hipping Ability of Soybean Proteins Solvent-extracted soybean flour dissolved in water whips to a stiff white foam greatly resembling egg white. The material whips best in concentrations of 7 to 8 per cent, and at pH values far removed from its isoelectric point of pH 4.1. Salt increases the whipping ability. The soy flours on the market at present do not show this foaming ability, either because they have not been solvent-extracted, or because the proteins have been denatured by treatment designed to rid them of their raw bean flavor. A method is here presented for removing the objectionable flavor without denaturing the proteins. I t is suggested that a soy product of the type described could be utilized to replace egg white in many culinary operations.
BETTY MONAGHAN-WATTS University of California, Berkeley, Calif.
T
HE soybean crop in the United States a t present is large and continually increasing ( 3 ) . The meal which remains after the extraction of soybean oil contains a high percentage of protein (40 to 50 per cent). So far, efforts toward the utilization of this meal as well as of the whole bean hatre been directed mainly toward the development of a soybean flour to replace, wholly or in part, wheat flour in the preparation of special types of baked products (8). If this flour or meal could be made to take the place in culinary operations of typical protein foods (milk and eggs) which it resembles dietetically rather than of the cereal grains, it would undoubtedly have a much wider range of usefulness. Horvath (7) points out that 100 grams of protein cost 45 cents in the form of eggs, 40 cents as milk, and about 4 cents as whole soy meal. The protein of extracted soy meal is even less expensive. So far, few suggestions have been made as to how these more expensive protein foods can be successfully replaced by soy meal. Osborne and Campbell (11) report that the chief protein present in soybeans, which they designate as glycinin, is salt soluble, is precipitated by acetic acid, and, when dissolved in 10 per cent sodium chloride, is not precipitated by heat. Smaller amounts of an albumin and a proteose are also present. Aside from this early work very little information has been obtained which can be applied practically in the utilization of the protein as a food. I n this laboratory it has been found possible to whip a solution or suspension of soy meal into a stiff white foam closely resembling beaten egg white. This property has consequently been investigated in some detail. Methods Whipping ability was measured by beating the flour and water suspension with a Hamilton Beach electric mixer, model D, in the quart-size glass bowl supplied with the mixer; this mixer has the advantage over other types of maintaining a constant speed of rotation regardless of the viscosity of the solution. The volume of the resulting foam was obtained by transferring it to short, wide, graduated cylinders. Various refinements of this simple method have been suggested from time to time ( I , 6) but the resulting increase in accuracy hardly justifies the extra time required to erform the experiments. %hen the stability of the foams was to be determined, a freshly beaten portion of the material was transferred to a glass funnel containing a square of cheesecloth and suspended in a graduated cylinder. The amount of liquid draining from the foam in a given time was considered to be inversely proportional to the stability of the foam. Here again greater accuracy could perhaps be obtained by plotting the slope of the drainage curve ( 2 ) but the simpler method seemed preferable. A quinhydrone electrode was used for pH determinations. All soy suspensions were diluted with distilled water to a concentration of approximately 1per cent before attempting to measure the pH, since much higher concentrations resulted in drifting potentials. The isoelectric point was determined by electrophoresis measurements carried out in a microscopic electrophoresis cell of the
Mattson type (9). The operation of this cell has been described elsewhere ( I O ) . For determinations at various pH values, a small pinch of the flour was suspended in 0.02 A4 acetate buffers. The pH was redetermined after the addition of the soy flour and in most cases was found to have shifted less than 0.2 pH unit. Stronger buffers are inadvisable in closed cells of this type, since the increased current may cause heating and formation of gas bubbles at the electrodes which interfere with the readings. When the current was applied, the particles of flour visible in the microscopic cell moved in the same direction and at the same speed a t any given pH value. When inert particles such as pulverized glass or oil droplets were added to the suspension, they also assumed the same charge as the flour particles. This is interpreted to mean that all of the particles present (whether of fiber or insoluble gummy material from the flour itself, or added glass or oil) are coated with the soybean protein and consequently assume the same charge as the protein itself. It is not necessarily true that the electrophoretic mobility measured in this way is that of any particular soybean protein. Probably the mobility is the result of an equilibrium adsorption of the various proteins present. In any case, the charge and isoelectric point of this protein mixture are significant in studies on foaming or emulsifying ability of the flour, and not the charge of any of the individual proteins which might be separated from the mixture and purified.
Preliminary Treatment of Soy Flour The soy flour used in these experiments was a commercial product ground by the manufacturer from beans of the Daizu variety in a steel ball mill without heat. About 80 per cent of this flour passed a 100-mesh screen. The flour 1009
INDUSTRIAL AND ENGINEERING CHEMISTRY
lOP0
l
was not debittered or treated in any way by the manufacturer. It had a. pronounced raw bean flavor. All commercial deflavored flours tested in this laboratory have little or no whipping ability, probably because of the denaturation of the proteins brought about by heat or chemicals in the deflavoring process. Removal of fat from the flour by solvent extraction is necessary before the flour will whip. The presence of even a small amount of fat (as in meal produced by hydraulic pressure or by the Anderson expeller process) prevents foaming. Similar observations have been reported on other protein solutions. The addition of a small amount of olive oil, for example, greatly decreases the whipping of egg white (1). For most of the present work fat was extracted from the flour by means of petroleum ether (boiling point, 30" to 60" C.). Preliminary storing of the petroleum ether for several days over concentrated sulfuric acid was necessary to remove nonvolatile impurities which otherwise left a perceptible taste on the extracted meal. The extraction was carried out by shaking a quantity of the flour with several volumes of petroleum ether, filtering, and repeating the process until no further yellow color was apparent in the filtrate. This generally required five or six washings. Other solvents may be used which do not denature the proteins present. Alcohol is unsuitable. Ethyl ether is as satisfactory as petroleum ether if the extractions are completed within the course of a few hours. If this solvent remains in contact with the flour for several days, however, the whipping ability is appreciably decreased.
Deflavoring Soy Flours without Denaturing the Protein Almost all firms which manufacture soybean flours for human consumption or soybean meal as a stock feed treat their products to remove the pronounced, undesirable beany flavor and also to increase the digestibility and biological value of the proteins (6). In view of this situation it became necessary to find a method of deflavoring which would not cause denaturation. Of several methods tested, only a combination of dry heat and reduced pressure yielded a product of neutral flavor and unimpaired whipping ability. The flour was spread out in a layer several millimeters thick and heated a t 130" C. in an electric vacuum oven which maintained a constant pressure of 45 mm. of mercury. The whipping ability was measured by dissolving 4 grams of the treated flour in 50 cc. of distilled water and whipping for 8 minutes a t room temperature (23"C,). The effects of such treatment on the flavor, color, and whipping ability of a sample of extracted flour are as follows :
Flavor 0 10
20
40 60
820 820
840
760 590
White White Faint yellow Light tan Tan
Noticeable raw-bean Slight raw-bean Neutral Noticeable haylike Pronounced haylike
Concentration of Flour for Optimum Whipping The concentration range within which the soy flour could be successfully whipped is somewhat limited. The following table shows the result of dissolving various weights of soy flour in the same amount of water and whipping both for 2 and for 8 minutes: Wt. of Final Vol. Flour as 70of Added to Min. of Original 50 Cc. HzO Beating Vol.
Wt. of Flour Added to Min. of 50 C c . Hz0 Beating
Final Vol. % of Original Vol.
The solution whips best in concentrations of 5 to 9 per cent, the optimum being between 7 and 8 per cent.
Effect of Beating Time The effect of time of beating on the foaming ability of the soy flour: Final Vol. as % of Original Vol. 460 750
Min. of Beating 2.5 5
Final Vol. Min. of Beating 8 10
?S.%
I
of
Original Vol. 830 800
With the beating apparatus used in these experiments the optimum foam volume is obtained in 8 minutes. Egg white under the same conditions reached its maximum volume in 6 minutes. The best whipping sample of egg white tested reached a maximum foam volume of 1000 per cent of the original liquid volume, but most samples of egg white are far inferior to this in whipping ability. In general, the soy foams appeared to be less brittle than the egg white foams, resembling more closely a stiff whipped cream in appearance.
Effect of Temperature Since temperature is generally considered to have a marked influence on the foaming ability of egg white, its effect on the final volume obtained and on the time required to reach the optimum volume was investigated for soy flour. The following table shows the effect of preheating the soy solution in a boiling water bath on the volume obtained in a short period of whipping; the whipping was carried out immediately ate room temperature (21' C.) : Temp,
e
C.
Foam Vol. as of , Liqm Vol
.?
Temp., 40
45
50 60
O
C.
Foam Vol. 88 7 of LiquiK Vol. 400 420
490 440
A marked temperature effect was obtained with this procedure, the optimum whipping temperature being 50" C. However, when the whipping was continued until the optimum foam volume was obtained, the temperature appeared to have little or no effect either on the final volume obtained or the time required to reach this optimum volume : Min. of Beating
A heating period of 20 minutes produced a product of neutral flavor which retained its whipping ability. A longer heating period not only decreased the whipping ability, but also developed a roasted haylike flavor which was as pronounced and almost as undesirable as the original raw-bean taste. The flour "deflavored" by this method still retains a slight bitterness which cannot be described as raw-beanlike and which could not be removed by any method tried.
VOL. 29, NO. 9
Foam Vol. as % of Liquid Vol.
25OC.
50'
C.
Min. of Beating
Foam Vol. as 70of Liquid Vol. 2 5 O C. 50'C.
It is quite possible that, if the whipping also were carried out in rooms heated to the temperatures specified, a temperature effect m7ould be obtained with the longer whipping time, but such an observation would seem to be of little practical importance.
SEPTEMBER, 1937
INDUSTRIAL -kh*D ENGINEERING CHEMISTRY
Effect of pH on Electrophoresis and Whipping The isoelectric point of the mixed soy proteins in acetate buffers (determined as previously described) was found to be a t approximately pH 4.1. Figure 1 shows the results of a typical electrophoresis experiment on a petroleum-etherextracted sample. Similar determinations were run on other samples, both extracted and unextracted, as well as on commercially deflavored flours. The point of zero charge In every case lay somewhere between pH 4.0 and 4.2. Cooking in boiling water for 30 minutes shifted the isoelectric point of one sample from pH 4.1 to 4.4. The isoelectric point of glycinin (extracted from soybeans with 10 per cent sodium chloride and purified by dialysis and treatment with methyl and ethyl alcohol and ether) is reported by Hartman and Cheng (4) to be a t pH 5.02, considerably higher than the value of 4.1 here obtained. It should again be emphasized that the charge and isoelectric point obtained in this investigation are those of an equilibrium mixture of untreated proteins adsorbed on inert surfaces, whereas the value obtained by Hartman and Cheng presumably represents the true isoelectric point of a single protein fraction whose electrical properties may, moreover, have been changed by the extraction and purification process. The effect of pH on the whipping of the soy suspenivon was as follows: Co. Liquid Added t o 4 G. Soy Flour HzO Vinegar 50 47.5 40 25 0
PH 6.40 5.10 4.32 3.94 3.68
0 2.5 10
25 50
Foam Vol. a8 yo of Liquid Voi.
Go. ad Drainaae in 20 h4in
820 640 580 500 700
5 13
14 18 9
The foaming ability is least a t the isoelectric point of the soy proteins, increasing both above and below this pH. Foam stability closely parallels foam volume. This relation is just the reverse of that obtained with egg white, which ie reported to increase both in volume and stability (especially the latter) when acid is added to bring it nearer its isoelectric pH ( 2 ) . Probably the difference in solubility of egg albumen as compared to soy protein a t their respective isoelectric points is responsible for this difference in the whipping behavior of the two. Soy protein resembles casein in being almost completely precipitated by the addition of the proper amount of acid, whereas egg albumen is quite soluble a t its isoelectric pH. Since egg white is reported to require a longer time t o whip to maximum volume after the addition of acid ( I ) , the effect of whipping time on foam volume was repeated on samples of soy flour suspended in the vinegar mixture. The greatest volume was again obtained in 8 minutes.
Effect of Salt Sodium chloride markedly improves the whipping ability (8 minutes) of the soy proteins in concentrations up to 2 per cent. Higher salt concentrations are of no practical iniportance in cookery. This effect is probably due to the fact that the chief protein, glycinin, is salt soluble: Concn. NaC1. % 0.00
0.25 0.50
Foam Vol. as 7 of
LisuiJ Vol.
820 830 880
Concn. NaC1, % 1.00
Foam 1701. as
7
of
Liquidl Voi.
2.00
920 980
Possible Uses of Soy Whips AS a foaming agent, extracted soy flour has a numbel oi advantages over egg white: ( a ) It is extremely cheap. ( b ) It keeps indefinitely and can be transported and handled easily and inexpensively. (c) It can be more easily standardized. (d) Whereas egg albumen is already diluted a h o t t
1011
to its optimum whipping concentration, soy flour is in a very concentrated form, so that it may be whipped with any of a number of liquids (skim milk, fruit juices, meat extracts, etc.), thus greatly increasing its possible usefulness in recipe combinations, As a leavening agent for dishes prepared in the cold there should be little difficulty in utilizing whipped soy protein. It should be very valuable in incorporating air into frozen desserts, especially in unchurned (electric refrigerator) products. It might also be expected to combine readily with gelatin and with cooked starch to form a variety of JThips, sponges, and molded desserts, or with cooked sugar sirups to form confections of the divinity type. It is d o u b t f u l whether soy foams can take the place of w h i p p e d egg white in baked d i s h e s ( c a k e s of the angel food and sponge v a r i e t y , souffl6s, fondues, p o p o v e r s , meringues, etc.) without many changes in existing recipes a n d m e t h o d s of rn a n i p u 1 a t i o n , since dishes of this t y p e depend not only on the foaming of egg white, but also upon its a b i l i t y to coagu3 4 5 6 late a t a definite PH temperature a n d FIGURE1. THE ELECTROPHORETICthus hold in the POINTOF MOBILITYAND ISOELECTRIC a i r . Since t h e PETROL^ UM-ETHER-EXTRACTED SOYearly report of OsBEAN FLOUR borne and Campc0.l gram of flour in 100 00. of 0.02 M acetate buffers) bell that glycinin in 10 per cent salt solutions is not precipitated by boiling, this protein has generally been considered noncoagulable by heat. Nevertheless it has been possible to set soy foams permanently by baking in a slow oven. Further investigation is needed on this point.
Acknowledgment The author is indebted to the following companies which cooperated in this investigation by supplying samples of their soy products and information concerning their methods of manufacture : Archer-Daniels-Midland Company, Minneapolis, Minn.; Glidden Company, Chicago, Ill.; Allied Mills, Inc., Peoria, Ill.; A. E. Staley Company, Decatur, 111.; American Soya Products Corporation, Evansville, Ind.
Literature Cited ;1) Bailey, IND. ENG.CHEM.,27,973 (1935). (2) Barmore, Coll. Agr. Expt. Sta., Tech. Bull. 9 (1934). (3) Burlison, IND. ENQ.CHEY.,28,772 (1936). (4) Hartman and Cheng, J. Phys. Chem., 40,453 (1936). (5) Hayward, Steenbook, and Bohstedt, S.Nutrition, 11,219 (1936). (6) Henry and Barbour, IND.ENG.CHEM.,25, 1054 (1933). (7) Horvath, “A Perfect Ration at Low Cost,” Newark, Del., Agr.
Expt. Eta. (8) Horvath, Food Manuf., 11, 336 (1936). (9) Mattson, S.Phys. Chem., 32, 1532 (1928): (lo) Monaghan and White, S. Gm. Physiol., 19,715 (1936). (El) Osborne and Campbell, Conn. Expt. Sta. Rept., 21, 374 (1897). R ~ C E W BMay D 27, 1937.
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