyanins of wine grapes, is bactericidal toward Escherichia coli. Previous work in our laboratory (Pratt, Powers, and Somaatmadja, 196; Powers, 1963, 1964; Somaatmadja and Powers, 1964) has shown that anthocyanin compounds and breakdown products of anthocyanin compounds (Hamdy et al., 1961; Powers et al., 196) inhibit microorganisms. Fellers, Redmond, and Parrott (1933) showed that cranberries contain quinic acid and appreciable quantities of benzoic acid; Bodel, Cotran, and Kass (199) suggested that cranberry juice might be used to treat urinary infections, because quinic acid is excreted in the form of hippuric acid which is bacteriostatic at ph.. Forty-three phenolic acids, including protocatechuic, vanillic, and homovanillic acids and p-hydroxybenzoate, have been shown to be excreted from the urine unchanged (Williams, 199; Barness et al., 1963; Armstrong, Shaw, and Wall, 196; Numerof, Gordon, and Kelly, 19). The purpose of this study was to determine whether syringic, vanillic, gallic, and protocatechuic acids, which are breakdown products of various anthocyanins, would inhibit E. coli, Proteus vulgaris, Aerobacter aerogenes, and Pseudomonas aeruginosa in urine. The four organisms are known to invade the urinary tract (Gershenfeld, 1948; Grayhack, 1964; Michel-Pascal, 1964). The action of methyl-p-hydroxybenzoate and propyl-p-hydroxybenzoate was also tested, be- I Approved as Journal Paper no. 39, College Experiment Station, College of Agriculture, University of Georgia, Athens. cause these compounds and other phenolic esters or salts have been used as food preservatives (Ingram, 196; Morse, 191; Schimmel and Husa, 196; Chemo-Puro Manufacturing Corp., 199). Downloaded from on September 11, 218 by guest MATERIALS AND METHODS Vanillic and protocatechuic acids (C grade) were purchased from Calbiochem; gallic acid, from Nutritional Biochemicals Corp., Cleveland, Ohio; syringic acid, from Mathenson Coleman & Bell, East Rutherford, N.J.; methyl- and propylp-hydroxybenzoates (USP), from Chemo-Puro Manufacturing Corp., Newark, N.J. A Warburg respirometer was used to measure the effects of vanillic, protocatechuic, gallic, and syringic acids, and methyl- and propyl-p-hydroxybenzoates on the respiration of E. coli, P. vulgaris, A. aerogenes, and P. aeruginosa in fresh human urine. The urine was heated for 2 min at 121 C before use. In use, it was diluted to one-third strength by the addition of phosphate buffer (ph 6.2) and the cells used for the manometric studies. The cells were grown in Tryptone Glucose Extract broth for 18 hr. They were then washed, suspended in.8% saline, and stored at -3 C until used. All cell suspensions were less than 2 weeks old. A 2-mg amount of cells was added to 2. ml of substrate. The compounds were at a final concentration of.11 and.22 plmole/ml of phosphate-buffered urine or buffered substrate at 37 C. A few runs were made with 9-day urine to determine whether different results would be obtained with old urine. The most probable number technique was used to determine the effect of gallic and protocatechuic acid on growth of A. aerogenes and of syringic and protocatechuic acid on P. aeruginosa. Cells grown in nutrient broth (Difco) for 18 hr were placed in 38

2 VOL. 13, 196 EFFECT OF PHENOLIC ACIDS ON BACTERIA IN URINE 39 fresh urine to which,, and,umole of acid per ml had been added. Nonsterilized urine was used to determine whether compounds which might affect growth were being volatilized or changed by heating, and to observe microbial growth in pure and mixed culture. The urine was buffered to ph 6.2 with a phosphate buffer. Cell counts were made at, 2, 4, 6, 24, 3, 48, 4, and 78 hr at 37 C. A Bausch & Lomb Spectronic-2 colorimeter was used to measure the turbidity of cultures grown at 37 C in the presence of compounds at,, and,umole/ml. Tryptone Glucose Extract broth was used to grow the 18-hr cells used for inoculations. Syringic and vanillic acids at ph 7,.6, 4., and 4. were placed in urine diluted 1:1 and inoculated with E. coli and P. vulgaris., vanillic acid, methyl-p-hydroxybenzoate and propyl-p-hydroxybenzoate were added to urine diluted 1:1 and containing.2% added dextrose. Phosphate buffer was added to establish ph levels of 7, 6.2, and.6. These solutions were inoculated with E. coli and P. vulgaris and with A. aerogenes. in 1:1 urine plus.2% sugar at ph 7. and.6 was inoculated with E. coli, P. vulgaris, and E. coli plus P. vulgaris. Readings were taken every hour for the first 2 hr, at 1-min intervals for the next 6 hr (during the logarithmic-growth phase), and then Organism Escherichia coli Proteus vulgaris Aerobacter aerogenes Pseudomonas aeruginosa every hour for 4 hr; finally, a 24-hr reading was taken. RESULTS Manometric experiments. Table 1 shows the results of six replications with human urine diluted to one third and buffered to ph 6.2. Inhibition is indicated by a negative sign. Table 2 shows that the organisms were generally able to utilize the compounds when no other carbon source was available. The positive sign indicates utilization and the negative sign indicates that oxygen uptake was less than in the buffered substrate with no urine. Figure 1 illustrates the effect of methylp-hydroxybenzoate, which was inhibitory toward A. aerogenes. In Fig. 2 is shown the effect of protocatechuic acid toward E. coli; the acid was inhibitory in the presence of urine, but was utilized by the organism when no other carbon source was available. In the evaluation of these results, the entire period of oxygen uptake, not just the 12- and 21-min readings, was considered. In urine, respiration of E. coli was inhibited by all compounds at the.22,um level and by all except vanillic acid and propyl-p-hydroxybenzoate at TABLE 1. Per cent change in oxygen uptake compared with control substrate Compound Mlethyl-p-hydroxybenzoate Vassillic acid Syriisgic acid 12 min pmole/ml.22 Ismole/ml min min min Downloaded from on September 11, 218 by guest

3 31 KEITH AND POWERS APPL. MICROBIOL. TABLE 2. Per cent change in oxygen uptake compared with control buffered substrate Organism Compound.11 ;&mole/ml.22,umole/ml 12 min 21 min 12 min 21 min Escherichia coli Proteus vulgaris Aerobacter aerogenes Pseudomonas aeruginosa the.11 AM level. When only vanillic acid and buffer were present, the level of respiration was even lower than the endogenous level. However, E. coli was able to utilize syringic acid, protocatechuic acid, and methyl-p-hydroxybenzoate if these were the only carbon compounds present. P. vulgaris in urine was also inhibited by all compounds at the.22,am level but was inhibited by only vanillic acid at the AM concentration. Syringic, gallic, and protocatechuic acid stimulated growth at.11 AM. The organisms were able to utilize syringic, gallic, and protocatechuic acids and methyl-p-hydroxybenzoate at both levels, but propyl-p-hydroxybenzoate only at the higher level, when these compounds were the only carbon source available. A. aerogenes was least affected by the compounds. Inhibition occurred with vanillic acid, syringic acid, methyl-p-hydroxybenzoate, and propyl-p-hydroxybenzoate at.22 AM, and with gallic acid and with methyl- and propyl-phydroxybenzoate at.11,um. A. aerogenes was able to utilize gallic and protocatechuic acids if they were the only carbon substances present. P. aeruginosa was inhibited by syringic acid, gallic acid, and propyl-p-hydroxybenzoate at the.22,um level, and by vanillic and syringic acids and propyl-p-hydroxybenzoate at the.11,m level. However, the bacterium was stimulated by vanillic acid at.22 Mm. P. aeruginosa was able to utilize all compounds except syringic acid if these compounds were the only carbon source available. There was no noticeable difference in the respiration of E. coli with propyl-p-hydroxybenzoate when 9-day urine instead of fresh urine was used as the substrate. Turbidimetric determination. Table 3 shows the effect of these compounds on multiplication in urine buffered at ph.6. At ph 7 the effect of vanillic and syringic acids on E. coli in urine diluted 1:1 was slight. At ph.6 the effect was pronounced. At ph 4. and 4., the effect of the acid was not as evident, as would be expected, because the controls showed rather limited growth at these ph levels. However, it could be seen that, as the concentration of the acid increased, inhibition of growth also increased. P. vulgaris in urine at ph 7, 4., and 4. with vanillic and syringic acid was inhibited according to the concentration of the acid;,am caused the least inhibition and Mm, the most. At Downloaded from on September 11, 218 by guest

4 VOL. 13, 196 EFFECT OF PHENOLIC ACIDS ON BACTERIA IN URINE ph.6, the lag phase was lengthened, but after 12 hr of incubation organisms at the,um level of vanillic acid treatment grew better than the control. This was true also for the,um level of syringic acid when the 1:1 urine plus.2% glucose substrate was inoculated with E. coli plus P. vulgaris. In mixed culture, E. coli and P. vulgaris did not grow well in the control medium at ph 7. Growth of E. coli and P. vulgaris was appreciable at ph.6, 4., and 4., and E. coli P. vulgaris A. aerogenes TABLE 3. E. coli + P. vulgaris the inhibition by both vanillic and syringic acids could be easily observed. A. aerogenes grown in 1:1 urine plus.2% sugar at ph 7., 6.2, and.6 in the presence of,, and,um vanillic acid, syringic acid, methyl-p-hydroxybenzoate, and propyl-p-hydroxybenzoate was inhibited with increasing concentration, in both the lag and the logarithmic phases. One exception to this was at ph.6; at this ph, the lag phase of A. aerogenes in the Effect of phenolic compounds on the lag phase and growth of Escherichia coli, Proteus vulgaris, Aerobacter aerogenes, and E. coli plus P. vulgaris Organism Compound Amt Length lag phase of Absorbance after 12 hr pmoles hr Downloaded from on September 11, 218 by guest

5 312 KEITH AND POWERS APPL. MICROBIOL. w us n z w to w11 t: ai a 2I1 ISO.U method as compared with that obtained turbi-./' dimetrically. / DISCUSSION ISO,' The ingestion of flavonoid compounds does not Ito,' / /necessarily mean that phenolic acids will be excreted in urine. The particular metabolic degradation of these compounds varies with the species 9 -Ml,, of animals. Masri, Booth, and DeEds (199) I '.1 found that when quercetin was fed to rabbits 3 I =(.22) protocatechuic acid was not excreted. However,.1 when protocatechuic acid was fed to the animals ua -a.- -,-.- -, -s s b Igo 21 it was excreted unchanged. Ribereau-Gayon MINUTES (1963) demonstrated that free phenolic acids FIG. 1. Effect of methyl-p-hydroxybenzoate on exist in grapes; phenolic acids could thus be in- a w n z x.-w Aerobacter aerogenes. U = urine; U + MB = urine gested as such in foods or be liberated in the body plus methyl-p-hydroxybenzoate; MB = methyl-p- as more complex compounds, such as antho- hydroxybenzoate; the numerals in concentration in pmoles/ml. parentheses show cyanins, are degraded. Armstrong et al. (196) demonstrated that more than 4 phenolic acids could exist in the urine of humans ingesting ordinary diets. Beveridge and Hugo (1964) found that,,,,,-,u P. convexa was able to utilize gallic acid as a sole U- PC("I carbon source, but P. flavorescens and P. demou lpc(22)yticum were unable to utilize the acid. The effect on P. aerugenosa was not tested. We found that P. aerugenosa could use gallic acid as the sole carbon source. Based upon the present results,...-(.. c which show free phenolic acids (in the presence PC(22) of other nutrient material) to be generally inhibitory to E. coli, P. vulgaris, A. aerogenes, and P. aeruginosa, and on the observation of Bodel et al. - ~ (199) that hippuric acid is bactericidal, there is the possibility that anthocyanin-containing foods 1 IGO 21 may suppress in vivo organisms which invade the MINUTES urinary tract, since free phenolic acids and hipic acid on Esch- puric acid would be among the end products. FIG. 2. Effect of protocatechui -l7a r TT _ 7I Ti D/ ertonza co(t. = urtne; u -t- rt = ursne plus protocatechuic acid; PC = protocatechuic; the numerals ACKNOWLEDGMENTS in parentheses show concentration in umoles/ml.,um level of propyl-p-hydroxybenzoate substrate was less than at the,um concentration. However, absorbance at 12 hr of incubation was the same for all levels. in 1:1 urine plus.2% sugar was inoculated with E. coli, P. vulgaris, and E. coli plus P. vulgaris, but, because the gallic acid substrate turned dark during incubation, turbidimetric results for gallic acid treatment were useless. Most probable number determination. The most probable number results agree in general with the turbidimetric determinations. Only at the highest level did gallic acid slightly inhibit A. aerogenes; at the other levels, it was without effect. No additional information was obtained with this This investigation was supported by Public Health Service grant EF-349- from the Division of Environmental Engineering and Food Protection, and by California Wine Advisory Board Contract M-4. LITERATURE CITED ARMSTRONG, M. D., K. W. F. SHAW, AND P. E. WALL The phenolic acids of human urine. J. Biol. Chem. 218: BARNESS, L. A., W. J. MOLLMAN, T. TEDESCO, D. G. YOUNG, AND R. NOCHO A quantitative method of determining urinary phenols. Clin. Chem. 9:6-67. BEVERIDGE, E. G., AND W. B. HUGO The resistance of gallic acid and its alkyl esters to attack by bacteria able to degrade aromatic ring structures. J. Appl. Bacteriol. 24: BODEL, P., R. R. COTRAN, AND E. H. KASS Downloaded from on September 11, 218 by guest

6 VOL. 13, 196 EFFECT OF PHENOLIC ACIDS ON BACTERIA IN URINE 313 Cranberry juice and the antibacterial action of hippuric acid. J. Lab. Clin. Med. 4: CHEMO - PURO MANUFACTURING CORP Chemocides MK and PK. Technical Data Sheet No. D-13F, Chemo-Puro Manufacturing Corp., Newark, N.J. FELLERS, C. R., B. C. REDMOND, AND E. M. PARROTT Effect of cranberries on urinary acidity and blood alkali reserve. J. Nutr. 6: GERSHENFELD, L Urine and urinalysis. Romaine Pierson Publishers, Inc., New York. GRAYHACK, J. T The year book of urology, Year Book Medical Publishers, Inc. Chicago. HAMDY, M. K., D. E. PRATT, J. J. POWERS, AND D. SOMAATMADJA Anthocyanins. III. Disc sensitivity assay of inhibition of bacterial growth by pelargonidin-3-monoglucoside and its degradation products. J. Food Sci. 26: INGRAM, M. A The preservative action of acid substances in food. Chem. Ind., p MASQUELIER, J., AND H. JENSEN. 193a. Recherches sur l'action bactericide des vins rouges. I. Bull. Soc. Pharm. Bordeaux 91: MASQUELIER, J., AND H. JENSEN. 193b. Recherches sur l'action bactericide des vins rouges. II. Bull. Soc. Pharm. Bordeaux 91:1-19. MASRI, M. S., A. N. BOOTH, AND F. DEEDS The metabolism and acid degradation of quercetin. Arch. Biochem. Biophys. 8: MICHEL-PASCAL, B Urologic aspects, from experience in four hundred seventy cases seen in eight years. Presse Med. 72: MORSE, R. E., 191. Mode of action of sodium benzoate. Food Res. 16:1. NUMEROF, P., M. GORDON, AND J. M. KELLY. 19. Metabolism of reserpine in the mouse. J. Pharmacol. Exptl. Therap. 11: POWERS, J. J The antibiotic values of wine anthocyanins. Bull. Soc. Med. Friends Wine (2) -6. POWERS, J. J Action of anthocyanins and related compounds on bacterial cells. Proc. Intern. Symp. Food Microbiol., 4th, Goteborg, Sweden, p POWERS, J. J., D. SOMAATMADJA, D. E. PRATT, AND M. K. HAMDY Anthocyanins. II. Action of anthocyanin pigments and related compounds on the growth of certain microorganisms. Food Technol. 24: PRATT, D. E., J. J. POWERS, AND D. SOMAATMADJA Anthocyanins. I. The influence of strawberry and grape anthocyanins on the growth of certain bacteria. Food Res. 24: RIBEREAU-GAYON, P Les acides-phenols de Vitis vinifera. Compt. Rend. 26: SCHIMMEL, J., AND W. J. HUSA The effect of various preservatives on microorganism isolated from deteriorated syrups. J. Am. Pharm. Assoc. Sci. Ed. 4: SOMAATMADJA, D., AND J. J. POWERS Anthocyanins. V. The influence of anthocyanins and related compounds on glucose oxidation by bacteria. J. Food Sci. 29: WILLIAMS, R. T The fate of phenolic compounds in the body. In J. W. Fairbairn [ed.], The pharmacology of plant phenolics. Academic Press, Inc., New York. Downloaded from on September 11, 218 by guest