Isolation, Separation, and Characterization of Beer Proteins -.
B. L, SCALLET, J. J. STANSBREY, F. W. SMALL, JR., AND P. F. GIBBSl Anheuser-Busch, Inc., S t . Louis, Mo.
T
HE “proteins” of beer are actually, for the most part,
was introduced into t h e bottom and overflowed through the spout near the top. Distilled water removed color as me11 as low molecular weight material from beer. T a p water (pH 9.5) removed little color. Beer contains about 50 mg. of solids per ml. Dialysis reduced the solids to 11.9 mg. per ml. in 24 hours and to 2.9 mg. per ml. in 240 hours in this apparatus (at 10’ C.). The protein wae recovered from the material in the dialyzing bags by saturating with ammonium sulfate, redissolving in a small amount of water, redialyzing t o remove ammonium sulfate, and freeze-drying. Beer proteins isolated in this manner ranged from 5 to 10% nitrogen. The method used for isolation of larger quantities of beer proteins was saturation of the cold beer with ammonium sulfate (Mallinckrodt black label). This was accomplished on a laboratory scale ( 5 to 10 liters) in battery jars or on a larger scale (40 liters up) in stainless steel tanks. The latter method will be described. Beer (44 liters) was transferred into an open stainless steel tank. Ammonium sulfate (41.3 pounds) was added slomly, and the mixture stirred constantly. This quantity of ammonium sulfate was just sufficient to saturate the beer. When protein precipitation was complete, the contents of the tank 15 ere transferred to an empty 16-gallon stainless steel barrel by means of vacuum. The mixture was then forced through a small Hercules pressure leaf filter, which consisted of a glass cylinder containing three stainless steel filter plates of total area 0.5 square feet. The plates were precoated with a mixture consisting of 7.5 grams of the finest chrysolite asbestos, 7.5 grams of the coarsest chrysolite asbestos, and 50 grams of Johns-Manville Hyflo FilterCel. The carbon dioxide pressure required for filtration gradually increased from 5 to 20 pounds per square inch during the filtration. The filtrate was recycled until it was perfectly clear. At the end of the filtration the plates were removed and the protein was stripped carefully from the precoat, after which it mas extracted three times each with water, 85% ethanol, and 0.2% sodium hydroxide in the 10” C. room. WATER-9o L U B L E PROTEINS. The combined aqueous extracts Figure 1. Refractive Index-Concentration were dialyzed free of ammonium Curves for Whole Beer Proteins sulfate, and were then ready for 0 In distilled water fractionation. K h e n 7% sodium 0 In standard succinate buffer before dialysis 0 In succinate buffer after dialysis against succinate chloride x a s used as the exbuffer
degradation products formed during the brewing process from the original proteins in the raw materials. About 3% of the nitrogen-containing components (0.0270 of whole beer) consists of fragments large enough to be retained by cellophane. This s~nallquantity of material, however, has a profound effect on the stability of beer. It is thus of interest to make a detailed study of these protein fragments. Most of the work on brewing proteins has been done in Europe. During the last 15 years, application of modern instruments has served to clarify somewhat the chemistry of barley and malt proteins. Quensel (8) used the ultracentrifuge t o separate barley globulin into four components of molecular weights 26,000, 100,000, 166,000, and 300,000. Lundin (7) and Sandegren (10) extended these studies t o the albumins and globulins of barley and malt. Sandegren has also isolated whole beer proteins in small quantities by dialysis, and studied them by means of polarography. St. Johnston (9) separated wort proteins into four fractions having different properties. Biserte and Scriban ( 1 ) made an electrophoretic study of barley proteins and their modification during germination. Bishop ( 2 ) fractionated wort and beer proteins by successive additions of phosphotungstic acid. The proteins were, of course. denatured by this reagent. Hartong, Mastenbroek, and Mendlik (6) made a very brief electrophoretic study of a protein isolated from beer by chilling and centrifuging. They concluded that this material was homogeneous. No one has reported the isolation and separation of undenatured beer proteins in substantial quantities, probably because of the low concentration of these materials in beer. The present work represents a direct attack on this problem. EXPERIMENTAL ISOL.4TION O F BEER PROTEINS. Isolation by dialysis was done in an apparatus consisting of three borosilicate tubes in a wooden frame. The tubes had a volume of 150 ml. each. They were constructed with a spout on one side near the top, and were tapered to a small opening at the bottom. Inside diameter was 22 mm. (to accommodate 19-mm. cellophane tubing). The cellophane bags were spaced within the tubes by means of small projections pushed into the glass. Dialyzing liquid 1
Present address, Great Lakes Carbon
Go., S t . Louis, Mo.
1016
May 1953
I N D U S T R I A L A N D E N G I N E E R I NG
tractant, dialysis led t o a clear solution, indicating that, no globulins were present. ALCOHOL-SOLUBLE PROTEINS.After filtration of the combined alcohol extracts through the Hercules filter, the clear brown solution was concentrated by evaporation through a cellophane membrane. During this operation precipitation occurred because of the change in alcohol concentration. The residue was
Ascending+
+Descending
Figure 2.
Electrophoresis Patterns for Whole Beer Protein
c H E M I S-TR Y
1017
ethanol concentration and a further precipitate, fraction IV, settled out. T h e remaining solution contained fraction V, which was obtained in dry form by dialysis followed by freeze-drying. NITROGENAND SULFUR ANALYSES. These were made by micromethods (Dumas and dry combustion or Carius), by Clark Microanalytical Laboratories, Urbana, 111. ELECTROPHORESIS. A Tiselius-Klett apparatus was used. Most of the fractions were run in standard succinate buffer, pH. 5.6, ionic strength 0.1, at a current of 18 ma. The time was usually 300 minutes, and the cylindrical lens was used in making photographs. Exposures ranged from 10 t o 20 seconds. An AH4 lamp was usually used with Kodak Contrast Process Ortho film. For highly colored solutions, a tungsten filament lamp was used with Kodak Process Pan film or with a red Corning Pyrex filter (No. 2418) and infrared film. Rough concentration measurements were made by means of refractive index readings. Figure 1 shows the working curves used. More nearly exact concentrations were determined by drying and weighing. ULTRACENTRIFUGE. A Specialized Instruments Corp. ultracentrifuge, Model E, was used. Analytical runs were made a t 59,780 r.p.m. at room temperature. Preparative runs were usually made at 20,000 t o 40,000 r.p.m. a t 0" C. Rotors A and D were used. Exposures were 30 to 45 seconds on Kodak Process Pan film. A traveling microscope (Gaertner Scientific Co.) was used t o measure sedimentation distances.
A. Chloroacetate buffer, pH 2.4 B. Succinate buffer, pH 5.6 C. Verona1 buffer, pH 8.5 D. Carbonate buffer, pH 9.6 Ionic strength, 0.10; current, 18 ma.; time, 300 min.; concn., about 1%
"
w
recovered by centrifugation, washed with acetone and ether, and vacuum dried. The remaining alcohol-soluble material was recovered by adding absolute alcohol (3 volumes), and the white creamy precipitate was removed by centrifugation, washed with acetone and ether, and vacuum dried. The supernatant solution still contained nitrogen and was concentrated on the steam bath, after which a third fraction was obtained on addition of absolute alcohol. ALKALI-SOLUBLE PROTEINS.The residue after alcohol extraction was washed with water and extracted with 0.2% sodium hydroxide solution. After filtration, the extracts were acidified to p H 3.9 with dilute hydrochloric acid, a t which point the protein precipitated. It was recovered by centrifugation, dissolved in dilute sodium hydroxide, and brought t o p H 7 with hydrochloric acid. The solution was dialyzed for 2 days t o remove sodium chloride and was then dried in the freeze-dryer. The protein remained in solution for several days after dialysis. FRACTIONATION OF WATER-SOLUBLE PROTEINS. The procedure finally adopted was as follows:
A beer protein water extract, volume 1480 ml., protein content 11.8 grams (N x 6.25), p H 4.8, ionic strength 0.02 in acetate buffer, was brought t o 16% ethanol concentration by volume. Fraction I was recovered by ultracentrifuging at 20,000 r.p.m. for 30 minutes. The supernatant liquid (1705 ml.) was brought t o p H 3.9 by addition of glacial acetic acid. The ethanol concentration was then raised t o 45% and the temperature allowed to rise t o 10" C. A heavy precipitate (fraction 11) settled out and was removed a t 2000 r.p.m. The supernatant solution was cooled t o 0' C., a t which point another precipitate appeared. This was removed in the ultracentrifuge a t 20,000 r.p.m. at 0' C. (fraction IIA). The supernatant solution (2518 ml.) was brought t o GO% alcohol concentration. Fraction I11 wa8 removed a t 2000 r.p.m. The supernatant liquid from fraction I11 was brought to 80%
Ammonium Sulfate Concn., GJ100 M1. Solution
Figure 3. Precipitation Curves for Ammonium Sulfate Fractionation of Water-Soluble Beer Proteins
0 Originally in distilled water
0 In 7% sodium chloride solution
OSMOTIC PRESSURE MEASUREMENTS.The Fuoss-Mead osmomwith certain modifications, was used in all osmotic preseter (6), sure measurements. The membranes were undried cellophane supplied by the Sylvania Cellophane Corp. Molecular weights of the fractions were measured in 0.2 M sodium chloride solution at various concentrations. It was found necessary t o change membranes for each protein fraction and t o start with the lowest concentration of each fraction. Equilibrium was approached from both sides, and each ascending and descending run required about 25 minutes with the 1-mm. diameter capillaries employed.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1018
Heights were read by cathetometer t o the nearest 0.1 mm. Corrections for surface tension were made in each case by immersing the capillary in the protein solution contained in a small beaker. The capillary rise was then noted. The capillary rise of the buffer was measured in the same way. RESULTS AND DISCUSSION
p H 5.6). Much of the work discussed in this paper was concerned with fractionation of these water-soluble components. Ammonium sulfate fractionation was tried first. Successive additions of ammonium sulfate resulted in gradual precipitation, but the precipitation curve (Figure 3) showed no distinct plateaus. Indications were t h a t there might be slight breaks in the curve at 15.3 and 18.3 grams ammonium sulfate per 100 ml. Prelim-
Isolation of Beer Proteins. This vias accomplished by two methods. The first, dialysis, was successful but cumbersome and time consuming, especially when relatively large quantities of proteins were required. I n t h e second method, proteins and high molecular weight carbohydrate materials were precipitated by saturation of beer with ammonium sulfate. After washing of the precipitated material with saturated ammonium sulfate solution, t h e proteins were separated from the carbohydrate by successive peptization in water, 85y0 ethanol, and 0.2% sodium hydroxide in the cold. Most of the carbohydrate material was insoluble in these reagents, but a small amount of i t was dissolved with the water-soluble proteins. The residual carbohydrate material was soluble in water at 50" C. The water-soluble proteins represented the largest part of the protein material. Electrophoresis showed this portion to consist of at least four components (Figure 2); the three largest peaks were labeled alpha, beta, and gamma (reading from left t o right on the ascending pattern in standard succinate buffer,
I
SODIUM
I
'ION
A. Alpha-rich fraction B. Beta-rich fraction C. Gamma-rich fraction Succinate buffer, pH 5.6; ionic strength, 0.10; current, 18 ma.; time, 300 min.; concn., about 1 %
SOLUTION
(DISCARD)
4
CAR84HYPRATE
,
I I SOLUTION JAR
I ,
inary fractionations at these levels indicated some concentration of gamma a t 15.3, alpha a t 18.3, and beta at 32.5 grams per 100 ml. The fractionation scheme of Figure 4 was then devised to obtain some quantities of fractions. However, fractionation according to this scheme was only partially successful. Electrophoresis patterns of the final fractions obtained are shown in Figure 5. The alpha concentrate contained much beta and gamma, and the gamma concentrate turned out to be largely alpha. Purity of the beta fraction was fairly good. Electrophoretic fractionation in the large Tieselius cell (150 ml.) resulted in three fractions, each of which appeared to be as complex as the starting material. The method was so tedious that it was discarded as a major source of protein fractions. However, it indicated that a t least ten components were present. An ethanol precipitation method, adapted from the method devised by Cohn and coworkers ( 3 ) for blood plasma, proved to
1
SOLUTION
I
,B
Figure 5. Electrophoresis Patterns for Fractions Isolated by .4mmonium Sulfate Precipitation
BEER
CHLORIDE
Vol. 45, No. 5
OF OPTIMUM pH TABLE I. DETERMINATIOX OF BEERPROTEIN
FOR
PRECIPITATION
(Fraction I)
PH 3.8
4.9
6.3 7.5 9.5
L
GAMMA CONCENTRATE
L
-.
1
(.
ALPHA CONCENTRATE
Figure 4. Flow Sheet for Fractionation of Salt-Soluble Beer Proteins with Ammonium Sulfate Ammonium sulfate concentrations, grams/100 ml.
4.0 4.5 4.8 5.1
5.5 5.5
Buffer 25-MI. Aliquots Acetate Acetate Phosphate Phosphate Carbonate 5-N1. Aliquots Acetate Acetate Acetate Acetate Acetate Phosphate
Volume of Ethanol, 1\11. 5.1
3.9 8.1 7.5
11.0
0.69 0.64 0.63 0.66 0.75 0.94
INDUSTRIAL AND ENGINEERING CHEMISTRY
May 1953
be the most satisfactory method of fractionation. The precipitation curve, showing two definite plateaus at 16 and 36 t o 50% alcohol concentrations, is given in Figure 6. The p H optima for precipitation of t h e first two fractions at these alcohol levels were determined b y buffering aliquots of whole beer protein solution at various p H values, Absolute FRACTION 2 .0
B
5
m
1*5
j 1
'E4 P .m
k
1.0
.-I
1019
tubing suspended in front of a fan. When the volume had decreased t o one twelfth of the original volume, t h e clear, sirupy concentrate was dialyzed. During dialysis proteins precipitated in a heavy floc. T h e material remaining in solution was predominantly carbohydrate, which could be fractionated by the scheme used for proteins. Table I1 gives the quantities of carbohydrate fractions isolated from beer. They represent a considerable proportion of t h e beer. No further information on t h e carbohydrate fractions is available at the present time. Properties of the Protein Fractions. Table I11 lists some of the physical properties of the protein fractions. They varied from snow white t o dark brown in color; some were dull and amorphous; some tended toward a shiny, crystalline appearance; some dissolved only with difficulty, while others dissolved almost instantly. Some were viscous in solution, others were not. Table I V gives information on the relative quantities of the fractions present in beer, along with nitrogen and sulfur contents. The quantities of the fractions as well as the nitrogen and sulfur contents vary considerably for different beers. Values in Table IV represent a typical set of fractions. The alcohol-soluble protein (fraction A) is normally present in the smallest quantity, while water-soluble fraction V is usually
E
E a f
0.5
: FRACTION
0
I
20
LO
Ethanol Concentration, Vol. Yo
Figure 6. tionation
Precipitation Curve for Ethanol Fracof Water-Soluble Beer Proteins in Acetate Buffer Original pH 4.8; ionic strength
0.02
I
ethanol was added from a buret to each aliquot until all solutions were equally cloudy. The buffer requiring the smallest amount of ethanol for precipitation was then used in the fractionation scheme. Table I gives some typical results. Fractions I11 and I V were taken when convenient quantities of material had precipitated, and fraction V included all material remaining in solution at 80 % alcohol concentration. T o summarize, the six fractions were obtained under the following conditions:
h4
Fraction I. Ethanol concentration, 16% by volume; p H 4.8, sodium acetate-acetic acid buffer, ionic strength 0.02; temperature, 0" C.; protein concentration, 1% Fraction 11. Ethanol concentration, 45%; p H 3.9; sodium acetate-acetic acid buffer, ionic strength 0.02; temperature, 10" c. Fraction IIA. Cooling supernatant liquid from fraction I1 t o 0" c. Fraction 111. Increasing the ethanol concentration of the supernatant liquid from fraction I I A t o 60% Fraction IV. Increasing the ethanol concentration of the supernatant liquid from fraction I11 t o 80% Fraction V. Dialyzing the supernatant liquid from fraction IV against water These six fractions, along with the alcohol-soluble fraction (called fraction A ) and the alkali-soluble fraction (called fraction B) represented the major portion of the high molecular weight proteins of beer. Although the fractions were not homogeneous, each had characteristic and distinct properties. Fractionation of Carbohydrates. During the course of the work, it was found t h a t t h e proteins could be altered by evaporation of water and alcohol from beer contained in cellophane
Figure 7. Electrophoresis Patterns of Beer Protein Fractions Obtained by Ethanol Precipitation Fractions I-V. Succinate buffer, pH 5.6; ionic strength, 0.10; current, 18 ma.; time, 300 min. ; concn., about 1 % Fraction A. 60% ethanol; acetate buffer,pH 5.4; ionic strength, 0.16; current, 0.75 ma.; time, 1325 min.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1020
I1
I
111
IIA
V
IV
Vol. 45, No. 5
present in the largest quantity. The nitrogen contents of the fractions vary considerably, since some carbohydrate material and perhaps other substances are carried along with the proteins. Fraction I I A in particular is predominantly carbohydrate; I1 and I11 are high in carbohydrate and low in nitrogen content. The sulfur contents also vary considerably (from 0 in fraction A t o 1.4in fraction IV). Electrophoresis patterns of the various fractions are shown in Figure 7. Fraction I was a mixture of several components (three show up clearly, and there are slight indications of one or two more). The mobility of the small center peak of fraction I was the same as that of the tall spike of fraction 11. At least three components show in fraction 11, but the tall spike predominates. Fraction I I A shows the same components as fraction 11, but the tall spike is even more prominent. Fraction I I A was the most nearly homogeneous one obtained. Fraction I11 was also relatively homogeneous, but the tall peak had a mobility different from that of the main peak of fractions I1 and IIA4. Fraction IV had a reIatively large secondary peak and a small third peak. The mobility of the main peak was different from the mobilities of the main peaks of fractions 11, IIA, and 111. Fraction V, as would be expected from its method of isolation, consisted of a mixture of a number of components. Four or five individuals show up, all with mobilities different from those of the components in the preceding fractions. Fraction A, run in 607, ethanol, appeared to contain reIatively extensively degraded material.
Figure 8. Ultracentrifuge Patterns for WaterSoluble Beer Protein Fractions 0.2 M s o d i u m chloride at 59,780 r.p.m.; t i m e , 208-224 min.
FRACTIONS IN BEER TABLE 11. CARBOHYDRATE Fraction I
Concentration, % '
I1
IIA
I11 IV
0.004 0.02 0.004 0.32 0.14
0.C
d u TABLE 111. PHYSICAL PROPERTIES OF BEERPROTEIN FRACTIONS \ Fraction
Ethanol Conon. for Pptn.. %
Color
I
16
Light tan
I1
45
White
IIA
45
White
60
White Light t a n Tan Dark brown Medium brown
I11 IV
.. ..b
V
"a
B
.
A
. C
Appearance Fluffy, amorphous Fluffy, definite sheen Fluffy, amorphous Amorphous Amorphous Crystalline sheen Horny Fluffy amorphoh
Rate of Soln. Fairly rapid
e1 2 0.L e CI .*0
-.
0.2
Slow
Slow Slow Rapid Fairly rapid Rapid
C.6
6 I
0
Rapid
Soluble in 80% ethanol, obtained by dialysis and freeze-drying. a Water-insoluble 85% ethanol-soluble. c Water- and ethAno1-insoluble, 0.2 72 sodium hydroxide-soluble.
a
Figure 9.
I
I
5 10 15 Concentration, G . Solute/Kg. Soln.
20
Osmotic Pressure-Protein Concentration Curves for Fraction I11
T w o separate r u n s Fitted statistically according to e q u a t i o n ?r = qc D r a w n with intercept, q, and slope, r (bottom)
+
rc2
(top)
TABLE IV. ANALYSISOF BEERPROTEIN FRACTIONS Concn. in Beer, Fraction
%
Xitrogen Content,
%
Sulfur Content,
%
Ultracentrifuge patterns of the fractions are shown in Figure 8. The patterns for fractions I and V are broad and smooth, indicating polydispersity. The pattern for fraction I1 shows a main peak and two smaller peaks, while the pattern for fraction I I A shows a relatively large peak accompanied by a small secondary peak. Fractions I11 and I V also show secondary peaks, but the broad outlines of the main peaks indicate less homogeneity than is the case for fractions I1 and IIA,
INDUSTRIAL AND ENGINEERING CHEMISTRY
May 1953
1021
CONSTANTS AND MOLECULAR TABLE V. SEDIMENTATION WEIGHTSOF BEERPROTEIN FRACTIONS Fraction
Sedimentation Constanta
Rlolecular Weight
1.84
Corrected t o water a t 20' C. By osmotic pressure measurement; plus and minus values represent 95% Confidence limits. a b
d
B
G
Figure 10.
Electrophoresis Patterns for Whole Beer Proteins
E. 1.25 grams/lOO ml. A. 0.25 gram/100 ml. F. 1.75 gramdl00 ml. B. 0.50 gram/100 ml. G. 2.00 grams/100 ml. C. 0.75 eram/l00 ml. D. 1.00 sam/100 ml. Succinate buffer, pH 5.61 ionic strength, 0.10; current, 18 ma.; time, 300 d n . I
Molecular weights vary from 34,000 in fraction I to 73,000 in fraction IIA. Although the osmotic pressure measurements were not so precise as could be desired, they gave results indicating the general size ranges for the fractions. Crystalline bovine serum albumin by this technique had a molecular weight of 64,000 f 13,000 (95y0 confidence limits). This range brackets the probable value of 69,000 for bovine serum albumin. However, there is still considerable uncertainty about the osmotic pressure results, since the sedimentation constants are very low for molecules of the size indicated. Sedimentation constants in the neighborhood of 1 are ordinarily found for protein molecules in the 10,000 t o 20,000 weight range. However, the considerable carbohydrate content of some of the fractions may have an effect. Further work.wil1 be necessary t o explain the discrepancy. There appeared to be some correlation between sedimentation constants and osmotic pressure molecular weights for fractions I, 11, and IIA. The water-soluble fractions appeared to separate into two distinct groups. The first, containing fractions I, 11, and IIA, increased in molecular weight from 34,000 to 73,000. The second, containing I11 and IV, had molecular weights of 38,000 and 50,000. During the fractionation there was a distinct gap between fractions I I A and 111. No material precipitated between alcohol concentrations of 36 and 50% (Figure 6). Color of the solution disappeared after precipitation of the first group. Fraction I, in particular, was gummy, dark in color, slow t o precipitate, and difficult t o centrifuge. The second group was entirely different in character; precipitates were granular and fell readily to the bottom of the container. I1
IIA
I11
IV
V
The ultracentrifuge patterns, in general, show t h e same polydispersity t h a t the electrophoresis patterns show, b u t with less sensitivity. Sedimentation constants for the main peaks are given in Table V, along with molecular weights obtained from osmotic pressure measurements. In calculating molecular weights, the osmotic pressure was plotted against protein concentration. A curve, x = qc rcs, where x is osmotic pressure in cm. of solution, c is concentration in grams protein per liter of solution, and q and r are constants, was fitted statistically t o the points obtained. The slope, q, of the curve a t zero concentration was related to
+
molecular weight at zero concentration, since = q
+ rc.
x
-C
Thus g is the intercept and r the
slope of the usual plot of
2n- versus
c.
These
relationships are shown in Figure 9, which gives a typical set of data (fraction I11 in this case). T h e plus and minus values in Table V are 95% confidence limits, calculated according to the methods of Fisher ( 4 ) . Data for two separate runs are given in the figure.
Figure 11. Foam Properties of Water-Soluble Protein Fractions Immediately after uniform shaking ( t o p ) Twelve minutes later with no further agitation ( b o t t o m )
1022
INDUSTRIAL AND ENGINEERING CHEMISTRY
There is evidence that the beer protein and carbohydrate components react with one another in solution. Figure 10 shows the effects of concentration on electrophoresis of whole beer protein (containing some carbohydrate). At low concentration (0.25 grams per 100 ml.) the beta peak was large, the alpha peak relatively small. At higher concentrations, the alpha peak increased rapidly in size and became relatively large compared with the beta peak. The alpha peak also increased in area relative to the beta a t low ionic strength (ionic strength = 0.02). Figure 11 shows the foaming properties of the various fractions in water (all concentrations were 0.02%). The first photograph was taken immediately after the solutions were given uniform shakings. Fractions I, IV, and V produced the most foam, fraction I11 the least. The lower photograph was taken 12 minutes later, with no further agitation of the solution. The foam on Fractions I1 and IIA persisted, while the foam on Fraction V collapsed almost completely. Fractions I and IV lost a large proportion of their originally high foam. SUMMARY
The proteins of beer have been isolated by ammonium sulfate precipitation follon~edby selective peptization of the precipitated materials. The water-soluble proteins have been separated into six fractions by alcohol precipitation from solutions at controlled temperature, pH, and ionic strength. Alcohol-soluble and alkalisoluble fractions have also peen obtained by peptization in the appropriate solvents. Progress of separation of the various constituents has been followed by means of electrophoresis and ultracentrifugation, and estimates of molecular weight have been obtained from osmotic pressure data.
Vol. 45, No. 5
Some carbohydrate material is carried along with the proteins and is concentrated in certain of the water-soluble fractions. A method for removing proteins and fractionating the residual polysaccharides has also been worked out. The compositions and properties of the protein preparations differ markedly. ACKNOWLEDGMENT
The authors wish to thank Frank Heckler, Rosalind Dean, Donald E. Heitmeier, and W. A. ITeber for assistance in various phases of the work. LITERATURE CITED
(1) Biserte, G., and Scriban, R., Brasserie, 5, 348 (1950). (2) Bishop, L. R., “Progress in Brewing Science,” Vol. 11, p. 244,
Ken. York, Elsevier Publishing Co., 1949.
( 3 ) Cohn, E. J., Strong, L. E., Hughes, W. L., Jr., Mulford, D. J.,
Ashworth, J. N., Melin, M . , and Taylor, H. L., J.Am. Chem. Sac., 68,459 (1946). (4) Fisher, R. A , , “Statistical Methods for Research Workers,” 8th ed., p. 153, Edinburgh, England, Oliver and Boyd, 1941. ( 5 ) Fuoss, R. M., and Mead, D. J , J . Phus. Chem., 47.59 (1943). (6) Hartong, B. D., Mastenbroek, G. G. A,, and Mendlik, F., “Progress in Brewing Science,” Vol. 11, p. 250, Ne%,York, Elsevier Publishing Co., 1949. (7) Lundin, H., Ibid., p. 229. (8) Quensel, O., “Untersuohungen Oeber die Gerstenglobuline,” Uppsala, Inaugural-Dissertation, 1942. (9) St.Johnston, J. H., J . I n s t . Brewing, 54, 305 (1948). (10) Sandegren, E., “Progress in Brewing Science,” Vol. 11, p. 78, New York, Elsevier Publishing Co., 1949. RECEIVED for review August 7, 1952. ACCEPTED FEBRUARY 4 , 1953. Presented before the Fermentation Subdivision of the Division of Agricultural and Food Chemistry at the 121st Meeting of the AMERICAN CHEMICAL SOCIETY,Milwaukee, Wis.
Antimicro ial Activity of uaternary Ammonium Chlori J
DERIVED FROM COMMERCIAL FATTY ACIDS R. A. RECB AND H. J. HARWQOD Research Division, Armour and Co., Chicago, I l l .
I
NCREASING utilization of higher aliphatic quaternary
ammonium salts as germicides and antiseptics has emphasized the need for a careful scrutiny of the economics of their manufacture and use. Two important factors must be considered with respect t o individuaI compounds-cost of manufacture and biological activity. Considering commercial fatty acids as the primary raw material, two major processes are applied to the production of quaternary ammonium salts. These processes are represented by the following series of reactions, in which palmitic acid is shown as the starting material. Process I (opposite) is .P four-step process and is especially useful for the introduction of substituent groups, such RS benzyl, which differ from those contained in the tertiary amine. Process I1 is a three-step process and of the two is more economical. I t can, however, be applied only to the manufacture of quaternary ammonium salts in which three of the substituent groups are methyl. A survey of the literature in the field ( 5 )reveals a large amount of work relating to the germicidal activity of quaternary- ammonium salts. A preponderance of this work pertains to compounds which contain aromatic nuclei, usually the benzyl group. The
inference might be drawn that the benzyl group is essential to high germicidal activity. Recent work in this laboratory has indioated that proximity of the phenyl group to the quaternary nitrogen actually results in decreased bactericidal activity ( 2 ) .
L
1 CH,
1
