THE EFFECT OF THE CURING PROCESS UPON THE VITAMIN A AND D CONTENT OF ALFALFA.*

Transcription

1 THE EFFECT OF THE CURING PROCESS UPON THE VITAMIN A AND D CONTENT OF ALFALFA.* BY WALTER C. RUSSELL. (From the Department of Agricultural Biochemistry, New Jersey Agriculturul Experiment Station, New Brunswick.) (Received for publication, August 8, 1929.) 1. Vitamin A Studies. Early in the history of vitamin studies, alfalfa (l-3) was recognized as an important source of vitamin A. In view of the extensive use of dried alfalfa as a roughage for domestic animals, it was of interest to determine whether curing processes affected the vitamin A content. The samples investigated were dried on the ground, as in the usual field curing process, and by a mechanical drying process. Curing of Samples. Both samples were taken from the same portion of a field of alfalfa on the same day. The cutting was the second of the season, July 31, Sample A.-This sample was cut at 3 p.m. and taken from the field at 5 p.m. It was spread out on a lawn at 6 p.m. and allowed to remain there, consecutively,% through 4 nights and 34 days. The 1st day was rainy and partly cloudy but the remaining days were hot and sunshiny. At the end of this period the hay was brownish green in color, much of the green color having disappeared. It would be classed as a poorly cured hay, as far as greenness is concerned, but it was typical of the alfalfa that is obtained in regions where cloudy and rainy weather often prevails during the haying season or under conditions of haying management whereby the greater part of the green color is lost even during clear weather. * Journal Series paper of the New Jersey Agricultural Experiment Station, Department of Agricultural Biochemistry. 289

2 290 Vitamins A and D in Alfalfa Sample B.-This was cut at 1 p.m. and left on the field in windrows until 3 p.m., when it was taken to the drier. The drying was done by the Mason process (4) at the Walker-Gordon Farms, Plainsboro, New Jersey. With this particular machine, the period of drying was 30 to 35 minutes and the temperature of the heated air as it passed from the furnace to the drying tunnel was The dried product was as green as the fresh material but the shade of green was a little darker than that of the undried material. Experimental Procedure. The procedure for the determination of vitamin A was essentially that outlined by Sherman and Munsell (5) except where otherwise indicated. The breeding colony ration was the Sherman Diet 13, consisting of 2 parts of ground whole wheat, 1 part of whole milk powder, and 2 per cent of the weight of the wheat as sodium chloride (6), modified by the incorporation of 10 per cent meat scrap (Swift s) at the proportionate expense of the wheat and milk. Young white rats, weaned at 28 days of age and weighing between 35 and 55 gm., were placed upon a basal, vitamin A- free diet, which was essentially that described by Sherman and Munsell, Diet 380, but modified to include the antirachitic factor. It was composed of purified casein 20 per cent, Osborne and Mendel (7) salt mixture 4 per cent, sodium chloride 1 per cent, dried yeast 10 per cent, corn-starch 63 per cent, and 2 per cent of olive oil, containing 25 mg. cholesterol per cc., the oil and dissolved cholesterol having been irradiated with a quartz mercury vapor lamp for 30 minutes at 60 cm. The vitamin B complex was determined in the yeast and if sufficiently potent the amount was decreased to 5 per cent and the corn-starch increased correspondingly. During the pretest period the animals were kept in groups but during the test period they were confined in individual cages. After transfer to the individual cages, yeast and the olive oil solution of cholesterol were removed from the basal diet and fed as separate supplements, 300 to 500 mg. of the former, depending upon its potency, and 5 drops of the latter per day (except Sunday). Corn-strach replaced these two ingredients in the basal diet. All animals used in the test groups were represented by litter mates in the control group. Not more than one animal from a litter

3 W. C. Russell 291 was used in a test group and in six cases litter mates appeared in each test group. In so far as possible an equal distribution of sexes was made. The depletion period averaged 5 to 6 weeks. An individual was ready for the test period when growth had definitely ceased and no gain was made in 3 to 5 days, and it was considered desirable to have some evidence of ophthalmia. In practically every case an eye disturbance was observed which varied from the whitening of the edge of the lid and loss of the usual beady appearance to a complete closure of the eye with marked reddening. The ophthalmias were cured during the 1st week of the test period, in the case of Samples A and B. Preparation and Feeding of Samples. On account of the dropping off of the leaves which results from the handling of the dried plants, a more accurate comparison of the vitamin A content can be made by comparing the leaves and stems separately. The present study is concerned with leaves only, but later studies are to be made of the stems. The leaves were picked from the stems, or sorted from those which had fallen off, and ground to a fine powder preparatory to feeding. The material was fed as a separate supplement, daily, except Sundays. Preliminary trials indicated that the mechanically dried product was 6 or 7 times as potent as that cured on the field. The daily supplement of Sample A was 150 mg. and of Sample B, 20 mg. Difficulty was experienced in inducing the animals to eat the dry, powdered leaves, but they consumed the material readily when the olive oil supplement, noted above, was added to it. Results. The average gain in weight in the 8 week test period was practically the same for both test groups (Table I), but the amount of the increase was more than the 25 gm. gain in 8 weeks recommended by Sherman and Munsell (5). The coefficient of variation, 24 per cent, however, for Sample B is practically the same as that reported by Sherman and Burtis (8), for an 8 week test period in which an average gain of gm. was obtained, with a larger number of animals. In the case of Sample A, the variability is higher, the coefficient being 30 per cent. Although the

4 292 Vitamins A and D in Alfa.lfa delicacy of the test may not be as great as when there is an average gain closer to that recommended by Sherman and Munsell, the results indicate that the vitamin A potency of the machine-dried material is at least 7 times that representing a field sample, cured so that the greater part of the green color is lost. Attention has been called in a number of instances to the association of vitamin A potency and greenness in plant tissue. Thus Coward (9) has noted the loss of vitamin A when a leaf dries up and dies, and Dye, Medlock, and Crist (lo), in a paper which summarizes studies of this relationship, have shown an association between greenness in lettuce leaf tissue and vitamin A potency. Also Steenbock, Hart, and their associates (11) found that alfalfa TABLE I. Summnry of Rat Growth Records. Vitamin A in Alfalfa. Sample A, field-cured, 150 mg. Sample 1 1, machinedried, 20 mg. Controls, no supplement. No. of animals Average weipht,at b;s;t;;g period. AXWag Average weight at gain in 8 end of test wk. test period. period. gm. gm.!lm Coe%ient variation. pm cent Average weight at death, 79 gm., average survival period, 13 days. which was completely bleached by long exposure to sunlight lost its vitamin A potency. The findings in the case of alfalfa confirm these observations in that the fine powder obtained by grinding the leaves of the field-cured sample was brownish green in color, as compared with the distinctly green color of the machine-dried sample. In a number of other tests with alfalfa, of a qualitative nature, made in this laboratory, the same relationship between color and vitamin A potency was found to hold. Dutcher (12) in a review article remarks that : Practical feeders have observed that bright green alfalfa, properly dried and cured, is superior to the light colored crops, bleached out by unsatisfactory climatic conditions or careless agricultural practice. 24

5 W. C. Russell 2. Vitamin D Studies. Steenbock, Hart, and their associates (11) have demonstrated that, The antirachitic properties of hays are related to their exposure to sunlight. Clover hay, for instance, made in the sunlight showed considerable calcifying power, whereas a sample of the hay made in the dark was inactive, the white rat being used as the test animal. They also pointed out that the antirachitic potency of clover hay which had been excessively weathered was reduced as compared with hays less exposed to dew and rain. Steenbock and Hart had noted as early as 1913 (13) that the feeding of green grasses to goats and keeping them out of doors greatly improved the assimilation of lime as compared with keeping the animals indoors on a ration of straws and grains. In view of these findings the problem naturally presented itself as to the antirachitic value of alfalfa hay dried artificially by the Mason process (4), mentioned above, a few hours after cutting, as compared with that of a hay cured in the sun. Curing of Samples. The samples were collected at the same time and from the same portion of the field as those described for the vitamin A studies. Sample C.-This sample was cut at 3 p.m. and taken from the field at 3.30 p.m. It was transported to the laboratory and spread on the floor overnight. On the following morning it was exposed to weak sunshine for 13 hours and then taken indoors on account of rain. During each of the following 2 days, it was spread on canvas in bright sunshine from 9.30 a.m. until 4.30 p.m. At other times it was kept indoors, out of sunlight. The total exposure to direct sunlight, after cutting, was about 16 hours. Sample D.-The handling of this sample was the same as that of Sample C, except that it was allowed to dry on the laboratory floor in diffused daylight, but not in direct sunlight. Sample B.-This sample was obtained by the same process as Sample B of the vitamin A studies. Experimental Procedure. The procedure for the assay of vitamin D has been described previously (14) and is based on that outlined by Bills, Honeywell, and MacNair (15). The diet used for the production of rickets

6 Vitamins A and D in Alfalfa was the Steenbock Ration 2965, a high calcium-low phosphorus ration (16). Young rats, weaned at 24 days, developed rickets in 20 to 22 days. After a 5 day test period, the degree of cure produced by the material under examination was estimated by the Shipley line test (17). Preparation and Feeding of Samples. As in the case of the vitamin A studies, only the leaves were used, in order to obtain a more accurate comparison. They were ground to a fine powder and incorporated in the rickets-producing diet, at the expense of the yellow corn, so that they constituted 15 per cent of the diet. Sample D was irradiated with a Cooper Hewitt Uviarc (poultry treater) quartz mercury lamp, operated on 110 volts, A.C. The exposure was for 3 hour at 60 cm. The question might be raised as to the disturbance of the calcium-phosphorus ratio in the rickets-producing diet by the introduction of alfalfa. Table II shows the calcium-phosphorus ratio of the Steenbock Ration 2965 and of this diet after the incorporation of 15 per cent of each of the alfalfa samples. The test diets are still high calcium-low phosphorus in character and the variation from the rickets-producing diet is probably not greater than would be found among diets prepared from different lots of yellow corn and of wheat gluten. Furthermore, the tests ot the three samples are comparable in that the ratios of the te+r diets are practically the same. Results. Table III is a summary of the rat records. Details of food consumption and growth are not given for each individual but an animal s record was rejected if the food consumption was less than 2 gm. for any one day of the test period or if the average consumption was less than 4 gm. per day. Also a loss of weight during the test period resulted in the discarding of an animal. The readings obtained in the case of Sample C, sun-dried, are higher than those of Samples D and B, air-dried in the absence of sunlight and machine-dried, respectively. In the case of the latter two samples, the degree of the cure is slightly greater in the case of Sample D, but the difference between the two samples is not considered significant.

7 W. C. Russell To determine whether the material exposed to sunlight had reached its maximum antirachitic potency, a portion of Sample D, TABLE Calcium-Phosphorus Ratios of the Rickets-Producing and Test Diets. II. Diet. Steenbock Ration Sample C incorporated., D I B.,,,.., _..._..., I Ca:P 4.9:1 5.6:1 5.6:1 5.3:1 TABLE III. Summary of Line Test Readiv ZQ. s. Vitamin D Determinations. Sample C, sundried. Sample D, airdried in absence of sunlight. Sample B, machine-dried. Sample F, irradiated portion of Sample D. Controls, at beginning of test period. gm gm Line test readings. 3+, 2+, 2+, 2+, 2+, +, f, +, - 2+, +3, +, f, +, +, +, +, -, - -> , +, f, -9 -, , 3+, 3+, 2+, 2+, 2+ 2+, 2+, 2+ 2+, 2+. All negative. air-dried in the absence of sunlight, was exposed to the radiations of a quartz mercury lamp, as described above. That still more activatable substance was present is evident from the higher

8 296 Vitamins A and D in Alfalfa degree of curing which resulted in the case of the irradiated sample, referred to as Sample F in Table III. Although the method for the determination of the antirachitic factor used in the present investigation is not directly comparab,le with that employed by Steenbock, Hart, and their associates (11) in their studies of clover hay, the results in the cases of Samples C, D, and F are in general agreement. * The results of the vitamin A studies described in the first part of the paper would lead to the question of the vitamin A content of Sample C, which lost the greater part of its green color during the sun-drying process. In Table IV is recorded the result of the feeding of a 50 mg. supplement of leaves of Sample C. The result is not directly Supplement. TABLE IV. Vitawlin A in Sample C. Average AIWage No. of weight at weight at animals. beginning of end of test test period. period. Average gain test in period, 8 wk CJm. gm. gm. Sample C sun-cured, mg. Controls. Same as in Table I. - Coefficient of variation. per cent comparable with that of Sample B, Table I, because an equal gain was not made in the 8 week period, but a loss of vitamin A has taken place owing to the process of curing. Also the eye disturbance persisted for a longer time than in the case of Sample B. Hence the increase in vitamin D potency, obtained by drying in the sun, was accompanied by a decrease in vitamin A. 46 SUMMARY. 1. Alfalfa leaves from plants dried by artificial heat, by the Mason process, were found to contain at least 7 times as much vitamin A as the leaves from hay that was cured in the field so that the greater part of its green color was lost. 2. The sample which contained the larger amount of vitamin A was green as compared with the brownish green color of the fieldcured sample.

9 W. C. Russell 3. The leaves of the artificially cured plants contained only a small amount of the antirachitic vitamin. 4. When the alfalfa was dried in the sun, without exposure to dew or rain, there was an increase in the antirachitic potency of the leaves, but it was accompanied by a decrease in vitamin A content. BIBLIOGRAPHY. 1. McCollum, E. V., Simmonds, N., and Pitz, W., Am. J. Physiol., 41,333 (1916). 2. Osborne, T. B., andmende1, L. B., J. Biol. Chem., 3 7,187 (1919). 3. Steenbock, H., and Gross, E. G., J. Biol. Chem., 41,149 (1920). 4. Hanke, 0. A., Farm J., 49,lO (1925). Mason, A. J., Farm J., 60,10, 13 (1926). 5. Sherman, H. C., and Munsell, H. E., J. Am. Chem. Sot., 47,1639 (1925). 6. Sherman, H. C., and Campbell, H. L., J. Biol. Chem., 60,5 (1924). 7. Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 37,572 (1919). 8. Sherman, H. C., and Burtis, M. P., J. BioZ. Chem., 78,671 (1928). 9. Coward, K. H., Biochem. J., 19,500 (1925). 10. Dye, M., Medlock, 0. C., and Crist, J. W., J. BioZ. Chem., 74,95 (1927). 11. Steenbock, H., Hart, E. B., Elvehjem, C. A., and Kletzien, S. W. F., J. BioZ. Chem., 66,425 (1925). 12. Dutcher, R. A., J. Znd. and Eng. Chem., 13,1102 (1921). 13. Steenbock, H., and Hart, E. B., J. BioZ. Chem., 14,59 (1913). 14. Russell, W. C., Button, F. C., and Kahlenberg, 0. J., J. Dairy SC., 12, 231 (1929). 15. Bills, C. W., Honeywell, E. M., and MacNair, W. A., J. BioZ. Chem., 76,251 (1928). 16. Steenbock, H., and Black, A., J. BioZ. Chem., 64,263 (1925). 17. McCollum, E. V., Simmonds, N., Shipley, P. G., and Park, E. A., J. BioZ. Chem., 61,41 (1922).

10 THE EFFECT OF THE CURING PROCESS UPON THE VITAMIN A AND D CONTENT OF ALFALFA Walter C. Russell J. Biol. Chem. 1929, 85: 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