JPH04330273A - New mutant strain and preparation of soy sauce having high amino acid content using said strain - Google Patents

Abstract

PURPOSE:To provide a new mutant strain capable of taking and utilizing ammonia as nitrogen source in the growth and proliferation in unrefined soy sauce and scarcely taking amino acids existing in the sauce as taste substances and useful for the fermentation and ripening of soy sauce to obtain a soy sauce having excellent taste. CONSTITUTION:The objective mutant strain is a microorganism belonging to the genus Zygosaccharomyces, resistant to p-fluoro-DL-phenylalanine and having low activity to take amino acid, e.g. Zygosaccharomyces FPA-16 (FERM P-12221). The Zygosaccharomyces FPA-16 can be produced by using Zygosaccharomyces rouxii ATCC 13356 as parent strain and subjecting to conventional mutagenic treatment such as ultraviolet irradiation.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a novel mutant strain that mainly ingests ammonia as a nitrogen source when yeast grows and multiplies in soy sauce moromi, and hardly ingests amino acids useful as flavor components; The present invention relates to a method for producing soy sauce with high amino acid content and excellent taste using this mutant strain.

0002

[Prior Art] In the fermentation of soy sauce moromi, soy sauce yeast, especially Zygosaccharomyces rukisi (Zygosaccharomyces
aromyces rouxii) plays an important role in the quality of soy sauce, such as producing significant amounts of alcohol and other aromatic components. Therefore, in order to produce high-quality soy sauce with well-balanced flavor and flavor, it is necessary to stably and reliably carry out fermentation using soy sauce yeast, which has excellent properties.

On the other hand, in the traditional soy sauce production method,
Fermentation of soy sauce moromi is usually carried out in an open system, with almost no artificial addition of microorganisms such as yeast, and all soy sauce yeast active in moromi naturally settles in the brewery, facilities, equipment, etc. It was left to the natural contamination of bacterial groups (natural flora). This natural flora is extremely diverse, and some naturally mixed yeast bacteria may contain bacteria with properties that are not necessarily desirable in terms of the quality of soy sauce. In addition, because the content of soy sauce changes depending on the region or over time, there are problems such as differences in product quality depending on the brewing location and the inability to obtain soy sauce of consistent quality throughout the year.

[0004] For this reason, soy sauce yeast with excellent properties has been selected in advance or specially bred for the purpose of producing brewed soy sauce of constant quality regardless of regional or time constraints. It has become common practice to artificially add soy sauce moromi to stabilize yeast fermentation. As a result, the soy sauce moromi underwent a vigorous fermentation process as expected, and a significant amount of alcohol and various aromatic components were produced and accumulated in the soy sauce moromi.

[0005] However, in the brewing of soy sauce, since the moromi liquid is directly used as the product soy sauce, it is desirable that the useful components in the moromi do not undergo any changes or depletion during the fermentation and maturation of the moromi. In fact, this is not the case; with the active fermentation of yeast, a considerable amount of amino acids, which are useful as flavor components, are also consumed. This is because soy sauce moromi is rich in suitable nutrients such as amino acids and peptides, which are produced by the decomposition of proteins, which are raw materials for soy sauce, by enzymes derived from Aspergillus oryzae. This is to provide a nutritional source for their own growth and reproduction.

[0006] Soy sauce is a seasoning that is familiar to many people's daily diet, and the soy sauce brewing industry is trying to provide soy sauce that contains as high a concentration as possible of the most important amino acids as flavor components. Nowadays, it is desirable to reduce the consumption of amino acids by soy sauce yeast as much as possible, which has become an important issue. In addition, in order for yeast to grow and be active in soy sauce moromi,
It is true that some kind of nitrogen source must be consumed, but on the other hand, in soy sauce moromi, in addition to amino acids, ammonia, which has a low taste and usually accounts for 20 to 30% of the amino nitrogen, is also used as a nitrogen source. The fact that it exists must not be overlooked.

[0007]

[Problem to be solved by the invention] Therefore, the present inventors
In view of this current situation, when yeast grows and proliferates in soy sauce moromi, it ingests and uses ammonia other than amino acids as a nitrogen source, and hardly ingests useful amino acids as unpresented components.In other words, it consumes a large amount of amino acids. Even in the coexistence of
By searching for strains that can grow by selectively using ammonia as a nitrogen source, and using such special microorganisms as seed yeast, we focused on the possibility of significantly preventing the consumption of amino acids. As a result of conducting various mutation treatments on yeast belonging to the genus Zygosaccharomyces isolated from Zygosaccharomyces luxii,
A special new mutant strain belonging to the genus Tygosaccharomyces and having parafluoro-DL-phenylalanine resistance obtained by mutating the parent strain ATCC13356 (Ces rouxii) ATCC13356 ingests a large amount of ammonia in soy sauce moromi, Don't take amino acids
We found that this is a new mutant strain that ferments vigorously, that is, has a reduced amino acid uptake ability, and by using the mutant strain obtained above as the yeast in the soy sauce production method in which yeast is added to soy sauce moromi. It was discovered that soy sauce with a high amino acid content and excellent flavor could be obtained, and the present invention was completed based on these findings.

[0008]

Means for Solving the Invention That is, the present invention relates to a novel mutant strain belonging to the genus Thigosaccharomyces, having resistance to parafluoro-DL-phenylalanine and having less amino acid uptake activity; This is a method for producing soy sauce with a high amino acid content, which is characterized by using a new mutant strain during the fermentation and maturation process in soy sauce production.

[0009]

EXAMPLES The present invention will be explained in detail below.

First, in the present invention, the microorganism used is a parafluoro-fluorinated microorganism belonging to the genus Chigosaccharomyces.
Examples include mutant strains that are resistant to DL-phenylalanine and have low amino acid uptake activity. The amino acid uptake activity preferably has an initial amino acid uptake rate of 0.1 or less, particularly 0.05 or less in the test described below. Such strains include, for example, Tygosaccharomyces ATC
It can be created by using C13356 as a parent strain and subjecting it to normal mutation treatments. Chigosaccharomyces FP
A-16 is the strain obtained in this way.

Next, the above-mentioned Chigosaccharomyces FPA-
16 mycological properties are shown.

(a) Growth status in each medium ■ Y
Cream-colored colonies are formed on M agar medium, and the vegetative cells are spherical to oval with a main diameter of 3 to 7 μm and proliferate by budding. YM liquid medium does not form a thin film. ■ Pseudohyphae do not form on potato extract agar medium.

(b) Formation of ascospores It is difficult to form ascospores using a general yeast sporulation medium, but ascospores are difficult to form.
Form zygotic asci in YM medium containing 9% salt.

(c) Formation of extruded spores No extruded spores were formed in YM agar flat culture.

(d) Physiological properties ■Grows well at 15-37°C, optimal temperature is 30°C, pH
It grows well at pH 3 to 7, with an optimum pH of 5. ■Assimilation of nitrate: None. ■Sodium chloride tolerance: Grows well in the presence of 18% (W/W) or more sodium chloride. ■Vitamin requirements: biotin, pantothenic acid.

(e) Presence or absence of assimilability and fermentability

0017
] (f) Amino acid uptake activity parent strain Chigosaccharomyces
The following tests were conducted to compare ATCC13356 and the mutant strain of the present invention, Stygosaccharomyces FPA-16.

[0018] Chigosaccharomyces ATCC13356
and Stygosaccharomyces FPA-16 were precultured at 30° C. for 5 days in synthetic culture media each having the composition shown below.

(Synthetic culture medium) Difco Yeast Nitrogen Base (free of amino acids and ammonium sulfate) 0.2% (W/V), ammonium sulfate 1
0mM, glucose 5% (W/V), salt 5% (W/V
), pH 5.2

[0020] Next, the obtained culture solution was centrifuged to collect bacteria, and this was added to 1
After washing with 00mM phosphate buffer (pH 7.0),
L-leucine labeled with 14C in the above synthetic culture medium,
L-phenylalanine, L-valine, L-glutamic acid, and L-citrulline were added at 0.1 mM each, and a certain amount of bacterial cells was inoculated, and the uptake amount was measured using a scintillation counter.

[0021]
Table 1 ──────────
──────────────────────

Initial capture speed
(nmol/
min/mg dry cell weight)
Substrate (0.1mM) ──────
──────────────────
ATCC
13356 FPA-16
────────────────────────
──────── L-Leucine
0.52
0.02 L-phenylalanine 0.53
0.03 L-valine
0.59
0.03 L-glutamic acid 0.60
0.01 L-citrulline
0.67
NM* ──────────
──────────────────────
*NM indicates not measured

[0022] As a result, the amino acid uptake activity in the culture medium was
Compared to C13356, the mutant strain Thigosaccharomyces FP
A-16 is significantly reduced (less than 0.1)
It has been found.

[0023] The above Chigosaccharomyces FPA-16
The mycological properties of (a) to (e) in "The Yeasts," edited by N. J. W. Kreger-van Rij.
onomic study” Elsevier Sc
ience Pub. ) According to the 3rd edition, they are all the same as those possessed by strains belonging to the genus Thigosaccharomyces.

[0024] And this parent strain Chigosaccharomyces AT
CC13356 and mutant strain Chigosaccharomyces FPA-
In addition to (f) above, the difference between the 16 strains is that the latter mutant strain is different from the former parent strain in that it has parafluoro-DL-
It must be a parafluoro-DL-phenylalanine-resistant bacterium that can grow in a medium containing at least 100 μg/ml of phenylalanine, and must have an amino acid uptake activity of 0.1 or less.

[0025] Based on these facts, the strain Stigosaccharomyces FPA-16 was identified as a new strain. In addition,
This strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-12221.

[0026] Any method may be used for the above mutation treatment. For example, chemical methods include mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as MNNG), ethyl methanesulfonate, methyl methanesulfonate, 4-nitrosoquinone, nitrous acid, and bromouracil. or use physical methods such as ultraviolet irradiation, X-ray irradiation, and radiation irradiation treatment.

[0027] As a medium, a carbon source that can be used by yeast,
Nitrogen sources, inorganic salts, and other ingredients necessary for yeast growth,
A suitably formulated synthetic medium or natural medium is used.

Furthermore, when it is intended to be added to moromi with a high salt concentration such as soy sauce, the salt concentration of the medium should be adjusted to 6 to 18% (
It is desirable to adjust it to approximately W/V).

[0029] In order to grow only parafluoro-DL-phenylalanine-resistant bacteria, parafluoro-DL-phenylalanine was added to the yeast culture medium at 100 μg/day.
Add about ml.

[0030] This yeast culture is carried out by shaking culture, aeration culture,
An appropriate method is employed among aerobic or anaerobic culture methods such as agitation culture and static culture. The culture temperature can be within the range where yeast cells can grow, but it is usually 25 to 30°C.
The culture time is about 10 to 50 hours in the case of aerobic culture, and about 120 to 170 hours in the case of anaerobic culture.

[0031] In the case of solid culture, the colonies thus formed are separated, and in the case of liquid culture, they are separated according to normal operating methods such as centrifugation and filtration, and washed if necessary to obtain the present mutant strain. .

[0032] The thus obtained mutant strain may be cultured according to a conventional yeast culture method.

Next, a method for producing soy sauce with a high amino acid content using the soy sauce production process using this mutant strain will be described.

[0034] First, by a conventional method, ordinary protein raw materials, carbohydrate raw materials, etc. are subjected to raw material treatments such as steaming, puffing, and roasting, and then seed koji is inoculated and koji is made to obtain soy sauce koji.

[0035] Next, the soy sauce koji is mixed with a saline solution of an appropriate concentration by a conventional method, and the mixture is fermented and aged to obtain aged moromi.

[0036] In the present invention, at an appropriate time during the fermentation and maturation process from this preparation to obtaining aged moromi, the cells of this strain obtained by separate cultivation or the culture solution thereof are added,
After that, normal moromi fermentation and maturation management will be carried out.

[0037] At this time, the timing and amount of addition of the cells of this strain or its culture solution are not particularly limited, but it is preferably about 105 cells/g, usually 1 to 2 months after preparation.
That's about it.

[0038] The aged moromi obtained in this way is pressed by a conventional method, and the soy sauce with a high amino acid content can be obtained by subjecting it to standard adjustment, pasteurization, etc.

[0039] In addition, when fermenting and maturing the raw acid or enzymatically decomposed liquid by contacting it with immobilized lactic acid bacteria, yeast, etc. by a conventional method to obtain a fermented liquid with a rich flavor, it is possible to use some or all of the usual yeast strains. Alternatively, this mutant strain can also be used.

[0040]

[Effects of the Invention] The novel mutant strain of the present invention has the property that yeast ingests ammonia other than amino acids as a nitrogen source in soy sauce moromi, and can grow without ingesting most of the useful amino acids as unpresented components. In the soy sauce manufacturing method in which yeast is added to soy sauce moromi, by using the mutant strain obtained above as the yeast, it is possible to obtain an excellent flavor that has a high amino acid content and does not change other component analysis values, color luster, pH, etc. You can get soy sauce.

[0041]

[Examples] The present invention will be explained in more detail with reference to Examples below. Example 1 Parent strain Tygosaccharomyces ATCC13356 was statically cultured at 30° C. for 5 days in 50 ml of an ammonium sulfate medium having the following composition containing ammonium sulfate as a nitrogen source.

(Ammonium sulfate medium) Difco Yeast Nitrogen Base (free of amino acids and ammonium sulfate)
0.2%
(W/V) Ammonium sulfate
10mM NaCl

5% (W/V)
pH
5.2

[0043] Next, the obtained culture solution was centrifuged to collect bacteria, which was washed with 100 mM phosphate buffer (pH 7.0) containing 5% (w/v) sodium chloride. MNNG was suspended in 100 mM phosphate buffer (pH 7.0) containing 5% (w/v) glucose and 5% (w/v) glucose, and then MNNG was added to the solution at 1 mg/ml. Mutation treatment was performed with slow shaking for hours.

[0044] Subsequently, this was centrifuged to collect bacteria, and this was incubated in 50 ml of the above ammonium sulfate medium at 30°C for 1 hour.
After static culture for 0 hours, parafluoro-DL-
It was spread on a medium similar to the above ammonium sulfate medium except that it contained phenylalanine at a final concentration of 100 μg/ml and agar at 2% (W/V), and was incubated at 30°C for 10 minutes.
Stygosaccharomyces FPA-16 was obtained from a large number of resistant strains that grew after culturing for days.

Example 2 100 kg of defatted soybeans denatured by steaming and 105 kg of wheat
kg of roasted soybean paste was mixed, inoculated with seed koji, and subjected to ventilation koji for 42 hours to obtain soy sauce koji.

[0046] 25% (W/V) cooled to 15°C
360 liters of brine was added and charged into a 600 liter sealed charging tank. At that time, the number of viable bacteria of Pediococcus halophilus IAM1693 was 1× per 1 g of moromi.
105 pieces were added.

[0047] After addition, the mixture was stirred occasionally and heated from the 14th day after preparation, and on the 60th day after preparation, a culture solution of Chigosaccharomyces FPA-16 separately obtained by the following method was added to the culture solution, with the number of viable bacteria per 1 g of moromi. They were added at a concentration of 1 x 105 pieces.

[0048] Culture solution of Chigosaccharomyces FPA-16: 20 days after preparation, the soy sauce moromi liquid was mixed with 15% salt.
(W/V) and then sterile filtered the medium (p
H 5.2), Chigosaccharomyces F obtained in Example 1
PA-16 was inoculated and cultured at 30° C. for 7 days in the case of static culture and 5 days in the case of shaking culture to obtain a culture solution.

On the other hand, the above-mentioned Chigosaccharomyces FPA-1
Instead of 6, Chigosaccharomyces ATCC13356
A culture solution (control product) was obtained in exactly the same manner as above except that .

Amino acid analysis of the above culture solution was carried out using an amino acid analyzer. The results are shown in Table 2.

[0051]
Table 2──────────────────
────────────────
Static culture
Shaking culture Amino acids ────────────────
─────── (mM)
ATCC FPA-16
ATCC FPA-16
13356
13356─
──────────────────────────
───────L-Leucine
42.97 45.31
33.72 42.26L-phenylalanine 22.07 23.35
19.40 22.16L-valine 35.02
35.77 31.11
35.13L-glutamic acid 8
2.26 82.73 83
．． 47 84.59L-Serine
40.52 41.20
38.35 41.26 Ammonia 97.26
91.70 84.95
63.42────────────────────
──────────────

The results in Table 2 show that, compared to the parent strain ATCC13356, the new mutant strain FPA-16 generally retains a larger amount of amino acids and consumes a larger amount of ammonia. ,
The difference is particularly noticeable in aerated culture.

Example 3 The above-mentioned moromi was then subjected to normal management for another 6 months to obtain aged moromi. After compressing this in a conventional manner, N
aCl 17.0% (W/V), T. N 1.57% (
W/V) and was heated at 80° C. for 4 hours to obtain heated soy sauce (product of the present invention) with a high amino acid content.

On the other hand, the above-mentioned Chigosaccharomyces FPA-1
Instead of 6, Chigosaccharomyces ATCC13356
Soy sauce (control product) was obtained in exactly the same manner as above except that .

Amino acid analysis of the above soy sauce was conducted using an amino acid analyzer. The results are shown in Table 3.

[0056]
Table 3 ────────────────
──────────── Amino acids
ATCC
FPA-16 (mM)
13356
───
────────────────────────
L-leucine
49.86 58.40
L-phenylalanine 2
2.09 24.58
L-valine
41.23 45.25
L-glutamic acid
67.97 69.09
L-serine
41.21 44.12
──────────────────────
──────

From the results in Table 3, in the soy sauce production method in which yeast is added to soy sauce moromi, as the yeast,
By using the mutant strain FPA-16, the parent strain A
It can be seen that a soy sauce with a higher amino acid content and better flavor than TCC13356 can be obtained. In addition, other component analysis values, color, pH, etc. were unchanged compared to the parent strain ATCC13356.

Claims (2)

[Claims] Claims: 1. A novel mutant strain belonging to the genus Thigosaccharomyces that is resistant to parafluoro-DL-phenylalanine and has low amino acid uptake activity. 2. A method for producing soy sauce with a high amino acid content, which comprises using the novel mutant strain according to claim (1) during the fermentation and maturation step in soy sauce production.