Studies on Enzymatic Liquefaction and Saccharification of Starch

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1 gricultural and Biological Chemistry ISSN: (Print) (Online) Journal homepage: Studies on Enzymatic Liquefaction and Saccharification of Starch Toshiaki Komaki & Noboru Taji To cite this article: Toshiaki Komaki & Noboru Taji (1968) Studies on Enzymatic Liquefaction and Saccharification of Starch, gricultural and Biological Chemistry, 32:7, , DOI: / To link to this article: Published online: 09 Sep Submit your article to this journal rticle views: 758 View related articles Full Terms & Conditions of access and use can be found at

2 [gr. Bio!. Chern., Vo!' 32, No.7, p , 1968] Studies on Enzymatic Liquefaction and Saccharification of Starch Part VIII. Liquefying Conditions of Corn Starch by Bacterial lphamylase By Toshiaki KOMKI and Noboru T JI rnagasaki Factory oj Nagase and Co., Ltd. rnagasaki city, Hyogoken Received December 9, 1967 Liquefaction of corn starch is generally considered to be difficult to achieve. double enzyme system, a combination of bacterial alphaamylase and fungal glucoamylase, was tested for the production of dextrose from corn starch. The former liquefies starch and the latter hydrolyzes further into glucose Particular! y various conditions for liquefaction of corn starch were examined. s criteria of solubilization of starch molecule, turbidity of filtrate obtained while the solution was hot, iodine coloration value, filterability and turbidity of the saccharified solution after the addition of alcohol were measured. Starch slurry containing bacterial alphaamylase was poured continuously in fine stream into hot water with vigorous agitation. Liquefaction at 92 C was better than at 87 C for dissolving corn starch. It was indispensable to apply heat treatment under high pressure after liquefaction followed by the second addition of bacterial alphaamylase at 90 C to render remaining tightly bound starch micelle to be broken down. Heat treatment was continued at 120 C or at higher temperature. Then, solution was cooled rapidly down to about 90 C. Immediately bacterial alphaamylase was added. In case the second addition of bacterial alphaamylase was carried out after cooling to 55 C or after holding for 10 min at 80 C, no effect was observed by the second addition. These findings were confirmed in the experiments on commercial production scale. In enzymatic hydrolysis for production of dextrose, corn starch is rather difficult to be liquefied by bacterial alphaamylase, as compared with sweet potato starch and potato starch. When corn starch is liquefied with alphaamylase by the instantaneous heating method and saccharified by glucoamylase in usual way, filtrate obtained from the hydrolyzate contains a small amount of insoluble starch particles of high molecular weight which shows blue iodine coloration after treatment with caustic soda solution. The insoluble starch particles are often carried into finished products such as dextrose powder or crystalline dextrose. Efficiency of the filtration is also rather low compared with that of sweet potato starch. One of the reasons for these disadvantages is the fact that corn starch granules have higher gelatinizing temperature and are more difficult in swelling than potato starch granules. nother reason is that corn starch originally contains about lo times as much insoluble starch particles as compared with sweet potato starch. These particles remain after liquefaction by the instantaneous heating method under the best conditions tested, using sufficient amount of bacterial alphaamylase as described in our preceding report. 1 I Corn starch granule generally contains acid. 1) T. Kornaki, gr. Bioi. Chern., 32, 127 (1968).

3 Studies on Enzymatic Liquefaction and Saccharification of Starch. Part VIII 861 Even when ph of slurry of corn starch is adjusted before liquefaction, the liquefied solution shows rather low ph. So there is also possibility that bacterial alphaamylase was inactivated by the acid which was liberated during the instant heating and the subsequent destruction of granules. In order to overcome these drawbacks that corn starch has, we have investigated the conditions in detail and have arrived at the optimum conditions. We applied these optimum conditions in the commercial production of dextrose from corn starch and obtained satisfactory results. MTERILS ND METHODS Corn starch. Corn starch commercially available, manufactured by Nihon Shokuhin Kako K.K. Bacterial alphaamylase. (EC alphai, 4glucan 4glucanohydrolase) NEOSPEEDSE K having activity of 7,500 DU per g, commercially available, was manufactured by Nagase and Co., Ltd. It is obtained by culturing Bacillus subtilis val'. Biotecus by tray culture method, followed by drying and pulverizing, and diluted with sodium chloride, calcium sulfate and calcium carbonate. One DU is defined as the amount of enzyme which is able to reduce 1 % of blue value of iodine color in 1 min in acting on 10 ml of 196 potato starch solution at ph 6.0 at 40 C. One DU corresponds approximately to the activity of 0.5pg of crystalline amylase. G1ucoamylase. (EC alphai, 4glucan glucohydrolase) This preparation manufactured by Nagase and Co., Ltd. was obtained by culturing Rhizopus niveus on wheat bran medium by tray culture method. The enzyme was extracted with water, precipitated with ammonium sulfate, dried, pulverized and then diluted with diatomaceous earth. ctivity was 3000 CU per g. One CU is defined as that amount of enzyme which produces pg true glucose from 5 ml of potato starch solution at ph 4.5 at 40 C in 1 min. mount of true glucose is determined using glucose oxidase. Liquefaction of starch by the instantaneous heating method. In order to minimize error caused by experimental operations, the experiments was conducted in pilot plant scale. Five kg of corn starch (moisture content 13%) was dispersed in 5liters of water containing g of calcium hydroxide to make it slurry. ph of the slurry was 6.0. Then 17.5 g of NEOSPEEDSE K (30 DU per g of dry starch) was added. The total volume was 8.5 liters. The mild steel jacketted reaction tank for liquefaction of starch was 230 mm in diameter and 380 mm in depth. Heating and cooling were made by introducing steam or water into the jacket. t the bottom of the tank, a horizontal agitator was equipped. Five liters of water were placed in the tank and heated to a certain temperature. With agitation, the above slurry containing amylase was poured continuously with a constant feeding pump through five thin nozzles at the rate of 170 ml per min maintaining a certain temperature. fter 3 liters of the slurry was poured, 3 liters of the liquid in the tank was discharged while pouring of the slurry was continued at the same time. fter the total volume of the slurry was poured (content in the tank was about 10.5 liters), the liquefied material was allowed to stand for a certain time. The liquefied material had ph of the 6.0, same as that of the original slurry (calcium carbonate in NEOSPEEDSE K has neutralizing action). Then ph of the liquefied material was adjusted to 5.1 with IN hydrochloric acid (about 50 ml) and boiled for 5 min. In the experiments in which heating at 120 C is needed, the liquefied material was heated in the autoclave at pre3sure of 1.5 kgjcm2c for 10 min. In the experiments in which the liquefied material was treated again with alphaamylase after heat treatment, addition of alphaamylase was made when temperature of the liquefied material dropped to a certain temperature. The saccharification was performed at 55 C, at ph 5.1, using 15 CU of glucoamylase per gram of dry starch. nalytical methods 1. Filterability. round filter paper, Toyo Filter paper No.2, of diameter 18.5 em folded in four was set on a casted glass funnel of 9 em diameter. Then 150 ml of sample at 30 C was transferred to on the filter paper and the filtrate obtained in 30 min was measured. Filterability was expressed in ml amount of the filtrate. Standard deviation of this experiment was Turbidity of filtrate obtained while the solution was hot. Sample was boiled and filtered immediately under suction with a No.2 glass filter. The filtrate was cooled to room temperature. The optical density of the filtrate was measured at 720 mp

4 862 Toshiaki KOMKI and Noboru TJI using a 10 mm cuvette. Turbidity was expressed in the optical density value. 3. Iodine coloration value. One ml of the hot filtrate obtained in the above test was added to 1 ml of 2N caustic soda solution. The mixture was heated C for I min. Then, 80 ml of water, 2 ml of 2N hydrochloric acid and 10 ml of the iodine reagent (0.5% KI, 0.05% 1 2 ) were added, and total volume was made to ml with distilled water. The aliquot was determined for optical density at 660 mfl using a 10 mm cuvette. Iodine coloration value was expressed in the optical density value. In case of saccharified solution, conditions were the same as above except for the amount of sample, 2N caustic soda solution and 2N hydrochloric acid solution which were 3 ml, 3 ml and 4 ml, respectively. The result was calculated for 1 ml of the sample. Hydrolysis degree, D/T. Reducing sugar was 4. measured by Somogyi's method. Total sugar was measured after 5 ml of approximately 15'0 sugar solution and 5 ml of IN hydrochloric acid were mixed and was hydrolyzed in boiling water and neutralized. Heating time was 2 hr for a liquefied starch solution, or I hr for a saccharified starch solution. D/T was expressed as per cent of reducing sugar in total sugar. 5. Turbidity in alcohol. From 0.1 to 0.5 ml of the filtrate obtained as described in 1 was mixed with 15 ml of 95% ethyl alcohol. Optical density of the mixture at 720 mp was measured using a 10 mm cuvette. Turbidity value was expressed as optical density value for 0.5 ml of the sample. 6. nalysis of variance. nalysis of variance technique was adopted to isolate and estimate the variations associated in these sets of designed experiments. The results of the saccharified material after 24 and 48 hr saccharification were calculated for significance of each source of variance, estimation of population mean, and their confidence limit. RESULTS 1. Effect of reaction time of alphaamylase, the second addition of alphaamylase and temperature of heat treatment after liquefaction The experiment was conducted using three way crossclassification: Factor () was the effect of alphaamylase reaction time in the liquefaction tank: (l) for 30 min and (2) for 60 min. Factor (B) was the effect of the second addition of alphaamylase after heat treatment: (El) without alphaamylase and (B2) with 5 DU per g of dry starch of alphaamylase. Factor (C) was the effect of heating temperature after liquefaction: (CI) at 120 C and (C2) at C. Liquefaction temperature was 92 C. fter pouring of the starch slurry containing alphaamylase was over, the mixture was allowed to stand for 30 min (I) and 60 min (II) at 92 C. Then imediately they were heated to boil. Each half of I(III) and II(IV) were heated in an autoclave at 120 C for 10 min. Then each half of I, II, III and IV were cooled to 55 C and glucoamylase was added. nd the remaining halves were cooled to 80 C and was added 5 DU per g dry starch of bacterial alphaamylase. fter the reaction with alphaamylase for 30 min, they were cooled to 55 C and glucoamylase was added. ll these samples were saccharified. Table I shows degree of hydrolysis (D/T), before addition of glucoamylase and after 48hr of saccharification. s to D/T figures before saccharification, the longer the alphaamylase action the higher D/T was obtained. nd in the second addition of alphaamylase after heat treatment the high D/T was obtained. But in D/T figures after saccharification, no significant difference was detected. Table II shows filterability, turbidity of filtrate obtained while the solution was hot, iodine coloration, turbidity of the solution after the addition of alcohol before saccharification, and after 24 hr and 48 hr of saccharification. Table III shows the results of significance of each factor. In case of filterability, the samples treated at high temperature showed high significance. The best result was obtained in the sample heated at 120 C and alphaamylase was added again. In case of turbidity of filtrate obtained while the solution was hot, heat treatment and second addition of alphaamylase were the most suitable means to reduce turbidity. The second addition of alphaamylase was also effective in reducing the iodine coloration value.

5 Studies on Enzymatic Liquefaction and Saccharification of Starch. Part VIII 863 TBLE I. 1 Reaction time for alphaamylase, 30 min 2 Reaction time for alphaamylase, 60 min TBLE II. 1 Reaction time for alphaamylase, 30 min RESULTS OF DEGREE OF HYDROLYSIS, BEFORE ND FTER SCCHRIFICTION C 1 Heat treatment at 120 C C 2 Heat treatment at C C 1 Heat treatment at 120 C C 2 Heat treatment at C B 1 Without the second addition of alphaamylase,., B 2 With the second addition of alphaamylase,., Before fter Before fter saccharification saccharification saccharification saccharification RESULTS OF FILTERBILITY, TURBIDITY OF FILTRTE, I 2 COLOR INTENSITY ND TURBIDITY FTER THE DDITION OF LCOHOL C 1 Heat treatment at 120 C C 2 Heat treatment at D C I C 1 I' Heat treatment. 2 R eac t lon at 1200C time for alpha) C 2 amylase, 60 min I Heat treatment at C F, Filterability T, Turbidity H, Saccharification time in hours, H o o o o TBLE III. B 1 Without the second addition of alphaamylase J ", F T 12 l , I 2 colour intensity I, Turbidity after the addition of alcohol NLYSIS OF VRINCE B 2 With the second addition of alphaamylase '' F T 12 l Filterabili ty Turbidity I 2 Color Intensity.,,,,, S V Fo S V Fo S V Fo (Reaction time * for alphaamylase) B (2nd addition of ** ** alphaamylase) C (Heat treatment ** after liquefaction) xb * xc ** BxC e T * Significant, Probability point ** Significant, Probability point 0.01.

6 864 Toshiaki KOMKI and Noboru TJI s to the continued action of alphaamylase in the liquefaction tank, a little significance was observed only in the turbidity of the filtrate obtained while the solution was hot. No significance was observed in any factors for turbidity after the addition of alcohol. 2. Effect of temperature of the second addition of alpha amylase after heat treatment and alphaamylase concentration The experiment was conducted using two way crossclassification: Factor () was effect of temperature at the second addition of alphaamylase after heat treatment: (I) at 90 C, (2) at 80 C and (3) at 55 C. Factor (B) was the effect of concentration of alphaamylase activity during the second addition after heat treatment: (Bl) without alphaamylase, (B2) with 3 DU per g of dry starch and (B3) 6 DU per g of dry starch. Liquefaction temperature was 92 C. fter starch slurry was poured, the mixture was kept for 30 min. Then ph of the mixture was adjusted at 5.1 by addition of IN hydrochloric acid. The mixture was autoclaved at 120 C for 10 min. The liquefied solution was divided into three portions. They were cooled to 90 C (I), 80 C (2) and 55 C (3) respectively. Each portion was further divided into three parts; To B2 and B3, 3 DU and 6 DU per g of dry starch of alphaamylase was added respectively. No enzyme was added to Bl. In the cases when alphaamylase was added, the portions were kept for 30 min at the constant temperature respectively. fter cooling them to 55 C, they were saccharified by adding glucoamylase. Table IV shows D/T just before saccharification and after 48 hr of saccharification. In D/T after saccharification, no significance was observed. In D/T before saccharification, the portions with addition of alphaamylase showed higher D/T. D/T was the highest when the alphaamylase action was conducted at 55 C. t 80 C, D/T was still almost as high as that at 55 C, but at 90 C alphaamylase did not appear to be acting. This means, alphaamylase was inactivated at 90 C at ph 5.1. Table V shows filterability, turbidity of filtrate obtained while the solution was hot, iodine coloration, and turbidity when alcohol was added to the hydrolyzates at saccharification time of 0, 24 and 48 hr. Table VI shows the result of significance of each factor. In case of filtrability, no significance was observed. In Experiment 1, significance was observed in temperature of heat treatment, but in this experiment heating was made at 120 C. Further no significance was observed in any factors for turbidity when alcohol was added. s for temperature of the second addition of alphaamylase, significance was observed in decrease in turbidity of filtrate while the solution was hot and decrease in iodine coloration. The second addition of alphaamylase was the best at 80 C and the worst at 55 C. fter heat treatment, 6 DU perg of dry starch of alphaamylase was added at 90 C, 80 C and 55 C. Estimation of population mean of iodine coloration were 0.053, and 0.138, respectively. t 55 C, where D/T was the highest gave the worst results. t 80 C the best lowest figure was obtained. nd at 90 C though action of alphaamylase was small iodine coloration became lower. fter heat treatment at 80 C at 0, 3 and 6 DU TBLE IV. Temperature for the second addition of alphaamylase RESULTS OF DEGREE OF HYDROLYSIS, BEFORE ND FTER SCCHRIFICTION B 1 o DUjg starch B 2 3 DUjg starch B 3 6 DUjg starch " " _..._ Before fter Before fter Before fter saccharifi saccharifi. saccharifi saccharifi saccharifi saccharifi catlon cation cation cation cation cation

7 Studies on Enzymatic Liquefaction and Saccharification of Starch. Part VIn 865 TBLE V. Temperature for readdition of alphaamylase RESULTS OF FILTERBILITY, TURBIDITY OF FILTRTE, I 2 COLOR INTENSITY ND TURBIDITY FTER THE DDITION OF LCOHOL B 1 0 DU/g starch B 2 3 DU/g starch B 3 6 DU/g starch '', '' H F T I2 l F T 12 l F T I2 o l a C o a C a C o F, Filterability T, Turbidity H, Saccharification time in hours l I 2, I 2 color intensity I, Turbidity after the addition of alcohol, l TBLE VI. NLYSIS OF VRINCE Filterabili ty Turbidity I.Color Intensity, '. S 1> V Fo S V Fo S V Fo (Temperature for the second addition of alphaamylase) ** * B (Concentration of alphaamylase) ** ** xb e T * Significant, Probability point ** Significant, Probability point per g of dry starch of alphaamylase was added. Estimation of population mean of iodine coloration were 0.205, and 0.023, respectively. It is concluded from above results that addition of alphaamylase after heat treatment was very effective, and enough effect was observed by addition of 3 DU per g of dry starch of alphaamylase. S. Effect of reaction time of alphaamylase and readdition of alphaamylase after heat treatment The experiment was conducted using two way crossclassification: Factor () was effect of alphaamylase reaction time in the liquefaction tank: (l) for 3 min, (2) for 15 min, (3) for 30 min and (4) for 60 min. Factor (B) was the effect of the second addition of alphaamylase after heat treatment: (BI) without alphaamylase and (B2) with 6 DU per g of dry starch of alphaamylase. In Experiment 1, reaction time of alphaamylase in the liquefaction tank was 30 min or 60 min. nd D/T before heat treatment was 8.0 or In this experiment, their ranges were further broadened. Liquefaction temperature was 92 a C. fter complete pouring of starch slurry, action of alphaamylase was continued for the reaction periods of 3 (I), 15 (2), 30 (3) and 60 (4) min. Portions of the reaction mixture were taken and made to ph 5.1 and boiled to stop the reaction. Further they were heated in an autoclave at 120 C for 10 min. The reaction mixtures were divided into two portions respectively. To one portion, 6 DU of alphaamylase per g of dry starch was added and it was held at 80 a C for 30 min.

8 866 Toshiaki KOMKI and Noboru TJI Then it was cooled to 55 C and glucoamylase was added to perform saccharification (B2). To the other portion, no alphaamylase was added and the portion was cooled to 55 C and saccharified in the same manner (BI). Table VII shows hydrolysis degree D/T before saccharification and 48 hr of saccharification. D/T figures before saccharification were proportional to the reaction time of alphaamylase. In the cases of the second addition of alphaamylase after heat treatment, D/T increased by %. In D/T after saccharification, no significance was observed. Table VIII shows filterability, turbidity of filtrate obtained while the solution was hot, iodine coloration, and turbidity after the addition of alcohol of the hydrolyzates at saccharification time of 0, 24 and 48 hr. TBLE VII. Reaction time for alphaamylase min 15 min 30min 60min RESULTS OF DEGREE OF HYDROLYSIS, BEFORE ND FTER SCCHRIFICTION B 1 Without the second addition of B 2 With the second addition of alphaamylase alphaamylase <, Before saccharification fter saccharification Before saccharification fter saccharihcation TBLE VIII. RESULTS OF FILTERBILITY, TURBIDITY OF FILTRTE, 12COLOR INTENSITY ND TURBIDITY FTER THE DDITION OF LCOHOL B 1 Without alphaamylase Reaction time for > alphaamylase on B 2 With 6 DU of alphaamylase " H F T 12 l F T I. l min min , min min F, Filterability 12,I2color intensity T, Turbidity I, Turbidity after the addition of alcohol H, Saccharification time in hours. TBLE IX. NLYSIS OF VRINCE Filterability Turbidity 1 2 Color Intensi ty / " S 1> V Fo S 1> V Fo S 1> V Fo (Reaction time for 1809 al phaam y lase) * ** B (The second addi tion of alphaamylase after ** ** ** heat treatment) xb e T * Significant, Probability point ** Significant, Probability point 0.01.

9 Studies on Enzymatic Liquefaction and Saccharification of Starch. Part VIII 867 Table IX shows the resuits of significance of each factor. No significance was observed among the factors regarding turbidity after the addition of alcohol. But clear significance was observed in turbidity of fiiterate obtained while the solution was hot. With higher DjT of liquefied solution before heat treatment, the turbidity was lower. significanc,e was also observed. In case of filterability With higher DjT before heating, the filterability of saccharified solution was better. In case of iodine coloration no significance was observed among each levels. The second addition of alphaamylase after heat treatment showed effect in filterability, turbiditv of filtrate obtained while the solution was hot, and iodine coloration, in this experiment. 4. Effects of temperature of the liquefaction, temperature of the heat treatment after liquefaction and the second addition of alphaamylase after the heat treatment The experiment was conducted using three way crossclassification: Factor () was effect of temperature of the liquefaction: (I) at 92 C and (2) at 87 C. Factor (B) was effect of the second addition of alphaamylase: (Bl) without alphaamylase and (B2) with 6 DU per g of dry starch of alphaamylase. Factor (C) was effect of heating temperature after liquefaction: (Cl) at 120 C and (C2) at C. In the preceding report, it was described that insoluble starch particle was definitely formed in liquefied solution when temperature of liquefaction was below 80 C, in case of sweet potato starch. In case of corn TBLE X. 1 Temperature C 1 Heat treatment for (l20 C) liquefaction C 2 Heat treatment (92 C) ( C) 2 Temperature C 1 Heat treatment for (120 C) liq uefaction C 2 Heat treatment (87 C) ( C) DEGREE OF HYDROLYSIS, BEFORE ND FTER SCCHRIFICTION B 1 Without alphaamylase B 2 With 6 DU of alphaamylase. Before fter Before fter sac chari fica ti on saccharification saccharification saccharification TBLE XI. RESULTS OF FILTERBILITY, TURBIDITY OF FILTRTE, I 2 COLOR INTENSITY ND TURBIDITY FTER THE DDITION OF LCOHOL B 1 Without double dose B 2 With double dose of of alphaamylase a I phaam y lase H F T 12 l F T h l C I Heat treatment I Temperature (120 C) for liquefaction C 2 Heat treatment (92 C) ( C) C 1 Heat treatment Temperature (120 C) for liquefaction C 2 Heat treatment (87 C) (l00 C) F, Filterability 1 2,I 2 color intensity H, Saccharification time in hours. T, Turbidity I, Turbidity after the addition of alcohol.

10 868 Toshiaki KOMKI and Noboru T JI TBLE XII. NLYSIS OF VRINCE Filterability Turbidity I.Color Intensity, S 1> V Fo S 1> V Fo S 1> V Fo (Temperature for ** liquefaction) B (2nd addition of ** ** ** alphaamylase) C (Heat treatment ** after liquefaction) xb xc BxC * ** e T * Significant, Probability point 0.05, ** Significant, Probability point starch, temperature of gelatinization was higher, so it was desirable to liquefy it at higher temperature (this was understood from experience.). Therefore in the experiments 13 liquefaction was performed at g2 e. But in this experiment 4, 87 C and g2 e were adopted as liquefaction temperature, keeping period for alphaamylase action at 30 min l20'e and loooe were adopted as temperature of the heat treatment. The respective parts were divided into two portions, and 6 DU per g of dry starch of alphaamylase was again added at 80 C in the one group, and no alphaamylase was used in the other group. fter cooling down to 55 C, the solutions were saccharified with glucoamylase. Table X shows DjT values before saccharification and after 48 hr of saccharification. DjT before saccharification of the solution liquefied at g2 e was lower by about 3% than that at 8re. This means that inactivation of alphaamylase at g2 e was larger than that at 87 C. In DjT after 48 hr of saccharification, there was no significance. Table XI shows filtrability, turbidity of the filtrates obtained while the solution was hot, iodine coloration, turbidity after the addition of alcohol solution of the hydrolyzates at saccharification time of O. 24 and 48 hr. Table XII shows the results of significance of each factor. In case of filterability, clear significance was observed in the second addition of alphaamylase after the heat treatment and the heat treatment at 120 C. These results can support the results obtained in the other Experiments. nd in case of turbidity of the filtrate obtained while the solution was hot, significance was observed in the second addition of alphaamylase after the heat treatment. In case of iodine coloration, clear significance was observed in correlation between liquefaction temperature and the second addition or nonaddition of alphaamylase after the heat u u '" "'" " ' / 0.3 / ' 0 / / 0 0 /, / / 0 ;/ U U, N 0.2 N 0 0. > '" '" " " o 120 Liquefaction temp. (OC) Heat treatment temp. (OC) FIG. 1. Effects of Temperature for the Liquefaction Temperature for Heat Treatment after Liquefaction and the Second ddition of lphamylase after Heat Treatment on Intensity of Iodine Color Reaction of Saccharified Liqued. e Without the second addition of alphaamylase after heat treatment. 0 With the second addition of alphaamylase after heat treatment.

11 Studies on Enzymatic Liquefaction and Saccharification of Starch. Part VIII 869 treatment. Significance was also observed in correlation between liquefaction temperature and iodine coloration. Liquefaction temperature at 92 C and the second addition of alphaamylase showed good results. For respective cases, estimation of population mean and confidence limit were calculated, and the results were shown in Fig Inftuence of ph for heat treatment, pressure of autoclaving, temperature during the second addition of alpha:amylase and holding time before the second addition of alphaamylase after heat treatment The experiment was conducted using the following classification: Factor C) was influence of ph for heat treatment after liquefaction: (3) at ph 6.5. (I) at ph 5.1, C2) at ph 5.8 and l T BLE XIII. RESULTS OF FILTERBILITY, TURBIDITY, Iz,COLOR INTENSITY, TURBIDITY FTER THE DDITION OF LCOHOL ND DfT Factors 24 hr saccharification 48 hr saccharification,,...ro, 2 3 B Bl B2 Bl B2 Bl B2 C Cl C2 Cl C2 Cl C2 Cl C2 Cl C2 Cl C2 D F. ', 78 T l O F 83 T Iz l DfT

12 870 Toshiaki KOMKI and Noboru TJI Filterability, S 1> V Fo S B ** 2 C * 80 D ** 78 e T Factor * Significant, Probability point ** Significant, Probability point TBLE XIV. NLYSIS OF VRINCE Turbidity hcolor Intensity '.., 1> V Fo S 1> V Fo * ** * ** ** ** ** Factor (B) was influence of pressure of autoclaving after liquefaction: (Bl) at 1.5 kg/cm 2 G and (B2) at 2.5 kg/cm 2 G. Factor (C) was effect of temperature during the second addition of alphaamylase after heat treatment: (CI) at 90 D C and (C2) at BODC. Factor (D) was influence of holding time before the second addition of alphaamylase after heat treatment: (DI) for 0 min, () for 10 min and () for 25 min. Liquefaction temperature was 92 C. fter starch slurry was poured, the reaction mixture was kept for 30 min and then boiled for 5 min. The liquefied solution was divided into three portions: they were adjusted ph at 5.1 (I), 5.B (2) and 6.5 (3) respectively. Each of them divided into two portions, then they were autoclaved at 1.5 kg/cm 2 G (Bl) and 2.5 kg/cm 2 G (B2) for 10 min respectively. The autoclaved solution was divided into two portions. They were cooled to 90 C (Cl) and BO C (C2), respectively. Further each portion was divided into three parts: then 5 DU per g of dry starch of alphaamylase was added again () immediatly, () after 10 min, and C) after 25 min, respecti vely. Table XIII shows the results of filterability, turbidity of filtrate obtained while the solution was hot, iodine coloration, and turbidity after the addition of alcohol of the hyhrolyzates at saccharification time of 24, and 4B hr. Table XIV shows the result of analysis of varience. In case of filterability factor B, D and C were significant, in case of turbidity factor, C and B were significant, in case of iodine coloration all factors were significant. From this we conclude that the ph of the liquefied solution should be adjusted to about ph 5.B and heat treated at 1.5 to 2.5 kg/cm 2 G pressure. Then the solution should be quickly cooled to 9O D C, and the second addition of alphaamylase should be done as quick as possible. The above conditions are proved to be the best conditions to solubilize corn starch by alphaamylase. DISCUSSION Corn starch is usually considered to be difficult to liquefy by bacterial alphaamylase. s criteria of solubilization of starch, turbidity of filtrate obtained while the solution was hot and iodine coloration value of the filtrate were determined. t the same time, filterability and turbidity of the saccharified solution after the addition of alcohol were determined. In case of liquefaction temperature, it is considered that higher temperature is desirable for the swelling of associated starch molecules to facilitate the hydrolysis by alphaamylase but there is a limit in the heat stability of alphaamylase. This experiment clearly indicated that liquefaction at 92 C is better than at BrC for dissolving corn starch, but in case of progression of the degree of hydrolysis during the liquefaction process, it was better at BrC than at 92 C, Even though under the best conditions of liquefaction of corn starch with bacterial alphaamylase, still now some amount of insoluble

13 Studies on Enzymatic Liquefaction and Saccharification of Starch. Part VIII 871 starch particles remain in hydrolyzate. It is absolutely necessary to apply the heat treatment under the pressure after liquefaction for swelling of insoluble starch particles so as to be easily liquefied by alphaamylase. Significant difference was observed in filterability between I.S kg(cm 2 G and 2.S kg(cm 2 G of autoclaving pressure. In this experiment and also in industrial experiment, heat treatment in continuous heating device at 3.2 kg(cm 2 G for 10 min was better than at 2.S kg(cm 2 G for 10 min for good filterability. lso the second addition of alphaamylase at about 9Q C immediately after heat treatment was indispensable to liquefy insoluble starch particles which were resistant to alphaamylase action. The most probable explanation on is, the starch molecule which resist to the action of alphaamylase are changed into the molecules which can easily be subjected to the action by the heat treatment under pressure, but when the molecule is cooled to SsoC, or kept at high temperature of 80 C for 10 min after the heat treatment without the second addition of alphaamylase, the starch molecules again turn to those which are resistant not only to the action of alphaamylase but also to that of glucoamylase. This transformation is assumed to be of the same type as that in retrogradation of straight chain fraction of starch. The higher temperature for addition of alphaamylase is better but in case of acid and heat inactivation of amylase will be large. So ph S.36.0 and temperature 9093 C are the best conditions for the second addition of amylase. Such tendency was also observed in the production trials in factory. The following two experiments were made in dextrose manufacturing plant: Experiment 1. fter liquefaction of starch with alphaamylase the liquefied solution was heated to 2.0 kg/cm 2 G by introducing pressurized live steam into the autoclave. It took 10 to IS min. Then, the solution was discharged into an open vessel. It took ls,...,20 min. It was cooled with plate cooler to STC. Then, alphaamylase and glucoamylase were added. Experiment 2. fter the liquefaction, the liquefied solution was treated in an autoclave in the same way. Then the solution was discharged into an open vessel and it was allowed to cool to 93 C. (ph was S.7S.9). Immediately about S DU of alphaamylase per g of dry starch was mixed, and the mixture was introduced continuously into a plate cooler to lower the temperature of the solution to STC. Then, glucoamylase was added. These results were compared. In Experiment 1, after saccharification for 70 hr, the solution was purified. The product dextrose powder showed clear blue iodine coloration. n aqueous solution of the product showed turbidity. Whereas, in Experiment 2, after saccharification for 40 hr, the solution did not show any iodine coloration and filtration in the followed purification was carried out well without any trouble. Experiment 3. fter liquefaction, the liquefied solution (which has slightly dirty blue iodine coloration) was heated to 13SoC using a continuous heating equipment. Then the solution was cooled down to 9Q C using a continuous flash cooler. The solution obtained showed bright blue iodine coloration. lphaamylase was added again and the mixture was held in a vessel at 8S90 C for 30 min. Then temperature of the solution was brought down to SsoC by plate cooler and saccharification was performed. ll the crystalline monohydrate lots which were produced by this procedure passed pharmaceutical specifications. In conclusion, the best conditions for Droduction of dextrose from corn starch which were obtained in this report are: In the liquefaction step, ph of starch slurry was made to To prevent ph change, a small amount of calcium carbonate was added. The amount of alphaamylase used was 2S 30 DU per g of dry starch. Liquefying temperature was 9092 C. lphaamylase was acted until hydrolysis

14 872 Toshiaki KOMKI and Noboru T JI degree reaches 1O 13. Then, ph of the solution was adjusted to about 5.3 to 6.0. Heat treatment (heating) was followed at least at 120 C or at higher temperature. The solution was cooled rapidly down to about 90 C. Immediately 36 DU of alphaamylase per g of dry starch was added. The mixture was held for about 30 min. fter cooling down to 5YC the solution was saccharified with glucoamylase. ll the above processes should be done continuously.