KR100469332B1 - Method for production of glycinebetaine by yeast - Google Patents

Abstract

The present invention relates to a glycinebetaine production method used in osmotic pressure regulators of plants, arterial blood disease improving agents, stress relief agents of livestock and fish, dementia treatment agents and the like.

In the method for producing glycinebetaine using yeast according to the present invention, a process of culturing yeast in a nutrient medium to which copper sulfate is added and producing the seed by culturing the cultured yeast three to five times in a nutrient medium excluding copper sulfate The seed is inoculated with sugar beet molasses or sugar beet extract containing inorganic salts, and the step of culturing, lyophilizing the culture solution, extracting glycine betaine from the lyophilisate.

Description

Glycinebetaine production using yeast {METHOD FOR PRODUCTION OF GLYCINEBETAINE BY YEAST}

The present invention relates to a method for producing glycine betaine used in osmotic pressure regulators of plants, arterial blood disease improving agents, stress relieving agents of animals and fish, dementia treatment agents and the like.

Glycinebetaine has the following structural formula.

Glycine betaine has been conventionally extracted from sugar beet, but the sugar beet has a low homogeneity because the glycine betaine content of sugar beet is not constant, and storage facilities are needed for continuous supply of raw materials. As an alternative to this, a method of producing glycinebetaine by culturing microorganisms in a reactor has been developed, which is a space required for freezing storage of sugar beet, which can stably obtain raw materials, and is susceptible to decay when produced by fermentation through microbial culture. Maintenance costs, as well as continuous fermentation can produce a homogeneous product. The only microorganism that produces glycinebetaine is Actinopolyspora halophilia, the only known aerobic heterotrophic eubacteria. Although known, these microorganisms are photosynthetic microorganisms and must be cultured under photo autotroph conditions for irradiating high-intensity light for efficient photosynthesis to induce glycine betaine accumulation in the body. In addition, in order to produce glycine betaine by fermentation by these microorganisms, an initial substrate, choline, should be added to the culture solution. However, choline is expensive and conventional methods have not been commercialized due to low profitability.

The present invention produces glycinebeta at low cost by using a yeast that can synthesize glycinebetaine without including high intensity light and corin, in order to solve the high cost problem when producing glycinebetaine using the microorganisms as described above. The purpose is to provide a way to.

Glycine betaine production method using the yeast of the present invention for achieving the above object, the process of culturing yeast once in a nutrient medium with copper sulfate added, the cultured yeast in a nutrient medium without copper sulfate 3 to 3 Process of producing spawn by five consecutive cultures, inoculating the spawn with beet molasses or sugar beet extract containing inorganic salts, culturing, lyophilizing the culture, extraction of glycinebetaine from the freeze-dried Can be achieved by.

1 is a process chart showing a production process of glycinebetaine using yeast.

Hereinafter, a method for producing glycinebetaine using yeast according to the present invention will be described in detail.

Figure 1 shows the production process of glycine betaine using yeast fermentation.

In the method for producing glycinebetaine using yeast according to the present invention, a process of culturing yeast in a nutrient medium to which copper sulfate is added and producing the seed by culturing the cultured yeast three to five times in a nutrient medium excluding copper sulfate The seed is inoculated with sugar beet molasses or sugar beet extract containing inorganic salts, and the step of culturing, lyophilizing the culture solution, extracting glycine betaine from the lyophilisate.

Yeast used for the production of glycinebetaine is Saccharomyces cerevisiae, and preferably any one of KCTC 1552, 1813 and 7928 is used. The KCTC 1552, 1813, and 7928 can be purchased from the Korea National Institute of Biotechnology Gene Bank.

In the copper sulfate addition culture process, the copper sulfate is preferably added 10mM copper sulfate. The reason for adding copper sulfate is to apply artificial environmental stress to the yeast to allow the yeast to synthesize glycinebetaine. It is understood that this is due to yeast's self-protective action against environmental stress. In addition, it is preferable to use a standard YPD liquid medium of the composition consisting of yeast extract, peptone and glucose commonly used in yeast culture.

In the spawn production process, the yeast cultured in the process is continuously cultured in a nutrient medium excluding copper sulfate to produce spawn. The number of cultures is preferably 3 to 5 consecutive cultures.

In the beet molasses or beet extract inoculation cultivation process, the incubation time is inoculated into the molasses or extract of the yeast, and then cultured for 48 to 120 hours, it is preferable to incubate until the end of the logarithmic phase or early phase of the growth phase .

The freeze-drying process is intended to extract not only glycinebetaine produced by the yeast cells, but also glycinebetaine in the remaining amount of glycinebetaine or beet extract remaining in the sugar beet molasses.

In the glycine betaine extraction process, the lyophilisate is dissolved in a minimum volume of water, and then about 4 times the volume of ethanol is added to the solution and heated to completely dissolve the yeast cells, and then under 12,000 × g. Glycinebetaine is obtained by centrifugation to remove the residue, extracting only the supernatant and drying.

Hereinafter, the present invention will be described in detail by examples.

The following examples illustrate the invention in detail and are not intended to limit the invention.

Example 1

This embodiment includes a process of producing yeast seed from yeast, a process of producing yeast cells by culturing the yeast seed in a medium, and extracting glycinebetaine from the yeast cells.

The yeast seed production process is prepared by the addition of 10mM copper sulfate to the standard YPD liquid medium of the yeast extract, peptone and glucose prepared sterilization medium for the stress price, inoculated KCTC 7928 in the stress price sterilization medium and 30 ℃ Incubated in a shaker incubator up to the mid-log phase, and then incubated part of the culture in a standard YPD liquid medium without copper sulfate to incubate to the mid-log phase. .

The yeast cell production process consists of inoculating a portion of the culture solution in 100ml standard YPD liquid medium to incubate until the end of growth (stationary phase) and centrifugation to recover the yeast cells.

The glycine betaine extraction process was added to the recovered yeast cells 4 times the volume of boiling ethanol and then further heated at 80 ℃ for 20 minutes to completely disassemble the yeast cells, centrifuged at 12,000 × g Separation removes the yeast cell residues, take only the supernatant and dry at 80 ℃ to extract glycinebetaine.

In the same manner as above, the yeast was cultured once in a standard YPD liquid medium to which 10 mM copper sulfate was added, followed by two to five successive incubations in a standard YPD liquid medium without copper sulfate, followed by a standard YPD liquid excluding 100 ml of copper sulfate. Inoculated to the medium and cultured until the end of growth. Then, the yeast cells are recovered and extracted by the same centrifugation method as described above.

Number of Continuous Cultures in Copper Sulfate Exclusion Medium Glycinebetaine production (mg) per 100 ml of culture 0 36.2 One 40.1 2 59.8 3 83.2 4 81.3 5 78.4

Table 1 shows the glycine betaine production according to the number of continuous cultures in the copper sulfate exclusion medium.

In Table 1, it can be seen that the production of glycinebetaine is significantly increased in the case of three consecutive cultures in a standard YPD liquid medium excluding copper sulfate compared to the two consecutive cultures.

In addition, the production of glycinebetaine is similar to that of three consecutive cultures in the case of 4, 5 consecutive cultures.

Example 2

This Example is the same process as used in the above [Example 1] the same process as in the yeast (KCTC 7928) cultured in a sterile medium for stress price added 10mM copper sulfate in a standard YPD liquid medium excluding copper sulfate three times, The cultured culture medium was inoculated into 100 ml of standard YPD liquid medium to which 10 mM (154.6 mg / 100 ml) glycinebetaine was added as a yeast seed, and then the inoculum was inoculated at 24 ° C shaker in 24, 48, 72 and 96 After culturing for a period of time, the yeast cells and the supernatant are centrifuged and separated and recovered.

Incubation time (hr) Glycinebetaine-free medium (mg) Glycinebetaine 154.6 mg addition medium (mg) Intracellular Content Contains supernatant 24 47.8 55.1 143.8 48 69.75 91.44 148.4 72 78.0 90.9 147.2 96 83.7 78.3 146.7

Table 2 shows the results according to the above examples. As a control, the same yeast seedlings were inoculated into 100 ml of standard YPD liquid medium without glycinebetaine and incubated for the same time, and the yeast cells were recovered by centrifugation to produce glycinebetaine.

In [Table 2], the production of glycinebetaine through yeast fermentation in a medium to which glycinebetaine was added 154.6 mg was higher than the production of glycinebetaine through fermentation of glycinebetaine in a medium without glycinebetaine. You can see the output increase.

Example 3

In this example, yeast spawn was prepared according to the same method as in [Example 1], and the yeast spawn was inoculated into 100 ml of beet extract with the addition of a suitable salt and nutrient source, and then designated according to the method of [Example 2]. After shaking the culture at 30 ℃ for a period of time, the yeast cells and the supernatant were recovered by centrifugation in the same manner as in [Example 1], and the process consists of extracting glycinebetaine.

Incubation time (hr) Glycinebetaine content (mg) B / A Culture supernatant (A) In supernatant [supernatant + bacteria] (B) 0 145.2 141.3 0.97 24 140.5 193.8 1.38 48 136.8 248.4 1.82 72 154.5 229.1 1.48 96 159.4 228.7 1.43

Table 3 shows the amount of glycine betaine produced by yeast fermentation in the sugar beet extract medium.

In Table 3, it can be seen that the high glycine betaine production when the incubation time is 48 hours.

The method for producing glycine betaine using the yeast fermentation of the present invention does not need to irradiate high-intensity light, unlike conventional photosynthetic microorganisms such as cyanobacteria. Since it is not added to the furnace medium, glycinebetaine can be produced at low cost. In the present invention, when the beet extract is produced as a medium by fermentation method, glycine betaine contained in sugar beet and sugar contained in sugar beet to be separated and removed as an impurity during the sugar beet purification process are converted into glycine betaine by yeast fermentation. By conversion can be obtained further, the yield of glycinebetaine can be increased.

Claims (3)

A step of culturing the yeast resistant to the metal in a nutrient medium containing copper sulfate once; Producing yeast spawn by continuously culturing 3 to 5 times in a nutrient medium excluding copper sulfate; Inoculating the yeast spawn in beets molasses containing inorganic salts or beets extract containing inorganic salts and incubating the same; Lyophilizing the culture solution; Glycine betaine production method using the yeast, characterized in that the process consisting of extracting glycine betaine from the lyophilisate. The method of claim 1, wherein the yeast is any one of KCTC 1552, 1813, and 7928. The method for producing glycinebetaine using yeast according to claim 1 or 2, wherein the copper sulfate is 10 mM copper sulfate.