JPH0458959B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for efficiently and inexpensively continuously producing high-concentration, high-purity glucose. (Prior Art) Currently, the process for producing starch sugar can be roughly divided into the following steps. (1) Starch milk production process (2) Starch liquefaction process using α-amylase (3) Starch liquefaction saccharification process using glucoamylase, β-amylase, maltooligosaccharide-forming amylase, isoamylase, pullulanase, etc. (4) Purification and concentration process of sugar solution (5) Isomerization process of sugar solution using glucose isomerase Out of the above five steps, except for the saccharification step (3), the other steps are produced by a continuous method. Only the saccharification process is carried out using the batch method. Moreover, when producing glucose using this batch method, the DE of the sugar solution is 93.
~98, it takes a long time of 48 to 72 hours in a huge tank to carry out saccharification until the amount of glucose produced is 91 to 96%. On the other hand, in order to make the saccharification process a continuous process during the production process of starch sugar, various saccharification methods have been proposed in which saccharification is performed by passing the liquefied starch solution through a fixed bed reactor in which glucoamylase is immobilized on a porous adsorbent. (For example, Japanese Patent Application Laid-Open No. 53-86086, Patent No. 1162764)
No., Japanese Patent Application Publication No. 55-144890, Patent No. 1120531, Patent
No. 1120532, Patent No. 1117041, JP-A-59-130193
issue). (Problems to be solved by the invention) However, these methods are rarely put into practical use due to the low concentration of the treatment solution, the short life of the immobilized glucoamylase, and the low purity of the glucose produced. has not yet been reached. There is a small amount of solidified glucoamylase (developed by Tate & Lyle) on the market, in which glucoamylase originating from Aspergillus niger is cross-linked and immobilized with glutaraldehyde in bone ash, but this is not possible by passing a high-concentration starch liquefaction solution through it. Even if saccharification is performed using this method, the maximum amount of glucose produced does not reach the amount of glucose produced by the batch saccharification method (approximately 91% or more).
For example, the above-mentioned immobilized glucoamylase has a solid content.
When saccharification is carried out by passing a 30 w/w% high concentration starch liquefied liquid (DE11.8) at a superficial velocity (SV) of 0.5 hr ^-1 , the maximum amount of glucose produced is 87-88%. In comparison, the amount of isomaltose produced increased to about 5%. Therefore, as a result of repeated experiments and research on the relationship between the amount of glucose and isomaltose produced when starch liquefaction is passed through immobilized glucoamylase for saccharification, the inventors of the present invention found that the starch liquefaction It was discovered that when glucose, which is hydrolyzed and produced in contact with amylase, comes into contact with glucoamylase, isomaltose is produced through retrosynthesis, suppressing the yield of glucose. At the same time, the present inventors have discovered that the amount of isomaltose produced increases as the amount of glucose produced increases, or as the contact time between the produced glucose and glucoamylase increases, leading to a decrease in the yield of glucose. I'm finding out. On the other hand, there is also a method of producing glucose by passing a starch liquefaction solution through an enzyme-immobilized membrane reactor in which glucoamylase is adsorbed and immobilized.
No. 57-59758, etc. The enzyme-immobilized membrane used here has small pores on one side and large pores on the other side, and glucoamylase is immobilized on the surface and inside the membrane on the large pore side, and when the starch liquefaction solution is passed through it, the starch The liquefied liquid is hydrolyzed by contact with glucoamylase, and when it is hydrolyzed to a low-molecular substance, such as glucose, that can pass through the small pores, it passes through the small pores and is discharged to the permeate side, passing through the small pores. Undecomposed oligosaccharides, dextrins, etc. that cannot be removed are circulated through the feed liquid side, brought into contact with glucoamylase, hydrolyzed to a size that can pass through the small pores, and discharged to the permeate side. According to this method, the contact time between glucose and glucoamylase is very short, so there is no production of isomaltose due to the reverse reaction. is only glucose, making it possible to produce highly pure glucose. However, in reality, a membrane that decomposes glucose and disaccharides has not been developed. Therefore, when the starch liquefaction liquid is passed through a fixed membrane reactor, the permeate contains many oligosaccharides other than glucose. Therefore, to increase the glucose yield in the permeate, the permeate must be passed through the fixed membrane several more times through the reactor. According to the experiments conducted by the present inventors shown in Control Examples 1 and 2 below, the yield of glucose (G ₁ ) in the first pass of the starch liquefied solution through the enzyme-immobilized membrane was less than 70%;
By the third pass, we finally achieved a value exceeding 90%. In addition, if the treated solution is passed through the enzyme-immobilized membrane many times, the contact time between glucose and glucoamylase becomes longer, and the production of isomaltose due to the reverse reaction increases, resulting in the suppression of the amount of glucose produced. Become. According to the above-mentioned experiments conducted by the inventors of the present application, isomaltose is produced in the first pass.
It is about 0.05%, but in the third pass it decreases to about 1%,
It increases to about 2% in the 5th pass, and along with this, the glucose yield also reaches a maximum value of 91-92% in the 4th pass, but decreases to 90-91% in the 5th pass. Furthermore, when starch liquefaction is directly saccharified in an enzyme-immobilized membrane reactor, the productivity is very poor in the first and second passes, and in the experiments conducted by the inventors of the present invention, the average flux in the first pass was 114 ml. /hr・
0.1m ² , second pass flux averaged 210ml/hr・
It was about ^0.1m2 . Therefore, the present invention aims to continuously produce, for example, 93% or more glucose with good production efficiency. (Means for Solving the Problems) In order to achieve the above-mentioned object, the present invention was developed as a result of extensive research, and it was discovered that a starch liquefied liquid was transferred to a fixed bed reactor with a superficial velocity of 3 to 3. Glucose content 65 after passing for 6hr ^-1
85% and the isomaltose content is 1% or less, the saccharified solution is passed through an enzyme-immobilized membrane reactor in which glucoamylase is adsorbed and immobilized, and the produced glucose is taken out of the reaction system. This paper proposes a manufacturing method. Glucoamylase is known to originate from fungi such as Rhizopus, Aspergillus, Mucor, and Piricararia, yeast from Endomyces, Trichoderma, and Satucharomyces, and from various bacteria. Among these, glucoamylases originating from Rhizopus delemar and Aspergillus niger are suitable for use in the fixed bed reactor or enzyme fixed membrane reactor of the present invention. In particular, when immobilized, glucoamylase originating from Rhizopus deremer (product name: Sumiteam, manufactured by Shin Nippon Kagaku Co., Ltd.) has an optimum temperature of approximately 50°C, compared to glucoamylase originating from niger (product name: AMG Novo). Although the temperature was lower than 58°C, it was found that the retrosynthesis effect was weaker and the accumulation of isomaltose was more than 1% lower. However, due to the low operating temperature, the rate of hydrolysis is slow and therefore the superficial velocity needs to be low. Porous adsorbents constituting the fixed bed reactor include weakly acidic porous resins, weakly acidic cationic resins,
Porous chitosan, natural chitin, bone char, cationized starch, etc. can be used, but among these, porous chitosan produced by deacetylating bone ash, chitin, or natural polymer chitin is particularly effective. There was found. In the case of bone char, glucoamylase and maltooligosaccharide-producing amylase are adsorbed very efficiently, but because they are gradually desorbed by passing a high-concentration substrate through, it is necessary to cross-link and immobilize with glutaraldehyde after adsorption. There is. Furthermore, since the activity of the immobilized enzyme is very high, the life of the enzyme is long, but on the other hand, there are drawbacks such as a large accumulation of isomaltose and the inability to reuse the adsorbent. Among porous chitosan, JP-A-61-40337;
Disclosed in Special Publication No. 61-76504 (Product name:
Chitopearl (manufactured by Fujibo) has excellent PH stability, chemical resistance, and heat resistance. "Chito Pearl" consists of porous beads (#3000 type) in which chitosan is cross-linked with dicarboxylic acid or dialdehyde and then diisocyanated aliphatic functional groups are introduced as spacers (#3000 type), and porous beads in which aromatic functional groups are introduced. (#3500 type), and glucoamylase and maltooligosaccharide-producing amylase are well adsorbed to both types. Furthermore, the membrane constituting the enzyme-immobilized membrane reactor is, for example, an asymmetric capillary membrane in which a skin layer (dense layer) with molecular fractionation and a sponge layer (porous layer) with many voids are integrated. After the membrane is activated with, for example, glutaraldehyde, it is treated with a buffer containing glucoamylase as described above to immobilize glucoamylase on the membrane. On the other hand, raw materials for starch liquefied liquid include corn starch, potato starch, cane starch,
One or more tapioca starches can be used. These starches are liquefied in a liquefaction process to, for example, DE5 to 20 of a liquefied liquid with a starch concentration of 30%. This starch liquefied liquid is passed through a fixed bed reactor in which glucoamylase is immobilized on a porous adsorbent as described above at a superficial velocity of 3 to 6 hr ^-1 to obtain a glucose content of 65 to 85%, preferably 70 to 6 hours. Carry out saccharification to reduce the isomaltose content to 80% and 1% or less. Next, this saccharified solution is passed through an enzyme-immobilized membrane reactor in which glucoamylase is adsorbed and immobilized, so that the produced glucose is taken out of the reaction system. The temperature conditions in the fixed bed reactor or enzyme fixed membrane reactor are preferably around the optimum temperature for glucoamylase.
When glucoamylase originating from Niger is used, the temperature is 55-60°C, and when glucoamylase originating from Rhizopus deremer is used, the temperature is about 40-50°C. Furthermore, the pH condition is preferably around the optimum pH of glucoamylase, for example around pH 4.0 to 7.0. (Effect of the invention) That is, according to the present invention, the starch liquefied liquid is saccharified in a fixed bed reactor to a glucose content of 65 to 85% and an isomaltose content of 1% or less, and then passed through an enzyme fixed membrane reactor. By doing so, for example, the yield of glucose can be increased to 93% or more by passing the saccharified solution through the enzyme-immobilized membrane reactor three times, and at the same time, the amount of isomaltose produced can be suppressed to 1.5% or less. can. Furthermore, by saccharifying the starch liquefaction liquid in the fixed bed reactor, the productivity in the enzyme fixed membrane reactor can be dramatically improved. Further, in the present invention, the starch liquefaction liquid is passed through the starch solution at a superficial velocity (SV) of 3 to 6 hr ^-1 , and high-concentration glucose can be obtained by passing through the enzyme-immobilized membrane reactor about three times. Therefore, according to the present invention, high-concentration glucose can be produced continuously with good productivity. The glucose obtained according to the present invention is
It satisfies the minimum glucose concentration of 93% required to continuously produce isomerized high-fructose corn syrup with an isomerization rate of 42% or more to fructose. It can be applied to (Example) Examples of the present invention are described above. <Preparation of starch liquefied liquid> (1) Preparation of corn starch liquefied liquid 11.4 kg of corn starch (10.0 kg of absolute dry solid content) was suspended in 20 kg of water, and after adding 8.0 g of calcium chloride (0.08% to corn starch), 1N -PH7.0 with Na0H
and liquefied enzyme (product name: Termamil 60L,
Originated from Bacillus licheniformis, manufactured by Novo),
Add 8.0ml (0.08w/w% to cornstarch),
1/ with a small continuous gelatinization device
After instantaneous heating to 105℃ at a flow rate of min, 15 to 105℃
It is passed through a gelatinization retention tube where it can be retained for minutes and discharged under normal pressure. After that, it passes through a liquefaction retention tube where it stays at 95-98°C for 120 minutes to complete gelatinization and liquefaction. Furthermore, adjust the pH to 2.5 to 3.0 with 1N-HC1, hold at 90℃ for 10 minutes to inactivate α-amylase, adjust the pH to 4.5 with 1N-NaOH, and filter at 60℃ or higher to remove aggregates. was removed to obtain a clear liquefied liquid.
The DE of the obtained liquefied liquid was 11.8. Hereinafter, this liquefied liquid will be abbreviated as CSL-11.8. (2) Preparation of liquefied solution of potato starch Enzyme liquefied dextrin of potato starch is commercially available under the trade name “NSD” (manufactured by Nippon Shiryo Kogyo Co., Ltd.) and “Amicol” (manufactured by Nippon Shihoku Kogyo Co., Ltd.). ,
In this example, NSD (DE13.2) was used after being dissolved at a predetermined concentration. Below, this dextrin is NSD
Abbreviated as −13.2. <Immobilization of enzyme onto chitosan beads> 50 g (wet 70 ml) of chitosan beads (product name: Chito Pearl 3010, manufactured by Fujibo Co., Ltd.) and 50 g of glucoamylase (product name: AMG (300 AGU/ml), originating from Aspergillus niger, manufactured by Novo). (Contains 5.4g of enzyme protein) and stirred at room temperature for 2 hours.
After suction filtration, the beads were washed with 100 ml of water, and washing and filtration were repeated until no enzyme protein was eluted in the washing solution. In the obtained Chitopal #3010 adsorption-immobilized glucoamylase, 1.91 g of enzyme protein was adsorbed to Chitopal (38.1 mg/g-Chitopal). <Preparation of enzyme-immobilized membrane reactor> Activate the membrane module with a 3.1% glutaraldehyde solution, and after activation, add a glucoamylase solution (5%) dissolved in 0.02M acetate buffer (PH4.5).
glucoamylase was immobilized on the membrane module by treatment with 1 mg/ml) (originated from Asp. niger, trade name: Gluczyme, manufactured by Amano Pharmaceutical Co., Ltd.). Example 1 CSL-11.8 was placed on a φ15 x 300 mm column of chitosan bead-immobilized glucoamylase prepared previously.
The sample solution CSL-CP-4 was obtained by passing through the solution using SV4hr ^-1 . This test solution contained 81.09% G ₁ , 0.98% IM, and 13.2% undegraded oligosaccharides. This CSL-CP-4 was adjusted to PH4.6 and Bx31, and placed in Membrane Master BM-1 (product name: Nitto Denko Corporation, enzyme-immobilized membrane reaction device) at a temperature of 57.0~
58.5, circulation pressure 1.45~1.55mg/ ^cm2 , circulation flow rate 0.96~
Flux of permeate for each treatment after repeated flow treatment at 1.04/min, glucose (G ₁ ),
The isomaltose (IM) content was as follows.

[Table] Control example 1 CSL-CP-4 in Example 1 was replaced with CSL-11.8, and Membrane Master BM- was used at Bx30.8 and PH4.6.
1. The treatment conditions were the same as in Example 1, and the flux, G ₁ , and IM content of the permeate obtained in each treatment were as follows.

[Table] Example 2 After dissolving NSD-13.2 and setting it to PH4.6 and Bx31.0,
As in Example 1, SV6hr ^-1 was passed through the xanbead-immobilized glucoamylase column, and the test solution was
NSD-CP-6 was obtained. This test solution has G ₁ 70.61%,
IM was 0.49% and undegraded oligosaccharide was 22.56%. Furthermore, this NSD-CP-6 was treated in the same manner as in Example 1, and the flux, G ₁ and IM content of the permeate obtained for each treatment were as follows.

[Table] Control example 2 The test solution was NSD-CP-6 of Example 2.
The same procedure as in Example 1 was carried out except that 13.2 was used. The resulting permeate flux for each treatment,
The G ₁ and IM contents were as follows.

[Table] Control example 3 CSL-11.8 and NSD-13.2 were applied to a chitosan bead-immobilized glucoamylase column at Bx30 and PH4.5.
The amounts of G ₁ and IM produced when the solution was passed at SV1hr ^-1 are shown below.

【table】

Claims (1)

[Claims] 1. A high-concentration starch liquefied liquid with a concentration of 30% or more is passed through a fixed bed reactor in which glucoamylase is immobilized on a porous adsorbent at a superficial velocity of 3 to 6 hr ^-1 to determine the glucose content. 65-85%, and the isomaltose content is 1% or less, and the saccharified solution is passed through an enzyme-immobilized membrane reactor in which glucoamylase is adsorbed and immobilized, resulting in high purity glucose with a purity of 93% or more. A method for continuous production of glucose, characterized by taking glucose out of the reaction system. 2. The continuous production method according to claim 1, wherein the starch is one or more of corn starch, potato starch, sweet potato starch, and tapioca starch.