SOME NUTRITIVE PROPERTIES OF NUTS.

Transcription

1 SOME NUTRITIVE PROPERTIES OF NUTS. II. THE PECAN NUT AS A SOURCE OF ADEQUATE PROTEIN.* BY F. A. CAJORI. (From the Department of Chemistry, Stanford University, Palo Alto.) (Received for publication, October 13, 1921.) It has been recently observed that young rats would grow at a normal rate and attain adult size on diets in which the essential source of the protein of the ration was derived from various nuts.1 From these results it was coneluded that the proteins of these nuts furnished suitable amounts of those amino-acids necessary for growth and that they can be regarded as complete from the point of view of nutrition. With the exception of the pecan nut successful feeding trials resulted with all the nuts investigated, a list that included many of our important protein-rich nuts. The rations containing the pecan nut as the source of protein were complete in every other known dietary essential. Two causes may account for the failure of rats to grow at a normal rate on such diets. The proteins of this nut may yield insufficient amounts of those amino-acids that determine the nutritive value of a protein; or the pecan nut may contain some substance which renders rations of which it is an important component distasteful or injurious to rats. At the time of these observations there were on record neither detailed studies of the type of protein existing in the pecan nut nor studies of their chemical make-up. In view of the importance that this nut is assuming as a food crop in the United States,2 * The results reported in this paper were presented before the Pacific Coast Division of the Society of Experimental Biology and Medicine at the meeting of October 15, Cajori, F. A., J. Biol. Chem., 1920, xliii, In 1909 the production of pecan nuts in the United States was 9,890,769 pounds. In 1919, 45,619,OOO pounds, a gain in 10 years of 400 per cent. These figures were taken from the 1910 United States Census Reports and the United States Bureau of Crop Estimation report,

2 Nutritive Properties of Nuts. II it seem.ed desirable to study the chemical character of the proteins of the pecan nut, and with such a basis for evaluation of their nutritive properties, to repeat feeding experiments on animals. Chemical Experiments. Methods for the isolation and purification of plant proteins have been developed by Osborne3 and more recently by Johns and his coworkers4 in their investigations of seed proteins. We have followed these methods in our work on the proteins of the pecan nut. The Van Slyke method5 was used for protein analysis. Preliminary Experiments. Several kilos of shelled pecan nuts (1.5 per cent nitrogen) were passed through a meat grinder and the finely divided mass put in a laboratory press. This process, followed by repeated extraction with petroleum ether, removed all but traces of oil. The oil-free residue was ground in a mortar; the resulting product was a fine gray powder containing 4.3 per cent nitrogen. Small samples of the pecan meal were extracted over night with sodium chloride solutions of different strengths, and aliquots of the clear filtrate analyzed for nitrogen. Table I shows the amount of protein extracted from 2.5 gm. samples of the meal. Fractional Precipitation with Ammonium i%lfate.-solid, finely powdered ammonium sulfate was added to 10 cc. of a 9 per cent sodium chloride extract of pecan meal. The salt was added in amounts calculated to gradually saturate the solution, each addition increasing the degree of saturation by 0.5 per cent. After each qddition of ammonium sulfate the solution was shaken and the salt completely dissolved before the next quota of salt was added. The solution was examined for precipitated protein at the various stages of saturation. When the solution was 0.2 saturated a slight cloud appeared. At 0.4 saturation the solution became turbid and at 0.45 saturation a flocculent precipitate formed which was removed by filtration. Further addition of ammonium sulfate to the filtrate caused no change until 0.8 saturation was reached when the solution again became slightly turbid. This turbidity persisted, unchanged, when the solution was completely saturated. Examination of the saturated solution after it had stood over night showed that a small flocculent precipitate had formed. 3 Osborne, T. B., The vegetable proteins, London, New York, Bombay, and Calcutta, Johns, C. O., and Waterman, H. C., J. Biol. Chem., 1920, xlii, 59. Johns, C. O., and Gersdorff, C. E. F., J. Biol. Chem., , XIV, Van Slyke, D. D., J. Biol. Chem., , x, 15; 1915, xxii, 281.

3 F. A. Cajori 391 To 5 cc. of the 9 per cent sodium chloride extract, 1.86 gm. of ammonium sulfate were added so as to make the solution 0.5 saturated. A heavy precipitate formed. After 2 minutes this was filtered and 0.74 gm. of ammonium sulfate was added, making the solution 0.7 saturated. A very slight turbidity was noticed. To completely saturate the solution 1.16 gm. of the salt were added. A marked turbidity resulted. Temperature of Coagulation.-5 cc. of the 9 per cent sodium chloride solution were acidified with a drop of very dilute acetic acid. A thermometer was inserted in the test-tube containing the acidified solution and the tube heated in a double jacketed water bath. The temperature was so regulated that there was a rise of not over 1 in 2 minutes. At 55 C. a slight turbidity was noted but no flocculent coagulum appeared until the temperature of 70%. was reached. The solution was kept at 71 C. for 1 hour. After cooling, the precipitate was removed by filtration and the clear filtrate again heated. At 70 C. the solution became slightly cloudy, and at C. a precipitate began to form. A definite TABLE I. Extraction of Pecan Proteins with Sodium Chloride Solutions. N&l N extracted. per cent mj Protein extracted (N X 6.25) per cent coagulum formed at 86 C. and the solution was kept at this temperature for 1 hour. The heavy coagulum present at the end of this time was removed by filtering, and the heating continued. At 90 C. a slight cloudiness appeared. Long heating at C. caused a slight coagulation to form. The preliminary experiments indicate that pecan meal contains, as its principal protein, a globulin, salted out by 0.5 saturation with ammonium sulfate and coagulated at 7946 C. There is evidence of a trace of an albumin, which starts to coagulate at 55-60%. and is precipitated by ammonium sulfate only at a point of complete saturation. The fact that at the temperature of boiling water a slight coagulum forms, in addition to that caused by lower temperatures, is evidence, berhaps, of a second globulin, present in very small amounts.

4 Nutritive Properties of Nuts. II Preparation of a Pecan Globulin. Several hundred grams of pecan meal were extracted with seven times their weight of 10 per cent sodium chloride solution. The undissolved residue was separated from the solution by squeezing the mass through cheese-cloth. A solution free from suspended particles was obtained by filtering it several times through thick layers of paper pulp on a Buchner filter. This slightly opalescent solution was made 0.5 saturated with ammonium sulfate by the addition of the calculated quantity of the salt and allowed to stand over night. As much as possible of the clear supernatant solution was syphoned off from the precipitated globulin before it was transferred to a filter. The protein was removed from the filter paper and redissolved with distilled water and a little 10 per cent sodium chloride. It was again filtered clear and then dialyzed in parchment paper bags against running water for 120 hours. After that time it gave no more marked a test for sulfates and chlorides than did the tap water against which it was dialyzed. The contents of the bag were transferred to a Buchner filter and washed with distilled water and alcohol. The globulin was dehydrated by suspending it in absolute alcohol over night. It was filtered on a hardened filter paper, washed with anhydrous alcohol and ether, dried in a Freas oven at llo C., and finally placed in a vacuum desiccator over concentrated sulfuric acid. The pecan globulin, prepared in this way, was a light gray powder, with no evidence of crystalline structure, containing per cent nitrogen and 0.83 per cent sulfur, calculated on a moisture- and ash-free basis. It gave the usual protein tests including a strongly positive test for tryptophane with the Hopkins-Cole reagent. With a-naphthol a very faint color developed due undoubtedly to slight contamination of the preparation with traces of filter paper. The distribution of the nitrogen in this globulin as determined by the Van Slyke method, after complete hydrolysis with 20 per cent hydrochloric acid, is shown in Table II. In general this analysis agrees fairly well with the recently published results of Dowel1 and Menau1.G These authors determined the nitrogen distribution of the mixed proteins extracted from pecan meal by barium hydroxide and sodium hydroxide. The fact that our analyses are similar would indicate that the globulin constitutes the large part of the proteins of the pecan nut. It may be noted that there is no evidence either in our 6 Dowell, C. T., and Menaul, P., J. Biol. Chem., 1921, xlvi, 437.

5 F. A. Cajori results or in those of Dowel1 and Menaul of the unusually high content of histidine or a low arginine content in pecan proteins, reported by Nollau, in an analysis published some years ago. In considering the nutritive value of the pecan nut, the large amounts of basic amimo-acids yielded by the globulin are significant. TABLE Distribution of Nitrogen in Pecan Globulin. After hydrolyzing gm. of the protein, the solution contained mg. of N. Amide N. Humin N Arginine N... Histidine N... Cystine N... Lysine N Monoamino N.. Non-amino N... II. Total Feeding Experiments. mg. per cent There is every indication from a chemical examination of the globulin of the pecan nut that this nut is of high biological value as a source of protein. It seems highly improbable that the failure of rats to grow, when the pecan nut furnishes the protein of their ration, can be attributed to an amino-acid deficiency of the diet. The shelled pecan is bitter and astringent because there is a large quantity of tannin in the outside cuticle. In a recent study, Friedemann* has shown that tannins constitute 2.57 per cent of the total carbohydrates of the nut, and are present in sufficient amounts to give a tannin content of 0.33 per cent to the whole nut. The astringency that this amount of tannin gives to diets made up in large parts of pecan nuts or pecan press-cake may render such rations unsuitable for rats, and we have endeavored to remove as much tannin as possible from the nuts before incorporating them in our diets. I Nollau, E. H., J. Biol. Chem., 1915, xxi, 611. * Friedemann, W. G., J. Am. Chem. Sot., 1920, xlii, 2286.

6 394 Nutritive Properties of Nuts. II Such a color test as the ferric chloride test shows that tannins are present exclusively in the outer layers of the pecan kernel, and removal of this layer should render the nut tannin-free. The integument of smooth nuts, such as. the almond, can be removed by blanching with hot water. But in the case of the pecan nut this procedure fails to detach the membranous skin from the numerous crevices of the wrinkled surface of this nut. An endeavor was made to remove the outer layer by laboriously scraping each kernel with a knife blade. Again the irregular surface of the nut made it difficult to remove all the parts containing tannin. Leaching pecan meal with water did not prove successful as a means of removing tannin, and the possible loss of proteins during the leaching made this process undesirable. With many fruits astringency has been buccessfully combated by the use of solutions of lye, and we have found that exposure of pecan nuts to hot solutions of sodium hydroxide would loosen the integument completely, and remove the layer containing tannins. Our procedure was as follows: Pecan halves were added to a boiling 1 per cent solution of sodium hydroxide and the boiling solution was stirred vigorously for a minute. The mass was quickly transferred to a brass screen whose mesh was large enough to permit the alkaline solution to pass through readily but which would retain the pecan halves. Without delay the nuts were washed several times with boiling water, once with 1 per cent hydrochloric acid and again with hot water. In this way the sodium hydroxide was completely removed. When this process was carried to completion rapidly the solvent action of the alkali, and consequent loss of protein, was restricted to the outer layer of the nut and a white non-astringent product was obtained. The nuts, after the lye treatment, were dried, passed through a meat grinder, and subjected to pressure to remove enough oil so as to obtain a press-cake of sufficiently high protein content to be incorporated in a diet at an 18 per cent level. Young rats were fed on rations containing this pecan press-cake, butter fat, and inorganic salts. In addition to this diet 1 gm. of pecan nut was fed daily to each rat to insure an adequate supply of watersoluble vitamine. Cajoril has shown that this dietary essential is furnished in sufficient amounts for normal nutrition in rats by that amount of pecan nut.g I We are indebted to Mr. C. J. Moore of San Antonio, Texas, who kindly supplied us with some of the pecan nuts used in these feeding experiments.

7 F. A. Cajori As will be seen in Chart 1, the animals on this diet grew at a normal rate, indicating that the proteins of the pecan nut furnish adequate quantities of those nitrogenous complexes essential for growth. The weekly food intake records are given in Table III CHART 1. Growth of young rats on diets in which the pecan nut furnished the sole source of protein in the diet. The pecan nut was incorporated in the diet in the form of a press-cake after the tannin had been removed from the surface of the nut by treatment with hot sodium hydroxide. The composition of the pecan nut diet was as follows: per cent Pecan press-cake, 3.2 per cent N Saltmixture* 3 Butter. 5 Lard... 2 Pecan nuts l gm. daily * The salt mixture used is that described by Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1917, xxxii, 317. The failure of normal growth on diets containing pecan nuts from which the tannins have not been removed, is shown in Chart 2. In all respects except tannin content, these animals received the same diet as those whose growth is pictured in Chart 1.

8 396 Nutritive Properties of Nuts. II TABLE III. Weekly Consumption of Pecan Nut Diet. Week. Rat 61. Rat 62. Rat 63. Rat m gm chart 2. Growth Experiments with i(ats. ET,-ect of Tamlna.in Pecan PTUt Diets. CHART 2. Failure of normal growth of young rats on diets in which the pecan nut furnished the sole source of protein in the diet. The tannin in the surface of the nut had not been removed completely before the nut was incorporated in the diet. Rats 30 and 31 received a ration containing pecan press-cake made fromshelled pecans. Pecan meal was leached with water before being incorporated in the diet of Rats 57 and 58. The integument of the pecan nuts used in the ration of Rat 44 was partly removed by scraping each nut with a knife. The composition of these diets was the same as described in Chart 1. 0m m

9 F. A. Cajori The difference in growth of the two lots is a striking example of the effect that some distasteful or injurious substance in the food may have on the growth of rats. Unless the components of a ration are carefully scrutinized for the presence of substances that may render the diet u&it for the animals for which it is prepared, there is always danger of incorrectly explaining the response of animals to such diets. The care that must be taken in interpreting the cause of failure of feeding experiments cannot be emphasized too often. SUMMARY AND CONCLUSIONS. The principal protein of the pecan nut is a globulin. This globulin has been isolated and the distribution of its nitrogen determined by the Van Slyke method. Normal growth has been observed in young rats whose dietary protein was derived from the pecan nut, indicating that the nut is a source of adequate protein. The presence of tannins in pecan diets has been shown to be a limiting factor for the growth of rats.

10 SOME NUTRITIVE PROPERTIES OF NUTS: II. THE PECAN NUT AS A SOURCE OF ADEQUATE PROTEIN F. A. Cajori J. Biol. Chem. 1921, 49: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1