JPS61254159A - Production of soy sauce - Google Patents

Abstract

PURPOSE:To carry out well-balanced lactic acid fermentation and alcoholic fermentation moderately and prevent the impartment of uncomfortable falvor thereto, by adding a killer factor, a material containing the killer factor, microorganism producing the killer factor, etc., to a suitable period of producing soy sauce. CONSTITUTION:A killer factor, a material containing the killer factor and/or microorganism producing the killer factor, culture thereof or treated material thereof is added to soy sauce KOJI, unrefined soy, raw soy sauce, product soy sauce, etc., in producing the soy sauce to control the yeast existing in the production process of the soy sauce. The microorganism producing the killer factor may be a microorganism belonging to any genus, and either one of novel and well-known microorganisms. The time of adding or inculating the above- mentioned killer factor, etc., may be suitably selected, but is preferably before mixing KOJI or activity of many kinds of yeast living in a charging tank, particularly Zybosaccharomyces rouxii in the unrefined soy becomes remarkable.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a rational method for producing soy sauce.

More specifically, the present invention relates to a method for rationally producing soy sauce while controlling soy sauce yeast using a killer factor.

[Prior art]

Generally, soy sauce is produced by roasting heat-denatured soybeans or defatted processed soybeans, sprinkling them with crushed wheat, making the entire mixture (raw material for koji) into koji, mixing it with brine, and making soy sauce moromi. shall be. Sometimes, a part of the koji raw material is made into koji, the rest of the koji raw material that was not made with koji is added and digested, and if necessary, salt is added to make soy sauce moromi, or the koji raw material is not made into koji at all and is made with a commercially available enzyme agent. After digestion, salt may be added to make soy sauce moromi. The soy sauce moromi obtained in this way undergoes lactic acid fermentation.
Alcoholic fermentation then takes place and further ripening takes place. The general outline of the entire process is to press these ripening methods to obtain raw soy sauce, pasteurize it, sterilize it, remove the fire-heated soy sauce, and then make it into a product immediately or after storing it.

However, it has long been pointed out that there are problems at each stage of this process. What is the problem?

(1) During koji making, koji is contaminated by yeast from the air or from utensils, etc., and the yeast is brought into various methods.

Similarly, yeast adhering to brewing warehouses and tanks are often introduced into various methods. If the number is small, the problem is small, but if there are many, it will seriously affect the quality yield of soy sauce. In other words, in the typical pattern of microorganisms used in various methods, lactic acid fermentation by lactic acid bacteria is first started, the pH is lowered by the produced lactic acid, and when the pH approaches the optimum pH for yeast, yeast proliferates and alcohol fermentation occurs, and then The flavor was further adjusted through the action of aging yeast and other ingredients.

However, now that heated brewing and low-salt brewing have become popular,
Although the brewing period has been shortened and efficiency has improved, it has also become more sensitive to bacterial contamination, and changes in bacterial growth require delicate adjustments to maintain an appropriate balance. Ta.

In terms of the relationship between yeast and lactic acid bacteria, it is preferable for lactic acid bacteria to proliferate in parallel to yeast, albeit at a slightly earlier stage. These are the methods in which yeast is used first.9 Yeast inhibits the growth of lactic acid bacteria, resulting in insufficient lactic acid fermentation, resulting in soy sauce with quality problems. In other words, yeast that performs alcoholic fermentation is necessary for soy sauce brewing, but if the timing of propagation and alcoholic fermentation is too early, it will inhibit the growth of necessary lactic acid bacteria.
This will reduce the quality of soy sauce.

(2) Also, among the yeast groups, there are not only those that act during the first half of preparation as mentioned above, but also those that act on salt-tolerant film-forming yeast (hereinafter referred to as film-forming yeast), commonly known as white mold, during the second half of the preparation. If contaminated, the quality of the soy sauce may deteriorate and the flavor of the soy sauce product may deteriorate. Film-producing yeast is present in the soy sauce even at the early stage of preparation, but it does not appear on the surface of the soy sauce. It starts to become noticeable from the ripening stage.

This bacterium has a high tolerance to alcohol and grows in raw and heated soy sauce, etc. - When it grows in the form of a white mold, it produces an unpleasant odor or gas, which significantly impairs the quality of soy sauce products. I think it would cause a significant loss of value.

[Purpose of the invention]

The present invention uses killer factors at appropriate times during soy sauce production.

By adding killer factor-containing substances or killer factor-producing bacteria, their cultures, or their processed products, the number of yeast in various methods at the initial stage of preparation,
Reducing the number of yeast in various aging methods (particularly the number of salt-tolerant film-producing yeast in this case), reducing the total number of yeast in raw soy sauce, finished soy sauce that has been fired and released, or reducing only a specific group of yeast. By reducing this, lactic acid fermentation and alcohol fermentation can be carried out in a well-balanced and appropriate manner, and unpleasant flavors can be prevented.

The term "killer factor" as used herein means an anti-yeast substance produced by yeast.

[Description of the invention]

In the production of soy sauce, the present invention involves adding a killer factor, a killer factor-containing substance, and/or a killer factor-producing bacterium, a culture thereof, or a processed product thereof to soy sauce koji, soy sauce moromi, freshly fried soy sauce, finished soy sauce, etc. This is a soy sauce manufacturing method characterized by controlling the yeast present in the process.

The present invention is characterized in that a killer factor is applied at an appropriate time during soy sauce production, and the killer factor may be any killer factor, a killer factor-containing substance, a killer factor-producing bacterium, a culture thereof, or a processed product thereof. good.

The killer factor-producing bacteria may belong to any genus, and may be new or known. Examples include the following bacteria:

Hansenula anom
ala)Kh-I FERM P-8159, Hansenula anomal
a) Kh-1 [FEBM P-8160, Hansenula murakii
IFO0895, No1゜Cizenula anomala (Hans
enula anomala) NCYC522, Saccharomyces cerevisiae (Saccharomyces
escerevisiae) IFO1661, Kluyveromyces lactis
s Iactis) IFO The killer spectra of these killer factor-producing bacteria are shown in Table 1 below.

The killer factors produced by each of the killer factor-producing bacteria shown in Table 1 have slightly different properties, and the conditions for expression of activity are also slightly different.

Hansenula anomala Kh-I F'ERM P-
8159, Hansenula anomala Kh-I FERM
P-8160 is a salt-tolerant yeast isolated from soy sauce moromi and can grow at concentrations of 0 to 25 g/100 d of salt. The killer factor produced by these two yeasts is 0 to 2 salt!
It is produced at l/100mA', but the salt is og71oom.
l shows no killer effect.

Furthermore, no killer factor is produced at a culture temperature of 28° C. or higher.

Also, Hansenura Muraki IFO0895,? Tucharomyces cerevisiae IFO 1661 and Culiveromyces lacteis IFO 1267 cannot grow or are very weak in salt concentrations of 5 g/100 ml or more. The killer factor was produced under conditions that allowed bacteria to grow, and the produced killer factor showed a killer effect up to 0 to 25 g/10[1-] of sodium chloride.

Furthermore, Hansenula anomala NCYC522 is an osmotic resistant yeast that grows well in a 10 g/100 d sucrose-containing medium or a 10 g/10 DrL1 salt medium. The killer factor was always produced under conditions that allowed the bacterium to grow. The killer factor produced is 0-25 g of salt.
/ IQ It showed a killer effect in Oml.

Next, the kiss in the presence of 15% salt was
Shown in the table.

Table 2 Note that the killer factors produced by the six strains shown in Table 2 were tested by Aspergillus ori7- (Aspergillus ori7-).
yzae) + AsR Lukil Su Sawyer (Asperg
It was degraded, albeit slowly, by contacting it with proteases produced by 100 Ius 5 oyac.

Furthermore, the killer factor had no effect on lactic acid bacteria.

In the present invention, in order to apply the killer factor during soy sauce production, the killer factor-producing bacteria can be cultured in YEPD medium (glucose 2 g/IDO ml, polypropylene 2 g/lDO
ml, yeast extract 1! ! /10o1nl, Na C13
0 to 8g/100ml), and this culture may be added as is to soy sauce koji, soy sauce moromi, etc., or this culture may be concentrated using an ultrafiltration membrane, an evaporator, chromatography, etc. It is also possible to prepare f# from this by chromatography, isoelectric focusing, etc., and then add this S product.

When the present invention is applied to soy sauce moromi or koji raw material digested material at the early stage of preparation, the addition of a purified product of the killer factor, a culture containing the killer factor, or a concentrate of the culture, reed mushrooms, killer factor-producing bacteria, etc. Either inoculation or addition of the culture or a processed product thereof may be used.

Generally, the produced koji is mixed with 20-25g/100mg of salt water and digested with the enzymes of the koji mold, but it is also possible to add a killer factor at the time of preparation, or to produce a salt-tolerant killer factor. Even if you inoculate yourself with bacteria: No.

The timing of addition and inoculation can be selected at the appropriate time;
Especially Zygoss
It is better to do this before the activity of Haccharomyces rouxii becomes prominent. After addition or inoculation, various management methods can be carried out as usual. The killer factor produced after addition or exposure to fm reduced the total number of yeast and reduced the growth inhibition of lactic acid bacteria by yeast, thereby improving the growth of lactic acid captives and inducing good lactic acid fermentation.

In addition, the killer factor that has completed its role is gradually degraded by the enzymes of Aspergillus oryzae, and eventually its effect is almost eliminated. At this point, it became possible to bring about very good alcoholic fermentation by adding the excellent Chigosatsuromyces luxi, which had been cultured separately. However, Hansenula anomala Kh -I FEJtM
P-8159, Hansenula anomala Kh -IF
When EJ (MP-8160 is inoculated into various methods, the temperature of each method is reduced to 2 when the total number of yeast to be reduced is reduced.
It is necessary to raise the temperature to 8°C or higher to stop the production of killer factors, and then inoculate the desired alcohol-fermenting yeast to carry out alcohol fermentation.

Thereafter, by going through a maturing process as usual, it is possible to obtain soy sauce with very good flavor and flavor.

After the main fermentation period ends, the ripening period begins, but during the ripening period, the intervals between operations such as aerating and stirring the various methods become longer and the various methods are less likely to move.

For this reason, there is little sap on the surface, and there is still a layer with little alcohol due to the sap coming from the surface. Here, the salt-tolerant film-forming yeast that survived in the various methods or lived in the warehouse grows as white mold on the surface of the various methods, deteriorating the quality of the various methods and, in turn, deteriorating the quality of the product. Sometimes I let it happen.

Even during this ripening period, the present invention can be carried out by adding a purified product of the killer factor, a culture containing the killer factor, or its concentrate, or inoculating the killer factor-producing bacteria, its culture, etc. Applicable. It is also possible to inoculate a killer factor-producing bacterium, but in this case, it is preferable to add a concentrate of a culture containing the killer factor or a purified product of the killer factor.

The addition may be made at any time from the end of the main fermentation to before the pressing, but preferably immediately after the end of the main fermentation and immediately after the fluidization due to the fermentation heat in the various methods has ended. Addition is 1D6/g moromi to 10'/! of killer factor crude. ! It is only necessary to add about 1 to 1 ttg/+nl or more based on the level of susceptible bacteria in moromi.

The addition of the killer factor is due to the fermentation of Candida etch, which is involved in the maturation of soy sauce.
ells), Camp Ida Parsadis (Can
dicla versatilis) etc., and because the competing film-producing yeasts are excluded, ripening is performed very well. As a result, by applying the method of the present invention, it is possible to obtain soy sauce that is free from off-taste and off-odor and is well aged.

In addition, when applied to raw soy sauce, it is the same as in the case of various methods, and a purified product of the killer factor, a culture containing the killer factor, or its concentrate is added, or a killer factor-producing bacterium, its culture, etc. is inoculated. Any method can be applied.

Ripening methods include pressing, filtering, leaving the sap, and removing the oil that floats on top.Nama-age soy sauce is usually stored at low temperatures, but salt-tolerant film-producing yeast live everywhere in soy sauce factories. In addition, there are many cases in which raw soy sauce is contaminated during various processing steps, and film yeast often occurs in raw soy sauce that is stored at low temperatures.

Such raw fried soy sauce is inoculated with killer factor-producing bacteria, and a culture containing the killer factor may be added, but it is preferable to add a concentrate of the culture or a purified product of the killer factor. The addition time may be at the stage of various methods before pressing, or at any stage after pressing. Preferably, immediately after pressing.

The amount added is 10°/ml of killer factor crude product []
It is sufficient to add ~1 μJ (approximately 7 ml or more) to the level of susceptible bacteria of 7/d.

By adding a killer factor in this way, no contamination by film-producing yeast is observed even without storing at low temperatures. Therefore, according to the present invention, it is possible to significantly prevent the production of soy sauce that has quality problems due to contamination with film-producing yeast.

Furthermore, any method of injecting a purified product of the killer factor, a culture containing the killer factor, a condensate thereof, or a killer factor-producing microorganism can be applied to the fire soy sauce, but it is preferable to use a purified product of the killer factor. Or concentrates of cultures containing killer factors are good.

Fired soy sauce (there are various products with different salt content, especially low-salt products are susceptible to being contaminated with film yeast, just like raw soy sauce. Soy sauce contaminated with film yeast is not a product) This significantly reduces the value.

When soy sauce is heated, it is kept at a temperature of 50 to 120 degrees Celsius for a certain period of time, then cooled or left to cool naturally, and then it is pulled down and becomes a product. It may also be kept in storage tanks for a limited time. In addition, it is necessary to pass through transport pipes, valves, etc. from the firing tank to the time it is packed into storage tanks or containers, and there are concerns about contamination within these pipes and valves.

In order to prevent this, a process called co-washing with a large amount of soy sauce is carried out, but this sometimes results in a poor product yield. In the present invention, in order to prevent this, the purpose is achieved by adding a purified product of killer factor or a concentrate of killer factor at an appropriate time after firing, preferably immediately after cooling.

In addition, the amount of killer factor added is 106 to 107/
The purpose can be sufficiently achieved by adding about 1μ97ml or more to 1nl.

According to the present invention, the occurrence of film-producing yeast in products is completely eliminated, and the stability of product quality is significantly improved.

Next, experimental examples and examples of the present invention will be shown.

Experimental example 1゜ Salt-containing YEPD medium (glucose 2 g 7100ml
, polypeptone 2j; l/1D Dd, yeast extract 1
g/IOC3ml, salt 89/ID 0ml) 20
0m! ! was placed in a 500 m Erlenmeyer flask, sterilized in an autoclave, and then strawberry-length Tucharomyces rukisi was added.
The cells were inoculated at Q'/ml and statically cultured at 20°C for 4 days. The number of viable bacteria on the fourth day was measured using the dilution plate culture method and was 107/rnl! Met.

Separately, YEPD medium (containing 0.5% salt, the same applies hereinafter) was added to Hansenula anomala (H), which is a killer factor-producing bacterium.
ansenula anomala) 'Kh-I
FPRM P-8159, Hansenula anomala (Ha
nsenula anomala ) Kh-X F
ER1'J P-8160, Hansenula muraki (1
-Jansenulamrakii) IFO089
5. Hansenula anomala
omala') NCYC522, Saccharomyces cerev
isiae) IFO1661, Krivellomyces
Kluyveromyces Iacti
s) IFO1267 was cultured individually, and 20 ml of the culture solution was directly added to the culture solution of Stigosatucharomyces rukisi which had been cultured on the 4th day. Changes in the number of viable bacteria of Romyces ruxi before and after addition of Hansenula anomala Kh-IFE M P-8
An example was shown in which 159 cultures were added. The results are shown in FIG. The control is a section in which no killer yeast culture was added.

Table 6 below shows the amount of Chigosactucharomyces before addition.
The number of viable bacteria on the 100th day after addition of the culture of Ruki-○ viable bacteria and other killer factor-producing yeast is shown.

Table 6 As is clear from Figure M1, Hansenula anomala Kh
-I By adding the culture of JERM P-8159, the number of dentition of S. rouxi was significantly reduced. Furthermore, as shown in Table 3, even when cultures of other killer yeasts were added, the growth of various yeasts was similarly inhibited.

fsn) These results show that the killer factor effectively acts on suppressing the alcohol-fermenting yeast of soy sauce brewing, Stigosatucharomyces ruxi.

Experimental Example 2: Salt water was added to the koji made using the conventional method, and the final various method sap salt content was 10g/100M, 15g/100M, 20. ! i
'/1oomg. 1 of each juice that was squeezed two weeks after preparation and further filtered and sterilized using a membrane filter with a pore size of 0.45 μm.
to DOm (salt 98~19-711/10'0rttl
, blood sugar 6-12 g/100rnl.

Tegosaccharomyces ruxii (total nitrogen 0.5 to 2.097100 m1) and Tegosaccharomyces ruxii variant halomembranis (Zygosaccc
haromyces rouxHvar, halom
embranis) were inoculated individually or in combination, and statically cultured at 30°C for 7 days. At this time, the number of viable bacteria determined by the dilution plate culture method was 106 to 10'/m (in the case of mixed culture, Stigosatucharomyces rouxi and Stigosatucharomyces rouxi variant Halomengrani can be counted separately based on the differences in colony morphology).

Separately, Hansenula anomala Kh-I FERM' P-8159, which is a killer factor-producing bacterium in YEPD medium,
Hansenula anomala Kh-In FERM P-8
160゜Hansenula muraki IF00B95, Hansenula anomala NCYC522, Satucharomyces cerevisiae IF0 166L After statically culturing Clyveromyces lactis IFO1267 at 25°C for 7 days, the culture solution was centrifuged to remove bacterial cells, and the pore size was reduced to 0. Sterile culture solutions were prepared by passing through a 45 μm membrane filter (6 types in total). 1 []mA' of this culture solution was added to the above-mentioned culture of Stygosaccharomyces ruxi and Stygosaccharomyces ruxi Variant Halomembranis, either alone or in combination. Table 84 shows the number of live cocoons before addition and the number of live cocoons 10 days after addition.

As shown in Table 4, Chigosatsucharomyces lukis, Chigosatsucharomyces lukin-variant.
By adding a culture solution containing a killer factor to each individual or mixed inoculation section, the growth of Chigosatsucharomyces rukii and Chigosatsucharomyces rukii variant Halomenplanis was significantly suppressed, and after the addition of Higuchi's has almost completely died out.

Experimental example 3゜Fresh fried soy sauce (salt 17.1 g/l 00ml, total nitrogen 1
．． 75&/100 punishment, direct reducing sugar 5.0g 7100ml
, alcohol 2. ”+ml/ID O+aA')
was placed in a 500 mA Erlenmeyer flask, and separately inoculated with pre-cultured Shitachigosatsuromyces lucyvariant Halomembranis at a concentration of 10'/ml, and statically cultured at 60°C for 7 days. Seven adjustments were made. At this time, the number of viable bacteria by dilution plate culture was 10.
'/m#. (Separately, YE
In PD medium, killer factor-producing bacteria Hysenula anomala Kh-I FERM P-8159, Hansenula
Anomala Kh-1 [FERM P-8160, Hansenula murakicho FO0895, Krivellomyces laftii"01267 were cultured at 20°C for 10 days] 200 volumes of the culture solution was centrifuged to remove bacterial cells, and further After filtering with Celite, the culture solution was made sterile by passing through a membrane filter with a pore size of 0.45 μm, and then further filtered using Amicon Holofiber HI-P.
After concentrating 10 times by treating with 10
150 m of a culture solution obtained by inoculating and culturing M in the above-mentioned raw soy sauce with Chigosatsu Ryomyces Variant Halomenplanis.
Added to 1.

The control is a section in which no concentrate of culture containing Killa-factor was added.

Chigosatsuromyces lukin after 10 days of addition
Variant: Table 5 shows the degree of romenbu membrane (formation of fungal cap by yeast).

Table 5 As is clear from Table 5, no film formation was observed in the group to which the concentrate of the culture containing the killer factor was added.

Experimental Example 4 Soy sauce after cooking (salt 17.09/10 CNn1.Total nitrogen 1.56.!i+/100ml, direct reducing sugar 4.0
P/100mf alcohol 2.097100m/ ) was put into a 50 QmJ Erlenmeyer flask, and here,
Separately precultured Stigosatucharomyces rouxi variant halomembranis was inoculated at a concentration of 10'/ml and statically cultured at 30°C for 7 days. At this time, the number of viable bacteria determined by the dilution plate culture method was 10'/d.

Separately, the killer factor producing bacterium Hansenula anomala Kh-I IR,MP-8159゜Hansenula anomala Kh-II FFiRM P,-
8160, Hansenula muraki IFO0895, and Krivellomyces laftii IFO1267 at 20°C for 10
200 TL of the culture solution cultured for 1 day was centrifuged to remove the blanks, treated with Celite, passed through a membrane filter with a pore size of 0.45 μm to make the culture solution sterile, and then filtered with Amicon Holo. After concentrating fiber 1 10 times (by treating with I-Plo), the IDd was inoculated with Stigosatuuromyces luxiiba IJ 7 nt. and added to the culture.

The control is a section in which no culture concentrate containing the killer factor was added.

Table 6 shows the degree of film formation 10 days after addition.

Table 6 As is clear from Table 6, no film formation was observed in the adult soy sauce group in which the concentrate of the culture containing the killer factor was added.

Example 1 25.
! i'/IDDml! Three bottles were prepared by adding 1401 brine and preparing in a conventional manner to make soy sauce and moromi.

Separately, Hansenula anomala Kh-I in YEPD medium
FERM P-8159, Hansenula anomala Kh-
After culturing I FERM P-8160 with aeration for 4 days,
The bacterial cells were collected by centrifugation.

These microbial cells were separately inoculated one week after the preparation of the soy sauce moromi at a concentration of 101/g moromi.

Changes over time in the total number of yeast (excluding killer yeast) in various methods and added Hansenula anomala Kh -IpERMP-
8159 and Hansenula anomala Kh-I FERM
The changes over time of P-8160 were separately counted and the second
It is shown in Fig. 3. The control is a section in which no bacteria were added.

As is clear from Figures 2 and 6, on the 20th day in the killer yeast-added plots, the number of killer yeasts was around 106/y, whereas the total number of yeasts excluding killer yeast was 1a' per y.
After reaching 7g moromi, it decreased rapidly and on the 40th day it was 102/
g It was less than moromi.

Hansenula Anomala Kh-I FERM p”81
59 and Ha:yce5Z 5 ・7 / Mala Kh-It
FERM'P-81 (S.

Thereafter, it increased slightly and reached equilibrium.

At this point, the process temperature was raised to 28-60°C to stop the production of killer factor. In addition, the remaining killer factor was decomposed by Aspergillus protease and was almost inactivated 5 to 7 days after the temperature was raised.

Lactic acid fermentation was carried out very well because the yeast that was present at the beginning of fermentation was killed and reduced by the action of killer factors.

After inactivation of the killer factor, the main fermentation yeast of soy sauce, Chigosatsucharomyces luxi, which was cultured separately using conventional methods, was cultured 5X.
10"/!il of moromi moromi was added. As a result, good alcoholic fermentation was carried out.

After that, the ripening process begins with the ripening yeast Candida parsatiris, which is insensitive to killer factors.
The soy sauce of Candida etchel (A versatilis) was also successfully processed, and a soy sauce with very good flavor and flavor was obtained.

In the control methods in which killer yeast was not added, yeast multiplied from the early stage of preparation and alcoholic fermentation was active, but after that, fermentation and ripening went smoothly.

Table 7 shows the analytical values and sensory test values for the finished soy sauces, those to which FEBM P-8159 was added and those for the control. The sensory test was expressed as the total ranking of 20 trained professional testers. FEBM P-81 in sensory evaluation
The product to which 59 was added had a better taste than the control (-1), roundness, and aroma, and was well received.

Example 2 Soy sauce koji SSO# obtained by a conventional method using defatted soybeans, wheat, and Aspergillus tsuya was added with 25 g/1ooy salt water 1
．． Add 2 kl and season with soy sauce and moromi.

Separately, Hansenula anomala Kh-I in YEPD medium
FERM P-8159, Hansenula anomala Kh
-I FERM P-8160, Hansenula muraki IFO0895, Hansenula anomala NCYC522
.

2 each of Satucharomyces cerevisiae IFO1661 and Culiveromyces laftii IF'0 1267
After culturing with aeration at 5°C for 5 days, the culture was centrifuged to remove the bacterial cells, filtered through Celite again, and treated with Amicon Holofiber HI-P10 to remove the culture. It was concentrated 100 times.

200ml of this concentrate! was added to the mouth for one week after preparing the soy sauce moromi. The total number of yeast immediately before addition and the total number of yeast 10 days after addition are shown in Table 8.

Table 8 Table 8 shows that by carrying out the method of the present invention, the number of yeast present at the initial stage of preparation could be significantly reduced.

As a result, lactic acid fermentation was performed very well without inhibition of the growth of lactic acid bacteria by yeast.

(M) After waiting for the killer factor to be degraded by Aspergillus protease, the cells were cultured separately in a conventional manner. Chisosatucharomyces luxi, which is the main fermenting yeast for soy sauce, was added at 5 x 10'/g moromi. As a result, good alcoholic fermentation was performed.

Thereafter, the aging process was carried out smoothly, and soy sauce with very good flavor and flavor was obtained.

Example 6゜ Fresh fried soy sauce (salt 16.9 g/100g, total nitrogen 1.8
1 i/100mJ, direct reducing sugar 4.8g/100g,
Pour 1 Kl each of alcohol (2.1d/100rILl) into two IKA capacity enamel tanks with open tops.
It was stored at room temperature for several days. At this time, the number of viable bacteria of the salt-tolerant film-producing yeast Chigosacuromyces luxi-paraant Halomembranis was measured using the dilution plate culture method.2
In both the book tanks, the number was 10s/WLl.

Separately, Hansenula anomala Kh-I FERM P
-8159 was sterilized in an autoclave (120°C, 1
5 minutes) Slant inoculate 200 m of YEPD medium, and
This culture was cultured with shaking at 0°C for 4 days, and then this culture was placed in a 201 volume jar fermentor containing similarly sterilized YEPD medium 101, aerated and stirred, and cultured at 20°C for 5 days. This culture was centrifuged to remove bacterial cells, further filtered through Celite, and concentrated to 5Qml by ultrafiltration using Amicon Fluorofiber I (I-Plo). A partially purified product was obtained by treatment with Graphie and elution.This partially purified product was freeze-dried to obtain partially purified powder 10DQi'.

Add 51n of this partially refined powder to the above-mentioned raw soy sauce I Kl.
g and stirred to mix.

Table 9 shows the measured values and sensory evaluation of the number of viable salt-tolerant film-producing yeast in the freshly fried soy sauce 10 days after addition.

The control is a section in which the 8-minute purified product containing the killer factor was not added. The sensory test was expressed as the sum of the rankings of 18 experienced sensory testers.

Table 9 As shown in Table 9, yeast could not be detected in the section to which the partially purified product containing the killer factor was added.

On the other hand, it was pointed out that in the control group where no addition was made, the number of viable bacteria increased and the quality of the rice deteriorated.

Example 4 Product soy sauce (salt content IZO, !li'/100 fresh, total nitrogen 1
55,! i'/1 oml, direct reducing sugar 3.8,9
/100 Go, A#: 7-R25m1! / 100m)
I prepared two bottles of ID Kl placed in an enamel tank covered with a mesh net at the top. The number of live yeast cocoons in the soy sauce product stored in these two enamel tanks after 20 days was measured by dilution plate culture method and found to be 10' cocoons/ml. When the contaminated yeast was observed using a scanning electron microscope, it was estimated from its morphological characteristics that it was S. lucys.

Separately, Hyzenula anomala Kh4 FERM 'P-
8160 was cultured with shaking in YEPD medium 20 Dd at 60° C. for 4 days, and this culture was further cultured with aeration at 20° C. for 5 days in a 201-volume jar fermentor containing YEPD medium 101. This culture was centrifuged to remove bacterial cells, and the resulting supernatant was further filtered through Celite, followed by ultrafiltration using Holofiber HI-P10 manufactured by Amicon, and concentrated to 50 ml.

This concentrate was treated with Sephadex G25 and further 0M
A partially purified product was obtained by treatment with Sephadex C25 chromatography and further elution with a sodium chloride gradient method. 3 This partially purified product was freeze-dried to obtain 10+ng of partially purified powder. This part is 40m of f# powder in 10Kl of the above-mentioned soy sauce? Add and stir to mix.

Table 10 shows the measured values and sensory evaluation of the number of viable yeast cells in the product soy sauce 10 days after addition.

The control was a section in which the partially purified product containing the killer factor was not added. The sensory test was determined by the total ranking of 18 experienced sensory testers.

Table 10 As shown in Table 10, yeast could not be detected in the section to which the partially purified product containing the killer factor was added (Mo9).

On the other hand, it was pointed out that the number of viable bacteria increased and the quality deteriorated in the control category where no addition was made.

[Brief explanation of the drawing]

Figure 1 shows F”ERM 13-815 in Experimental Example 1.
9 is a diagram showing the change in the number of viable bacteria of Chigosatsuromyces ruxi after adding the culture of No. 9. FIG. 2 is a diagram showing changes in the total number of yeast (excluding killer yeast) in various methods after inoculating FEBM P-8159 cells in Example 1. FIG. 3 shows FEBM P-81 in Example 1.
60 is a diagram showing changes in the total number of yeast (excluding killer yeast) in various methods after inoculating 60 bacterial cells. C control, A total yeast count (excluding killer acid J), B-f
Gosatsucharomyces rukisi Viable Bacteria Count Agent Patent Attorney 1) Oya Danmai (F/ken) "Koshizuka (Utsusho 1/Shuku) ■Meitechnological Procedures Amendment Document July 11, 1985

Claims (1)

[Claims] In soy sauce production, killer factors, killer factor-containing substances, and/or killer factor-producing bacteria, their cultures, or their processed products are added to soy sauce koji, soy sauce moromi, raw fried soy sauce, finished soy sauce, etc., and are present in the soy sauce manufacturing process. A soy sauce production method characterized by controlling yeast.