Nitrogenous Constituents
of Flaxseed PEPTIZATION EDGAR PAGE PAINTER AND L. L. NESBITT N o r t h D a k o t a Agricultural College and E x p e r i m e n t S t a t i o n , Fargo, N . Dale. Salt solutions disperse from 43 to 81% of the nitrogen of finely ground oil-free flaxseed meal. In aqueous solution from 85 to 90% of the nitrogen is dispersed at a pH near 11; from 15 to 21%, at pH 3.5 to 4; and from 53 to 65%, between a pH of 1.0 to 1.6. Formic acid disperses €rom 96 to 99 % of the nitrogen. More nitrogen is dispersed from the endosperm than from whole meal or hull. Most of the protein dispersed in alkaline solution can be recovered after precipitation at a pH of 4, but in salt dispersions considerable protein remains in solution. Approximately 20% of the nitrogen of flaxseed is nonprotein; thus protein yields based on the total nitrogen are limited.
imately 24% of the nitrogen of flaxseed extracted by water, whereas the other groups found approximately 65% extractable by water. The percentage of nitrogen extracted by sodium chloride solutions ranged from about 65 t o 80. Dilute salt solutions (0.2 t o 0.3 N ) extracted approximately as much nitrogen from flaxseed meal as more concentrated (3.0 N ) salt solutions (10, 12). When Staker and Gortner compared the percentage of nitrogen in flaxseed meal which went into solution in potassium salts, they found the peptizing power of the anions t o rank as follows: SOc > Br > I > C1 > F. EXTRACTION OF MEAL
Four lots of flax meal were usea in this study. Meal 1 was from a sample of Redwing variety grown in 1941; meal 2 was a composite of several new brown-seeded varieties grown in 1944; meal 3 was from Viking, a yellow-seeded flax, grown in 1942; meal 4 was a composite of several yellow-seeded flaxes grown in 1944. T h e seed lots (7.7t o 8.6% moisture) were ground in a roller mill, and the oil was extracted for 20 hours in a large Soxhlet apparatus with petroleum ether (Skellysolve F). Since the roller mill used in grinding the flaxseed does not give a finely powdered meal, portions of the extracted meals were further ground in a ball mill with flint pebbles or in a Wiley mill. Portions of the meals were separated into hull and endosperm fractions in order t o compare the nitrogen solubility in each fraction with that of the whole meal. A fairly effective separation, although it is not complete, was made by grinding the fat-free meals for approximately 1.5 hours in a ball mill and then sifting through a No. 40 (0.42-mm. opening) sieve. T h g bulk of the hull flakes is retained by the sieve. By further grinding the endosperm in a ball mill and sifting it through finer sieves (No. 60 or No. SO), it is possible t o obtain a fraction almost free of hull. I n the fractions extracted, a No. 40 sieve was used and care was taken t o collect all of the original meal into the two fractions. All peptizations were carried out in 250-ml. centrifuge bottles. One hundred milliliters of solvent were added t o 3 grams of meal, and t h e bottles shaken mechanically at room temperature (from 22' t o 26" (3.). After shaking, the mixture was centrifuged and aliquots of the supernatant liquid were taken for analysis. Nitrogen was determined by the micro-Kjeldahl method of Ma and Zuazaga (8). The p H of a portion of the supernatant liquid was determined at the same time samples were taken for nitrogen determination. The p H values are those recorded with the Beckman glass electrode, no corrections being made when the solutions were alkaline. Although several extractions of flaxseed meal remove slightly more nitrogen than single extractions, for the purpose of comparing different reagents, a single extraction seemed adequate. The additional amounts at the meal weight-volume ratio used are small.
T
HE proteins of many oil-seed meals have been studied for
their nutritive value because the meal has been essentially a by-product which has gone into animal feeds. Recently interest has been aroused in oil-seed meals because they may be sources of useful proteins for other than livestock feed. Current work on these proteins has therefore been focused on industrial utilization. Several oil-seed meal proteins, notably those of soybeans, have been studied extensively; b u t little information is available on the properties and composition of flaxseed proteins as no satisfactory methods have been despribed for separating them from the meal. An efficient method for separating the bulk of the proteins is the major objective in studies designed t o separate the nitrogenous constituents of flaxseed. Before attempting to separate the proteins of flaxseed, it appeared essential t o learn first how much nitrogen can be dispersed in several of the reagents used for dispersing proteins. Observations on preparing the meal before extraction, on t h e amount of nitrogen extracted from whole meal and from separated meal fractions by protein dispersing agents, and on the precipitation of proteins, are recorded in this report. Osborne (11) attempted t o isolate the proteins of flaxseed in 1892. When he dialyzed water or salt solution extracts of flaxseed meal, proteins precipitated. These he classified as globulins. After the salt-soluble proteins were removed, the meal contained protein which went into solution in dilute alkali and precipitated when the solution was neutralized. Osborne also reported albumin and a proteose and peptone fraction in his extracts. His yields of protein were much lower than expected, and he expressed disappointment in being unable to account for the major portion of the nitrogen in t h e meal in isolated protein. Foreman (6) separated a protein fraction by neutralizing an alkaline extract of flaxseed meal. Hamilton and co-workers (7) reported composition analyses upon whole meal. Staker and Gortner (13),O'Hara and Saunders (IO),and Smith et al. (12), in studies of the peptization of nitrogen from a large number of seeds, recorded a few values for the nitrogen of flaxseed peptized by water and salt solutions. O'Hara and Saunders found approx-
9.5
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
96
TABLEI. EFFECTOF GRIKDIKGo s - ~ M O U K T OF NITROGEN PEPTIZED“ IN FLAXSEED MEAL Reagent 7c .x Treatment of N e a l
Original meal Ground in ball mill 1.5 hr. Ground in ball mill 6 hr. Ground in ball mill 50 * hr.
,
Ground in Wiley mill (0.5-mm. sieve)
(1 N )
Peptized
SaCl MgC12 NaCl hlgClz NaCl >lgCI* NaCl MgClz NaCl MgClz
58.8 61.6 73.5 74.7 79.0 79.6 77.0 81.1 58.9 60 6
a Peptizations lasted 2 hours: meal 1 was used,
TABLE11. EFFECTO F TIME O F S H A K I N G ON NITROGENPEPFLAXSEED M E A L A S D p H CHANGES OF SUSPENSION 95 ,N Hours PH of
TIZEDa FROM
a
Reagent
Shaken
1 iV NaCl
1 2 6 24 48
Suspension 6 6 6 5 5
2 2 2 5 3
Peptized 75 7 79 0 79 7 81 79 J
Peptizations made on meal 1, ground in ball mill 6 hours.
Vol. 38, No. 1
dispersing agents. In one case an extract of flax meal maintained for 3 to 4 hours a t a p H near 10.5 decreased to a p H 6.5 in approximately 36 hours. When flax meal is extracted by alkaline solutions, it is necessary t o add base a t intervals to maintain the desired pH. I n water solutions the change in pH is accompanied by precipitation of protein as would be predicted from Figure 1. I n salt solutions (sodium chloride in Table 11) the decrease in p H may also be accompanied by precipitation of protein, but precipitation occurs only after long standing and far more protein remains in solution than in water alone a t the same pH. Figure 1 shows the nitrogen prptized in aqueous solutions a t different p H values for meal 1. Sodium hydroxide was used in the alkaline solutions and hydrochloric acid in acid solutions. Some peptizations were attempted in buffers, but phosphates as strong as 0.2 M failed to hold the pH constant. Minimum solubility occurs between pH 3.5 and 4.0. The nitrogen peptized increases on either side of the minimum solubility point, but far more nitrogen goes into solution in alkaline than in acid solutions. The flaxseed meal used in these studics when suspended in water gives an initial p H of around 6.5. Upon standing the p H decreases t o near 5.0. The amount of nitrogen which remains in solution follows closely the curve shown in Figure 1. The solubility of nitrogen in flaxseed somen-hat resembles the solubility of nitrogen in soybean and peanut meals (4). Nitrogen solubility in these meals is not only greater than that of flaxseed in alkaline and acid solutions, but also increases much more rapidly on either side of the p H of minimum solubility.
EFFECT OF GRINDIVG
EFFECT O F VARIOUS IONS
The amount of nitrogen in the fat free meal which could be peptized in normal salt solutions (Table I ) was increased about 20% by grinding the meal in a ball mill with flint pebbles. Little is to be gained by grinding for more than 6 hours. Grinding in a Wiley mill, which has a cutting action, failed t o increase the amount of nitrogen peptized. After grinding in a ball mill for 6 hours, most of the endosperm passes through a No. 60 (0.25-mm. opening) sieve, but a large part of the hull still remains as thin flakes. Routh and eo-workers (3)and Cohen ( 2 ) have shown that long grinding of proteins in a ball mill modifies their properties and composition. When ground in a ball mill for long periods, proteins are cleaved to give water-soluble fragments which may differ in composition from t h a t of the original protein. A loss of cystine and tryptophan takes place during grinding. When measurable 3) resulted, the materials used were changes in Qolubility (i?$ essentially purified proteins or materials predominately keratin in nature such as u-001, and it was necessary to grind much longer than the flax meal was ground. It is reasonable to assume that flax proteins are not modified in solubility by grinding the whole meal for 6 hours (a total of about 8000 revolutions) in a ball mill, since 50 hours of grinding did not materially increase their solubility. Flax meals from which proteins were to be isolated were not ground for more than 2 hours in a ball mill. O’Hara and Saunders (IO) found as much nitrogen in sodium chloride solutions after 30 minutes of shaking as after 4 hours. We found nearly the maximum amount of nitrogen in sodium chloride solutions after 2 hours (Table 11). However in water the maximum was not reached until about 8 hours. I n other trials on meal fractions after separation into t u o parts, one predominantly hull and the other predominantly endosperm, only slightly more nitrogen was in solution after 16 hours of shaking than after 1 hour (Table V).
Salt solutions differ greatly in the amount of nitrogen they peptize (Table 111). The range in the salts used is from about 43 t o 80% of the total nitrogen. When the results in Table 111 are compared with those in Figure 1, the peptizing action of specific ions is evident. I n some cases more nitrogen is peptized in salt solutions than in water alone when meal extracts of both are a t the same p H ; in other cases the presence of salt reduces the amount of nitrogen in solution. Sodium sulfate, sodium fluoride, and sodium acetate peptize less nitrogen than water when extracts of the latter are adjusted t o the p H of the extracts of these salt solutions. Mignesium chloride is one of the better salt dispersing agents used, yet the p H of the meal extracts of this salt solution is near t h a t of the minimal solubility in water alone. The salt-soluble protein of flaxseed which precipitates upon dialysis was called “globulin” by Osborne (If). Salts differ greatly in the amount of nitrogen they peptize, and the amount of nitrogen dispersed in salts in excess of t h a t dispersed in water
EFFECT O F pH
The pH of a suspension of flaxseed meal in water or dilute alkali decreases upon standing (Table 11). Similar changes in p H take place when neutral or alkaline salt solutions are used as
TABLE 111.
PEPTIZATION O F NITROGEN O F FLAXSEED LfCAL‘ B Y NORMAL SALT SOLUTIoNb
x
Salt
Suspension PH
.N Peptized
Viea1 1 Sodium chloride Sodium sulfate Monosodium phosptlate Sodium salicylate Sodium acetate Calcium chloride Magnesium chloride Magnesium sulfate Potassium sulfate
6.2 6.7 4.8 6.7 7.2 6.5 5.4 5.9 6.7
79.0 48.3 43.3 77.3 50.4 74.4 79.6 75.4 63.6
Meal 2 Sodium chloride Potassium chloride Lithium chloride Sodium bromide Sodium iodide Sodium fluoride Potassium bromide Potassium iodide
6.1 6.2 5.7 6.0 5.8 7.3 6.2 6.3
76.2 72,9 77.0 77.9 77.7 51.1 77.0 79.8
a Meal shaken u i t h solvents 2 houis, meal 1 ground G hours, meal 2 ground 2 hours in ball mill.
INDUSTRIAL AND ENGINEERING CHEMISTRY
January, 1946
alone is not a dependable estimation of globulins as Gortner (6) emphasized. Precipitation of protein by dialysis results from both a decrease in salt concentration and a change in pH. Most of the protein nitrogen in sodium chloride extracts of flax meal precipitates upon addition of acid t o a p H near 4. This precipitated protein is sparingly soluble in salt solutions.
’OI I
2
3
4
5
6
7
8
9
1011
12
PH
Figure 1. Peptization of Nitrogen in Flaxseed Meal in Aqueous Solutions at Different pH Values
Flax meals have been extracted with 50 and 70% ethyl alcohol, but less than 7% of the nitrogen was extracted. The nitrogen extracted was nonprotein, so flax does not contain alcohol-soluble proteins. SEED COAT AND ENDOSPERM
Flaxseed contains a mucilage which swells in aqueous solutions. Apparefitly more work has been done on the mucilage of flaxseed t h a n on the proteins. Tipson et al. (14) cite earlier work on this subject. Solutions containing this substance defy filtration through filter paper. Osborne was the first t o comment upon the difficulties encountered while working with highly viscous suspensions of flax meal extracts. The bulk, if not all, of the mucilage is in the seed coat, so Osborne partially separated the hull from the rest of the seed. H e then carried out his studies on the meal from which the bulk of the seed coat had been removed. Later workers studying flaxseed proteins also partially remove the seed coat. T o compare the solubility of the nitrogen in the seed coat and endosperm with that of the whole meal, each of the four lots of meal were separated into these two fractions. The separations by sifting should be considered only approximate because part of the hull goes into the endosperm fraction and a coating of endosperm can be seen on some of the flakes of hull. Table I V lists the relative amounts and nitrogen contents of hull ahd endosperm. Table V gives the results of peptizations of the endosperm and hull fractions of meal 1 in several reagents. Nitrogen in the endosperm is far more soluble than that in the hull. The hull gives a suspension of lower pH. When the values in Table V are compared with those obtained from whole meals in Tables I, 11, and 111, it is evident that the nitrogen of the endosperm is more soluble than t h a t of the whole meal. The difference in solubility in sodium sulfate solutions is surprisingly large. The greater solubility of the endosperm nitrogen may be due in part t o the fact t h a t it is more finely powdered. Further grinding of the separated hull in a Wiley mill, however, increases the nitrogen solubility very little. It is also possible that the increased solubility of the nitrogen of the endosperm over that of the hull is due t o the large amount of the mucilage in the latter
97
fraction and not t o different nitrogen compounds in the two fractions. Comparison of the results in Tables 11, V, and VI and in Figure 1 may explain the divergent results reported on the solubility of the nitrogen in flaxseed. Most workers (5, 10, 11, I S ) screened their meals, and there is no way of knowing how much was discarded. T h e major part discarded must have been hull. The small amount of nitrogen O’Hara and Saunders (10) found dispersed in water indicates they made their determinations on an extract near the p H of minimum solubility. The three other meals and their endosperm and hull fractions were peptized with five reagents t o determine whether great differences in the solubility of nitrogen in the four meals,would be found. The reagents chosen were 1 N sodium chloride (one of t h e better salts), 0.1 N hydrochloric acid which gives a p H near that of maximum solubility in acid solution, 0.2 N hydrochloric acid which gives a p H near t h a t of minimal solubility, 1/28 N sodium hydroxide which gives a p H near t h a t of maximum solubility in aqueous solution (Figure l), and formic acid. These results (Table VI) are similar t o those in Table V and Figure 1, but a few notable differences stand out. None of the endosperm fractions in Table V I give as much sodium-chloride-soluble nitrogen as the endosperm fraction i n Table V. The nitrogen of meal 3 and its separated fractions is more soluble than t h a t of the others in 0.1 N hydrochloric acid (compare with Figure 1). The nitrogen of the three samples in Table V I is-a little more soluble at a p H slightly below 4 than t h a t of sample 1 (Figure 1). Thus we can expect differences in the solubility of nitrogen in different lots of flaxseed. SEPARATION O F DISPERSED PROTEIN
Studies in this laboratory on the separation of the dispersed protein are far from completion, but some preliminary results will be presented. Flax meal ground 2 hours in a ball mill has been stirred for 1 t o 2 hours in a protein dispersing solution and centrifuged. The meal is then extracted three or four more times by stirring for a few minutes, followed by centrifugation t o give a total volume of approximately 4 liters per 100 grams of meal.
TABLE IV. NITROGENDISTRIBUTION IN AND PROPORTION OF ENDOSPERM AND HULLSEPARATED FROM FL.4XSEED MEAL Proportion. % Meal 1 Endosperm Hull Meal 2 Endosperm Hull Meal 3 Endosperm Hull Meal 4 Endosperm Hull
..
64 36
63 37
..
65 35
..
73 27
Nitrogen,
70
6.20 7.51 4.14 6.02 7.08 4.01 6.89 8.37 4.21 5.78 6.96 2.56
When the meal is extracted with sodium hydroxide solution maintained at a p H near 10.5 and the protein precipitated by the addition of acetic acid to a p H of 4.0 to 4.5,as much as 65% of the total nitrogen in the meal has been recovered as protein. When sodium chloride extracts are treated in a similar manner the protein isolated contains from 35 to 40% of the total nitrogen. Proteins from sodium chloride extraction, after dehydration with alcohol and ether, have contained as much as 17.0% nitrogen (moisture- and ash-free basis). Similarly treated proteins from sodium hydroxide extraction have not contained more than 15.6% nitrogen. When proteins are dried without organic solvents, ‘the nitrogen content is approximately 1.5% lower. Since alkaline solutions extract more pigment from flaxseed then neutral or acid solutions (except concentrated formic acid), crude proteins from sodium hydroxide extracts are darker than those
98
INDUSTRIAL AND ENGINEERING CHEMISTRY
TABLE V. PEPTIZATION OF NITROGENIN HULLSAND ENDOSPERM OF FLAXSEED MEAL1 % N Peptized Endosperm Hull 86.1 53.0 87.5 57.7 .. 79.0 .. 81.4 6:O .. 32:2 82.3 6:3 68.8 39:3 97.0 89:s i6:i 93:o 56.0 58.2 5.9 88.5 59.0 5.3 88.9 59.4
H~~~~ P H of Suspension Treatment of Sample Ground i n ball mill 1.5 hr. and separated
Hull and endosperm ground separately in ball mill 4.5 hr. Hull ground in Wiley mill
TABLEVI.
Reagent 1 sodium chloride 1 N sodium chloride 1 N sodium sulfate 1 N sodium sulfate 1 N sodium sulfate 1 N sodium salicylate Distilled water Formic acid approx. 87%) Formit acid {approx. 87%) 1/28 N sodium hydroxide 0.1 N hydrochloric acid 1 N sodium chloride 1 N magnesium chloride 1 N sodium chloride
1
16 2 1 4 1 2 1 1 2 2
Endosperm 7.1 6.3 7.6 7.4
2
..
7.6 6.9
.. ii:i
1.7 6.3 5.9
Hull 5.8 5.8
..
..
..
5.4
% N
..
..
VARIOUS MEALS“ AXD THEIRENDOSPERM HULLFRACTIONS 0.1 N HC1 0.02 N HC! 0.036 iV NaOH ~~~~i~
PEPTIZ.4TION OF AND
1.0 N NaCl
Meal 2 Endosperm Hull
Shaken 1 16
pH
peptized
pH
6.1 6.1 5.9
76.2 81.4 59.8
1.5 1.7 1.05
%K
peptized
pH
% N
peptized
pH
52.2 3.8 20.4 10.7 55.3 4.1 23.4 10 9 44.5 3.7 16.2 10.3 Meal 3 6.1 77.5 1.6 65.8 3.9 19.5 10.8 Endosperm 6 . 3 82.6 1.7 72.5 4.2 23.1 11.1 Hull 6.0 62.2 1.5 55.9 3.6 19.9 10.5 Mea! 4 6.2 81.5 1.3 58.8 3.9 20.9 11.4 Endosperm 6 . 3 81.4 1.4 60.5 4.1 22.9 11.6 Hull 6.0 61.3 1.3 48.5 3.4 19:9 10.6 a Meals ground in ball mill 2 hours and shaken 2 hours in reagent. b Meal 1 gave 96y0 in formic acid.
% ,N
peptized 85.4 90.6 63.5 86.5 90.4 67.5 90.3 92.4 70.0
from salt solutions. Air-dried proteins darken on the surface, so the appearance of these products depends t o a large extent upon the surface area exposed. w h e n flax proteins are dehydrated with alcohol and ether, white products are obtained. When the amount of nitrogen extracted is taken into account, protein yields from sodium chloride extraction are disproportionately lower than from sodium hydroxide extraction. The chief reason is t h a t far more nitrogen remains in salt solutions at a p H of 4. From about 30 t o 40% of the total nitrogen (based on nitrogen of the meal) remains in sodium chloride extracts while 21 t o 24% remains in sodium hydroxide extracts. Flax contains a surprisingly large amount of nitrogen (approximately 20% of the total) not precipitable with trichloroacetic acid. This fraction, which is considered nonprotein, remains in solution when flax proteins are precipitated. The amount of nonprotein nitrogen extracted by differcnt nitrogen dispersing agents is essentially the same. Thus protein precipitations are far more complete from alkaline extracts than from sodium chloride extracts. Since formic acid extracts nearly all of the nitrogen of flaxseed (Tables V and VI), it seems t h a t this might be a desirable solvent for the separation of flax proteins. Albanese and coworkers (1) applied this reagent, previously used by Mazur and Clarke (9), for the extraction of the nitrogenous constituents of fresh vegetables. When formic acid extracts of flax meal are neutralized, protein precipitates and the yield is higher than obtained by other procedures. The protein does not settle well and is highly pigmented. From limited experience with formic acid it seems that this reagent is not suitable for large-scale isolation of proteins. When proteins of flax meal are extracted with neutral or acid aqueous solutions the extracts are not nearly so viscous as when flax meal is extracted by alkaline solution or formic acid. When flax meal is extracted by alkaline solution, i t is possible to prepare a concentrated suspension so jelly-like it will not pour evenly. The chief compound which makes these solutions slimy is the mucilage of flax. Extracts of endosperm samples contain much less mucilage than extracts of whole meal. If the endo-
61.7
Vol. 38, No. I
sperm were entirely free of hull particles, we believe no mucilage would b e present. The presence of mucilage does not, however, greatly interfere with separation of crude protein from flaxseed. When proteins are precipitated, the curd settles much more rapidly from sodium hydroxide extracts than from sodium chloride extracts although the former contains more mucilage. Some manipulative difficulties could be partially obviated by using dehulled seed, but these can be largely overcome by dilution of the extracts. Protein yields from endosperm are slightly higher than those from whole meal. SUMMARY
The amount of nitrogen in finely ground, oil-free flaxseed meal peptized in normal Acida, % N salt solutions ranged from 43:3% irr, Peptized monosodium phosphate t o 79.6% in mag97 0 nesium chloride in one sample and from 51.1 with sodium fluoride t o 79.8 with potassium iodide in another sample. 98.7 Two-hour shaking of flaxseed meal extracts almost as much nitrogen as longer 99 5 shaking. The p H of suspensions of flaxseed meal in neutral and alkaline water or salt solutions decreases upon standing. The change in p H is usually accompanied by precipitation of protein. The minimum solubility of the nitrogen in flaxseed in aqueous solution is at a p H of 3.5 t o 4.0 when about 15 to 21% is, peptized. I n alkaline solution the solubility increases to a maximum in different samples of about 85 t o 90% at a pH of 11, and on t h e acid side t o a maximum of about 53 t o 65% at a p H between 1 and 1.6. When concentrated formic acid (87%) is the solvent, from 96 t o 99.5% of the nitrogen goes into solution. Flaxseed meals were separated into two fractions: one chiefly hull and the other chiefly endosperm. Nitrogen in the endosperm is much more soluble in all the reagents used than is that of the hull. The solubility of nitrogen - in different lots of seed varied slightly. Protein yields as high as 65% of the total nitrogen have been obtained by sodium hydroxide extraction. When sodium chloride is used, the yields are much lower but t h e product has a higher nitrogen content. Protein yields from flaxseed are limited because approximately 20% of the nitrogen is nonprotein. LITERATURE CITED
Albanese, A. A., Wagner, D. L., Frankston, J. E., and Irby, V., IND. ENQ.CHEW,ANAL.ED., 16, 609 (1944). Cohen, H. R., Arch. Biochem., 4, 151 (1944). Edwards, B., and Routh, J. I., J . Biol. Chem., 154, 593 (1944). Fontaine, T. D., and Burnett, R. S., IXD.ENO.C H ~ M36, . , 164 (1944). Foreman, F. W., J . Agr. Sci.. 3, 358 (1910). Gortner, R A., “Outlines of Biochemistry”, p. 417, New York, John Wiley & Sons, 1938. Hamilton; T. S., Uyei, N., Baker, J. B., and Grindley, H. S., J. Am. Chem. Soc., 45, 815 (1923). Ma, T. S., and Zuazaga, G., IXD. EXG.CHEM.,ANAL.ED., 14, 280 (1942). hlaaur, A,, and Clarke, H. T., J . Bio2. Chem., 123, 729 (1938). O’Hara, L. P., and Saunders, F., J . Am. Chem. SOC.,59, 352 (1937). Osborne, T. B., Am. Chem. J., 14, 629 (1892). Smith, A. K., Circle, S. J., and Brother, G. H., J . Am. Chem. SOC.,60, 1316 (1938). Staker, E. N., and Gortner, R. A., J. Phys. Chem., 35, 1565 (1931). Tipson, R. S., Christman, C. C., and Levene, P. A., J . Biol. Chem., 128,609 (1939). PUBLISHED by permission of the Director, North Dakota Agriculturs! Experiment Station. These studies were carried out under Purnell Project 95, “The Chemistry of Flaxseed”.
