JPH06153896A - Production of refined sake (rice wine) - Google Patents

Abstract

PURPOSE:To obtain refined SAKE reduced in amount of phytin and high in content of inositol having preventing action on hepatic disease, arteriosclerosis, etc., and growth promoting action, by producing refined SAKE from rice KOJI prepared by multiplying a rice KOJI mold having high phytase activity as a raw material. CONSTITUTION:Aspergillus-oryzae-RIB128 is used as a parent strain, a spore suspension of the parent strain is treated with nitrosoguanidine at 30 deg.C for 10 minutes, washed, applied to a KOJI essence medium, cultured at 28 deg.C for 5-7 days, the deposited spores are collected, inoculated into a medium containing 2-deoxyglucose and subjected to shaking culture at 30 deg.C for 24 hours to form PHA2-4-3 strain (FERM p-13,062) of high phytase activity strain. The strain is added to boiled rice and KOJI is produced by a conventional procedure. The prepared KOJI is used, the whole rice is prepared at two stages and SAKE is produced at 20% KOJI yield at 140% water dipping ratio and at constant SAKE temperature of 15 deg.C to give the objective refined SAKE having a reduced amount of phytin and an increased amount of inositol.

Description

Detailed Description of the Invention

[0001] The present invention relates to a method for producing sake by using rice koji having a phytase highly active koji mold as a raw material.

[0002] Generally, inositol is known to have a preventive effect on fatty liver and liver cirrhosis, an arteriosclerosis preventive effect, a growth promoting effect and the like, and is a physiologically active substance used in pharmaceuticals such as multivitamin preparations, liver enhancers and antiaging agents. is there.

Inositol is a constituent of phytin, and phytin is contained in rice bran in a maximum amount of about 10%. Brown rice also contains about 1%.
And about 10% of those phytins are inositol.

Inositol is 0.1 to 0.12 in brown rice.
Although most of them are contained in the form of phytin, they are rarely used even if they are ingested as they are by humans, and the absorption of calcium and the like may occur due to the chelating action of phytin.

Phytase is a kind of acid phosphatase, which is an enzyme involved in a 6-step hydrolysis reaction in which one phosphate group is released from the myo-inositol nucleus using phytin as a substrate, and is present in plant seeds, molds, yeasts and the like. Has been confirmed.

When phytase contained in rice is decomposed into inositol by phytase, the amount of phytin in sake can be reduced and the amount of inositol can be increased.

[0007]

DISCLOSURE OF THE INVENTION An object of the present invention is to obtain sake having a high inositol content by using a koji mold having a high phytase activity.

[0008]

In the present invention, aspergillus oryzae is subjected to mutation treatment and grown in a medium containing 2-deoxyglucose.
Deoxyglucose resistant strain phytase Highly active koji mold is used as a koji mold to produce koji, and by producing sake using this koji as a raw material, the amount of phytin is reduced and the amount of inositol is increased. Was successful.

In the breeding of the koji mold having a high activity of phytase of the present invention, any koji mold may be used as long as it is a koji mold generally used for sake production.

Aspergillus oryzae is first subjected to mutation treatment, but it is exposed to ultraviolet rays,
Any of the physical mutagenesis treatment with rays, radiation, etc., the chemical mutagenesis treatment with a drug such as nitrosoguanidine (NTG), or the mutation treatment used in combination may be used.

The koji mold which has been subjected to the mutation treatment is inoculated with the microbial cells or spores into a basic medium containing about 1 to 1.5% of 2-deoxyglucose and cultured at 30 ° C. for 24 hours. A deoxyglucose resistant strain is obtained. Two
-Separate culture of deoxyglucose-resistant Aspergillus is more than twice 4
A stronger 2-deoxyglucose-resistant koji mold can be obtained by repeating the process about once.

In the present invention, it has been found that the higher the resistance to 2-deoxyglucose, the higher the phytase activity of the koji mold.

It is unclear why the koji mold having a high resistance to 2-deoxyglucose produces a large amount of phytase. I have to wait for future research.

In the present invention, NTG mutation treatment is carried out to
After culturing 4 times in a medium containing deoxyglucose, a koji mold having high resistance to this was obtained.
% PS calcium phytate (described below) was inoculated and cultured at 30 ° C for 24 hours to form halos, and phytase highly active Aspergillus oryzae was isolated. The strains and halo values are shown in Table 1 below. It was In Table 1, RIB 128
Is an ordinary koji mold that has not been subjected to any treatment such as mutation treatment.

[0015]

[Table 1]

The phytase highly-active koji mold used in the present invention produces a larger amount of phytase than conventional koji molds, and thus can be used for sake production to produce sake having a high inositol content.

Further, it is noted that phytin, which is a substrate of inositol, is large in rice bran, brown rice, and low-polished rice, and by producing a koji by breeding strains with high activity of phytase, they contain more inositol than conventional sake. It is also possible to brew sake.

[0018]

Example 1 Method for constructing mutant strain with high activity of phytase Aspergillus oryzae RIB 128 was used as a parent strain. R
3 ml (2 × 10 ⁶ / ml) of IB 128 spore suspension was treated with 4 mg / ml of NTG (nitrosoguanidine) at 30 ° C for 10 minutes, washed, applied to a koji extract medium, and cultured at 28 ° C for 5 to 7 days. To do. Collect the settled spores and add 2-deoxyglucose to 1
Inoculated into a medium containing 0.5% to 0.5% (glucose 0 to 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.8%, magnesium sulfate 0.05%, peptone 0.2%). ℃
After 24 hours of shaking culture, the cells were inoculated in the same medium. After repeating this 4 times, it was cultured in a koji extract medium, and the resulting spores were PS medium (glucose 1.5%, calcium phytate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.05%, 0.05% potassium chloride, 0.001% ferrous sulfate, 0.001% manganese sulfate, Trit
on X-100 0.3%, agar 2%) Apply 50 plates per plate and incubate for 4-5 days.

The halo of the colonies obtained in the above was measured, and a strain with a large halo was selected as a phytase highly active strain. Among the mutant strains thus obtained, the PHA2-4 strain, which had a particularly large halo, was used as the parent strain, and the same operation was performed again.
A PHA2-4-3 strain with favorable spore settlement was obtained. P
The phytase activity of the HA2-4-3 strain was superior to that of the parent strain.

The phytase activities of the mutant strain PHA2-4-3 and the parent strain RIB128 are shown in comparison in Table 2 below.

[0021]

[Table 2]

The PHA2-4-3 strain has been deposited in the Institute of Microbial Technology, National Institute of Industrial Science and Technology as Microorganism Research Institute No. 13062.

[Example 2] Production of inositol by PHA2-4-3 strain Each spore suspension of RIB128 and PHA2-4-3 was adjusted to 1 x 10 ² cells / ml, and a Burkholder synthetic medium was prepared. From inositol, calcium chloride, phosphoric acid 2
Excluding potassium hydrogen, sodium phytate 0.5%,
Inoculate 30 ml of medium containing 0.15% potassium chloride to
Shaking culture was carried out at 8 ° C for 5 days, and inositol in the culture solution was quantified by gas chromatography. The following Table 3 shows inositol production in comparison.

[0024]

[Table 3]

[Example 3] Production of koji by PHA2-4-3 strain The raw material used was steamed by adding rice bran produced at the time of rice polishing before steaming to rice with 95% rice polishing rate. 350g steamed rice
On the other hand, seed koji (RIB128 strain and PHA2-4-3, FE
2 g of RM P-13062 strain) was added and koji was made by a conventional method. α-amylase, glucoamylase, and acidic protease were assayed by the National Tax Agency, and phytase was quantified by Nakamura's method, using 10 mM sodium phytate as a substrate and inorganic phosphate produced by the reaction at 50 ° C. for 30 minutes. The results are shown in Table 4 below.

[0026]

[Table 4]

[Example 4] Production of sake by PHA2-4-3 strain Using the two types of koji obtained in Example 3, 200 g of total rice was charged in two stages. Koji ratio 20%, drawing water ratio 140
% And the product temperature were constant at 15 ° C. Glutase 6000 (0.1%) was added to the water drawn during the distillation. The charge formulation is shown in Table 5 below.

[0028]

[Table 5]

The sensory evaluation of the sake brewed was not much different from that of the parent strain. The ingredients of the sake liquor were analyzed, and the results are shown in Table 6 below. In addition, general analysis of sake liquor was determined by the analysis method prescribed by the National Tax Agency, and inositol was quantified by gas chromatography.

[0030]

[Table 6]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Mitsuhiro Shindo 4-4-14 Chikusa, Takarazuka City, Hyogo Prefecture

Claims (4)

[Claims] 1. A method for producing sake with a high inositol content, which comprises producing rice sake with the use of rice koji having a phytase highly active koji mold as a raw material. 2. The method for producing sake having a high inositol content according to claim 1, wherein the Aspergillus oryzae having high activity of phytase is obtained by subjecting Aspergillus strain to mutation treatment and collected as a 2-deoxyglucose-resistant strain. 3. Aspergillus oryzae highly active in phytase is PHA2-4.
-3, FERM P-13062, The method for producing sake having high inositol content according to claim 1. 4. Sake with a high inositol content produced by the method according to any one of claims 1 to 3.