JPH0544267B2 - - Google Patents

Description

[Detailed description of the invention]

<Industrial Application Field> The present invention relates to a method for culturing yeast, and in particular to a method for efficiently and continuously obtaining yeast with a large number of viable cells suitable for producing brewed foods such as soy sauce and miso. <Prior Art> Conventionally, in the brewing of soy sauce, miso, etc., a method has generally been practiced in which yeast that has been separately artificially isolated and cultured is added during the brewing process. There are generally three methods for culturing yeast: a batch culture method, a fed-batch culture method, and a continuous culture method. Yeast used in brewing soy sauce, miso, etc. is cultured by the batch culture method. In addition, research on yeast culture conditions includes
Those that administer a carbon source (for example, JP-A-49-
12087, JP-A No. 49-101584, etc.), those that adjust the residual alcohol concentration (for example, JP-A No.
50-121483, etc.), those that adjust pH (e.g., JP-A-51-35472, etc.), and those that maintain dissolved oxygen concentration (e.g., JP-A-57-58881, etc.). . <Problems to be solved by the invention> As mentioned above, in the brewing of soy sauce, miso, etc.
The batch culture method, in which all nutritional sources are added from the beginning, is simple and often used, but this method has the drawbacks of low yield and poor productivity. There is also a fed-batch culture method in which nutrients such as sugar are added little by little to the yeast culture solution over the course of culture time. It is unique in that it can be adjusted, but in order to obtain yeast with a large number of viable bacteria in a high yield and efficiently, it is necessary to always control the concentration of nutrients added to the medium within a certain range. The problem is how to easily and reliably perform this control. In any case, in the conventional cultivation of yeast used in the brewing of soy sauce, miso, etc., in both the batch culture method and the fed-batch culture method, the culture is terminated at the final stage of yeast growth, and the subsequent culture is It is not a method for culturing by controlling the liquid. On the other hand, in the cultivation of baker's yeast, etc., a continuous culture method is used in which yeast is cultured in a basal culture medium and a fed-batch medium is added during the cultivation, but the carbon source concentration of the culture medium after the end of yeast growth is This method does not focus on culturing yeast by controlling the culture solution. The present invention has been made to solve the above-mentioned problems, and its purpose is to easily and reliably control the concentration of nutrients in the culture solution always within the optimum range when culturing yeast. To provide a yeast culture method for efficiently and continuously obtaining yeast having a large number of viable bacteria suitable for producing brewed foods such as soy sauce and miso. <Means for solving the problem> When culturing yeast, the present inventors set the carbon source concentration in the culture solution to 0.05% after the final stage of growth.
(W/V) The present invention was completed based on the knowledge that the above-mentioned problems could be solved by adding a liquid medium and culturing. That is, when culturing yeast, the present invention reduces the carbon source concentration in the culture solution to 0.05% after the final stage of growth.
(W/V) This is a continuous yeast culture method characterized by adding a liquid medium and culturing so as to achieve the following. The present invention will be explained in detail below. First, yeasts used in the present invention include yeasts of the genus Zygosaccharomyces, saccharomyces, Candida, Pichia, Hansenula, etc. Without limitation, Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) ATCC 13356, belonging to Zygosaccharomyces rouxii, Candida etskelsii and Candida versatilus;
ATCC14679, IAM4114, Candida etchellsii IFO1229, Candida versatilis
Yeasts commonly used for brewing soy sauce, miso, etc., such as IFO10038 and IFO10056, are preferably used to prevent bacterial contamination due to their salt tolerance. As the yeast culture medium, a synthetic medium or a natural medium containing a carbon source, a nitrogen source, inorganic salts, and other necessary components for the growth of yeast that can be used by yeast are appropriately blended. Note that when it is intended to be added to moromi with a high salt concentration such as soy sauce, miso, etc., it is desirable to adjust the salt concentration of the medium to about 5 to 18% (W/V). Next, for culturing the yeast, appropriate methods such as aerobic or anaerobic culture such as shaking culture, aeration culture, stirring culture, static culture, etc. are employed, and aerobic culture is particularly preferred. For example, when performing aerated culture,
The aeration rate is 2 to 20/min per 10 culture fluids.
It is desirable to have some ventilation. For other culture conditions such as culture temperature and pH of the medium, the conditions normally used for yeast culture can be applied, and if necessary,
It is also possible to expand the culture as appropriate, such as seed culture or main culture. When yeast is cultured in this manner, it passes through a lag phase at the initial stage of culture and then enters a growth phase, where the number of bacterial cells increases significantly. Normally, proliferation stops approximately 18 to 24 hours after the start of culture, and after the final stage of proliferation, the cell enters a stationary phase. In the batch culture method, the culture ends at approximately this stage, but in the present invention, the carbon source concentration in the culture solution after this final stage of growth is reduced to 0.05.
% (W/V) or less, preferably 0.002 to 0.02%
(W/V) Add the liquid medium so that it becomes (W/V). The liquid medium for this addition, like the above-mentioned first yeast culture medium (hereinafter referred to as the basic medium), contains carbon sources, nitrogen sources, inorganic salts, and other components necessary for yeast growth as appropriate. Compounded synthetic media and natural media are used. The medium for this addition may be a salt-free medium or a solid medium, but in order to prevent bacterial contamination and ease of addition, it is a liquid containing 5% or more, preferably 10% or more of salt. A medium is preferred. Further, the medium for addition may have the same composition as the basal medium or may have a different composition. When adding the liquid medium for this purpose, it is added continuously or intermittently using means such as a pump, but the key is to control the carbon source concentration in the culture medium after the end of growth. is 0.05% (W/
V) Add in the following amounts. The carbon source concentration in this culture solution was set to 0.05% (W/V).
The reason why the amount is set below is that if it exceeds 0.05% (W/V), the amount of yeast cells per unit decreases. On the other hand, the carbon source concentration in the culture solution was 0.002% (W/
V) Below, the ability to produce alcohol becomes weak. Therefore, the carbon source concentration in the culture solution is 0.05% or less,
The medium for addition is added preferably in a range of 0.002 to 0.02% (W/V). Then, by continuously or intermittently adding an additional medium to the culture solution after the end of the growth stage, and culturing by continuously or intermittently taking out the culture solution in an amount corresponding to the added amount, viable bacteria can be grown. A large number of yeast can be obtained efficiently. The present invention will be explained below with reference to experimental examples. Experimental example 1 Comparison of batch culture method and continuous culture method Raw soy sauce: 15/(V/V), glucose: 7%
(W/V), yeast extract: 0.01% (W/V),
KH ₂ PO ₄ : 0.05% (W/V), MgSO ₄ 7H ₂ O:
0.025% (W/V), CaCl ₂ 2H ₂ O: 0.005%
(W/V), salt 11% (final, W/V) A liquid medium (PH5.5) containing NaCl was used as the basal medium, and this was heat sterilized at normal pressure using an autoclave.・Inoculate the culture solution of Zygosaccharomyces rouxii ATCC13356 at 10 ⁷ cells/ml, and stir for several times.
Culture was carried out at 500 rpm and 30°C. 20 hours after the start of this liquid culture (end of growth stage)
From then on, use the same composition as the above basal medium (however, PH5.0)
This medium was used as an addition medium, and this was heat sterilized in an autoclave at normal pressure, and the dilution rate was 0.03V/
Continuous culture is carried out by continuously supplying the culture solution from the supply port of the jar fermenter at a rate of V・hr., and continuously collecting the same amount of culture solution as the supplied amount from the jar fermentor outlet. I did it. The continuous culture described above was carried out at a culture temperature of 30° C., a medium pH of 5.0, and a stirring speed of 500 rpm. Then, the carbon source (glucose) concentration of the culture solution after 20 hours (end of growth phase) after the start of the first liquid culture was adjusted to 0.004 to 0.007%.
(W/V) and cultured. Table 1 shows the results of measuring the number of viable yeast cells using the culture solution collected above as a test sample. In addition, from 20 hours after the start of the above liquid culture, the temperature was 30℃ without adding the supplementary medium.
A control sample was obtained by batch culturing at an agitation rate of 500 rpm from the time of the initial culture start until 48 hours, and the same information was given.

[Table] As shown in Table 1, from 20 hours after the start of culture (end of growth), supplementation medium was continuously added to maintain the carbon source (sugar) concentration at 0.004 to 0.007% (W/V). The test sample of the present invention, which was continuously cultured using the same method, showed a viable cell count approximately three times higher than the control sample (batch culture) 48 hours after the start of culture, and maintained a high level during subsequent culture. , after 144 hours of culture, approximately 1 billion cells/
It showed a high number of viable bacteria per ml. Experimental Example 2 Influence of carbon source concentration in the culture solution after the end of growth In the continuous culture method described in Experimental Example 1, except for culturing at a dilution rate of 0.02 to 0.08 V/V hr. and culturing to an almost steady state. The test was carried out in the same manner as the continuous culture method described in Experimental Example 1, and the continuously collected culture solution was used as a test sample, and the number of viable bacteria and carbon source (glucose) concentration were measured. The results are shown in Table 2. Ta.

[Table] As shown in Table 2, after 20 hours after the start of culture (end of growth), supplementation medium was added to maintain the carbon source (glucose) concentration at 0.003 to 0.03% (W/V). The dilution rate is 0.02 to 0.08V/V, respectively.
When the glucose concentration was changed to 0.003 to 0.02 (W/V), the number of viable bacteria was 5.0 to ¹⁰⁸ cells/ml or more, but it was 0.02%.
(W/V) and becomes 0.03% (W/V),
The number of viable bacteria showed a tendency to decrease slightly. <Examples> Hereinafter, the present invention will be explained in more detail by showing examples. Example 1 Raw soy sauce: 15% (W/V), glucose: 7%
(W/V), yeast extract: 0.01% (W/V),
KH ₂ PO ₄ : 0.05% (W/V), MgSO ₄ 7H ₂ O:
0.025% (W/V), CaCl ₂ 2H ₂ O: 0.005%
(W/V), salt: 11% (final, W/V) 2 liquid medium (PH5.5) was used as the basal medium, and this was put into a 5-volume jar fermenter, and heated at 120℃ for 15 minutes. Sterilize by autoclaving for minutes.
A culture solution of Zygosaccharomyces rouxii ATCC13356 was inoculated into the medium at a concentration of 10 ⁷ cells/ml, and the temperature was 30°C.
Culture was started at an aeration rate of 1 V/V·m. and a stirring rate of 500 rpm. Then, 18 hours after the start of culture (end of growth), the culture solution was added with a medium [raw soy sauce: 15% (V/V), glucose: 7% (W/V)].
V), salt: A liquid medium (PH5.0) containing 1.1% (final, W/V) sterilized in an autoclave at 120°C for 15 minutes] was passed through a silicone tube at 80°C.
While supplying at a rate of ml/hr. (dilution rate 0.04V/V・hr.), the temperature was 30℃, the pH of the culture solution was 5.0, and the aeration rate was 1V/hr.
Continuous culture was carried out for 50 days at V.m. and stirring number of 500 rpm. During this period, the carbon source (glucose) concentration in the culture solution was maintained at 0.004 to 0.007% (W/V). The number of viable bacteria in the yeast culture solution thus obtained was 9.0×10 ⁸ cells/ml. Example 2 In the continuous culture method described in Example 1, continuous culture was carried out for 10 days in the same manner as in Example 1, except that the glucose in the supplementation medium was changed to 12% (W/V), and the yeast culture solution was I got it. The carbon source (glucose) concentration in the culture solution at this time was 0.032% (W/V). The number of viable bacteria in the obtained culture solution was 6.8×10 ⁸ cells/ml. Example 3 Yeast extract: 1% (W/V), polypeptone:
Liquid medium (PH5.30) containing 2% (W/V), glucose: 5% (W/V), and salt: 10% (W/V)2
This was used as a basal medium, which was placed in a 5-volume jar fermenter and sterilized in an autoclave at 120°C for 15 minutes. Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) was added to the medium.
A culture solution of ATCC13356 was inoculated at 5×10 ⁶ cells/ml, and culture was started at a temperature of 30° C., an aeration rate of 1 V/V·m, and an agitation rate of 500 r.Pm. 24 hours after the start of culture (end of growth), addition medium [yeast extract: 1% (W/V), polypeptone: 2% (W/V), glucose: 4% (W/V)] was added to the culture solution.
V), a liquid medium (PH5.0) containing 10% (W/V) salt, sterilized in an autoclave at 120°C for 15 minutes] was supplied at a rate of 80mlhr. through a silicone tube at a temperature of 30°C. , PH5.0 of culture solution,
Continuous culture was carried out for 40 days at an aeration rate of 1 V/V·m. and a stirring rate of 400 rpm to obtain a yeast culture solution. The carbon source (glucose) concentration of the culture solution during culture is 0.03 to 0.05.
% (W/V). The number of viable bacteria in the yeast culture solution thus obtained was 2.0×10 ⁸ cells/ml, and the number of viable bacteria 24 hours after the start of culture was 4.7×10 ⁷ cells/ml. . Application example The yeast culture solution obtained by the batch culture method of Experimental Example 1 (viable cell count: 2.2 × 10 ⁸ cells/ml) and the yeast culture solution obtained by the continuous culture method of Example 1 (viable Bacterial count: 9.0 x 10 ⁸ cells/ml) was added to regular dark soy sauce moromi after one month to give a concentration of 5 x 10 ⁵ cells/g moromi, and fermented and aged at 30℃ for 2 months. Then, we obtained soy sauce moromi. Table 3 shows the analytical values for the juice obtained by squeezing the obtained moromi using a conventional method.

[Table] As shown in Table 3, although the amount added was 1/4 of that of batch-cultured yeast, the analytical values of moromi soup to which continuous-cultured yeast was added by the method of the present invention were lower than that of batch-cultured yeast. There was no difference. <Effects of the Invention> According to the present invention, yeast with a significantly large number of viable bacteria can be obtained efficiently and continuously, and can be particularly suitably used in the production of brewed foods such as soy sauce and miso. is of great industrial significance.

Claims (1)

[Claims] 1. A method for continuous cultivation of yeast, characterized in that when culturing yeast, a liquid medium is added and cultured so that the concentration of carbon source in the culture solution after the end of growth is 0.05% (W/V) or less. .