JPH0353905B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Aspergillus oryzae and Aspergillus
awamori or Aspergillus awamori var.
This invention relates to an alcoholic beverage produced using a koji mold made from a cell fusion strain of M. kawachii. Generally, Aspergillus oryzae is well known as the koji mold used in producing sake. Also
Aspergillus oryzae has a high productivity of enzymes such as starch liquefaction and saccharification, but its citric acid production ability is low, and it has been unable to produce sake with a refreshing sour taste. Also, generally Aspergillus awamori or
Aspergillus awamori var. kawachii is well known as the koji mold used in producing shochu. Although they have high citric acid production ability, they have weak fertility on grains, starch liquefaction ability, and saccharification ability, so they take a long time to make koji, and they have disadvantages such as extremely low raw material utilization rate. It was being watched. Conventionally, breeding by protoplast fusion has been carried out in large numbers, and for the Aspergillus genus, intraspecific fusion using various strains of Aspergillus oryzae has been carried out (Furuya et al.: Noka 57 1 (1983)),
Aspergillus nidulans and Aspergillus fumigatus,
A heterogeneous fusion between Aspergillus nidulans and Aspergillus rugulosus has been carried out. (L. Ferenczy: Experientia 33 184 (1977), F.
Kevei et al: J.Gen.Microbiol.102 255 (1977))
However, Aspergillus oryzae and
Aspergillus awamori or Aspergillus
There is no report that a cell fusion strain with awamori var. kawachii was obtained. The inventor of the present invention developed a method for producing alcoholic beverages of new quality by combining the various characteristics of many Aspergillus bacteria into a single strain and using this as a koji mold. After intense research into fusion, we found that Aspergillus oryzae and Aspergillus awamori have excellent properties.
Or Aspergillus awamori var.kawachii
We were able to obtain a cell fusion strain. The cell fusion strain obtained in the present invention is
It retains the high enzymatic power of starch liquefaction and saccharification that is characteristic of Aspergillus oryzae, and
Aspergillus awamori or Aspergillus
It has high citric acid productivity, which is a characteristic of awamori var. kawachii. The present invention utilizes the cell fusion strain obtained in this manner as Aspergillus oryzae.
citric acid can impart a refreshing flavor to alcoholic beverages produced using Aspergillus awamori or Aspergillus awamori.
Since it is possible to increase the fertility, starch liquefaction power, and saccharification power of Aspergillus awamori var. kawachii in the koji, the koji making time can be shortened and the raw material utilization rate can be increased. To carry out the present invention, it is necessary to create a cell fusion strain.
A strain belonging to Aspergillus oryzae,
Aspergillus awamori or Aspergillus
It is necessary to make protoplasts from each strain belonging to awamori var. kawachii separately.
The strain belonging to Aspergillus oryzae may be a mutant species, each original strain, or an auxotrophic mutant strain. Also, Aspergillus
awamori or Aspergillus awamori var.
The strain belonging to P. kawachii may be a mutant, each original strain, or an auxotrophic mutant. Among these, the use of auxotrophic mutant strains is preferred because it is convenient for selectively growing only the fused strain after fusion treatment. Both strains to be fused are separately inoculated into a nutrient medium and cultured with shaking for about 24 hours. After culturing, the culture solution is filtered to obtain mycelium. The mycelium is suspended in a phosphate buffer containing stabilizers such as sugars, sugar alcohols, and salts, and treated with cell wall lytic enzymes.
Novozyme234 from Trichoderma harzianum
and Cellulase Onozuka R-10 at about 5 mg/ml each, and after gently shaking and stirring at about 30° C. for 4 hours, centrifugation was performed at 1500×g for 10 minutes to obtain each protoplast. For protoplast fusion, each protoplast was mixed, centrifuged at 700 x g for 10 minutes, and then mixed with 0.01M CaCl ₂ 2H ₂ O preheated to 30°C.
Add PH7.5 20% polyethylene glycol 6000 solution containing 0.05 M glycine and fuse for 10 min at 30 °C. The obtained fusion product is suspended in a phosphate buffer containing a stabilizer, diluted appropriately, spread on a nutrient-deficient medium, and cultured at 30°C for several days. grows, so by isolating it and culturing it,
A cell fusion strain of Aspergillus oryzae and Aspergillus awamori or Aspergillus awamori var.kawachii can be obtained. In the present invention, by the method shown in Fusion Example 1, the histidine auxotrophs of Aspergillus oryzae, RIB647, and IFO 30103 and Aspergillus awamori
Many fusion strains were obtained by fusing the lysine-requiring strain of IFO4033, and two strains were selected from among them, FF2 and FF9.
It was named. Similarly, Aspergillus oryzae RIB647,
Histidine auxotrophs of IFO 30103 and Aspergillus
We fused the asparagine auxotrophic strain of awamori var. kawachii IFO 4308 to obtain many fused strains, from which we selected four strains, FF6, FF7, FF10, F.
It was named F.12. The results of looking at the organic acid composition ratio of each fusion strain are shown in Figure 1, and (1) is Aspergillus
oryzae parent strain, (8) indicates the Aspergillus awamori parent strain, and (9) indicates the Aspergillus awamori var. kawachii parent strain. Further, Table 1 below shows the growth rate and produced enzyme activity (U/mg CO ₂ ) of each parent strain and each fusion strain.

[Table] From these cell fusion strains, enzyme productivity and citric acid production were examined, and cell fusion strains FF2 and FF7 were selected as the most preferred koji molds for producing alcoholic beverages. This cell fusion strain F.
F.2 and FF7 are FERM P-7214 and FERM respectively
It has been deposited as P-7211 at the Institute of Microbial Technology, Agency of Industrial Science and Technology. Cell fusion strains FF2 and FF7 were used as koji mold, and 2x/g was added to 30 g of cooled steamed rice at 30°C as a substrate.
¹⁰⁶ spores were added and koji was made using the Schare method. Table 2 shows the analysis results of the koji.

[Table] As is clear from this table, cell fusion strain FF2
and FF7 have slower cell growth than the Aspergillus oryzae parent strain, a moderate increase in citric acid production, and a considerable increase in glucoamylase, making them extremely desirable as Aspergillus for sake production. It is acceptable. In the present invention, various cell fusion strains are used as koji and produced in the same manner as sake, resulting in a wide variety of new types of alcoholic beverages having various citric acid concentrations. The alcoholic beverage of the present invention is similar to sake, but
Since it has a high citric acid content, it can be said to be a novel alcoholic beverage that has a refreshing sour taste and also has a refreshing aroma. Next, fusion examples and examples of the present invention will be shown. Fusion example 1 Aspergillus oryzae RIB647, IFO 30103,
Aspergillus awamori IFO 4033, Aspergillus
awamori var.kawachii IFO 4308 spores were suspended in 14mM nitrosoguanidine solution and incubated at 30°C.
The cells were treated for 20 minutes to generate mutations, and histidine-, lysine-, and asparagine-requiring mutant strains were isolated. Each of the mutant strains obtained here was grown separately at 3 x 10 ⁵ spores/ml in CM medium (Zapex culture medium supplemented with 0.2% Difco yeast extract).
The cells were inoculated and cultured with shaking at 28°C for 24 hours. The culture solution was filtered to obtain mycelium, which was washed with water. Separately, 75 mg (wet fungal cells) of each of the obtained mycelium was added to 4.6 ml of 2/15 M phosphate buffer (stabilizer added), and then
Novozyme234 5mg/ml and Cellulase
Onozuka R-10 (5 mg/ml) was added thereto, and the mixture was gently stirred at 30°C for 4 hours. The resulting treated solution was centrifuged at 1500 xg for 10 minutes and washed twice with stabilizer-containing water to obtain protoplast suspended in stabilizer-containing water. Mix approximately 5 x 10 ⁶ pieces of each protopst and add 700 x
Centrifuge for 10 min at
PH with 0.01M _{CaCl2.2H2O ,} 0.05M glycine
7.5, 20% polyethylene glycol 6000 solution 1.0
ml was added and fusion treatment was performed at 30°C for 10 minutes. After fusion treatment, MM medium containing stabilizer (PH6.0
6 ml of 0.6M KCl was added to the C.A. culture medium and centrifuged at 700×g for 10 minutes. Then, the Aspergillus
oryzae and Aspergillus awamori from FF2
(FERM P-7214), FF9, and Aspergillus
oryzae and Aspergillus awamori var. kawachii from FF6, FF7 (FERM P-7211), FF10, F.
Got F.12. Example 1 Cell fusion line FF2, FERM P-7214; FF7,
FERM P-7211 spores were cooled and steamed at 30°C.
2×10 ⁶ pieces per g were added to 300 g, cultured at 30°C for 14 hours, turned over, and cultured at 35°C for 26 hours to produce koji. The three parent plants were treated in the same manner and made into koji. Table 3 shows the analysis results of these koji.

[Table] Using this koji, sake was produced according to the commonly used sake brewing process. Sake brewing was carried out using the mixing ratio shown in Table 4.

[Table] The first addition of water is 7 ml of lactic acid diluted 10 times and 10 ⁹ /ml.
Add 2 ml of yeast suspension to make 210 ml, and adjust the initial addition temperature to
The temperature was 13℃, the middle addition temperature was 9℃, and the distillation temperature was 7℃.After that, the temperature was increased by 1℃ per day, and the fermentation was carried out at a maximum temperature of 15℃, after distillation addition.
The tank was replaced in 15 days. Table 5 shows the components and amount of lees of Jotansake.

[Table] As a result, the dissolution of steamed rice and the production of alcohol are
It was confirmed that this sake outperforms Aspergillus oryzae and has a refreshing acidity due to its high citric acid content, as well as a refreshing aroma.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the organic acid composition ratio of the parent strain and the cell fusion strain. Lac.; lactic acid, Mal.; malic acid, Suc.; succinic acid, α-KG.; α-ketoglutaric acid, Cit.; citric acid, others; other organic acids.

Claims (1)

[Claims] 1. Aspergillus oryzae and Aspergillus awamori
Or an alcoholic beverage produced using Aspergillus aspergillus made from a cell fusion strain of Aspergillus awamori var. kawachii. 2 Aspergillus oryzae and Aspergillus awamori
The alcoholic beverage according to claim 1, wherein the cell fusion strain is FF2. 3 Aspergillus oryzae and Aspergillus awamori
The alcoholic beverage according to claim 1, wherein the cell fusion strain of var. kawachii is FF7.