Saccharification of Wheat by Fungal Amylases for
Martin Roberts, Stephen Laufer, Earl D. Stewart, and Leonard T. Saletan SCHWARZ LABORA+ORIES,INC., NEW YORK, N. Y.
ALCOHOL PRODUCTION analysis of the grains is given in Table I. The mold bran was a commercial product called Polidase-C, prepared by Schwarz Laboratories. On a small scale it is prepared by mixing wheat bran with an equal weight of water and spreading the mixture in one-inch layers on aluminum trays. After sterilization in an autoclave for 30 minutes a t 20-pound pressure, the trays of bran are cooled and inoculated aseptically with a culture of Aspergillus oryzue. Incubation is a t 25 " C. until the mold begins to sporulate (about 4 days, as a rule). For the purposes of these experiments, a t this stage the moldy bran is mixed with l ' j ~ volumes of 95% alcohol (1500 ml. of alcohol to 500 grams of bran and 500 grams of water) to kill the mold spores. The alcoholic extract is pressed out with a hydraulic press. The dried, ground press cake, knowr as Polidase-C, was used in the experiments to be described. Two methods of cooking, pressure and atmospheric, are used by distilleries. Both were used in these experiments, and the procedures followed for wheat and 3% mold bran are given to illustrate the mashing methods:
Mold bran is compared with barley malt for the saccharification of mashes intended for production of industrial alcohol. Five grains were tested, one corn, two types of wheat, and two corresponding granular wheat flours. Mashes were made by both pressure (125' C.) and atmospheric cooking (66' C.); they were saccharified by using 8% malt, 2.5-4% dry mold bran, 3% undried mold bran, or 1% malt for cooking and 2% dry mold bran for saccharification. Wheat mashes cooked at atmospheric pressure and saccharified with 2.5-3.0% mold bran yielded 2-14% more alcohol than did similar mashes saccharified with 8% malt. Malt was superior for pressure-cooked mashes. Mold bran saccharification at 30' C. was equal or slightly superior to mold bran at 52.5' in both pressure- and atmospheric-cooked mashes. In pressure-cooked mashes malt saccharification at 5'7.5' C. was generally superior to mold bran at either 52.5' or .30°, but gave lower alcohol yields in atmospheric cooked mashes than mold bran saccharification at 52.5' or 30' C.
PRESSURE MASHING..Four hundred milliliters of 'water are heated t o 50" C., and 97 grams of wheat and 0.6 gram of dry mold bran are added. The temperature of the mash is held at 50' C. for 16 minutes while stirring constantly and then raised over a 15minute period t o 66" C. where it is held for 30 minutes. Durin the next 16 minutes it is raised to 93" where it is held for a seconj 30-minute period. Finally the mash is autoclaved for one hour a t 20-pound steam pressure before being cooled to the temperature chosen for conversion. ATMOSPHERIC MASHING. The procedure is the same as for pressure meshing until a temperature of 66" C. is reached. It is held a t 66' C. for 55 minutes and then cooled to the conversion temperature within 6 minutes.
T
t4
HE need for increased output of ethyl alcohol from grain for war purposes has made the saccharification of grain mashes by agents other than barley malt of great practical interest. Such agents would lessen the demands on the supply of barley malt, already somewhat strained. Any improved yield or increased output of grain industrial alcohol through their use would be highly desirable. Fungal amylases for saccharification of distillery mashes have been advocated by investigators (4) who have shown that the yield of alcohol from grain is inThe combined weight of grain and total saccharifying agent creased by using mold bran instead of malt. used was 100 grams in all experiments. One gram of malt or I n the work reported here, the use of mold bran for the sacwet mold bran was used for liquefaction in experiments involving charification of wheat mashes was investigated. Mold bran these materials. percentages from 2.5 t o 4.0 were employed, and the alcohol yields Both types of mashes are CONVERSION AND FERMENTATION. from these mashes were compared with those obtained when converted and fermented in the same way. A slurry of 2.5 8% malt was used as saccharifying agents. grams of mold bran is prepared and held a t the conversion temIn the course of the work it was noticed that malt gave better perature for 15 minutes before being added to the mash, which is liquefaction of the mash than did mold bran, whereas the latter then held a t the conversion temperature for another 30 minutes gave better conversion than malt. To see if these favorable efbefore it is cooled to 30" C. Conversion temperatures of 52.6" fects could be combined to advantage, mashes were made using and 30" C. were used. The mash is diluted with water:o a total 1% malt for liquefaction in cooking and 295 mold bran for conversion. In the pre.paration of mold bran, drying is necessary to preserve it. It has been suggested (4) that TABLE I. ANALYSIS OF GRAIN" mold bran could be used undried, provided it was Winter Spring obtained immediately after the growth gf the mold A.O.A.C. S ring Granular Granular Winter Wheat Method (1) Wheat $heat Corn Wheat was complete. This would save the drying cost and 13.12% 13.87% P.363,par.2 9.82% avoid possible losses of enzyme activity during the Moisture 10.79% 10.68% 0.55 0.56 P.354,psr.8 1.67 1.82 1 24 drying period. A series of undried mold bran mash$$tein 8.84 13.39 14.36 11.64 12 69 P.26,por.23 0.94 0.71 P.356,prr.22 1.74 1.89 4 14 ings was therefore made under conditions found 1.84 2.72 2.63 0.63 0.63 P.368,par.27 63.86 63.68 P.369,par.31 57.87 56.93 most favorable for saccharification by dried mold Starch 69.13 Reducing sugar bran. (oalcd. as dextroae) None None None None None P.368,par.28 The grains used were obtained through ComNonreduoing sugar modity Credit Corporation. Corn and wheat wyere (aalod. &s BU1.48 1.94 1.68 1.66 1.37 P. 369, par.29 orme) supplied by Norris Grain Company, the granular Diastatic activjty of malt on dry basis w ~ 194' s Lintner a t 20' C. ( 1 , p. 161, par. 48); wheat flour from Intern&iod Milling Company, dextrinogenic activlty of mold bran was 64 units per gram (3). and the malt from Pabst Brewing Company. An
fisbr
811
',
INDUSTRIAL AND ENGINEERING CHEMISTRY
812
Vol, 36, No. 9
way until the yeast becomes adapted as shown by normal appearance and absence of irregular-shaped and thick-walled cells. About three transfers are necessary. Winter Sorine Spring Winter Spring Granular Grhnulir Granular The final culture is kept in the refrigeraConWheat Wheat Flour Whext Flour version Corn, Wheat _ _ _ Pres- Atmos- Pres- Atmostor and used as a stock culture. Stock T t m p . , Pres- Pres- Atmos- Pres- Atmoscultures are transferred a t least once every C. sure sure pheric sure pheric sure pheric sure pheric Saccharifying Agent 2 weeks. To prepare the inoculum for a 57.5 10.37 10.05 9.37 9 . 6 8 9.53 11.69 1 1 . 1 9 11.58 10.90 mash, 1 ml. of stock culture is used to in52.5 10.36 8.92 1 0 . 4 5 9.04 10.09 11.89 1 2 . 2 5 11.67 12.17 oculate 20 ml. of wheat wort which is then 30.0 10.34 9.57 10.35 9.80 10.10 1 1 . 3 5 12.39 11.87 12.42 52.5 10.12 7.76 9.52 8 . 8 5 10.22 11.02 12.15 11.21 12.13 incubated at 30" C. for 20 hours. Avolume 30.0 10.43 9 . 5 8 9.82 9 . 3 8 10.18 11.19 12.22 11.66 12.43 of the 20-hour culture equal in milliliters 52.5 10.50 7 . 9 6 10.10 9 . 2 4 10.35 3% mold bran .. . 12.42 .. to 3% of the weight of the wheat mash 30.0 . . . . . . 10.34 . . . . . . ... .. .. ... 12.39 12.24 52.5 .. . . . . 10.17 . . . 9 . 9 7 . . . l2:40 12.70 4% mold bran in grams is centrifuged. The remainder 52.5 9.93 . . , 1 0 . 2 5 . . , 9 . 7 3 ,.. 12.47 . . . 1 2 . 3 5 3% wet mold branC of the 20-hour stock culture is used to pre30.0 10.24 .. . 9 . 8 3 . , . 9 . 2 0 . . . 11.96 .. . . 1 1 . 6 8 pare a new stock culture. I n this way the a Yields are corrected for alcohol produced from malt. Alcohol from mold bran is negligible. All adaptation is continued and a fresh stock yields are averages of two or more results. b For premalting. Sample contained 50% moisture, therefore 6 grams were used. culture is maintained. After centrifuging, the supernatant liquid is poured off and the yeast is resuspended in 10 ml. of water. The suspension is centrifuged and TABLE111. COMP~RISON OF MALTAND MOLDBRANYIELDS the wash water poured off. The mashed yeast cells are suspended (IKPERCENT) Winter Surine in 3 ml. of water and added to the wheat mash. The purpose of Granular G c a n u k r the washing is to eliminate errors due to alcohol or fermentable Winter Spring Wheat Wheat substances present in the yeast inoculum. Corn Wheat Wheat Flour Flour Grain For granular wheat flour mashes the yeast is prepared in a 10.37 10.05 9.68 11.69 11.58 Yield using ma14 similar way. For the corn mashes yeast from a tube slant less 10.50 10.46 1 0 , 3 5 12.47 12.70 Highest yield using moldbran than a week old is suspended in 3 ml. of water as for wheat, but Results elsewhere reptd.a the adaptation technique is omitted. The suspension is used as a Average 10.50 10.04 9.77 11.44 11.24b stock culture; the same procedure is followed as for wheat mashes Highest 10.71 10.59 10.20 11.82 11.24 Theoretical yieldC 11.72 11.31 11.17 12.93 12.99 except that the yeast is propagated in barley malt wort.
TABLE 11. YIELDOF ALCOHOL" IN PROOF GALLONS PER 100 POUNDS GRAINON DRYBASIS
% of theoretical yield
90 92 93 96 98 (mold-bran) a By other laboratories using the same grains: various mashing procedures were used but the saccharifying agent was 8?, malt,. b Only one laboratory reported yields on t is grain. Based on the starch a n d sugar content.
weight of 550 grams (600 grams for corn), making the mash concentration equivalent to 33 gallons per bushel of wheat or 34 gallons per bushel of corn. A sample of about 200 grams is removed for testing and analysis. The rest of the mash is inoculated with 3% yeast and placed in a n incubator a t 30" C. for 4860 hours, depending on the rate of fermentation. After completion of the fermentation, 100 grams of the fermented mash are distilled, and the remainder is used for testing and analysis. The alcohol content of the distillate is determined by a pycnometer and corrected for the alcohol yield due to the malt, when malt is used. The amount of alcohol obtained from the mold bran is negligible, and no correction was made when this sacchrifying agent was used. PREPARATION OF YEAST INOCULUM
.
Early in the experiments i t was noticed that the yeast propagated in wheat, and especially in granular wheat flour mashes saccharified by malt, had irregular-shaped and thick-walled cells. These abnormalities were probably due to toxic substances in the wort, as the presence of a heat-labile agent in wheat flour which inhibits the growth of yeast but not mold was reported by Balls and Harris ( 2 ) . Yeast grows normally in wheat mashes cooked under pressure or saccharified with mold bran. To eliminate possible experimental errors due to substances that might be toxic, the yeast was adapted to them by successive transfers in wort prepared from the grain for which the yeast was to be later used as an inoculum. Yeast Y567 (obtained from Northern Regional Research Laboratory) was used, and the inocula were prepared as follows:
INOCULUM FOR WHEATMASH. The yeast from a tube slant is suspended in 3 ml. of sterile water; 1ml. of the suspension is used t o inoculate 20 ml. of wheat wort previously only pasteurized in order not to destroy possible heat-labile toxins. (The wheat nutrient medium was made by heating 92 grams of ground wheat in 450 ml. of water to 70-75" C. for half an hour before saccharification with 8% malt. On cooling the mash the grains settle out and the supernatant liquid is used for the propagatin medium.) The seeded wort is incubated at 30" C. for 24 hours, Q%en 1 ml. of the culture is used to inoculate a second 20 ml. of wheat wort. Successive transfers after 24 hours of incubation are made in this
R E S U L T S AND CONCLUSIONS
The alcohol yield data obtained are given in Table 11, and some calculations derived from the data, in Table 111. The results obtained support the following conclusions: 1. hlold-bran-saccharified mashes yield from 2 to 140/, more alcohol than malt-saccharified mashes, on all five of the grains used. Favorable conditions were those in which 3-40/, dry mold bran was used on a mash cooked at atmospheric pressure and saccharified at 30' C. 2. I n most cases mashes saccharified a t 30" C. with mold bran gave alcohol yields equal to or slightly higher than those saccharified at 52.5" C. When using fresh, undried mold bran, the higher temperature gave better results, probably because at 30' these mashes are more easily infected than those saccharified at 52.5". 3. Mashes saccharified by mold bran gave higher alcohol yields with atmospheric cooking, whereas malt saccharification gave better yields when pressure cooking was used. 4. Mashes premalted with barley malt were more liquid than corresponding mashes premalted with mold bran, but the alcohol yields were not improved over those obtained by using 3-470 mold bran alone. o. While the yields of alcohol obtained by malt saccharification of the mashes are in good agreement with those obtained elsewhere by malt saccharification on the same grains, the alcohol yields from atmospheric cookin and 3% mold bran for saccharification were superior to those &tained from mashes saccharified by 8% malt. The superiority is particularly evident in the yields obtained from mold bran on granular wheat flour where the increase amounts to about 5% for granular winter wheat flour and about 10% for the granular spring wheat flour, over the highest results obtained elsewhere for 8% malt. 6. The simplified procedure possible and high yields obtainable by using 3 4 % dry mold bran in place of 8% malt should be of interest to those engaged in alcohol production from wheat or granular wheat flour. Elimination of pressure cooking and reduction of saccharification temperature plus higher alcohol yields a t the same time are the attractive possibilities offered by the mold-bran saccharification process. LITERATURE C I T E D
(1) Assoc. of Official Agr. Chem., Methods of Analysis, 5th ed., 1940. (2) Balls, A. K., and Harris, T. H., Cereal Chem., 21, 74 (1944). (3) Kneen, E., Beckord, 0. C., Sandstedt, R. M., Ibi& 1 8 , 7 4 1 (1941). (4) Underkofler, L. A,, and co-workers, IND. ENQ.CHEM.,31, 734 (1939), 32, 544 (1940), 35, 814 (1943); Brewers Pigest, 17, No. 12, 29 (1942). PRESENTED before the Division of Industrial and Engineering Chemistry a t the 107th Meeting of the AnfERIaAN CmmIcar, SOCIETYin Cleveland Ohio.
