JP2609580B2 - Novel mutant strain and method for producing soy sauce using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel mutant strain that highly produces 2-phenylethanol, one of important flavor components in soy sauce, and a soy sauce having an excellent aroma using the same. Related to manufacturing method.

[Conventional technology]

Higher alcohols and their esters produced together with ethanol by yeast are one of the important flavor components in soy sauce. Among these higher alcohols, 2-phenylethanol having a rose-like aroma is one of the ones that play a central role. These higher alcohols are produced during the fermentation process of yeast, and their production has been studied for sake yeast, brewer's yeast, and the like. For 2-phenylethanol, Ehrlich produced from phenylalanine, one of the amino acids, is also used. A biosynthetic pathway which is produced from an intermediate in the pathway and a pathway in which phenylalanine is biosynthesized by glucose is known.

Regarding the pathway for biosynthesizing 2-phenylethanol from glucose, sake yeast, beer yeast, wine yeast,
Saccharomyces cerevisiae such as shochu yeast
myces cerevisiae), the regulation of the biosynthetic pathway (the cause of the suppression of biosynthesis) includes deoxy-D-arabino-D-
Heptulosonate phosphate biosynthetic enzyme (deoxy
-D-arabino-D-heptulosonate phosphate synthas
e) Feedback inhibition of activity by phenylalanine and tyrosine, chorismate mutease
utase) activity inhibition by tyrosine, prephenate dehydratase activity inhibition by phenylalanine, and prephenate dehydrogenase
This activity is known to be inhibited by tyrosine (Lingens,
F. et al., Eur. J. Biochem., 1, 363 (1967)).

In this regard, it has been reported that Saccharomyces cerevisiae, a sake yeast or shochu yeast, was used as a parent strain, and a fluorophenylalanine-resistant mutant strain that produced 2-phenylethanol at least twice or at least 10 times that of the parent strain was obtained. (Heisei 1989 Annual Meeting of the Japanese Fermentation Engineering Congress,
(Pages 50 and 52, issued September 10, 1989).

However, such a thing is not known about yeast of the genus S. saccharomyces.

[Problems to be solved by the invention]

The present inventor aimed at producing even more abundant soy sauce flavor components, and produced Zygosacchromyc (Zygosacchromyc) which highly produces 2-phenylethanol biosynthesized from glucose.
es) A yeast mutant belonging to the genus was searched. That is, an object of the present invention is to provide a yeast belonging to the genus Tigosaccharomyces
-To obtain a mutant strain that produces high phenylethanol and to produce a soy sauce with excellent aroma using the mutant yeast.

[Means for solving the problem]

The present inventors have performed various mutation treatments on yeast belonging to the genus S. saccharomyces, thereby performing the above-described rate-limiting except for prephenate dehydrogenase in the pathway for biosynthesizing 2-phenylethanol from glucose. As a result of searching for a mutant having the ability to produce a large amount of 2-phenylethanol without inhibition of the enzyme, the target strain was obtained by mutating the parent strain Zygosaccharomyces rouxii ATCC 13356. -The inventors succeeded in obtaining a novel mutant belonging to the genus Tigosaccharomyces which produces phenylethanol at a high level, and completed the present invention.

That is, the present invention is a novel mutant belonging to the genus T. saccharomyces luxii and having resistance to fluorophenylalan, and the present invention is characterized in that the novel mutant is used during a fermentation ripening step in soy sauce production. This is a method for producing soy sauce.

 Hereinafter, the present invention will be described in detail.

First, as a strain used as a parent strain of the present invention, for example, S. saccharomyces luxii ATCC13356 and the like can be mentioned. Then, the parent strain is subjected to ordinary mutation treatment to obtain a novel mutant strain of the present invention, Tigosaccharomyces FPA-1.
Can be obtained.

Next, the bacteriological properties of the above-mentioned C. saccharomyces FPA-1 will be described.

(A) Growth state in each medium A cream-colored colony is formed on the YM agar medium, and the vegetative cells proliferate by budding in a spherical or oval shape having a main diameter of 3 to 7 μm. YM liquid medium does not form a thin film.

 No pseudohyphae are formed on the potato extract agar medium.

(B) Formation of ascospores It is difficult to form ascospores in a spore-forming medium for general yeast,
Form oocysts in YM medium containing 9% saline.

(C) Formation of ejected spores No ejected spores were formed on YM agar plate culture.

(D) Each physiological property Grows well at 15-37 ° C, optimal temperature is 30 ° C, pH 3-7
And has an optimum pH of 5.

 Nitrate homogenization: None.

Sodium chloride resistant ... Grows well in the presence of 18% (W / W) or more sodium chloride.

 Vitamin requirement: biotin, pantothenic acid.

(E) Assimilation, fermentability presence or absence assimilation fermentability D-arabinose--D-xylose--D-glucose ++ D-galactose +-assimilation fermentable maltose--sucrose + + lactose--raffinose- -Α-methyl-D-glucoside--arbutin--succinate--melibiose--cellobiose--trehalose--ethanol--D-mannitol + + (f) 2-phenylethanol production amount Parent strain Tigosaccharomyces luxii ATCC13356 was compared with the mutant strain S. saccharomyces FPA-1 of the present invention, and the following tests were performed.

Both strains of S. saccharomyces luxii ATCC13356 and S. saccharomyces FPA-1 were precultured in a synthetic culture medium having the following composition at 30 ° C. for 5 days.

(Synthetic culture medium) Difco yeast nitrogen base (excluding amino acids and ammonium sulfate) 0.2% (W / V), ammonium sulfate 20 mM, glucose 5% (W / V), salt 5
% (W / V), pH 5.2 Then, this was mixed with a synthetic culture medium having the above composition at 30 ° C., 7
The culture was allowed to stand for a day. As a result, the amount of 2-phenylethanol produced in the culture medium was 4 ppm for the parent strain, S. saccharomyces luxii ATCC13356, and 150 ppm for the mutant S. saccharomyces FPA-1.

(G) Prephenate dehydrogenase activity Comparing the parent strain S. saccharomyces luxii ATCC13356 with the mutant strain S. saccharomyces FPA-1 in the present invention, the activity of the prephenate dehydrogenase obtained from S. saccharomyces FPA-1 per total cell mass of the bacterial cell is: Parent strain S. saccharomyces luxii ATCC1335
Assuming that of 6 is 100%, it is reduced to 2% or less, which is significantly different from the parent strain.

That is, in the mutant strain T. saccharomyces FPA-1, as described above, the activity of prephenate dehydrogenase is significantly reduced to decrease the biosynthesis of tyrosine, and the feedback inhibition by tyrosine of the enzyme on the pathway of biosynthesizing 2-phenylethanol from glucose is suppressed. Is reduced, resulting in significant production of 2-phenylethanol.

(A)-in the above-mentioned T. saccharomyces FPA-1
The mycological properties of (e) are described in Kreger (NJWKreger-va
n Rij 「The Yeasts, A toxonomic
study ”Elsevir Science Pub.) According to the third edition, all are the same as those belonging to the strain belonging to T. saccharomyces luxii.

And this parent strain Chigosaccharomyces luxii ATCC13
The difference between both strains of 356 and the mutant strain S. saccharomyces FPA-1 is that, in addition to (f) and (g) above,
Unlike the former parent strain, 50
It is a fluorophenylalanine-resistant bacterium that can grow on a medium containing μg / ml.

From these facts, the strain S. saccharomyces FPA-
1 was identified as a new strain.

In addition, this Strawberry saccharomyces FPA-1 was supplied to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology.
MP-11115).

Any method may be used for the mutation treatment. For example, as a chemical method, a mutagenic agent such as N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as MNNG), ethyl methanesulfonate, methyl methanesulfonate, 4-nitrosoquinone, nitrite, bromouracil, etc. Is used, or physical methods such as ultraviolet irradiation, X-ray irradiation,
A radiation irradiation treatment or the like is performed.

As the medium, a synthetic medium or a natural medium appropriately containing a carbon source, a nitrogen source, inorganic salts, and other components necessary for the growth of yeast, which can be used by yeast, is used. In addition, like soy sauce,
When adding a high salt concentration to moromi, it is desirable to adjust the salt concentration of the medium to about 6 to 18% (W / V). Then, in order to impart fluorophenylalanine resistance, p-fluorophenylalanine, o-fluorophenylalanine or m-fluorophenylalanine, preferably p-fluorophenylalanine, is added to the yeast culture medium at about 50 μg / ml.

This yeast culture is performed by shaking culture, aeration culture, stirring culture,
Among aerobic or anaerobic culture methods such as stationary culture,
An appropriate method is adopted. The culturing temperature is possible within a range in which yeast cells can grow, but is usually 25 to 30 ° C., and the culturing time is 10 to 50 hours for aerobic culturing, and 120 to 50 hours for anaerobic culturing. Incubate for about 170 hours.

In the case of solid culture in this manner, the resulting colony is separated, and in the case of liquid culture, the colony is separated according to ordinary operation methods such as centrifugation and filtration, and washed as necessary to obtain the mutant strain.

The thus obtained mutant strain may be cultured according to a usual yeast culture method.

Next, a method for producing soy sauce using this mutant strain will be described.

First, a conventional protein raw material, carbohydrate raw material, or the like is subjected to a raw material treatment such as steaming, puffing, and roasting, and then inoculated with a seed koji to produce a soy sauce koji. Next, the soy sauce koji is charged together with a salt solution having an appropriate concentration by a conventional method, and this is fermented and aged to obtain an aged moromi. Then, in the present invention, at an appropriate time during the fermentation and ripening process from the preparation to the aging moromi, the cells of the present strain obtained by separately culturing or a culture solution thereof are added, and thereafter, ordinary moromi fermentation is performed. Perform aging management. At this time, the time of addition of the bacterial cell of the present strain or its culture solution, the amount added is not particularly limited,
Preferably, usually about 1 to 2 months after preparation, about 10 ⁵
Unit / g.

The aged moromi thus obtained is squeezed by a conventional method,
By performing standard adjustment and burning, a burned soy sauce with excellent aroma can be obtained.

Also, the acid or enzymatic decomposition solution of the raw material is immobilized by a conventional method, contacted with lactic acid bacteria, yeast and the like, and fermented and ripened to obtain a fermentation solution with a rich flavor, replacing some or all of the normal yeast strains, The present mutant strain can also be used.

〔The invention's effect〕

The novel mutant of the present invention is a conventional mutant of S. saccharomyces.
Compared to strains belonging to Ruxii, the ability to produce 2-phenylethanol is remarkably excellent. When soy sauce is produced using this strain, the obtained soy sauce contains higher alcohol components such as 2-phenylethanol. Contains a remarkably large amount,
It is remarkably fragrant.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 A parent strain S. saccharomyces luxii ATCC13356 was prepared by using a proline medium having the following composition and containing L-proline as a nitrogen source.
The cells were cultured at 50 ° C for 30 days at 30 ° C.

(Proline medium) Difco yeast nitrogen base (excluding amino acid and ammonium sulfate) 0.2% (W / V) L-proline 2 mM NaCl 5% (W / V) Then, the obtained culture solution was centrifuged. The cells were collected, washed with 100 mM phosphate buffer (pH 7.0) containing 5% salt (W / V), and the washed cells were washed with 5% salt (W / V) and 5% glucose (W / V). / V) suspended in 100 mM phosphate buffer (pH 7.0), and added with MNNG to a concentration of 1 mg / ml.
For 1 hour while shaking slowly. Subsequently, the cells were collected by centrifugation, and the cells were cultured in 50 ml of the above-mentioned proline medium at 30 ° C. for 10 hours, and then p-fluorophenylalanine was added to a final concentration of 50 μl.
g / ml and agar 2% (W / V) except that it was spread on a medium similar to the above-mentioned proline medium, cultured at 30 ° C. for 10 days, and grown from a number of resistant strains to obtain S. saccharomyces FP.
A-1 (FERM P-11115) was obtained.

Example 2 100 kg of defatted soybeans were steam-denatured and 105 kg of wheat crushed in a roasted mushroom were mixed, inoculated with seed koji, and subjected to 42 hours of ventilation koji making to obtain soy sauce koji.

To this, 360% of a 25% (W / V) saline solution cooled to 15 ° C. was added, and the mixture was charged into a 600-volume closed charging tank. At that time, Pediococcus halophilu
s) IAM1693 was added so that the viable cell count was 1 × 10 ⁵ per g of moromi. The mixture was stirred occasionally after the addition, heated from the 14th day after the preparation, and on the 60th day after the preparation, a culture solution of T. saccharomyces FPA-1 (FERM P-11115) separately obtained by the following method was added.
It was added so that the viable cell count was 1 × 10 ⁵ per g of moromi.

[Culture solution of C. saccharomyces FPA-1 (FERM P-11115): The soy sauce moromi sap on the 20th day after the above preparation was added to a 15% salt (W /
After adjusting to V), the medium obtained by aseptic filtration (pH 5.2)
Next, the strawberry Saccharomyces FPA-1 obtained in Example 1 (FREM
P-11115) and cultured at 30 ° C. for 4 days to obtain a culture solution. After that, normal moromi management was carried out for another 6 months to obtain an aged moromi.

After squeezing this by the usual method, NaCl 17.0% (W / V),
The TN was adjusted to 1.57% (W / V), and the mixture was heated at 80 ° C. for 4 hours to obtain a burned soy sauce (this product).

On the other hand, except that the above-mentioned T. saccharomyces FPA-1 is replaced with T. saccharomyces luxii ATCC13356,
Fired soy sauce (control product) was obtained in exactly the same manner as above.

A general analysis of the fired soy sauce was performed in accordance with "Soy sauce standard analysis method" edited by Soy Sauce Technical Society, and 2-phenylethanol was also quantified. Table 1 shows the results.

In addition, the gas chromatography was used for the quantification method of 2-phenylethanol by an ordinary method.

In addition, about the above-mentioned hot soy sauce, panel of 24 people,
Sensory tests were performed by the triangle method. Table 2 shows the results.

As shown in Table 1, alcohol is sufficiently generated,
Normal yeast fermentation was performed for all products. Then, it was confirmed that the amount of 2-phenylethanol was increased about 20 times in this product as compared to the control product.

Also, as is clear from the results in Table 2, this product was a product having a remarkably superior aroma compared to the control product.

Claims (2)

(57) [Claims] 1. A novel mutant strain belonging to T. saccharomyces luxii and having resistance to fluorophenylalanine. 2. A method for producing soy sauce, wherein the novel mutant strain according to (1) is used during a fermentation ripening step in soy sauce production.