Dec.,
1920
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERIlVG C H E M I S T R Y
There remains, of course, the fact that a well-standardized titrimetric procedure will indicate differences of not very clear theoretical significance, but nevertheless capable of aiding the judgment. Such procedures indicate in a very rough way that careless handling of the curd by methods which offer no control over the acid used in precipitating and washing may result in the inclusion of considerable quantities of free acid. On the other hand, the method for estimating the so-called free acidity which was included in the specifications was quite inadequate. It was directed that a given weight of casein be dissolved in an excess of alkali, and that the unneutralized acid be titrated with phenolphthalein as an indicator. When analyzed, this procedure consisted only in titrating to an arbitrary pH, a s indicated by phenolphthalein, a mixture of amphoteric protein, occluded salts, and the products of alkali hydrolysis of the protein. As was to be expected, it gave no consistent results, partly because of the difficulty of titrating to an arbitrary and insignificant pH and partly because of the hydrolysis of the casein. Some careful studies were made with the hydrogen electrode as indicator, and by extrapolating back t o zero time of contact between alkali and casein i t was found that a representative sample of grain-curd casein agreed very closely with the “free acid” in casein “nach Hammarsten.” Unfortunately the details of these experiments were lost in a fire.
1171
T h e 0 . 2 M borax solution at 30’ C. was added t o exactly I O g. of each casein and thoroughly stirred. This was permitted t o stand, with frequent stirring, for 30 min., when 7 5 cc. of distilled water a t 30° were added t o each mixture, and t h e mixture stirred until smooth. T h e solutions were then allowed t o stand in a constant temperature water bath a t 30’ for I hr. before t h e viscosity was determined in t h e MacMichael apparatus. The hydrogen ion was determined, when possible, with t h e hydrogen electrode. I t has been observed t h a t after definite time intervals t h e viscosity of caseins t h a t have been subjected t o pasteurizing temperature is always greater t h a n t h a t of caseins prepared a t lower temperatures. If t h e solutions of caseins are allowed t o s t a n d a great length of time (24 hrs. or more) t h e viscosities tend t o approach in value. I n cases of caseins prepared under excessive temperatures, t h e difference in viscosity was so marked t h a t careful control of reaction and time was unnecessary.
TABLEI-VISCOSITY AND PHYSICAL APPEARANCE OF SOMECOMMERCIAL AT 250 c. CASEINSIN BORAXAS SOLVENTS, Ash Viscosity Appearance Laboratory Per Angular of Solution No. Type cent Degrees S o h . 102-A Cooked curd 4.27 430.0 Flakes VI-C Argentine 3.69 47.5 8 . 8 Flakes and milky STUDIES ON TECHNICAL CASEIN. IV-STANDARDIZAA-133(1) Graincurd 0.64 61.0 9 . 0 Translucent D-14 Commercial TION OF THE BORAX SOLUBILITY TEST FOR graincurd 2.55 50 9 9 . 0 Translucent 97-D I‘-aturalsour 1.49 52.2 8 . 9 Smooth; slightly milky COMMERCIAL CASEINS1 104-B Commercial Flakes sulfuric 5.12 276.0 By Harper F. Zoller Flakes 103-B Cookedcurd 6 0 . 3 306.0 . RESEARCH LABORATORIES OF THE DAIRYDIVISION,BUR$AUOF ANIMAL M-10 “h’ach Hammarsten” 1.07 69.8 9 . 0 Clear OF AGRICULTURE, INDUSTRY, u. s. DEPARTMENT WASHINGTON, D . c. I n all cases, i n t h e preparation of solutions of caseins Received June 28, 1920
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T h e casein solubility test as conducted i n general practice has been as follows: “ T o 50 g. of casein (ground t o pass a 20-mesh sieve) are added 300 cc. of water containing 7 . 5 g. of borax. T h e mixture is stirred thoroughly and is immediately set i n a water bath controlled a t a temperature of 65’ C. With continuous stirring t h e casein should be completely dissolved in I O min.” This test2 is largely used t o determine whether casein is suitable for t h e coating of paper. T h e fact t h a t a borax solution buffers t h e H+-ion concentration i n a zone (roughly from 9 .o t o IO.o p H ) where there is scarcely any alkaline hydrolysis of casein suggests this as a valuable solvent in which caseins may be differentiated from t h e standpoint of physical constitution. From a s t u d y of t h e viscosity of a variety of caseins in borax solutions it was realized t h a t there was no parallelism between t h e viscosity and t h e contents of ash or acid. Sutermeister3 has since published results of a somewhat different nature, b u t materially confirms t h e above findings. F r o m a more careful s t u d y of t h e viscosity of certain caseins t h e author finds t h a t there is a correlation between t h e viscosity of caseins and certain physical properties (hydrophilic) which are a result of t h e heat t r e a t ment of t h e casein before or after separation from milk:. Table I includes t h e observed viscosity of some commercial caseins i n bo,rax as solvent. : Pubjished by permission of the Secretary of Agriculture 2 3
U. S. Department of Agriculture, Bulletin 661 (1918). Paper, 24 (1919), 217.
i n alkali solvents, temperatures above 30’ C. were avoided; t h e viscosities were usually read a t 25’ C. It should be mentioned i n this connection t h a t t h e failure t o apply successfully t h e viscosimeter t o t h e analysis of commercial casein i n t h e past may be a t tributed t o t h e high temperatures and uncontrolled conditions employed. A glance a t Table I will show t h a t in general these caseins yielding high viscosities contained high ash, whereas not all high-ash caseins gave high viscosities. High-ash caseins which have been subjected t o high temperatures always dissolve slowly a t low solution temperatures, and, as a rule, are t h e caseins which are unpopular commercially. From t h e viscosity curve (Fig. I ) of t h e best quality of casein (“nach Hammarsten”) on borax, i t is found t h a t t h e viscosity of casein-borax solutions reaches a maximum at a n Hf-ion concentration of about p H 8.15, or when 9 . j g. of anhydrous and ash-free casein are dissolved in 150 cc. of solution containing 40 cc. of 0.2 M sodium borate. T h e ascent of t h e curve is steep on approaching this point from t h e acid side. A complete solution is obtained with 9 . 5 g. of casein i n ~ j cc. o of solution containing I O cc. of 0 . 2 M borax, b u t t h e viscosity is much less. Upon t h e alkaline side of t h e maximum viscosity t h e drop is quite as precipitous, and a t a p H of 8 . g t o 9 . I t h e viscosity is practically constant, even though t h e quantity of solvent is increased in t h e order of 60 t o I O O cc. of o. 2 M borax t o 9 . 5 g. of casein. The concentration of casein was t h e same in each total volume of solution, t h e concentration of borax being t h e only variant.
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
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No.
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t h e writer will present d a t a t o show t h a t t h e hydrophylic nature of casein is sensitive t o pasteurizing temperatures. Accordingly, a working temperature of 30' C. is advocated. This is chosen also for another reason, Complex borax solutions change internally a t 3 5 . j o C.l Above this temperature the pentahydrate may exist and deposit crystals from such a supersaturated solution; below this point the decahydrate separates. The latter is ordinary borax. A 0 . 2 M solution of borax is unsaturated a t 30' C., but deposits crystals a t 2 5 " C. upon standing undisturbed. REVISED METHODS
F I G . 1-vlSCOSITY C U R V E OF CASEIN-BORAX SOLUTIONS. CONCENTRATION O F ANHYDROUS C A S E I N 6.5 P E R CENT. HYDROGEN-ELECTRODE VALUES
EXPRESSED IN pH AREGIVENFOR SEVERAL POINTSON
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The casein is ground t o pass a 40-mesh sieve; ~j g. of the casein are measured into a zjo-cc. beaker; IOO cc. of 0 . 2 M borax a t 30' C. ( 7 6 . 3 2 g. of Na2B407.-
I n choosing the correct reaction for the casein/BO borax solubility test i t is necessary t o avoid the region / I of greatest viscosity changes, since contaminating substances in the commercial casein naturally influence the reaction of t h e mix, and when this occurs in the aforementioned region erroneous interpretations would follow. For example, we might have a casein from heated milk checking t h e viscosity of a normal casein possessing a medium content of acid salts as a result of the natural-sour process of manufacture. This has also been t h e experience in several instances in applying t h e commercial solubility test mentioned above. Naturally, t h e zone in which least viscosity change occurs is in t h a t zone of maximum buffer action of the solution-beyond 9.0. The p H of a 0.2 M sodium biborate solution is approximately 9.13 (hydrogen electrode); the curve in Fig. I shows t h a t in this region t h e viscosity is constant. This is the zone t o which t h e reaction of t h e solutions in t h e improved method is adjusted. T h e concentration of casein chosen for the improved casein-borax test is about 12 per cent of anhydrous 1 5 per cent casein1 by weight. Chick and Martin2 showed t h a t t h e viscosity of casein in sodium hydroxide rapidly increased when t h e concentration approached I O per cent. This was verified with "nach Hammarsten" by the writer, and also with grain curd (C. P,). T h e curve i n Fig. 2 illustrates the point under discussion. The measurements were made with a MacMichael "universal" viscosimeter using torsion wire No. 0 0 0 . Each solution was adjusted to the same reaction before measurement (p H 9 . o), and temperaFIG.2-VISCOSITY-CONCENTRATION C U R V E O F C A S E I N IN NaOH. tures above 30" C. were avoided in preparing t h e solupH MAINTAINED CONSTANT AT 9.0 tions. The viscosity became unmeasurable a t concentrations of 14 per cent. Those caseins which tend 1oHz0 diluted t o I liter) are added with vigorous toward gel formation exhibit i t strongly a t this concentration, while those t h a t remain fluid a t this con- stirring. This is allowed t o stand for 30 min., with thorough stirring a t intervals of 5 min. During t h e centration still flow rather freely. A second important change from t h e ordinary pro- first 5 min. t h e mixture should be stirred rather frecedure is t o use much lower temperatures in t h e test. quently. A casein of known purity and conduct A t temperatures around 40' C. and above, casein should be used as control until thorough familiarity solutions change physically. I n a succeeding paper with t h e method is gained. Usually t h e character of t h e casein shows up during t h e first I O min., b u t 1 The method calls for 15 g. casein in 100 cc. borate solution. The 30 min. is advised for safety. Longer periods are uncommercial caseins contain about 10 per cent moisture, 4 to 5 pe: cent ash, and some fat and sugar; perhaps 20 per cent of the commercial sample will satisfactory because of difficulty i n interpretation. consist of material not casein. a Kolloid-Z., 11 (1902), 102.
1 Van't
Hoffand Blasdale, W e b . A k a d . Wiss Berlin, 1088 (1905).
