Recovery of Proteins from Wheat Mashes
with
Sulfite Waste Liquor lignin addition, the mash is heated to at least 85' C. and is preferably cooled before filtration. Under these conditions 85 to 90% of the total mash protein (or 80 to 85%of the aoluble protein) is readily recovered, and the filterability in markedly improved when compared with that of untreated mashes. The recovered protein shows promise a%a feed, when properly supplemented.
Sulfite waste liquor can be employed for the racovery of proteins from dilute solutions. A method is described for wheat mashes. For maximum recovery, protein hydrolysis should be avoided, and the acidity of the mash should be adjusted to a pH range of 1.5-2.5. Two parts of the sulfite waste liquor, calculated on the basis of its lignin content, are required to recover 5 parts of protein. During or after
James S. Wallerstein, Eduard Farber, Gertrude D. Maengwyn-Davies, and Arthur L. Schade OVERLY BIOCHEMICAL RESEARCH FOUNDATION, INC., NEW YORK 1, N. Y.
HEN cereals are used as the carbohydrate source in industrial fermentations, the protein content of the grain represents a valuable by-product. I n view of the shortage of protein under wartime conditions, its recovery is important for the national economy. Furthermore, by such recovery the amount of wastes from fermentation industries will be reduced and stream pollution diminished. Various methods for protein recovery. such as screening, spraydrying of slops, the sulfite process ( I ) , etc., are in use. A new method for protein recovery by precipitation with sulfite waste liquor is described here as applied to wheat mashes; it may be used with equal success for other cereals. The advantages are a high recovery of protein, low material and handling costs, and the use of conventional equipment. The ability of some constituents of sulfite waste liquor to combine with protein is made use of in the tanning industry. It has been shown that the lignosulfonic acid effects a chemical combination with hide proteins (3, 7 ) . A study of the depressive effect of lignin upon the decomposition of proteins by bacteria showed that a complex is formed when lignin and gliadin (6,6 ) , or casein, were allowed to interact. This lignin-protein complex possessed the various chemical, physicochemical, and biological properties characteristic of the major portion of the organic matter (humus) of the soil. Recently, proteins were used as a lignin precipitant in the purification of wood liquors (2). I n 1942 research was-started in this laboratory on the production of glycerol from wheat by fermentation. Initially, second clears flour, and later, granular wheat flour, were employed. Since sizable quantities of nitrogenous matter interfere with the distillation of fermentation glycerol in various ways, experiments for the removal of proteins with sulfite waste liquor were undertaken for prepurification of the mash prior to fermentation. When the maximum amount of protein was removed by sulfite waste liquor treatment, it was found that distillation difficulties were substantially eliminated, the amounts of residue and solids in the distillations were reduced, and a greater purity of end products was obtained. Initial experiments indicated that the quantity of protein re-
moved by sulfite waste liquor is inversely proportional to the degree of its disaggregation. Therefore a method was employed by which the protein in the mash undergoes as little breakdown RR possible prior to precipitation. MATERIALS AND METHODS
The granular wheat flour used had the following analysis: moisture l l . O % , protein 10.3%, carbohydrates (calculated as glucose) 77.9%, ash 0.6%. The flour was converted into a fermentation mash with a minimum of proteolysis by the following method: A premalt was prepared by mixing 50 grams of high-diastatic malt with 500 ml. of water, stirring the mixture a t 37' C. for 30 minutes to free the diastases for maximum activity, and then for an additional half hour at 60' C. to inactivate the proteases of the malt. One kilogram of granular wheat flour, suspended in 5 kg. of water, was liquefied by heating to 85" C. after addition of one fifth of the premalt. After liquefaction of the starch, the mash was cooled to 62" C., and the remaining four
TABLE I.
COMPARISON OF PROTEIN-PRECIPITATING ACTIONOF
SAMPLESF AND M
Total Nb in % Total Sol. Filtrate of LigninLignin Added. Mg. Treated Sample, and Insol. ProRemoved tein Recovered Sample F Sample M' Mg. 0 39 0 0 6.3 9.5 44.9 5.61 0.78 (3)O 54.2 . 4.72 25.0 1.56 (6) 54.8 4.66 26.0 2.6 (10) 3:9 (5.67) 4.17 33.5 59.5 5:i (20) 3.45 45.0 66.0 7:8(11.34) 2.99 52.5 71.0 7 : s (30) ... 2.39 62.0 76.8 10.4 (40) 1.22 80.5 88.1 15:6(22.68) 1.32 79.0 87.2 1.12 82.0 89.1 2 0 : s (80) 3i:i (c5.36) 1.23 80.5 88.1 3i:i (120) , ., 1.10 82.5 89.3 41.6 (160) 1.07 83 89.6 a Figures in parenthesea represent mg. of samples F and M used. b Calculated after deduction of nitro en present in amounts of samples F and M added ( F 0.86. M 0.70 mg.% per gram).
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August, 1944
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
fifths of the premalt was added with stirring. The mash was then malted for 2 hours a t 62' C. and for an additional 2 hours at 52' C. The malting time is not critical and may be appreciably shortened. Instead of malt, other amylolytic materials, such as bacterial amylase, can be employed. Two samples (F and M) of lime-neutralized, concentrated sulfite waste liquor were investigated with respect t o their protein-precipitating action. Sample F was obtained as a concentrated liquid for which the following percentage analysis was submitted by the Farber Laboratories: Water (as lose at 106O C.) Sulfur (calcd. total) As inorganic 80s
As so;
Fe Alkali metals (as Ne) Or anic matter (calcd. by difference) in organic compound sulfone Lignin Total eugars -4cetic and formic acids
2.55 0.10 2.39 0.75 0.13 0.002 0.011 0.09 2.47
49.17 2.71 5.30
46.47
26
11 1.8
Sample M was a dry powder with the following analysis (in per cent): MANVPACTVRER'S ANALYSIS (MARATEON PAPBEMILLS Co.) 6.24 Moisture Oven-dry basis 5.m Total S Sulfate 8 0.15 0.10 FelO: 3.42 (4.79 CaO) Total Ca 0.96 (0.58 MgO) Total M g Total methoxyl 9.98
CALCVLATED COHPOBITION CaS04.1/;HtO 0.68 0.10 Fer01 13 :70 Sulfonic SO; Sulfo*c c a 3.23 0.12 Sulfonic M g 0.23 Nonaulfonic M g Lignin 68.80 Nonlignin organic 13 14 (by die.) matter
-100.00
moved by filtration with Celite 512. The action of sulfite waste liquor was studied on the soluble 61% of the protein. VARIATION 1 N QUANTITY OF PROTEIN PRECIPITANTS
The malted mash was acidified to approximately pH 2.0, diluted
to four times ita original volume with distilled water, and filtered with Celite 512. Each 100 ml. of the clear filtrate used for the following experiments contained an equivalent of 6.25 grams of granular wheat flour (392 mg. of soluble protein). One milliliter of solution, containing different amounts of samples F and M by appropriate dilution of their stock solutions, was added to 10 ml.*of the filtrate. The mixture was heated to boiling, readjusted to the preboil weight with distilled water, and filtered with Celite 512. One milliliter of distilled water was added to 10 ml. of the filtrate as control, and the mixture treated in the same manner. The percentage of soluble protein removed with the addition of varying amounts of the sulfite wmte liquor samples, calculated on their lignin contents, is shown in Table I. The protein recovery rises to a maximum with the increase in the concentration of precipitating agent. The lignin content of the sulfite waste liquor samples is a satisfactory index of their proteinpreqipitating action. The protein-precipitating action of the sulfite waste liquor samples was compared with that of sulfosalicylic acid. Five milliliters of varying concentrations of sulfosalicylic acid made by dilution of the stock solution with 0.1 N hydrochloric acid were added to 10 ml. of filtrate; the mixtures were left in the incubator for 40 minutes (4) and then filtered with Celite 512. As control, 5 ml. of 0.1 N hydrochloric acid were added to 10 ml. of filtrate, and the mixture waa left in the incubator for 40 minutes. WLFOSAUC'ILIC ACID M6S.
I
r
Stock solutions of equal concentration with respect to lignin content were prepared from sample F (26%) and M (68.8%). The actual precipitating agent was lignosulfonic acid, expressed in terms of lignin in accordance with the common analytical terminology for sulfite waste liquors. A 7.5-gram portion of sample F was diluted to 25 ml. with distilled water; 1 ml. of this solution contained 0.3 gram of the sample-i. e., 0.078 gram of lignin, calculated on the basis of the above analysis. Sample M was made up to the same concentration on the basis of ita lignin content after conversion into free lignosulfonic acid (a procedure suggested by the manufacturer). To 22.675 grams of oven-dry sample M suspended in 100 ml. of water, 1.22 ml. (2.243 grams) of concentrated sulfuric acid (95.5%), diluted to 8 normal strength, were added under stirring. The mixture waa heated to 8 5 O C. and filtered, the precipitate was washed, and the combined filtrate and washings were made up to 200 ml. with distilled water. One milliliter of this stock solution corresponds to 0.078 gram of lignin, calculated on the basis of the manufacturer's analysis. For a more general comparison of the protein-precipitating action of the two samples of sulfite waste liquor, sulfosalicylic acid wm employed; as stock solution, 10 grams of this acid (reagent grade) were dissolved in distilled water and brought to a volume of 100 ml. (pH = 1). The semimicro-Kjeldahl method was used for the nitrogen analyses. The factor 6.25 was taken for the conversion of nitrogen into protein. The pH determinations were made with a Beckman meter. Experiments were undertaken to determine the optimum conditions for the precipitation of proteins ih the maah. When the malted wheat flour mash was acidified to pH 2.0, approximately the optimal pH for precipitation with sulfite waste liquor, 39% of the protein originally present was insoluble and could be re-
773
f a
x SAMPLE C 0
SAMPLE M
SULCOSAUCVUC ACI
UENlN MQS.
Figure 1. Comparison of Sulfosalicylic Acid and 2 Sulfite Waste Liquors (on Basis of Lignin Content) as Protein Precipitants
The percentage of soluble nitrogen removed from the mash filtrates with increasing amounts of sulfosalicylic acid and of the sulfite waste liquor samples in terms of atheir lignin contents are shown in Figure 1. These curves, which are representative of the experiments, indicate that sulfite waste liquor is a better precipitant for wheat proteins than sulfosalicylic acid; in order to achieve the same degree of precipitation, more than ten times as much sulfosalicylic acid was needed than lignin, and the maximal percentage of protein precipitated was considerably higher with the latter agent.
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INDUSTRIAL AND ENGINEERING CHEMISTRY VARIATION IN pH
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was 85-90% recovery' realized, but also the filterability of the
The effect of pH variation on the precipitation of protein was determined with the optimum addition of sample F. Samples of the mash used in the precipitation experiments were adjusted by addition of sulfuric acid or sodium hydroxide to the desired pH, made up to a final dilution of 1 to 4, and filtered with Celite 512. The pH values of the filtrates are shown in Figure 2. Ten milliliters of these filtrates were treated with 1 ml. of sample F containing 15.6 mg. of lignin. One milliliter of distilled water wm added to 10 ml. of the filtrate in each case and used as control. The treated samples and controls were heated to boiling, cooled, brought to original weight with distilled water, and filtered with Celite 512.
fermentation mash was greatly improved. The fermentation was not affected by this treatment. To recover 85-90% of the protein from a fermentation mash, the acidity was adjusted to a pH value in the range 1.5-2.5. Sulfite waste liquor in an amount equal to 2 parts of lignin for every 5 parts of protein was added to the adjusted mash. The treated mash waa heated to 85-90' C. for 10 minutes with thorough mixing. For the highest recovery of protein, it is preferable to filter after cooling the mash to mom temperature. If desired, the filter cake can be washed free of sugar and the wash water used for subsequent mashings. The protein-lignin cake can readily be converted to a dry form following the usual precautions for drying such materials. Proteins may be recovered by this method either from mashes prior to fermentation or from fermentation slops, such as slops from alcohol distilleries. If used for alcohol slops, the method not only provides for the recovery of a material high in protein but also leads to a considerable purification and thereby a lowering of the B.O,D. and solids content of the effluent. Preliminary tests on the feeding of the dried protein-lignin to rats indicated that this material is a satisfactory source of protein, provided the diet is supplemented by the essential amino acids known to be lacking in wheat protein. With such a supplement the rats maintained satisfactory growth rate. While the process described was developed for the recovery of wheat proteins, it may also be applied to proteins from other vegetable sources such aa corn and potatoes. Yields for the latter are of the same order as those for wheat. SUMMARY
SAMPLES
Figure 2 compares the nitrogen contents of the treated samples and controls at varying pH values. The upper curve (controls) indicates that the amount of soluble nitrogen (in other words, the amount of nitrogenous matter remaining in the filtrates) changes considerably with the pH of the mash; it is a t a maximum at pH 2.75. Maximum precipitation of this soluble nitrogenous matter by addition of the optimum amount of sulfite waste liquor is obtained between the pH range of 2 and 4. This fact is derived from an examination of the lower curve (treated samples). The yield of precipitated protein calculated on the basis of the s u m of soluble and insoluble nitrogen is greatest in the pH range of 1 to 3. Results of pH studies on sample M closely resemble those for sample F.
1. A method is described for the recovery of protein by the addition of sulfite waste liquor to hot, acidified wheat mashes. By this method 8540% of the protein is readily recovered and filtration of the mash is greatly improved. 2. Studies with two different sulfite waste liquor samples indicate that the protein-precipitating action is a function of lignin (lignosulfonic acid) content. For the precipitation of soluble wheat proteins, the optimum ratio of lignin to protein is 1 to 4. 3. The effectiveness of lignosulfonic acid for the precipitation of soluble wheat protein was compared to that of sulfosalicylic acid. The relative activity of lignosulfonic acid was more than ten times aa great as that of sulfosalicylic acid. Further, the maximum amount of precipitation achieved by lignosulfonic acid was considerably higher. 4. The optimal range of acidity for maximum precipitation of soluble protein is pH 2 to 4. 5. With fermentation mashes containing both soluble and insoluble protein, approximately 2 parts of sulfite waste liquor, calculated on the basis of its lignin content, are required to recover 5 parts of protein. The optimum pH range for this precipitation is 1.5 to 2.5. 6. Preliminary tests indicate that the recovered protein is suitable for feed purposes, when properly supplemented. ACKNOWLEDGMENT
The authors are indebted to Arnold Schein of the Flower and Fifth Avenue Hospitals for the rat assays. LITERATURE CITED
DISCUSSION
These studies on the precipitation of wheat proteins show that, with sulfite waste liquor, 80-85% of the soluble protein in a diluted mash is readily recovered. For experimental purposes, attention was directed chiefly to the soluble protein of the mash. However, when fermentation mashes of the usual concentrations containing insoluble as well as soluble protein were treated under optimal conditions for the recovery of soluble proteins, not only
(1) Balls, A. IC., Hale, W. S., Rose, W. G., Tucker, I. W., Axelrod, B., Schimmer, S., Peruzzi, J. J., and Walden, M. K., Commun. from Agr. Research Administration, Sept. 17, 1943. (2) Farber, Eduard, unpublished communication, 1942. (3) Gustavson, K.H.,Suensk Papperstidn., 44,193-200 (1942). (4) Mawaon, C.A . , Biochm. J . , 36, 273-80 (1942). (5) Waksman, S. A., and Iyer, K. R. N., Soil Sci., 34,43 (1932). (0) Ibid., 34,71 (1932). (7) Wilson, 3. A., and Porth, I. H., J . Am. Leather C h m . ASYOC., 38,
20-30 (1943).
