KR20250056810A - Method of cryprotection for seed culture using deep eutectic solvent - Google Patents

Abstract

The present invention provides a method for maintaining the stability of a starter using a eutectic solvent, comprising the steps of: (a) growing microorganisms in a growth medium and filtering the grown microorganisms; (b) subjecting the filtered microorganisms to a first osmotic pressure treatment with saline water; (c) mixing the microorganisms that have undergone the first osmotic pressure treatment with a water-soluble sugar and then subjecting them to a second osmotic pressure treatment; and (d) adding and mixing a eutectic solvent to the microorganisms that have undergone the first and second osmotic pressure treatments and freezing.

Description

{METHOD OF CRYPROTECTION FOR SEED CULTURE USING DEEP EUTECTIC SOLVENT}

The present invention relates to a method for securing the stability of a seed using a eutectic solvent, and more specifically, to a method for maintaining the stability of a seed using a eutectic solvent, which can increase the survival rate of microorganisms and enhance stability even when long-term storage is carried out by removing moisture from cultured microorganisms through an osmotic pressure treatment method and then freezing them.

The current problem with industrial fungal storage and use is that genetic mutations and death occur frequently during long-term storage.

There are short-term and long-term preservation methods for already known spawn storage methods, and in particular, there are short-term preservation methods such as subculture, immersion preservation, freezing and drying preservation, and long-term preservation methods such as ultra-low temperature freezing preservation and freeze-drying.

However, these methods have the problems of requiring a lot of labor due to short-term, regular subculturing, a high possibility of genetic mutation, and a high possibility of microbial contamination.

In addition, in the storage method by freezing, cell damage may occur during the freezing and thawing process, and there was a problem of decreased viability after long-term frozen storage.

The background technology or prior art described herein is merely intended to help understand the technical significance of the present invention, and does not mean technology widely known in the technical field to which the present invention belongs prior to the filing of the present invention.

Republic of Korea Patent No. 10-2220167

In order to solve such problems, the present invention has been made based on the aforementioned background technology, and its purpose is to provide a method for maintaining the stability of a seed using a eutectic solvent, which increases the stability of microorganisms that have undergone filtration and osmotic pressure treatment by treating the eutectic solvent with the microorganisms, thereby enabling the microorganisms to survive for a long time.

In addition, the present invention aims to provide a method for maintaining the stability of a starter using a eutectic solvent that increases stability, does not change during preservation, is free from microbial contamination, and can be used continuously or repeatedly.

In addition, the present invention aims to provide a method for maintaining the stability of seed using a eutectic solvent, which can be preserved at a low cost and provides an environmentally friendly preservation method by using an environmentally friendly solvent.

In addition, the present invention provides a method for maintaining the stability of a starter using a eutectic solvent, which can be applied and utilized in the fields of pharmaceuticals related to microbial pharmaceutical production and lactic acid bacteria, foods related to fermentation using microorganisms, agriculture such as microbial preparations, livestock farming including microbial feed, and environmental fields related to microorganisms for environmental purification.

In addition, the present invention aims to provide a method for maintaining the stability of seed using a eutectic solvent, which can secure the storage stability of microorganisms for culture, thereby minimizing problems such as long-term maintenance of cells and cell mass, prevention of inhibition of cell activation, and maintenance of quality during culture.

In addition, the present invention provides a method for maintaining seed stability using a eutectic solvent, which can be manufactured by mixing a hydrogen bond acceptor such as choline chloride or betaine and a hydrogen bond donor such as a sugar or an organic acid in the applied eutectic solvent treatment, and which can be manufactured in various ways, and has the advantages of a simple manufacturing method, being environmentally friendly, having low manufacturing cost, and being harmless to the human body, thereby maximizing the stability of the seed and the industrial use of microbial culture.

However, the purpose of the present invention is not limited thereto, and it goes without saying that purposes or effects that can be understood from the means for solving the problem or the embodiment thereof are also included herewith, even if not explicitly stated.

According to one embodiment of the present invention for achieving such a task, the present invention is characterized by including the steps of: (a) growing microorganisms in a growth medium and filtering the grown microorganisms; (b) subjecting the filtered microorganisms to a first osmotic pressure treatment with saline water; (c) mixing the microorganisms that have undergone the first osmotic pressure treatment with a water-soluble sugar and then subjecting them to a second osmotic pressure treatment; and (d) adding and mixing a eutectic solvent to the microorganisms that have undergone the first and second osmotic pressure treatments and freezing them.

According to one embodiment of the present invention, the microorganism is characterized in that it is one species selected from bacteria, microalgae, yeast, and mold, including Lacticaseibacillus casei and Weissella cibaria .

According to one embodiment of the present invention, the step (b) is characterized in that the primary osmotic pressure treatment of microorganisms is performed using saline water having a concentration of 2% to 8%.

According to one embodiment of the present invention, the step (b) is characterized by further performing a step of centrifuging at a temperature of 20°C and a speed of gravitational acceleration of 6,000 G for 5 minutes to remove a solution containing microorganisms.

According to one embodiment of the present invention, the step (c) is characterized by performing the steps of: (c1) mixing a water-soluble sugar having a concentration of 10% to 40% into microorganisms that have completed the first osmotic pressure treatment to perform a second osmotic pressure treatment, and (c2) centrifuging the microorganisms that have completed the second osmotic pressure treatment at a temperature of 20°C and a gravitational acceleration of 6,000 G for 5 minutes.

According to one embodiment of the present invention, the water-soluble sugar is characterized by being composed of one or more of monosaccharides or polysaccharides including sucrose, fructose, and glucose.

According to one embodiment of the present invention, the step (d) is characterized by including: (d1) a step of preparing a eutectic solvent using glycerol-sodium acetate; (d2) a step of mixing the eutectic solvent with microorganisms that have completed secondary osmotic pressure treatment; and (d3) a step of mixing the microorganisms that have completed mixing with the eutectic solvent with 10 mL of PBS, dispensing the mixture into a vial, and storing the vial in a freezer.

According to one embodiment of the present invention, the step (d1) is characterized by manufacturing the product by heating and mixing at a molar ratio of 1:1 to 1:10 an acceptor including sodium acetate, an amino acid, choline chloride, and imidazole, and a donor including glycerol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, and propylene glycol.

According to one embodiment of the present invention, the step (c1) is characterized by preparing a eutectic solvent by treating glycerol-sodium acetate and an acceptor at a weight ratio of 3:1 at 75°C.

The method for maintaining the stability of the seed using a eutectic solvent according to an embodiment of the present invention has the effect of increasing the stability of microorganisms, ensuring the quality of microorganisms during the main cultivation by preventing mutations in the microorganisms during long-term preservation.

In addition, according to an embodiment of the present invention, there is an effect of increasing and securing the stability of the seed according to the combination and composition of the manufactured eutectic solvent.

In addition, according to an embodiment of the present invention, there is an effect of increasing and securing the stability of the seed according to the application of the primary and secondary osmotic treatment methods and the applied materials.

In addition, according to an embodiment of the present invention, the eutectic solvent used has the effect of increasing commercial use by being a low-cost, environmentally friendly material that is harmless to the human body and diversifying the application areas depending on the composition combination.

In addition, the various beneficial advantages and effects of the present invention are not limited to the above-described contents, and will be more easily understood in the process of explaining specific embodiments of the present invention.

Figure 1 is a flow chart showing a method for maintaining seed stability using a eutectic solvent according to one embodiment of the present invention.
Figure 2 is a table showing the composition of an experimental group according to one embodiment of the present invention.
Figure 3 is a graph showing the survival rate of Lacticaseibacillus casei strains according to repeated freezing and thawing by experimental group according to one embodiment of the present invention.
Figure 4 is a graph showing the culture recovery ability of the Lacticaseibacillus casei strain after freezing and preserving the seed produced according to one embodiment of the present invention for 90 days.
FIG. 5 is a graph showing the culture recovery ability of a Weissella cibaria strain after freezing and preserving the seed produced according to one embodiment of the present invention for 90 days.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. First, when adding reference symbols to components in each drawing, it should be noted that identical components are given the same symbols as much as possible even if they are shown in different drawings.

In addition, it should be noted that the technical terms used in describing the present invention are only used to describe specific embodiments and are not intended to limit the present invention, and if a specific description of a related known configuration or function is judged to obscure the gist of the present invention, the detailed description will be omitted. In addition, in describing the present invention, general terms used should be interpreted as defined in the dictionary or according to the context, and should not be interpreted in an excessively narrow sense, and when a technical term used is an incorrect technical term that does not accurately express the idea of the present invention, it should be replaced with a technical term that a person skilled in the art can correctly understand and understand.

In addition, in describing the present invention, terms such as "comprise," "comprises," or "has," unless specifically stated to the contrary, mean that the corresponding component may be included, and should not be construed to necessarily include all components or multiple steps, and some of the components or some steps may not be included, or may further include additional components or steps, and all terms including technical or scientific terms, unless defined otherwise, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs.

In addition, when describing components of the present invention, identification codes such as first, second, A, B, (a), (b), etc. may be used. These identification codes are intended to distinguish the components from other components and are used only for convenience of description, and the nature, order, or sequence of the components are not limited by the identification codes.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.

As illustrated, the method for maintaining the stability of the seed using the eutectic solvent of the present invention performs the steps of growing microorganisms in a growth medium and filtering the grown microorganisms (S110).

Step S110 can use 1L MRS broth as a growth medium for culturing microorganisms, but is not limited thereto.

At this time, the microorganism to be cultured may be one species selected from bacteria, microalgae, yeast, and mold, including Lacticaseibacillus casei and Weissella cibaria .

This S110 step cultures microorganisms at a temperature of 37℃ for 8 hours and obtains a culture solution containing cultured microorganisms.

In addition, step S110 performs a step of centrifuging the obtained culture solution to separate the cultured microorganisms and the culture solution.

At this time, step S110 can separate the cultured microorganisms and water by centrifugation at a temperature of 20℃ and a speed of 6,000G of gravitational acceleration for 5 minutes to recover the cultured microorganisms, but is not limited thereto, and the temperature, rotation speed, and time of centrifugation can be performed in various ways depending on the type of microorganism being cultured.

Afterwards, a first osmotic pressure treatment step is performed to treat the microorganisms filtered by centrifugation with saline water (S120).

Step S120 performs a first osmotic pressure treatment step in which filtered microorganisms (bacteria) are subjected to osmotic pressure treatment using saline water having a certain concentration.

Step S120 is a step that improves survival in a specific environment by subjecting microorganisms to osmotic pressure treatment using salt water, dehydrating the water contained in the microorganisms to inhibit growth and proliferation, and inducing physiological stress in the microorganisms to increase the resistance required for survival.

This S120 step can use a saline solution having a concentration of 2% to 8% when osmotic pressure treating microorganisms.

At this time, if osmotic pressure treatment is performed with a brine concentration of less than 2%, the effect of regulating the metabolic activity and fermentation process of microorganisms is bound to be minimal, and this makes it difficult to induce physiological responses of microorganisms.

In addition, if osmotic pressure treatment is performed on the microorganism of the present invention using saline water having a saline concentration exceeding 8%, the cell structure may not be maintained smoothly, and there is a concern that this may cause changes in the physiological functions of the microorganism.

Meanwhile, at step S120, the recovered microorganisms are mixed with 20 mL of a NaCl solution having a concentration of 7%, and left for 10 minutes so that the first osmotic pressure can be achieved.

In addition, in step S120, when the osmotic pressure treatment step using brine is completed, a centrifugation step may be further performed at a temperature of 20°C and a speed of gravitational acceleration of 6,000 G for 5 minutes to remove the solution containing microorganisms.

Afterwards, a second dehydration step is performed to osmotically treat the microorganisms (bacteria) that have undergone the first osmotic pressure treatment with sugar (S130).

Step S130 is a step in which the microorganisms that have undergone primary osmotic pressure treatment are subjected to osmotic pressure treatment with a water-soluble sugar having a concentration of 10% or higher, thereby increasing the rate of osmotic pressure treatment outside the cells, thereby controlling the moisture content of the cells, thereby enhancing the inhibition of microbial growth and proliferation, increasing the microbial resistance, thereby enhancing the adaptability to extreme environments, and further enhancing the viability by affecting the cell membrane and cell wall of the microorganisms, thereby stabilizing the cell structure and regulating cell function.

Here, the water-soluble sugar is mixed at a concentration of 10% to 40%, and may include one or more of monosaccharides or polysaccharides including sucrose, fructose, glucose, etc.

This S130 step is bound to have a minimal effect on osmotic pressure treatment when osmotic pressure treatment is performed with less than 10% of soluble sugar, and when osmotic pressure treatment is performed with a concentration of soluble sugar exceeding 40%, there are problems such as death due to excessive dehydration of microorganisms, decreased growth rate due to decreased metabolic function of microorganisms, decreased fermentation rate, and the problem of rapidly decreasing the survival ability of microorganisms.

In the present invention, step S130 may be performed by mixing 20 mL of 30% water-soluble sugar and the recovered cells that have undergone primary osmotic pressure treatment, leaving the mixture for 10 minutes, and then centrifuging the mixture at a temperature of 20°C and a speed of 6,000 G for 5 minutes to remove the solution.

Thereafter, the present invention performs a step of freezing by adding and mixing a eutectic solvent to the microorganisms that have completed secondary osmotic pressure treatment (S140).

Step S140 first performs a step of preparing a eutectic solvent using glycerol-sodium acetate.

At this time, in the present invention, a eutectic solvent can be prepared by mixing glycerol and an acceptor including glycerol-sodium acetate, choline chloride, imidazole, etc. in a weight ratio of 1:1 to 5:1.

More preferably, the eutectic solvent can be prepared by treating glycerol-sodium acetate and the acceptor at a weight ratio of 3:1 at 75°C, but is not limited thereto.

That is, the eutectic solvent of the present invention may be manufactured by selecting one or more of sodium acetate, amino acid, choline chloride, and imidazole as acceptors and glycerol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, and propylene glycol as donors, and mixing them by heating at a molar ratio of 1:1 to 1:10.

In addition, step S140 performs a step of mixing the manufactured eutectic solvent and microorganisms that have completed secondary osmotic pressure treatment.

At this time, step S140 can mix 20 mL of eutectic solvent with microorganisms that have completed secondary osmotic pressure treatment.

In addition, step S140 can be performed by mixing the microorganisms that have been mixed with the eutectic solvent with 10 mL of PBS (Phosphate-buffered saline), dispensing them into vials, and storing them in a freezer.

[Example 1] Method for maintaining seed stability using a eutectic solvent

The eutectic solvent was prepared by mixing sodium acetate, a hydrogen bond acceptor, and glycerol, a hydrogen bond donor, in a ratio of 1:3 and stirring at 75°C for 24 hours.

Microbial culture was performed by receiving Lacticaseibacillus casei KCTC 3109 strain and Weissella cibaria KCTC 3817 strain, and shaking culture was performed at 37℃ for 8 hours using 1 L of MRS broth medium. The cultured microorganisms were centrifuged at 6,000 g for 5 minutes at 20℃ to recover the cells.

The recovered microorganisms were mixed with 20 mL of 7% NaCl and left for 10 minutes, then mixed with 20 mL of 30% sucrose and left for 10 minutes.

To remove the used solution, centrifugation was performed again at 6,000 g for 5 minutes at 20°C, 20 mL of glycerol-sodium acetate eutectic solvent prepared at 75°C in a 3:1 ratio was mixed, and then 10 mL of PBS was mixed, dispensed into vials, and stored in the freezer.

Afterwards, freezing at -80℃ and thawing at 25℃ were repeated 6 times, and the survival rate was confirmed through a viable cell count analysis (plate counting method). In addition, to confirm the culture recovery ability, the frozen inoculum of each experimental group was stored at -80℃ for 90 days, and then inoculated onto MRS medium, and the viable cell count analysis of the solution cultured at 30℃ for 8 hours was performed.

The composition of the experimental groups for the application of brine treatment as the first osmotic treatment, sugar treatment and eutectic solvent treatment as the second osmotic treatment is shown in Figure 2. Each experimental group was named A, B, C, D, E, F, and G.

As a result, as shown in Figures 3 to 5, the survival rate of the seed was different depending on whether brine treatment was applied as the first osmotic treatment, sugar treatment and eutectic solvent treatment were applied as the second osmotic treatment.

In particular, as shown in Fig. 3, when brine treatment was applied as the first osmotic treatment, and sugar treatment and eutectic solvent treatment were applied as the second osmotic treatment, the highest survival rate was observed after freezing and thawing. In particular, a higher survival rate was confirmed when the hydrogen bond acceptor and donor used in preparing the eutectic solvent were treated in the form of a eutectic solvent than when they were added as substances rather than in the form of a eutectic solvent.

In addition, as shown in FIGS. 4 and 5, when saline treatment was applied as the first osmotic treatment and sugar treatment and eutectic solvent treatment were applied as the second osmotic treatment, both Lacticaseibacillus casei KCTC 3109 strain and Weissella cibaria KCTC 3817 strain showed the highest growth recovery ability in the culture medium.

The above description is merely an illustrative description of the technical idea of the present invention, and those skilled in the art will appreciate that various modifications and variations may be made without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention but to explain it, and the scope of the technical idea of the present invention is not limited by these embodiments. The protection scope of the present invention should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims (9)

(a) a step of growing microorganisms in a growth medium and filtering the grown microorganisms;
(b) a step of first osmotic pressure treatment of the filtered microorganisms with saline water;
(c) a step of mixing microorganisms that have undergone primary osmotic pressure treatment with water-soluble sugars and then performing secondary osmotic pressure treatment;
(d) a step including the step of adding and mixing a eutectic solvent to the primary and secondary osmotic pressure-treated microorganisms and freezing them;
A method for maintaining the stability of a seed using a eutectic solvent, characterized by including:
In the first paragraph,
A method for maintaining the stability of a starter using a eutectic solvent, characterized in that the microorganism is one species selected from bacteria, microalgae, yeast, and mold, including Lacticaseibacillus casei and Weissella cibaria .
In the first paragraph,
Step (b) above,
A method for maintaining the stability of a starter using a eutectic solvent, characterized in that the primary osmotic pressure treatment of microorganisms is performed using a saline solution having a concentration of 2% to 8%.
In the third paragraph,
Step (b) above,
A centrifugation step to remove the solution containing microorganisms;
A method for maintaining the stability of a seed using a eutectic solvent, characterized by further performing the following.
In the first paragraph,
Step (c) above,
(c1) A second osmotic pressure treatment is performed by mixing a water-soluble sugar at a concentration of 10% to 40% with microorganisms that have completed the first osmotic pressure treatment,
(c2) A step of centrifuging the microorganisms that have undergone secondary osmotic pressure treatment;
A method for maintaining the stability of a seed using a eutectic solvent, characterized by performing the following.
In paragraph 5,
The above water-soluble sugars are
A method for maintaining the stability of a starter using a eutectic solvent characterized by being composed of one or more of monosaccharides or polysaccharides including sucrose, fructose, and glucose.
In the first paragraph,
Step (d) above,
(d1) a step of preparing a eutectic solvent using glycerol-sodium acetate;
(d2) a step of mixing the above eutectic solvent and microorganisms that have completed secondary osmotic pressure treatment; and
(d3) A step of mixing the microorganisms that have been mixed with the above eutectic solvent with 10 mL of PBS (Phosphate-buffered saline), dispensing them into vials, and storing them in a freezer;
A method for maintaining the stability of a seed using a eutectic solvent, characterized by including:
In Article 7,
The above step (d1) is,
A method for maintaining the stability of a seed using a eutectic solvent, characterized in that the method comprises mixing and heating one or more of an acceptor including sodium acetate, an amino acid, a choline chloride, and imidazole, and a donor including glycerol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, and propylene glycol at a molar ratio of 1:1 to 1:10.
In Article 7,
The above step (c1) is,
A method for maintaining the stability of a seed using a eutectic solvent, characterized in that the eutectic solvent is prepared by treating glycerol-sodium acetate and a receptor at a weight ratio of 3:1 at 75°C.