JPS6243676B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for treating an enzyme preparation for producing high maltose syrup, and more specifically,
- In the presence of 1,6 glucosidase, Bacillus
Activate the saccharifying enzyme agent produced by polymixer,
When producing high maltose syrup having a sugar composition of 80% or more maltose, 8 to 15% maltotriose, and 0.4% or less glucose, the saccharifying enzyme is treated in advance and incorporated into the saccharifying enzyme. The present invention relates to a method for treating an enzyme preparation for producing high maltose syrup, in which the activity ratio of β-amylase and α-amylase is 1:0.02 to 0.2. In recent years, when producing high maltose syrup on an industrial scale, β-amylase alone or β-amylase
Starch treatment is carried out using a combination of amylase and α-1,6 glucosidase. In the case of β-amylase alone, a product with a maltose content of 45 to 60% is obtained, and β-amylase is combined with bacterial α-1.
When 6-glucosidase is used in combination, products with maltose content of 70% or more have been obtained. However, if this method is used to increase the maltose content to 80% or more, the maltose will crystallize, which is inconvenient, and it will not be possible to obtain an amorphous maltose syrup with a maltose content of 80% or more. Although saccharified liquid with a maltose content of 80% or more has been obtained up to now, this method is for separating crystalline maltose, and the maltose content is 80% or more.
% or more, in some cases more than 90%, but since the remaining sugar is mainly sugar with a degree of polymerization of 4 or more, maltose tends to crystallize, making it difficult to make maltose syrup for food additives. It was. The present inventors have discovered that maltose is advantageous for food additives.
When conducting research to produce maltose syrup with over 80% maltose content, it was by chance that β-amylase from Bacillus polymyxa was converted into α-1, 6-amylase.
In the presence of glucosidase, when liquefied starch with D/E5 or less was treated, the maltose content was 80%.
They discovered that it is possible to produce the above amorphous high maltose syrup. However, in order to explain this phenomenon theoretically, we conducted further research and found that when Bacillus polymyxa is cultured in an appropriate medium and produces β-amylase, the bacterium produces α-amylase in addition to β-amylase. A very small amount of amylase is produced, and this small amount of α-amylase is moderately involved in the production of maltose and maltotriose. Even those that cannot be broken down by β-amylase alone are broken down to maltose or maltotriose, and maltose It has become clear that a maltose syrup that is difficult to crystallize and is mainly composed of oligosaccharides with a content of 80% or more and a degree of polymerization of 3 or less can be obtained. That is, in the presence of α-1,6 glucosidase to liquefied starch of D/E5 or less,
The desired high maltose syrup can be obtained by reacting with a culture in which the activity ratio of β-amylase and α-amylase produced by Bacillus polymyxa is 1:0.02 to 0.2, or an enzyme obtained therefrom. . Furthermore, the present inventors investigated the culture medium, culture conditions, etc. in order to increase the productivity of Bacillus polymyxa saccharifying enzyme agents, which have the advantages mentioned above.
- It was found that the amylase activity was further increased, and the ratio of β-amylase activity to α-amylase activity was more than 1:0.2, and that using this enzyme agent resulted in a decrease in the amount of maltose produced. did. Therefore, the present inventors were able to selectively inactivate α-amylase while minimizing the inactivation of β-amylase in a Bacillus polymyxa saccharifying enzyme preparation, and also to reduce β-amylase activity to α. - The present invention was completed as a result of intensive study on treatment conditions for adjusting the amylase activity ratio to 1:0.02 to 0.2. Conventionally, various methods have been described for selectively deactivating α-amylase from β-amylase including α-amylase, so-called saccharifying enzyme agents, and extracting β-amylase alone, such as (1) bentonite adsorption method. A method for extracting β-amylase from soybeans using (Japanese Patent Publication No. 47-31639), (2) extraction from soybeans at 60°C or lower in the presence of calcium acetate, salting out ammonium sulfate, dialysis, ion exchange resin, gel filtration, etc. (3) α- in malt amylase
A method for separating amylase and β-amylase by PH and temperature treatment (University of Tokyo Experimental Agricultural Chemistry Vol. 1, p. 279), (Enzyme Research Methods Vol. 2, p. 110), (4)
Starch adsorption method for microbial β-amylase (Special Publication 1972-
38112) etc. However, none of these methods describes conditions under which the activity ratio of β-amylase to α-amylase of the saccharifying enzyme produced by Bacillus polymyxa is 1:0.02 to 0.2. If the treatment method of the present invention is carried out, even if the saccharifying enzyme agent produced by Bacillus polymyxa contains a large amount of α-amylase, it will always be able to produce β-amylase containing α-amylase, which is optimal for maltose syrup production. The α-amylase activity ratio is 1:0.02 to 0.2.
It can be regulated. The treatment can be carried out on any Bacillus polymyxa culture, culture solution, intermediate purified solution, purified solution, etc. The pH of the culture solution and purified solution is almost neutral, so add an acid such as hydrochloric acid to adjust the pH to 4.0 to 4.5, raise the temperature to 4 to 40℃, and leave for 1 to 3 hours. be done. By using the treatment method of the present invention, it is possible to maximize β-amylase activity using Bacillus polymixa, and at the same time moderately reduce only α-amylase activity, which increases production. This makes it possible to provide an enzyme agent most suitable for producing high maltose syrup. Next, production examples, test examples, and examples of the present invention will be shown. Production example: 20 media containing 10% dextrin, 0.75% ammonium sulfate, 1% defatted soybean powder, 0.3% NaCl, and 0.1% ammonium phosphorus (PH6.5) were placed in a 50 volume fermenter and heated to 120°C.
Sterilize under autoclave for 30 minutes. Pre-soluble starch 0.5
%, Phosphoranium 0.1%, KCl0.02%, _MgSO4・_7H2O0.02
%, yeast extract 0.2% (PH7.3), put 100ml of medium into a 500ml Sakaguchi flask, and incubate at 120℃30.
Bacillus polymixa after high pressure sterilization
No. 55 and FERM-P No. 3952 were inoculated and cultured with shaking at 30°C for 20 hours. The entire amount of the seed culture was inoculated into the above fermenter, and cultured at 37°C throughout the entire culture period. During the culture, the pH was maintained at 6.5 ± 0.5 with 3N NaOH, and the culture was carried out with aeration for 100 hours. After the culture was completed, the bacteria was removed using a DeLaval centrifuge, and the sterilized solution was concentrated under reduced pressure. This concentrated crude enzyme solution was By performing ammonium sulfate salting out, DEAE-cellulose treatment, and CM-cellulose treatment, β
-Amylase and α-amylase were separated and purified. The purification diagram by CM-cellulose treatment is shown in FIG. Here, A...α-amylase, B...β
-Amylase, C...Protein, D...Salt concentration, respectively. Test Example 1 A 25% potato starch slurry was liquefied using a commercially available liquefaction type bacterial amylase to obtain a liquefied liquid with a DE of 2.3. 4 U per g of starch dry matter, and 7 U of α-1,6 glucosidase (Pulteam K2000: ABM Chemicals Limited product) per g of starch, and purified α-amylase from Bacillus polymyxa obtained in the production example against β-amylase. When saccharification was carried out at 60° C. for 30 hours while maintaining the pH within the range of 6.0 to 5.0 by adding various active ratios, the results shown in Table 1 were obtained.

[Table] To measure the sugar composition above, spot the saccharified solution on paper and add 3
The mixture was developed twice, and portions corresponding to each sugar were cut out, and the water extracted liquid was quantified using the phenol-sulfuric acid method. However, only glucose was directly quantified in the saccharified solution using the glucose oxidase method. The saccharified solutions having the sugar compositions obtained in No. 1 to No. 10 in Table 1 were purified by ion exchange resin treatment and activated carbon treatment, and then vacuum concentrated to a solid content of 75% and left at room temperature for one month. No crystals were precipitated in the syrups obtained from No. 2 to No. 7. Furthermore, as shown in Table 1, by adding α-amylase to β-amylase, the maltose content can be reduced to 80%.
% or more of high maltose syrup can be obtained, but if too much α-amylase is present, good results cannot be obtained, and therefore β-amylase vs.
- It has been found that an appropriate amylase activity ratio is 1:0.02 to 0.2. However, as it is currently not economically viable as a saccharification enzyme agent, in order to increase the β-amylase production ability of Bacillus polymyxa, culture media,
When culturing conditions were examined, it was found that although β-amylase activity increased, α-amylase activity increased even more, resulting in a ratio of 1:0.2 or more. Therefore, the present inventors investigated the treatment conditions for the saccharifying enzyme agent in order to maintain the ratio of β-amylase to α-amylase at 1:0.02 to 0.2 even in such cases. Test example 2 Pharmacopoeia dextrin 20%, phosphorus ammonium 0.75%, defatted soybean powder 1.5%, NaCl 0.1%, KCl 0.05%, MgSO ₄ .
Medium 20 with the composition of _7H2O0.05 % (PH6.5)
Place in a 30 volume fermenter and autoclave at 120℃ for 30 minutes.
In advance, soluble starch 0.5%, phosphorous ammonium 0.1%,
KCl0.02%, _MgSO4・_7H2O0.02 %, yeast extract
500ml 100ml medium with a composition of 0.2% (PH7.3)
Bacillus Polymixa No. 55, FERM-
P No. 3952 was inoculated and cultured with shaking at 30°C for 20 hours. The entire amount of the seed culture was inoculated into the above fermenter, and the culture was incubated at 39°C for 30 hours from the initial stage of culture, and then at 34°C for up to 82 hours. did. PH during culture was 6.5±0.5 3N−
The culture was maintained with NaOH and aerated with aeration. After the culture was completed, the bacteria was removed using a DeLaval centrifuge, and the sterilized solution was concentrated under reduced pressure. The concentrated crude enzyme solution contained β-amylase 1080 μ/ml and α-amylase 360 μ/ml. I got it. Next, take 20 ml of each of these concentrated crude enzyme solutions and adjust the pH of each.
After adjusting with 4N-HCl, changing temperature conditions, and treating for 3 hours, the β-amylase activity and α-amylase activity of each concentrated crude enzyme solution, and the ratio thereof, that is, α-amylase activity/β-amylase activity (hereinafter referred to as α/S) was determined as shown in Table 2.

[Table] As is clear from Table 2, α/S is in the range of 0.02 to 0.2 when treated at PH4.0 to 4.5 at 4℃, at PH4.5 at 20℃, and at PH4.5 at 40℃. It can be seen that α/S falls within the range of 0.02 to 0.2, respectively. Test Example 3 Each 20 ml of the concentrated crude enzyme solution obtained in the same manner as Test Example 2 was adjusted to pH 4.5 with 4N-HCl, and the remaining β-amylase activity and α-amylase were treated at 20°C for each time. The activity and α/S were determined as shown in Table 3.

[Table] That is, it is clear that α/S is within the range of 0.02 to 0.2 under the conditions of treatment at a temperature of 20° C. and a pH of 4.5 and a treatment time of 60 to 180 minutes. In this way, even if the Bacillus polymyxa saccharifying enzyme agent has a ratio of β-amylase activity to α-amylase activity of 1:0.2 or more, it can be treated at pH 4.0 to 4.5, temperature 4 to 40°C, and for 1 to 3 hours. By doing so, the ratio can be set to 1:0.02 to 0.2. Next, a method for measuring the activity of the enzyme used in the present invention will be described. 1 α-1.6 glucosidase activity: 1 g of pullulan in M/20 phosphate buffer (PH6.0)
Transfer 0.5 ml of the substrate solution to 100 ml by dissolving it in a test tube, preheat it at 40°C for 5 minutes, add 0.5 ml of the appropriately diluted enzyme solution, and let it react at 40°C for 15 minutes. After 15 minutes, add 1 ml of DNS reagent and mix to stop the reaction. Thereafter, the mixture was heated in boiling water to develop color, cooled in running water, 8 ml of purified water was added, and the mixture was mixed uniformly. The absorbance was then measured at a wavelength of 540 mμ. Activity (μ/ml) is expressed as (absorbance of sample - absorbance of control) x dilution factor. 2 β-amylase activity: 0.5 g of soluble starch was added to M/20 phosphate buffer (PH
7.0) Pour 10ml of the substrate solution dissolved in 100ml into an Erlenmeyer flask, place it in a constant temperature bath at 40°C, and after preheating for at least 5 minutes, add 1ml of diluted enzyme solution and react at 40°C for 30 minutes. After 30 minutes, 2 ml of Fehring's alkaline solution was added to stop the reaction, and then 2 ml of Fehring's copper solution was added. After boiling for another 2 minutes, cool and add 30% (W/W) potassium iodide 2.
ml, 2 ml of 25% (W/W) sulfuric acid, and immediately titrate with 0.05N sodium thiosulfate (A
ml), separately add the enzyme solution and Fehling's alkaline solution in the same manner but in the opposite order.
As a control (Bml), β-amylase activity (μ/ml) is expressed as 1.62×(B−A)/10×enzyme dilution factor. 3 α-Amylase activity: Amylase test “Daiichi” (Daiichi Chemical Co., Ltd.)
Sales) 1 tablet contains 2mM calcium chloride
10 Add to 6 ml of acetic acid buffer (PH6.0) to make a substrate solution, preheat at 40°C for 5 minutes, and then add 0.1 ml of diluted enzyme solution.
After 15 minutes, 1 ml of 0.5N sodium hydroxide was added to stop the reaction. After passing through this, the absorbance at 620 mμ is measured, and the same operation is carried out using water instead of the enzyme solution as a control. α-amylase activity (μ/ml) is expressed as (absorbance of sample - absorbance of control) x dilution factor. Example According to Test Example 2, Bacillus polymixa
No. 55 and FERM-P No. 3952 were cultured to obtain 20 crude enzyme solutions containing bacterial cells. After the culture was completed, bacteria were removed using a DeLaval centrifuge, and the sterilized solution was concentrated under reduced pressure.
-Amylase 1040 μ/ml, α-amylase 330
A concentrated crude enzyme solution 2 with a ratio (α/S) of 0.33 in μ/ml was obtained. This concentrated solution (PH6.3) was cooled to 4℃ and 4N HCl
to pH4.5, treated for 3 hours, and then adjusted the pH to 4.5 with 4N NaOH.
When the pH was returned to a stable pH of 6.3, the β-
Amylase is 930μ/ml, α-amylase is 150
μ/ml, and α/S is 0.16. The syrup produced using this product was amorphous high maltose.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a crude enzyme solution containing β-amylase and α-amylase produced by Bacillus polymixa in Test Example 1, purified by CM-cellulose treatment. A: α-amylase, B: α-amylase, C: protein, D: salt concentration.

Claims (1)

[Claims] 1. A saccharifying enzyme agent produced by Bacillus polymyxa is applied to liquefied starch with DE5 or less in the presence of α-1.6 glucosidase to obtain maltose content of 80% or more, maltotriose content of 8-15%, glucose content of 0.4% or less. When producing high maltose syrup having a sugar composition of 1. A method for treating an enzyme agent for producing high maltose syrup, which comprises treating at a pH of 4.0 to 4.5 and a temperature of 4 to 40°C for 1 to 3 hours.