JP4570067B2 - Process for producing β-amylase - Google Patents

Description

  The present invention relates to a method for producing β-amylase and a technique for producing maltose using the β-amylase. β-amylase is generally used for maltose production and starch aging prevention.

  Conventionally, what is manufactured by extracting and refine | purifying (beta) -amylase from soybean, wheat, and barley is used industrially. Among these β-amylases, those derived from soybean are known to have high enzyme activity and excellent heat resistance, but are expensive. Moreover, although β-amylase has been produced from a water-soluble fraction of soybean oil juice, in recent years, a production method for obtaining soybean oil by extraction using an organic solvent has become the mainstream, and β-amylase is produced by this production process. Since it is inactivated, it is not suitable for the production of β-amylase, and there is a situation that β-amylase derived from soybean is not so much produced at present. In addition, the wheat and barley-derived ones are inexpensive because of the availability of abundant raw materials and the like, but have a problem that they are severely restricted by usage and conditions because of their low heat resistance.

  Maltose is produced by adding an enzyme agent such as β-amylase to a starch material and saccharifying the resulting saccharified solution. In this case, the higher the saccharification temperature, the faster the production rate, but the enzyme is easily deactivated, and the enzyme reaction itself is not performed efficiently. Lowering the saccharification temperature makes it difficult to inactivate the enzyme, but the time required for the enzyme reaction becomes longer, the conditions for easy propagation of miscellaneous bacteria tend to be complicated, and the work process management tends to be complicated. The processing temperature differs greatly from the pre-processing step and the post-processing step, and there is a problem that it is not economical not only from the work step but also from the viewpoint of energy efficiency.

  Therefore, there is a demand for a method for producing β-amylase that is an alternative to β-amylase derived from soybean, wheat, and barley.

Therefore, the use of β-amylase contained in sweet potatoes that can be obtained from abundant raw materials has been studied, but the enzyme obtained from sweet potato (sweet potato, hereinafter referred to as sweet potato) contains α-amylase, Due to the difficulty of refining, it is in the current situation that it does not reach the industrially usable level.

  Therefore, in view of the above circumstances, an object of the present invention is to provide a technology for obtaining β-amylase having high enzyme activity and high heat resistance and efficiently obtaining high-quality maltose by the β-amylase. .

As a result of intensive studies on β-amylase in the enzyme contained in sweet potato, the present inventors have found that this β-amylase has relatively high heat resistance. Moreover, it has been found that the enzyme can selectively inactivate the α-amylase contained by selecting specific conditions, and a highly pure β-amylase can be obtained. The present invention has been made on the basis of the above-mentioned new findings, and the characteristic configuration of the method for producing β-amylase of the present invention for achieving the above object is as follows:
A pH adjusting step for obtaining an acidic treatment liquid obtained by adjusting the squeezed citrus juice to pH 3.9 to 4.0,
A heating step of heat treating the acidic treatment liquid at 59 to 61 ° C. for 35 to 45 seconds;
A neutralization treatment step for neutralizing the heat-treated liquid,
The point is to perform in order.

  The sweet potato is preferably composed mainly of at least one selected from Kyushu No. 140 and Sunny Red.

In addition, the method for producing maltose of the present invention is characterized in that an enzyme preparation containing β-amylase derived from sweet potato is mixed with a starch raw material to cause a conversion reaction from starch to sugar .

[Function and effect]
In other words, α-amylase and β-amylase are included in the enzyme derived from potato, and as a result of earnest research on the β-amylase, β-amylase derived from potato is currently widely used in β-derived from wheat and barley. Excellent heat resistance compared to amylase.

  Therefore, when a sugar conversion reaction or the like is performed using β-amylase having high heat resistance, problems such as a decrease in enzyme activity are less likely to occur during the heat treatment necessary for the reaction, and the temperature is higher than that in the past. Since the reaction can be performed at a lower temperature, the reaction rate can be increased, the reaction can be completed in a short time, and the productivity of the product by the reaction can be improved. In addition, when such a reaction is usually performed, it is a reaction derived from a natural organic matter, so it is necessary to pay attention to prevention of contamination and propagation of various germs, but by selecting a high temperature condition in which the germs are difficult to propagate. Such hygiene management can be facilitated, and there is an advantage that the quality of the product can be improved and the labor of production management can be saved.

  In addition, β-amylase derived from sweet potato has a high reaction activity for the conversion reaction from starch to sugar. If maltose production is carried out using this β-amylase, the conversion rate from starch to sugar exceeds 80%. From the experimental examples, it was found that this was particularly useful for the production of maltose. When maltose is obtained in this manner, a product with high quality in terms of hygiene can be obtained with high productivity as described above.

  Furthermore, the maltose syrup obtained by the above-described maltose production method has an extremely high sugar conversion rate as compared with the conventional maltose syrup, is high quality as a syrup, and is extremely high in hygiene. It is also useful for supplying products at low cost because they can be produced efficiently with inexpensive raw materials.

  Moreover, the above-mentioned enzyme can selectively inactivate only the said alpha-amylase by selecting specific conditions so that it may become clear from the below-mentioned experiment example. Therefore, highly purified β-amylase can be obtained, and a sugar conversion reaction based on the β-amylase can be selectively performed.

  As a further experimental fact, when various sweet potatoes were screened, it was found that Kyushu No. 140 and Sunny Red are sweet potatoes with a particularly high enzyme content and are suitable as raw materials for the production of β-amylase. all right.

  Moreover, since the activity of the α-amylase derived from sweet potato increases during storage, it was found that it is desirable to process it immediately after harvesting.

  Furthermore, although the sweet potato-derived β-amylase is highly purified, the selectivity of the enzyme reaction is increased, but the activity is found to decrease. If the sweet potato powder is used as an enzyme preparation without excessive purification, maltose production is possible. It has also been found that it can be suitably used for such applications.

  In the pH adjustment step, it is preferable to adjust the squeezed potato juice to pH 3.9 to 4.0. If the pH is less than 3.9 from the experimental examples described below, β-amylase may be inactivated. No.

  Moreover, it is preferable to heat-process at 59-61 degreeC for 35 to 45 second, and when it is 59 degrees C or less, the inactivation of (alpha) -amylase is not enough. Further, heating at 61 ° C. or higher is not preferable because there is a risk of inactivation of β-amylase.

  Furthermore, when the conversion reaction from starch to sugar using β-amylase is performed, it is preferable to perform the treatment at 65 ° C. to 70 ° C. for 22 to 26 hours. There is a problem, there is a problem that the time required for the conversion reaction becomes long and the reaction efficiency is lowered. On the other hand, when the temperature is 70 ° C. or higher, the activity of β-amylase tends to decrease, and the durability of the enzyme decreases. In particular, it is preferable to carry out the conversion reaction under the above-mentioned conditions since a particularly high-quality maltose syrup having a conversion rate from starch to maltose of 80% or more can be obtained efficiently.

Embodiments of the present invention will be described below.
<Β-amylase>
While obtaining tuberous root juice of sweet potato (Kyushu No. 140), hydrochloric acid is added to this juice and adjusted to pH 3.9 to 4.0 (pH adjustment step). In this step, it was possible to apply a continuous process in which the squeezed liquid was continuously supplied to the mixer at 80 mL / min and the hydrochloric acid was continuously supplied at 4 mL / min and mixed for about 1 minute 50 seconds.

  Next, the obtained acidic treatment liquid is heated at 60 ° C. for about 40 seconds (heating step). Here, if a heating plate is used for heating, a heating process can be performed continuously.

  By such treatment, α-amylase contained in the juice is inactivated, and β-amylase is selectively contained.

  Furthermore, the heat-treated heat-treated liquid is neutralized using ammonia water for neutralization (neutralization treatment step). In this step, it was possible to apply a continuous treatment in which the heat treatment liquid was continuously supplied to the mixer at 80 mL / min and the ammonia was supplied at 80 mL / min and mixed for about 1 minute 50 seconds. The treatment liquid obtained by this neutralization treatment is neutralized to about pH 6.0 to 6.2.

  The citrus water extract thus obtained is cooled to room temperature using a cooling plate using cold water, freeze-dried, and processed into a powdery β-amylase preparation.

  This β-amylase preparation obtained by such treatment contains 5233 U / gDS β-amylase, almost 0 U / gDS α-amylase, and has high β-amylase activity and low other enzyme activity. It turned out that it became the (beta) -amylase preparation which is the right.

  In addition, U / gDS is a unit which shows the activity amount of the enzyme with respect to each solid content weight here. U, which shows the activity of β-amylase, reacts with p-nitrophenyl maltopentaoside (final concentration 0.238%) at pH 5.8 and 40 ° C. in the presence of excess α-glucosidase. The amount of enzyme required to release 1 μmol of p-nitrophenol in the first minute is defined as 1 U. The activity U of α-amylase was 0.27 ml of a substrate solution (10 mmol / L sodium chloride, 2 mmol / L calcium chloride, amylose azure 5.4 mg containing 20 mmol / L sodium phosphate buffer (pH 5 8)) is added with 0.03 ml of enzyme solution and 40. The reaction is terminated for 10 minutes at C, 0.2 ml of 18% acetic acid solution is added to terminate the reaction, and the amount of enzyme required to increase the absorbance at 595 nm of the soluble dye released to the supernatant after centrifugation is increased by 2.5. 1 U of α-amylase was used.

  The amount of β-amylase contained in commercially available enzyme preparations and the like is as shown in Table 1, and it can be understood that the β-amylase preparation contains a high concentration of β-amylase.

<Maltose syrup>
A 37% corn starch milk adjusted to pH 5.5 with calcium hydroxide was mixed with a commercially available acid-resistant heat-resistant α-amylase (Chrytase Y7 manufactured by Daiwa Kasei Co., Ltd.) and mixed at 95 ° C. for 50 minutes. Thereby, the liquefied liquid which the said corn starch milk liquefied is obtained. The liquefied liquid is held at 130 ° C. for 10 minutes to inactivate the enzyme and obtain a pretreatment liquid adjusted to DE 2.0.

  The pretreatment liquid is cooled to 65 ° C. to 70 ° C., and 16 U / gDS of β-amylase, 1 U / gDS of heat-resistant pullulanase (Chrystase PL45 manufactured by Daiwa Kasei Co., Ltd.) and water are mixed, and the corn starch concentration is 35%. A saccharification reaction was performed.

  When the conversion reaction from starch to sugar was performed at 65 ° C. to 70 ° C. for 24 hours, it was found that the composition shown in Table 2 was obtained, and a high-quality maltose syrup having a maltose production rate corresponding to G2 exceeding 80% was obtained. . In this reaction, no problems related to hygiene such as a decrease in pH due to contamination with various bacteria were observed.

Below, the experiment example performed in order to select the manufacturing conditions used by the above-mentioned embodiment and confirm an effect is shown.
<Selection of sweet potato>
Examination of β-amylase activity contained in various potato tuber juices revealed that it was as shown in FIG. 1 and that potatoes such as Sunny Red and Kyushu No. 140 were suitable for β-amylase production.

  The β-amylase activity was obtained by adding 20 μL of juice, 20 μL of synthetic substrate p-nitrophenyl-α- [D] maltopentaoside (5 mmol / L) and 2 U of α-glucosidase to a 96-well microplate. After stirring for 1 minute, the enzyme reaction was performed at 40 ° C. for 10 minutes at a constant temperature, and 0.2 mL of trizma base was added to terminate the reaction, and then evaluated by measuring absorbance.

  According to such an activity measurement method, a variety of sweet potatoes can be evaluated quickly and easily with a small base mass.

  Furthermore, when storing the sweet potato tuber, the activity of α-amylase in sweet potato improves with time (FIG. 2 (a)), whereas the activity of β-amylase hardly changes (FIG. 2 ( b)) Therefore, it can be seen that the higher the β-amylase ratio is extracted, the more the sweet potato is processed immediately after harvesting.

In this embodiment, sweet potatoes are stored in a cool dark place at 12 to 15 ° C.
<Thermal resistance of β-amylase>
When the heat resistance of various β-amylase preparations was examined, it was as shown in FIG.
Moreover, when the heat resistance was investigated about the enzyme formulation from which the refinement | purification degree differs about (beta) -amylase of Kyushu 140, it came to show in FIG.

  From FIG. 3, it can be seen that the β-amylase preparation derived from sweet potato exhibits higher heat resistance than wheat bran, although it does not reach the enzyme preparation derived from soybean, and shows high resistance to high-temperature treatment during maltose production. I understood it. In addition, it can be seen from FIG. 4 that the heat resistance decreases as the degree of purification of the enzyme preparation increases, and the sweet potato juice is expected to contain other substances that improve the heat resistance of β-amylase. Is done. Therefore, when purifying the potato-derived β-amylase for the purpose of using it at a high temperature, it is understood that excessive purification is not preferable, and it may be desirable to use the citrus water extract as it is as an enzyme preparation.

  In addition, heat resistance shows the enzyme activity (residual activity) after heating up and hold | maintaining for 20 minutes by setting the activity in 40 degreeC of each enzyme formulation etc. to 100 as a relative value.

  Furthermore, when the β-amylase used in the above-described embodiment was used for maltose production under various temperature conditions, it was as shown in FIG. FIG. 5 shows that when barley-derived β-amylase is used, starch has a high maltose production rate of about 80% under the reaction conditions of 58 ° C., but the production rate decreases rapidly as the reaction temperature is increased. However, only 37% was exhibited at 65 ° C, whereas β-amylase derived from sweet potato maintained a high production rate of 80% or higher up to a high temperature range of 65 ° C, and was found to have excellent heat resistance. .

In addition, the test in FIG. 5 was performed by making reaction time into 48 hours on the manufacturing conditions of the maltose syrup in the said embodiment.
<Inactivation of α-Amylase> The stock solution of the sweet potato juice was stirred for 110 seconds under various acidic conditions, heat-treated for 40 seconds and then neutralized to pH 6.0 to 6.2 by adding aqueous ammonia. The activity was as shown in Table 3.

From this result, when β-amylase is extracted from sweet potato, it is preferable to adjust the pH of the juice to 3.9 to 4.0 and perform a heat treatment that is maintained at about 60 ° C. for 40 seconds after stirring. Recognize.
<Another embodiment>
For the production of β-amylase, instead of the sweet potato juice, sweet potato powder, a liquid extract from the powder, a concentrated liquid, or the like may be used. Note that it is particularly preferable to extract β-amylase from sweet potato processing waste liquid because it leads to reduction of waste and effective use of resources.

  Similarly, various starch materials can be applied to the production of maltose syrup.

The figure which shows the difference of (beta) -amylase activity between sweet potato varieties Figure showing daily changes in amylase activity during storage of sweet potato Diagram showing the difference in heat resistance of β-amylase between the types of raw materials The figure which shows the difference in the heat resistance of (beta) -amylase by the difference in a refinement | purification degree The figure which shows the difference in the maltose production rate by the difference in β-amylase

Claims (3)

A pH adjusting step for obtaining an acidic treatment liquid obtained by adjusting the squeezed liquid of sweet potato to pH 3.9 to 4.0,
A heating step of heat treating the acidic treatment liquid at 59 to 61 ° C. for 35 to 45 seconds;
A neutralization treatment step for neutralizing the heat-treated liquid,
A method for producing β-amylase, which is sequentially performed. The method for producing β-amylase according to claim 1, wherein the sweet potato is mainly composed of at least one selected from Kyushu No. 140 and Sunny Red. A method for producing maltose, wherein an enzyme preparation containing β-amylase produced by the method for producing β-amylase according to claim 1 or 2 is mixed with a starch raw material to cause a conversion reaction from starch to sugar.

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