Factors Affecting the Antimicrobial Activity of Vitamin K

APPLIED MICROBIOLOGY, Sept., 1965 Copyright @ 1965 American Society for Microbiology Vol. 13, No. 5 Printed in U.S.A. Factors Affecting the Antimicrobial Activity of Vitamin K LARRY S. MERRIFIELD2 AND H. Y. YANG Department of Food Science and Technology, Oregon State University, Corvallis, Oregon Received for publication 14 May 1965 ABSTRACT MERRIFIELD, LARRY S. (Oregon State University, Corvallis), AND H. Y. YANG. Factors affecting the antimicrobial activity of vitamin K5. Appl. Microbiol. 13:766-770. 1965.-Pure cultures of Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphylococcus aureus, and Pseudomonasfluorescens were used in this investigation. The bactericidal concentrations of vitamin K5 required for E. coli, B. subtilis, P. vulgaris, S. aureus; and P. fluorescens; the effect of an absence of oxygen; the effect of contact time with E. coli and S. aureus; and the effect of initial counts per milliliter of E. coli were studied. The bactericidal concentrations ranged from 60 ppm of K5 for S. aureus to 220 ppm for E. coli, with an initial count of 160,000 to 200,000 cells per milliliter and a contact time of 12 hr in nutrient broth. The gram-positive bacteria tested were more susceptible to the antimicrobial activity of vitamin K5 than the gram-negative bacteria. In the studies conducted under nitrogen atmosphere, the per cent inhibition showed an inverse relationship to the bactericidal concentrations required for complete inhibition in studies conducted under air atmosphere. This finding suggested that there might be different factors responsible for inhibition depending on the species of bacteria being tested, and it also might help explain the difference in concentrations necessary for inhibition. Cells of E. coli and S. aureus were not inhibited immediately on coming into contact with vitamin Kr; 50% inhibition occurred after 25 and 32 min, respectively. A rapid inhibition rate was maintained until approximately 90% inhibition occurred, after whch a rapid decrease in the rate was noted. Vitamin K5 (4-amino-2-methyl-1-naphthol hydrochloride) has been shown to possess an antimicrobial activity toward many bacteria, molds, and yeast, and much of the work reported in the literature is on its use as a food preservative. Vitamin K5 seems to be a promising food preservative, because it has a high antimicrobial activity towards a number of microorganisms (Pratt, Dufrenoy, and Pickering, 1949), it is relatively soluble in water compared with other vitamin K compounds (Pratt et al., 1950), it has a low order of toxicity (Pratt et al., 1950), and it belongs to a class of compounds which occur in normal human metabolism (Butt and Snell, 1941). There have, however, been several problems encountered in the use of vitamin K5 as a food preservative. Faggioli (1953) and Matsumoto and Komatsu (1952) mentioned the darkening 1 Technical Paper no. 1936, Oregon Agricultural Experiment Station. This paper is based on a thesis submitted by Larry S. Merrifield in partial fulfillment of the requirements for the M.S. degree. 2 Present address: Coca-Cola Export Corp., New York, N.Y. effect of vitamin K5 as a serious disadvantage in its application as a preservative. Yang et al. (1958) found a serious discoloration in crab meat. Tengerdy and Francia (1954) found vitamin K5 to be unsatisfactory as a beer preservative because of its poor stability. It was the purpose of this study to investigate some of the factors which might affect the antimicrobial activity of vitamin K5 to develop its more effective use as a food preservative. MATERIALS AND METHODS Microorganisms. Cultures of Escherichia coli (ATCC 11303), Bacillus subtilis (ATCC 9945), Proteus vulgaris, Staphylococcus aureus, and Pseudomonas fluorescens were obtained through the Department of Microbiology, Oregon State University. They were chosen to give a spectrum of food spoilage bacteria as well as to compare with bacteria previously used in conjunction with vitamin K5. Vitamin K5. Vitamin K5 was obtained by synthesis according to the procedure of Oneto and Sah (1949). The starting material was 2-methyl- 1,4-naphthoquinone obtained from Nutritional Biochemicals Corp. (Cleveland, Ohio) without specification. 766

10OL. 13, 1965 ANTIMICROBIAL ACTIVITY OF VITAMIN K5 767 Microbiological media. The cultures of bacteria were grown and maintained on nutrient agar (ph 6.8) and nutrient broth (ph 6.8; both Difco products). The cultures were also treated with vitamin K5 and subcultured in nutrient broth (ph 6.8). All the plates for total counts were poured with Plate Count (Difco) agar as a medium (ph 7.0). Growth of the bacterial cultures. The different bacteria were incubated throughout the investigation at their respective optimal temperatures. All of the tubes containing vitamin K5 and bacteria were maintained at the respective temperatures during contact time. The plates for total plate counts were incubated at the same temperatures as those used to maintain the cultures for 48 hr prior to counting. Preparation for inoculation. The bacterial inocula were standardized to contain 160,000 to 200,000 viable cells per milliliter. The culture was first incubated on a nutrient agar slant for 24 hr and then transferred into 20 ml of -sterile nutrient broth and incubated for 12 hr. After 12 hr, the mixture was read at 580 min in a Lumetron photoelectric colorimeter, model 400-A, with orange filter, and standardized to 100% transmission with nutrient broth. The results were determined as turbidity, and, from this, a dilution was made to contain the initial count in a 1-ml inoculum. Method of application. The inoculum was added to a screw-cap test tube containing 24 ml of broth, the tube was shaken 50 times, and the desired concentration of vitamin K5 was added. The tube was then shaken 100 times, and the tube was incubated for 12 hr before plating. Anaerobic studies. The study concerning the effect of vitamin K5 against bacteria in the absence of oxygen was carried out with E. coli, P. vulgaris, and S. aureus. These bacteria were chosen, since they are known to be facultative anaerobes (Bergey's Manual). The bacteria were grown and prepared for inoculation exactly as before, except each species was grown in nitrogen atmosphere, as well as in air, prior to inoculation. Thunberg tubes were used for the inoculation, with the desired concentration of vitamin K5 being weighed out and placed in the cap and 24 ml of nutrient broth being placed in the tube. The standard inocula were introduced, and a 76.2-cm (30 inch) vacuum was pulled on the tube, after which nitrogen was leaked back. This was repeated, except that the nitrogen was leaked back until a 5.08-cm vacuum remained (to keep the cap in place). The tube was tilted to get the vitamin K5 into the broth and then shaken 150 times and placed in the incubator for 12 hr. All tubes were run and plated in duplicate, and results were averaged. Contact time study. To determine the effect of contact time on the antimicrobial activity of vitamin K5 against E. coli and S. aureus, the following procedure was followed. Both bacteria were grown, prepared for inoculation, and inoculated as described before. Plates of the total count were prepared in duplicate at 1, 25, 50, 100, 150, 200, TABLE 1. Bactericidal concentrations of vitamin K5 Initia GramBacteri- Microorganism countmil Gram cidal coun/ml tain concn PPM Staphylococcus aureus... 160,000 + 60 Bacillus subtilis... 176,000 + 80 Proteus vulgaris... 173,000-120 Pseudomonas fluorescens. 186,000-140 Escherichia coli... 182,000-220 TABLE 2. Total counts per milliliter in the tubes after 12 hr of incubation Microorganism count/mi of Initial Concn Total vim t- count/mi min K, X 106 ppm Staphylococcus aureus 160,000 0 61.5 160,000 60 0 Bacillus subtilis 176,000 0 11.0 176,000 80 0 Proteus vulgaris 173,000 0 60.0 173,000 120 0 Pseudomonas fluorescens 186,000 0 13.4 186,000 140 0 Escherichia coli 182,000 0 118.0 182,000 220 0 250, 300, and 350 min. All plates were taken from the same tube. The tube was tilted 40 times so that a minimum of air oxidation would take place before each plating. RESULTS AND DISCUSSION Effect of vitamin K5 against different bacteria. The study to determine a bactericidal concentration of vitamin K5 against different bacteria was undertaken because previous investigations (Kowalski, 1952; Pratt et al., 1950; Verona, 1953) did not indicate the methods or the initial counts per milliliter of microorganisms used. The bactericidal concentrations of vitamin K6 necessary for the bacteria studied are presented in Table 1. Table 2 shows the bactericidal concentrations of vitamin Ks and the total counts per milliliter of the control tubes at the end of the 12-hr incubation period. To confirm that the results were truly bactericidal and not bacteriostatic, duplicate subcultures (1 ml in 20 ml of fresh broth) were prepared and incubated for four days. If no growth appeared in these tubes at the end of the 4-day incubation period, the concentration was termed bactericidal. S. aureus and B. subtilis, the gram-positive

768 MERRIFIELD AND YANG APPL. MICROBTOL. TABLE 3. Effect of nitrogen atmosphere on the antimicrobial activity of vitamin K5 against microorganisms grown in air Microorganism Initial Concn of Total Inhibition count/mi vitamin Ks count/mi X 104 Ihiito ppm % Staphylococcus aureus 200,000 0 1,290 0 200,000 60 3.14 99.76 Proteus vulgaris 164,000 0 6,000 0 164,000 120 1,015 83.08 Escherichia coli 160,000 0 12,200 0 160,000 220 5,825 52.25 TABLE 4. Effect of nitrogen atmosphere on the antimicrobial activity of vitamin K5 against microorganisms grown in nitrogen Microorganism Initial Conc of Total Inhibition count/mi vitamin K, count/mi X Inhbiio ppm S Staphylococcus aureus 182,520 0 5,000 0 182,520 60 0.4175 99.99 Proteus vulgaris 176,000 0 5,000 0 176,000 120 163.25 96.74 Escherichia coli 200,000 0 12,400 0 200,000 220 3,805 69.32 microorganisms, required the lowest concentrations of vitamin K5 for inhibition (Table 1), whereas the gram-negative microorganisms, P. vulgaris, P. fluorescens, and E. coli, required higher concentrations. This is in agreement with the findings of Shwartzman (1948) and Kitamikado (1959). Effect of anaerobic conditions. The antimicrobial activity of vitamin K5 under anaerobic conditions was investigated for the following reasons. (i) It was found that air oxidation enhanced the antimicrobial activity of vitamin K5 (Armstrong, Spink, and Kahnke, 1943; Shwartzman, 1948), and (ii) in an investigation of the sensitizing action of vitamin K5 in conjunction with y-radiation, vitamin K5 was found to be more effective against bacteria in the absence of oxygen (Noaman et al., 1964; Shehata, 1961; Silverman, Shehata, and Goldblith, 1962; Silverman, Davis, and Goldblith, 1963). Table 3 shows the effect of vitamin K5 against the bacteria grown in air, and Table 4 shows the results against bacteria grown in nitrogen. The bacteria grown in nitrogen and treated (with vitamin K5) in nitrogen were a control, showing that bacteria grown in air and treated in nitrogen did not represent a facultative adaptation. The concentrations of vitamin K5 that were bactericidal for the different species by use of the regular method (air oxidation, Tables 1 and 2) were not completely bactericidal under a nitrogen atmosphere. The vitamin K5 did, however, inhibit the growth of the different species in varying degrees in all cases. S. aureus was inhibited to the greatest extent, whereas E. coli was inhibited the least amount by both methods. The reasons for this inhibition could be that there was still some oxidation able to take place in the tubes, or that the inhibition was brought about by vitamin K5 alone. If the inhibition was due to vitamin K5, it is also noted that the three species of bacteria were inhibited in an inverse relationship to the concentration of vitamin K5 that was bactericidal in air. This would explain why E. coli, which requires a high concentration of vitamin K5 for inhibition in air (and therefore more oxidation and formation of oxidation products of vitamin K5), was inhibited only about half as much as S. aureus, which requires a low concentration for air inhibition (and therefore correspondingly lower amount of oxidation and oxidation-product formation). P. vulgaris, which was inhibited by a concentration between those required by S. aureus and E. coli in air, also showed a corresponding per cent inhibition between them when grown in nitrogen. Effect of contact time. E. coli was not inhibited immediately on coming into contact with vitamin K5 (Table 5). The lethal effect of vitamin K5, however, increased as the contact time was increased; 50% inhibition (I5o) occurred after 25 min. A rapid rate of inhibition was maintained

VOL. 13, 1965 ANTIMICROBIAL ACTIVITY OF VITAMIN K5 769 TABLE 5. Effect of contact time on the antimicrobial activity of 220 ppm of vitamin K5 against Escherichia coli Contact time Vitamin K5 (ppm) 0 220 Inhibition min % 0 5,175* 5,175 0 1 5,325 5,012.5 3.2 25 5,387.5 2,537.5 51.0 50 7,075 1,262.5 75.6 100 9,675 112.5 97.8 150 20,750 32.5 99.9 200 45,500 7.5 loot 250 177,500 3.75 loot 300 498,750 0.075 loot 350 975,000 0 100 * Results expressed as total counts times 1,000. t These values are approximate. TABLE 6. Effect of contact time on the antimicrobial activity of 80 ppm of vitamin K5 against Staphylococcus aureus Contact time min Vitamin K, (ppm) 0 80 Inhibition 0 5,250* 5,250 0 1 5,575 5,111.5 2.6 25 6,925 3,750 32.7 50 7,350 727.5 86.9 100 9,375 262.5 95.3 150 19,875 175.0 96.9 200 62,250 106.25 98.1 250 117,250 28.5 99.9 300 292,500 3.25 100t 350 737,500 0 100 * Results expressed as total counts times 1,000. t Approximate value. until I'o was reached; thereafter, it became quite slow. The same effect was noted with S. aureus (Table 6). The lethal effect of vitamin K5, again, increased with contact time, 150 occurring after 32 min. Rapid inhibition of the E. coli and S. aureus cells to the Igo level could be caused by the vitamin K5 itself and, thereafter, as most of the vitamin oxidizes, the inhibition becomes much slower. It can also be said that the rapid inhibition might be caused by the formation of the intermediate oxidation products (quinoneimine and vitamin K3) and their subsequent antimicrobial activity (if any); as the vitamin K5 and the intermediate products are exhausted due to fur- N TABLE 7. Effect of varying initial counts on the antimicrobial activity of vitamin K5 against Escherichia coli Initial count/ml Concn of vitamin K, (ppm) Bactericidal Growth 80,000-120 160,000 220 180 200,000 220 200 7,200,000 340 300 16,000,000 440 400 ther oxidation and condensation (Knobloch, 1949), the reduced inhibition rate after Igo occurs. It is also possible that a combination of these two explanations might be the actual method of inhibition. A similar study with cells of Saccharomyces cerevisiae plus a concentration of 100 ppm of vitamin K5 was reported by Rasulpuri et al. (1965). The results were very similar and showed an accelerated inhibition until Iso was reached, with I50 occurring after 50 min. Effect of initial number of microorganisms. As expected, an increase in the initial count of E. coli requires an increase in vitamin K5 concentration to achieve bactericidal results (Table 7). A concentration of 220 ppm of vitamin K5 was bactericidal to an initial inoculation of 160,000 cells per miuiliter of E. coli. Upon an initial inoculation of 80,000 cells per milliliter, a concentration of approximately 120 ppm was not bactericidal. When the initial inoculation was 100 times as great (16,000,000 cells per milliliter), the concentration of vitamin K5 required for complete inhibition was only twice as much (440 ppm). The results of this study show that the concentration of vitamin K5 is not the limiting factor involved in the inhibition of E. coli, since the initial counts per milliliter can be increased without a proportional increase in the vitamin K5 required for complete inhibition. This could mean that the intermediate oxidation products of vitamin K5 are quite important for inhibitiovr of E. coli, since we know that the higher the concentration of vitamin K5 the more rapidly it tends to oxidize in solution. It could also indicate that a tanning effect on the cell membrane, as mentioned by Hoffman-Ostenhof (1947), is the bactericidal factor in higher concentrations of vitamin K5. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant EF-00022 from the National Institutes of Health.

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