GB2099823A - Process for the production of glucose - Google Patents

Abstract

A process for the production in high yield of glucose of high purity, the process comprising enzymatically saccharifying a liquefied starch into glucose using a combination of glucoamylase (E.C. 3.2.1.3) and isoamylase (E.C. 3.2.1.68), the liquefied starch solution having a dextrose equivalent of less than 15, said liquefied starch solution being subjected to the action of glucoamylase and isoamylase at a pH of 3.5 to 4.8 to effect saccharification of the starch. The isoamylase is advantageously derived from a microorganism of the genus Flavobacterium, Cytophaga or Pseudomonas and the glucoamylase from Rhizopus or Aspergillus.

Description

SPECIFICATION Process for the production of glucose The present invention relates to a process for the production of glucose.

In this specification all percentages are given on a dry solids basis.

Conventionally, saccharification of starch into glucose has been carried out by a method in which a starch suspension is liquefied with an acid or a liquefying a-amylase, and is then subjected to the action of glucoamylase to obtain a saccharified starch solution with a glucose content of up to 9094% on dry solids basis. The solution is decolorised with activated charcoal, deionised with ion exchanger, concentrated, and crystallised, and the resultant mascuite is then prepared into a powder having a high glucose content, or separated into crystalline glucose and mother liquor, depending upon the intended use of the glucose.

Recently, in order to enhance the glucose content of the saccharified starch solution, several improvements have been proposed. Thus, Japanese Patent Publication No. 29,570/79 discloses the saccharification of a liquefied starch solution having a dextrose equivalent (abbreviated as DE hereinafter) of 1 5-20 using the action of an enzyme system consisting of glucoamylase and amylo1,6-glucosidase at a pH of 5.0 to 6.7, this range of pH being one in which the reversibie reaction by glucoamylase is suppressed. Japanese Patent Publication No. 23,894/81 discloses the saccharification of a liquefield starch into glucose by using a Bacillus or-1,6-glucosidase in combination with glucoamylase at a pH of 5.0 to 7.0.Japanese Patent Publication No. 23,895/81 proposes that in the course of the saccharification of a liquefied starch with glucoamylase, it is desirable to allow an a-1,6- glucosidase to act at a pH of 5.0 to 7.0 on a partially saccharified starch solution in which 3080% saccharification has already been performed.

However, in the above-mentioned processes saccharification is carried out at a pH higher than 5.0, and this results in certain disadvantages. For example, the resultant saccharified starch solution prepared by these processes are inevitably susceptible to turn brown in colour and to microbial contamination under conventional saccharification conditions when performed on an industrial-scale saccharification temperature, ca. 50-600C, andsaccharification time, ca. 40-90 hours.

Confronted with the above described disadvantages, we have investigated processes for the production of a saccharified starch solution having a high glucose content, in which saccharification of the starch is carried out at a pH lower than 5.0 so as to overcome the said disadvantages.

We have found that a saccharified starch solution having a high glucose content can be obtained easily by converting a starch suspension into a liquefied starch solution with a low DE, and subjecting the resultant liquefied starch solution to the action of glucoamylase (EC 3.2.1.3) and isoamylase (EC 3.2.1.68) at a pH of 3.5 to 4.8.

Accordingly the present invention provides a process for the production of glucose which comprises enzymatically saccharifying a liquefied starch into glucose using a combination of glucoamylase (E.C. 3.2.1.3) and isoamylase (E.C. 3.2.1.68), the liquefied starch solution having a dextrose equivalent of less than 1 5, said liquefied starch solution being subjected to the action of glucoamylase and isoamylase at a pH of 3.5 to 4.8 to effect saccharification of the starch.

The starch materials which may be used in the invention can be starches obtained from the roots or tubers of potatoes, sweet potatoes and cassava, or from grains of corn, rice or wheat. Also, the use of other starch materials, such as grits and tuber tips with a higher starch content, realises the same effect as obtained with the above described starch materials.

As regards the concentration of the starch suspension, any concentration can be used as long as the starch is sufficiently liquefied and dispersed. The concentration of the starch is usually 10-50%, preferably 2040%.

The liquefaction of the starch suspension can be carried out according to conventional methods, and the resultant liquefied starch solution is then subjected to the enzymatic action of glucoamylase anc isoamylase at a temperature of 50 to 600C and at a pH of 3.5 to 4.8. In the course of the saccharification of the liquefied starch solution, isoamylase should be added to the solution before the DE of the solution becomes higher than 1 5, preferably before it becomes higher than 1 0.

The glucoamylases (EC 3.2.1.3) suitable for use in the present invention are those which act on the liquefied starch solution at a pH of 3.5 to 4.7, and release glucose units. Examples of suitable such glucoamylases are commercial glucoamylases from molds, such as of the genera Rhizopus and Asperglllus,'and especially, glucoamylase from the genusAspergillus: are these may be used in the present invention due to their lower pH optima at pH 4.0 to 4.5.

The isoamylases (EC 3.2.1.68) suitable for use in the present invention are those from bacteria, such as of the genera Flavobacterium, Cytophaga and Pseudomonas; Pseudomonas isoamylases being preferred.

After completion of the saccharification, the resultant saccharified starch solution is heated to inactivate the enzymatic activities present therein, and subjected to purification, such as filtration, decolorisation with activated charcoal, and/or deionisation with an ion exchanger, and subsequently concentrated, to obtain a starch syrup having a high glucose content. The glucose content of the syrup is extremely high, i.e. 95 to 98%.

Unlike the saccharification according to conventional processes which is carried out at a pH of 5.0 or higher, the process according to the invention is carried out at a pH of 3.5 to 4.8 using a combination of glucoamylase and isoamylase, and provides a number of advantages such as shortening the saccharification time, and reducing the microbial contamination and browning which occurs during the saccharification. As a result subsequent purification of the saccharified starch solution can be carried out with much ease. Accordingly, the mass production of a saccharified starch solution having a high glucose content can be performed easily.

The present invention can be favourably employed for the production, in increased quantity and/or greater unity, of crystalline glucose, of fructose by isomerisation of glucose, of sorbitol by hydrogenation of glucose, and other products, such as ethanol and sodium mono L-glutamate which are produced by fermentation of glucose.

The following Examples illustrate the present invention.

In this specification, the enzymatic activities were assayed as follows: (1) Glucoamylase (EC 3.2.1.3): one unit of glucoamylase activity is defined as the amount of enzyme that releases 1 mg glucose over three minutes at 40CC in a reaction mixture consisting of 5.0 ml of 1.0 w/w % aqueous soiuble starch solution, 4.0 ml of 1 M acetate buffer solution (p 4.5), and 1.0 ml of a glucoamylase solution; and (2) Pullulanase (EC 3.2.1.41) or isoamylase (EC 3.2.1.68): one unit of pullulanase or isoamylase is defined as the amount of enzyme that increases the 610 nm absorbance by 0.01 per hour.More- precisely, to a solution, consisting of 5.0 ml of 1.0 w/w% aqueous soluble waxy-rice starch solution and 1.0 ml of 0.5 M acetate buffer solution in which the pH of the buffer solution is adjusted to 6.0 for pullulanase, of 3.5 for isoamylase, and the mixture is incubated at 400C for one hour. Then, 1 ml of the reaction mixture is collected, and added with 1 ml of 0.01 M aqueous l2-Kl solution and, subsequently, 0.02 N H2SO4 to give a final volume of 25 ml. Fifteen minutes after the addition of sulfuric acid, the absorbance of the mixture is measured at a wave length of 61 0 nm with 1 cm cell.

The glucose content in the saccharified starch solution was determined by conventional paper chromatographic analysis, and expressed as a weight percentage of the total amount of sugars.

EXAMPLE 1 To a 28 w/w % corn starch suspension was added calcium carbonate in an amount of 0.05 % per dry starch-solid and a commercial Bacillus licheniformis a-amylase "Termamyl 60L" (Novo Industri A/S, Copenhagen, Denmark) in an amount of 0.1 5 % per dry starch solid, and the mixture was liquefied at 980C for 30 minutes, and exposed to ambient conditions, thereby to obtain a liquefied starch solution with DE of 4.0.After-adjusting thepH to 4.5 and cooling to 550C, promptly, 500 ml aliquots of the solution were placed in 1 litre flasks, and then a commercial Aspergillus glucoamylase "XL--128" (Nagase a Company, Ltd., Osaka, Japan) was added thereto in an amount of four units, and an Enterobacter pullulanase or a Pseudomonas isoamylase (Hayashibara Biochemical Laboratories Inc., Okayama, Japan) was added thereto in an amount of 1 50 units. The aliquot mixtures were then subjected to saccharification for 40 hours at pH 4.5 and 550C.

A control experiment was carried out simultaneously. In the control, the saccharification was carried out similarly as described above, but the pullulanase or isoamylase was omitted.

After the saccharification, the glucose content in the saccharified starch solution aliquots was determined. The results are given in Table 1.

TABLE 1

Saccharifying enzyme tested Glucose content (%) Glucoamylase 93.9* Glucoamylase + Pullulanase Glucoamylase + Isoamylase 97.4** Note: *), control; and **), present invention The experimental results as shown in Table 1 confirm that a saccharified starch solution having an extremely high glucose content is easily obtainable by saccharifying a liquefied starch solution with glucoamylase in combination with isoamylase at a pH of 4.5.

EXAMPLE 2 Five hundred ml aliquots of a liquefied starch solution with DE of 4.0, obtained in a manner similar to that described in Example 1, were placed in 1 litre flasks, and adjusted to a pH of 3.0, 3.5, 3.8, 4.0, 4.5, 4.8, 5.1, 5.5, or 6.0 with hydrochloric acid, and cooled to 500 C, promptly. Thereafter, glucoamylase and isoamylase were added to the aliquot solutions in a manner similar to that described in Example 1, and the resultant aliquots were incubated at 500C and at the specified pH level for 48 hours to effect enzymatic saccharification.

A control experiment was carried out simultaneously, in which saccharification was carried out similarly as described above, but with the isoamylase omitted.

After the saccharification, the glucose content, and degree of coloration and microbial contamination in the saccharified starch solution aliquots were determined. The results are given in Table 2.

The experimental results shown in Table 2 confirm that saccharification using a combination of glucoamylase and isoamylase at a pH of 3.5-4.8 results in a saccharified starch solution having a very high glucose content, and that at a pH of 5.1 or higher the saccharified starch solution is very susceptible to browning and microbial contamination.

TBLE 2

Relative degree of Glucose content coloration Degree of micro Enzyme pH (o) (%) bial contamination 3.0 92.1 94 3.0 92.1 94 (-)* Glucoamylase 3.5 95.5 96 3.8 96.4 96 4.0 97.6 98 + 4.5 97.2 100 4.8 96.0 103 5.1 93.8 118 (+)* isoamylase 5.5 92.0 135 6.0 86.2 254 Glucoamylase 4.5 93.9 98 Notes: (1) "Relative degree of coloration" was calcuiated by the following equation; B Relative degree of coloration (%) > x 100 A where A is the absorbance of the filtrate at a wave length of 420 nm with 1 cm cell, which was obtained by saccharifying a liquefied starch solution at pH 4.5 using combination of glucoamylase and isoamylase, and filtering the resultant saccharified starch solution with filter paper; and B is the absorbance obtained similarly as A except that the liquefied starch solution was saccharified at other-pH level, and then adjusted to pH 4.5.

(2) "Degree of microbial contamination" was determined after completion of the saccharification by measuring the number of microbial colonies in the saccharified starch solution, and used as a criteria for evaluating the degree of the microbial contamination; (+) 1,000 or more colonies were found in 1 ml of the saccharified starch solution; (+). 1 00 or more, but less 1 ,000 colonies; and (-), less than 100 colonies.

(3) *), control; and **), present invention.

EXAMPLE 3 Thirty five w/w % liquefied tapioca starch solution aliquots were different DE, obtained similarly as described in Example 1, were adjusted to pH 4.5 with hydrochloric acid and cooled to 60"C, promp.tly.

Thereafter, a commercial Aspergillus glucoamylase "Amyloglucosidase 150 L" (Novo Industri A/S, Copenhagen, Denmark) was added to the said aliquots in an amount of five units and Pseudomonas isoamylase (Hayashibara Biochemical Laboratories inc., Oakayama, Japan) was added thereto in an amount of 75 units, and the resultant aliquots were incubated at 600C and pH 4.5 for 36 hours to effect enzymatic saccharification.

A control experiment was carried out simultaneously, in which the saccharification was carried out similarly as described above, but with the isoamylase omitted.

After the saccharification, the glucose content in the resultant saccharified starch solution aliquots was determined. The results are given in Table 3.

The experimental results as shown in Table 3 confirm that in the course of the conversion of starch into glucose using a combination of glucoamylase and isoamylase to obtain a saccharified starch solution having a very high glucose content, the DE of the starting liquefied starch solution should be adjusted to less than 1 5.

TABLE3

DE Glucoamylase Glucoamylase + Isoamylase 1.9 92.5%* 96.Z%** 4.2 93.80/6* 97.0%** 8.8 93.7%* 96.7%** 10.0 93.3%* 96.2%** 14.2 92.8%* 95.1%** 19.6 92.4%* 93.8%* 21.4* 92.1%* 93.2%* Note: *), control; and **), present invention

Claims (7)

1. A process for the production of glucose which comprises enzymatically saccharifying a liquefied starch into glucose using a combination of glucoamylase (E.C. 3.2.1.3) and isoamylase (E.C. 3.2.1.68), the liquefied starch solution having a dextrose equivalent of less than 1 5, said liquefied solution being subjected to the action of glucoamylase and isoamylase at a pH of 3.5 to 4.8 to effect saccharification of the starch.

2. A process according to Claim 1, wherein the isoamylase is one derived from the genus Fla vobacterium, Cytophaga or Pseudomonas.

3. A process according to Claim 1 or 2, wherein the glucoamylase is one derived from the genus Rhizopus or Aspergillus.

4. A process according to Claim 1, 2 or 3, wherein the DE of the liquefied starch solution is 10 or less.

5. A process according to any one of the preceding claims, wherein the liquefied starch solution is subjected to the enzymatic action of glucoamylase and isoamylase at a temperature in the range of 50 to 60"C for 30 to 50 hours.

6. A process according to Claim 1 substantially as hereinbefore described in any one of the Examples.

7. Glucose whenever prepared by a process as claimed in any one of the preceding claims.