INDUJSTRIALA N D ENGINEERING CHEMISTRY
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Vol. 15, No. 6
Inactive Malic Acid as a Food Acidulent’ By John M. Weiss, Charles R. Downs, and Harry P. Corson THEBARRETT Co., 40 RECTORST., NEW YORK,N. Y.
T H E commonly used
As a result of the manufacture of synthetic malic acid under ea1 with citric, tartaric, and food aciddents of the authors’ patents and its subsequent conversion to malic acid, malic acids. Ordinary care the Past, citric and in bottling and sterilizing a new outlet for the material was sought. The occurrence of malic tartaric acids, have been in certain frliit juices suggested its use as an acidulent in foods to prevents deterioration from 1argelY imported. T h e replace citric and tartaric acids, the supply of which is seasonal molds. Superior methods of hortiand for the most part imported. The toxicity of malic acid to rabbifs . METHOD culture used in California was determined, as well as other properties which might affect its as compared with those of Work on fumaric acid use as a food acidulent. The results show that it may be safely used in this way to replace the naturalfruit acids now employed. Malic, was included, not with the Italy Produce a much smaller Proportion of the citric, and tartaric acids showed no cumulatioe effects and in every idea of considering it as a citrus-fruit crop as C U 1 h way their actions were the same. food acidulent per se, but so that the production of because it is present in citric acid in this country small quantities in inachas never equaled the consumption. Tartaric acid has been tive malic acid. It is widely found in nature, existing in produced largely as a by-product of the wine industry. many edible mushrooms, although in small concentrations. The manufacture of the crude acids has been seasonal, and Its use as a food acidulent is not practicable because of its a more uniform source of food acidulents is desired. More- high insolubility in water. over, the imported acids are largely affected by tariff rates. Some of the most recent authoritative work on the physioDomestic production of a food acidulent would place the in- logical effect of citric and tartaric acids has been done by dustry of food-acidulent application on a more stable basis. Salant and Smith,2 Salant and Wise,3 and Underhill, Wells, For some time malic acid has been used commercially and Golds~hmidt.~ in the food industry on the ton, not the pound, scale. It has Although some work on the physiological action of malic proved very satisfactory as a food acidulent in jellies, soft and fumaric acids has been reported in the literature, the drinks, and candies, and, in the opinion of many manufac- limited quantities available and excessive cost of these acids turers, it is superior to tartaric acid for this purpose andatleast have prevented exhaustive feeding experiments, equal to citric acid in flavor, blending qualities, and general I n much of the experimental work on the relative toxicity of characteristics. Some manufacturers who have specialized chemical substances, the technic followed has been to adminin the production of such flavors as raspberry, pear, and apple ister the substance to small animals by subcutaneous, insay that malic acid gives a flavor superior to that obtained with travenous, and intraperitoneal injection. The specific citric acid. Malic acid has also been tried experimentally action of the substance can be studied more accurately by in effervescent‘salts and other medicinal laxatives in about the the injection method than by administration per os. With same manner that citric and tartaric acids and their salts have most animals, administration of large quantities of some subbeen used. This is under investigation a t present, and stances causes vomiting, thus upsetting the plan of experialthough progress of the work seems to give favorable indi- mentation. Moreover, the injection method is more rapid cations, the results are not sufficiently complete to form and requires much less material. any final conclusions. Onta5found that part of the levo-malic acid administered Before inactive malic acid could be offered for food pur- in sufficient doses to rabbits and dogs was excreted unPoses, it was necessary to determine its suitability. It has changed in the urine. Administered orally in large quantities, the advantage of being less hygroscopic than levo-malic the acid is toxic. Malic acid givenup to 10-20 g. per os to rabacid. Its taste apparently is the same as that of thelevo-acid, bits was entirely destroyed. When the doses reached 25 to a t least as good as that of citric acid, and superior to that of 30 g., about 5 per cent was excreted in the urine. Results tartaric acid. This superiority is more marked when the acid with subcutaneous injection were similar but gave less deis mixed with flavors. With tartaric acid two separate struction of the acid. tastes are apparent, while with malic or citric the essences Wise6 obtained results differing only in degree. *He recombine with the acid into one flavor. ports that with doses of 1 g. per kg. of body weight, which were not toxic, rabbits excreted from 3 to 21 per cent of the PRELIMINARY EXPERIMENTS malic acid unchanged. Doses of 3.3 g. per kg. body weight Malic acid acts on tin, tin-coated iron, and lead parts of produced toxic symptoms and death. containers in much the same way as, but probably is less Tomita7 investigated the behavior of racemic-malic acid corrosive thqn, citric, tartaric, and lactic acids, It may by subcutaneous injection of the sodium salt into rabbits. He therefore be handled in containers suitable for the other food found that the dextro form was destroyed to a less extent acidulents. than the levo form. No mold growth took place in citric, tartaric, and malic Administration per os, commonly used for substances to be acid-water solutions of 50 per cent strength. even after stand- introduced into the stomach, was used throughout the work ing for one month exposed to the air, This concentration reported in this paper. represents the approximate strength of solutions used in I n therapeutics and toxicology, results obtained by experithe manufacture of sirups. The Concentration’a t which mold a A m . J. Physiol.. 35 (1914), 239; J . Pharmacal , 6 (1914), 123. growth occurs is between 10 and 20 Der cent. and the three 8 J . Pharmacol.. 8 (1916). 123. 4 J . E x @ . Med.9’18‘(1913j,317, 322, 347. acids are alike in this respect. The rate of mold growth in 6 Biochem. Z.,44 (1912), 481. solutions of from 0.1 to 1per cent strength is practically identi0 J . Biol. Chem., 28 0 9 1 0 ) , 185. 1
Received September 22, 1922.
Biochem. Z.,123 (1921), 231.
IiVD USTRIAL A X D ESGILYEERISG CHEMISTRY
June, 1923
mentation on animals of this type can be transferred to the human organism in the same way that other fields of experimental medical research have been transferred, and as a rule the same toxic agent affects similar structures in a similar way, no matter in what animal they are studied8 Within reasonable limitations, therefore, it seems fair to consider that the relative results for toxicity as obtained on the rabbits are in the same relative proportion as the toxicity of the same substances when ingested by man.
ANIMALS SELECTED The rabbit, which is easily handled and retains whatever dose is administered, was selected for experimentation. Growing animals of the Belgian hare and Flemish Giant breeds weighing from 3 to 5 lbs. each, were fed hay and oats, supplemented by green vegetables. LfATERIALS
USED
CITRICACID-U. S.P . crystals were used when the free acid was fed. When the sodium salt was fed, the acid was first dissolved in water and then made neutral to phenolphthalein paper by adding the proper volume of sodium hydroxide solution. TARTARIC ACID-U. S.P. crystals were used for the free acid dose. The neutral sodium salt was prepared as under citric acid. LACTICAcm-The U. S.P. product was used. The quantities administered refer to total acid (free titratable acid and available acid present as anhydride). The sodium salt was prepared by adding the calculated quantity of standard sodium hydroxide solution to a previously weighed and assayed quantity of U. S. P. lactic acid and boiling the mixture for 10 min., thus insurini the conversion of both free and available lactic acid into the sodium salt. PHOSPHORIC ACID-A high-grade C. P. reagent, o-phosphoric acid, was used. SULPURIC Acm-Free sulfuric acid was not administered. C. P. reagent grade of sodium sulfate was the salt administered. XA’I’URALMALIC AcID-’I’he product used was white, free from hydrogen sulfide metals, and completely soluble in a 50 per cent solution except for the presence of a slight opalescent turbidity, SYNTHETIC MALIC ACID-TWO samples of inactive malic acid were used, one (“i-malic acid from fumaric acid”) prepared from fumaric acid, and another (“i-malic acid from maleic acid”) prepared from maleic acid. Both of these 4-malic acids were prepared in the laboratory from the corresponding source acid by autoclaving with water under the conditions found necessary to prevent the presence of maleic acid in the product, as described in a paper on the equilibrium conditions of fumaric and malic acids by’ Weiss and Downs. The autoclaved product was cooled to 25’ C., the fumaric acid which crystallized out was filtered off, the solution was decolorized by boiling with a decolorizing carbon, and the filtrate was evaporated to a 55 per cent solution in vacuo. The cooled solution was again filtered to remove further quantities of dissolved fumaric acid. The filtrate was evaporated to a heavy sirup in vacuo, cooled to solidification, and dried in vacuo. The acid was white and had an acidity of 98 per cent and an ash content of approximately 0.3 per cent. The sodium salts were quantitatively prepared from these acids. FUMARIC ACID-Because of the fact that 4-malic acid usually contains very small quantities of fumaric acid, this acid and its sodium salt were included in the list of materials to be investigated. The acid was prepared by acidifying a colorless solution of its sodium salt with sulfuric acid and filtering off the precipitated fumaric acid. It contained about 1 per cent of ash, consisting chiefly of sodium sulfate, and showed an acidity of approximately 98 per cent. It was white. The sodium salt was quantitatively prepared from the acid. HYDROCHLORIC Acm-The free acid was not fed, but a C. P. reagent sodium chloride was used.
ADMINISTRATION OF DOSES The doses were administered in a solution, or in a suspension when the solubility of the substance did not permit complete solution, using a total volume of 30 to 35 cc. The 8 9
Sollmann, “Manual of Pharmacology,” 2nd ed., 1922, p. 82. J . Am. Chem. Soc., 44 (1922), 1118.
629
solution was introduced through a rubber tube which was inserted down the throat into the stomach. The animal was placed on its back on a board, the feet being securely tied. A stick in which a hole had been bored was placed between the animal’s teeth, the hole coming ovcr the mouth. A soft rubber tube was slipped through the hole in the stick and thence into the stomach. By means of a funnel the solution was passed through the tube into the stomach, after which the animal was released and returned to its cage, which contained food and water. The animal was observed a t least once an hour for 24 hrs.
RESULTS The following three tests will show the type of the experiments: Rabbit No.
Body DOSE Wt. G. G / K g . EFFECT Citric Acida (Free Acid) 17 F 1675 6.4 3.8 9 1677 None visible: animal shows no abnorF 7.7 F mal symptoms 25 2166 11.0 F 32 1860 1 2 . 0 6 . 5 Animal showed unfavorable symptoms 2090 1 4 . 6 7 . 0 F 34 15 min. after feeding; nervous tremors, violent twitching, convulsions. and death 2 hrs. after feedinc Tartaric Acida (Free Acid) 15 F 2312 11.3 5 . 0 Ate, but very depressed; dead 5 hrs. after feeding 1869 1 0 . 0 5 . 4 20 M Lay down breathing rapidly, 1 hr. after feeding; greatly improved the following day 26 M 1647 8.2 5.0 Plainly in distress soon after feeding; lay down with head thrown back rigidly; dead 3 hrs. after feeding 28 F 1384 8.0 5.8 Very ill soon after feeding; lay down; dyspnea; struggled violently; dead 2 hrs. after feeding i-Malic Acida (Free Acid) F 1755 10.0 5 . 7 None visible; animal apparently normal 9 8 F 1745 12.0 7 . 0 Soon after feeding animal seemed depressed; lay down with eyes shut; dead 3 hrs. after feeding; autopsy showed normal lungs, heart, liver and kidneys; intestines distended with watery fluid 33 F 1470 8.8 6.0 Apparently normal; no visible ill effects. 27 F 1525 11.0 7 . 0 uiet but ate; next day normal and fully recovered a Weights refer to anhydrous free acid. Sex
2:;
j
I
*
A summary of the results is given in Table I. TABLE I
ESTIMATED
ACID Citric Tartaric i-Malic from maleic
;-Malic ~- ... from
-
fumaric
- --
Srrmmsrv for
i-malic 2-Malic Fumaric Lactic Sulfuric Phosphoric Hydrochloric
RANGEIN BODY INITIAL WEIGHTIN WHICH TIME FROM FEED- MINIMUM DEATHTOOKPLACE ING TO DEATH FATAL DOSE Free Acid Free Acid N a Salt Free Acid N a Salt G./Kg. G./Kg. Hrs. Hrs. G./Kg. 6 . 5 t o 7 . 0 3 . 4 t o 4 . 7 01 to 2 12 to 3 7.0 5 . 0 t o 5 . 8 4 . 8 t o 5 . 2 3 t o 2 11 to 8 5.0 5 . 7 t o 7.0 6.0t07.0
5 . 5 to 8 . 0 5.0
01
to 3
a to
01
22 to 1.5 14
6 . 0 to 7 . 0 5 . 0 to 8 . 0 a to 3 14 t o 1.5 5.5 to 6.0 5 to 3 5.0 4.5’to 5 . 0 O1 O1iO’lOO 4 . 0 t 0 6 . 4 5 . 0 t o 6 . 0 01to 2 ato 4 4.0 19 3. O‘to 3 . 5 ,‘Io 12 , .. 5:0 5
..
7.0 7.0+
7.0+ 5.5 5,0+ 5.0 3:b(?)
...
The results, which represent experiments on forty animals reported in Table I, make possible a close estimate of the minimum fatal dose for the free acids, as slight differences in the quantities produce marked differences in results. I n these cases the rabbits either lived indefinitely or died shortly after the dose was administered. This was not the case when the salts of the acids were fed where even wide variations in the quantities administered produced death. One rabbit lived for 100 hrs. It is evident that the actions of acids and their salts are of an entirely different nature. No estimate of the minimum fatal doses for the salts has been made. The results have been arranged in an ascending scale of dose per unit of body weight in Table 11.
INDUSTRIAL A N D ENGINEERIIVG CHEMISTRY
630
TABLE I1
G./RG.
ACID
TIMEPROM FEEDINC
OR SALT
BODY WEIGHT
Citric Malic NazSO4 Fumaric Citric Tartaric NaCl Malic Fumaric Lactic Tartaric Malic Malic
3.4 3.8 4.0 4.5 4.7 4.8 5.0 5.0 5.0 5.0 5.2 5.5 8.0
TO DEATH
HRS.
12
n
19
(I
3 11 5 14 100 (I
8 22 1.5
More work should be done to determine the comparative physiological effect of the salts of these acids, using basic radicals other than sodium. After determining the initial minimum fatal doses of the several acids, an investigation was made to ascertain the effect of feeding 2.5 g. of the free acids daily in order to determine whether or not tolerance or cumulation would be exhibited. The acids used were i-malic, citric, and tartaric. The type of results is shown by the record of one of the rabbits fed on malic acid. DATE February 28 March 1 2
3 4 5 6 7 8 9 10 11 12 13 14 15
Doss G./KG. 2.5 2.5 2.5 2.5 2.5 2.5 None None 2.5 2.5 2.5 2.5 2.5 None None 6.0
BODY WEIGHT G.
REMARKS
1843
1917 Apparently normal
1951 Dead 12 hrs after feeding
Vol. 15, No. 6
Three rabbits (2 female and 1male) were given malic acid, 2 rabbits (1 female and 1 male), citric acid, and 5 rabbits (3 female and 2 male), tartaric acid. The data show that of the 3 animals which received 2.5 g. of i-malic acid per kg. of body weight on 5 consecutive days per week for 2 wks., 2 survived and 1 died after the first week. The two survivors were then given doses of 6 g. per kg. One died and the other survived. After 2 days the latter was given a dose of 8 g. per kg., with a fatal result. The 2 rabbits which received 2.5 g. of citric acid per kg. of body weight on 5 consecutive days per week for 2 wks. survived. After an interval of 2 days, a dose of 7 g. per kg. was administered. This proved fatal to one animal. The survivor was given a dose of 9 g. per kg. after a second interval of 2 days. This proved fatal. Of 5 rabbits in the series which received 2..5 g. of tartaric acid per kg. of body weight, 4 failed to survive even the first week. Three, and perhaps all of the 4,died from acute lung infection. The fifth survived for 2 wks. and after an interval of 2 days was fed 5 g. per kg. of body weight with no unfavorable symptoms. After a n interval of 2 days, 7 g. per kg. were fed, with a fatal result. Whether tartaric acid weakens the resistance of the animals and thus predisposes to infection, or whether the fatalities were accidental and due to infection started by irritation of the throat with the stomach tube, is open to question. It should be stated, however, that all the animals were confined in the same room in separate cages and the same care and technic was used with all. Less favorable results might have been obtained with malic and citric acids if the daily doses had been 50 per cent of the predetermined fatal dose, as was used in the case of tartaric, instead of approximately 35 per cent. In any event, the results obtained show the equivalent actions of malic and citric acids and their superiority over tartaric acid,
Conference on Scientific Instruments and Apparatus A conference of men interested in the use or the manufacture of scientific instruments and apparatus was held recently a t the headquarters of the National Research Council, in Washington, D. C., a t the call of the Council. It was attended by members of government scientific bureaus, scientific societies, universities, the Association of Scientific Apparatus Makers, and individual firms. G. K. Burgess, director of the Bureau of Standards, presided. A committee appointed by the chairman brought in a report which was adopted. At its suggestion a Committee of Apparatus Makers and Users is t o be formed under the Research Extension Division of the Council. This committee is to consist of one representative from each of the following technical societies and organizations: American Chemical Society, American Physical Society, American Instit u t e of Electrical Engineers, Optical Society of America, American Electrochemical Society, American Ceramic Society, the Association of Educational Buyers, the American Society for Testing Materials, the Engineering Chemists’ Association, the American Society for Steel Treating, the Society of Automotive Engineers, two representatives from the Bureau of Standards and t h e National Research Council, and six representatives from the Association of Scientific Apparatus Makers of the United Statrs of America, with members-at-large t o be appointed by thia committee.
The following were appointed to serve as members-at-large until their successors are chosen: Hermann Rellner, Bausch & Lomb Company; A. L. Day, Corning Glass Works; H. B. Williams, Society of Automotive Engineers; H. E. Ives, Western Electric Company, and C. E. K. Mees, Eastman Kodak Company.
The following were selected to act as an executive committee until their successors are chosen by the main committee : W. M. Corse, National Research Council, chairman; Paul Moore, National Research Council, secvetary; W. D. Collins, Geological Survey; F. K. Richtmyer, Cornell University; M. E. Leeds, president of the Association of Scientific Apparatus Makers; John Roberts, secretary of the Association of Scientific Apparatus Makers; and G. K. Burgess, director of the Bureau of Standards.
It was agreed that there should be held a t least one meeting a year of the general committee, and it is hoped that this can be arranged to take place on the third Thursday in April. This meeting is to be open to all interested. Vernon Kellogg, in welcoming the conference, spoke of the importance of little things, and indicated the progress science had made with the development of the instruments for learning the truth. The discussion-for the whole purpose of the conference was to bring face to face in a frank conference men interested in the use, manufacture, and the sale of apparatus-centered around (1) apparatus supply, under the heads-importation, domestic apparatus manufacture, instrument shop of research and college laboratories, manufacture of special apparatus not now made in the United States; ( 2 ) standardization of apparatus, under the heads of limitation of types and sizes, standardization of parts, standardization of methods; (3) information service, under the heads-sources of information, finding list, scientific bulletin concerning apparatus and use; and (4)inspection service. I t was unanimously agreed that the conference had served a very useful purpose, that it would bring about a better understanding between makers and users of the peculiar conditions which face them and lead to more cooperation between them.
