JPH06339368A - Fused cell strain - Google Patents

Abstract

PURPOSE:To prepare an alcoholic beverage having refreshing acid taste and flavor by brewing with a malt consisting of fused cell of Aspergillus oryzae and Aspergillus awamori, etc. CONSTITUTION:Fused cell strain F.F. (FERM P-7214) derived from Aspergillus oryzae and Aspergillus awamori or mixed fused strain F.F.7 (FERM P-7211) derived from Aspergillus oryzae and Aspergillus awamori var. kawachii is used as the malt for brewing. The malt-preparation can be quickened and the raw material yield can be increased by the use of the strain.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to Aspergillus.
oryzae and Aspergillus awamor
i or Aspergillus awamori
var. It relates to a cell fusion strain of kawachii.

And, according to the present invention, Aspergi
llus oryzae and Aspergillus a
wamori or Aspergillus awa
mori var. By using Aspergillus oryzae consisting of a cell fusion strain of kawachii, an alcoholic beverage having a refreshing sourness and emitting an aroma can be efficiently produced.

[0003]

2. Description of the Related Art Generally, Aspergillus ory
Zae is well known as a koji mold used when producing sake. See also Aspergillus oryza
Although e has a high enzyme productivity such as liquefaction and saccharification of starch, it has a low citric acid production ability and cannot produce sake having a refreshing sourness.

Further, in general, Aspergillus a
wamori or Aspergillus aw
amori var. Kawachii is well known as a koji mold used in producing shochu.

Although they have a high citric acid producing ability, they have a weak reproductive ability on cereals and a liquefying ability and a saccharifying ability of starch,
There were drawbacks such as the time required for making koji was long and the raw material utilization rate was extremely poor.

Conventionally, breeding by fusion of protoplasts has been
Aspergillus has become popular
Regarding the genus, Aspergillus oryzae
Intraspecific fusion using each of the strains (Furuya et al .: Agricultural Science 571 (1983)), Aspergillus n.
idlens and Aspergillus fumig
atus, Aspergillus nidulans
And aspergillus rugulosus have been cross-linked. (L. Ferenc
zy: Experientia 33 184 (197).
7), F.I. Kevei et al. Gen. Mi
crobiol. 102 255 (1977)) However, Aspergillus oryzae and As
pergillus awamori or Asp
ergillus awamori var. kawa
There is no report that a cell fusion strain with chii was obtained.

[0007]

The problem to be solved by the present invention is that Aspergillu which could not be obtained conventionally.
s oryzae, which has the high enzymatic activity of starch liquefaction and saccharification, which is a characteristic of soryzae, and Aspergillus
awamori or Aspergillus a
wamori var. It is to create an excellent cell fusion strain having high citrate productivity which is a characteristic of kawachii.

[0008]

As a result of examinations from various aspects in order to solve the above problems, the present inventor found that many Asp
We obtained a new idea that we can produce a new quality alcoholic beverage by combining various characteristics of the bacterium of the genus ergillus into one strain and using it as a koji mold, and we are keen on cross-species fusion. Aspergillus ory has excellent properties when studied.
zae and Aspergillus awamori or Aspergillus awamori va
r. We succeeded in obtaining a cell fusion strain of kawachii.

The cell fusion strain obtained in the present invention is As
Pergillus oryzae has the characteristic of starch liquefaction and high saccharification enzymatic activity as it is, and Aspe
rgillus awamori or Asper
gillus awamori var. kawach
It has a high citric acid productivity which is the characteristic of ii.

When the cell fusion strain obtained in the present invention is used as a koji mold, Aspergillus oryz
A refreshing flavor due to citric acid can be imparted to an alcoholic beverage produced using the malt of ae, and As
pergillus awamori or Asp
ergillus awamori var. kawa
Since the breeding power, starch liquefaction power and saccharification power of chii koji can be increased, the koji making time can be shortened and the raw material utilization rate can be increased.

The creation of the fusion strain according to the present invention is as follows.
a strain belonging to pergillus oryzae and A
spergillus awamori or As
pergillus awamori var. kawa
It is necessary to separate each strain belonging to achii into protoplasts. Aspergilluso
The strain belonging to ryzae may be a mutant strain, each original strain, or an auxotrophic mutant strain. In addition, Aspergillus awamori
Or, Aspergillus awamori
var. The strain belonging to kawachii may be a mutant strain, each original strain, or an auxotrophic mutant strain. Among them, if an auxotrophic mutant is used, only the fused strain can be selectively grown after the fusion treatment.
It is more convenient and is preferred.

Both strains to be fusion-treated are separately inoculated into a nutrient medium and shake-cultured for about 24 hours. After the culture, the culture solution is filtered to obtain mycelium. Mycelia are sugars, sugar alcohols,
Suspended in a phosphate buffer containing stabilizers such as salts, and added to this as Trichoderm as a cell wall lysing enzyme.
Novozyme 234 from a harzianum
And Cellulase onozuka R-10 were added at about 5 mg / ml, and the mixture was gently shaken and stirred at about 30 ° C. for 4 hours and then centrifuged at 1500 × g for 10 minutes to obtain each protoplast.

Upon fusion of the protoplasts, the respective protoplasts were mixed, centrifuged at 700 × g for 10 minutes, and then preheated to 30 ° C. with 0.01 M CaCl2.2H.
A 20% polyethyleneglycol 6000 solution containing 20% and 0.05 M glycine at pH 7.5 is added and fusion is performed at 30 ° C. for 10 minutes. The obtained fusion-treated product is suspended in a phosphate buffer containing a stabilizer and appropriately diluted,
If it is spread on a nutrient-deficient medium and cultured at 30 ° C. for several days, only the auxotrophic complementary fusion strain grows.
lus oryzae and Aspergillus aw
amori or Aspergillus awa
mori var. A cell fusion strain of kawachii can be obtained.

In the present invention, Aspergillus oryzae, RIB is prepared by the method shown in Example 1.
647, a histidine-requiring strain of IF030103, and As.
pergillus awamori IFO4033
Many lysine-requiring strains were fused to obtain many fusion strains, two strains were selected from among them, and F. F. 2, F. F. I named it 9.

Similarly, aspergillus
oryzae RIB647, IF030103 histidine-requiring strain, and Aspergillus awamo
rivar. The asparagine-requiring strain of kawachii IFO4308 was fused to obtain a large number of fused strains, 4 strains were selected from among them, and F. F. 6, F.I. F. 7, F.I.
F. 10, F.I. F. I named it 12. The results of looking at the organic acid composition ratio of each fusion strain are shown in Fig. 1. (1) shows Asp
ergillus oryzae parental strain, (8) is As
pergillus awamori var. (9) is Aspergillus awamori var. k
Awachii parent strains are shown.

Further, Table 1 below shows the growth rate and the production enzyme activity (U / mgCO2) of each parent strain and each fusion strain.

[0017]

[Table 1]

From the cell fusion strain of the present invention, the productivity of the enzyme and the amount of citric acid produced were examined, and the cell fusion strain F. F. 2 and F.I. F. 7 was selected. This cell fusion strain F. F. 2 and F.I. F. 7 is FERM P-72
14, FERM P-7211 has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.

Cell fusion strain F. F. 2, F. F. 7 was used as a koji mold, and 2 × 10 6 spores per g were added to 30 g of the cooled steamed rice at 30 ° C. as a substrate, and koji was made by the Schaire method.

The results of koji analysis are shown in Table 2 below.

[0021]

[Table 2]

As is clear from this table, the cell fusion strain F. F. 2 and F.I. F. 7 shows that the bacterial growth is Asperg
Since it is superior to the parent strain of Illus oryzae, the amount of citric acid produced is moderately increased, and the amount of glucoamylase is considerably increased, it is recognized as an extremely preferable koji mold for sake production.

According to the present invention, various cell fusion strains are used as koji and produced in the same manner as sake, and a new variety of alcoholic beverages having various citric acid concentrations can be obtained.

The alcoholic beverage produced using the cell fusion strain according to the present invention is similar to sake, but since it has a high citric acid content, it is a novel alcoholic beverage having a refreshing sour taste and a refreshing aroma. You can

Next, examples and applications of the present invention will be described.

[0026]

Example 1 Aspergillus oryzae
RIB647, IFO30103, Aspergill
us awamori IFO4033, Asperg
illus awamori var. kawachi
i IFO4308 Each spore is nitrosoguanidine 1
The cells were suspended in a 4 mM solution and treated at 30 ° C. for 20 minutes to cause mutation, and histidine, lysine, and asparagine-requiring mutant strains were isolated. Each of the mutant strains obtained here was separately inoculated into CM medium (Tuapekk dox medium supplemented with 0.2% Difco yeast extract) at 3 × 10 ⁵ spores / ml, and at 28 ° C. Shaking culture was carried out for 24 hours.
The culture solution was filtered to obtain mycelium, which was washed with water.

Each of the obtained mycelia was 75 m apart.
g (wet cells) was added to 4.6 ml of 2 / 15M phosphate buffer (with stabilizer added), and then Novoz
ime 234 5 mg / ml was added, and the mixture was gently stirred at 30 ° C. for 4 hours. The obtained treatment liquid was centrifuged at 1500 × g for 10 minutes, washed twice with stabilizer-containing water, and suspended in the stabilizer-containing water to obtain protoplasts.

Approximately 5 × 10 ⁶ protoplasts were mixed, centrifuged at 700 × g for 10 minutes, and preheated to 30 ° C. with 0.01 M CaCl ₂ .2H ₂ O, 0.05.
Add 1.0 ml of PH 7.5, 20% polyethylene glycol 6000 solution containing M glycine, and add 10 ml at 30 ° C.
Fusion processing was performed for a minute. After the fusion treatment, 6 ml of MM medium containing stabilizer (pH 6.0 6.0 Tuapeck Dox medium to which 0.6 M KCl was added) was added, and 7
Centrifuge at 00 × g for 10 minutes. Then, it is washed twice with stabilizer-containing water, suspended in stabilizer-containing water, diffused on MM medium, and cultured at 30 ° C. for several days to obtain Asp.
ergillus oryzae and Aspergill
from us awamori. F. 2 (FERM P-
7214), F.I. F. 9 again, Aspergillus
oryzea and Aspergillus awamo
ri var. kawachii to F.I. F. 6, F.I.
F. 7 (FERM P-7211), F.I. F. 10,
F. F. I got 12.

[0029]

[Application Example 1] Cell fusion strain F. F. 2, FERM P-7
214; F. F. 7. The spores of FERM P-7211 were added to 300 g of cooled steamed rice at 30 ° C., 2 × 1 per g.
Add ⁶⁰ cells, incubate at 30 ° C for 14 hours, turn back and turn 35
Culturing was carried out at 26 ° C. for 26 hours and koji was carried out. The parent strains of the three strains were treated in the same manner and were then koji.

The results of analysis of these koji are shown in Table 3 below.

[0031]

[Table 3]

[0032] Using these koji, sake was produced in accordance with the ordinary brewing of sake.

Sake brewing was carried out with the formulation shown in Table 4 below.

[0034]

[Table 4]

The initial addition pumping water was made up to 210 ml by adding 7 ml of 10-fold diluted lactic acid and 2 ml of 10 ⁹ / ml yeast suspension, and the initial addition temperature was 13 ° C., the intermediate addition temperature was 9 ° C., and the distillation temperature was 7 ° C. so,
Thereafter, the temperature was raised by 1 ° C. per day, and the fermentation was carried out at the maximum temperature of 15 ° C., and 15 days after the distillation, the upper tank was used.

Table 5 below shows the components and the amount of lees of the upper tank liquor.

[0037]

[Table 5]

As a result, it was confirmed that the sake has a refreshing sourness and a refreshing scent because the dissolution of steamed rice and the production of alcohol surpass Aspergillus oryzae, and the content of citric acid is high.

[0039]

Industrial Applicability According to the present invention, Aspergillus oryzae (Aspergillus oryza) is not used.
e) and Aspergillus awamori (Aspergill)
us awamori) or Aspergillus awakawa
mori var. kawachii) cell fusion strain was successfully created. For example, koji-making using these cell fusion strains, the obtained koji was used to efficiently produce alcoholic beverages having a refreshing sourness and aroma. Can be manufactured in a simple manner.

[Brief description of drawings]

FIG. 1 is a diagram showing an organic acid composition ratio of a parent strain and a cell fusion strain.

[Explanation of symbols]

 Lac. Lactic acid Mal. Malic acid Suc. Succinic acid αKG; α-ketoglutaric acid Cit. ; Citric acid Others; Other organic acids

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 665 7804-4B 1:69) 7804-4B (72) Inventor Eiji Ichikawa Fushimi-ku, Kyoto 247 Minamihamacho Okura Sake Brewing Company (72) Inventor Tetsuyoshi Mizutsu 247 Minamihamacho, Fushimi-ku, Kyoto Okura Sake Brewing Company

Claims (3)

[Claims] 1. Aspergillus oryzae (Asper)
gillus oryzae) and Aspergillus awamori or Aspergillus awamori bal kawachi (Asp)
ergillus awamori var. kawa
chii) cell fusion strain. 2. Aspergillus oryzae
The cell fusion strain of Aspergillus awamori and Aspergillus awamori is F. F. The number is 2.
Cell fusion strain. 3. Aspergillus oryzae (Asper)
gillus oryzae) and Aspergillus awakawa (Aspergillus awawa)
mori var. cell fusion strain of F. kawachii). F. 7. The cell fusion strain according to claim 1, which is 7.