JPH0757161B2 - Method for producing hydrolyzed gluten by immobilized complex protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to an average molecular weight using an immobilized complex protease.
The present invention relates to a method for producing 5,000 to 20,000 hydrolyzed gluten.

[Conventional technology]

In order to effectively use plant proteins, various attempts have been made in recent years to treat and process gluten, which is a wheat protein, to improve its properties and to develop its use as a new material. In particular, there have been many reports of partially hydrolyzing wheat gluten with a protease, and it is also known to produce a foaming agent by treating gluten with a protease. Most of the reports, however, use a batch method to hydrolyze gluten using a free protease, and there are problems in controlling the reaction, making it continuous, and separating the enzyme from the product. In order to overcome such problems caused by the batch method, the use of so-called bioreactor system in which protease is immobilized on various carriers has been studied.

[Problems to be solved by the invention]

However, gluten mainly contained in wheat is a giant polymer mainly composed of gliadin and glutenin and having a molecular weight of several million.Therefore, when it is attempted to treat with a protease immobilized on a carrier, contact between the enzyme and the substrate occurs. Was hindered by steric hindrance, etc., and the expression of enzyme activity was suppressed. Therefore, there was a problem that the reaction efficiency was lowered and a satisfactory productivity could not be obtained. Moreover, the foaming power and foam stability of the hydrolyzed gluten obtained by the treatment with immobilized protease were not sufficient, and it was not satisfactory as a foaming agent for foods and the like.

Therefore, an object of the present invention is to improve the reaction efficiency when producing a gluten hydrolyzate using an immobilized protease. Further, the present invention has an average molecular weight of excellent foaming characteristics.
It also aims to produce 5,000 to 20,000 hydrolyzed gluten.

[Means for solving problems]

The present inventors have continued research on effective use of plant proteins, and one of such studies is hydrolysis treatment of gluten contained in wheat and the like with a protease. As a result of research on the hydrolysis treatment of gluten by protease, when peptin immobilized on the carrier was used to hydrolyze gluten, other specific proteases were fixed to the carrier at a fixed ratio along with pepsin. When it is used after being immobilized, gluten can be efficiently hydrolyzed while retaining the above-mentioned excellent properties inherent to the treatment with immobilized protease (eg, easy reaction control, continuity, and easy product separation). Furthermore, they have found that the hydrolyzed gluten produced has excellent foaming properties, and completed the present invention. That is, the present invention provides pepsin 60-70.
% By weight and at least one other acidic protease 40
A method for producing a hydrolyzed gluten, characterized in that gluten is hydrolyzed using an immobilized complex protease in which a complex protease consisting of ˜30% by weight is immobilized on a carrier.

Gluten is a mixture of proteins mainly composed of glutenin and gliadin, which is obtained mainly from wheat, and its composition is slightly different depending on the kind of raw material and the preparation method. The "raw material gluten" used in the present invention may be any of them, regardless of composition and preparation method. The raw material gluten may be raw gluten or powdered gluten.

Further, in the present invention, gluten that has been subjected to other modification treatment such as deamidation treatment or reduction treatment in advance may be used.

And, when those gluten are treated with the immobilized complex protease, the peptide bond of the main chain is hydrolyzed,
It is cleaved to lower the molecular weight and its solubility in water increases.

In the present invention, as another protease to be used in combination with pepsin, the reason for adopting another acidic protease is that pepsin is an acidic protease that has activity under acidic conditions, and therefore hydrolysis treatment of gluten by immobilized pepsin is performed. This is because it is performed under acidic conditions, and other proteases also need to exhibit activity under acidic conditions. Therefore,
The term "other acidic protease" in the present invention means an enzyme that is a protease other than pepsin and has a protein (gluten) degrading ability at least under acidic conditions. A protease having gluten degrading ability under acidic or neutral conditions, or a protease having gluten degrading ability in a wide pH range from an acidic region to an alkaline region is capable of degrading gluten under acidic condition. It is included in “acidic protease”. The origin of the other acidic proteases may be any, and may be animals or plants or microorganisms.

Examples of other acid proteases include acidases of oyster mushroom origin such as lapidase (trade name: Takeda Pharmaceutical Co., Ltd.), acid proteases of Asp.saitoi origin such as morphine, acid proteases of Asp.niger origin, trypsin, chymotrypsin, Many kinds such as papain and bromelain can be used, and they may be complexed with pepsin alone or in combination with plural types to complex with pepsin. Among them, lapidase and morphine are preferable, and particularly lapidase significantly improves the hydrolysis efficiency.

In the present invention, it is necessary to use pepsin in a larger amount than other acidic proteases. Especially pepsin 60 ~
When 70% by weight and 40 to 30% by weight of other acidic proteases, the hydrolysis efficiency is good.

As a carrier for immobilizing pepsin and other acidic proteases, various forms such as beads, membranes, nets and filaments can be used. As the material of the carrier, any of organic polymer materials, ceramics, glass, metals, sand, and inorganic materials such as carbon can be used. It is desirable to use a carrier that can firmly support pepsin and other acidic proteases without degrading them without deteriorating their activity. Carriers having amino groups such as chitosan beads, alkylaminated glass beads, allylaminated glass beads, and aminosilanized alumina can firmly support pepsin and other acidic proteases,
Moreover, it is particularly preferable in that the activity is not reduced.

When immobilizing pepsin and other acidic proteases on a carrier, any of the conventionally known carrier immobilization method, cross-linking method, and entrapment method can be adopted.

Pepsin and other acidic proteases may be immobilized on the carrier at the same time or sequentially. It is convenient and convenient to include pepsin and other acidic protease in a solution at the same time and then bring the solution into contact with a carrier.

After supporting pepsin and other acidic proteases on a carrier, it is preferable to crosslink and fix using a crosslinking agent such as glutaraldehyde, glyoxal, and dialdehyde starch.
More preferably, a glutaraldehyde crosslinking agent is used.

The complex protease is usually added in an amount of about 10 to about 1 g of the wet weight of the carrier.
It is preferable to immobilize it on a carrier at a ratio of 50 mg.

The hydrolysis treatment of gluten with immobilized complex protease is usually carried out at a temperature of about 30 to 50 ° C. and a pH of about 1.5 to 4 with a gluten-containing solution or dispersion at about 1.0 to 6.0 hr ⁻¹ (retention time of about
It is carried out by passing the immobilized complex protease at a rate of 10 to 60 minutes). The gluten content in the liquid to be treated is usually about 20 to 60 g / l.

When the treatment temperature is 60 ° C. or higher, the protease is inactivated, and when the gluten content in the treatment liquid is too high, the yield is lowered.

Next, the pH and temperature of the solution that has undergone protease treatment are adjusted to precipitate undegraded substances. After removing the precipitate, the hydrolyzed gluten contained in the liquid is recovered by an appropriate method such as drying to obtain the desired product. Precipitation and separation of undecomposed matter are usually carried out by heating at a pH of about 4.0 to 9.0 and a temperature of about 70 to 100 ° C for about 10 to 30 minutes.

The hydrolyzed gluten obtained by the method of the present invention is usually about
It has an average molecular weight of 5,000-20,000, is soluble in water,
It is generally white and has no unpleasant taste or odor.

Next, the relationship between the weight% of pepsin contained in the protease and the yield of hydrolyzed gluten when the gluten was hydrolyzed using an immobilized protease in which one or both of pepsin and lapidase was immobilized was investigated. . Gluten was hydrolyzed as follows.

Hydrolysis treatment of gluten with immobilized protease Chitopearl (Fuji Spinning Chitosan beads) with pepsin
(Manufactured by Amano Pharmaceutical) and Lapidase (manufactured by Takeda Pharmaceutical Co., Ltd.)
A solution containing one or both of them is contacted with pepsin or
After adsorbing one or both of
Immobilize the protease by cross-linking and immobilizing it with taraldehyde.
Prepared. Pack this in a column and add 5 wt% gluten water.
Solution (pH3) at 40 ℃, space velocity 2hr^-1It was passed at. Profit
Adjust the pH of the hydrolyzed solution to 4.8 and then at 85 ℃ for 20 minutes.
After heating for a while to remove the precipitate, the liquid is dried.
The gluten hydrolyzate was obtained. Yield of hydrolyzed gluten
The rate was calculated by the following.

The results obtained are shown in the drawing. In the figure, the horizontal axis represents the weight% of pepsin relative to the total weight of pepsin and rapitase, and the vertical axis represents the yield (%) of hydrolyzed gluten.

From the results of the drawings, it will be apparent that the yield of hydrolyzed gluten is high when the pepsin content is 60 to 70% by weight.

Next, examples of the present invention will be shown, but the present invention is not limited to the examples.

In the examples, the yield of hydrolyzed gluten was measured in the same manner as above.

The gluten hydrolysis rate, foaming power and foam stability were measured as described below.

Hydrolysis rate of gluten Mix 1 ml of gluten decomposition solution after protease treatment with 4 ml of 0.4 M trichloroacetic acid aqueous solution, leave at 40 ° C for 20 minutes, and then use Toyo Paper No.2. Obtained liquid 1
Centrifuge at 2,000 rpm for 5 minutes to obtain 280 nm of the supernatant.
The absorbance was measured to determine the protein concentration.

Foaming power Dissolve the sample (hydrolyzed gluten) in 30 ml of pH 4.0 acetate buffer so that the protein concentration is 0.1% w / v, and use Excel Auto Homogenizer (Nippon Seiki) at 10,000 rpm for 1 minute at 25 ±. Stir at 1 ° C. to form a foam. The foam was transferred to a graduated cylinder, and the whole volume after 1 minute of foam preparation was measured and used as the foaming force.

Foam stability The change in foam volume with time after foam preparation was measured, and the time required for the residual foam volume to become half of the foam volume after 1 minute of foaming was calculated.
The foam stability was evaluated by taking it as the foam half-life.

Example 1 Chitopearl Carrier made of (Fuji Boseki), pepsin
Contains Amano Pharmaceuticals and Lapidase (Takeda Pharmaceuticals)
Aqueous solution (protease concentration 30 mg / ml, in protease
Pepsin-containing 60% by weight) was added per 1 g of carrier to 1 ml of aqueous solution
Contact each other and absorb 26 mg of complex protease per 1 g of carrier.
The coated immobilized complex protease was prepared. This fixation
Packed with 15 ml of complex protease in a φ16 × 200 m / m column
Containing 5% by weight of gluten prepared from wheat
Add 1000 ml of gluten dispersion adjusted to pH 3.0 to 40
℃, space velocity 2hr^-1Gluten partial water
A solution was obtained. Adjust the pH of the decomposition solution with a 5N aqueous sodium hydroxide solution.
After adjusting to 4.8, heat at 85 ℃ for 20 minutes to form a precipitate
Let Freeze the supernatant by centrifugation at 6500G for 10 minutes.
After drying, 34.7 g of hydrolyzed gluten was obtained. Hydrolyzed glue
The average molecular weight of ten was 10,000.

Comparative Examples 1 to 3 The complex proteases in Example 1 were the same amounts of pepsin (Comparative Example 1), lapidase (Comparative Example 2) and pepsin (40 wt%) / rapidase (60 wt%) complex protease (Comparative Example). Hydrolyzed gluten was produced in the same manner as in Example 1 except that the substitution in 3) was performed.

Example 2 Hydrolyzed gluten was produced in the same manner as in Example 1 except that the complex protease in Example 1 was replaced with the complex protease consisting of the same amount of pepsin (60% by weight) / molcin (40% by weight).

When the hydrolysis rate of gluten, the yield of hydrolyzed gluten, the foaming power, and the foam stability in Examples 1 and 2 and Comparative Examples 1 to 3 were examined, the results shown in Table 1 below were obtained.

From the results shown in Table 1 above, Example 1 of the present invention using the immobilized protease consisting of 60% by weight of pepsin and 40% by weight of lapidase showed that the hydrolysis rate of gluten, the yield of hydrolyzed gluten, and the foaming rate. Significantly improved in all of force and foam stability as compared with Comparative Example 1 in which only pepsin was immobilized, Comparative Example 2 in which only lapidase was immobilized, and Comparative Example 3 in which 40 wt% of pepsin and 60 wt% of lipase were immobilized. You can see that In addition, in Example 2 of the present invention which uses an immobilized protease consisting of 60% by weight of pepsin and 40% by weight of morphine, the yield of hydrolyzed gluten, the foaming power and the foam stability are the same as those of Comparative Examples 1 to 3. It can be seen that it is greatly improved in comparison.

〔The invention's effect〕

As described above, the present invention using an immobilized complex protease containing pepsin and at least one of other acidic proteases in a specific ratio is superior to that using an immobilized protease consisting of only one acidic protease. As a result, the hydrolysis rate of gluten is greatly improved, and hydrolyzed gluten can be produced with a high decomposition rate and yield.

Furthermore, since the hydrolyzed gluten produced by the present invention has excellent foaming properties, as a highly safe foaming agent derived from wheat or the like, cakes, cuties, meringues, confectionery and bread such as icing, It is effectively used in the production of kneaded products such as kamaboko and rice paste, and other foods. Also,
The hydrolyzed gluten produced according to the present invention is in the form of powder,
Whether stored in paste or solution form,
Alternatively, it may be used, and can be used in the processing of foods in the same manner as conventional additives of this type.

[Brief description of drawings]

The drawing shows the change in the yield of hydrolyzed gluten with respect to the change in the ratio of pepsin in the immobilized complex protease immobilized with pepsin and lapidase.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kengo Ogawa 356 Ishii, Sakado City, Saitama Prefecture (72) Inventor Takaki Yamagata 1052-602, Kamimae-cho, Nagahama City, Shiga Prefecture (72) Inventor Toshio Tanaka Shiga Prefecture No. 52-105, Kamimae-cho, Nagahama-shi (72) Inventor Jun Nakamura 8-5-5, Okusawa, Setagaya-ku, Tokyo (56) Reference JP-A-59-11195 (JP, A) JP-A-59-11194 (JP, A) JP 59-11193 (JP, A) JP 59-11192 (JP, A) edited by Ichiro Chibata "Immobilized Enzyme" Kodansha P. 186-191, P.I. 145-149 (Showa 5/11)

Claims (1)

[Claims] 1. Hydrolysis of gluten using an immobilized complex protease in which a complex protease consisting of 60 to 70% by weight of pepsin and at least 40 to 30% by weight of at least one other acidic protease is immobilized on a carrier. A method for producing hydrolyzed gluten having an average molecular weight of 5,000 to 20,000.