Browning of Autoclaved Milk Chemical Factors Involved J. P. I(ASS1 AND L. S. PALMER Division of Agricultural Biochemistry, University of Minnesota, St. Paul, Minn.
An explanation for the tan color of autoclaved and evaporated milk was sought in a study of the effect of inorganic and organic buffers on lactose a t autoclave temperatures. The discoloration is accompanied by the development of acidity, pronounced fall of optical activity, comparatively slight loss of copper-reducing ability, and a n appreciable but constant conversion of lactose to ketoses, or substances not oxidized by sodium hypoiodite. Although the pH a t which any buffer produces an equivalent color or loss of optical activity depends upon its nature, connected with its concentration, dissociation, and buffer capacity, the trend of the caramelization reaction is the same for all buffers, and the coloration developed is a logarithmic function of the optically inactivated lactose. This loss of optical activity and resulting discoloration is a complex function of the buffer concentration and duration of heating, and is directly proportional to the initial concentration of lactose. The colora-
I
NTENSIVE or prolonged heating of milk produces the gradual appearance of a characteristic tan or brown dis-
coloration, the intensity of which depends upon the duration and temperature of heating, the lactose concentration, and the initial pH of the milk. This coloration is a p parently irreversibly transmitted to the proteins precipitated from the heated milk, and its origin has been variously attributed to the caramelization of lactose or to the condensation of the lactose with the milk proteins. However, both explanations have been based more upon an inferred similarity of the development of the color in milk to the discoloration of solutions of lactose heated in the presence of various buffers and amino acids, rather than upon a direct demonstration of the occurrence of either reaction in strongly heated milk. The object of the present investigation is therefore to determine the chemical reactions which characterize the caramelization of lactose and to establish whether either of the proposed processes actually occurs in autoclaved mixtures of lactose with the individual components of milk.
Historical Richmond and Boseley (21) were the first to find that the intensity of the brown color of heated milk varies to some extent inversely as the rotation of the residual lactose, with little or no change in the copper-reducing ability of the sugar, They therefore suggested the formation of an optically inactive caramel, which Kometiani (10) believed to result from the caramelizing effect of the dissolved milk salts, especially the phosphates. Wright (SI), however, stated that the colloidal calcium caseinate is the caramelizing agent 1 Present address, D e p a r t m e n t of Botany, University of Minnesota, Minneapolis, Minn.
tion can be inhibited with formaldehyde which, however, does not prevent the optical inactivation of lactose. Lactocaramel can be decolorized by bromination. Three per cent sodium caseinate sols affect lactose like other buffers, and casein adsorbs caramel according to the Freundlich isotherm. The extent of adsorption is determined by the pH of the sol and is complete a t pH 11. The brownish protein precipitated from autoclaved milk is also decolorized by bromination. The increase in coloration due to increased initial pH’s is independent of the quantity of free amino nitrogen, and the characteristic color of autoclaved milk is ascribed to the caramelization of the lactose by, and the adsorption of the lactocaramel on, the caseinates of heated milk, with no stoichiometric bifunctional reaction between the aldose and amino groups forming definite lactoseprotein compounds involved.
and the protein remains essentially unaltered, as proved by the identity of the racemization curves of the caseins isolated from raw and autoclaved milks. The latter observation receives some support in the data of Kometiani (IO), which show no change in the amino nitrogen content of heated milk and only a relatively slight rise in the Sq4rensen titration values. Many other workers (11, 66, 32) also ascribed the discoloration to caramelization. An alternative explanation of the origin of the brown color was first suggested by Orla-Jensen and Plattner (16), who pointed out that solutions of either casein or lactose heated separately do not discolor a t all. Since a mixture of the two does become brown a t elevated temperatures, these workers postulated an interaction of unspecified nature between lactose and casein as the cause of the discoloration. More recently Ramsey, Tracy, and Ruehe (19) believed that they had entirely discredited the caramelization theory and proposed an aldose-amino group interaction, possibly similar to the reaction investigated by Neuberg and Kobe1 ( I @ , von Euler and Brunius ( 5 ) ,and others (4,18). They based their conclusion on the fact that several amino acids produce a browning of solutions of aldoses and ketoses but not of sucrose, the production of the discoloration being aided by alkalies and inhibited by formaldehyde. This, as well as their inability to remove the color from the precipitated casein (which they claim loses its biuret reaction, acquires the ability to respond to Benedict’s reagent, and becomes difficult to precipitate after prolonged heating in the presence of sugars) led them to state that “the brown discoloration of sweetened condensed skim milk and possibly many other food products is due to sugar-protein condensation products instead of caramelization. Caramelization plays no role in the discoloration of dairy products. The similarity of this defect to
1360
OCTOBER, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
caramelization no doubt explains why the brown discoloration has always been explained on the basis of caramelization." Webb (29) studied the effect of various amino acids and buffers at different p H ranges on solutions of pure lactose and found t h a t the intensity of the color produced on heating depends upon the pH and the nature and concentration of the buffer, phosphates apparently having a specific caramelizing effect'. Webb concluded t h a t a sugar-amino combination accounts for a major portion of the color formed in autoclaved milk, although the contribution of the caramelizing effect of the milk phosphates is probably considerable. Other factors t h a t contribute t o the discoloration are the p H [previously ascribed b y K e b b and Holm (SO) t o its effect in the formation of substances in milk t h a t increase the susceptibility of the lactose t o caramelization], the lactose concentration, the c,oucentration of the phosphate ion, the normal tendency of lactose t o caramelize at the p H of normal milk, the gases in the container, and certain catalysts.
Experimental Procedure The chemical changes which characterize and accompany the caramelization of lactose were studied by following the development of color in relation to the reducing, optical, and acidforming properties of lactose solutions heated in the presence of various buffers at extended ranges of pH, concentration, and time. The concentrations of total reducing substances were determined by the method of Munson and Walker, the weight of the cuprous oxide being checked by thiosulfate titration (1). The iodometric method of Hinton and Macara (8) was applied to determine t,he concentration of aldose. Changes in optical activity were followed with a Jellet-Cornu compensated half-shadow glucometer, using a white light source. Since the specific rotations of glucose and lactose are practically identical (+52.3" and +52.5', respectively), the direct reading of the instrument was taken as equivalent to the lactose concentration expressed as per cent. With the exception of two experiments in which the quinhydrone electrode was used, all pH measurements were made with the Coleman glass-electrode electrometer. The relative concentrations of the caramel were determined with a Bock-Benedict colorimeter, using an arbitrarily selected member of a series as the standard; all concentrations were thus referred t o the selected standard to which unit intensity was assigned. It should be kept in mind, therefore, that the numerical identity of intensities as given in the various tables of experimental results does not necessarily indicate equivalence of color depth. The buffer solutions em loyed were as follows: the phosphate buffer of Clark and Lubs YS), the phosphate-citrate buffer of McIlvaine (II),the veronal buffer of Michaelis ( I S ) , the borate buffer of Palitzsch (IY), the glycine buffer of SZrensen ( 8 4 , mixtures of milk salt,s as postulated by Soldner (IS) and by Van Slyke and Bosworth (68),and 2-2.5 per cent sodium caseinate sols a t pH 6.6-6.8, approximating milk both in concentration of casein and in pH. The standard buffer solutions were usually prepared as directed by the authors, except that their concentrations were doubled, so that dilution of selected volumes with equal volumes of an 8 per cent lactose solution produced buffered lactose solutions with the sugar content of an average milk. Aliquots were then sealed in tightly stoppered pressure flasks of 60-ml. capacity and heated in an upright, gas-fired autoclave for a stated eriod after the desired temperature and pressure were reached. ince the amount of water in the autoclave frequently varied, the time required to attain the selected temperature also varied somewhat with each experiment, The total time during which a given solution was maintained above 100° C. is therefore stated where necessary. That the caseinates of milk are largely res onsible for the discoloration of lactose on heating was shown t y studying the behavior of the sugar in solutions of synthetic milk salts mixtures, in whey powder free from casein, and in milk serum obtained by ultrafiltration. The adsorption of lactocaramel on casein was demonstrated by applying the familiar Freundlich adsorption isotherm t o data obtained by comparing the relative color intensities of the filtrates from clarified mixtures of progressively diluted caramel solutions with constant quantities of sodium caseinate. Proof of an interaction between the aldehyde function of lactose and the amino groups of casein was sought by means of determinations of the free amino nitrogen and the total nitrogen of the protein in heated lactose-caseinate sols, the coloration of which was varied by controlling the initial pH.
I361
All reagents em loyed were of the highest purity :ivail;ihle commercially, incfuding Eastman Kodak Company's and Pfannstiehl highest purity lactose. The casein was prepared according to the method of Van Slyke and Baker ( 2 7 ) .
Results
h comparison of the effects of various buffers on approximately 4 per cent lactose solutions a t autoclave temperatures is summarized in Table I. It is evident t h a t the caramelization of lactose by buffers is characterized by a partial conversion of lactose t o acids, a transformation of the aldose t o ketoses, and the formation of a colored substance from the transformation products, the latter supposition being indicated by the disproportionately large drop in rotation compared to the surprisingly constant loss of aldose. The general trend of the caramelization reaction is apparently similar for all buffers examined. In evaluating these data, it should be kept in mind t h a t the effects of the same p H established by different buffers are not necessarily comparable, since the concentrations of the buffering agents are not the same at equal hydrogen-ion activities. Similarly, it is possible that the influence of rising pll in the same buffer series may be modified by changes in the concentration of the effective ion. To mention a specific example, the glycine buffer at pH 1.86 contains only half the volume of 0.05 M glycine solution of the same buffer at p H 7 . 3 . Furthermore, the effects of high temperatures are undoubtedly different on the various buffers, which mere checked at room temperature. Thus, while the p H of a given phosphate buffer is probably only a little lower a t 120" than at 25" C., that of a borate buffer is decreased markedly by increasing temperatures, because of a shift of equilibrium between boric acid and its polymolecular complexes. Nor are the effects of the initial p H on the final p H (of particular interest in the case of the phosphate buffers) necessarily comparable among the several series since their buffer capacities are certainly not identical,
TABLEI. EFFECTOF AUTOCLAVING 15 MINUTESAT 130" C. ON 4 PER CENT LACTOSESOLUTIONS IN THE P R E ~ E NOF CE VARIOUS BUFFERS Buffer Clark-Lubs, phosphate Clark-Lubs, phosphate
MoIlvaine, qhosphatecitrate
pHa Raw Heated 6 50 6.45 6 75 6.45 6.90 6.45 7.20 6.45 5.80 5.60 6.40 6.15 6.80 6.45 7.20 6.45 7.60 6.45 8.00 6.45
3 4 5 6
7 Spensen, glycine Michaelis. veronal Palitzsch, borate
8 1.85 3.98 7.30 7.65 6.65 6 90 7.50 7.90 6.75 7.05 7.55 8.05
.. . ...
..... . ... ... 2.00 4.00 5.70 6.15 6.40 6.60 7.00 7.20 5 50 5.80 5,ss 6.05
% Lactose Lost CuproIod?metricb metricc Polarimetric 4.14 ... 41.4 4.25 . .. 48.1 6.76 60.5 7.75 . 64.6
... ... ... .. . .. .
... .. . ... .... .. ... ... ... ... ... ...
... ...
. .. ..
5.0 9.4 15.3 15.3 15.3 15.3 10.3 10.3 10.3 10.3 10.3 10.3
...
...
...
.
,.
65,O
70.0 72.5 0 0 2.5
5.0 22.5 37.5 67.5 2.5 (gain) 2.5 5.0 7.5 7.5 12.5 37.5 52.5 12.: 17.0 35.0
... ... ... ... ... ... ...
... ... ... ...
5.0 25 0 45 0
... ... ...
60.0
Relative Colord
.. . .. . ...
... I 0.36 1.00 1.33 1.51 0.00 0.00
... ... ... 0.00 0.00
0.118 0.142 I 0.05: 0,285
0.57 0.084
0.220 0,439
Determined potentiometrically x i t h glass electrode. Munson and Walker method ( 1 ) . Hinton a n d Macara method ( 8 ) . d Determined colorimetrically, using t h e color developed b y t h e phosphate solution ( p H 7.2) as standard. e Barely perceptible color. f Perceptible color too weak t o be measured i n colorimeter. Q
h
c
1362
INDUSTRIAL AND ENGINEERING CHEMISTRY
as Cianci ( 2 ) has already emphasized. The final pH measurements of the lactose solutions autoclaved with the borate buffers especially are of questionable significance, for Michaelis (13) showed that in such a mixture the pH is perceptibly shifted and none of the substances is present in the orlginai state. F i g u r e 1, in which the data of the last two columns of Table I are plotted, 0' 1.0 .4 * shows the essencJ .e .b tially logarithmic u 2 .6 .eg relation between I1 . 0 4 .4 the relative color w .2 1.2 developed and the a percentage of opti0 PO 40 60 80 100 20 40 60 80 cally inactivated Z LACTOSE DESTROYED lactose. With the exception of the FIGURE 1. RELATION BETWEEN PER CEST OPTICALLACTOSE DESTROYED BY glycine solutions, BUFFERAND RELATIVE INTENSITIES OF t h e r e s u l t s of COLORDEVELOPED BY 4 PER CENT which are not L.4CTOSE SOLTJTIOSS ON AUTOCL.4VING given in Figure 1. the intensitv ok the color is h general a function of the quantity of sugar destroyed, irrespective of the agent causing the transformation. Although the pH a t tvhich equivalent intensities are produced differs v i t h each buffer, there is probably no reason to ascribe a specific caramelizing effect to any one of them. The colors developed by the glycine-lactose mixtures, however, are much deeper than can be accounted for by the relatively slight loss of optical activity. This may point to a different type of reaction, although the data a t hand are too meager to permit a definite conclusion. S o l u t i o n s of pure lactose do not d i s c o l o r a t 70 a u t o c 1a v e tema m peratures, in W a g r e e m e n t with 0 p50 the findings of a I-40 w VI Orla-Jensen and 0 % Plattner (16) and W of Ramsey, Tracy, VI 0 zo and Ruehe (19). 5 10 W r i g h t ' s stateh . 025 IO 15 20 ment (31) to the MOLS PHOSPHATE contrary may unFIGURE2. EFFECTOF PHOSPHATE doubtedlY be athiCONCENTRATION ON THE OPTICAL buted to the imACTIVITYO F A 0.3 M LACTOSE SOLUpu ri ty 0f hi s TION BUFFERED AT PH 6.68 AND AUTOfor a CLAVED FOR 30 MINUTESAT 120" C. domestic preparation of "pure milk sugar", solutions of which discolored merely on boiling for a few minutes, was found to contain 0.75 per cent protein and to produce 0.15 per cent carbonate ash in which phosphates were present.
8
Effect of Phosphate Concentration Constant portions of a 0.3 M lactose solution were treated with progressively smaller volumes of a 0.2 M phosphate buffer a t pH 6.68; the mixtures mere made up to twice the volume of the lactose solution and autoclaved a t 120' C. and 15 pounds per square inch (1kg. per sq. cm.) pressure for 30 minutes. Table I1 and Figure 2 summarize the results; they show that, although the color again bears an apparently logarithmic relation to the fraction of optically inactivated
VOL. 32, NO. 10
lactose, the phosphate may have a t least a double function in the caramelization reaction. The loss of optical activity in a 0.15 Jf lactose solution apparently becomes a straightline function of the phosphate concentration when the latter exceeds 0.075 M per liter. No account was taken of the slight changes in the pH of the buffer due to dilution. 11. EFFECT OF CONCENTRATION OF PHOSPHATE BUFFER (PH 6.68) ON OPTICALACTIVITYAND RELATIVE COLOROF 0.15 .$f LACTOSE SOLUTIONS AUTOCLAVED 30 MINUTESAT 120" C.
TABLE
PO1 Ion
% Lactose Before 5.40 5.40 5.40 5.40 5.40
Concn. Mole/Liter 0.20 0.15 0.10 0.075 0.050 0,025 0.0125
5.40 5.40
by Glucometer After Loss 1.65 1.90 2.10 2.20 2.35 2.80 3.25
69.4 64.8 61.1 59.3 56.5 48.1 39.8
Relative Color 1.oo 0.78 0.54 0.53 0.39 0.22 0.13
Effect of Lactose Concentration Table I11 shows the effect of the concentration of lactose on the loss of optical activity in solutions heated 30 minutes at 120' C. and buffered with 0.1 M phosphate at pH 6.3 and the effect on color a t pH 6.6. The color developed a t the lower pH was too light for satisfactory measurement. It is evident that the loss is directly proportional to the initial concentration; that is, the mole fraction of lactose optically inactivated is essentially independent of the concentration. TABLE111. EFFECT OF LACTOSE CONCENTR.4TION ON OPTICAL ACTIVITY OF L.4CTOsE SOLUTIONS AUTOCLAVED 30 MINUTES AT 120" C. WITH 0.1 M CLARK-LUBS BUFFER(PH 6.3) AND EFFECT ON COLOR AT PH 6.6 Lactose Concn.
Lactose Concn. by Glucometer Before After Loss
Mole/l.
70
%
Mole
0.20 0.10 0.09 0.07 0.05 0.03 0.02
7.20 3.60 3.24 2.52 1. s o 1.08 0.72
4.15 2.1 1.8 1.4 1.0 0.65 0.4
O.OS5 0.042 0.0403 0,0308 0.0224 0.0120 0.0084
Lactose Lost
Lactose Concn.
Mole fraction
Mole/l.
0.425 0.420 0.447 0.440 0.448 0.440 0.420
0.20 0.18 0.14 0.10 0.08 0.06 0.04
Relative Color after Heating at pH 6.6
1.00 0.96 0.93 0.86 0.83 0.80 0.65
Effect of Duration of Heating The constancy of the mole fraction of lactose optically inactivated (Table 111) suggested that the reaction may be monomolecular. Portions of a 0 2 M phosphate buffer (pH 6.6) were diluted with equal volumes of an approximately 0.4 M lactose solution, and the mixtures heated in stoppered pressure flasks in a stirred calcium chloride bath maintained a t 120" C. Individual samples were removed at intervals, quickly cooled, and examined for residual optical activity and relative color as usual. Figure 3 shows the complexity of the reaction. The nature of the curve may, however, be due to the fact that a t least three simultaneous reactions are involved in the loss of optical activity-namely, the production of acids, ketoses, and caramel-so that the "residual lactose" actually represents the composite optical activity of the resulting mixtures rather than the concentration of unreacted sugar.
Effect of Formaldehyde on the Caramelization Reaction The inhibiting action of formaldehyde on the production of coloration in autoclaved milk was used by Ramsey, Tracy, and Ruehe (19) as proof of their theory that the brown color of heated milk is due to an aldose-amino conden-
OCTOBER, 1940
IKDUSTRIAL AKD ENGINEERING CHEMISTRY
1363
made to dissolve in the readily available solvents. LactoTABLEIT'. EFFECTOF AUTOCLAVING 1 5 MISUTESAT 137" c. caramel solutions prepared by heating lactose in the presence ON THE OPTICAL ACTIVITYOF LACTOSE SOLUTIONS Ix o.osi ~~1 of buffers took up considerable quantities of bromine with r+ PHOSPHATE BUFFERSIN THE PRESENCEOF FORMALDEHYDEtluction in color, although in this case decolorization was not 70Lactose by Glucometer p H b y Quinhydrone Electrode complete. Before After Loss Before AfterG 6.48 6.60 6.70 7.00
6.35 6.40 6.48 6.50
0.13 0.20 0.22 0.50
4.15 4.15 4.15 4.15
'22 9 30 0 32 3
3 20 2.90 2.80 2.60
-1gent Responsible for Lactose Discoloration in
Autoclaved Milk
37.4
EFFECTOF MILE SALTS. The following suspensions were prepared : sation, since formaldehyde may obviously be assumed to block the amino group and thus prevent the reaction with the aldehyde function of the lactose. However, Webb (29) showed that, although small quantities of formaldehyde increase the color of autoclaved buffered lactose solution>. larger quantities seem to decrease the discoloration, ant1 '/z4 M sodium bisulfite prevents it completely. Spoehr and Wilbur (26) had previously ascribed this inhibiting effect of reducing agents to their reaction with the sugar split products, which consequently cannot polymerize t o caramel. If, therefore, it can be shown that sufficient quantities of formaldehyde will entirely prevent the discoloration of buffered lactose solutions, the observation of Ramsey, Tracy, and Ruehe (19) may be used equally well in support of the caramelization theory, To test such a possibility, " g r a m s a m p l e s of lactose were made up t o 50 ml. each with .i ml. of neutral formaldehyde and 45 ml. w 30 v) of 0.05 M Clark-Lubs p h o s p h a t e buffers. y LO 2 These were autoclaved f o r 15 m i n u t e s a t 137" C. and 30 pounds per square inch (2.1 kg. w 10 20 SO 40 50 per sq. cm.) pressure. TIME IN MINUTES Despite the intensity FIGURE 3 . EFFECTO F TIME0s of the heat treatment, THE OPTICAL ACTIVITY ASD coLoR OF 0.2 lv L~~~~~~ all solutions remained SOLCTION HEATED AT 120" C. colorless. When 0.5 WITH PHOSPHhTE BUFFER AT to 1 gram of trioxyPH 6.6 methylene was used in place of formaldehyde, the effect \\ah identical. Table IT shows, however, that formaldehyde does not inhibit the formation of acid and the loss of optical activity, although the latter is much smaller than in the absence of formaldehyde.
e
Decolorization of Solid Lactocaramel by Bromine A sample of pure lactose was heated for 72 hours a t lT5" C. in a vacuum-drying pistol. The resulting light-brown powder was practically insoluble in n-ater and swelled into a flocculent form. The method of preparation pointed to obvious unsaturation, which vias confirmed by triturating the material with saturated bromine water. The marked absorption of bromine was followed by an almost complete disappearance of the caramel color. The brominated substance was considerably more friable than the parent substanoe, but neither could be TABLEv. EFFECTO F AUTOCLAVING 15 M I N C T E S
AT
% Lactose Concentration B Y Copper Reduction B y Glucometer Salts Before After Before After Loss Loss 13.792 3.748 1.1 3.7 3.0 19.0 I1 4.092 4.092 0.0 3.6 12.2 4.1
120"
I (Soldner, ZS),added t o 300 cc. of water t o give a suspension of p H 6.68: CaHPOn 0 . 5 2 6 gram NasCeHsO? 0.666 gram CaCL 0.357 KsCsHsOi 0.156 AlgH4PiOs 0.309 KtHPOi 0.690
h.i:t;r
%Its I1 (Van Slyke and Bosworth, ZS), added t o 300 cc. of water t o give a -uspension of p H 6.49, adjusted with 5 cc. 0.1 N sodium hydroxide t o p H 6.70: KaCaHsOi
0,1008gram 0.2505 0,3468 0.2490 0.2886
KeHPOi
KHPOi KC1 NaCl
Samples of lactose were made up to 100 ml. with the rebpective suspensions. For polarimetric and gravimetric analyses, they were filtered through Gooch crucibles into receiving flasks, both previously dried a t 100" C. for 30 minutes. Table V shows that milk salts play only a minor part in the discoloration. The pronounced drop in p H on heating the suspensions, accompanied by only an insignificant loss of reducing substances, suggested a change in the nature of the buffers. Their acid-base balance \vas therefore determined, the resulb in Table VI showing that the greater portion of the acidity noted in the preceding experiment was due to the milk salts rather than the lactose. No drift in pH, similar to that observed in heated milk, was noted after 24 hours. The validity of any conclusions drawn from the preceding two experiments is questionable on the grounds that the salt mixtures bear only an analytical similarity to the actual composition of the inorganic components of cow's milk. However, the probable ineffectiveness of the soluble milk salts in the discoloration of heated milk was borne out by additional evidence. Three grams of a whey powder containing 75.58 per cent lactose were dissolved in 50 ml. of water to give a suspension of p H 6.4, which was adjusted to p H 6.6 with 0.4 ml. of 0.1 N sodium hydroxide. The concentration of lactose and pH thus correspond to average milk. Autoclaving for 15 minutes a t 120" C. changed the opalescent solution to a colorless liquid containing a white precipitate, the pH dropping to 6.05. This rise in acidity is greater than is usually developed in a similarly treated whole milk, and is presumably due to the absence of the buffering action of the colloidal caseinates and calcium phosphates. The concentration of lactose, determined polarimetrically after clarification of the sols with equal volumes of 5 per cent phosphotungstic-sulfuric acid, dropped from 4.6 t o 4.2 per cent, the loss corresponding to that in an identically heated whole milk. As final proof of the essential role of casein in the discoloration reaction, some skim milk was filtered through a porous Berkefeld cylinder (porosity 1') and the filtrate refiltered
c. O S
Total Before 6.68 6.70
0.6399 gram 0.2418 0.2013 0.1101 0.1108
Caa(CsHsO7)z Caa(PO4)z CaHPO4 Algz(CaH6Oi)n XIgHPOd
THE
L.4CTOSE
p H of Suspension After Loss 6.13 0.75 5.90 0.80
IS
SOLCTION
WITH
SYNTHETIC MILK S.4LTS
Vol. 0.1 N NaOH for 10 Cc. Gooch Filtrate t o pH 8.3 Before, After, Gain, cc. % CC. 0.60 1.08 80 0.78 65 0.50
Color Before None None
After Faint yellow None
1364
INDUSTRIAL AND ENGINEERIKG CHEMISTRY
VOL. 32, NO. 10
ture was diluted to 200 ml. and filtered. The reaction of three equal portions was adjusted as indicated in Table VI11 and the colorless sols Acidity as 0.1 S X a O H for 100 Cc. Suspension were then autoclaved in pressure flasks. Twentyf__ o p H 8.3Q milliliter portions of the resulting colored sols Cnoxalated Oxalatedb pHC After Aftpr Gain Imore Salts Before Gal; Before After Loss were diluted with 60 ml. of water in order to preMZ. M1. c c .l!fl. 'M1. % vent the formation of unwieldy coagula, and Van 10.0 12.0 :333 7.0 42.8 I 9.0 6.65 6.22 0.43 Slyke amino nitrogen determinations were run on 10.0 11.1 7.5 0.5 15.4 I1 9.0 6.70 5.90 0.80 10-ml. aliquots of the diluted solutions, using dia Phenolphthalein used as indicator. b 2 cc. s a t u r a t e d K2C206 solution added t o 100 cc. suspension prior t o titration. phenyl ether as foam depressant (14). It was C Quinhydrone electrode. found that the voIume of nitrogen obtained with the ether was only a fraction of that obtained when octyl alcohol was used, and in both cases through an ordinary Berkefeld candle (porosity A') to give it varied to such an extent that i t was considered necessary a slightly opalescent serum. The opalescence disappeared to average the results of a t least six determinations. upon slight acidification with acetic acid. The unacidified Although these data are a t best only semiquantitative, filtrate neither changed in color upon autoclaving, nor acthey are sufficiently accurate to invalidate the assumption quired the characteristic odor of cooked milk; the only noticeof a simple aldose-amino condensation, unless the rate of able effect was the inability of acid to clear the opalescence, hydrolysis of the peptide linkage is greater than the rate of undoubtedly because of the coagulation of albumen. Under condensation. Another approach to this problem also failed similar treatment, filtrate V , which still contained appreciable to furnish conclusive evidence. It was reasoned that a proquantities of casein precipitable with acetic acid, became disgressive reaction, or else adsorption, should result in a regular tinctly brown and on addition of acid produced a characterisdecrease of total nitrogen in proportion to the amount of lactically tinted precipitate. tose bound by the casein. Sixty grams of casein were therePrecipitation of the phosphates of milk by calcium salts fore triturated with 375 ml. of 0.1 N sodium hydroxide, diluted diminishes the production of color, whereas the liberation of phosphates by the precipitation of the milk calcium with oxalates intensifies it. This effect, however, is due entirely TABLE VII. EFFECTO F h C T O s E COSCENTR4TION ON TIIE Loss OF LACTOSE A N D COLOR DEVELOPED IN 2 PERCENTSODIUM to changes in pH, which is lowered by calcium salts and CASEINATE SOLS (PH 6.8) AUTOCLAVED 30 MINUTESh T 120" raised by their precipitation, and may be duplicated by adjusting the pH to similar levels with acid or base prior to To Lactose Concentration B y GlucometPr Loss heating. TABLE VI. EFFECT OF AUTOCLAVING 15 MINUTESAT 120" C. ON THE ACID-BASEB.%LASCE OF ARTIFICIALMILK SALTS SUSPENSIONS
Beforeb
Interaction of Lactose and Casein Attempts to show a parallelism between the caramelizing effects of phosphates and sodium caseinate produced some contradictory results. When portions of a 4 per cent sodium caseinate sol, prepared by triturating pure casein with sufficient 0.1 N sodium hydroxide to bring the diluted sol to pH 6.8, were mixed with diminishing quantities of a 14 per cent lactose solution, made up to constant volumes containing 2 per cent casein, and autoclaved, the amount of lactose inactivated was found to be directly proportional to the initial concentration; that is, as in the case of phosphates, the mole fraction destroyed was constant. Table VI1 gives the polarimetric data obtained after clarification of the sols with 5 per cent phosphotungstic-sulfuric acid (20). However, when the quantity of casein mas varied and the concentration of lactose kept constant a t 4 per cent, the color after autoclaving a t 120" C. for 30 minutes varied with the amount of protein, as expected, but the relative loss of lactose was surprisingly constant a t 19.6 per cent, irrespective of the wide variations in the concentration of the protein. Similarly, when identical mixture? containing 2.5 per cent casein and 4.5 per cent lactose were heated a t 120" C. for 10, 25, and 45 minutes, the color again increased with time, but the relative loss of lactose throughout was constant a t 11.1 per cent; that is, the concentration fell from 4.5 to 4.0 per cent, irrespective of the duration of heating. Moreover, an inverse relation could not be demonstrated between increasing color due to using initial pH and amount of free amino nitrogen, as would be required by the interaction theory. Table VI11 shows that not only does increased alkalinity with its concomitant greater coloration fail t o reduce the number of amino groups, but on the contrary it leads to a marked increase, no doubt due to hydrolysis. In this experiment 6 grams of purified casein were triturated with 36 cc. of 0.1 AT sodium hydroxide, the sol was diluted somewhat, 8 grams of pure lactose were added, and the mix-
4.55
3.64 Z.L.3
After
3.50 2.80
... 1.40 0.70 0.35
Actual 1.05
0.84
... 0.42
Relative 23 23
Relative Colore
..
1.00 0.83
...
23 0.64 1.82 0.91 0.21 23 0.30 0.105 23 0.455 0.20 a T o t a l time over 100° C. was 1 hour. b Calculated. c These measurements are only approximate, since a slight opalescence d u e t o a n accidental admixture of some NaCl made accurate comparisons inipossible.
VIII. EFFECTO F INCREASING ALKALINITYON THE COLORAND FREEAMINO NITROGEN CONTENT OF 3 PER CENT SOLS AUTOCIAVED 20 MINUTES C A S E I N A T E 4 P E R C E N T LACTOSE -4T 120" c.
TABLE
Composition of Sol, 111. 3% caseinate4 % lactose 0.5 .T NaOH 0.00 50 0.15 50 50 0.30
HtO 0.30 0.15 0.00
Relative Color a f t e r Heating 1.00 1.33 1.66
Amino K a f t e r Heating. llg./MI, 0.0975 0.2694 0.3790
to 1000 ml., and filtered. The clear tan sol had a p H of 6.85. One-hundred-milliliter portions were treated with 6 grams of lactose and with increasing volumes of 0.5 N sodium hydroxide. These, with a suspension of 6 grams of casein in a 6 per cent lactose solution, were autoclaved; they became progressively browner with pH. The mechanically stirred sols were precipitated with dilute acetic acid, and the coagula repeatedly washed with water and centrifuged. The precipitates were then transferred to filter funnels and washed with hot water, This treatment, which instantly turned the material to gummy masses much browner on the surface, failed to free the proteins of reducing substances, even after the use of several liters of liquid. The samples were consequently extracted for 24 hours with boiling water in Soxhlet extractors and again copiously washed by decantation until free from reducing substances. That the washing was still incomplete was indicated by the fact that after several negative
OCTOBER. 1940
INDUSTRIAL AND ENGINEERIKG CHEMISTRY
Fehling tests, additional wash waters would frequently again respond to the test. A 1 samples were finally washed with purified alcohol and ether, and then dried a t 110' C.; the dry materials, ranging in color from a golden brown to a dark chocolate, were analyzed by the usual Kjeldahl method. The average of three determinations is given in Table IX.
TABLE X. ADSORPTION OF LACTOCARAMEL BY CASEIN hI1. Caramel Soln. in 20 Ml. of M i x t . Containing 10 M I . 3% Na Caseinate 10 5 3
Depth of S o h . Standard Tesi 1.5 20.3 20.7
2 1
TABLEIX. TOTALA-ITROGEN CONTENT OF CASEINISOLATED FROM SODIUM CASEINATE-LACTOSE SOLS AUTOCLAVED15 MISUTESA T 125' c. AT INCREA4SINGPH pH of Sols Before After 4.60 4.40 6.85 6.10 7.10 6.15 7.55 6.15 8.20 6.19
1363
32.8
Relative Concn. of Test S o h . 0.739 0.725
Fraction of Caramel Adsorbed 0.261
0.762
Total N a f t e r Heating, $5 14.41 14.56 14.37 11.15
14.18
The lack of uniformity in the nitrogen content of the reisolated protein disposes of the possibility that a single substance of definite composition but of variable color is formed when casein is heated with lactose. However, the irregularity of the values is also not in harmony with the adsorption theory or with the progressive reaction hypothesis, and is undoubtedly due to incomplete extraction of soluble substances, partial elution, and hydrolysis. T h a t the latter takes place on autoclaving casein has already been shown by Hammarsten ( 7 ) and Komatsu and Okinaka ( 9 ) . Furthermore, the1 centrifugates and washings from the precipitates were all pale yellow and contained considerable material precipitable with copper sulfate and trichloroacetic or phosphotungstic acids, to yield filtrates having strong reducing power. The substance contained in the water extracts was very soluble in hot water but precipitated on cooling; it was also soluble in acids and bases. Its solutions responded readily to the biuret, Adamkiewicz, and Millon reactions. Hence the calculation of the extent of reaction or adsorption on the basis of nitrogen determination is futile. The adsorption of caramel by casein was, however, quantitatively demonstrated in a direct manner.
Adsorption of Caramel by Casein Pure lactose contained in a dry flask was caramelized by immersion for a few minutes in an oil bath heated to 280" C. Sufficient water was added to give a brownish-yellow solution of pH 3.4, decreasing portions of which were added to 10-ml. samples of a 3 per cent sodium caseinate sol a t pH 7.0, and the mixtures were made up to 20 ml. with water. Standards were similarly prepared, using 10 ml. of water in place of the protein. After 1.5 hours each solution was treated with 5 ml. of 20 per cent trichloroacetic acid and filtered through dry paper into a dry flask, and the filtrate was compared with the corresponding standards in the colorimeter, ten readings being taken and averaged for each pair. The results are shown in Table X. When the logarithms of the relative amounts of caramel adsorbed are plotted against the logarithms of the fractions of caramel remaining in the corresponding solutions (Figure 4), the graph shows that the adsorption of caramel by sodium caseinate follows the familiar Freundlich isotherm, x / m = aCb the mass of adsorbing casein m remaining constant and consequently being disregarded. The extent of adsorption is governed by the initial pH of the sol, increasing with rising alkalinity, in agreement with the experimental fact that the color of the casein precipitated from autoclaved milk depends upon the pH of the unheated milk. Figure 5 shows €he results of a colorimetric experiment in which the p H of 20ml. portions of a 3 per cent sodium caseinate sol prepared from
80U 0
5,
,
1 ,
1 ' 2
; 7
I/ l
1.1. E x t r a p o l a t i o n of the curve indicates that adsorption is completeat an initial pHof 11.
1366
INDUSTRIAL AND ENGINEERING CHEMISTRY
Conclusion While the possible occurrence of a stoichiometric reaction between lactose and the amino groups of the proteins in heated milk has by no means been disproved, there can be little doubt that the development and properties of the brown substance in autoclaved milk are not in harmony with the assumption that i t is a casein-lactose condensate. The data presented in this paper do not permit the interpretation of the “interaction” of casein and lactose as a bifunctional reaction other than a complex, indefinite, and progressive formation of a melanoid. The exact parallelism between the reactions of caramel, prepared either through the agency of heat alone or heat in the presence of buffers, and those of the brown substance present in heated milk point to the conclusion that the origin and behavior of this coloration may be satisfactorily accounted for on the basis of the caramelization of lactose by the casein and the adsorption of the lactocaramel by the colloidal caseinates. The effect of the soluble phosphates and the other dissolved milk salts is probably negligible in this reaction, since their concentration is usually too low to induce an appreciable discoloration.
Literature Cited Assoc. Official Agr. Chem., Methods of Analysis, 3rd ed., 1930. Cianci, V., Boll. SOC. ital. hiol. sper., 8 , 1684-94 (1933). Clark, W. M., and Lubs, H. A., J. Bact., 2, 1-34 (1917). Englis. D. T.. and Dykins, F. A., IND. ENG.CHEM.,Anal. Ed., 3, 17-21 (1931). (5) Euler, H. von, and Brunius, E., Ber., 60, 992-9 (1927). (6) Garino-Canina, E., Notiz. chim. ind., 2, 133-6 (1927). (7) Hammarsten, O., Arch. ne‘erland. physiol., 2 , 658-63 (1918). (1) (2) (3) (4)
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(8) Hinton, C. L., and Macara, T.. Analyst, 49, 2-24 (1924). (9) Komatsu, Sh., and Okinaka, Ch., Bull. Chem. SOC.J a p a n , 1, 102-8, 151-7 (1926). (10) Kometiani, P. A., Milchw. Forsch., 12, 433-54 (1931). ( 1 1 ) Leeds, A. R . , J . A m . Chem. Soc., 13, 34-43 (1891). (12) McIlvaine, T. C., J. Biol. Chem., 49, 183-6 (1921). (13) Michaelis, L., Ihid., 87, 33-5 (1930). (14) Mitchell, H. H., and Eckstein, H . C., Ihid., 33, 373-5 (1918). (15) Neuberg, C., and Kobel, M., Biochem. Z.,174, 464-79 (1926). (16) Orla-Jensen and Plattner, Rev. g e n . Eait, 4, 361-8, 388-97, 41924 (1905). (17) Palitzsch, S., Biochem. Z., 70, 3 3 3 4 3 (1915). (18) Quagliariello, G . , and De Lucia, P., Arch. sci. biol. (Italy), 10, 237-44 (1927). (19) Ramsey, It. J., Tracy, P. H., and Ruehe, H. A,, J . Dairy Sci.. 7 (1910). .
.
(23) SBldner, F., Landwirt. Vers. Stat., 35, 351-436 (1888). (24) Sgrensen, S.P. L., Biochem. Z., 7 , 45-101 (1907). (25) Splittgerber, A . , 2. Untersuch. Nahr. u. Genussm., 24, 493-507 (1912). (26) Spoehr, H. .4.,and Wilbur, P. C., J . Biol. Chern., 69, 421-34 (1926). (27) Van Slyke, L. L., and Baker, J. C., Ibid., 35, 135 (1918). (28) Van Slyke, L. L., and Bosworth, A. W., N . Y. Agr. Exp. Sta., Tech. Bull. 37, 4-6 (1914). (29) Webb, B. H., J. Dairy Sci., 18, 81-96 (1935). (30) Webb, B. H., and Holm, G. E., Ibid., 13, 25-39 (1930). (31) Wright, N. C., Biochem. J.,18, 247-51 (1924). (32) Wroblewski, A., Oesterr. Chem.-Ztp., 1, 5-6 (1898). PRESENTED before t h e Division of Agricultural a n d Food Chemistry a t the 99th Meeting of t h e American Chemical Society, Cinoinnati, Ohio. Contribution from t h e Division of Agricultural Biochemistry, University of Minnesota. Paper 1802, Journal Series, Minnesota .4gricultural Experiment Station.
RECLAIMED RUBBER Application of the T-50 Test HENRY F. PALMER AND ROBERT H. CROSSLEY, Xylos Rubber Company, Akron,
T
HE T-50 test was first proposed by Gibbons, Gerke, and Tingey (4) in 1933 as a means of determining the state of cure of a rubber compound. Its use depends primarily upon the relation which exists between the combined sulfur of a rubber compound and its resistance to “freezing”. Test specimens of the cured compound are subjected to very low temperatures (-50” to -70’ C.) while elongated from 300 to 800 per cent of their original length. At this temperature the specimens become “racked”; i. e., they lose their elasticity. As the temperature is slowly raised at a constant rate, the specimens gradually retract. The temperature a t which they reach 50 per cent of the elongation at which they were frozen is known as the T-50 value. The effect of several compounding ingredients on the T-50 value of a rubber compound has been studied. Haslam and Klaman (6) in 1937 observed the effect of various zinc oxides and concluded that the T-50 test is a satisfactory tool for the study of curing rates. I n 1938 the effect of various accelerators with zinc oxides was studied by Gibbons, Gerke, and Cuthbertson (9) who found that in the absence of zinc oxide there is close correlation between T-50 values and the amount of combined sulfur for vulcanizates with different accelerators and times of cure, but that in the presence of zinc oxide this relation varies with the amount and kind of accelerator used.
Ohio
These data and data presented by Vila (16) in 1939 indicate that there is no general relation between T-50 and amount of combined sulfur for all compounds, but that a specific relation does exist between these properties for any one compound. Various laboratories (6, 12) now employ this test as a means of evaluating carbon black. Roberts (12) concluded that the test was useful in evaluating the curing rate of carbon blacks. The effect of some accelerators, antiscorch materials, and antioxidants on the T-50 value of rubber compounds was reported by Tuley (14) in 1937. He points out that the test may be used as an aid in compounding rubber goods to obtain definite states of cure at desired times and temperatures; hence, i t is useful in the design of new compounds as well as in factory control work. Coe (2) determined the temperature coefficient of vulcanization for reclaim compounds by means of the T-50 test, and reports that the values compare favorably with those obtained by free sulfur or modulus methods. The purpose of this work is to determine whether the T-50 test can be used for evaluating reclaimed rubber.
Experimental Method Apparatus manufactured by the Henry L. Scott Company was used throughout this work. It differs from that of t h e original investigators in that the open-pan type of racking vessel is
