T H E EFFECT OF PREHEATING ON THE DJSPERSITY OF CALCIUM CASEINATE I N SKIM MILK* BY J. B. NICHOLS,'
E. D. BAILEY,' G. E. HOLM,* G. R. GREEIVBANK,~ AND E. F. DEYSHER?
Casein is suspended in milk in the form of highly dispersed calcium caseinate which may be separated from the serum by means of a clay or collodion ultrafilter or by high-speed ~entrifuging.~ Attempts to estimate the degree of dispersion of calcium caseinate have been made by Svedberg, Wiegner, and B e ~ h h o l d . ~Svedberg and Fbhraeus, from preliminary ultracentrifugal studies made in 1924, concluded that the particles were of the order of magnitude of I O to 70 mpradius (mp= IO-7cm). Wiegner considers that the major proportion of the particles are amicrons and that the probable range in size is from 5 mp to IOO mp in diameter. He found the number of particles to be constant even when the milks had been heated or slight amounts of acid had been added. Bechhold considers, from ultrafiltration data on a series of proteins and inorganic colloids, that the calcium caseinate particles in milk are probably greater than 40 m p in diameter. The dispersity of calcium caseinate is of considerable practical as well as theoretical interest. One of the chief problems of the evaporated-milk industry, as well as of other branches of the dairy industry, is the develop ment of proper stability to heat. Little is known concerning the mechanism involved in coagulation, though the industrial methods used for its prevention are well established. I n the manufacture of evaporated milk, the milk is first heated to 95°C. for a short time prior to its concentration. This practice insures a greater stability of the finished product in the sterilization process. If temperatures of approximately 7ooC. are used, the tendency seems to be to decreme the ~ t a b i l i t y . ~ These heat treatments also seem to affect certain physical properties of the dispersion. For example, heating milk to 70" lowers the viscosity of the milk, whereas heating to higher temperatures increases the viscosity. These changes in viscosity are accompanied by changes in density (unpublished data). Though stabilization is affected largely by the electrolytes present * Paper presented at the Cincinnati (September 1930) meeting of the American Chemi-
cal Society. Contribution No. 47 from the Experimental Station of the E. I. du Pont de Nemours & Company and The Research Laboratories of the Bureau of Dairy Industry U. S. Department of Agriculture. Experimental Station, E. I. du Pont de Nemours & Company. * Bureau of Dairy Industry, U. S. Department of Agriculture. Friedenthal: Ber., 44,904 (191 I); Van Slyke and Bosworth: New York (Geneva) Agr. Exp. Sta. Tech. Bull. 39. ' Svedberg: Kolloid-Z., 51, I O (1930); Wiegner: Z. Nahr. Genussm., 27, 425 (1914); Bechhold: Z. physik. Chem., 60, 257 (1907). 5 Deysher, Webb, and Holm: J. Dairy Sci., 12, 80 (1929).
DIGPERSITT O F CALCIUM CASEINATE IN SKIM MILK
1305
The apparent density of the calcium caseinate was taken as the reciprocal of the partial specific volume. In order to calculate the partial specific volume from the pycnometric data, the concentration of the suspended solids, composed mainly of calcium caseinate, must be determined accurately. Skim milks are quite constant in the amount of lactalbumin and lactoglobulin they contain, this value being approximately 0.55’%.’ The serum
FJO-arY, ln miirmiCmni
FIG.2. “Weight optical” distribution curves of calcium caseinate from milk ( I ) Heated to 9j”C. Curve 1 4 7 % (2) Heated to 65°C. (3) Unheated I sq. = 2 0 5
TABLE I Skim Milk
Serum
Solids* 9.40% 6 . 22yo Proteins 3.39% 0.40% Density a t 3oOC. 1,0317 1.022 Apparent density of calcium caseinate = 1,504 Viscosity of serum a t 3 0 T . = 0.0116 Viscosity of 4:1 serum-skim milk a t 3 o o C . = 0.0120 * Solids content determined at 100°C and 2 5-27 in. vacuum
used contained 0.40%~ of protein, hence a portion of the soluble proteins was retained by the filter. Had no lactalbumin or globulin been retained by the filter the protein content of the serum would have been approximately 0.55% and the resulting solids content approximately 6.3770. Thus 3.03% (=9.40 - 6.37) of the solids of skim milk represents the content of suspended solids, mainly calcium caseinate. I n addition to calcium caseinate the fraction undoubtedly contains a small amount of highly dispersed fat, some leucocytes, and perhaps some colloidal phosphates. From other data on these samples the weight of the calcium caseinate has been calculated as 2.94%. From these considerations and for the purpose a t hand 3 . 0 3 7 ~has been chosen as the total amount of the suspended Rogers: “Fundamentals of Dairy Science,” p. 43 (1928).
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J. B ~ C H O L SE.D. , BAILEY, G. E. HOLM, G. R. GREENBANK, E.F.DEYSHER
phase. The value found for V, the partial specific volume, was 0.665, or 1.504 for the apparent density of the calcium caseinate. The three samples of skim milk ( ( I ) control, ( 2 ) sample heated to 65'C. for ten minutes, and (3) sample heated to 95" for ten minutes), which contained approximately 3% of solids, were diluted to one-fifth the original concentration with the milk serum prepared by ultrafiltration and centrifuged a t a speed of 4,000 r.p.m. Eastman Process Plates were used to record the effect of centrifuging. Since the illumination came from a Pointolite lamp, the effective radiation was the blue region of the spectrum. Exposures ten and fifteen seconds in length were made every ten minutes, each exposure yielding data on which a complete distribution could be calculated. Figure I represents a series of photographic exposures taken of the sedimenting calcium caseinate in the control sample. The exposure a t the left represents the sedimentation which has occurred in the first ten minutes, and the succeeding exposures represent the condition a t ten-minute intervals up to sixty minutes from the start. The weight-optical distribution curves obtained for the three samples are shown in Fig. 2 . The three distribution curves are similar enough to be considered almost identical within the limit of experimental error, although there is an indication of a slight shift in the mean radius to a smaller particle size as the temperature of forewarming is raised -from a mean size of 45mp for the control sample to a mean size of 41 m p for the sample heated to 95°C. This slight shift could hardly account for the rather large differences in stability observed for the samples when subjected to the coagulation test subsequent to forewarming.
Discussion The results indicate that the calcium caseinate particles in milk range in size from about IOO mp radius (zoo m p diameter) down to molecular sizes, which, according to recent work of Svedberg, Carpenter, and Carpenter' are of the order of 4 mp radius for casein. The mean radius is 40 to 50 m p (80 to go mp diameter). However, an appreciable portion (ca. I j% on the basis of its light absorption) of the suspended material had left the field of observation between the time of the original exposure of the photographic plate and the second exposure ten minutes later. This fraction undoubtedly consists of small amounts of fat, leucocytes, some colloidal calcium phosphate and perhaps some large aggregates of calcium caseinate. I n a polydisperse system having the range of sizes of the calcium caseinate system under consideration, the light absorption of particles increases rapidly with increasing radius in accordance with the Rayleigh scattering of small particles; therefore, the curves presented in Fig. 2 do not represent the true relation of weight of material to radius but the so-called weighhptical relation, the term "weight-optical "referring to an apparent concentration which is the product of the absorption constant IC of the given radius by the concentration c of material of that radius. If the distribution curves had been 1
Svedberg, Carpenter, and Carpenter: J. Am. Chem. SOC.,52,
241,701 (1930).
DISPERSITY OF CALCIUM CASEINATE IN SKIM MILK
I307
markedly d s e r e n t this variation of light absorption with particle size would have rendered difficult the comparison of the different samples. However, since the curves are so nearly identical in spite of the large variation of light absorption with particle size, we may safely conclude that the particle-size distribution of calcium caseinate is very little affected by forewarming up to 95OC. for a short time. Prolonged heating of milk results in flocculation; thus it would be entirely reasonable to suppose that the particles would increase progressively in size with the time and temperature of heating.' Although this may occur in the later stages, the results presented indicate that particle-size changes are not of primary importance in the phenomenon of the stabilization of a milk to heat by forewarming, that is, unless the ad hoc assumption is made that the small shifts in the distribution curve to smaller particle sizes are capable of producing the large changes in stability observed. Hydrolytic changes may be responsible for the stabilization.
Summary I. The weight-optical particle-size distribution curves have been determined for samples of the calcium caseinate of skim milk subjected to different temperatures of preheating. 2. The majority of the material is less than zoo mp in diameter with a mean size of about go mp. 3. A small amount of coarser material, probably colloidal calcium phosphate and large aggregates of calcium caseinate, is also present. 4. Preheating up to 9j°C. has little effect on the distribution curve of particle size.
Wilmington, Delaware, U'ashington. n. c. Svedberg, Carpenter, and Carpenter: loc. cit. p. 708, have observed an increase in size of the casein molecule at 40%; if such a low-temperature chan e occurs in the calcium caseinate suspension in skim milk, the control sample would unfergo the same change as the heated samples because the milk was warmed to 40°C. previous to skimming.
