1142
INDUSTRIAL AND ENGINEERING CHEMISTRY
(20) Irvine and Hynd, J . Chem. SOC.,99, 101 (1911). (21) Isbell, Ann. Rev. Biochem., 12,206 (1943). (22) Isbell, Piaman, and Frush, J . Research Natl. Bur. Standards, 24, 241 (1940). (23) Jackson and Gillia, Bur. Standards, Sci. Paper 375 (1920). (24) Kilp, 2. Spiritusind., 55, 188 (1932). (25) Kostytchev and Brilliant, 2. physiol. Chem., 127,224 (1923). (26) Kuzin and Polyakova, Biokhimiya, 6, 113 (1941). (27) Lobry de Bruyn and Alberda van Ekenstein, Rev. trav. chim., 16,257,262, 284 (1897): 18,72 (1899). (28) Maillard, Compt. rend., 154,66(1911);Ann. chim., 5,258(1916). (29) Mathews and Jackson, B u r . Standards J . Research, 11, 619 (1933). (30) Maurer, Ber., 59,827 (1926). (31) Maurer and Schiedt, 2. phgsiol. Chem., 206,125 (1932). (32) Nagel, Arch. Suikerind.,38,111,1139(1930). (33) Pictet and Chavan, Helv. Chim. Acta, 9,809(1926). (34’1Prinsen-Geerligs, Intern. Sugar J., 40,345 (1938).
Vol. 37, No. 11
(36) Proefstation Java-Suikerind., “Methoden van Onderzoek”, 6th ed., p. 365 (1931). (36) Proefstation Java-Suikerind., Verslag 1929,p. 241. (37) Reindel and Frey, 2. Spiritusind., 57,237 (1934). (38) Ripp, 2. Ver. deut. Zucker-Ind., 76,627 (1926). (39) Sattler and Zerban, Facts About Sugar, 23,686 (1928). (40) Schlubach and Bohre, Ann., 508,16 (1933). (41) Schlubach and Elsner, Ber., 61,2358 (1928). (42) Shiga, J . Biochem. (Japan), 25,607 (1937); 27,307 (1938). (43) Spoehr and Strain, J.Biol. Chem., 85,365 (1929). (44) Spoehr and Wilbur, Ibid., 69,421 (1926). (45) StanBk. Z.Zuckerind. Bdhmen, 41,298,607(1916-17). (46) Stoltzenberg, Ber., 49,2021,2675 (1916). (47) Waterman and van der Ent, Arch. Suikerind., 34,11, 942 (1926). (48) Weast and McKinney, IND.ENG.CHEM.,33,1408(1941). (49) Wohl, B ~ T .23, , 2084 (1890). (50) Zerban. J . Assoc. Official Agr. Chem., 23,562 (1940). ENG.CHEM.,34, 1180 (1942). (51) Zerban and Sattler, IND.
Evaluation of Malts for Production of Alcohol from Wheat C. B. THORNE, R. L. EMERSON, W. J. OLSON, AND W. H. PETERSON University of Wisconsin, Madison, Wis.1 Knowledge of the amylase content of a malt is helpful but is not adequate for complete evaluation of the malt. Other factors also operate, and probably only a fermentation test can give an accurate evaluation. A possible method for evaluating malts for production of alcohol from wheat is to employ a 5% level of malt in a 36-hour fermentation. If two fermentations are run, 24- and 72hour periods give both the rata of conversion and the final degree of conversion of the wheat starch to alcohol. In laboratpry fermentations of wheat, malt in the amount of 8% of the total grain bill, as commonly used in industrial plants, was found to be in excess. Many malts at a 5% level gave maximum yields of alcohol in 36 hours. Under the conditions employed, the limiting factor in speed of fermentation appeared to be the action of the malt rather than the fermenting power of the yeast,
T
HE tremendous increase in the demand for industrial alcohol, together with the use of grains as raw material, placed a great load on malting facilit#iesand barley supply. In the industrial production of alcohol from starchy mashes, the amylase activity of the saccharifying agent is probably of primary concern. The index used classically in the evaluation of malts is the Lintner value; other indices are the alpha- and beta-amylase activity of the malt. The final test must be the yield of alcohol obtained. In this paper the amylolytic criteria are compared with the results obtained by a suitable fermentation procedure t o determine which gives the best indlcation of the malt value. Dark Northern Spring Wheat No. 2 and Hard Winter Wheat No. 2 were obtained from the Commodity Credit Corporation through the Narris Grain Company. These grains are referred to here as spring wheat and winter wheat. The wheat was ground and sifted to determine the fineness of the meal. All of it passed through a 2-mm. sieve, 63% passed through a I-mm. sieve, and 38% passed through a 0.5-mm. sieve. The moisture content was determined by drying the ground material at 100-105 O C.for 10-12 hours. The starch content of the ground material was determined by the malt-acid method of the A.O.A.C. (3). The values 1
Barley and Malt Laboratory and Departments of Agricultural Bacteri-
ology and Biochemistryin cooperation with the O 5 c e of Production Research
and Development.
obtained on the dry basis were 66.7% for spring wheat and 65.8% for winter wheat. A barley malt set aside by the Pabst Brewing Company for research on the Wheat Alcohol Project was used as a reference standard, The other barley malts, prepared under varying conditions of malting, represent a number of different barley varieties which are produped commercially and some hybrid selections which are being developed by plant breeders. Tables 1 and I1 give information on the varieties of barley, malting conditions, and amylolytic properties of the malts. FERMENTATION PROCEDURE
Seven liters of a 22.25% suspension [weight equivalent to 22.25 grams of dry grain per 100 ml. of mash or 30.2 gallons per bushel (56 pounds) of dry grain] of meal in water were held at room temperature for 15 minutes. The p H was adjusted to approximately 5.6 with 1 N sulfuric acid. With continuous stirring, the mash was heated in a boiIing water bath for 1 hour and then cooled, and water was added to make up for loss due to evaporation. Aliquot8 of 600 ml. of the mash were autoclaved in I-liter flasks a t 15 pounds per square inch pressure for 1.5 hours with 5 dropa of Vegifat Y to prevent loss by frothing. This mashing and cooking procedure was used for both spring and winter wheat, except that with winter wheat 1% malt was added at the beginning of the procedure t o accomplish liquefaction. The spring wheat contained enough amylase to produce autoliquefaction, but the amylase was not so abundant in the winter wheat. For conversion the mash was cooled to 65’ C. The desired amount of finely ground malt was made into a 10% slurry with water and added to each of the flasks of mash. The flasks were shaken vigorously for 2 minutes and then cooled quickly under the water tap. The p H was adjusted to approximately 4.8 with 1 N sulfuric acid, and 10 ml. of yeast suspension were added. The yeast (No. 47 of our collection) was a strain of Saccharomyces cereuisiae from the Lawrenceburg plant of Joseph E. Seagram and Sons, Inc. It was transferred from an agar slant into a tube containing 10 ml. of medium containing 3% malt sprouts extract and 5% glucose, and was allowed to grow 24 hours a t 30”C. The contents of the tube were then transferred to a bottle holdinq 200 ml. of the malt
INDUSTRIAL AND ENGINEERING CHEMISTRY
November, 1945
1143
TABLEI. EFFECTOF LOW MALTLEVELSON ALCOHOL YIELDS FROM SPRING WHEAT I N 7zHOtlR FERMENTATIONS WITH MALT^ PRODUCED UNDER DIFFERENTCONDITIONS Malt No. 4270-71 4278-79 4274-75 4282-83 4268-69 4276-77 4272-73 4280-81 4207-09 4228-30 4234-35 Pabst
Variety or Selection Wis. Barblew
Location Grown Clinton, Wis.
Manch., N. D. 2121
Langdon, N. D.
Trebi Minn. 11-31-19 Kindred Control
Edgeley. N. D. Waseca, Minn. Xinnesota
......
Malting Moisture, % 44 44 48 48 44 44 48 48 48 48 48
..
Malting Time, Days 5 7 5 7 5 7 5 7 5 5 5
..
Diastatic Power Maltose O L. equiv. 146 584 145 578 159 636 168 672 196 786 208 830 219 874 902 225 280 920 89 356 261 1044 183 732
Mljtoae Eqmvalent 8aamylase amylase 547 37 42 536 597 39 45 627 746 40 53 777 47 827 850 52 874 46 38 318 88 956 43 689
Alcohol Yieldhb. Proof Ga1./100 17 malt 7.76 7.93 7.91 8.21 8.21 8.22 8.14 8.37 8.80 7.46 8 .91 8.01
27
47
malt
ma%
8.46 8.87 8.65 9.12 9.09 9.10 9.13 9.49 8.94 89 . 4 3 54 8.65
9.88 9.57 9.57 9.85 9.65 9.30 9.56 9.85 9.72 8 . 9 08 9 9.66
VALUESON MALTSFROM DIFFERENT BARLEY VARIETIES AND ALCOHOL YIELDSFROM SPRINGWHEATWITH TABLE 11. AMYLASE
5% LEVELOF MALT I
Malt No Pabst 4228-30 mal-33 42 19-21 4207-09 4222-24 4189-91 4174-70 4195-97 2070 4573 4574 4575 4576 4577 4578 4579 4580 4581 4582 4583 4589 4467 4479 4484 4609 4620
Variety or Selection
Location Grown
...... Waseca Minn.
Madiso; Wis. Fargo Id IY. Edge& 'N.D. Brookinks 9. D. Davis, Caiif. Fargo N. D. Linooin, Nebr. Lincoln , Nebr. Waseoa. Minn. Wasecs, Minn. Waseca, Minn. Waseca hlinn. Waseca' Minn. Waseoa: Minn. Waseca, Minn. Waseca, Minn. Waseca Minn. Waaeca: Minn. Waseca, Minn. Madison Wis. Have. KAnsas Caiada Davis Calif. Maddon Wis. Madison: Wis.
Tregal Iowa 15 Peatland Oderbrucker Reno Winter Garton Rojo Trebi Kindred
Malting Moisture,
%
_
Malting Time, Days
48 48 48 48 48 48 48 48
46
45 45 45 46 45 45 45 45 45 45 45 45 45 45 45 45 45
8
6
6
6 6
6
6
6 6 6 6 6 6 6 6 6 6 6
sprouts extract-glucose medium and grown for another 24 hours at 30' C. The cells were removed from the medium by centrifugation, and were resuspended in water to make a volume of 20 ml. Ten milliliters of such a suspension were used to inoculate each 600-ml. portion of mash. After inoculation the volume was made to 750 ml. (equivalent to 17.8 grams of wheat per 100 ml.). Du-
TABLE 111. EFFECT OF HIGHMALTLEVELON ALCOHOL YIELDS FROM SPRING WEEAT I N 72-HoUR FERMENTATIONS Malt Psbat
4228-30 4234-35 4231-33 4207-09
Yields Proof Ga1./100 Lb. D r y Grain -Experhe& 1-Experiment 2* 5% 6% 7% 8% 5% 6% 7% 8% 10.32 10.29 10.45 9 . 9 5 10.00 9 94 9.92 9.93 10.28 10.44 10.33 10.51 10.21 10 29 10.01 10.16 10 60 10 42 10.33 10 60 10.52 10.40 10.33 10.27 10.29 10.29 10.23 10.39 10.31 10.25 i o : h 9 85 9 . 8 7 9 . 5 4 10.15 10.03 10 06 10.10 10.17
TABLE IV. ALCOHOLYIELDSFROM WINTERWHEATWITH 5% MAL^
IN
3 6 - H o u ~FERMENTATIONS
Proof Ga1./100 Lb. Dry Grain Malt No. Expt. 1 Erpt. 2 Pabst 9.31 9.26 10.21 4467 9.77 10.47 4470 10.38 4484 8.63 8.66 8.63 4577 8.79 10.16 4580 9.92 4589 8.98 8.77 4609 8.86 8.80 4620 10.16 10.21 * Of the 5% total, 1% was Pabst malt used for preconverting.
Diastatic Power Maltose O L. equv. 183 732 89 356 139 556 248 992 230 920 230 920 80 320 150 600 133 532 220 880 540 135 123 492 112 488 105 420 150 600 106 784 246 984 203 812 146 584 158 632 179 716 161 644 258 1032 159 636 91 364 162 648 226 904
Maltoae Equivalent 8aamylase amylase 689 43 318 38 484 72 89 908 874 46 73 847 60 260 37 563 486 46 87 793 46 494 57 a 5 73 415 77 343 54 546 88 696 93 891 86 726 52 532 43 589 126 590 48 598 108 924 538 98 325 39 612 36 817 87
Alcohol Yield Proof Gal./lpO kb: at Fermentation Time of: 36 hr. 72 hr. 9.84 8.95 10.19 9.55 10.39 9.96 10.44 10.07 9.87 8.53 9.84 9.39 9.84 8.80 9.39 8.10 8.63 9.62 9.39 10.23 9.83 10.18 9.82 10.42 10.22 10.43 10.12 9.98 9.03 10.36 9.92 10.45 9.96 10.42 10.14 10.24 9.95 10.50 9.80 10.44 10.27 .. 9.17 9.99 10.28 .. 8.54 8.91 10.08
...
.... ... ...
plicate 350-ml. aliquots were placed in 500-ml. Erlenmeyer flasks. A scrubber, consisting of a glass tube leading into an &inch test tube, about three fourths full of water, was attached to each of the flasks to catch the alcohol carried over during the evolution of carbon dioxide, The flasks were incubated a t 30" C. for the desired time with ocoasional shaking. At the end of the fermentahion period the contents of each flask were diluted to 500 ml. with tho water from the scrubber tube and more tap water. A 200-ml.aliquot of the diluted mash waR placed in a Kjeldahl flask, and about 95 ml. distilled off. This distillate was diluted to 100 ml., and alcohol was determined by means of a Westphal balance. Weight of alcohol was read from a curve made with known solutions. Yields were calculated aa proof gallons of ethyl alcohol per 100 pounds of total dry grain. The results of duplicate fermentations (duplicate flasks in the same run) agreed closely. The data are not given in the tables, but such flasks usually checked within 0.05 proof gallon per 100 pounds of dry grain. The results obtained with a malt in successive runs did not check so well. The variation was of the order of 0.0 to 0.5 proof gallon per 100 pounds of dry grain. Tables I11 and I V give data for two runs at different times. EVALUATION OF MALTS
Various malts differ A L C O H OYIELDS ~ FROM SPRING WHEAT. in rate as well as in ultimate degree of conversion of wheat starch Therefore, a method for evaluating malts should provide for comparison of these factors. The reduction of the quantity of malt to a limiting level or the use of short fermentation times are two evident Drocedures. Several fermentation exneriments in which
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
1144
TABLE V . EFFECT OF LENGTHOF FERMENTATION TIMEON ALCOHOLYIELDSFROM SPRING WHEAT Malt No. Pabat
4228-30 4234-35 4231-33 4207-09 4222-24 2070 4189-91 4195-97 4174-76
Yield, Proof Ga1./100 Lb. Dry Grain after Fermentation for: 36 hr. 48 hr. 72 hr. A . 8% Malt Level 9.81 8.50 9.50 9.71 10.19 9.85 9.17 10.00 10.25 10.06 10.17 9.74 10.15 10.18 9.81 10.19 9.73 8.91 9.41 7.77 10.16 10.00 10.10 9.39 10.00 10.26 10.26 9.00 10.00 10.26 10.26 8.95 9.47 10.20 10.10 8.54 10.00 9.85 9.32 7.75
24 hr.
E. Psbst
4228-30 4231-33 4219-21 4207-09 4222-24 2070 4189-91 4174-76 4195-97
7.54 8.36 9.60 9 66 7: 09 7.96
7.26 7.75 6.17 6.72
5% Malt Level 8.95 9.72 9.54 10.09 9:95 10.28 10.07 10.32 8.62 9.38 9.39 9.84 9.39 9.46 8.80 9.45 8.10 8.71 8.63 9.32
9.84 10.18 10.39 10.43 9.86 9.84 9.62 9.84 9.39
malt level and fermentation time varied were carried out to determine the most suitable combination for comparing malts. Pabst malt was used as a control throughout. A yield of 10 proof gallons of alcohol per 100 pounds of total dry grain was taken as a reasonable final standard in evaluating the fermentations. Such a yield was obtained regularly when proper conditions were observed and has been reported for the same batch of spring wheat by several alcohol producers. Table I shows the alcohol yields obtained with several malts at levels of 1, 2, and 4% and a fermentation time of 72 hours. The yields are averages of two runs. From approximately thirty malts tried in this manner, data were also arranged to show the influence of malting conditions on alcohol yields. The relation will be discussed later. None of the fermentations with these low levels of malt reached the standard yield of 10 proof gallons. Table I11 presents results obtained with higher levels of malt (5, 6, 7, and 8%) in 72-hour fermentations. Two of these were new malts. The yield of 10 proof gallons was obtained with all levels. Therefore levels above 5y0 appeared to be excessive in a 72-hour fermentation. Table V A shows the effect of shorter fermentation times with a malt level of 8%. None of the fermentations reached the standard yield of 10 proof gallons before 36 hours. The data in Tables I11 and V A seem to show that a concentration of malt less than Syo and a time of fermentation shorter than 72 01 even 48 hours would give the best conditions for evaluating a malt. To test this assumption, fermentations with 5y0 malt for various periods were tried. Table V B gives averages of two runs, The figures show that the earliest time a t which any of the fermentations reached a 10-gallon yield was 36 hours. At 48 hours three of the fermentations reached this figure and two came within 0.3 gallon of it. At 72 hours three more fermentations were close to the standard figure. The three fermentations yielding 8.4 gallons or better at 24 hours all reached a 10-gallon yield in 48 hours. Table V B seems to indicate that, if a single fermentation is to be relied on to evaluate a malt, a 5% concentration and 36-hour period are best. These conditions permitted some fermentations to reach the 10-gallon level but were rigorous enough to sort out slow fermentations. If two periods are to be used in rating the malt, a 24- and a 48-hour fermentation give a better basis for rating. If i t IS assumed that industrial practice calls for fermentation lasting approximately 48 hours, three of the malts qualified as excellent, two were good, three rated fair, and one was poor. Comparisoii of the data in Table V, A and B , suggests that malt activity was the limiting factor in the speed of the fermentations. With 8% malt the yield a t 24 hours was from 0.21 to 2.58 gallons (average 1.27) higher than at the 5% level. From these data it
Vol. 33, No. 11
appears that if the rate of starch conversion could be increased, the yeast would be adequate to convert the sugar to alcohol. ALCOHOLY m m s FROM WINTERWHEAT. Two runs were made with winter wheat, A total of 5y0malt was used of which 1% (Pabst malt) was required for liquefaction. Alcohol determinations were made a t the end of 36 hours. Table I V gives results of these fermentations. The yields correlated in most cases with those obtained with spring wheat when the same malts were used (Tables I1 and IV). This was expected since the starch contents of the two wheats were approximately equal. AMYLOLYTIC ACTIVITY OF MALTS AND ALCOHOL YIELDS
Table I1 records the characteristics of the malts in the conventional terms used in the malting industry. Diastatic activity was determined according to the Official Method of the American Association of Cereal Chemists (1). The results are expressed as degrees Lintner and maltose equivalent (degrees Lintner X 4). The a-amylase dextrinizing activity and ~-ataylaseactivity were determined by the methods of Sandstedt, Kneen, and Blish (6) and Kneen and Sandstedt (a), as modified by Olson, Evans, and Dickson (4). The analytical data are given in Tables I and 11. Table I shows the influence of malting moisture and germination time on the amylase values of the malts and the alcohol yields. Increased malting moisture and longer time increased the amylase values somewhat and, in most cases, the alcohol yields. As indicated earlier, alcohol yields were obtained with thirty-one malts a t a 2% malting level and 72-hour fermentation time. Correlation coefficients were calculated from the data as an aid to interpretation. I n this experiment alcohol yield correlated significantly with both p- and a-amylase when low malt concentrations and long fermentation times were used. Table I1 presents analytical data on another group of malts representing several m e r e n t varieties and selections grown a t different locations, and the alcohol yields obtained with 5% malt at 36- and 72-hour fermentations. Significant correlations were found between alcohol yields and a-amylase only a t the 36-hour fermentation. No relation between alcohol yield and diastatic power or ,??-amylasewas indicated. When higher malt concentrations and short fermentation times were used, the relations between alcohol yield and amylase values on the malts were different from those given for the previous series of malts. Although only suggestive, the data indicate that a-amylase may be of more importance in the rate of fermentation; in longer fermentations a t low malt concentrations, both amylase components assume importance. With a malt concentration of 5% and a 72-hour fermentation, the values level off sufficiently so that no relations with the original amylase values of the malts are indicated. While the amylaae activity of a malt is informative, it alone is not adequate for evaluating the malt for alcohol production; other undetermined factors also play an important role. Therefore, the best evaluation of a malt is the determination of the amount of alcohol produced in a fermentation with the malt. ACKNOWLEDGMENT
The authors are indebted to M. J. Johnson, A. D. Dickson, W, C. Frazier, P. W. Wilson, and J. G. Dickson of the University of Wisconsin for counsel in the planning and execution of the work, Others who participated in the laboratory work are H. J. Koepsell, Ruth Evans, F. B. Gailey, and B. A. Burkhart. LITERATURE CITED
(1) Am. Assoc. of Cereal Chem., Cereal Laboratory Methods, 1941. (2) Assoc. of Official Agr. Chem., Methods of Analysis, 5th ed., p. 359 (1940). (3) Kneen, E.,and Sandstedt, R. M., Cered Chem., 18,237 (1941). (4)Olson, W.J., Evans, R., and Dickson, A. D., Zbid., 21,533 (1944). (5) Sandstedt, R. M., Kneen, E., and Blish, M. J., Zbid., 16, 712 (1931). PUBLIBHED with approval of the Director,Wisconsin AgriculturalExperiment Station.
