JPS6358559B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing yeast, and more specifically to a method in which a culture solution is extracted from a culture tank, the culture solution is sterilized, the yeast is then revived, and the yeast is cultured while being returned to the culture tank. be. In the fermentation industry, bacteria prevention technology is indispensable for pure fermentation of bacteria producing useful substances, and even a small amount of bacterial contamination makes it impossible for the producing bacteria to grow, thereby inhibiting the production of useful substances. You will end up being rejected. For this reason, when culturing is actually carried out, strict sterilization or sterilization treatment is performed on the culture solution, culture equipment, ventilation air, etc. On the other hand, methods for eliminating bacteria in the culture solution (for example, intervening drugs to eliminate the activity of bacteria, making the pH acidic, etc.) have been proposed (for example,
39511, Special Publication No. 47-38988, Special Publication No. 30-4440, Special Publication No. 30-4439). However, all of these methods have the disadvantage that they cause the activity of various bacteria to be lost and at the same time, the activity of yeast to decrease, resulting in a decrease in the productivity of the culture operation and an increase in production costs. The balance between prevention of germs and culture productivity becomes a major issue. Therefore, it is a challenge in the art to create a yeast culture method that is highly productive while appropriately eliminating contaminant bacteria, and the present invention has successfully solved this challenge. That is, as a result of intensive research by the present inventors on a method for culturing yeast that is not affected by bacterial contamination and that achieves high productivity, the present inventors extracted the culture solution, performed sterilization treatment, and further performed yeast rejuvenation treatment. discovered that by culturing yeast while returning it to the culture tank, it was possible to prevent a decrease in yeast activity caused by strong sterilization treatment, and completed the present invention. The present invention will be explained in more detail below. Although FIG. 1 shows a schematic process diagram of the present invention, this is an example of the present invention and does not show the entire process. 1
2 is a culture tank, 2 is a sterilization treatment device, and 3 is a revival treatment tank. The sterilization device No. 2 is a device for sterilizing the culture solution introduced from the culture tank No. 1. As a sterilization treatment method, methods such as chemical treatment, ultraviolet sterilization, radiation sterilization, and pasteurization can be used, but it is generally preferable to perform chemical treatment because it is economically advantageous. Therefore, as a sterilization treatment, chemical treatment will mainly be described below. The sulfur treatment must be conducted under conditions such as the amount of chemicals added and the treatment time that kill or deactivate bacteria but do not kill yeast. This also applies to other sterilization methods. These conditions cannot be defined unambiguously because they differ depending on the type of bacteria, yeast, and drug, but these conditions should be determined by testing the activity of yeast and bacteria by the drug in advance. This will help you find the appropriate conditions. Other sterilization methods can also be tested in advance to determine appropriate conditions. Needless to say, these conditions can be determined by focusing only on miscellaneous bacteria without considering the reduction in yeast activity. For example, when performing acidic treatment as a chemical treatment, the pH is generally 1.5 to 3 and the treatment time is 20 to 30 minutes.
All you need to do is add acid and process for 120 minutes.
When sterilizing with sodium hypochlorite, the effective chlorine concentration is 40 ppm or less and the treatment time is 10 minutes or less. As another example, in the case of a surfactant such as lauryldimethylbenzylammonium chloride, it is effective if the treatment time is 500 ppm or less and the treatment time is 20 hours or less. The types of drugs include acids, bases, hydrogen peroxide, sodium hypochlorite, formalin, caffeine, aldehydes, surfactants, ozone, gases such as chlorine, and antibiotics. Appropriate processing conditions can be determined by prior testing. When a chemical is used, a tank may generally be used as the sterilizer, and any type of sterilizer can be used, regardless of the presence or absence of a stirrer. For other sterilization methods, any known device suitable for each treatment may be used. The culture solution sterilized as described above is introduced into the revival tank 3, which is an area where the yeast that has been deactivated by the sterilization treatment is revived again. If the inactivated yeast is directly returned to the culture tank and cultured, the overall growth of the yeast will decrease and productivity will decrease.
Therefore, this restoration process is an essential process in the present invention. The method conditions for performing the revival treatment cannot be unambiguously defined because they vary depending on the degree of inactivation by the sterilization treatment in 2, the type of yeast, etc., but in general, the conditions are PH4~
8, treatment time is 10 minutes or more, more preferably PH4-6
In order to revive the yeast, a treatment time of 20 to 120 minutes will be more effective if the main carbon source and other nutrient sources are supplied (line 6), or if aeration is performed (line 7) in the case of aerobic culture. It is true. However, appropriate conditions can be determined by testing the degree of resurgence in advance. For example, conditions for revival (PH, temperature, treatment time, etc.) may be selected based on the respiration rate, growth rate, etc. of the yeast. For example, when yeast is acid-treated (pH is about 2 and treatment time is about 20 minutes), a small amount of the main carbon source and other nutrients is added, and while aeration is performed, the pH is raised to 4. If the revival process is performed in steps 6 to 6 with a processing time of about 60 minutes, most of the yeast will be revived. Similar calculations can be made for other chemical treatments and sterilization treatments. As a device for performing the restoration process,
Tanks are generally used, and any type of tank may be used, with or without a stirrer. The yeast revived in this way is returned to culture tank 1.
and cultured. As described above, the present invention is characterized in that it is cultured while performing two treatments, sterilization treatment and revival treatment, and only by performing these treatments can the effects of bacterial contamination be eliminated and high productivity can be achieved. It will be done. As is clear from the principle, the method of the present invention can be applied in any case, regardless of the type of yeast or the type of culture (batch method, semi-batch method, continuous culture method, etc.). Of course, it can be combined with a multi-stage culture tank, and a two-stage type is shown in FIG. 2 as an example. Next, the present invention will be further explained with reference to Examples. Example 1 Using the process shown in Figure 1, Sa
- Continuous culture was performed using ccharomyces cerevisiae (baker's yeast). A 30-volume jar fermenter was used as the culture tank, and the culture was carried out at a temperature of 33°C and a pH of 4.5. As a sterilization treatment, a chemical treatment is performed, using sulfuric acid as the chemical, and the treatment conditions are PH2.0 for about 30 minutes.
Ammonia water is used for the revival treatment, and the treatment conditions are PH.
4.5 for about 30 minutes and provide a small amount of the main carbon source,
Air was also ventilated. The treatment temperature was 30°C in both cases, and 6% molasses was used as the main carbon source, which was supplied to the culture tank and revival tank. The main carbon source supply to the culture tank is 2.0
/h, and the product was removed so that the liquid level in the culture tank remained constant. Comparative Example 1 is an example in which acid treatment is performed as in Example 1 as a sterilization treatment, but no revival treatment is performed, and Comparative Example 2 is an ordinary chemostat-based continuous culture without sterilization treatment or revival treatment. In each case, the other conditions and methods were the same as in Example 1. The experimental results are shown below.

[Table] A microscopic examination was performed at each time point during the course of the culture, and almost no bacteria were observed, and there was no abnormality in the growth of yeast. In Comparative Example 1, there was not much growth of bacteria, but the activity of yeast was also reduced, so there was a tendency to wash out after about 5 hours. Furthermore, in Comparative Example 2, the number of miscellaneous bacteria increased after about 24 hours, and the growth of yeast also decreased significantly. From the above, high productivity can be obtained without being affected by germs by performing two treatments: chemical treatment and revival treatment. The effect of the method of the present invention was demonstrated. Example 2 Using the process shown in Figure 1, as yeast
Continuous culture was performed using Candida utilis IAM4215. The same culture tank, sterilization equipment, and resurrection tank as in Example 1 were used, and the culture temperature was 30°C and the pH was 4.6. The main carbon source is 4.2% molasses, approximately 4.0/h
The product was removed so that the liquid level in the culture tank remained constant. Sodium hypochlorite is used as a sterilization agent, and the effective chlorine concentration is 40 ppm or less and the treatment time is 10 minutes or less. For the revival treatment, a small amount of the above-mentioned main carbon source nutrient solution is added, and the pH is raised to 4.5 while aeration is performed. Processed for about 30 minutes while adjusting the temperature. Comparative Examples 1 and 2 were under the same conditions as Example 1. The results are shown below.

[Table] A microscopic examination was performed at each time of the culture progress, but Example 2
Almost no bacteria were observed, and there was no abnormality in the growth of yeast. In Comparative Example 1, as in Comparative Example 1 in Example 1, there was not much growth of bacteria, but the washout phenomenon was observed from about 5 to 6 hours due to the decrease in yeast activity. Ta. Comparative Example 2 is also similar to Comparative Example 2 in Example 1,
After about 24 hours, the number of miscellaneous bacteria increased rapidly and inhibited yeast growth. From the above, the effects of the present invention were proven.

[Brief explanation of the drawing]

FIG. 1 is a process explanatory diagram when the culture tank of the present invention is a single tank, and FIG. 2 is a process explanatory diagram when a two-stage culture tank is used as a multistage culture tank. 1... Culture tank, 2... Sterilization treatment device, 3... Resurrection tank, 4... Drug addition line, 5... Base addition line, 5'... Liquid feeding line from the culture tank to the culture tank,
6... Main carbon source and nutrient addition line, 7... Ventilation line, 8... Liquid extraction line as a product.

Claims (1)

[Scope of Claims] 1. A method for culturing yeast, in which a culture solution is extracted from a culture tank, the culture solution is sterilized, the yeast is revived, and the yeast is cultured while being returned to the culture tank. 2 Claim 1 that uses chemicals for sterilization treatment
Culture method described in section. 3. The culture method according to claim 1, wherein the yeast is revived at pH 4 to 8 for a treatment time of 10 minutes or more. 4. The culture method according to claim 3, wherein the yeast is revived at pH 4 to 6 for a treatment time of 20 to 120 minutes. 5. The culture method according to claim 1, wherein the revival treatment is carried out while supplying the main carbon source and other nutrient sources. 6. The culture method according to claim 1, wherein the revival treatment is performed while aeration is performed.