I N D U S T R I A L A N D ENGINEERING CHEMISTRY
January, 1924
49 -
The Amino Acid Distribution in Proteins of W h e a t Flours’ With a Note on a n Improved Method for the Preparation of Aldehyde-Free Alcohol By Robert J. Cross and Robert E. Swain STANFORD UNIVERSITY, CALIF.
VERY
baker
and
ferent flours produce quite different results in making bread. Some wheat flours are found to be suitable for making macaroni and pastry but quite unsuitable for making bread. Millers have learned, mainly by experience, how to blend the socalled ‘(hard” and “soft” wheatti so as to produce a flour havingthe desired baking Properties. C e r t a i n chemical and physical tests have been developed which help in estimating the value of a given wheat or the flour made from it, but as to the leavening process, and the reason for many of the results obtained we are still much in the dark. The marked differences in behavior of different flours are popularly supposed to be due mainly to differences in the gluten content. Differences in behavior cannot be attributed entirely to quantity of gluten present, and it seemed doubtful whether the differences in glutens could be laid entirely to differences in the ratio in which the two proteins, gliadin and glutenin, occur in them. Many investiggitions have been undertaken in recent years, especially by nutrition experts, in an endeavor to throw light on the complicated chemistry of the proteins, and the proteins of wheat and other cereals have not been neglected. Osborne, Voorhees, and others have published numerous papers dealing with this subject, and Osborne has more recently collected many of these data, together with a review of the work which had been done up to that time,2 in a single publication. This publication gives results of careful investigations of the amino acid distribution of the different proteins of the wheat kernel by the best methods then available-those of Kossel, E. Fiecher, and others. Van Slyke,3 in developing his now well-known method for the determination of amino acid distribution, chose gliadin as one of the proteins easily obtained in a pure condition. I n a later paper4 additional analyses were made, primarily with the object of developing the method and of learning the composition of this protein from a nutrition standpoint. So far as the present authors are aware, no comparative study of the amino acid distribution in the proteins isolated from different varieties of wheat has been published. For the present investigation four flours were prepared by the Sperry Flour Company. The sample “Idaho” is a straight flour made from a hard wheat from that State. “Patent” is their regular, high patent, family flour made from a mixture of wheats. “Club” is a straight flour made from pure Club Wheat, a soft wheat from California, while “FortyFold” is a straight flour made from this variety grown in Received September 24, 1923. Curnegie Ins!. Pub. 04. * J . b i o l . Chem., 10, 15 (1912). 4 l b i d , 22, 259 (1915). 1
2
Washington. Since, of the wheat proteins, gliadin, the alcohol-soluble protein, is present in much the larger amount, examination of this protein wasfistundertaken. Preparations of gliadin from each of these flours were made and examined by triplicate determination according to the Van Slyke method. Then preparations of glutenin were made from each flour, and these were examined by the same genera1 methods, with slight modifications as noted later. A third phase of this investigation consisted in an attempt to ascertain whether or not gliadin from different parts of the same wheat kernel is identical in amino acid distribution. For this phase of the investigation four flour samples were taken from different mill streams at the same time while grinding the same kind of wheat. These are designated in Table I11 as Patent, Second Crush, Clear, and Third Tailings. The Patent is the regular, high-class, patent family flour, while the Second Crush is a still higher grade and contains a larger percentage of material from the interior of the wheat kernel than any other mill stream. The Clear was taken from the regular clear flour spout, while the Third Tailings is the lowest grade of flour, much of the substance of which comes from the kernel adjacent to the bran portion. PREPARATION OF GLIADIN The method used in the preparation of the gliadin was essentially that recommended by Osborne. One thousand grams of flour were mixed with 600 cc. of water; the resulting dough was allowed to stand from one-half to one hour, after which the starch was washed from the gluten with tap water. The moist gluten was then treated with a quantity of alcohol sufficient to bring the alcohol concentration up to about 65 per cent, assuming the moist gluten to be twothirds water. The moist gluten was cut into small pieces by passing it twice through a meat cutter. After standing over night or longer the solution was decanted off and allowed to stand some time until the bulk of the suspended matter had settled as a slimy deposit. This liquid was decanted off and filtered through several close filter papers until clear. Some of these solutions, especially the later washings, were never entirely transparent. The first liquid drawn off could be obtained perfectly transparent. It seemed as though the slimy deposit formed carried down the finely suspended matter and a solution was obtained which readily filtered perfectly clear and transparent. This solution became cloudy upon standing for several days. This residue, which, as Osborne has previously observed, fails to redissolve in alcohol, was collected in the course of the preparation of the various samples of gliadin and a Van Slyke determination was run on the combined lot. The results appear in Table I11 as Residue.
Four flours were prepared from varieties of wheats which had been found to giue markedly different results in the bakery. From these flours gliadin and glutenin preparations were isolated and carefurry purified. Gliadin preparations were also isolated from different mill streams while grinding the same variety of wheat. Nitrogen distribution was then determined in these proteins by the methods of V a n Slyke. Tyrosine and tryptophan were determined by the methods of FoNn and Looney. The variations noted in results of analyses of different preparations fall well within the variations shown i n separate analyses on the same individual preparation, which leads the authors to conclude that the eoidence oflered points to the chemical identity of gliadin in different wheats. and that differences in behaoior. if in fact they are attributable to the proteins present, are to be ascribed to differences in physical state rather than chemical constitution. The same comment is made with respect to the glutenins.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
50
All results are corrected for solubility of the bases IdahoNITROGEN COWPOUND 1 2 3 Ammonia 26.57 26.80 27.02 Acid-insoluble humin. 0.08 0.06 0.11 Acid-soluble humin ...... 0 . 3 9 0.48 0.46 Phosphotungstic humin.. 0 . 0 8 0.35 0.27 Histidine. bv Koessler method.. (3.66) Histidine. 5.85 4.46 6.30 0.65 Cystine. 0.78 0.70 5.02 Arginine. 4.77 4.86 0.42 Lysine.. ............... 0.94 0.64 Amino, filtrate from bases. 51.97 51.21 53.60 Nonamino. filtrate from bases 4 . 9 2 3.97 4.43
...............
..
I
............ .............
Vol. 16, No. 1
TABLEI-ANALYSES o# GLIADINS and are expressed as percentage of nitrogen based on the total nitrogen of the protein as 100 per cent -Forty-Fold---Patent-ClubAv. 1 Av. 1 2 3 Av. 1 2 3 2 3 Av. 2 6 . 7 9 2 6 . 7 4 2 5 . 9 6 26.60 2 6 . 4 0 2 6 . 6 4 2 5 . 5 4 2 6 . 4 0 2 6 . 2 0 24.84 2 6 . 0 5 27.70 26.20 0.08 0.17 0.19 0.18 0.19 0.11 0.15 0.13 0.09 0.11 0.44 0.67 0.48 0.57 0.44 0.34 0.39 0.39 0.41 0.40 0.23 0.38 0.14 0.08 0.20 0.23 0.08 0.09 0.13 0.10 0.18 0 . 1 4
--
... ...
--
... ...
... ... ...
_
............... ..............
...............
5.54 0.71 4.89 0.66
95.95
3.77 0.84 5.12 0.59
...
(3.10) 7.05 0.84 4.86 0.60
5 2 . 2 6 5 3 . 4 1 5 3 . 2 0 51.59
................ - - - - - - _ _ _ - - -4 . 4 4 TOTAL .........
7.15 0.85 5.19
93.82 98.39 96.04
3.60
5.99 0.84 5.06 0.59
7.56 0.86 5.03 0.56
5.24 0.51 4.81 0.57
(3.08) 6.45 0.94 5.80 0.56
6.41 0.77 5.21 0.56
5.03 0.69
52.73
54.58
52.63
52.58
53.26
2 96
7.09
4.55
3.95
4.39
9 7 . 3 2 93 42
99.38
97.11
99.41
94.40
...
0.52
5.21 0.71 4.75 0.51
(3.56) 7.27 0.79 4.81 0.61
52.97
53.19
48.16
3.29
10.68
51.44 5.65
101.51 9 8 . 6 0 8 7 . 0 4 9 4 . 3 3
100.70
95.84
.24 2.99 -8 - 5-. 5 2- -
5.84 0.73 4.78 0.55
TABLE 11-ANALYSES O F GLUTENINS AI! results are corrected for solubility of the bases and are expressed as percentage of nitrogen based on the total nitrogen of the glutenin as 100 per cent ___... Club -Forty-Fold---Tdaho, PatentNITROGEN COMPOUND
1
2
-
Av.
Ib: e 2
0.66 0.80 0.49 (3.70) 7.06 0.72 10.10 4.74
1 2 3 Av. 24 36 48 1 5 . 9 4 1 5 . 8 5 16.16 15:98 0 . 2 3 0.63 0.69 0.52 0.96 0.88 0.91 0.90 0.44 0.30 0.37 ... (3.91) 8.97 8.18 9.22 10.51 0.70 0.70 0.71 0.73 7.97 8.18 8.17 8.37 5.22 5.63 4.93 3.94 58.70 52',90 53',i5 54'.b7 2.72 4.91 3.81
...
... ...
1 24 14.17 0.63 0.94 1.20 (4.30) 11.42 0.65 9.23 4.72
1 24 12.88 0.73 1.10 0.90 (3.70) 7.12 0.65 13.47 6.22
2 36 13.35 0.62 1.04 0.83
95.89
PREPARATION O F REAGENTS-Reference should here be made to several precautions in preparing the reagents used. One of these is that of making sure that aldehyde-free alcohol is used in the preparation of the protein, on account of the well-known fact that addition products are formed by union of aldehyde with the amino group, a reaction which may give rise to misleading results if this precaution is not observed. A considerable quantity of 95 per cent alcohol was treated t o free it from aldehydes. The method of preparation was that used by the Bureau of Chemistry,6 and tests were made as there described to be sure that the resulting product was aldehyde-free. The distillation was made from the m-phenylenediamine hydrochloride in faintly acid solutions to be sure that no ammonia would be given off, and this distillate was again distilled over lime. This was found to be a tedious and wasteful method, as only the intermediate distillate was found to be aldehyde-free. A modified method was then resorted to. Raw 95 per cent alcohol was boiled for 2 hours under a reflex condenser with some sticks of sodium hydrate in order to polymerize the aldehydes. About 1 gram of silver sulfate was then added to each 2 liters of alcohol and the boiling was continued for several hours. Upon distillation the first 500 cc. were found to give a decided test for aldehydes, the following 500 cc. gave only a very faint test, and the remainder, or over one-half of the total, gave no test for aldehyde. Unlike the former method, the last runnings were aldehyde-free, so the yield of pure alcohol was greater. The first runnings by this method were treated by the nz-phenylenediamine method and a further yield was obtained. The resulting product was tested and found to give a neutral reaction. The isoamyl alcohol subsequently used in some of the Van Slyke determinations was also prepared aldehyde-free by this method. The isoamyl alcohol was shaken out repeatedly with dilute sulfuric acid solution and finally distilled t o free it from pyridine bases.
The alcoholic solution of gliadin was concentrated under reduced pressure in a flask placed in a water bath and kept a t a temperature between 40" and 50" C. Additional portions of strong alcohol were added whenever frothing began to take place, as directed by Osborne. The thick sirup finally obtained was poured in a thin stream into ice-cold water containing a few grams of sodium chloride. The resulting precipitate was washed with water, dissolved in alcohol of about 70 per cent, and the excess of alcohol again distilled off. The precipitation was then repeated without the addition of sodium chloride. This precipitate was again dissolved in dilute alcohol, the excess alcohol removed, and the resulting sirup poured into 99 per cent alcohol. The precipitate was broken up under this alcohol, allowed to stand some time, 6
U.S.Dept. Agr., Bur. Chemistry, Bull. 107.
97.87
100.06
98.42
99.07
99.19
102.15
100.95
ii:ii
0.67 1.07 0.86
i :27 0.65 12.42 6.62
.54: b2 ... 56'.b5 5b:i4 5g.'60 3.66 ... - 2.64 2.14 ---5 . 8 3 - --
TOTAL . . . . . . . . . . . . 100.00
Av.
102.13
$:io
0.65 12.94 6.42
5i. 62 3.98 101.52
filtered off, and finally washed with 99 per cent alcohol and anhydrous ether (over sodium), and dried in a desiccator over sulfuric acid. This process was used for the gliadin samples from the different varieties of wheats. Since, however, the removal of the excess water by addition of alcohol and repeated distillation was found to be a very tedious process, in the subsequent preparation of the gliadin samples from the mill stream flours the excess of alcohol was removed by distillation until the liquid began to cloud and frothing commenced. The liquid was then poured from the distilling flask onto large dinner plates, and the blast from an electric fan was sent over the surface of the liquid. The resulting evaporation together with the cooling effect caused the gliadin to precipitate in a short time. This precipitate was washed in water and the process repeated as described above. Snow-white preparations were obtained which were reduced to a fine powder when dry. The gliadin preparations gave the following results by the Kjeldahl method for nitrogen: Digested with HzS04 KzSO4 and CuSOa.. HPSOI: KrSO,: and HgO
....
Idaho
Patent
Club
%
%
%
17.81 17.41
17.14 17.21
17.63 17.41
Forty-Fold
%
17.66 17.91
DETERMINATION OB' AMINO ACIDSI N GLIADIN The method used for the determination of the amino acids in the gliadin samples was the amyl alcohol modification of Van Slyke6 with the modification of Plimmer' for arginine. For 'this determination an inverted U-tube was used with % condenser on each arm, one a reflux and the other a straight condenser replacing the Folin bulb described by Van Slyke. A small electric heater was used surrounded by an asbestos and tin jacket. Bumping in this case was not nearly so troublesome as when a flame was used. It was thought well to continue the distillation with lime for 45 minutes or a little longer, instead of for half an hour as directed by Van Slyke, since the distilling flasks used were thick-walled, the heat transfer poor, and the distillation consequently slow. A total of 200 cc. of the amyl alcohol-ether mixture was used in all cases when this modification was followed. An improved quantitative Buchner funnel was used in the filtration of the phosphotungstate precipitate, separate 8
7
J . B i d . Chcm., 22, 281 (1915). Biochem. J . , 10, 115 (1916).
IhTDUSTRIALA N D ENGINEERING CHEMISTRY
January, 1924
51
TABLE 111-ANALYSES OF GLIADINS FROM MILL STREAM SAMPLES All result!; are corrected for solubility of the bases and are expressed as percentage of nitrogen based on the total nitrogen of the gliadin as 100 per cent NITROGEN COMPOUND
Ammonia .............................................. Acid-insoluble humin.. .................................. Acid-soluble humin., .................................... Phosphotungstic humin. ................................. Histidine, by Koesder method..
..........................
Arginine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino, filtrate from the bases.. .......................... ............ Nonamino, filtrate from the bases
TOTAL
mention of which is made elsewhere* on account of its adaptability to other filtrations.
PREPARATION OF GLUTENINS The glutenin preparations were made according to the method of Osborne from the residue remaining after the extraction of the gliadin by alcohol. The residue was washed with water and dissolved in weak alkali as described. The clearer portion of the alkaline liquor was decanted off, and the glutenin was precipitated by the addition of hydrochloric acid as directed. On redissolving in alkali the solution was carefully filtered until clear. The glutenin was finally dried with 99 per cent alcohol and washed with anhydrous ether (over sodium). The glutenin preparations gave the following results by the Kjeldahl method for nitrogen using copper sulfate as catalyst. Nitrogen, per cent..
.......
Idaho
Patent
Club
16.81
16.80
16.60
Forty-Fold
16.23
The gliadin samples made from mill streams gave the following results: Patent
Second Crush
Clear
Third Tailings
....... 17.05 17.10 16.73 16.85 DETERMINATION OF HISTIDINE It was noted in the course of the analyses that the histidine values obtained for gliadin were higher than those obtained by Van Slyke. Histidine determinations were therefore made on some of the samples as noted by the method of Koessler and Hanke.9 It was rather difficult to get a dye to match exactly the color produced, especially as the color of the test sample was continually changing. A standard solution containing a known quantity of pure histidine monochloride was finally used, with very good results. Duplicate sets of pipets and of other apparatus were used, the standard being manipulated with the left hand while the test sample was taken care of with the right. I n this way the colors in test sample and standard were developed together and readings taken over considerable ranges in time were in close agreement. The determinations of histidine by the Koessler method were made on the solution of the bases fraction of the Van Slyke scheme of analysis. The results by the Van Slyke method in this instance were in most cases undoubtedly too high. This may have been due to the phosphotungstic acid used precipitating more than it is supposed to do. All the phosphotungstic acid used in these analyses came from the same bottle, so that the results are comparative. It was thought that the high histidine values might be due to the amyl alcohol used so that the analyses on the glutenins were made by Van Slyke's original procedure. Nitrogen, per cent..
DISCUSSIONOF ANALYSESOF GLIADINS In the analyses of gliadins in Table I, no account was taken of the nitrogen retained in the amyl alcohol-ether mixture,
Patent
26.38 0.10
0.33 0.11
....
7.3s 0.67 4.64 0.28 52.97 7.62
Clear
Third Tailings
26.10 0.14 0.40 0.14
26.40 0.12 0.43 0.35
26.70 0.15 0.41 0.26
25.70 0.64 0.50 0.27
(3.16) 6.35 0.67 5.07 0.30 52.24 10.01
(3.43) 5.67 0.67 4.92 1.20 52.30 7.46
(3.06) 7.71 0.73 5.00 0.00 53.25 5.82
(3.18) 5.09 0.47 4.97 0.73 53.65 6.02
-
-
-
-
100.45
101.42
99.52
100.03
Residue
98.04
and in consequence the analyses do not total as near to 100 per cent as they would with this fraction added. The portion here designated as phosphotungstic humin is that portion insoluble in the amyl alcohol-ether mixture. This determination seems to be of little value, for it was found that by rubbing this precipitate or emulsion carefully with a rubber-tipped stirring rod it could all be broken up and it went into solution or suspension sufficiently to go completely through the filter. As the paper of Menaul'o came to hand while the analyses of the glutenins were being made, the procedure was modified somewhat in so far as the humin portions were concerned. The hydrolysate was filtered and the insoluble residue taken as the acid-insoluble humin. The acid-soluble humin was that portion absorbed by lime in the ammonia distillation. After filtering off the excess of lime the solution was made acid with 18 cc. of concentrated hydrochloric acid as directed by Van Slyke, about 0.5 gram of phosphotungstic acid was added, the mixture heated to boiling and filtered boiling hot through a funnel heated by a jacket of boiling water. If too large an amount of phosphotungstic acid is added not all the phosphotungstate of the bases will go into solution even a t boiling temperature, and the phosphotungstic acid humin portion will be too large. This is what happened in the first analysis of the Idaho glutenin. After filtering off the phosphotungstic humin insoluble in acid solution, 14.5 grams additional phosphotungstic acid were added and the procedure directed by Van Slyke was followed. It was noted that the ammonia fraction was lower in the case of the glutenin from Forty-Fold; therefore, another run was made to ascertain if this was due to more extensive hydrolysis. Also, two further tests were made on Patent glutenin to ascertain the effect of prolonged hydrolysis. The time of hydrolysis of all gliadin samples was 30 hours.
TYROSINE AND TRYPTOPHAN CONTENTOF GLIADINSAND GLUTENINS In addition to the foregoing determinations, samples of the various gliadins and glutenins were hydrolyzed with barium hydroxide solution for 48 hours and the tyrosine and tryptophan content was determined according to Folin and Looney.'' The tyrosine values are doubtless a little high owing to the residual color of the reagent, but are comparative as the same weights of sample and same procedures were employed throughout. Per cent Gliadins Idaho Patent Tyrosine 5.10 5.10 Tryptophan . . . . . . . . . . . . . . . . . . 1.11 1.19 Per rent Glutenins Tyrosine .................... 5.34 5.52 Tryptophan .................. 1.59 1.61
....................
C!ub
Forty-Fold
5.04 1.03
5.04 1.13
5.47 1.55
5.92 1.61
CONCLUSION The variations noted in the results for the different gliadin samples fall well within the variations shown in separate
* THISJOURNAL,
10
9
11
15, 910 (1923). J . 13ioI. Chem., 89, 497 (1919).
Second Crush
J . B i d . Chem., 46, 351 (1921). Ibid., 61, 420 (1922).
I X D USTRIAL AND ENGINEERING CHEMISTRY
52
analyses on the same individual sample, so that the evidence offered points to the chemical identity of the gliadins from the different varieties of wheat and to the conclusion that the differences in behavior, if in fact they are attributable to the proteins present, are to be ascribed to differences in physical state rather than chemical constitution. The close agreement of the results of those determinations, such as cystine, tyrosine, and tryptophan, in which the probable error is small, lends particular support to this conclusion. The same comment may be made with respect to the glutenin samples with the possible exception of the ammonia fraction which seems
Vol. 16, No. 1
to be somewhat lower in the Forty-Fold sample than was the case with the other samples examined. A new preparation of lime was used on this sample, which may have had something to do with the lower result noted here. ACKNOWLEDGMENT The authors wish to extend their thanks to the Sperry Flour Company for its cooperation in providing the fellowship under which this work was done, and to Bert D. Ingels of this company for selecting the wheats and preparing the flours for this investigation.
Vitamin C in Canned Foods' By Walter H. Eddy and Edward F. Kohman COLUMBIA UNIVERSITY,NEWYORK,N. Y.,AND NATIONALCANNERS' ASSOCIATION,WASHINGTON, D. C.
HE experiments reported herein are the first of a series of joint researches undertaken to establish to what extent vitamin C is destroyed in present canning methods, the causes underlying this destruction, and to evolve better methods for its prevention. Recently, evidence was presented by Eddy and associates2tending $0 show that when cabbage is cooked to palatable form, either by boiling in an open kettle or by cooking in a pressure cooker, 20 grams per day is the amount necessary to protect a guinea pig from scurvy, whereas 1 gram of raw cabbage per day suffices for this purpose. From control experiments reported a t that time it followed that home cooking methods result in practically 95 per cent destruction of vitamin C in both cabbage and the rutabaga turnip. It has been the practice t o infer from such experiments that if canned material is subjected to similar temperatures for similar periods of time the vitamin destruction in canned foods will be of the same order. Such inferences ignore the possible influence of other factors concerned in vitamin C destruction, among which are the presence or absence of oxygen, variation in reaction (pH), and possibly the presence or absence of oxygen activators. Kohman3 has already called attention to the desirability of investigating the oxygen content of canned foods as bearing on this point. Still more recently, Zilva4 has produced evidence tending to show that the destruction of vitamin C is an oxidation process which proceeds rapidly in alkaline solutions even a t room temperature, and also, but more slowly, in acid medium.
T
EXPERIMEXTAL I n beginning this series it seemed particularly desirable first to determine the actual destruction of vitamin C in a foodstuff subjected to commercial canning in which the only variables should be the degree of temperature used in the processing and the duration of the heating process. For this purpose cabbage was used. This was harvested about November 15, 1923, and canned a few days after as follows: Cabbage heads were cut in quarters, all the bad outer leaves stripped off, and the hearts cut out. Then a solid piece was cut out of the quarter to fit approximately into a No. 2 can. If this did not make 11 ounces, sufficient loose material was filled in t o make that weight. After the cabbage was packed in cans, these were filled with hot water drawn from the factory tubs and through which live steam was passed to boil it actively for 2 or Received September 26, 1923. J. Home Econ., 15, 15 (1923). a THISJOURNAL,16, 273 (1923). 4 Biochem. J . , 17, 410 (1023). 1
2
3 minutes. It required 5 t o 10 minutes to water the various lots of cans, which were then passed through an exhaust box at a temperature of about 95' t o 99' C. (200' to 210' F.). This required 4 to 5 minutes and the cans were then sealed. The experimental pack thus prepared was divided into lots processed as follows: Lot 1 2 3 4 5 6
Time Minutes 30 60 15 30 45
30
-Temperature-
c.
100 100 115 115
115 126
(" F.)
.
(212) (212) (240) (260
These cans were then shipped to Columbia University laboratory and there used for feeding tests after the routine established by Sherman, La Mer, and Camphel16 for scurvy testing. Two sets of control animals were used. One group received the basal diet alone and died from scurvy in about 30 days. The second control group received 1 gram of raw cabbage per day plus the basal diet. These animals grew normally and a t the end of 80 days were still free from any symptoms of scurvy. A point in regard to the second control group should be noted. For purposes of accurate comparison, an attempt was made to use throughout the 80day period cabbage taken from the lot that was canned. This was kept in the cellar room in the university. The feeding experiments were b e u n about December 1and about January 10 it was found that 1 gram per day of this lot began to lose potency. The outer leaves were wilted, but the inner leaves, which were fed, seemed crisp. However, by substituting fresh cabbage obtained in the market a potency was secured from 1 gram. The writers interpret this to mean the destruction of the vitamin in storage. Zilva's experiments4 showed that in the presence of air decitrated lemon juice made pH 12.5 in reaction lost nearly 80 per cent of its tiitamin C content a t room temperature in half an hour. It might well be expected that cabbage with pH of 5.6, even a t a lower temperature, would suffer considerable vitamin C destruction if air was not excluded. A third control group, representing a series of animals t o which were given 20 grams daily of open-kettle cooked cabbage (cooked 45 minutes a t 100' C.), is included in the chart. The feeding experiments fall into several series. The first series were fed 40 grams, 30 grams, and 20 grams daily of the canned cabbage weighed on a raw basis. It soon became evident, however, that these amounts were in excess of what was needed for protection, and after 40 days the 6
J . A m . Chem. Soc., 44, 165 (1922).
