FISH AND WILDLIFE SERVICE ALBERT M. DAY, Director FISHERIES REVIEW FE RVICE A REVIEW OF DEVELOPMENTS AND NEWS OF THE FISHERY INDUSTRIES A. W. Anderson, Editor R. T. Whiteleather, Associate Editor Wm. H. Dumont and J. Pileggi, Assistant Editors Applications for COMMERCIAL FISHERIES REVIEW, which is mailed tree to members of the fishery industries and allied interests, should be addressed to the Director, Fish and Wildlife Service, United States Department of the Interior, Washington, 25, D.C. FEEDING TESTS WITH GALLIC ACID ESTER ANTIOXIDANTS, BY H. W. NILSON, M. BENDER, AND D. B. DARLING .. 19 The report is a record of experiments conducted in an investigation at the College Park Laboratory as a wartime project to obtain information on the development of and the effectiveness of various gallic acid ester antioxidants in preventing the development of rancidity in fish oils and in inhibiting the decomposition of vitamin A in fish oils. A number of gallic acid ester antioxidants were prepared in the laboratory and tested with various fish oils. Several of the gallic acid esters served as good antioxidants. The alkyl gallates offered some protection to shark liver oils against the loss of vitamin A when the oils were blown with oxygen. INTRODUCTION Considerable losses are suffered yearly in stored fishery products through deterioration of the oils contained therein. This degradation is manifested in the development of a rancid flavor, off odor, and discoloration of these products. Rancidity also is associated with the loss of vitamin A of fishery products, particularly in liver oils. A large number and variety of compounds have been proposed as inhibitors of the development of rancidity in fats and oils. Some are particularly effective in vegetable oils, while others are more adaptable for use in animal fats. Most of these antioxidants have been developed for use in lard, shortenings, oleomargarine or other foods which usually contain a high percentage of fat. These generally contain little if any highly unsaturated fatty acids. Comparatively little work has been done on preventing or retarding rancidity in fish oils. These oils contain highly unsaturated fatty acids and are subject to serious losses because of rancidity. Therefore, an investigation was begun at the Service Laboratory in College Park, Maryland, as a wartime project to obtain information on the effectiveness of various antioxidants in preventing the development of rancidity in fish oils. The objects of the experiments were: (1) to develop materials which would be effective antioxidants for highly unsaturated oils and (2) to develop materials which would inhibit decomposition of vitamin A in fish oils. During the course of some earlier investigations conducted by the author and associates in another laboratory, it was found that protection was given to natural seed oils and to crude vegetable oils by substances of a phenolic nature which were related to the tannins. Various concentrates of tannins from tea and other sources were found to be quite effective in preventing rancidity in these vegetable oils. Later, gallic acid, which had been reported suitable by Golumbic and Mattill (1942), was found to be very effective as an *Former Chemist, Fishery Technological Laboratory, Branch of Commercial Fisheries, U. S. . Fish and Wildlife Service, College Park, Maryland. NOTE: Chronic toxicity experiments with some of the se antioxidants have been conducted at this Laboratory and are reported in the next article in this issue, "Feeding Tests With Gallic Acid Ester Antioxidants," pp. 19-20. antioxidant but had the disadvantage of being rather insoluble in oil. It was found that 10 percent gallic acid in cottonseed oil, which had been partially hydrogenated as a mixture, had a strong antioxidant effect when added in small amounts to vegetable or animal oils. It is probable that under conditions of the hydrogenation, a triglyceride was formed with the gallic acid radical introduced in place of one or more of the fatty acid radicals. In the present investigation, the more promising antioxidants were incorporated into preparations of similar nature. No conclusive evidence of the exact chemical nature of the autoxidation of fats and oils is available. It has been shown that rancidity of fats is associated with the formation of an ozone at the ethylenic double bond, or formation of monohydroperoxides, which in turn can and probably do act as catalysts to accelerate the production of additional unstable peroxides followed by the formation of aldehydes and ketones. This is a chain reaction involving the activation of further molecules of the autoxidizable substance with the attendant liberation of energy in excess of that necessary to activate the same number of subsequent molecules. That a series of reactions induced at an ever-increasing rate takes place has been shown by the characteristic curves for rate of peroxide formation in natural fats. For fats containing no antioxidants, either natural or otherwise, the rate of peroxide formation immediately begins to increase logarithmically. The peroxide formation in fats containing antioxidants increases at a constant rate until the so-called end of the induction period is reached, at which time the antioxidant is largely destroyed. Thereafter, the rate of increase in peroxide formation is about the same as for a fat containing no antioxidant. The mechanism of inhibition of rancidity is probably as follows: The esters of gallic acid and fatty acids combine to form triglycerides which are large molecules similar in size to the fatty glycerides themselves. These probably preferentially absorb the great amounts of the activation energy which are released and which normally cause the formation of peroxides. With efficient antioxidants there should be a constant slow rate of oxidation which is indicated by a straight-line relationship of peroxide formation with time. Synthetic fatty triglycerides have been prepared successfully by direct esterification of fatty acids and glycerol. This method was used in these studies. Dry carbon dioxide was used as a catalyst for the reaction and to remove the water formed during esterification. The glyceride gallate antioxidants are thick, viscous liquids. In making them, however, it is very possible that a small quantity of pyrogallol, or an ester of pyrogallol is formed during the synthesis. This was indicated by a brown tint which formed when they were treated with ferric chloride, as well as the blue-black color which developed with the gallates. The presence of the small quantity of substances containing pyrogallol was discovered late in the progress of this work. Pyrogallol probably has no toxic properties in the amounts present since no adverse effects were noted in rats fed the antioxidants in levels approximating five times the quantity used in oils as antioxidants. These chronic toxicity tests were then in progress for more than a year. Chemically these gallic acid esters are similar to fatty acid modified alkyl resins. Substances of this nature are not crystallizable and do not distill without decomposition even under high vacuum. They are, therefore, practically impossible to separate in pure form. It was recognized that there was a good possibility that migration of the hydroxyl groups might occur, and that the esterification reaction as indicated by formula would not be complete. There was also the possibility of cross polymerization and esterification of fatty acids with gallic acid hydroxyl groups. The characteristic blueblack color formed by the gallic acid radical with ferric chloride was depended upon as an analytical index to reveal the presence of this group. The insolubility of free gallic acid in oil and the values of the acid numbers found were used as an indication of the completeness of esterification. The direct esterification procedure was used as it is a comparatively simple technique and could be carried out with equipment generally available, such as in a varnish kettle. These types of esters probably could be produced with fewer side reactions by esterifying triacetyl gallic acid or 4-methyl gallo-etheric acid with mono- and di-glycerides of fatty acids, followed by reestablishment of the gallic hydroxyl groups through hydrogenation or reduction with zinc and acid. The shorter and more direct method of production was chosen because any materials produced would have to be tested for chronic toxicity regardless of their composition. If no toxicity is indicated, it is the preferred method. In the tests reported herein, all of those materials which contain gallic acid or its esters were added to the substrates in amounts equivalent to 0.1 percent of gallic acid radical. The other materials tested for comparative purposes were added at the 0.1-percent level unless otherwise stated. During the course of oxidation of various deodorized fish oil substrates using a modified Swift test (King et al, 1933), it was found under the conditions of testing that the oils uniformly became rancid to taste at a peroxide value (p.v.) of about 20 millimoles per kilogram of oil. This peroxide value, therefore, has been taken as the value for comparison in calculating the protective factor for any particular antioxidant. The protective factor equals the time to reach p.v. 20 for the treated oil divided by the time to reach p.v. 20 for the untreated oil. In evaluating antioxidants as inhibitors of vitamin A destruction, two protective factors were calculated. These were based on the ratio of the time necessary to destroy 10 and 20 percent of the vitamin content in a treated oil, to the time necessary to destroy 10 and 20 percent of the vitamin content in the untreated oil. PEROXIDE VALUES: METHODS OF ANALYSES Peroxide values were measured by a modification of the Wheeler method (1932). Samples of oil from 0.1 to 1 g. in weight, and which required a titration of not more than 15 ml. sodium thiosulfate solution, were weighed into small weighing vials. The vials plus samples were placed directly into dry, glass-stoppered 200 ml. Erlenmeyer flasks containing 50 ml. of a mixture of two parts of glacial acetic acid and one part of chloroform. After thorough mixing, one ml. of saturated potassium iodide was added, and the flask was held in the dark for three minutes. Fifty ml. of water were added together with a little starch solution, and the mixture was titrated with .002 N sodium thiosulfate. The data were expressed as millimoles of peroxide per kilogram of oil. VITAMIN A: The vitamin A determinations were made by direct solution of the oil in isopropyl alcohol and a photometric estimation of absorption in a United Drug Company Vitamin A Meter. Some determinations were made using the unsaponifiable portion of the oil to rule out possible absorption by saponifiable materials added to the oils. The RAPID TEST FOR OXIDATION: apparatus used embodies the principles of the well-known Swift stability test apparatus (King et al, 1933) except that in these experiments U.S.P. oxygen from a pressure cylinder was used (Fig. 1). The apparatus consists of a metal container fitted with a reflux condenser to return the moisture and to maintain a constant temperature of 100° C. within the boiling water bath. Large test tubes containing mineral oil are fitted into this metal container. These serve as oil baths in which the smaller test tubes containing the test sample are immersed. A manifold system is provided for introducing oxygen at a pressure of about 14 inches of water. The pressure is controlled by a water column manometer. Capillaries 0.1 mm. or less in diameter are placed in the bubbling-tube lines and are calibrated to allow a flow of 50 ml. of oxygen per minute at the before-mentioned manifold pressure. Pyrex test tubes, 25 mm. in diameter, are used as sample tubes. To these are sealed, near the top, horizontal short lengths of 7 mm. tubing which serve to limit the depth of immersion of the tubes in the oil bath and which are used as exit tubes for the oxygen. The gas flowing from these tubes is sniffed for rancid odors to determine how oxidation is proceeding. All glass parts of the apparatus that came in contact with the samples were thoroughly cleaned with a wetting agent, acetone, and tap water. The parts were immersed in concentrated nitric acid at a temperature of about 90° C. for at least 3 hours, rinsed at least 10 times with tap water, and at least 6 times with distilled water, and oven-dried. The various antioxidants tested were dissolved in the oil used as substrate by stirring with a clean glass tube through which passed a stream of hydrogen gas. Very gentle heat was used only with those samples which would not dissolve otherwise. The oxidation apparatus was brought to operating temperature, which required about 10 minutes, before any samples were placed in it. The sample tubes were filled to a depth of 9 cm. and the bubbling tubes were inserted. The samples were allowed to remain in the apparatus without bubbling for 15 minutes to allow them to reach the required temperature. The oxygen system had been previously turned on without a connection with the sample tubes in order to flush out the air. At the end of the 15 minutes, the tubes were connected with the oxygen system. Samples for the determination of peroxide value were taken periodically by momentarily interrupting the oxygen flow and using the bubbling tube as a sampler. Two samples were run simultaneously in this apparatus. The temperature of the oil in the sample tubes was determined periodically by using a similar tube which contained a thermometer. The temperature was found to remain constant at 99.5° C. |