AN ABSTRACT OF THE THESIS OF Larry Stanford Merrifield for the M.S. in Food Science (Name) (Degree) (Major) Date thesis is presented June 26, 1964 Titie FACTORS AFFECTING THE ANTIMICROBIAL ACTIVITY OF VITAMIN K 5 Abstract approved (M^jor Tp&Sfesso/) Vitamin K, 4-amino-2-methyl- 1-naphthol hydrochloride, a 5 water soluble analog of vitamin K has been shown to possess an anti- microbial activity toward many bacteria, molds, and yeast. Much of the work reported in the literature is on its use as a food preserva- tive, and it was the purpose of this study to investigate some of the factors which might affect the antimicrobial activity of vitamin K in order to add insight into its more effective use as a food preserva- tive. Pure cultures of Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphlococcus aureus, and Pseudomonas fluorescens were utilized. The effect of the method of application of vitamin K on Escherichia coli; the effect of purity of vitamin K against Escherichia coli; the bactericidal concentrations required for Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphlococcus aureus, and Pseudomonas fluorescens; the effect of an absence of oxygen; the effect of contact time with Escherichia coli; the effect of initial count/ml of Escherichia coli; and the synergistic action in combination with propylene glycol were studied. The results demonstrated that air oxidation of vitamin K was 5 necessary to obtain maximum inhibitory activity against Escherichia coli. The use of white, crystalline vitamin K synthesized in the laboratory, as compared to partially oxidized commercial prepara- tions, gave better results against Escherichia coli. It was shown that some species variation, with the five micro- organisms tested, did exist with respect to the antimicrobial activity of vitamin K . The bactericidal concentrations ranged from 60 ppm 5 for Staphlococcus aureus to 220 ppm for Escherichia coli, using an initial count of 160, 000 to 200, 000 cells/ml and a contact time of 12 hours in Bacto-nutrient broth. The gram-positive bacteria tested were more susceptible to the antimicrobial activity of vitamin K 5 than the gram-negative bacteria tested. In the studies conducted under nitrogen atmosphere the percent inhibition showed an inverse relation to the bactericidal concentra- tions 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 also might help explain the difference in concentra- tions necessary for inhibition. Cells of Escherichia coli were not inhibited immediately on coming into contact with vitamin K . Fifty percent inhibition occur- 5 red after 25 minutes. A rapid inhibition rate was maintained until approximately 90 percent inhibition occurred, afterwhich, a rapid decrease in the rate of inhibition was noted. An increase in the initial number of cells of Escherichia coli utilized. required an increase in the concentration of vitamin K for complete inhibition, although the concentration of vitamin K was not 5 the limiting factor. A synergistic effect does exist between vitamin K and propylene glycol, and the bactericidal concentrations necessary for the different bacteria can be reduced depending on the species of bacteria and the concentration of propylene glycol utilized. FACTORS AFFECTING THE ANTIMICROBIAL ACTIVITY OF VITAMIN Kc by LARRY STANFORD MERRIFIELD A THESIS submitted to OREGON STATE UNIVERSITY in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 1964 APPROVED: Associate Prof^ssoiybf Fcpd Science and Technology In Charge of Major Head of Department of Footl^Science and Technology Dean of Graduate School Date thesis is presented June 26, 1964 Typed by Ilia W. Atwood ACKNOW LEDGMENT This study was conducted under the guidance of Dr. H. Y. Yang, Associate Professor of Food Science and Technology. The author is greatly indebted to him for his help and advice during the course of this investigation and preparation of this manuscript. Special thanks are also due to Dr. C. R. Heisler for his helpful suggestions during the latter part of this study. Acknowledgment is due to the National Institutes of Health which supported this work in part by funds supplied to the research grant EF-22. TABLE OF CONTENTS Page Introduction 1 Review of Literature 4 Methods and Materials 21 Microorganisms 21 Vitamin K5 21 Microbiological Media 25 Bac to-Nutrient Agar 25 Bacto-Nutrient Broth 25 Bacto-Plate Count Agar 26 Propylene Glycol 26 Microbiological Methods 27 Method of Application Study 31 Anaerobic Studies 32 Contact Time Study 33 Propylene Glycol Study 33 Results and Discussion 36 Effect of Method of Application 36 Effect of Purity of Vitamin K5 38 Effect of Vitamin K,- Against Different Species of Bacteria 39 Effect of Anaerobic Conditions 42 Effect of Contact Time 46 Effect of Initial Number of Microorganisms 49 Synergistic Effect of Vitamin K and Propylene Glycol 51 Summary and Conclusions 54 Bibliography 56 LIST OF FIGURES Figure Page 1 Effect of contact time on the antimicrobial activity of vitamin K against Escherichia coli 48 5 LIST OF TABLES Table Page 1 Antihemorrhagic activities of certain derivatives of 2-methyl-l, 4-naphthqu.inone 6 2 Vitamin K activities of preparations of certain micro- organisms, expressed in 2-methyl- 1, 4-naphtho- quinone units per gram 7 3 Vitamin K content of some materials 8 4 Bactericidal concentrations of vitamin K against certain microorganisms 14 5 Synergistic effect of vitamin K with some antibiotic compounds 17 6 Bacteria used for study 21 7 Incubation temperatures for bacteria studied 27 8 Typical turbidity readings and inoculations used to give an initial count of 160, 000 to 200, 000 cells/ml with the bacteria studied 29 9 Effect of different concentrations of propylene glycol on the total microbial count after 12 hours 34 10 Effect of different concentrations of propylene glycol on the percent inhibition after 12 hours 35 LIST OF TABLES (Continued) Table Page 11 Effect of different methods of application on the antimicrobial activity of 220 ppm vitamin K against Escherichia coli as determined by subcultures stored at 320C for four days 37 12 Bactericidal concentrations of vitamin K in ppm 40 5 13 Total counts/ml in the tubes at the end of the 12 hour incubation period 41 14 Effect of nitrogen atmosphere on the antimicrobial activity of vitamin Kg against microorganisms grown in air 43 15 Effect of nitrogen atmosphere on the antimicrobial activity of vitamin Kg against microorganisms grown in nitrogen 44 16 Effect of contact time on the antimicrobial activity of 220 ppm vitamin Kc against Escherichia coli (total counts) 47 17 Effect of varying initial counts on the antimicrobial activity of vitamin K against Escherichia coli 50 18 Bactericidal concentrations as effected by the synergistic action of vitamin K and propylene glycol 52 19 Total counts /ml of control tubes after 12 hours 53 INTRODUCTION Vitamin K seems to be a promising food preservative because 5 it has a high antimicrobial activity towards a number of microorgan- isms (79, p. 862); it is relatively soluble in water compared with other vitamin K compounds (80, p. 128); it has a low order of toxicity (80, p. 128); and it belongs to a class of compounds which occur in normal human metabolism (17, p. 2). There have, however, been several problems encountered in the use of vitamin K as a food preservative. Faggioli (37) and 5 Matsumoto and Komatsu (64) mentioned the darkening effect of vitamin K as a serious disadvantage in its application as a preserva- tive. Yang et al. (115, p. 504) likewise found a serious discoloration in crab meat. Tengerdy and Francia (103) found vitamin K to be 5 unsatisfactory as a beer preservative because of its poor stability. Gomez (40, p. 63) found that dipping fresh meats, fish, fruits, and vegetables in solutions of vitamin K was an unsatisfactory method 5 of obtaining the contact with the microorganisms which is necessary for food preservation. It was the purpose of this study to investigate some of the factors which might affect the antimicrobial activity of vitamin K in order to add insight into its more effective use as a food preservative. The following factors were investigated to determine their affect on the antimicrobial activity of vitamin K : 5 Purity of vitamin K5r : The study to determine the affect of purity of vitamin K on the antimicrobial activity was undertaken because all 5 of the commercial preparations of crystalline vitamin K exhibit a 5 definite pink to red-brown coloration, indicating the presence of some oxidation products, whereas Oneto and Sah (75) outlined a method for purifying it to shiny white crystals. Type of microorganisms: Pure cultures of Escherichia coli, Bacillus sub tills, Proteus vulgaris, Staphlococcus aureus, and Pseudomonas fluorescens were used for this study. It was desired to find the bactericidal concentration of vitamin K against these different species of microorganisms, as well as to utilize them in studies of the other factors, since they provide a spectrum of food spoilage bacteria. Action in combination with propylene glycol: Pratt e_t aj.. (80, p. 133- 134) found propylene glycol to be an excellent solvent for pharma- ceutical applications of vitamin K and suggested its use as a solvent 5 for vitamin K in food preservation work. Since it is also known that 5 propylene glycol has a low toxicity (41; 42), is an allowable chemical additive in foods (45, p. Ill), has some antimicrobial proper ties itself (86; 87; 88; 89; and 90), and has recently been demonstrated to exert a synergistic effect with certain antibiotics against Escherichia coli and Staphlococcus aureus (70), its study in combination with vitamin K was undertaken.