JP6393898B2 - Yeast culture method - Google Patents

Description

  The present invention relates to a yeast culture method. More specifically, the present invention relates to a yeast culture method that can promote the growth of yeast and efficiently perform yeast cell production, ethanol production by yeast, and the like.

  Yeast is used for the production of industrial ethanol as well as fermented foods such as beer, wine, shochu, sake, soy sauce, miso and bread. Furthermore, the yeast is used as a food raw material, a pharmaceutical raw material, a cosmetic raw material, etc. as dry yeast or yeast extract. Thus, yeast is an indispensable microorganism for human beings, and yeast cell production and ethanol production by yeast are important technologies that support modern society.

  Under such a background, it is very important to construct a technique for efficiently producing yeast cells or producing ethanol with yeast in a short time. In order to improve the productivity of yeast cells and the productivity of ethanol by yeast, it is effective to mutate or modify the yeast to be used. However, this method requires complicated operations for each yeast to be used. Therefore, there is a drawback in terms of versatility. On the other hand, if the growth of yeast can be promoted by controlling the culture environment, there is no need for complicated operations for each yeast to be used. It can be an effective method with versatility in performing efficiently.

  Conventionally, it has been known that the growth of yeast is affected by the culture method, dissolved oxygen concentration, carbon source concentration, etc. in the culture medium. In commercial yeast culture, yeast growth is promoted by controlling them. Is planned. However, conventional methods for controlling culture conditions are limited in terms of the effect of promoting yeast growth, and further development of technology is desired.

  On the other hand, since microorganisms cannot grow in the absence of sulfur sources, sulfur sources are an essential nutrient source for culturing microorganisms. Sulfate is generally used as a sulfur source in the culture of microorganisms, but the use of sulfur sources other than sulfate may be effective when producing sulfur-containing compounds efficiently in microorganisms. Has also been reported. For example, Patent Document 1 discloses that a bacterium belonging to the family Enterobacteriaceae that has L-cysteine-producing ability and has been modified so that the expression of a gene involved in sulfite reduction is increased is contained in a medium containing thiosulfate. It has been disclosed that the production efficiency of L-cysteine or a related substance thereof is improved by culturing in the above.

  However, except for the case of producing sulfur-containing compounds, the effect of the type of sulfur source used on the culture of microorganisms has not been clarified. Furthermore, in the culture of yeast, sulfuric acid and / or a salt thereof are generally used as a sulfur source. However, the relationship between the type of sulfur source used and the growth of the yeast has not been studied. It is also not clear whether and / or salts thereof can be used as a single sulfur source.

International Publication No. 2012/137689

  An object of the present invention is to provide a yeast culture technique that can promote the growth of yeast and efficiently perform yeast cell production, ethanol production by yeast, and the like.

  As a result of intensive studies to solve the above problems, the present inventors can promote yeast growth by culturing yeast using a medium containing thiosulfuric acid and / or a salt thereof as a sulfur source. It has been found that it is possible to efficiently produce microbial cells and ethanol by yeast. Furthermore, by increasing the proportion of thiosulfuric acid and / or its salt in the sulfur source contained in the medium, particularly by using thiosulfuric acid and / or its salt as a single sulfur source, the effect of promoting the growth of yeast It has been found that the production of yeast cells and the production of ethanol by yeast can be performed more efficiently. The present invention has been completed by further studies based on such knowledge.

That is, this invention provides the culture method and culture medium of the aspect hung up below.
Item 1. A method for culturing yeast, comprising culturing yeast in a medium containing thiosulfuric acid and / or a salt thereof.
Item 2. Item 2. The culture method according to Item 1, wherein the yeast is ethanol-producing yeast.
Item 3. Item 3. The culture method according to Item 2, which is applied to ethanol production.
Item 4. Item 4. The culture method according to any one of Items 1 to 3, wherein thiosulfuric acid and / or a salt thereof is 1% or more by molar ratio based on a total amount of sulfur sources contained in the medium.
Item 5. Item 5. The culture method according to any one of Items 1 to 4, wherein in the medium, thiosulfuric acid and / or a salt thereof is a single sulfur source.
Item 6. A culture medium for yeast culture comprising thiosulfuric acid and / or a salt thereof.
Item 7. Item 7. The medium according to Item 6, which is used for culturing ethanol-producing yeast.
Item 8. Item 8. The medium according to Item 7, which is used for producing ethanol.
Item 9. Item 9. The medium according to any one of Items 6 to 8, comprising thiosulfuric acid and / or a salt thereof in a molar ratio of 1% or more per total amount of sulfur source.
Item 10. Item 10. The medium according to any one of Items 6 to 9, wherein thiosulfuric acid and / or a salt thereof is a single sulfur source.

  According to the present invention, yeast growth can be promoted by culturing yeast using a medium containing thiosulfuric acid and / or a salt thereof as a sulfur source, and yeast cell production and ethanol production by yeast can be efficiently performed. It becomes possible to do. Therefore, according to the present invention, it is possible to efficiently perform inoculation yeast preparation and fermentation process in various fermentation productions using yeast, and further, the production of yeast-derived substances such as yeast extract can be made more efficient. It can contribute to cost reduction in the industrial field using yeast.

  Although a limited interpretation is not desired, the mechanism of action by which the growth of yeast is promoted by using thiosulfuric acid and / or a salt thereof as a sulfur source is presumed as follows. Yeast has a metabolic pathway that converts sulfate in the order of adenosine 5′-phosphosulfate, 3′-phosphoadenosine-5′-phosphosulfate, sulfite, hydrogen sulfide, and homocysteine (see FIG. 1). In this metabolic pathway, the energy required to produce 1 mole of homocysteine corresponds to 14 moles in terms of ATP. On the other hand, from the research results of the present invention, it is predicted that thiosulfuric acid sulfotransferase (GlpE) that converts thiosulfuric acid into sulfite or hydrogen sulfide exists in yeast, and thiosulfuric acid is used as a sulfur source. Thus, it can be inferred that 1 mol of homocysteine can be generated with an energy of about 6 mol in terms of ATP (see FIG. 2). Therefore, when thiosulfuric acid is used as the sulfur source, it is considered that the energy efficiency is improved as compared with the case of using sulfuric acid, and the growth of yeast is promoted, and hence the growth rate and the ethanol productivity are improved.

It is a figure which shows the metabolic pathway of the sulfate in yeast. It is a guess figure of the metabolic pathway of thiosulfate in yeast. In Test Example 1, the growth of yeast was measured using thiosulfate alone or sulfate alone as a sulfur source. In Test Example 2, as a sulfur source, using thiosulfate alone, a mixture of thiosulfate and sulfate, or sulfate alone, the results of measuring yeast growth, glucose consumption, and ethanol production were measured. It is.

1. Culturing method The culturing method of the present invention is characterized in that yeast is cultured in a medium containing thiosulfuric acid and / or a salt thereof. Hereinafter, the culture method of the present invention will be described.

[Culture medium]
In the culture method of the present invention, a medium containing thiosulfuric acid and / or a salt thereof is used as a sulfur source. As described above, by using thiosulfuric acid and / or a salt thereof as a sulfur source, it becomes possible to promote the growth of yeast and efficiently produce yeast cells or produce ethanol by yeast.

  The thiosulfate used in the present invention is not particularly limited as long as it can liberate thiosulfate ions in the medium. For example, alkali metal salts such as sodium salt and potassium salt; calcium salt and magnesium salt Alkaline earth metal salts such as ammonium salts and the like. These thiosulfates may be used individually by 1 type, and may be used in combination of 2 or more type. Among these thiosulfates, an alkali metal salt is preferable, and a sodium salt is more preferable.

  In addition, the medium used in the culture method of the present invention may contain only one of thiosulfuric acid or thiosulfate, or both of them.

  In the culturing method of the present invention, the concentration of thiosulfuric acid and / or a salt thereof in the medium may be appropriately set according to the type of medium, the culture method, etc., for example, 0.001 to 100 mM, preferably 0. 01-50 mM, More preferably, 0.1-10 mM is mentioned.

  Furthermore, the medium used in the culture method of the present invention may contain a sulfur source other than thiosulfuric acid and / or a salt thereof. Such a sulfur source is not particularly limited, and examples thereof include inorganic sulfur compounds such as sulfuric acid, sulfurous acid, hyposulfite, and salts thereof. The type of sulfate, sulfite, and hyposulfite is not particularly limited. For example, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salt and the like Is mentioned.

  However, sulfur sources other than thiosulfuric acid and / or its salt do not contribute to the growth promotion of yeast, and may diminish the growth promotion effect by thiosulfuric acid and / or its salt. From the viewpoint of efficient promotion, it is desirable that the proportion of thiosulfuric acid and / or its salt is high in the sulfur source contained in the medium. More specifically, thiosulfuric acid and / or a salt thereof is 1% or more, preferably 10% or more, more preferably 50% or more, more preferably 70% or more, in terms of molar ratio, based on the total amount of sulfur source contained in the medium. More preferably, it is desirable to occupy 80% or more, particularly preferably 90% or more, and it is particularly preferable that thiosulfuric acid and / or a salt thereof is a single sulfur source. Here, “thiosulfuric acid and / or a salt thereof is a single sulfur source” means that a sulfur-containing compound other than thiosulfuric acid and / or a salt thereof is inevitably mixed in a trace amount as a sulfur source. It means that it is not substantially contained except.

  In addition to thiosulfuric acid and / or salts thereof, the medium used in the culture method of the present invention includes nutrient sources required for yeast growth. Specific examples of such nutrient sources include carbon sources, nitrogen sources, minerals, and the like.

  As the carbon source, a carbon-containing compound that can be assimilated by the yeast may be appropriately selected according to the type of yeast to be used, the purpose of culture, and the like. For example, glucose, fructose, sucrose, lactose, maltose, molasses Saccharides such as hydrolyzate of starch and hydrolyzate of biomass; organic acids such as fumaric acid, citric acid and succinic acid; alcohols such as methanol and the like. These carbon sources may be used individually by 1 type, and may be used in combination of 2 or more type. Moreover, what is necessary is just to set the density | concentration of the carbon source in a culture medium to the same range as the case of normal yeast culture | cultivation.

  Regarding the nitrogen source, a nitrogen-containing compound that can be assimilated by the yeast may be appropriately selected according to the type of yeast to be used, the purpose of culturing, etc., for example, ammonia; ammonium sulfate, ammonium chloride, ammonium phosphate, etc. Inorganic ammonium salts of: amino acids, peptone, meat extract, yeast extract, casein hydrolyzate, organic nitrogen such as soybean hydrolysate, and the like. These nitrogen sources may be used individually by 1 type, and may be used in combination of 2 or more type. Further, the concentration of the nitrogen source in the medium may be set in the same range as in the case of normal yeast culture.

  The mineral may be appropriately selected according to the type of yeast to be used, the purpose of culture, and the like, and examples thereof include salts such as magnesium, calcium, potassium, iron, manganese, and zinc. These minerals may be used individually by 1 type, and may be used in combination of 2 or more type. Further, the mineral concentration in the medium may be set in the same range as in the case of normal yeast culture.

  Furthermore, the medium used in the culture method of the present invention may contain vitamins such as vitamin B1 as necessary. Moreover, if it is a case where the culture method of this invention is used for a fermentation process, according to the kind of fermented material used as the manufacture objective, various food materials should be included.

  Moreover, about the pH of the culture medium used with the culture | cultivation method of this invention, what is necessary is just the range which can grow the yeast to be used, For example, pH 5-10, Preferably pH 6-9 is mentioned.

  The medium used in the culturing method of the present invention may be liquid, solid, or semi-solid, and may be set as appropriate according to the purpose of culturing.

[yeast]
The type of yeast to which the culture method of the present invention is applied is not particularly limited. For example, yeast for brewing (including ethanol-producing yeast) such as beer, wine, whiskey, shochu, sake, miso, soy sauce; baker's yeast Ethanol-producing yeast used for the production of industrial ethanol; organic acid-producing yeast;

  Specific examples of yeasts to which the culture method of the present invention is applied include the genus Saccharomyces, the genus Zygosaccharomyces, the genus Schizosaccharomyces, the genus Candida, the genus Pichia, Examples include yeast belonging to genera such as Hansenula. More specifically, yeasts belonging to the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces mikatae, Saccharomyces bayus ces Examples include Sevicharomyces kudriavzevii, Saccharomyces pastorianus, Saccharomyces carlsbergensis, and the like. Specific examples of yeast belonging to the genus Tigosaccharomyces include Zygosaccharomyces rouxii and Zygosaccharomyces mellis. Specific examples of the yeast belonging to the genus Schizosaccharomyces include Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces japonicus, and Schizosaccharomyces octosporus. More specifically, the yeasts of the genus Candida include Candida utilis, Candida tropicalis, Candida lypolitica, Candida sake, Candida versatis ( Candida versatilis). More specific examples of the Pichia yeast include Pichia pastoris. More specifically, examples of Hansenula yeast include Hansenula polymorpha.

  According to the culture method of the present invention, it is possible to efficiently perform ethanol production by yeast by promoting the growth of yeast. Therefore, in view of the effects of the present invention, the culture method of the present invention. The yeast to which is applied is preferably an alcohol-producing yeast, more preferably a yeast of the genus Saccharomyces, particularly preferably Saccharomyces cerevisiae.

  In addition, the culture method of the present invention can be applied to both wild strains and mutant yeasts. Here, the “wild strain” means a yeast that has not been subjected to artificial mutation treatment on the gene and has existed in nature, and the “mutant strain” means an artificial mutation for the gene. It means yeast that has been subjected to mutation treatment.

[Culture conditions]
The culture method in the culture method of the present invention may be any of batch culture, fed-batch culture, continuous culture, and the like, and may be appropriately set according to the type of yeast used, the culture purpose, and the like.

  The culture temperature in the culture method of the present invention is not particularly limited as long as the yeast can grow, and may be appropriately set according to the type of yeast used, the purpose of culture, and the like. In addition, the culture time in the culture method of the present invention may be appropriately set according to the concentration of yeast, the concentration of carbon source remaining in the culture solution, the purpose of culture, and the like.

  Since the culture method of the present invention can improve the productivity of yeast cells by promoting the growth of yeast, the production of yeast for brewing, the production of baker's yeast, and the production of ethanol for industrial use It can be applied to yeast culture performed for the purpose of production of yeast cells such as production, production of yeast used for the production of organic acids, and production of yeast for raw material of yeast extract.

  In addition, the culture method of the present invention can improve ethanol production efficiency by ethanol-producing yeast, so it can be applied to yeast culture for the purpose of producing ethanol, and further, beer, wine, whiskey, shochu, It can also be applied to yeast culture performed in the fermentation process in the production of alcoholic beverages such as sake.

2. Medium for Yeast Culture The medium of the present invention is a medium used for culturing yeast and is characterized by containing thiosulfuric acid and / or a salt thereof. By using the culture medium of the present invention, it is possible to promote the growth of yeast, and to efficiently produce yeast cells and ethanol by yeast.

  In the medium of the present invention, the components to be blended, the yeast to be applied, and the like are as described in the column of “1. Culture method”.

  Next, the present invention will be specifically described, but the present invention is not limited to the following.

Test example 1
As a sulfur source, sodium thiosulfate or sodium sulfate was used, and the growth characteristics and ethanol production ability of budding yeast were evaluated.

First, as a preculture, budding yeast (Saccharomyces cerevisiae) was shaken at 30 ° C. for one and a half days in a YPD medium and cultured to a stationary phase. Thereafter, the cells were collected at 5,500 rpm for 5 minutes at 4 ° C., and the bacteria were washed three times with deionized water. Then, OD ₆₀₀ was measured and inoculated into 30 mL of SD medium having the composition shown in Table 1 so that OD ₆₀₀ = 0.1, and cultured at 30 ° C. At 0, 6, 12, 18, 24, and 30 hours after inoculation, 1 mL was sampled, 100 μL of which was mixed with 900 μL of deionized water, and the degree of growth was measured with OD ₆₀₀ .

  In Test Example 1, the composition of the SD medium used for budding yeast is as follows, and the sulfur source is not contained other than sodium sulfate or sodium thiosulfate.

  The obtained results are shown in FIG. From these results, it was clarified that budding yeast could not grow on a medium containing no sulfur source (Comparative Example 1), but that sodium thiosulfate was used as the sulfur source, and that budding yeast showed good growth. (Example 1). Furthermore, when sodium thiosulfate is used as the single sulfur source, the growth of yeast is promoted and the growth rate is faster than when sodium sulfate is used as the single sulfur source. (Example 1 and Comparative Example 2).

Test example 2
As a sulfur source, sodium thiosulfate alone, sodium sulfate alone, or a mixture thereof was used to evaluate the growth characteristics and ethanol production ability of ethanol-producing yeast. The ethanol-producing yeast used in Test Example 2 is the same as the budding yeast (Saccharomyces cerevisiae) used in Test Example 1.

Specifically, after pre-culture of budding yeast under the same conditions as in Test Example 1 and collecting the cells, OD ₆₀₀ was measured, and 30 mL of SD medium having the composition shown in Table 2 so that OD ₆₀₀ = 0.1 was obtained. Was inoculated and cultured at 30 ° C. At 0, 6, 12, 18, 24, and 30 hours after inoculation, 1 mL was sampled, 100 μL of which was mixed with 900 μL of deionized water, and the degree of growth was measured with OD ₆₀₀ . The remaining 900 μL was centrifuged at 5,500 rpm for 5 minutes at 4 ° C., and the supernatant was collected and stored at −20 ° C. for glucose and ethanol measurements.

  A glucose kit Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.) was used for measurement of the remaining amount of glucose in the medium. For ethanol measurement, 150 μL of one sample and 450 μL of 1% propanol as an internal standard are placed in a vial, warmed in a thermostatic bath at 50 ° C. for 30 minutes, and measured by gas chromatography (GC-14B, SHIMADZU). did.

  In Test Example 2, the composition of the SD medium used for budding yeast culture is as follows, and the sulfur source is not contained other than sodium sulfate or sodium thiosulfate.

  The obtained results are shown in FIG. From this result, when sodium thiosulfate is included as a sulfur source (Examples 2 and 3), the growth of budding yeast is promoted as compared with the case where sodium sulfate is included alone as a sulfur source (Comparative Example 3). In addition, the growth rate and the glucose consumption rate in the medium were improved, and the ethanol production was also improved. In addition, when sodium thiosulfate is used as the sole sulfur source (Example 2), the growth rate of budding yeast, glucose is higher than when sodium thiosulfate and sodium sulfate are used in combination as the sulfur source (Example 3). It has also been clarified that the consumption rate and the ethanol production amount are improved, and that the use of sodium thiosulfate as a sole sulfur source can further improve the growth of budding yeast and the ability to produce ethanol.

Claims (6)

A method for culturing yeast, comprising culturing yeast in a medium containing 0.001 to 100 mM thiosulfuric acid and / or a salt thereof as a single sulfur source .   The culture method according to claim 1, wherein the yeast is ethanol-producing yeast.   The culture method according to claim 2, which is applied to ethanol production. A culture medium for yeast culture comprising 0.001 to 100 mM thiosulfuric acid and / or a salt thereof as a single sulfur source . The culture medium of Claim 4 used for culture | cultivation of ethanol production yeast. The culture medium of Claim 5 used for manufacture of ethanol.