JPH0824564B2 - New koji mold and its use - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hybrid strain by cell fusion of Aspergillus oryzae and Aspergillus oryzae and a method for brewing mirin using the same.

[Conventional technology]

Conventionally, in Mirin brewing, Aspergillus oryzae (hereinafter, abbreviated as A. oryzae) is usually used to prepare koji as a raw material. It is considered that A. oryzae was used as a koji mold which has strong α-amylase and protease in the bed in order to dissolve starch and protein components in rice grains into mirin components in mirin moromi. However, mirin, which is mainly used as a seasoning, contains a large amount of sugar components and nitrogen-containing components, has a high content of mildly sweet oligosaccharides, and also has a high content of amino acids and low-molecular peptides as taste components. Was desired.

For the production of oligosaccharides in mirin, koji mold, which has a high transglucosidase (hereinafter abbreviated as T Gase) production ability and a low glucoamylase production ability, is desirable. But α
-Aspergillus niger having high ability to produce amylase, protease and T Gase and low ability to produce glucoamylase has not been obtained.

[Problems to be Solved by the Invention]

At present, strains of Aspergillus oryzae (A. oryzae) having strong α-amylase and protease have been obtained, but the T Gase-producing ability is low and the glucoamylase-producing ability is high. On the other hand, Aspergillus usamii (hereinafter, abbreviated as A. Usami) koji mold has high TGase-producing ability and low glucoamylase-producing ability, but produces acid, which results in imparting sourness to mirin.

The object of the present invention is to effectively enhance non-fermentable oligosaccharides without increasing sourness, and to increase the number of amino acids and low-molecular peptides as taste components by acid carboxypeptidase (hereinafter abbreviated as ACPase). An object of the present invention is to provide a koji mold and a method for brewing mirin using the koji mold.

[Means for solving the problem]

Briefly explaining the present invention, the first invention of the present invention is that the cell fusion of A. oryzae and A. usami has almost the same acid-producing ability and improved TGase activity as compared with the original strain A. oryzae. The second invention relates to a method for making koji using the koji mold of the first invention, and brewing mirin using the obtained koji mold.

The koji mold of cell fusion according to the present invention is T Gase or T Ga
Aspergillus oryzae effective for brewing mirin, which has significantly improved se and ACPase production ability compared to A. oryzae. By using the Aspergillus oryzae of the present invention for brewing mirin, the content of non-fermentable oligosaccharides having a mild sweetness is also increased, and it becomes possible to brew mirin without cold crystallization due to precipitation of glucose at low temperature. Also, ACPa
By using Aspergillus oryzae by cell fusion with strong se-producing ability, the amount of palatable amino acids and low-molecular-weight peptides is high and the protein-based turbid mother substance can be reduced without compromising the balance of total nitrogen components and total sugars in mirin.

The novel Aspergillus oryzae of the present invention may be any cell fusion hybrid strain of A. oryzae and A. usami, and there is no limitation on the method for obtaining it.
For example, it is acquired as follows.

Aspergillus usamii mutant shiro-usamii (IFO
6082) (hereinafter, abbreviated as white-rabbit) was irradiated with UV, and then a mutant strain with proline auxotrophy as a marker and an AP strain with arginine auxotrophy as a marker (FERM P-9863)
One example of the Aspergillus oryzae of the present invention can be obtained by screening a hybrid strain in which the above cells are fused with TGase-producing ability as an index.

One strain of the microorganism newly isolated by the above method has the following mycological properties, is a cell fusion hybrid koji mold of Aspergillus niger and Aspergillus niger, has a high TGase-producing ability, and is also desired to produce glucoamylase. Since it is low and has a high ACPase-producing ability, it was identified as a hybrid strain of them and designated as Aaspergillus sp.45.
It has been deposited as P-9862).

 Its mycological properties are as follows.

Morphological characteristics The above morphology was measured at 25 ° C. for 7 days.

Growing state (cultivated at 25 ° C and 34 ° C for 7 days) (1) Malt extract agar medium (i) Cultured at 25 ° C The diameter is about 42.0 mm, a flat huge colony is formed, the central part is green and the outer periphery is white. Presents, forms spores, has no folds on the reverse side of the shale, and has a cream color. (Ii) Culture at 34 ° C. Approximately 43.0 mm in diameter, forming a flat huge colony, green in the center and white in the outer periphery It forms spores and has a pale yellow color without folds on the reverse side of the dish. (2) Czapek agar medium (i) Culture at 25 ° C Approximately 22.0 mm in diameter, forming a flat colony with a green central part Are white and form spores, and are white with no folds on the back. (Ii) 34 ° C culture The diameter is approximately 25.0 mm, a flat colony is formed, the central part is green and the outer periphery is white, and the spores are It has no creases on the back and has a cream color.

Physiological properties (1) Growth temperature: 10 to 45 ℃ (2) Optimal growth temperature: 25 to 45 ℃ (3) Growth pH: 2 to 9 (4) Optimal growth pH: 4 to 8 (5) Origin of TGase The production capacity of TGase is remarkably improved compared to the strain A. Olise (6) Generation of glucoamylase The production capacity of glucoamylase is decreased compared to the strain A. Olise (7) Generation of ACPase ACPase production ability is significantly improved compared to strain A. oryzae (h) Acid production Acid production almost equal to that of the original strain Olyse. Based on the above properties, this strain clearly does not belong to Aspergillus oryzae. , Since it does not belong to Aspergillus oryzae from the trait of the ability to generate TGase, it is appropriate to use a hybrid strain of cell fusion, and a new hybrid strain of Aspergillus oryzae and Aspergillus oryzae, the Aspe
It was named rgillus sp. 45 (hereinafter abbreviated as F-45).

The new hybrid strain according to the present invention has high TGase-producing ability and ACPase-producing ability, and not only low glucoamylase producing ability but also α-amylase and protease producing ability is maintained at the same level as the original strain Olyse, which is extremely useful. However, if it is used for mirin brewing after koji-making, the content of non-fermentable oligosaccharides increases, and cold crystallization due to glucose at low temperatures can be prevented. In addition, the content of amino acids and low molecular weight peptides in mirin increases, the protein-based cloudy mother substance decreases, and it is particularly useful in that it can brew only phosphorus to prevent clouding. Therefore, the industrial applicability of the new hybrid strain according to the present invention is extremely remarkable.

[Examples] Examples of the present invention are specifically described below, but the present invention is not limited to these examples.

Example 1 Acquisition of Amino Acid-Required Strain A sterilized aqueous solution containing 0.01% Tween 80 was added to spore-forming colonies of white-rabbit (IFO 6082), and spore suspension was performed by shaking at 30 ° C for 30 minutes. A suspension is obtained. Add 10 W of 15 WUV lamps to this spore suspension from a distance of 30 cm.
Irradiated for minutes. The survival rate at this time was 2.1 × 10 ^-3 %. To 5 ml of the spore suspension, 45 ml of Tuapeck's medium shown in Table 1 below was added, and the mixture was cultured at 30 ° C for 48 hours to grow non-amino acid-requiring bacteria, and a glass filter was used.
The cells were sterilized with 3G2.

After growing the spores passing through the filter in Tuapetsk agar medium, the medium containing proline in the case of white-rabbit and arginine in the case of A. oryzae was overlaid, and further at 30 ° C.,
After culturing for 48 hours, the newly grown strain was picked up. After inoculating slants with these strains and forming spores,
This was inoculated into the Tuapetsk agar medium containing proline and the medium not containing it, and one proline-requiring strain was obtained from white-rabbit due to the difference in growth. In addition, one arginine-requiring strain (AP strain) was obtained from A. oryzae in the same manner.

To the agar medium was added 1.5% of Agerpurified, manufactured by Difco.

Table 1 Tuapetsk medium pH 5.0 to 5.5 Sucrose 30 g NaNO ₃ 2 K ₂ HPO ₄ 1 MgSO ₄ / 7H ₂ O 0.5 KCl 0.5 FeSO ₄ / 7H ₂ O 0.01 Distilled water 1 Obtaining protoplasts Spores of a rabbit strain were inoculated into 10 ml of Tuapetque medium supplemented with proline, and 40 mg of washed bacterial cell pellets obtained by shaking at 30 ° C for 48 hours were added with chitinase and zymolyase, and reacted at 30 ° C for 30 minutes, About 5 × 10 ⁶ protoplasts were obtained. Similarly, about 5 × 10 ⁶ protoplasts are obtained from the AP strain. 0.5 ml of 0.5 ml of 10 mM CaCl ₂ -containing buffer solution (pH 7.5) in which 10 ⁷ of each protoplast of each strain was suspended
40% polyethylene glycol (molecular weight: 6000) was mixed and maintained at 30 ° C. for 30 minutes for cell fusion treatment, and then washed to recover protoplasts. The cell fusion-treated protoplasts were cultivated in Tuapetsk agar medium at 30 ° C. for 48 hours for regeneration to obtain hybrid strains. The regeneration rate was 0.01% for each protoplast used. Of the obtained hybrid strain
Bacteria with strong TGase and ACPase activities were screened to obtain hybrid strains FO and F-45 koji mold.

Example 2 1,000 g of polished rice (polished rate 85%) was washed according to a conventional method, soaked in water for 8 hours, drained for 5 hours and then steamed for 40 minutes. did. FO, F-45 (FERM P-
9862) and as a control, the original strain A. oryzae, the original strain Shiro-Usami, the AP strain, and the proline-requiring Shiro-Usami were seeded, respectively, and koji-making was carried out for 42 hours.

Regarding the enzymes considered important in mirin brewing, α-amylase, glucoamylase, protease, ACPase and TGase in the obtained koji were measured and the results shown in Table 2 were obtained. As can be seen from this table, FO koji has high TGase-producing ability and weak glucoamylase-producing ability, while F-45 koji has
Furthermore, the ability to generate ACPase is also enhanced.

Using the rice malt of FO and F-45 strains, the control was used as the rice malt of the original strain Olise, and a small preparation test of 1 kg moromi was performed. The charge composition was as shown in Table 3.

In the case of ordinary moromi, koji and steamed rice under atmospheric pressure were mixed in 35% alcohol and aged at 30 ° C for 30 days. In the case of low-alcohol, enzyme-added moromi, koji, steamed rice under atmospheric pressure and enzyme preparation are mixed in 12.5% alcohol, ripened at 30 ° C for 5 days, and the remaining 93.2% alcohol is added, and then continued for 25 days. 30
Aged at ℃. Aged moromi obtained mirin and lees using a small press.

The general analytical value of mirin obtained by this method is
Shown in the table. As is clear from this result, in the case of using F-45 koji according to the present invention, the index value amino nitrogen content of amino acids and low molecular weight peptides in mirin significantly increased compared with the case of A. oryzae koji, and total nitrogen was significantly increased. The content also increased somewhat.

Furthermore, the protein turbidity matrix substance in mirin decreased, and the turbidity prediction tests, such as shochu, water and heating tests were also negative.

In addition, the characteristic that the ratio of reducing sugar in soluble sugar is low is
Found in FO and F-45 koji mirin.

Next, the increased amino acids were examined. That is,
Using the resulting mirin as a sample, the amount of free amino acid in mirin was determined by an automatic amino acid analyzer. The results are shown in Table 5. As is clear from this result, when the Aspergillus oryzae F-45 according to the present invention is used, the free amino acid which is a taste component in mirin is significantly increased as compared with the case where the original strain A. oryzae is used. Is clear.

The sugar component in mirin was also quantified by separating the sugar by paper chromatography. The results are shown in Table 6.
When the Aspergillus oryzae FO and F-45 according to the present invention are used, the original strain
The content of non-fermentable oligosaccharides in mirin was remarkably increased and the content of glucose was also decreased as compared with the case of using A. oryzae, and no cold crystallization was caused by precipitation of glucose at low temperature.

In addition, from the sensory test, it was evaluated that mirin using Aspergillus oryzae FO and F-45 has a wide range of umami and sweetness.

[Effects of the Invention] As is clear from the above, when mirin is brewed using this strain, the content of non-fermentable oligosaccharides with a mild sweetness also increases, the sweetness has a wide range, and glucose is produced at low temperatures. Cold precipitation that precipitates can also be prevented by reducing the glucose concentration. In addition, amino acids, which are the taste components in mirin,
The low peptide content is increased, the inconvenient component as a product, the content of the protein turbid mother substance is also reduced, and a remarkable effect of preventing turbidity can be obtained.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:69 1:66) (56) Reference JP-A-60-58065 (JP, A) JP-A-60 -188057 (JP, A) JP-A-60-188058 (JP, A) JP-A-50-157584 (JP, A)

Claims (3)

[Claims] 1. Aspergillus oryzae, which has substantially the same acid-producing ability and improved transglucosidase activity as compared to the original strain Aspergillus oryzae by cell fusion of Aspergillus oryzae and Aspergillus oryzae. 2. The koji mold according to claim 1, wherein the koji mold is Aspergillus sp 45 produced by cell fusion having the following mycological properties. (A) Morphological properties (The above-mentioned form was measured at 25 ° C. for 7 days) (B) Growth state (25 ° C. and 34 ° C. for 7 days) (a) Malt extract agar medium (i) 25 ° C. culture diameter is about 42.0 mm , A huge flat colony, green in the center, white in the outer periphery, forming spores, no folds on the back of the sheare, and a cream color. (Ii) Culture at 34 ° C, diameter is approximately 43.0 mm, flat Form a huge colony, the central part is green, the outer periphery is white, spores are formed, and there are no folds on the back of the sheare, and it is a pale yellow color. (B) Tuapetsk agar medium (i) 25 ° C culture diameter is about 22.0 mm, flat colonies are formed, the central part is green, the outer periphery is white, spores are formed, and there are no folds on the back surface, which is white. (ii) 34 ° C Culture diameter is approximately 25.0 mm, flat It forms a colony, the central part is green, the outer periphery is white, spores are formed, and the back surface is free of folds.
(C) Physiological properties (a) Growth temperature: 10 to 45 ° C (b) Optimal growth temperature: 25 to 45 ° C (c) Growth pH: 2 to 9 (d) Optimal growth pH: 4 to 8 (E) Production of transglucosidase Compared with the original strain Aspergillus oryzase, the ability to generate transglucosidase is remarkably improved. (F) Glucoamylase Compared to the original strain Aspergillus oryzae, the production ability of glucoamylase is decreased. (G) Acidic carboxypeptidase production Compared to the original strain Aspergillus oryase, the ability to produce acidic carboxypeptidase is significantly improved. (H) Acid production Almost the same acid production as the original strain Aspergillus oryzae. 3. A method for brewing mirin, which comprises making koji using the koji mold according to claim 1 and brewing mirin using the obtained koji.