KR0135543B1 - Enzymatic hydrolysis of starch derivatives - Google Patents

Abstract

The present invention relates to a method for enzymatic hydrolysis of fiber-containing starch materials such as cassava, potatoes, and sweet potatoes. Conventionally, glucoamylase-based enzymes have been used to hydrolyze starch into glucose, but the contact between glucoamylase and starch is hindered by the fibers around the starch, resulting in a long reaction time and a problem in the treatment of residual solids by residual fibers. The present invention hydrolyzes by adding cellulase and cellobiase together with glucoamylase at pH 4.0 to 5.0 and a reaction temperature of 60 ° C. The fiber is degraded by cellulase and cellobiase and consequently the starch saccharification reaction time is increased. It has a shortening effect, minimizes residual fiber and increases the glycosylation efficiency.

Description

Enzymatic Hydrolysis Method of Starch-Based Substances

The present invention relates to a method for enzymatic hydrolysis of fiber-containing starch materials such as cassava, potatoes, sweet potatoes and the like.

Cassava is one of the starch ingredients used as fermentation raw material and is widely used in fermentation of alcohol ethanol for alcohol in Korea. Conventionally, in order to produce glucose, which is a fermentable form of microorganisms from starch, a conventional two-step enzyme reaction is performed. The two-step reaction is a liquefied enzyme reaction and a glycated enzyme reaction. Alphaamylase, which is used as a liquefaction enzyme during liquefaction, breaks down the starch chain structure, and glucoamylase, a glycosylation enzyme used in the glycosylation reaction, is used for non-reducing ends. It acts and breaks down into exo form, finally producing glucose. Several methods have been reported to improve the hydrolysis process of starch using enzymes, some of which are typical.

British Patent No. 2,029,432A discloses liquid glycosylation using a tubular continuous reactor, in which the tubular reactor is divided into three parts of gelling, liquefaction, and saccharification to perform liquefaction and saccharification continuously. Here, alpha-amylase was added to a slurry containing 30% of cassava starch, and heated to 105 ° C. using steam. After gelation and liquefaction were completed, the solution was cooled to 90 ° C. and glycosylated to obtain 8-12% of glucose conversion (Dextrose Equivalent, D.E.).

At this time, the residence time of the reactor was liquefied, saccharified part 3 minutes, 2 hours respectively.

Another British Patent No. 2,045,764A discloses an enzyme recycling method using ultrafiltration and reacted for 55 ° C., pH 5 to 5.5, 18 to 19 hours to obtain a glucose conversion rate of 80 to 91%.

In addition, there is a method of promoting the enzymatic reaction by changing the structure of the raw material by pretreating the raw material with acid or alkali.

In the acid treatment, the reaction time is an important factor. In the short reaction time, the amount of reducing sugar increases as the reaction time increases. For example, when treating starch of 35 to 40% by weight in HCl solution at pH 9.1, the conversion was reported to be 42% after 8 minutes and 55% after 10 minutes.

However, when the reaction time is longer, side reactions occur and the produced glucose is decomposed, which is rather disadvantageous. After acid treatment, 35% by weight of starch in the HCl solution of pH 2.0 was converted to 15-20% after pretreatment. The conversion was 95% when the glucoamylase was added after pH and temperature adjustment.

Alkaline pretreatment was performed for 12 hours at 0.2 N NaOH, 50 ° C., followed by saccharification with glucoamylase to obtain a conversion of 90% or more, and fermentation with the treatment solution yielded 93% yield after 4 days. (Biotechnology Bioengineering, Vol. 26 pp 627-630)

However, the conventional methods of the ring have the disadvantage that the enzyme reaction takes a long time, difficult operation and control in the continuous process and low conversion rate of glucose, especially when using the ultrafiltration membrane depending on the state of the starch-based material as a raw material Foreign substances such as fibers are stuck in the membrane, which adversely affects the recycling action, thereby reducing the membrane separation efficiency. In addition, the method by acid-alkali pretreatment has a problem in the treatment of inorganic salts which may occur after the adjustment of pH for the continuous saccharification reaction. In addition, the conventional method has difficulty in treating residual solids mainly made of fibers after saccharification of starch-based materials.

Accordingly, it is an object of the present invention to provide an improved enzymatic hydrolysis method of distilling starch-based moles to decompose the fibrous part of the starch-based material to shorten the glucoamylase enzyme reaction time, thereby increasing reaction efficiency and minimizing residual solids. .

An object of the present invention is a method of hydrolyzing a fiber-containing starch-based material into fermentable polysaccharide by using a glucoamylase-based enzyme, wherein the starch is added by adding cellulase and cellobiase in a constant temperature bath at pH 4.0 to 5.0 and 60 ° C. It is achieved by providing a hydrolysis method characterized by saccharifying a systemic material.

Starch-based materials such as cassava, potatoes, and sweet potatoes contain a large amount of fiber, and the fiber is a tissue that surrounds starch. Thus, in the saccharification reaction of starch using enzymes of the glucoamylase system, the fiber is composed of enzyme and starch. Interfering with the contact caused a long reaction time, and the fibers which were not dissolved by the glucoamylase enzyme remained as a residual solid, and there were many problems in the treatment. In particular, cassava contained 25 to 35% by weight of fiber, which caused a problem such as long residual fiber treatment and saccharification reaction time.

According to the present invention, when hydrolysis is performed by adding cellulase and cellobiase together with glucoamylase in a thermostat at pH 4.0 to 5.0 and 60 ° C, fiber around starch is decomposed by enzymatic reaction by cellulase and cellobiase. As the residual is minimized and the contact between the glucoamylase and the starch is larger than before, the saccharification reaction of the starch becomes active and thus the reaction time is shortened, thereby increasing efficiency.

Cassava is composed of 65 to 76 wt% starch and 25 to 35 wt% fiber, in which case glucoamylase is 1 g of glucose at 25 g / L soluble starch at a temperature of 60 ° C., pH 4.3 and a reaction time of 60 min. When defined as 1SGU (Specific Glucoamylase Unit), 300SGU / ml of glucoamylase of 0.15% by weight or more, cellulase was saturated in carboxymethyl cellulose for 40 minutes at a temperature of 40 ° C, pH 4.8, and reaction time. When defined as 1NCU (Novo Cellulase Unit) to produce 1 micromole per poly, 1500 NCU / g of cellulase 0.2 to 1.0% by weight, cellobiase is 250 CbU (Cellobiose Unit) / at a temperature of 40 ℃, pH 5.0 It is preferable to use it in the ratio of 0.04-0.2 weight% of g cellobiase.

Example 1

The saccharification conditions were pH 4.3 to 4.5 and a temperature of 60 ° C., and 100 g of cassava and 400 ml of water were added to a flask in a thermostat, followed by stirring using a magnetic rod. The amount of enzyme added was based on the ratio of starch 閨 flow rate in the case of glucoamylase, but the amount of enzyme was determined based on the remaining portion except for the content of cassava starch. Glucoamylase 0.1g of 300SGU / ml of glucoamylase, defined as 1SGU (Specific Glucoamylase Unit), is the amount of glucose produced from soluble starch 25g / L at 60 ° C, pH 4.3 and 60 min reaction time for starch content. Experiment with only% and adding cellulase and cellobiase, but cellulase produced 1 micromole of glucose in carboxy methyl cellulose for 1 min. As defined in the unit), 1.0% by weight of cellulase at 1500 NCU / g and cellobiase were added to 0.2% of 250 CbU (Cellobiose Unit) / g cellobiase at 40 ° C. and pH 5.0 to examine the saccharification reaction. On the other hand, the reaction time to the saturation concentration of glucose was reduced by about 40% compared to the case without the addition of cellulase and cellobiase. Yield 93%.

Example 2

The amount of cellulase and cellobiase and other glycosylation conditions were the same as those of Example 1, and then the amount of glycoamylase was changed. Glucoamylase was dissolved in starch at a temperature of 60 ° C., pH 4.3 and a reaction time of 60 minutes. 25 g / When 1g of glucose was produced in L as a specific glucoamylase unit (SGU), 300SGU / ml of glucoamylase was reacted with 0.06%, 0.1%, and 0.2%, respectively, and the reaction time was the same as that of Example 1. When the glucoamylase was 0.15% or more, the concentration of glucose produced was almost the same.

Example 3

The glycosylation conditions were the same as in Example 1, and the reaction was carried out by changing the pH to 4.0, 4.3, 4.5, 4.7, 5.0, and the highest pH conversion rate of 94% was found in glucose at 4.3.

Example 4

The saccharification conditions were the same as those of Example 1, and the cellulase and the cellobiase were added first, and glucoamylase was added every 30 minutes (0,30,60,90,120). In all five cases, the time to reach the final glucose concentration was the same, but the reaction rate increased rapidly from the time of glucoamylase.

Example 5

The saccharification conditions were the same as those of Example 1, and the amount of cellulose and cellobiase was 1NCU. The amount of cellulose produced 1 micromole of glucose in carboxymethyl cellulose for 40 minutes at a temperature of 40 ° C. and pH 4.8 with respect to the fiber content. When defined as (Novo Cellulase Unit), 1500NCU / g of cellulase and cellobiase are 250CbU (Cellobiose Unit) / g of cellobiase at temperature of 40 ° C. and pH 5.0, respectively (0.5, 0.1%), (0.4 , 0.8%), (0.3, 0.06%), (0.2, 0.04%). The time to reach final glucose was the same within 10%.

Therefore, hydrolysis by addition of cellulase and cellobiase together with glucoamylase at pH 4.0-5.0 and reaction temperature of 60 ° C can reduce the reaction time, minimize residues due to residual fiber, and increase glycosylation efficiency. have.

Claims (2)

In the method of hydrolyzing starch-based substances such as cassava, potatoes, and sweet potatoes containing fiber into glucose which can be fermented using glucoamylase-based enzymes, cellulase and cellobiase at a pH of 4.0 to 5.0 and a reaction temperature of 60 ° C. Hydrolysis method characterized in that the addition of. The method according to claim 1, wherein the glucoamylase is 1SGU (Specific Glucoamylase Unit) to produce 1 g of glucose at 25 g / L of soluble starch at a temperature of 60 ° C., pH 4.3, and a reaction time of 60 minutes with respect to the starch content of the starch-based material. By definition, 300SGU / ml of glucoamylase is used at 0.15% by weight or more, and cellulose is 1NCU (1 micromole of glucose produced in carboxy methyl cellulose at a temperature of 40 ° C., pH 4.8, and reaction time for 20 minutes. As defined by Novo Cellulase Unit), 0.2 to 1.0 wt% of cellulase at 1500 NCU / g, and cellobiase at a temperature of 40 ° C. and pH 5.0 to 0.04 to 0.2 wt% of 250 CbU (Cellobiose Unit) / g of cellobiase Hydrolysis method characterized in that the addition.