Saccharification of Starchy Grain Mashes for the Alcoholic Fermentation Industry J
Comparison of Several
LORIN SCHOESE, ELLIS I. FULMER, 4ND L. A. UNDERKOFLER
Saccharifying Agents'
Iowa State College, Ames, Iowa
Three amylolytic substances-malt, moldy bran, and soybean meal-have been employed alone and i n combinations for saccharifying tw-o types of corn mashes, differing i n concentration at cooking, for alcoholic fermentation. When used alone, moldy bran was most effective while soybean meal was of little worth. Combinations of two of the enzymic materials gave somewhat increased alcohol yields over those obtained with a single preparation in some cases; in other instances there was no change.
Lower alcohol yields are generally obtained from grain mashes saccharified with dilute mineral acids rather than with amylase; addition of active moldy bran to such acid-hydrolyzed corn mash resulted in normal alcohol yield. The greater variety of enzymes present explains the greater effectiveness i n this respect of moldy bran over the other amylolytic materials tested. Soybean meal employed in combination with a very small proportion of moldy bran also effectively increased alcohol yield from acid-saccharified corn mash.
I
K T H E first paper of this series ( I S ) studies were reported
nearly optimum ratio. I n view of the above facts, saccharification studies are here presented on the three amylolytic materials, soybean meal, malt, and moldy bran, alone and in combination. The soybean meal was prepared by grinding the whole fresh bean. Two commercial barley malts were employed, and the moldy bran was prepared as described in the previous paper (13) by growing a strain of Aspergillus oryzae on moistened wheat bran in a rotating drum. An improvement was made in the method of drying the moldy bran. After suitable growth had occurred, instead of the moist material being spread on papers to dry, it was allowed to remain in the drum, the air-humidifying flask was removed, and dry, sterile air was passed into the slowly rotating drum until the moldy bran mas dried. The air-dried moldy bran and the malts were ground before using.
on mold amylase preparations and their use in the saccharification of corn mash for the alcoholic fermentation. Of the eight species of bacteria and twenty-two species or strains of molds studied, two strains of Aspergillus oryzae were found to be most satisfactory. Optimum conditions were described for the preparation of highly active saccharifying preparations by growing the mold on wheat bran in rotary drums; best results were obtained by employing the moldy bran in the same manner as malt. On the average, the use of the moldy bran gave yields of ethanol about 12 per cent higher than those obtained with a good dried barley malt. Amylase is an enzymic complex, and the amylases from different sources may differ considerably in their effect upon starch. I n general, amylase possesses an amyloclastic or starch-liquefying enzyme which converts starch into dextrins and a saccharogenic or sugar-forming enzyme which hydrolyzes the starch or dextrins to maltose. These factors have been separated (2,6,11, 15) and the observed differences in amylases are in large part associated with differing proportions of the two fractions; other factors may be involved, such as coaction of specific natural activators or inactivators of the starch degradation (3, 7 , 14, 16). Since amylase is a complex, the use of different amylolytic materials in combinations might give better saccharification for alcoholic fermentation than when employed alone. This effect would be associated either by complementary action or by furnishing the various fractions of the complex in more 1
Mashing Procedures Before actual fermentation tests were made with the amylolytic materials, investigations were carried out to determine the best methods for the preparation of the mash. As prepared in the laboratory, mashes containing 15 to 20 grams of corn meal per 100 ml. of water, proportions frequently employed in industrial practice, are so thick and lumpy after gelatinization of the starch that the hydrolytic enzymes cannot exert optimum activity. Hence premalting-i. e., preliminary liquefaction of the raw starch-is often resorted t o in the laboratory to provide a workable mash. The problem of lumping is less acute in commercial practice owing to the use of efficient stirring devices in the cookers; hence, premalting is rarely used on the plant scale.
The first paper i n this series appeared in 1939 (23).
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INDUSTRIAL AND ENGINEERING CHEMISTRY
The following procedure was used in studies on premalting: For two series of 300-ml. Erlenmeyer flasks containing the proportions of amylolytic materials indicated in Table I and 20 grams of corn meal per flask, 100 ml. of water a t 75' C . were added t o each flask, and the resulting temperature of 55 C. was maintained by a constant-temperatnre bath. One series wis removed from the bath a t the end of 30 minutes and the other after 60 minutes. Before cooking, both series were steamed for about 10 minutes to effect initial gelatinization; then the flasks were shaken thoroughly to give a smooth suspension. The mashes were cooked a t 20 pounds per square inch (1.4 kg. per sq. cm.) steam pressure for 30 minutes. After cooling to 55" C., both series were malted at this temperature for 60 minutes with 10 per cent malt based upon the weight of corn in the mash. Observations on the condition of the mashes, after completion of malting, are given in Table I. O
TABLE I.
PRELIMINARY
Amylolytic Material Malt .Moldy bran Soybean
LIQUEFACTION O F CORN MASH BY THREE
AMYLOLYTIC MATERIALS (Normal Mash)
meal
Proportion of Material, 7*of Corn 0.3 0.6 1.0 0.3 0.6 1.0 1.0 2.0 3.0
Condition of Malted Mash after Heating at 55' C. befoye Cooking for:
--
30 min. Lumpy
Smooth Smooth
Lumpy
Smooth Smooth Very lumpy
Lumpy Lumpy
-
60 min. Smooth Smooth Smooth Slightly lumpy Smooth Smooth Very lumpy
Lumpy
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The thick mashes were treated as above except that they were diluted with an equal volume of water after cooking. The acid concentrations varied from 0.03 N to 0.20 X. The thick mashes cooked with acid of concentration less than 0.10 N could not be mixed readily with water and gave lumpy mashes on malting. The use of 0.12 N or stronger acid gave mashes which mixed readily with water on stirring and had a smooth consistency after malting. That is, the preliminary treatment of the thick mashes with 0.12 -V or stronger acid accomplished a liquefaction not attainable by premalting. In view of the above findings, the following procedures mere adopted for preparing the mashes for fermentation studieb: NORMAL MASHES. Into each 1000-ml. Erlenmeyer flask 100 grams of ground yellow corn meal and 500 ml. of 0.02 N hydrochloric acid were introduced. The mashes were steamed, shaken thoroughly, and cooked for 30 minutes a t 20 pounds per square inch (1.4kg. per sq. em.) steam pressure; the pH was adjusted to 5.0 by the addition of concentrated ammonium hydroxide. The mashes were then saccharified in the presence of the amylolytic material a t 55 C. for 60 minutes; the flasks were frequently shaken during this period. THICK MASEES. In each 1000-ml. Erlenmeyer flask 100 grams of corn meal and 167 ml. of 0.12 N hydrochloric acid were placed, steamed, and cooked as above. To the contents of each flask while still hot (70' to 90 C.) were added 333 ml. of distilled water a t 60" C., and the mash was mixed by shaking or with a stirring rod. The above procedure gave a smooth mash with a final concentration exactly that of the normal mash. The pH was adjusted to 5.0 and saccharified in the same manner as described above. Initial reducing sugars, before malting, ran about 5 grams per flask. O
Slightly lumpy
Comparison of Malt, Moldy Bran, and Soybean Meal as Saccharifying Agents
It is evident that the soybean meal is inefficient in the preliminary liquefaction. For the 30-minute period 0.6 per cent of malt and of moldy bran are required; for the 60-minute period 0.3 per cent of malt is sufficient. I n commercial practice the cost of cooking and cooling large volumes of mashes reaches a considerable figure. Reduction in the volume of mash cooked by increasing the concentration may be resorted to in order to eliminate part of this cost, and the resulting thick mash is subsequently diluted with cold water t o the proper concentration for fermentation. Hence, other series were run, similar to those described above, with thick mashes. The thick mash contained 60 grams of corn meal per 100 ml. of water; that is, the mash contained about three times the concentration of corn meal employed in the previous studies. The malt concentrations varied from 0.5 to 3.0 per cent, but in no case was a smooth mash obtained on malting. Evidently the premalting technique was not a p plicable to the thick mash employed. I n view of the ineffectiveness of premalting of the thick mash, experiments were made with hydrochloric acid as the agent for preliminary liquefaction both of normal (20 grams of corn meal per 100 ml. of water) and of thick (60 grams of corn meal per 100 ml. of water) mashes. The normal mashes, containing 0 01 N to 0.1 N acid were steamed, shaken thoroughly, and cooked for 30 minutes a t 20 pounds per square inch steam pressure. Preliminary studies had shown that best saccharification and subsequent highest yields of ethanol were obtained at p H 5.0; hence all the cooked mashes were adjusted to this value by the addition of concentrated ammonium hydroxide. The mashes were then malted for 60 minutes at 55' C. using 10 per cent of malt. With 0.05 N acid or stronger, the mashes were liquid without malting. The mashes cooked with 0.02 N to 0.03 N acid gave smooth, liquid mashes upon malting; with 0.01 N acid the mash was slightly lumpy after malting. That is, cooking with 0.02 N to 0.03 N hydrochloric acid accomplished the same purpose as the premalting.
The procedure employed in the fermentation of the mashes was described in detail in the previous publication ( I S ) . After saccharification the contents of each flask were cooled to 30 'C. and inoculated with 50 ml. of a n active, 24-hour culture of Saccharomyces cerevisiae growing in beer wort. The alcohol was determined after 3-day incubation a t 30' C. The alcohol yields are given as per cent of theoretical. Fermentation experiments are often characterized by some uncertainty due to the fact that duplicate fermentations, run under seemingly the same conditions but a t different times, frequently give different results. Hence quantitative cornparisons of yields of alcohol produced in different runs csnnot be relied upon. I n each case in which comparisons are to be made, it is necessary to run a complete series. Moreover, to verify trends, during this investigation each fermentation series was run a t least twice and only conclusions are reported which were confirmed in duplicate series.
TABLE 11. ALCOHOL YIELDS WITH MALTA, MALTB, BRAN F
hmylolytio Materid, % of Corn
-Normal Malt
A
4VD
MOLDY
Alcohol Yield, yo of Theory------Thick MashesMashesMalt Moldy Malt Moldy B bran B bran
A preliminari comparison was made of yields of alcohol from mashes saccharified with soybean meal, with malt, and with moldy bran. I n quantities equivalent to 10 per cent, of the corn in the mash the soybean meal gave such poor yields that in future work it was used only in combination with the other two materials.
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Table I1 shows the yields of ethanol, from normal and thick mashes, employing malt and moldy bran as saccharifying agents. The superiority of the moldy bran is evident. Since malt B proved to be more effective than malt A, the former was employed in the subsequent studies. It is of special interest t o note that the proportions of malt and of moldy bran required for maximum alcohol yields are less with the thick than with the normal mash. The reasons for these results are being investigated. Table I11 presents the effect upon alcohol yield of combinations of moldy bran, soybean meal, and malt as saccharifying agents, both in normal and in thick mashes. As noted above, the soybean meal is not efficient when used alone and hence was employed only as a complementary material. I n the experiments involving moldy bran or malt as the principal saccharifying agent, such concentrations were employed as to give maximum yields; hence any significant change in alcohol yield will be associated with the specific effect of the added agent. The addition of soybean meal with the moldy bran shows a slight trend toward improved yields in both types of mashes; the addition of malt gives a small increase in yield with the normal mash but is without appreciable effect in the thick mash.
OF TABLE 111. ALCOHOLYIELDSFROM USE OF COMBINATIONS MOLDYBRAN(MB), SOYBEAN MEAL(S), AND MALT (M)
-Kormal MashesProportion of Material, yc of Corn MB S If 10 0 0 10 1 0 10 2 0 10 3 0 0 10 0 10 0 1 2 10 0 10 0 4 12 0 0 0 1 12 12 0 2 0 4 12 n 12
Alcohol Yield.
%
83.1 85.2 85.3 84.2 78.6 82.0 82.8 82.2 70.7 72.1 73.5 77.0 67.2 70.6 73.9
-Thick MashesProportion of Material, yo of Corn MB S &I 8 0 0 8 1 0 s 2 0 8 4 0 8 0 0 S O 1 8 0 2 8 0 4 0 0 10 0 1 10 10 0 2 0 4 10 0 0 10 1 0 1( 2 0
Alcohol Tield,
%
81.8 84.1 84.5 85.6 85.4 83.8 85.4 86.8 64.5 65.0
65,O 65.0
VOL. 32, h-0.4
Severson (12) studied the saccharification of mashes of various grains with hydrochloric acid. The maximum alcohol yields were not obtained from mashes which showed the highest titer for reducing sugars. It is apparent that a distinction must be made between the reducing sugar content, as determined by analysis, and the fermentable sugars as determined by actual yields of alcohol. Severson found that for corn maximum alcohol yields were obtained by heating the mash for 2 t o 3 hours a t 25 pounds per square inch (1.8 kg. per sq. cm.) steam pressure in the presence of 0.10 N hydrochloric acid. However, the maximum alcohol yields were, on the average, 12 per cent higher with malt than with acid as the saccharifying material. The lower alcohol yield with acid hydrolysis is probably associated with one or more of the following factors: (a) presence of nonfermentable carbohydrates due to either incomplete saccharification or reversion of dextrose, ( b ) lack of nutrients or growth stimulants, (c) formation of toxic or inhibitory substances during the hydrolysis. Studies are here presented on the above factors and on the development of methods for obtaining maximum alcohol yields from corn mashes saccharified by means of hydrochloric acid. The following procedure, using the conditions found by Severson to give maximum alcohol yields, was employed in the preparation of the acid-saccharified corn mashes. Into each 1000-ml. Erlenmeyer flask were placed 100 grams of corn meal and 500 ml. of 0.10 N hydrochloric acid, and the mixture was cooked for 2.5 hours a t 25 pounds per square inch (1.8 kg. per sq. cm.) steam pressure. After cooking, the pH of each flask was adjusted to 5.0 with concentrated ammonium hydroxide. With this procedure the conversion of the starch to reducing sugars (as dextrose) was about 90 per cent of theory. When amylolytic materials were employed, the technique was as described above.
O N ALCOHOL YIELDSOF ADDINGAMYLOLYTIC TABLE IV. EFFECT M.4TERIALS TO ACID-HYDROLYZED C O R E MASH
63.5 Amylolytic Material Added None (control) Malt Moldy bran Inactivated malt Inactivated moldy bran
The addition of soybean meal to the normal mash saccharified with malt gives considerable increase in alcohol yield but is without effect in the thick mash. The addition of moldy bran to both types of mashes gives the expected increase in yields, but there is no advantage in using combinations over using the moldy bran alone.
Comparison of Hydrochloric Acid and Amylolytic Materials as Saccharifying Agents Dilute mineral acids are extensively employed (8, 9) in the production of corn sugar (dextrose) and have been suggested for use as saccharifying agents in the fermentation industry. However, it has been found in practice that alcohol yields are lower in mashes saccharified with acids than for those treated with malt. The conversion of starch by means of acid goes from starch t o dextrins t o maltose to dextrose to polysaccharides. The reversion of dextrose to polysaccharides accounts for the fact that approximately one third of the carbohydrate content of hydrol, the final mother liquor from the commercial manufacture of crystalline dextrose, is not fermentable by yeast. About one iifth of this nonfermentable sugar has been shown (1, 4) to be the reducing disaccharide, gentiobiose.
Proportion of Material, % of Corn
Alcohol Yield, % of Theory
Increase in Yield,
0
81.8 83.1 92.9 81.0 82.1
....
5
5 5 5
%
1.59 13.58 -0.98 0.37
Reduoing Sugar in Mash. G./100 M1.
9.61 9.92 10.01 9.75 9.60
Table IV shows the effect upon alcohol yield of treating the acid-saccharified mash with 5 per cent malt or moldy bran as amylolytic agent. The reducing sugar values of the mashes, calculated as dextrose, are also included in Table IV. Treatment with malt gave some increase in alcohol yield while the increase with the moldy bran was considerable; these findings are confirmed by the data in Table V. One series (Table IV) included fermentations in which the amylolytic agents were added t o the mash a t the boiling temperature and the enzymes were destroyed by heating the mash for 15 minutes a t 15 pounds per square inch (1.0 kg. per sq. cm.) steam pressure. The inactivated materials had no appreciable effect upon the yield of alcohol. Evidently the high yield with the moldy bran is not associated with the addition of nutrients or of growth stimulants, since these would not have been destroyed by heat treatment, nor can the lower yields in acid-saccharified mashes be due to the formation of toxic or inhibitory substances during the hydrolysis. Another point to confirm these conclusions is that in the control the yield of 81.8 per cent of theory, based on total starch, represents a yield of 91.9 per cent based upon reducing sugars actually present.
APRIL. 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
This is a satisfactory result and indicates that neither nutrients nor growth factors are lacking, and that toxic or inhibitory substances are not present.
547
in near optimum proportion of about 5 per cent of corn used, again confirming the superiority of the enzymic system of the mold preparation. The effect of the addition of soybean meal with emaller quantities than the optimum of moldy bran is of special interest since this resulted in increased alcohol yields. The addition of soybean meal was so effective that a combinaTABLEV. EFFECTOF MALT,SOYBEAS MEAL,AND MOLDY BRAN UPON T H E ALCOHOL YIELDS FROM ACID-SACCH~RIFIED CORS~'IASH tion of 1 per cent soybean meal with 1 per cent moldy bran gave nearly as high an alcohol yield as that given by 5 per Mold) Bran a n d cent moldy bran alone. Malt and So\ bean Meal Moldy B r a n and 3Ialt Soybean Meal ,-' The frequently repeated suggestion that acid sacchariProportion of Proportion of Proportion of Material, Material, Material, fication of starchy mashes be used in the fermentation industry % of Corn Yield, % of Corn Tield, 70of Co-n Tleld, to replace the malting process seems, on the basis of the exhl S 70 MB hl 70 MB 5 % perimental findings, to be little warranted unless this process 0 0 0 76.8 76.3 0 0 0 84.0 1 0 0 1 0 76.4 82.5 2 87.1 is used in combination with mold amylase. The saving in 2 0 2 0 0 88.6 76.0 3 82.9 0 3 0 3 0 malt cost with such a process would be considerable, but 89.8 75.5 82.9 5 92.4 0 5 equipment which is acid resistant would be required for cook2 0 2 78.0 75.7 0 1 .3 2 83.0 0 0 3 87.8 78.5 ing the mash. The mash itself resulting from the acid hydroly0 1 1 1 5 8 2 . 2 9 0 . 9 77.0 3 1 1 82.5 78.5 1 2 1 sis is quite liquid in consistency and would be easier to 90.5 1 2 3 1 90.7 78.3 handle than the thick, gelatinized mash obtained in the 2 1 3 2 81.2 76.4 2 2 1 1 82.6 2 3 90. 7 76.5 customary mashing process. This would result in a saving 2 2 2 2 2 91.2 82.7 5 77.5 2 in power needed for mixing the mashes; on the other hand, 2 3 3 91.3 76.6 the longer periods of cooking required for the acid method would require more power. It might be less difficult and cheaper, in the long run, to employ complete saccharification From the data obtained it is evident that the increased by means of moldy bran, as outlined in the previous paper yields of alcohol from acid-saccharified mashes t o which (IS), rather than preliminary acid hydrolysis followed by moldy bran is added must be due to enzymatic action. A completion of saccharification with moldy bran. wide variety of enzymes is produced by the mold. WohlgeOn the other hand, another possibility for employing acid muth ( I 6 ) , Nishimura (IO), and Harada ( 5 ) have identified hydrolysis for fermentation mashes involves the separation of a total of a t least twenty-two different enzymes in preparathe starch from the remainder of the corn by the customary tions from Aspergillus oryzae. The increased yields of alcohol wet milling process and subsequent fermentation of this obtained by adding moldy bran are probably largely due t o starch after acid saccharification. More complete and more more complete conversion to fermentable sugars of the nonrapid hydrolysis of the separated starch is to be expected fermentable polysaccharides remaining in the acid-saccharified under these conditions. However, the probIem of reversion mashes. The reducing sugar values given in Table IV tend of dextrose to nonfermentable polysaccharides will still be to confirm this conclusion although such analyses are of little encountered, as well as a new problem of yeast nutrition. value for comparative purposes. The end product from the These factors might still make desirable the supplementary action of malt is largely maltose while the mixed enzymes of use of mold enzyme preparations. moldy bran produce dextrose; with either, a mixture of sugars is certainly present in the mashes, allowing no possibility of Acknowledgment a n accurate calculation. The increase in sugar content of the mash to which malt was added was approximately that The authors wish t o thank Julian Banzon for his assistance to be expected from the carbohydrgtes of the malt itself. with much of the routine work involved in this investigation. Practically no utilizable carbohydrate was added with the The study 'was supported in part by a grant from the research moldy bran. funds of the Science Division of Iowa State College for the The more favorable action of moldy bran over malt in instudy of the fermentative utilization of agricultura,l products. creasing alcohol yields from acid-saccharified mashes may be associated with the greater variety of enzymes present; for instance, the emulsin of the moldy bran would hydrolyze to Literature Cited dextrose such carbohydrates as gentiobiose] which possess Berlin, H., J . Am. Chem. Soc., 48,2627 (1926). 0-glucoside linkages, while malt would be unable to acCaldwell, M. L., and Doebbeling, S. E., J. B i d . Chem., 110, 739 complish this conversion. Moreover, it is possible that the (1935). mold proteolytic enzymes produce more available nitrogen Chrzasscs. T., and Janicki, J., Biochem. Z., 260, 354 (1933); 264, 192 (1933) ; 265, 260 (1933). for the yeast. The presenre of ammonium salts from the Coleman, G. H., Buchanan, M. A,, and Paul, I'. T., J . Am. neutralization of the acid and the fact (mentioned above) Chem. Soc., 57, 1119 (1935). that the alcohol yield based upon reducing sugars actually Harada, T., IND. EXG.CHEM.,23, 1424 (1931). present in the acid-saccharified mash amounted t o 91.9 per Kuhn, R., Ber., 57B, 1965 (1924); Ann., 443,1 (19251. Myrback, K., and Myrback, S., Biochem. Z., 258, 158 (1933). cent of theory, however, relegate this possibility t o a place of Newkirk, W. B., IND. ENQ.CHEY.,16, 1173 (1924). minor importance. (9) Ibid., 31, 153 (1939). Table V presents the effect of combinations of malt, soy(10) Nishimura, S., Chem. Zelle Gewebe, 12, 202 (1925). bean meal. and moldy bran upon the yield of alcohol from (11) Ohlsson, E., Compt. rend. soc. biol., 87, 1183 (1922); Compt. Tend. trav. lab. Carlsbera, 16, No. 7. 1 (1926); 2. physiol. Chem., acid-saccharified mashes. I n these series no significant in189, 17 (1930). crease in alcohol yield was obtained with malt alone. There (12) Severson, G., Iowa State Coll. J . Sci., 11, 215 (1937:l. was a slight trend for the addition of soybean meal to augment (13) Underkofler, L. A., Fulmer, E. I., and Schoene, L., IND. ENQ. the yield, but the increases were not of practical significance. CHEM.,31, 734 (1939). (14) Waldschmidt-Leita, E., and P u r r , A,, 2.physiol. Chem., 203, 117 There were no advantages in combinations of malt and soy(1931); 213, 63 (1932). bean meal over the soybean meal alone, nor in combinations (15) Waldschmidt-Leits, E., Reiohel, M., and Purr, A., Naturwissenof moldy bran and malt beyond that of the moldy bran itself. schuften, 20, 254 (1932). The latter gave a 10 per cent increase in yield when employed (16) Wohlgemuth, J., Biochem. Z., 39, 324 (1912). . 7 -
