lune, 1941
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crawling are different from those which give rise to the defects produced upon baking, A change in the balance of the simple mechanical forces involved in the equilibrium between adhesion and cohesion probably accounts for these differences. Changes in polarity of the materials during baking which, in turn, would affect the interfacial tension a t varnish-metal interfaces, also probably play an important part. This work is being continued.
for his disinterested contributions through his communications. They also wish to thank H . A. Stobbs, of the Wheeling Steel Company for the palm oils used.
Acknowledgment
PBBIEBINTBID before the Division of Paint and Varnish Chemistry at the 100th Meeting of the American Chemioal Society, Detroit, Mich. Based upon a part of a thesis submitted by J. E. Hanle to the Graduate Faculty of the Polytechnic Institute of Brooklyn in partial fulfillment for the requirements for the degree of master of chemical engineering.
~h~ authors wish to express their appreciation to the &furand faphy Varnish Company for the use Of their cilities in carrying out this investigation, and to G. H. Young
Literature Cited (1) American Can Co., Canned Food Reference Manual, 1939. (2) Hade, J. E., unpublished investigation. (3) Stobbs, H. A., private communication. (4) Young, G . H.,private communications.
Fungistatic Properties of
Antiseptics and Related Compounds EFFECT OF pH CHARLES HOFFMAN, T. R. SCHWEITZER, AND GASTON DALBY Ward Baking Company, New York, N. Y.
URING a study of the fungistatic properties of the fatty
D
acids (7) considerable data on the effect of pH on the fungistatic properties of some of the common antiseptics were accumulated. Although much work has been done on the effect of antiseptics on fungi, comparatively little attention has been paid to pH in these studies. Smith ( 2 1 ) reviewed the effect of a number of inorganic and organic substances on mold. Schamberg et al. (20) and Kingery et al. (8) made extensive studies on fungicides from the point of yiew of the relation of structure to fungicidal properties; however, little attention was given to pH. Degering and co-workers ( 1 , 2, 3,6, 6) published a series of articles on the effect of pH and substituent groups on the bacteriostatic and bactericidal properties of certain antiseptics. The work of Degering on the critical role of pH in bacteriological studies is of special interest in view of the effect of pH on the fungistatic properties of various compounds. Molds grow readily over a wide range of pH, and consequently any effect of pH on fungistatic properties is not due solely to hydrogenion concentration. This is not generally true of bacteria. Degering pointed out (3) that solutions of high acidity are bactericidal in themselves as a consequence of hydrogen-ion concentration alone. I n general, therefore, bacteria prefer neutral pH ranges for optimum growth, whereas the common molds grow best under acid conditions. Degering showed increased potency with an increase in the hydrogen-ion concentration of the menstruum for most of the compounds tested, and therefore postulated a “hydrogen-ion effect”. Although many substances show enhanced fungistatic properties with an increase in hydrogen-ion concentration, a
The pH of the medium has no appreciable effect on the fungistatic properties of mercuric chloride, formaldehyde, phenol, copper sulfate, and iodine which are apparently due to the inherently poisonous nature of the materials. In the case of sodium 1hiosulfate, sodium sulfite, and hexamine, the decomposition of the molecule at certain pH values, with the consequent liberation of toxic products, indicates an indirect effect of pH on fungistatic properties. The pH of the medium has a significant effect on the fungistatic properties of benzoic acid, salicylic acid, acetylsalicylic acid, propionylsalicylic acid, catechol (to some extent), resorcinol, and hydroquinone. The pH of the medium also has an effect on the fungistatic properties of boric acid. A suggested relation of structure to fungistatic power is made.
corresponding postulate for fungistatic properties, based on the evidence now available, cannot be made. The variation of fungistatic effect with pH in case of a number of common antiseptics is of interest from a practical point of view in the explanation of the action of antiseptics on fungi, and also from theoretical considerations in that some interrelations between fungistatic properties and molecular structure are apparent. The effect on fungi of a number of compounds chemically related to some of the common antiseptics has been determined in order to study the relation of structure to fungistatic properties.
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3
4
5
6
7
PH
It must be emphasized that an antiseptic is not necessarily a fungicide, but in many cases those substances which are antiseptics because of their poisonous nature are also effective against molds. Smith (11) called attention to the extreme resistance of fungi to toxic substances and the variation in the effect of antiseptics on bacteria and on molds. This study was limited to a determination of fungistatic properties; no attempt was made to determine the critical fungicidal concentrations of the antiseptics and related compounds. The fungistatic effect-that is, the power of a substance t o inhibit the growth of mold rather than kill the organism-appears to be a t least as interesting physiologically as a determination of the fungicidal properties. In the field of bacteriostats, Cowles (4)remarked that "from the standpoint of the physiology of bacteria, bacteriostasis is probably quite as important as is the actual death of the cell". The method previously reported for the determination of the fungistatic properties of the fatty acids was followed (7). The points on the graphs represent the minimum molar concentrations a t each pH value necessary t o inhibit all visible mold growth for 48 hours a t 37.5" C. The spores used for seeding were from a mixed culture of common molds occurring on foodstuffs, and consisted of representatives of the usual types such as Aspergillus niger, Aspergillus glaucus, Rhizopus nigricans, Penicillium frequentans, etc. The media consisted of nutrient agar containing one per cent sucrose, and the buffers were mixtures of citric acid and disodium phosphate. An incubation temperature of 37.5" C. was
chosen because it has been a practice over a long period of years in this laboratory to incubate daily samples of bread for keeping properties. This temperature gives an acceler= ated mold growth and therefore serves excellently for routine control. On the plates without inhibitor (media plus buffer) there was always a vigorous growth even in 24 hours, and in 48 hours sporulation was heavy a t this temperature.
Inorganic Chemicals The inorganic substances tested were iodine, copper sulfate, sodium thiosulfate, sodium bisulfite, mercuric chloride, and boric acid. Iodine showed excellent fungistatic properties over the range from pH 2 to 8. Copper sulfate had a similar type of curve (Figure 1). Sodium thiosulfate was effective a t pH 4 and below, but above that point rapidly lost its effect. Sodium sulfite was also effective a t p H 2 and 4, but not above pH 4. The value of sodium thiosulfate has generally been considered due to sulfur dioxide liberated from the thiosulfate in the acid ranges. These results appear to bear out this conclusion. Mercuric chloride has a powerful fungistatic effect over the entire pH range of these experiments as shown in Table I. Chlorine was not included in these experiments because it was extremely difficult to maintain constant concentrations in a medium containing nutrients. Unquestionably this situation also applied in a smaller degree to iodine, but reproducible results with iodine were obtained. I n this connection Lever (9) pointed out that iodine combined with the protein material of the body and was thus
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acid. Formaldehyde is a powerful disinfectant and a powerful fungistat. The effectiveness of hexamine as an urinary antiseptic is considered to be due to the liberation of formaldehyde under acid conditions, and in view of the powerful effect of formaldehyde on mold, the inhibiting properties of hexamine were studied over the pH range 2 to 8. As the curve indicates, hexamine is effective in the acid ranges apparently because of the liberation of formaldehyde under acidic conditions. These facts are in agreement with the knowledge conOF INORGANIC COMPOUNDS cerning hexamine as an urinary antiseptic. Since mandelic TABLEI. MOLARCONCENTRATIONS REQUIRDD TO INHIBIT MOLDAT VARIOUSPH VALUES acid is coming into more general use as an urinary antiseptic, Chemical pH2 pH4 pH5 pH6 p H 7 pH8 its action on mold was studied to compare i t with hexamine. ... 0.00157 ... 0.00390 0.00390 Iodine 0.00078 Mandelic acid, however, had no fungistatic properties. 0.12 0.10 0.10
rendered partially inert when used as an antiseptic. At pH 2 boric acid showed fungistatic properties, but at pH 4, 5, and 6 it was ineffective up to the limit of solubility. At pH 7 and 8 it showed fungistatic properties. I n the neutral pH ranges boric acid would exist in the form of the sodium salt, and these results apparently indicate that borax is more effective against mold than boric acid.
Co per sulfate S&um thiosulfate Sodium sulfite Merouric chloride Boria acid
0.10
0.0024 0.0056
0.00007
0.29
Ineff&iive above pH 4 {Ineffective above pH 4{ 0.0022 0.0026 (IheffeativeatpH4,6:6) 0.25 0.15
0.0806
0.0056
0.00037
Organic Compounds The first group of related organic compounds tested were phenol, benzoic acid, salicylic acid, nicotinic acid, m-hydroxybenzoic acid, and p-hydroxybenzoic acid. The effectiveness of phenol is scarcely affected by pH, and its graph is essentially a straight line. Benzoic and salicylic acids are markedly affected by pH. Salicylic acid contains both the hydroxyl of phenol and the carboxyl of benzoic acid in the ortho position, but its fungistatic curve more nearly resembles that of benzoic acid (Figure 1). It is generally less effective than benzoic acid, however. The shift of the hydroxy group to the meta or para position eliminated the fungistatic p r o p erties of the compound. m-Hydroxybenzoic and p-hydroxybenzoic acids were tested in very high concentrations (in many cases to the limit of solubility) without showing mold inhibition. Nicotinic acid lacked fungistatic properties. Nicotinic acid has a structure analogous to benzoic in that in the former a nitrogen replaces a carbon in the benzene ring. This change, however, markedly affects the fungistatic properties. The second related group of organic compounds tested were the acetyl and propionyl derivatives of salicylic acid. The acetylation of the hydroxy group somewhat changed the character of the curve of salicylic acid (Figure 1). Propionylsalicylic acid was considerably more effective than the acetyl derivative. The amount of propionylsalicylate required to inhibit a t pH 8 could not be determined because of the limiting factor of solubility. The third related group of organic compounds tested were the three dihydroxybenzenes-catechol, resorcinol, and hydroquinone (Figure 1). The curve of phenol, monohydroxybenzene, is included for comparison. Catechol, the ortho compound, rather closely resembles phenol in the character of its curve. Hydroquinone, the para compound, is much less effective in the acid pH ranges, but on the alkaline side, the effectiveness increases sharply. This apparently indicates that the quinone form is a more powerful fungistat than the corresponding reduced hydroxy form. The shift to the quinone is facilitated by a neutral or alkaline pH, and the development of color in the plates is indicative of this shift. The meta compound, resorcinol, is intermediate in its action between catechol and hydroquinone. It is interesting to note the relation between the hydroxy and carboxyl substituents in the benzene ring as, for example, the curves of salicylic acid and catechol. Salicylic acid is more effective than catechol a t high acidities and much less so in the neutral ranges. The presence of a carboxyl in the compound markedly changes the effect of pH on the fungistatic power of such substances. The fourth group of organic compounds tested were formaldehyde, hexamethylenetetramine (hexamine), and mandelic
Discussion Substances such as mercuric chloride, formaldehyde, and phenol which are poiBonous in nature are not markedly affected in their fungistatic action by a change in pH. Substances such as hexamine and sodium thiosulfate are affected by pH in that, at some definite pH value, decomposition of the molecule takes place with the liberation of toxic products. I n the type of compound represented by benzoic acid, the fungistatic power is closely associated with pH. I n the acid pH ranges benzoic is a fungistat whereas in the alkaline range it is not. The molecule as such, except for salt formation under alkaline conditions, is not affected by pH changes. It is suggested that the fungistatic power of such a molecule depends on the presence of both water- and oil-soluble groups in the molecule in some degree of balance. The benzene ring group is oil soluble (nonpolar), and the carboxyl is water soluble (polar). The introduction of a hydroxyl group in the ortho position, to form salicylic acid, weakens the molecule as a fungistat. I n this case the oil-soluble portion of the molecule has been lessened and the water-soluble portion augmented. When the hydroxyl is introduced in either the meta or para positions of benzoic acid, the resulting compounds are no longer fungistatic. In p-hydroxybenzoic acid, for example, there is a polar group on each end of the benzene nucleus, and the balance between the polar and nonpolar groups has apparently been affected in such a way as to cause the loss of fungistatic properties. An analogous relation holds for phenol, catechol, resorcinol, and hydroquinone; that is, if the polar groups are concentrated in one area of the molecule, a more powerful fungistat results than if the polar groups are spaced around the nonpolar nucleus. Apparently the hydrogen-ion concentration of the media affects the relation of polar and nonpolar groups and thus affects fungistatic power. For example, a carboxyl group in the alkaline pH range, with resultant salt formation, mav act more stronelv than a - ” Dolar . carboxyl in the acid pH”range.
Literature Cited Bittenbender, W. A., and Degering, E. F., J. Am. Pharm. Assoc., 28,514 (1939). Bittenbender, W. A., Degering, E . F., and Tetrault, P. A., IND.ENO. CHQM., 31, 742 (1939). Bittenbender, W. A., Degering, E. F., Tetrault, P. A . , Feasley, C. F.,and Gwynn, B. H . , Ibid., 32, 996 (1940). Cowles, P. B., Yale J . Biol. Med., 11, 33 (1938). Goshorn, R. H . , and Degering, E. F , J. Am. Pharm. Assoc., 27,865 (1938). Goshorn. R. H., Degering, E. F., and Tetrault. P. A.. IND. ENQ.C H I M . , 30,646 (1938). Hoffman, C., Schweitzer, T. R., and Dalby, G., Food Reseurch, 4,539 (1939). Kingery, L. B., Williams, R., and Woodward, G., Arch. Dermatol. Syphilol., 31,452 (1935). Lever, W. F.,Ibid., 40, 19 (1939). Schamberg, J. F., Brown, H., and Harkins. M. J., Ibid., 24, 1033 (1931). Smith, G., “Introduction to Industrial Mycology”, pp. 269-74, London, Edward Arnold and Co., 1938.
