Industrialization of a Noncooking System for Alcoholic Fermentation from Grains

Transcription

1 Agric. Bio!. Chem., 46 (6), , Industrialization of a Noncooking System for Alcoholic Fermentation from Grains Nobuya Matsumoto, Osamu Fukushi,* Masanobu Miyanaga,* Katsushi Kakihara, Etsuko Nakajima and Hajime Yoshizumi Central Research Institute, Suntory Ltd., 1-1, Wakayamadai, Mishima-gun, Osaka 618, Japan * Usuki Plant, Suntory Ltd., 920, Fukura, Usuki-shi, Ohita 875, Japan Received November 2, 1981 Alcoholic fermentation from grains with a noncooking system was successfully carried out for the first time on an industrial scale. The results were comparedwith those with a conventional hightemperature cooking system and a low-temperature cooking one and it was found that: (1) The fermentation efficiency is equal or superior to that of the high-temperature cooking system. (2) Mashing at a concentration high enough to obtain an average 14.2% final alcohol concentration can be very easily done on an industrial scale. (3) The need for heavy fuel oil for the mashing process is eliminated. (4) The noncooking system allows much energy saving in industrial production of alcohol from starchy materials. In the industrial production of alcohol from starchy materials, production costs have risen extremely due to the great energy consumption in the cooking, sterilization and distillation processes. In the case of alcohol production from cereal grains such as maize, the method widely adopted for obtaining alcohol in a high yield is to cook the mash at a high temperature of nearly 140 C for batch mashing or around 180 C for continuous mashing.1 ~3) The mash is cookedto rupture the structure of the cereal grits to elute starch which enhances the action of liquefying and saccharifying enzymes on the starch and also to sterilize the mash. However, the recent rise in the price of petroleum has prompted many research programs on energy saving in the cooking process in alcohol production from starchy materials, especially since the oil crisis of About thirty years ago, pioneering studies on energy saving in the cooking process in alcohol production from starchy materials were reported by Yamasaki et al^~n) They reported successful alcoholic fermentation from sweet potato starch without cooking on using Black-koji amylase on a laboratory scale. However, in a study on a pilot scale, bacterial contamination was observed In 1963, alcoholic fermentation from ground rice and maize without cooking was successfully carried out on a laboratory scale by Yamasaki et al8) Since 1979, Ueda et al.9~12) have investigated in detail the alcoholic fermentation from starches without cooking by using Blackkoji amylase, acidification of the mash (ph 3.5), dialysis of the fermenting broth and vacuum distillation on a laboratory scale. They clarified that alcohol can be produced with high efficiency from various starches such as those of maize, sweet potato and cassava. Kumagai et al.13) have reported a method of sake brewing without cooking the rice. We have been studying alcoholic fermentation from starchy materials such as maize for a long time. In 1974, we14'15) proposed a lowtemperature cooking system in which mashes of ground cereal grains are cooked at 75 C to 85 C, which is lower than the gelatinization

2 1550 temperature of starch and higher than the sterilization temperature for undesirable microorganisms in the mash which grow during fermentation with yeast. If the lowtemperature cooking system is used on an industiral scale* heavy fuel oil consumption in the mashingprocess can be reduced to about 40% of that needed for the high-temperature cooking system. We nowreport the industrialization of the noncooking system for alcoholic fermentation from grains on the basis of laboratory-scale data. The fermentation efficiency was equal or superior to those of the high- and low-temperature cooking systems. By adopting the noncooking system, a large amount of energy can be saved in industrial production of alcohol from cereal grains such as maize. MATERIALS AND METHODS N. Matsumoto et al. Materials. (a) Grain. Whole kernels of yellow dent maize (grown in the U.S.A.), which are used on an industrial scale, were employed. Content analysis of the grains, given in Table I, showed that the average starch content was 64.6%. (b) Enzyme. The enzyme from Rhizopus sp. was selected on the basis of experimental results for saccharifying enzymes from various microorganisms. The details will be reported in the future. Details of the enzyme preparation, given in Table II, show that other than saccharifying power, various enzymeactivities such as liquefying power and those of protease (acid, neutral and alkaline), cellulase and pectinase were also present. (c) Mashing water. In the noncooking system, yellow dent maize was ground as described below and mixed with mashingwater containing stillage to obtain a mashwhich was then pumpedinto a fermenter. Stillage was used at the ratio of from 20% to 30% of the total mashing water volume to increase buffer capacity, prevent bacterial contamination and modify product quality. Typical analytical data for the stillage used are shownin Table III. Analytical methods. (a) Total sugars, direct reducing sugars, ph, total acidity, ash, crude protein, crude fiber and crude fat of materials and mashes were analyzed by the methods prescribed by the National Tax Administration.16) (b) The yeast population in the starter and the mash was counted using a Thoma haemocytometer. Yeast cells stained with Rhodamine B solution were judged as being dead cells. (c) The bacterial number was counted by the agar cultivation method.17) The medium contained Kabisaijin Table I. Analysis of Maize Grains Used Table II. Enzyme Activities in the Saccharifying Enzyme Preparation Used Enzymepreparation (1 g) was added to distilled water (1000ml) and then occasionally stirred for 1 hour at 30 C. The supernatant obtained by centrifugation (3000 rpm, lomin) was used as enzyme solution. Various enzyme activities were determined as described in Materials and Methods. Table III. Analysis of the Stillage Used Whole stillage obtained from the base of a beer still was centrifuged to removethe coarse grain solids and the thin stillage was analyzed. from Daigo Eiyou Kagaku Co. and bromocresol green as a ph indicator. (d) Enzyme assay. Liquefying power, dextrinizing power, saccharifying power, and protease, cellulase and pectinase activities in the enzymepreparation were assayed by the methods of Wohlgemuth as modified,18) Tsujisaka,19) JIS K 7001,20) Kunitz,21) Miller22) and Willstatter-Schudel,23) respectively. For the assay of the saccharifying power in the mash, the supernatant obtained

3 Noncooking Alcoholic Fermentation from Grains on an Industrial Scale 1551 by centrifugation was used as the enzyme solution and its saccharifying power on soluble starch and on raw starch was assayed. The former was assayed by the method ofjis K ) The latter was assayed by the following method. A mixture of 9ml of 0.05m acetate buffer (ph 4.5), 0.9 grams of raw corn starch and 1 ml ofenzyme solution was incubated at 40 C. After 60 min, the reducing sugars in the reaction mixture were determined by a modified form of the method of Somogyi.24) One unit of amylase i«defined as the activity equivalent to lomg of glucose liberated from 0.9 grams of raw corn starch in 60 min at 40 C. (e) Fermentation efficiency was calculated as: Mash volume* (liter) x Alcohol content* (%) x 100 Weight of maize used (kg) x Total sugars in maize (% as glucose) x RESULTS Mash ing (a) Outline of the mashing process. The ground maize and mashing water (1:2 by weight) were mixed in a mash tank with an agitator to obtain a mash. The saccharifying enzymepreparation was added to the tank and then the mash was directly pumpedup to the fermenter containing the yeast starter. The fermentation temperature was controlled at 26~32 C and the fermentation period was about 96 hours. This noncooking system is shown in Fig. 1. (b) Grinding of the maize. The grinding conditions for the maize were preliminarily studied for the effect on the fermentation rate and fermentation efficiency. Dry milling was found to be the most suitable for the noncooking system. Table IV shows typical particle size distribution of ground maize, measured by using sieves for testing purposes of JIS.25) The details will be reported in the future. Starter The mash for the yeast starter was prepared according to the following procedure. After Table IV. Size Distribution of the Ground Maize The size of granules was measured by using sieves for testing purposes of JIS Z Fig. 1. Flow Diagram of the Noncooking Fermentation System for Use on an Industrial Scale. Capacities: mill, 6 tons/hour; mash tank, 5 kl; fermenter, 140kl. * At the end offermentation.

4 1552 N. Matsumotoet al saccharification of a noncooking maize slurry with the enzyme preparation shown in Table II, the mash (saccharified slurry) was sterilized for 60 min at 135 C and cooled to 30 C. The precultivated yeast was inoculated into the mash. Cultivation was carried out for 24 hours Table V. Analytical the Starter Before inoculation Data for After cultivation ph Total acidity (ml)* Total sugars (% as glucose) Direct reducing sugars (w/v %as glucose) Viable yeast (cells/ml) x 108 Dead yeast (cells/ml) - 2 x 106 Bacterial number (cells/ml) 0 0 In the cases of total acidity and direct reducing sugars, the mash filtrate was used for the analyses, the others were analyzed using the whole mash. Total acidity was expressed as milliliters of n/10 NaOHrequired for neutralizing 10 ml of the filtrate. at 30 C under aerobic conditions. The proportion of the starter used was about 7% of the final mash volume. The analytical data for the starter before and after cultivation are shown in Table V. No bacterial contamination was found, which is essential for adopting the noncooking system on an industrial scale. The yeast used was a strain of Saccharomyces (No. SU-3851) selected from many strains in our laboratory as the most suitable for the alcoholic fermentation with the noncooking system. Fermentation (a) Fermentation rate. Figure 2 shows the changes of alcohol content and residual direct reducing sugar in the fermenting mash. A fermenter became full at about 10 hours after the beginning of mashing, as indicated in Fig. 2. By 48 hours, about 12.9% alcohol, equivalent to about 90% of the final alcohol content, was produced. The content of direct reducing sugars in the mash rapidly decreased with the progress of fermentation, and after 24 hours, "1.6 i 14- q ^-" ]A "5" o 8- \f -0.8 /\ ^ =4~ /V ' 4s 2- à" 6 o o o- -2 n r^l i i i u o u u Fermentation Time (Hours) Fig. 2. Changes of Alcohol Content and Residual Direct Reducing Sugar Content in the Fermenter during Fermentation with the Noncooking System on an Industrial Scale. %-. alcohol content in the whole mash; O-O, R.D.S. content* in the mash filtrate; (< >, the time required to fill up the fermenter with mash. * Residual direct reducing sugar content.

5 Noncooking Alcoholic Fermentation from Grains on an Industrial Scale 1553 remained constant 0.2%. at a very low value of about (b) Changes ofsaccharifying power. Figure 3 shows the changes of saccharifying power on soluble starch and on raw starch in the supernatant of the uncooked mash during fermentation. Saccharifying enzyme preparation was added to the mash at the ratio of4.3 units per gramof maize as saccharifying power by the JIS K-7001 method before the mash was pumped up to the fermenter. The saccharifyingpower on soluble starch was 0.9 to 1.1 units per milliliter of the supernatant (u/ml-sup) from the beginning of mashing to the end of fermentation. The saccharifying poweron raw starch was 0.5u/ml sup at 10 hours after the beginning of mashing, but rapidly increased to 1.35 u/ml-sup, equivalent to 2.7 times the value at 10 hours, and then gradually decreased. (c) Changes of ph and total acidity. Figure 4 shows these changes in the fermenting mash. Total acidity slightly increased from the initial 2.7ml to 3.3 ml at 96 hours after the beginning of mashing. The ph decreased with the progress of fermentation, reaching 4.75 at 24 hours and then remaining at about this level thereafter. These results indicated that acidproducing bacterial contamination had not occurred. (d) Changes of yeast population and bacterial number. The viable yeast population in the fermenting mash changed as shown in Fig. 5. At 10 hours after the beginning of mashing, the yeast population in the mash had already reached the maximumof about 1 x 108 cells/ml and remained almost the same thereafter. One of the greatest technical problems with noncooking alcoholic fermentation is the inhibition of alcohol fermentation caused by contamination by harmful bacteria. Therefore, the success of alcoholic fermentation with a noncooking system, particularly on an industrial scale, depends on whether bacterial contamination can be prevented or not. As 1.5, : :-å. 1.4 t 1.4- /\ J / X -c 1.3- / \ -1-2 fe / V -!.!%~: OD. / U -O 5 O. Hl.o-*r I ^v^ " -9 B".=w 0.9- y ~ -8 E " ^ / ^ X 0.8- / o / "g / 0.6- O ~ N H 1 I I lj Fermentation Time (Hours) Fig. 3. Changes of Saccharifying Power on Soluble Starch and Raw Starch in the Fermenter during Fermentation with the Noncooking System on an Industrial Scale. Mash supernatant obtained by centrifugation (3000 rpm, 10 min) was used as the enzyme solution and the saccharifying power was determined as described in Materials and Methods. 0-O, saccharifying power on soluble starch; O-O, saccharifying power on raw starch; < >, the time required to fill up the fermenter with mash.

6 1554 N. Matsumoto et al <c ^ +-> o C=5l 1 1 I U Fermentation Time (Hours) Fig. 4. Changes of ph and Total Acidity in the Fermenter during Mashing and Fermentation with the Noncooking System on an Industrial Scale. à"-à", ph of the whole mash; O-O, total acidity; (< >j, the time required to fill up the fermenter with mash. Total acidity, see Table V f\ O io3o <^-»l 1 I I L_ Fermentation Time (Hours) Fig. 5. Changes of Viable Yeast Population and Bacterial Number in the Fermenter during Mashing and Fermentation with the Noncooking System on an Industrial Scale. 0-#, viable yeast population in the whole mash; O-O, bacterial number in the whole mash; (< >, the time required to fill up the fermenter with mash.

7 ll Noncooking Alcoholic Fermentation from Grains on an Industrial Scale 1555 shown in Fig. 5, the number of bacteria in the mash was initially about 103 cells/ml and rapidly increased with the progress of fermentation up to 24 hours, reaching about 107 cells/ ml. However, after 24 hours, the bacteria stopped increasing and were about 4x lo5 cells/ml at 96 hours, that is, at the end of fermentation. Because the maximumnumber of bacteria of 107 cells/ml was only one-tenth of the yeast population (108 cells/ml) in the fermenting mash, we considered that the bacterial metabolites scarcely affected the mash from the viewpoint of the difference of metabolic activity per cell between the bacteria and the yeasts in the fermenting mash. This was supported by the results for the changes of ph and total acidity in the mash shown in Fig. 4. At 24 hours after the beginning of mashing, about 8% alcohol had already been produced. In addition, after 24 hours, reducing sugar that is necessary for bacterial growth was scarce because the large active yeast population decomposed reducing sugar as soon as it was liberated from raw starch. Thus, it is considered the bacteria could not increase under such unfavorable environmental conditions. Fermentation results Table VI shows the results of alcoholic fermentation with the noncooking system with a mash volume of about 120kl per mashing unit. The ph of4.8, the total acidity of 3.3ml and the bacterial number of 3.5 x 105 cells/ml show that no distinct contamination by harmful bacteria occurred. In alcohol production from starchy materials such as maize, a mashing concentration of about 14.5% final alcohol is very difficult to attain with the conventional high-temperature cooking system. Therefore, adoption of this noncooking system can greatly increase the quantity of alcohol produced per fermenter. The results of a long-run test on an industrial scale indicated that this alcoholic fer- Table VI. Analysis of the Mash after Fermentation with the Noncooking System on an Industrial Scale Air agitation was conducted for 20 min before sampling. In the cases of total acidity and residual direct reducing sugars, the mash filtrate was used for the analyses, the others were analyzed using the whole mash. See Table V. Table VII. Comparison of ph and Alcohol Content of the Fermented Mash and Fermentation Efficiency among H.T.C., L.T.C. and N.C.S. on an Industrial Scale The results were obtained from the following mash bills. H.T.C., maize 30 tons, water 74 kl, thin stillage 25kl, initial total volume 120kl; L.T.C, maize 31 tons, water 73 kl, thin stillage 25 kl, initial total volume 120 kl; N.C.S., maize 40 tons, water 64kl, thin stillage 28 kl, initial total volume 120kl. phof the whole mash Alcohol content in the whole mash (v/v %) Fermentation efficiency (%) H.T.C.* L.T.C.** N.C.S.*** * High-temperature cooking, cooking temp., 140 C. ** Low-temperature cooking, cooking temp., 80 C. *** Noncooking system. Data are averages of thirty 120kl mashing units.

8 1556 N. Matsumoto et al. mentation with a noncooking system is superior to the high- and low-temperature cooking systems. The ph and alcohol content at the end of fermentation and fermentation efficiency are compared amongthese systems in Table VII. The fermentation efficiency of the noncooking system was the best. The content of alcohol produced by the noncooking system was also the highest (14.2%) compared with those of the high-temperature (10.8%) and low-temperature (1 1.0%) systems. These data show that the alcoholic fermentation with the noncooking system was successful. Energy consumption In the alcohol production industry, most of the energy required for the processes prior to distillation is for grinding and transportation of materials, cooking, agitation and transportation of mash, supplying of cooling water and sterilization of equipment such as fermenters. Reduction of energy consumption at any of these steps is desirable. Our noncooking system eliminates the need for fuel for mashing which requires 128 and 52 liters/kl of alcohol in the high- and low-temperature cooking systems. DISCUSSION Industrial production of alcohol from cereal grains such as maize, is usually done by cooking the mash at a high temperature of over 140 C prior to saccharification and fermentation. The mash is cooked to rupture the cereal grit structure to elute starch for enhanced liquefying and saccharifying activity on it as well as to sterilize the mash. However, heating to over the gelatinization temperature of the mash results in a rapid and extreme increase of viscosity making transportation and agitation of the mash difficult. If the mash temperature is raised above 100 C, such as to 140 C, mash viscosity does decrease but increases again on cooling prior to saccharification. Thus, a liquefying enzyme must be added to the mash during cooling and, at a temperature nearly optimum for saccharification, a saccharifying enzyme is added. Although rupturing the cereal grit structure with high-temperature cooking seems to improve the fermentation efficiency due to increased saccharification efficiency, K. Lorenz et al.26) reported that such high-temperature cooking results in excessive decomposition of starch into unfermentable sugars and other compounds. Therefore, high-temperature cooking may not be good from the viewpoint of effective utilization of starch. The increase in the price of energy in recent years has forced the alcohol production industry to search for energy-saving mashing systems, especially after the oil crisis of We developed an energy-saving lowtemperature cooking system, which has provided satisfactory results on an industrial scale. However, the increase in the price of energy is continuing and an even more economical system is needed. Weconsidered alcoholic fermentation with a noncooking system. The problems with this method were that an enzymecapable of digesting raw starch could not be supplied on an industrial scale and obtaining alcohol in a high yield from an uncooked mash, that is, unsterilized mash, without contamination by harmful bacteria has been very difficult. We have previously studied various saccharifying enzymes produced by many kinds of microorganisms such as fungi, yeasts and bacteria, and found an enzymepreparation from Rhizopus sp. which efficiently digests raw starch in cereal grits. This enzyme preparation, shown in Table II, was added to an uncooked mash in a quantity which synchronized the saccharification with the fermentation rate by the yeast. Therefore, the fermentable sugars liberated from raw starch were immediately converted into alcohol by the yeast and very little fermentable sugars remained in the fermenting mash. Adopting this procedure led to successful alcoholic fermentation with the noncooking system because such extremely low concentrations of fermentable sugar in the mash prevented both the inhibition of the

9 Noncooking Alcoholic Fermentation from Grains on an Industrial Scale 1557 enzyme reaction for raw starch digestion by sugar27'28) and the utilization of sugar as a carbon source by bacteria present in the mash. The saccharifying power on soluble starch shown in Fig. 3 can be considered to be due to the combined activities of glucoamylase I and II as reported by Ueda et al.29) and the saccharifying power on raw starch can be considered to be due to the activity of glucoamylase I. Figure 3 shows that the saccharifying power on raw starch increased with the progress of fermentation. The low saccharifying power on raw starch at the beginning of fermentation seems to result from the adsorption of the enzymeon the raw starch, which is present in a high concentration in the mash. This phenomenon can be explained by Ueda's27'29~33) theory which shows in detail the relation between the susceptibility to digestion and the adsorption of glucoamylase on raw starch. One of the reasons that alcoholic fermentation with the noncooking system could be successfully used is that the high saccharifying power on raw starch, which causes rapid digestion of raw starches and oligosaccharides, could be maintained in the mash during fermentation. With this noncooking system, mashing at an extremely high concentration (maize-mashing water=1 :2) could be carried out, because starch in the uncooked mash did not gelatinize. Of course, such high concentration mashing improves productivity on an industrial scale. Another reason for the success of the alcoholic fermentation with the noncooking system was the adoption of such a high concentration for mashing, as the high concentration of alcohol prevented bacterial contamination. This alcoholic fermentation technique with a noncookingsystem can be expected to contribute greatly to reducing production costs of fuel alcohol, which is being considered as an alternative energy source to petroleum, and to decreasing the energy needed in industrial production of alcohol, in particular, that from starchy materials. Acknowledgments. The authors are grateful to Emeritus Professor K. Arima, Tokyo University, Emeritus Professor M. Hongo, Kyushu University, and Emeritus Professor M. Funatsu, Kyushu University, for their encouragement. Wealso wish to thank Mr. S. Miyata, Dr. T. Amachi, Mr. N. Ohta and Mr. O. Fukuda, Suntory Ltd., for their helpful advice and encouragement. REFERENCES 1) W. H. Stark, "Industrial Fermentation," Vol. 1, ed. by L. A. Underkofler and R. J. Hickey, Chemical Publishing Co., Inc., New York, N. Y., 1954, p ) E. D. Unger, H. F. Willkie and H. C. Blankmeyer, Trans. Am. Inst. Chem. Eng., 40, 421 (1944). 3) R. Smith, Food Can., 29, 23 (1969). 4) I. Yamasaki and S. Ueda, Lecture delivered at the Annual Meeting of the Agricultural Chemical Society of Japan, April, ) I. Yamasaki and S. Ueda, Lecture delivered at the Annual Meeting of the Agricultural Chemical Society of Japan, April, ) I. Yamasaki and S. Ueda, Lecture delivered at the Annual Meeting of the Agricultural Chemical Society of Japan, April, ) I. Yamasaki, /. Ferm. Ass. Jpn., 10, 319 (1952). 8) I. Yamasaki, S. Ueda and H. Shimada, J. Ferm. Ass. Jpn., 21, 83 (1963). 9) S. Ueda and Y. Koba, Abstracts of Papers, Annual Meeting of the Agricultural Chemical Society of Japan, Tokyo, April, 1979, p ) Y. Koba, S. Kusano and S. Ueda, Abstracts of Papers, Annual Meeting of the Agricultural Chemical Society of Japan, Fukuoka, April, 1980, p ll) S. Ueda and Y. Koba, /. Ferment. Technol, 58, 237 (1980). 12) Y. Koba and S. Ueda, /. Brew. Soc. Japan, 75, 858 (1980). 13) C. Kumagai, I. Suzuki, S. Koh, M. Miyairi, T. Tanaka and Y. Akiyama, Abstracts of Papers, Annual Meeting of the Society of Fermentation Technology of Japan, Osaka, Nov., 1980, p ) H. Yoshizumi, N. Matsumoto and O. Fukushi, Japan Kokai Tokkyo Koho, (1976). 15) H. Yoshizumi, N. Matsumoto and O. Fukushi, U. S. Patent, (1978); British Patent, (1978); Canadian Patent, (1979). 16) "Kokuzeicho Shotei Bunsekiho Chukai," the Brewing Society of Japan, ) H. Yoshizumi, Nippon Nogeikagaku Kaishi, 37, 326 (1963). 18) F. Egami, "Hyqjun Seikagaku Jikken," Bunkodo, 1953, p ) Y. Tsujisaka, Report of the Osaka Municipal Technical Research Institute, 23, 6 (1960). 20) "Japanese Industrial Standard," JIS K 7001,

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