August 1947
INDUSTRIAL AND ENGINEERING CHEMISTRY
dried 8 hours a t 80" C. and 50 mm. pre-sure. T h e ammonium salt was not dried above room temperature because of its lolv h m t stability. The amorphous sodium and ealciuni salts nere dried overnight a t 80" C. and 50 mm. pressure. The stoppers Ivere left out of the neighing bottles during the drying period aad replaced as soon as the bottles were removed from the oven. Tests in this laboratory indicate that the above method of drying gives substantially water-free material x h e n tested according to the mcthod set, forth by the Food and Drug .Idministration ( I ) , exrept that the ammonium salt may contain 0.1 to 0.2cp water. The weighing bottles were xeighed when cool and placed in the respective humidity desiccators n i t h t h e top out of the bottle. They were removed from the hunlidity chambers at intervals and weighed to drtermine the amount of moisture absorbed. The hygroscopicity data for a single salt species were plottcd as per cent gain in n-eight against time in the humidity chamber (Figures 1 to 5 ) . I t is apparent that potassiuni penicillin (Figure 3) is the least hygroscopic of the salts tested. The gain in rveight for 48, 58, and 70% humidities n-as less than O.-lnC at the end of 12 days. Cr)-stalline sodium penicillirl absorhed Ie?s than o.3cG nioisture at 48 and 58c;'0 humidities in 12 days. .in interesting characteristic was exhibited by animoniuni, calcium, and amorphous sodium salts. The dry salts absorbed 11-ater rapidly the first day to a limiting value and then remained a t a constant n-eight or absorbed much more slo~vly. The absorption at 1007, humidity for all thwe salts and a t 80% for the amorphous jotlium salts did not exhibit this characteristic. The absorption of a liniiting amount of water was not ail indication of hydrate iormation because the limiting amount of v-ntcr v a s incrcnscd with increased humidities. This is idpally exhibited by the calc % ~ salt m (Figure 4).
1023
The 100% humidity curves for the amorphous and crystalline salts shoived a marked difference in geueral slope. The slopes for the amorphous salts are much steeper than those of the crystalline salts, being alnlost perpendicular as plotted in Figures 2 and 4. When the percentage increase in \wight was plotted against the relative humidities for a given time period, ths increase in hygroscopicity with increasing humidity became apparent. -klI penicillin salts tested exhibited a decided break in this relation (Figure 6'1 a t one of the test humidities. The humidity a t which the slope of the curve increased appreciably has been called t,he "critical humidity point." Both sodium salts shoned a critical The other three salts !jho\ved this charhumidity point a t 705;. acteristic a t 81%. The differences between the humidity values studied are rather large. The critical humidity point as given represents the experimental humidity at which there is a decided change in the rate of absorption of moisture h v the salt. This point ma:; not represent the highest humidiiy at which this change occurs. Figure 6 points out clearly that crystalline sodium and potassium penicillins nmy be handled or storcd for at least 5 days in any space where the humidity is below the critical humidity point \I-irhout absorbing more than a, fraction of a per cent of 1r:tter. I t is not advisable to handle or stow, even for a short time, any of tlie penicillin salts a t R humidity above their critical humidity point because of the rapidity n i t h xhich they absorb nioisturt:. LITERATURE CITED
(1) Food and Drug Administration, Federal Register, p. 11483 (Sept 8 , 1945). (2) Hodge. Senkus. and Kiddick, Chem. Eng. News, 24. 21i7 (1846). ( 3 ) Spencer, International Critical Tables, s'ol. I, p. 67 (1926).
Effect of Dry Heat on Proteins J DALE IC. MECHAM AND HAROLD S. OLCOTT
V k s t e r n Regional Research Laboratory, tinited Strrtps D e p a r t m e n t of 4griculture, .4lbany, Calif.
1""
successful application of proteins t o iridastriul uses m a and Gersdorff (6) found no diflercrice b e t w e n the lysiiw contents require modification of the prot.eiiis Ijy simple physical or of hydrolyzates of heated and unheated casein. Stwgcrs and chrniical treatment. For example, Bruther, Binkley, and Brandon Mattill ( I S ) observed that beef liver suffered considerable loss in ( 7 ) report that chicken feathers and hoof meal, after bring heated iiutritive value as a protein source by being heated a t 120" for to 210-220" C. for 1 hour, can be u d adr:tntageously to modify 72 hours or by extiaction x-ith boiling &hano1 for 130 hourd; Balcelite-type molding plastics. The preient investigntion was but acid hydrolyzates of trested and untreated samples were undertalcen when it appeared that only a limited amount of insimilar in nutritive value. Harris and Mattill (11) found the formation as available concerning thi, chunges that occur in free amino nitrogen content of liver and kidney globulins to be proteins when they are suhjccted to dry heat. The data obdccreased (by half) by hot ethanol extraction. Sincc there was tainwl may assist in the modificatiori of proteins for .;u 1.pno loss in total nitrogen, they suggested that tlie dvcrense WLS t h r plirations as plastics, coatings, adlle;livc%s,and fibw5. result of the formation of new enzyme-resistan1 linkagcs involving 1 f o A t of the previous \\ 111.11 011 tile effect of heat L u ' < L i inent 011 proteins has Wheat gluten, casein, zein, egg w-hite, cattle hoof, arid soybean proiein w-ere heated bvt-11 rvportcd by investiin boiling inert hydrocarbons at temperatures from 110" to 203" C. for 18 hours. Solugstors intcrested in the bilitj decreased marhedly with increase of heating temperature up to l53", but there w a s no change in total nitrogen and little change in the amide nitrogen contents. niotlification oi iiutritive 4bove 1%' eutensiTe degradation occurred, with continuing loss of water and the pro~ivrties: tlic literature Lias heeii summarized by formation of more soluble products. With wheat gluten and zein, total and amide W:tisman a n d E l v e h j e m nitrogen contents were decreased. The amino and total basic groups of all ,*roteins were ( 2 1 ) a n d hy G r e a v e s , found to be decreased b j the heat treatment. Wheat gluten and cattle hoof, after being llorgan, and Loveen ( I O ) . heated abobe 153", were not digestible by pancreatin. The cystine content of cattle hoof I>>-sine was reported to was markedly decreased by heat treatment above 153". Equilibrium moisture cnntenta at be the first amino acid iO% relative humidit? were decreased by heat treatment at all temperatures. The equilibrium moisture contents continued to decrease at temperatures above 153" dedamaged by heat treatrnrLnt ( I O ) , b u t Block, Jones, 3pite the increasing solubility of the heated products.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1024 woo,
Figure 1.
TEMPERATURE I N O C . Effect of Heat on Solubility of Proteins
the e-amino groups in lysine. I n accord with this suggestion, Eldred and Rodney (8) found less free lysine in enzyme digests of a casein that had been heated at 150' for 70 minutes than in siniilar digests of the unheated protein. Barker ( 2 ) reviewed previous work cin the effect, of heat on egg albumin and determined the temperatures and times a t which egg albumin samples of varying moisture content became half insoluble. For constant time of heating, the temperature required was a linear function of the relative humidity with which the protein had been in equilibrium. Egg albumin that had been stored over phosphorus pentoxide became half insoluble after 60 minutes at 140" or 10 minutes a t 162" C. Bernhart (4) studied t'he effect of heat on dry egg albumin by measuring t,he rate of formation of insoluble protein. The curves resembled those characteristic of autocatalytic reactions. Beckel, Bull, arid Hopper ( 3 ) reported that solvent-extracted soybean heated at 120" and 0% relative humidity for 2.5 hours contained only 490; xs much water-extractable protein ae did unheated meal.
Vol. 39, No. 8
Dried egg white was prepared from fresh egg white by t~uposiiig a thin layer to a n air stream at room temperature. Denatured egg white was prepared as follows: .1 filtered solution of dried egg white was adjusted to p H 5 wit,h dilute hydrochloric acid, heated to 70" C. for 15 minutes, and filtered. The filter c:tkc, was washed with water, dried in a n air stream (room temperature) and finally in a vacuum oven (65 C,). Powdered hoof was 01)tained from R-ashed cattle hoofs. These were dried a t 70" C. until brittle, broken into small pieces by being pounded in an iron container, ground for 24 to 48 hours in a porcelain ball mill, arid sieved. Material passing a 60-mesh and retained by H I I 80-mesh screen was used. Heated samples were prepared from a single lot of each protein. The hydrocarbons used, together with their HYDROCARBOSS. observed boiling points, were: toluene, 110 "; xylene, 140'; cumene, 153 "; cymene, 177"; diethylbenzene and n-butylbenzene, 182 '; diisopropylbenzene, 203 O C. They were commercial products. Traces of water were removed by a prcliminary distillation. REMOVAL OF VOLATILE SUBSTANCES
Tlie tollowing technique was ubed in the preparation of most of the heated protein samples. Thirty grams of protein were suspended in about 250 ml. of the hydrocarbon held in a 500-ml. round-bottomed flask connected through ground-glass joints to x Ridwell-Sterling type of moisture-determination tube of 5 a I . capacity and to a reflux condenser. With the hydrocarboils of
18.0
,
\
17.0
16.0
HEATING TECHNIQUES 15.0
In the present work several proteins were heated a t teriiptbratures from 110" to 203" C., and t,he effects of such treatment on loss of water, total nitrogen content, amide nitrogen content, solubility, equilibrium moisture content in a n at,mosphere of 70C;; relative humidity, and, in some cases, on amino riitrogeii, acaid: and basic groups, cystine content, and pancreat,in digestibility were determined. Most of the data were obtained with samples heated in boiling aromatic hydrocarbons; the temperaturc of t'reatment then could be controlled by the choice of hydrocarboil, the rate of removal of water from the sample could be followed 1 ) ~ the use of a Bida-ell-Sterling moisture determination tube ( 5 ) and in addition any possible oxidative effect of air was effectively prevented. After t,he time periods indicated the samples x e w filtered and washed with pet,roleum ether. Traces of residual hydrocarbon were removed in a vacuum oven a t 60" C. 0thi.r techniques for ht.ating the proteins were sometimes used. as described below. PROTEINS.TVheat gluten (gum gluten), soybean pi.ott.iri (a-protein), acid casein, and zein were conimercial product>.
14.0 W K
?
-0 I 3.0 I
+ 2
12.0
%
\
\
\
\
\
\
\
\
---
GLOBULIN
Q
\
W
"
I 1.0
a
W 0
10.0
9.0
8.0
TABLE I. ELIMISATIOS O F IYIATER FROM J$-HE.4T G L C T E A~ h 0 CATTLE HOOFHEATED IX BOILISGHYDROCARBOXS" lemp., C.
I \
Wheat Gluten, To 18 h r .
2 hr.
Cattle Hoof, % 2 hr.
9.5 9.7 9.9 182 9.3 11.8 203 11.8 15.6 0 P e r cent loss i n a n air oven a t 105' C. for 16 hours, wheat gluten 6.0, hoof 9.3. Compare Sair and Fetzer ( 1 7 ) . 110 140 153
6.6 6.8 7.2 8.0 9.3
6.7 6.9
7.2
9.3 9.4 9.9 10.7 12.3
7.0
18 I n .
6.0
I
I
I
IO0
140
180
TEMPERATURE I N 'C. Figure 2. Effect of Heat on Moisture Content of Proteins in Equilibrium with 7 0 7 ~Relative Humidity at Room Temperature
August 1947
INDUSTRIAL AND ENGINEERING CHEMISTRY
high boiling point it was found advantageous to insulate the upper part of the boiling flask to ensure continual elimination of the water being formed. The duration of heat,ing was measured from the time the liquid started to boil. The amounts of water obtained from wheat gluten and cattle hoof at various temperatures after 2 and 18 hours are given in Table I. The data indicate that, a t temperatures of 153" and below, any reactions that result in a chemical loss of water from the protein are practically completed in 2 hours. At the higher temperatures, additional dehydrating reactions continue during the entire period of heating. At 153' C. and above, the samples darkened rapidly, a n otfensive odor was produced, and, with some of the proteins, a deposit formed in the condenser above the Bidwell-Sterling t'ube. These deposits, usually yellow, were partially soluble in ethyl ether; the white crystalline remainder was found to be chiefly ammonium carbonate. The ether-soluble material also contained nitrogen and may be assumed to consist at least in part of pyrroles. In order to deterniine whether xater-soluble nitrogen compounds were being eliminated a t lower temperatures and from samples giving no condenser deposit, some samples of t,he water collected in the Bidwell-Sterling tubes were analyzed for nitrogen. The results indicated that traces of nitrogen were eliminated even a t 110"; a t 153" as much as 0.3% of the total nitrogen of soybean protein, hoof, and casein was found, although collection of evolved nitrogenous compounds would be espected to be incomplete. SOLUBILITY OF HEATED PROTEINS
Solubilities a t room temperature were determined in solvents appropriate for each protein, The procedures used were as follo\vs. \THEATGLUTEN. One gram of sample was suspended in 5 nil. OF ethanol, 25 ml. of 0.1 N acetic acid n-ere added and the inisture was permitted to stand, with occasional shaking, for 16 to 18 hours (14). ZEIN. Two grams o f sample were suspended in 15 mi. of 9557; ethanol, 10 nil. of water were added, and the mixt,ure \vas permitted to stand 16 to 18 hours. CATTLE HOOF. T o 1.75 grams were added 25 ml. of 0.5 M sodium sulfide. The mixture lvas shaken a t 15-minute intervals for 4 hours. The solutions were then at p H 10.8-10.9. SOYBEAX PROTEIS, (?.&SEIS, AXD SATIVE AXD DENATURED EGG WHITE. T o 1.00 gram were added 25 ml. of a phosphate buffer (800 ml. of 0.2 disodium hydrogen phosphate and'132 ml. of 1 N sodium hydroxide per liter). The mixtures n-ere shaken mechanically for 16-18 hours, The solutions were t,hen a t p H 10.6-10.8. I n all cases the undissolved residue vias removed by centrifugation and filtration ( S o . 4 Whatman paper), and thrl extent of solution was determined by nitrogen analyses of aliquots of the filtrate. The results obtained are plotted in Figure 1. All figures are for a heat treatment of 18 hours in boiling hydrocarbons at the temperatures indicated on the graphs. I n general, minimum solubility was obtained with those samples that had been heated a t 153" C. The esceptions, zein and hoof samples, rvere those whose solubilities were determined in solvents of quite different nature than were used for the other protein
11. EFFECT O S TOTAL NITROGES CONTEST O F HEATING PROTEINS IN BOILING HYDROCARBOKS FOR 18 HOURS
TABLE
Temp., C. Control 110 140 153 182 203
0
K h e a t gluten 14.4 14.2 14.2 14.0 12.0 11.5
Moisture-free basis.
T o t a l Xitrogen Contenta, % Zein Denatured egg u hite 15.7 15.6 15.5 15.2 12.8 13.2
15.0 15.1 15.2 15.0 15.1 15.0
1025
Figure 3. Effect of Heat on Amide Nitrogen Content of Proteins
samples. daniples of these protein- ihowed a definite niiriiniuni solubility when heated at temperatures belox the highest usrtl. The occurrence of minimum solubilities in samples heated a: 153" C. and the continuing loss of water from samples hcated a! temperatures above 153" C. suggest that the increased solubilities found for the latter samples are the rewlt of th(s fornifition of wluble degradation products. ANALYSES I I O I S T G R E ONT TENT AT 7070 RELATIVE HumDIw. h i l p k S o f the heated proteins and unheated controls wert: permitted to conic to constant weight at room temperature in a n atmosphere of 70"; relative humidity maintained within a closed container by mean. of a solution kept sat,urated with both ammonium chloride anti potassium nitrate. The moisture cont,ent of the equilibrated sample was then determined by drying to iconstant weight in a n Abderhalden dryer heated with boiling toluene (110"). Tlir drying chamber contained fresh phosphoroh pentoxide, and vacuum was maintained with a mechanical pump. Results a w shown in Figure 2 . Significant decreases in the equilibrium moisture contents tit 70% relative humidity were found in sample3 heated a t l l O ' , and the amounts of water adsorbed continued to decrease as the temperature of treatment was raispd. Samples heated at 203' showed thc unusual properties of having decreased moisture content in spite of the fact that they were more water soluble than the samples heated at lower temperatures. The sharpest decrease in water adsorption occurred in samples heated above 182" C. Gluten appeared to differ from the other proteins by showing no lowring of water adsorption until it was heated above 153 TOTAL SITROGES ASD . ~ I D E - S I TCONTESTS. R O O E ~Total X nitrogen was determined by the Kjeldahl-Gunning-Arnold method. Wheat gluten and zein were the only proteins t,hat showed apparent change in total nitrogen content, and these only when heated a t the higher temperatures (Table 11). Denatured egg white, as a n example of the other proteins, a t corresponding temperatures underwent no appreciable change in nitrogen content. Amide nitrogen was determined by measuring the ammonia produced by heating 0.500-gram samples with 12.5 ml. of 1.2 iY .sulfuric acid for 40 minute5 in an autoclave a t 20 pounds steam pressure. The autoclaved sample was made to 25 ml. volume with watw and filtered. The ammonia present in a n aliquot of filtrate was then distilled from a suspension of magnesium oxide. This procedure has been found to give satisfactory results for amide nitrogcn in protrins. S o additional ammonia is cvolwtl
1026
INDUSTRIAL AND ENGINEERING CHEMISTRY w 100 m -1
3
-I
5:
80
z w
60
a
t lL
40
0 #-
20 0
a W
n
o
0
6
18
12
24
30
36
42
48
HOURS Figure 4. Effect of Heat o n Digestibility of Wheat Gluten by Pancreatin
until the samples are heated for a n appreciably longer timca. Results are plotted in Figure 3. T h e proteins of relatively high amide nitrogen content, glututi a n d zein, shon-ed marked decreases in amide nitrogen content when heated to temperatures above 153" C. The loss of amide nitrogen would appear to be responsible for the definite lolt.ering of the total nitrogen content of these proteins a t the higher tcmperatures. A m N o YITROGES,. ACID, ATD BASIC GROUPS. The a!nin? nitrogen contents of some of t,he heated proteins n-ere determined by the manometric method of Van Slyke with a 15-minute reaction period ( 2 0 ) . Total acid and basic groups Tverc determined for some of tlie samples by the dye methods of Fraenkel-Conrat and Cooper (9). The data are given in Table 111. Consistent decreases in amino nitrogen were found, in agreement with the observations of earlier investigators ( 2 1 ) . The decrease in basic groups was similarly in proportion to the severity of the heat treatment. However, the data are not of sufficient accuracy to indicate whether basic groups other than tlie amino groups &re involved. T h e increase in the number of acid groups of heated n-hc,it gluton and zein can be attributed t o the transformation of the neutral amide groups t o carbosyl groups through the loss of ammonia. The losses in acid groups in other proteins are probably the result of the interaction of basic with acid g r o u p to form new amide-type linkages. If these two reactions occur to a somelvhat similar estent, the total change in acid groups might be small, as was found with dried egg white (Table 111). CYSTISE. Preliminary results of Binkley and Joncs of this laboratory had indicated t h a t the cystine content of fcathms that had been heated a t 210' for 2 hours \vas negligible. In the present study attempts were made to determine the cystine contents of the ' r . \ B L E 11'. samples of hoof meal that. had been given various heat treatments. Three different methods (12, IS, 19) \yere found to give agreeing values for unheated hoof (approsimately 6%) but could not be used satisfactorily Tvith the heated s a m p l w With each method no appreciable loss of cystitif5 appeared to have occurred in those saniplas vihich had been heated at 153' or below. At the higher temperatures erratic results were obtained. Thus, for one sample heated a t 203" for 18 hours, the apparent cystine content was 2.8'T0 by the Sullivan method and 0.3% bv the modified Vassel method (fa). It, may be concluded t h a t a n unknown b u t ap;>reciable amount of cystine is destroyed in keratins t h a t have been heated t o temperatures above 150". Unheated hoof meal I
.
and the sample that had been heated 18 hours a t 153" contained 2.0% total sulfur, but the sample that, had been heated to 203" for 18 hours contained only 0.856 sulfur; this indicated that sulfur was lost by volatilization. The increase in flow observed with heated feathers ( 7 ) is possibly ascribable t o the decrease in disulfide cross links in the keratin structure. DIGESTIBILITY B Y PANCREATIS: One gram portions of the prcrein samples were incubated 6, 21, and 48 hours a t 30" C. with 25 ml. of a 0.2 J4 phosphate buffer, p H 7.5, containing 0.3 mg. pancreatin nitrogen per ml. The digests xwre filtered ( S o . 4 Whatman paper), and aliyuots were analyzed for nitrogen. ,411 filtrates n'ere a t pH 7.1 to 7.5. The dat,a shown in Figare 4 were corrected by subtracting the amounts of nitrogen soluble in the buffer alone. I n the case of v h e a t gluten samples, the initial rate of digestion was decreased markedly by 18 hours of heating a t 110 ', 130 ', and 153" C . ; each increment in temperature resulted in a further decrease in the initial rate. rlftcr 48-hour digestion, however, these samples and an unheated samplc had become solublc to approximately the same extent. Samples heated a t 182" and 203' C. were practically not digested under the conditions used. Hoof samples were digested at much slower rates, so that even after %hour digestion the data permitted no estimation of the extent to which digestion might eventually have gone. The rate of digestion was again definitelv reduced by heat treatment of the hoof a t 110" or 140", and treatment at 153" C. or higher prevented any dutcctahle digestion. EFFECT OIZ VAHIAIlLES
Protein? t h It hsd haen TI\[[;O F Hr:irTRCtT\rEvT 4 ~ 2 0 3 C. " hraterl for 18 hours at trmperatims near which degrad ttion hcgan
T.~BI.E111. S L W B E R O F -%IIIYO, TOTAL ACID, A S D TWAL n.4SIC GROLTPS' I S USHEATED . i S D HEATED PROTEISSb
'rr
Protein !lriril epn xhittL
p.,
C.
Amino Groups
Basic Groups
3.6
7.0 7 0 6.5 6 3 3.9 2.9 3.2 1.3 1.3 0 0.4 1.8 8 5 4.6 6.3
('oltlol
3.3 2.6 2 4 1.4 2.1 1.8 1.1
11(1
140 1.53
182 203
\!-heat
Control
qliiteii
182
Zein
Co?trol
,..
Hoof
Cont .ol
POLbPan proti'in
Control
Casein
Coqtrol
3:3 1.2 4.9 1.4 5.3
182
182 182
n
i n
182
x
Acid Groups 12.1 11.6 10 3 10.1 12 n 13 8 5.5 Q - 7 . 5 2 9.2 8 6 5 6 14 9 12 0 17 8 13 6
lIoiztnre-fret! tmis. b T h e tii'ated sanrnles had been refluxed f o r 18 houm in hyrlrorarbons t h e tomr,eratiires indicated. '2
E'liiit .ilbnts p e r g x n i p v t e i n
k;FFE(.T
OF
\v I l N i t gl utell
0
1
,
11 18
0 1
3
7 11 18
'Total S,
FIl?\TrVG AT
CASEIN hrnide Sa,
%
'7a
14.4 14 2 13.0 11.5 10 7 11.8
21.4
13.8 13 9 14 13 i 13.8 14.1
2
104.
'21.8
19.5 14.8 12 i 11.8 0.8 9.6 8 3 7. 8 7.3 6.8
203' So1y.b c"
73 24
28
23 18
30
96 15 18 19 20 20
c;.
at
ON ifT€iIIEAT G I . U T R N
AIoisture Contentc,'
'70
hinirro
Nd,
?
11.0 10 2 9 7 8 0 6.3
...
11.4 8.6
0.74 0.14
8.0 7.2
0.12
7.3
8 3
6.8
...
... ...
...
0.08
total nitrown. b Soluble nitrogen a8 pcr r e n t of total nitrogen. c After equilibration a t 7OC< rFlati1.e Iirimidity. d T h e arnino nitrogen content of a.heat gluten is too low (0.25%) for determinatione of changes. 0
. i s ~ > ecent r of
TJMEO F
AND Tiint, of Heating Hr.
-
Vof. 33, No. 8
August 1947
INDUSTRIAL AND ENGINEERING CHEMISTRY
showed minimum solubility. T h e possibility t h a t heating a t higher temperatures for a shorter time might give even less soluble products was investigated. Samples of casein and lvheat gluten were heated at 203" C. for various periods of time and examincd as described. Results are given in Table IV. The data indicate that samples heated a t 203" for 18 hours were more soluble than those heated for shorter tiines, but the minimum solubilities found were not so low as those of saniples heated 18 hours a t 140" or 153". However, in tho cnse of casein partieularly, heating times less than 1 hour might have given loner values. HEATISGIS AIR ASD IS Vacr-o. T h e possibility existed t h a t heating t h r protein samples in contact n i t h the hydrocarbons had led t o results of a different type than might have been obtained if contact with hydrocarbon hati heen avoided. This possibility \vas investigated by examining samples of protrins which had heen heated a t 110", l 5 3 " , and 182" C. in a n oven in open flasks. Analyses of these samples shon-ed the same trtnds observed with samples heated in contact n i t h hydrorarbons, although the samples heated in an oven a t 110"showed somewhat less, and those at 133" and 1'32" somenhat niorc, change than samples heated in hydrocarbons a t corresponding temperatures. Protcins hratcd in air ivert, darker than thow heated in hydrocarbons. .A furthcr set of prolein samples wts heated iu side-arm test tubes, fitted Ivith thermometers, connwted to a water aspirator (20-inch vaccum), and inimersed in boiling diisopropyl benzene (203" C.) for various periods of time. This assembly was used to decrcbase the possibility of oxidative changes and t o aid in the removal of volatile substances formcd. Examination of these samples shovvcd the trends in all analyseh to be the same as those found after heating in hydrocarbon; the changes appeared t o oec:ir more slowly, however, and t h e deereases in solubility were less marked. D a t a are presented in Table \-. K h e n wheat gluten was heated at 1.53' C. in vacuo for periods of 1 , 3, and 7 hours, relative solubilities of 43%36, and 2 7 7 , respectively, ~vvcreobtained: the relative solubility of gluten heated for 3 hours whil(9 immersed in cumene (153" C.) \vas 2 5 7 . DISCUSSIO\
\Vhen crateins are heated in the dry state, the Iollommg reactioni occur in turn as the trmperature and time of heating are increased: At relatively low temperatures (lOO-llOo C . ) there are only slight losses of water and nitrogenous compounds, but readily measurable changes in solubility and rate of digestion by pancreatin. Even samples heated a t 110" C. show significant dccrcwses in moisture content after equilibration at 7 0 7 relative humidity. This may be interpreted as a loss of some polar groups (16),possibly by internal ester or amide formation, which reaction may also be the source of some water. At 150" C. and above, definite signs of decomposition occur. Foul odors are evolved, the proteins darken, considerable amounts of ammonia are lost from proteins containing relatively large amounts of amide nitrogen, and solubility falls t o a minimum. At temperatures around 200" C. decomposition occurs t o the extent that the residual material has become again more soluble. The amounts of water absorbed at 70% relative humidity b y samples which had been heated a t 153" t o 203" C. decrease, irrespective of the increase in solubility occurring in most samples at the higher temperature. There are consistent decreases in amino and basic groups with increase in heating temperature. T h e changes in number of acid groups are complicated b y the conversion of amide groups to carboxyl groups by loss of ammonia, a s indicated particularly by the behavior of zein and wheat gluten. If the content of polar groups determines &he water absorption of the heated samples, the basic groups would appear t o exert considerably more influence than the acid groups. A similar suggestion has already been made (16),based upon different lines of evidence.
1027
TABLE V. EFFECTOF HEATTREATMENT AT 203" C. ON PROTEIXS
Wheat gluten
a
Time, HI. 0 1.5 3 7 11
Total N,
70
14.4 13.5 13.5 13.1 13.1
Amide No,
S d y . b,
70
21.4 22.2 21.4 17.4 15.5
IN
VACUO
Moisture Contentc.
70
470
73 33 40 48 40
11.0 10.6 10 3 9.0 8.6
As p e r cent of total nitrogen.
b Soluble nitromen as per cent of total nitrogen. C
After equilibGtion a t 70% relative humidity.
T h e digestibility of whoat gluten and hoof by pancreatin decreases in proportion t o the severity of the heat treatment. Preliminary experiments with egg white (by H . Lineweaver) indicate t h a t this material is more readily digested by papain after being heated 18 hours at 60" C. than is t,he unheated protein. This is in accord with the concept t h a t denatured proteins are more readily digested than are native proteins ( 1 ) . However, treatment a t higher temperatures caused egg Tvhite to become 0. progressively more resistant to digestion. I n studies in this laboratory yet to be reported, Lundgren, High, Liudquist, and K a r d have sliown that dry heat treatment of synthet'c protein fibers results in a n increase of tensile strength-addit,ional evidence for the formation by heat of nen' cross linkage, presumably of a n amide type. A method of differential thermal analysis was used to determine the optimal trmperature (165 ") for this reaction. ACKVOWLEDGMEVT
The authors are indebted to H. Fraenkel-Conrat for the detcrminations of total acid and basic groups by the dye technique, to H. Lineweaver for making available the results of experiments with papain on heated egg white samples and, to A. H. Elder for valuable technical assistance. LITERATURE CITED
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