KR20200072886A - MEDIUM COMPOSITION FOR PRODUCING BIOPOLYMERS FROM Aureobasidium - Google Patents

Abstract

The present invention relates to a medium composition for manufacturing a biopolymer, containing beta-glucan derived from Aureobasidium pullulans, wherein the composition includes: lignosulfonate which is a carbon source; at least one selected from an ammonium salt which is a nitrogen source, potassium hydrogen phosphate (K_2HPO_4) and magnesium sulfate (MgSO_4) which are inorganic substances; and vitamin C. Accordingly, polycan, a biopolymer containing beta-glucan derived from Aureobasidium pullulans, can be produced in high yield.

Description

Medium composition for producing biopolymer derived from black yeast and a method for producing biopolymer derived from black yeast using the same{MEDIUM COMPOSITION FOR PRODUCING BIOPOLYMERS FROM Aureobasidium}

The present invention relates to a medium composition for producing a biopolymer derived from black yeast and a method for producing a biopolymer derived from black yeast using the same, and more specifically, a biopolymer containing beta glucan derived from black yeast that can be used as an environmentally friendly soil reinforcement material, etc. It relates to a medium composition capable of significantly improving the production yield of phosphorus polycans and a method for producing polycans using the same.

Biopolymer, which is in the spotlight as an eco-friendly material, is a high-molecular substance produced by organisms, and various types of polymers that are carbohydrates, fats, proteins, nucleic acids, and complexes of them are synthesized in organisms and secreted outside the body. Refers to a substance.

Among the industrially important biopolymers are xanthan, gellan, and chitosan, which are obtained by deacetating chitin, which are produced by microorganisms. In addition, water-soluble or insoluble polysaccharides (polysaccharides) is a polymer produced by more than 10 monosaccharides or inducing monosaccharides by glycosidic bonds, and is a representative biopolymer useful in humans while being most abundant in nature.

Unlike chemical synthetic polymers, they are environmentally friendly, and are used in various industries due to functional properties such as gel formation, emulsification stability, surface tension control, water absorption, adhesion, lubrication, and biofilm formation resulting from structural properties. It is used as the main material in the field. These various biopolymers are also used as additives to improve functionality such as concrete and dry-mix mortar in civil engineering and construction.

In concrete, biopolymers such as Welan gum and Curdlan are used as eco-friendly high-mobility stabilizers, and they exhibit excellent performance as high-mobilization stabilizers despite their relatively high cost. Is forming.

As such, biopolymers are eco-friendly materials, unlike petroleum-based synthetic polymers, and can be used as important material materials in the future in terms of free from environmental and social problems caused by harmful substances emitted from cement-based and synthetic resin-based materials. .

In Korea, beta-glucan is used as a soil reinforcement to prevent erosion of soil. It is about the germination and vegetation enhancement technology of environment-friendly vegetation using biopolymers, which are polysaccharides produced from living organisms as a method of promoting germination or growth of vegetation using biopolymer It has been used as a biopolymer polycan as a main component.

Glucan, which is produced from yeast, is a polysaccharide and is used by extracting from microorganisms, mushrooms, or grains, and there are various types. As a microorganism that produces glucan, the Scharomyces cerevisiae R4 line, which produces 1,3-linked beta-glucan, is known, and a variant strain of Agrobacterium that produces 1,3-linked beta-glucan Is known.

Glucan, a type of polysaccharide, is a glucose polymer composed of only D-glucose and is classified into beta-glucan or alpha-glucan depending on the type of linkage. In particular, beta-glucan (beta-glucan) is a 4 (1,4 bond) or 6 (1,6 bond) carbon of the basic skeleton in which the glucose unit is linked by a beta (beta)-glucoside bond at positions 1 and 3 Has side chains, and physicochemical properties may be changed along with structural differences due to the presence or absence of side chains.

Beta-glucan isolated from living organisms such as mushrooms or black yeasts are generally widely distributed and are polymer polysaccharides with a broad molecular weight range from kilo-Daltons to mega-Daltons. Beta-glucan is used only in the small-scale food industry due to its gelling ability and high viscosity in aqueous solution.

In order to utilize the polycan, a biopolymer based on beta-glucan, in various fields such as soil reinforcing materials, it is necessary to develop a high-yield, low-cost biopolymer manufacturing method.

Korean Registered Patent No. 10-1551920 U.S. Patent No. 5504079

An object of the present invention is to provide a medium composition capable of producing polycans, which are biopolymers containing beta-glucan derived from black yeast, with high yield.

Another object of the present invention is to prepare a biopolymer derived from black yeast that produces a polycan, a biopolymer containing beta glucan derived from black yeast, in a high yield by using a medium composition containing a carbon source, nitrogen source, mineral, and vitamin of a specific component. In providing a way.

According to one aspect of the invention,

Carbon source lignosulfonate; Nitrogen source ammonium salt, mineral; And vitamin C; a media composition for preparing a biopolymer comprising beta-glucan derived from black yeast (Aureobasidium).

The ammonium salt is ammonium chloride (NH ₄ Cl), ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), ammonium nitrate ((NH ₄ )NO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and ammonium acetate (CH ₃ COONH ₄ ) It may be one or more selected from the group consisting of.

The inorganic material may be at least one selected from potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (MgSO ₄ ).

The medium composition, based on 100 parts by weight of the lignosulfonate as a carbon source,

2 to 10 parts by weight of an ammonium salt that is a nitrogen source;

1 to 10 parts by weight of minerals; And

Vitamin C 1 to 10 parts by weight; may include.

The nitrogen source and the mineral may be in a weight ratio of 1:0.1 to 1:1.

The vitamin C and the mineral may be in a weight ratio of 1:0.1 to 1:1.

According to another aspect of the invention,

(a) One or more selected from lignosulfonate as a carbon source, ammonium salt as a nitrogen source, potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (MgSO ₄ ), and black yeast (Aureobasidium) containing vitamin C Preparing a media composition for preparing the biopolymer containing beta-glucan;

(b) inoculating the medium composition with black yeast (Aureobasidium) and culturing to prepare a culture solution; And

(c) filtering the culture solution after sterilization; a method for preparing a biopolymer containing beta-glucan derived from black yeast is provided.

Step (b),

(b-1) inoculating the medium composition with black yeast (Aureobasidium) and culturing to prepare a seed culture solution; And

(b-2) inoculating the seed culture medium with 1 to 5% by weight of the separate medium composition prepared in step (a) to incubate for 2 to 4 days under aerobic conditions to prepare a culture medium; Can.

The black yeast may be Aureobasidium pullulans.

The medium composition for producing a biopolymer derived from black yeast of the present invention can produce polycans, which are biopolymers containing beta glucan derived from black yeast, with high yield.

In addition, the method for producing a biopolymer derived from black yeast of the present invention uses a medium composition containing a carbon source, a nitrogen source, minerals and vitamins of a specific component to produce polycans, which are biopolymers containing beta glucan derived from black yeast, in high yield. can do.

1 is a photograph of the culture medium cultured according to Examples 4 to 6, and Comparative Examples 5 to 8.
2 is a photograph measuring the polycan content in the culture medium according to Test Example 1.
Figure 3 is a graph comparing the polycan content in the culture medium according to Test Example 1.
4 is a photograph measuring the content of beta glucan compared to the carbon source according to Test Example 2.
5 is a graph comparing beta glucan content with respect to a carbon source according to Test Example 2.

The present invention can be applied to various transformations and can have various embodiments, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In the description of the present invention, when it is determined that a detailed description of related known technologies may obscure the subject matter of the present invention, the detailed description will be omitted.

Hereinafter, a medium composition for producing a biopolymer derived from black yeast of the present invention will be described.

The medium composition for producing biopolymer derived from black yeast of the present invention includes lignosulfonate as a carbon source; Nitrogen source ammonium salt, mineral; And vitamin C; beta glucan derived from black yeast (Aureobasidium).

The ammonium salt is ammonium chloride (NH ₄ Cl), ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), ammonium nitrate ((NH ₄ )NO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and ammonium acetate (CH ₃ COONH ₄ ) It is preferred that at least one selected from the group consisting of, more preferably, it may be ammonium chloride (NH ₄ Cl).

The inorganic material may be at least one selected from potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (MgSO ₄ ), preferably magnesium sulfate.

The medium composition, 2 to 10 parts by weight of ammonium salt as a nitrogen source, based on 100 parts by weight of lignosulfonate as a carbon source; 1 to 10 parts by weight of minerals; And vitamin C 1 to 10 parts by weight.

When the content of the ammonium salt is less than the lower limit, the amount of cells in the culture medium using the medium composition of the present invention may not increase, and consequently, the content of the biopolymer including polycan, that is, beta-glucan derived from black yeast, may not increase.

More preferably, the nitrogen source and the mineral in the medium composition may be in a weight ratio of 1:0.1 to 1:1, and even more preferably in a weight ratio of 1.0.2 to 1:0.4. When the content of the mineral is less than the lower limit based on the nitrogen source, the content of the polycan may not be improved and physical properties may not be improved.

The vitamin C and minerals are used together with the lignosulfonate and ammonium salts to serve to improve the yield of beta-glucan.

More preferably, in the medium composition, the vitamin C and the mineral may be in a weight ratio of 1:0.1 to 1:1, and even more preferably, 1:0.2 to 1:0.4. At this time, when the content of the mineral based on the vitamin C is less than the lower limit or exceeds the upper limit, the content of the polycan may be lowered.

Hereinafter, a method for producing a biopolymer containing beta-glucan derived from black yeast of the present invention will be described.

First, the cause of carbon Lignosulfonate , Nitrogen source ammonium salt, mineral potassium hydrogen phosphate ( K ₂ HPO ₄ ) And Magnesium sulfate ( MgSO ₄ ), one or more selected from, and black yeast containing vitamin C ( Aureobasidium ) Origin Beta glucan Containing Biopolymer A preparation medium composition is prepared (step a).

Since the medium composition is the same as the medium composition for preparing a biopolymer containing beta-glucan derived from the black yeast of the present invention described above, the detailed description will be referred to the above description.

Next, to the medium composition Black yeast (Aureobasidium) After inoculation, cultured to prepare a culture medium (step b).

Preferably, this step is performed in the following order.

First, after inoculating the medium composition with black yeast (Aureobasidium) and culturing it to prepare a seed culture solution (b-1).

The black yeast is preferably the black yeast is Aureobasidium pullulans.

Biopolymers produced using Aureobasidium pullulans, which is a black yeast used in the present invention, may be prepared as food raw materials.

When aureobasidium pullulans are cultured using the medium composition of the present invention, the content of beta-glucan, which is a main component of polycane, is increased, and as a result, the content of polycane, which is a biopolymer derived from black yeast, is increased. It can also be used as a reinforcing material.

Preferably, after inoculating the black yeast with the medium composition, a culture for 1 to 4 days at 150 to 250 rpm at 20 to 30°C can be obtained to obtain a seed culture medium.

When performed in a range outside the culture conditions, the content of the polycan obtained later may be lowered.

Next, a culture medium is prepared by culturing the seed culture medium in a separate medium composition prepared in step (a) (b-2).

The culture in step (b-2) is preferably inoculated with the seed culture medium at 1 to 5% by weight in the separate medium composition, and cultured under the same conditions as the pre-culture in step (b-1).

When performed in a range outside the culture conditions, the content of the polycan obtained later may be lowered.

Finally, the culture solution is sterilized and then filtered (step c).

In this step, the culture solution was sterilized at 110 to 130°C for 10 to 30 minutes to kill the mycelium and filter it with an ultrafilter (UF) to obtain a biopolymer derived from black yeast.

Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited thereto.

[Example]

Example 1: Preparation of medium composition

The medium composition contains 8 parts by weight of ammonium chloride (NH ₄ Cl), 2 parts by weight of magnesium sulfate (MgSO ₄ ), and 8 parts by weight of vitamin C based on 100 parts by weight of lignosulfonate sterilized with a carbon source. Used.

Example 2: Preparation of medium composition

A medium composition was prepared in the same manner as in Example 1, except that ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ) was used instead of ammonium chloride (NH ₄ Cl).

Example 3: Preparation of medium composition

A medium composition was prepared in the same manner as in Example 1, except that ammonium nitrate ((NH ₄ )NO ₃ ) was used instead of ammonium chloride (NH ₄ Cl).

Example 4: Biopolymer Produce

All cultures were inoculated into 100 ml of the medium composition of Example 1 prepared by taking sterilized Aureobasidium pullulans SM-2001 (KCCM 10307) from a solid medium and sterilized in a 250 ml flask. , Cultured by shaking for 36 hours at a shaking speed of 220 rpm at 25 ℃ to prepare a seed culture. This culture was inoculated with 2% by weight of the same medium composition used in the previous culture of 200 ml prepared by sterilizing the seed culture medium in a 500 ml flask, and cultured for 3 days in the same manner as the previous culture to obtain a culture solution. . The obtained culture solution was sterilized at 1.2 atmospheres for 20 minutes at 121? and the filtrate filtered through an ultrafiltration membrane (UF) was collected to obtain a biopolymer.

Example 5: Biopolymer Produce

A biopolymer was prepared in the same manner as in Example 4, except that the medium composition of Example 2 was used instead of the medium composition of Example 1.

Example 6: Biopolymer Produce

A biopolymer was prepared in the same manner as in Example 4, except that the medium composition of Example 3 was used instead of the medium composition of Example 1.

Comparative example 1: Preparation of medium composition

Glucose was added to 10.0g/L as a carbon source, and 2.0g/L yeast extract (yeast extract, Difco Lab., USA) was used as a nitrogen source, and 2.0g/L of vitamin C and 0.5g/mineral minerals were used. L K ₂ HPO ₄ , 0.5g/L MgSO ₄ ·7H ₂ O was mixed and sterilized to prepare a medium composition.

Comparative example 2: Preparation of medium composition

A medium composition was prepared in the same manner as in Example 1, except that yeast extract (yeast extract, Difco Lab., USA) was used instead of ammonium chloride (NH ₄ Cl) as a nitrogen source.

Comparative example 3: Preparation of medium composition

A medium composition was prepared in the same manner as in Example 1, except that the inorganic magnesium sulfate (MgSO ₄ ) was not used.

Comparative example 4: Preparation of medium composition

A medium composition was prepared in the same manner as in Example 1, except that inorganic magnesium sulfate (MgSO ₄ ) and vitamin C were not used.

Comparative example 5: Biopolymer Produce

A medium composition was prepared in the same manner as in Example 4, except that the medium composition of Comparative Example 1 was used instead of Example 1.

Comparative example 6: Biopolymer Produce

A medium composition was prepared in the same manner as in Example 4, except that the medium composition of Comparative Example 2 was used instead of Example 1.

Comparative example 7: Biopolymer Produce

A medium composition was prepared in the same manner as in Example 4, except that the medium composition of Comparative Example 3 was used instead of Example 1.

Comparative example 8: Biopolymer Produce

A medium composition was prepared in the same manner as in Example 4, except that the medium composition of Comparative Example 4 was used instead of Example 1.

Pictures of the cultures cultured according to Examples 4 to 6 and Comparative Examples 5 to 8 are shown in FIG. 1.

[Test Example]

Test example 1: yeast culture Polycan Content check

The contents of polycans contained in the cultures prepared according to Examples 4 to 6 and Comparative Examples 5 to 8 were measured for the content (% (w/v)) of polycans compared to the total culture solution produced, The method for measuring the content of polycans is shown in FIG. 2, which is measured by the alcohol immersion method, and the results of the content are shown in FIG. 3 and Table 1 below.

division Example 4 Example 5 Example 6 Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Polycan
%(w/v) 1.8 1.6 1.6 1.2 1.1 1.3 1.2

According to this, the content of polycans in the culture medium in Examples 4 to 6 using the medium of Examples 1 to 3 using the lignosulfonate as the carbon source and the ammonium salt as the nitrogen source and containing minerals and vitamins is relatively Appeared high.

Test example 2: Polycan Yield Check

The yields of beta-glucan (beta-1,3/1,6-glucan), which is a main component of polycans contained in cultures prepared according to Examples 4 to 6 and Comparative Examples 5 to 8, are carbon sources of lignosulfonate. The beta glucan content compared to 1 g was measured and confirmed.

The yield of beta-glucan (beta-1,3/1,6-glucan) is measured by using the megazyme kit to measure the total amount of glucan and the amount of alpha-glucan in the strain culture in powder form. A photograph confirming the amount of glucan is shown in FIG. 4, and the content results are shown in FIG. 5 and Table 2 below.

division Example 4 Example 5 Example 6 Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Beta glucan
(g/g) 0.75 0.64 0.64 0.48 0.44 0.52 0.48

According to this, Examples 4 to 6 of the present invention was found to have higher beta glucan yields compared to Comparative Examples 5 to 8. In particular, Example 4 was found to have a beta glucan yield of 5% or higher compared to Examples 5 and 6.

Considering that other global companies also obtain beta glucan in a yield of 0.67 to 0.7 g/g, it is understood that the method for preparing a biopolymer according to Example 1 of the present invention can obtain beta glucan with a very high yield. Can.

The embodiments of the present invention have been described above, but those skilled in the art can add, change, delete, or add components within the scope of the present invention as set forth in the claims. It will be said that the present invention can be variously modified and changed by the like, and this is also included within the scope of the present invention.

Claims (6)

Carbon source lignosulfonate; Nitrogen source ammonium salt, mineral; And vitamin C; a media composition for preparing biopolymers including beta glucan derived from black yeast (Aureobasidium). According to claim 1,
The ammonium salt is ammonium chloride (NH ₄ Cl), ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), ammonium nitrate ((NH ₄ )NO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and ammonium acetate (CH ₃ COONH ₄ ) biopolymer production medium composition comprising beta-glucan derived from black yeast, characterized in that at least one selected from the group consisting of. According to claim 1,
The mineral is a medium composition for preparing biopolymers containing beta-glucan derived from black yeast, characterized in that at least one selected from potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (MgSO ₄ ). According to claim 1,
The medium composition,
With respect to 100 parts by weight of lignosulfonate, the carbon source,

2 to 10 parts by weight of an ammonium salt that is a nitrogen source;
1 to 10 parts by weight of minerals; And
Vitamin C 1 to 10 parts by weight; Biopolymer production medium composition comprising beta-glucan derived from black yeast, characterized in that it comprises a. (a) One or more selected from lignosulfonate as a carbon source, ammonium salt as a nitrogen source, potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (MgSO ₄ ), and black yeast (Aureobasidium) containing vitamin C Preparing a media composition for preparing a biopolymer according to claim 1 comprising beta-glucan;
(b) inoculating the medium composition with black yeast (Aureobasidium) and culturing to prepare a culture solution; And
(c) filtering the culture solution after sterilization; a method for preparing a biopolymer containing beta-glucan derived from black yeast. The method of claim 5,
The black yeast is aureobasidium pullulans (Aureobasidium pullulans) characterized in that the production method of biopolymers containing beta-glucan derived from black yeast.

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