KR20170133687A - Fermented liquid fertilizer containing chitooligosaccharides and preparing method thereof - Google Patents

Abstract

The present invention relates to a liquid manure composition, which comprises a chitooligosaccharide culture solution having a molecular weight of 10,000 or less obtained by decomposing chitosan with a chitosanase-degrading enzyme culture and chitosan added in a proportion of 5 to 15 wt%, and to a manufacturing method thereof, in which the chitosan is decomposed into chitooligosaccharides having a relatively low molecular weight with a certain degree of polymerization which is easily absorbed into crops, and the chitosan is added at a high concentration to store microorganisms in a dormant state, so that a change in products is not caused, the shelf life is extended, the volume becomes small due to the high concentration of the products to be easily stores and used, a long-lasting effect of the products is maintained, and the crops cultivated using the liquid manure composition of the present contain a large amount of chitooligosaccharide.

Description

TECHNICAL FIELD The present invention relates to fermentation liquid compositions containing chitooligosaccharide and fermented liquid fertilizer containing chitooligosaccharides and preparing method thereof,

The present invention relates to a fermentation broth composition containing chitooligosaccharide, and more particularly, to a composition for fermentation broth for cultivating crops using chitooligosaccharide reduced in molecular weight using microorganisms and a method for producing the same.

Chitosan is obtained by deacetylation of chitin, which crushes, deproteinizes, and dechlorinates the bones of crustaceans such as crabs, shrimp, and other mollusc bites such as squid and squid, and N-acetyl-D-glucosamine -acetyl-D-glucosamine) and D-glucosamine are β- (1,4) -bonded polysaccharides, and the ratio of D-glucosamine in the molecule is 60% or more. Chitosan is degraded by gastric acid (pH 0.9 ~ 1.5), Lysozyme, intestinal bacteria, chitinase, chitosanase and the like present in the body at the time of ingestion and finally decomposed by acetylglucosamine, glucosamine, chitosan, Oligosaccharides and the like.

Chitooligosaccharide is obtained by decomposing chitosan into oligosaccharides in the form of an oligomer having 2 to 10 molecules by acid treatment or enzymatic treatment. The chitooligosaccharide has a smaller molecular weight and is easier to absorb than chitin and chitosan, which are high molecular substances. These chitooligosaccharides can have both characteristics of chitosan and oligosaccharide, which is very helpful for the growth of beneficial bacteria in the intestines.

Chitosan is a polysaccharide that is not digested by animal digestive enzymes. It is characterized as an animal dietary fiber. It is charged with a weak cation charge and excreted in association with charged bile acid charged with an anion charge. In this process, it is necessary to synthesize bile acid as much as the excreted amount. In order to synthesize bile acid, cholesterol accumulated in the body is used to control the concentration of cholesterol in blood, and thus it has been confirmed that cholesterol is improved.

In addition, chitosan penetrates into bacterial cells and adsorbs on DNA, which interferes with DNA transcription or adsorbs on the surface of bacterial cells, thereby inhibiting cell division and inhibiting mass transfer through the cell membrane. In this regard, chitooligosaccharide and water-soluble chitosan have been found in E. coli , Staphylococcus ( Staphylococcus aureus) aureus), cholera, salmonella (Salmonella cholesterol and cholera (Chung et al., 2002), chitooligosaccharide has antibacterial effect on fish-borne vibrio ( Vibrio ) (Chung et al., 2005) and chitooligosaccharides with molecular weight of 3,000 or less ( Candida albicans ) and mycorrhizal Trichophyton rubrum ( Phytophthora infestans ) were effective in preventing tomato and potato blight caused by Phytophthora infestans (Choi et al., 2011) apoptosis, and so on (Ji et al., 2006). These antimicrobial properties are limited and effective in the molecular weight range of 10,000 to 100,000. Effective antibacterial activity is shown at low molecular weight of 40,000 or less for pathogens and antibacterial activity against beneficial bacteria at high molecular weight of 150,000 or more.

Chitosan is also known to exhibit tumor suppression and immune enhancing effects. In this regard, chitosan oligosaccharides of hexamer have been shown to be highly potent tumor suppressor when administered intravenously to rats induced by solid tumors (Suzuki et al., 1986; Tokoro et al., 1988) Oligosaccharides have been shown to inhibit the metastasis inhibition of LLC (Lewis lung carcinoma) as well as lung cancer (Tsukada et al., 1990). In terms of macrophage activation in terms of immunological activity of chitin derivatives, 30% deacetylated chitin, 70% deacetylated chitin, chitosan and carboxymethyl chitin have macrophage activation ability, among which 70% deacetylated chitin The ability to activate macrophages was found to be the strongest (Nishimura et al., 1985). In addition, 70% deacetylated chitin exhibited effective activity against NK cells (natural killer cell), helper T cell (helper T cell) and cytotoxic T lymphocyte in addition to mouse peritoneal macrophage activation ability, .

Chitosan is a high molecular weight material with a molecular weight of over 200,000 and is not easily absorbed by crops because of its high molecular weight. In the case of chitooligosaccharides obtained by decomposing polymer chitosan, it is known that when the molecular weight is relatively high, the chitooligosaccharide is not easily absorbed by the crop. It is known that chitooligosaccharides that can be absorbed in plants have an optimum molecular weight of 10,000 or less (Korean Chitosan Society).

There is also a technology in which a cultivar of Rizofocus is fermented to produce a liquid containing chitooligosaccharide having a molecular weight of 10,000 or less and applied to crops, but the level of chitooligosaccharide in actual crops is reported to be very low. In addition, it is known that a commercially available polymer chitosan or chitooligosaccharide product takes a long period of 3-5 years until the plant absorbs it.

Korean Patent Registration No. 10-0396833 Korean Patent Registration No. 10-1357814 Korean Patent Registration No. 10-1384470

In order to increase the content of chitooligosaccharides in crops, relatively high concentration of chitooligosaccharide should be contained in the product to be treated with the crops using the livestock and the like, and a relatively low degree of polymerization (DP, Degree of Polymerization) The development of a technology capable of producing an oligomer having a low molecular weight is desired.

In addition, when the microorganism is mixed with the microbial culture to produce chitosan, when the microorganism is not stored properly, the microorganism grows due to the characteristics of the culture medium, and if it is placed in a place exposed to sunshine or sunshine, the microorganism is over- cultured, There is a problem that such a problem can not be expressed.

In order to solve the above problems, the present invention provides a chitooligosaccharide having a relatively low molecular weight and a high degree of polymerization, which can be easily absorbed into a crop by fermentation decomposition using a chitinolytic enzyme microorganism. To prevent microbial contamination, To provide a chitooligosaccharide fermentation broth composition capable of being stably stored for a long period of time and capable of containing a large amount of chitooligosaccharide in cultivated crops.

In order to achieve the above object, the present invention includes a culture solution containing chitooligosaccharide having a molecular weight of 10,000 or less obtained by decomposing chitosan into a chitosanase-degrading enzyme culture, and chitosan added in a proportion of 5 to 15 wt% ≪ / RTI >

In the composition of the present invention, it is preferable that the chitosanase is a lysozyme oligosporus.

The present invention also provides a method for producing a chitosan-degrading enzyme, comprising: culturing a chitosanase in a liquid medium for 40 to 56 hours to obtain a liquid seed culture; Culturing the liquid seed culture on a solid medium and culturing the solid medium for at least 40 hours to obtain a solid culture; Drying and pulverizing the solid culture to obtain a powdery culture; Chitosan powder and sterilized water are mixed at a ratio (w / v) of 1: 1 ~ 3 and stirred at 2,500 ~ 3,500 rpm for 1 ~ 3 hours. Acetic acid is added to the above- And stirring the mixture at 2,500 to 3,500 rpm for 0.5 to 2 hours to obtain a chitosan solution; Mixing the chitosan solution with the liquid seed culture and stirring at 2,500 to 3,500 rpm for 1 to 3 hours; Culturing the agar-cultured product by adding the above-mentioned culture medium and sterilized water, stirring the mixture at 2,500 to 3,500 rpm for 40 to 56 hours, and filtering the supernatant to obtain a primary fermentation broth; And adding chitosan to the primary fermentation broth so that the final chitosan concentration is 5 to 15% by weight, and then allowing to stand at room temperature for 20 to 50 days.

In the method of the present invention, it is preferable that the chitosanase gene is a lysozyme oligosporus.

In the method of the present invention, it is preferable that the solid medium is a mixture of rice bran, zeolite and sterilized water.

In the method of the present invention, it is preferred that the pulp-seed culture is pulverized to a size of 50 to 70 mesh.

The present invention relates to a method for producing a chitosan-degrading enzyme microorganism, which comprises obtaining a liquid seed culture obtained by culturing a microorganism strain of chitosanase, obtaining a solid culture by solid-culturing the liquid seed culture, drying and pulverizing the solid culture, After mixing and stirring the powder and sterilized water, acetic acid was mixed and stirred to obtain a chitosan solution. The chitosan solution was mixed and stirred with the liquid seed culture, and the seed culture and the sterilized water were added to the stirred culture By producing the primary fermentation broth, the chitosan is decomposed into chitooligosaccharides having a relatively low molecular weight, which is easy to absorb into the crop, with a certain degree of polymerization.

In addition, by adding chitosan to the primary fermentation broth at a high concentration to keep the microorganism in a dormant state, it is possible to solve problems such as the growth of microorganisms, the overgrowth of microorganisms, and the microbial death due to the fermentation broth containing conventional microorganism culture solution. Therefore, it is possible to extend the shelf life more than 2 years without the deterioration of the product even if it is not kept at the farmhouse or stored at low temperature, and it is easy to store and use because the volume is low due to the high concentration of the product. , It is effective to contain a large amount of chitooligosaccharide in the cultivated crops.

1 is a graph showing the amount of reducing sugar obtained when a chitin substrate is hydrolyzed with a ribofusol oligosporoschitin degrading enzyme.
2 is a graph showing the amount of each of N- acetyl-chitooligosaccharide (N -Acetylchitooligosaccharides) that is generated when sikyeoteul hydrolyzed with spokes Russ chitin-decomposing enzyme oligonucleotide crispus rayijo 0.6% colloidal chitin by time.
3 is a manufacturing process diagram showing one embodiment of a process for producing a primary fermentation broth containing chito oligosaccharide.
FIG. 4 shows TLC analysis results of each standard solution and fermentation broth, wherein (1) is N-acetyl-D-glucosamine, (2) is D-Glucosamine, (3) 4) is a 5X fermentation broth, and (5) is an acid-decomposed 5X fermentation broth.
5 shows the result of HPLC analysis of each standard solution and the fermentation broth, wherein (A) N-acetyl-D-glucosamine, (B) D-glucosamine, (C) , And (E) is an acid-decomposed 5X fermentation broth.
6 shows the results of analysis of the molecular weight of chitooligosaccharide in the Pullulan standard solution and the fermentation broth.
7 is a test report verifying the content of chitooligosaccharide of bean sprouts cultivated with the fermentation broth ratio of the present invention.
FIG. 8 is a test report verifying the content of chitooligosaccharide of tomatoes grown with the fermentation broth ratio of the present invention.
FIG. 9 is a test report verifying the content of chitooligosaccharide of an apple cultivated with the fermentation broth of the present invention.

The fermentation broth composition of the present invention comprises a chitooligosaccharide culture solution having a molecular weight of 10,000 or less obtained by digesting chitosan with a chitosanase-degrading enzyme culture, and chitosan added in a proportion of 5 to 15 wt% in the total weight of the composition.

As the chitosanase, strains commonly used for chitosan degradation may be used, and it is preferable to use strains belonging to the genus Rizofus, and it is most effective to use a strain of Rizofocus oligosporus.

The method for preparing the fermentation broth composition of the present invention is as follows.

(1) Production of liquid seed culture

First, the chitosanase is cultivated in a liquid medium for 40 to 56 hours to obtain a liquid seed culture.

As the chitosanase, strains commonly used for the decomposition of chitosan can be used. It is preferable to use strains belonging to the genus Lyapofus, and it is most effective to use strains of Lyapofus Oligosporese.

When the density of the soil after treatment with Rizofusol oligoporus was measured, the density was 0.73 × 10 ⁶ cfu / g after 48 hours of treatment, 8.7 × 10 ⁶ cfu / g after 120 hours of treatment, 3.5 × 10 ⁷ cfu / g after 720 hours of treatment, and 8.3 × 10 ⁷ cfu / g after 720 hours of treatment, and the density is stably maintained until 720 hours (about 1 month) after fixing in soil for about 48 hours.

As the liquid medium for culturing the chitosanase, for example, liquid mediums commonly used can be used. Examples of the liquid medium include PDA (Potato dextrose agar) medium, PDB (Potato dextrose broth), Nutrient broth, A culture medium, a culture medium, and the like.

(2) Preparation of solid culture

The liquid seed culture is spread on a solid medium and cultured for 40 hours or more, preferably 40 to 60 hours to obtain a solid culture.

The solid medium is preferably a mixture of rice bran, zeolite and sterilized water, and the rice bran, zeolite and sterilized water are mixed preferably in a weight ratio of 2: 3: 6 to 8: 4 to 6.

It is preferable to spray the liquid seed culture at a ratio of 15 to 25 (v / w) to the solid medium.

(3) Preparation of a culture of a seed culture

The solid culture is dried and then pulverized to obtain a pulmonary seed culture. As the drying method, it is preferable to use a ventilation drying method, and the dried solid culture is preferably pulverized to a size of 50 to 70 mesh.

(4) Preparation of chitosan solution

Chitosan powder and sterilized water are mixed at a ratio (w / v) of 1: 1 ~ 3 and stirred at 2,500 ~ 3,500 rpm for 1 ~ 3 hours. Acetic acid is mixed with the above-mentioned cross-linking solution in a proportion of 10 to 20% by weight of the sterilized water and stirred at 2,500 to 3,500 rpm for 0.5 to 2 hours to obtain a chitosan solution.

(5) Mixture of chitosan solution and liquid seed culture

The chitosan solution is mixed with the liquid seed culture and stirred at 2,500 to 3,500 rpm for 1 to 3 hours.

(6) Mixing of Chromosomal Cultures

The above-mentioned culture medium is added to the culture medium and sterilized water, and the mixture is stirred at 2500 to 3,500 rpm for 40 to 56 hours. The supernatant is filtered to obtain a primary fermentation broth.

(7) Addition of chitosan

Chitosan is added to the primary fermentation broth. The chitosan is preferably added so that the concentration of the final chitosan in the composition is 5 to 15% by weight, more preferably 10 to 1% by weight. After adding chitosan, the mixture is allowed to stand at room temperature for 20 to 50 days to obtain a fermentation broth composition. It is more preferable to leave it for 30 days.

When the product is not properly stored, the product produced by adding the microorganism culture solution to decompose the chitosan grows in the nature of the culture liquid and the microorganism is over-cultured when exposed to high temperature or direct sunlight, resulting in gas generation and microorganism death, have.

To solve these problems, chitosan is added to the primary fermentation broth fermented with chitosan and microorganism culture solution at a high concentration and left for 20 to 50 days to make the microorganism into a dormant state. If the concentration of chitosan is 5 wt% or more, the microorganism becomes dormant. If the chitosan concentration is more than 10 wt%, most microorganisms become dormant. If the concentration is 15 wt% or more, all microorganisms become dormant, .

The chitooligosaccharide and the microorganism which are brought into a low molecular state by the above process are kept in a dormant state without losing their activity. When they are diluted with water and sprayed in water during use, microorganisms are adsorbed on the soil and proliferate, , A larger amount of chitooligosaccharide can be absorbed into the crop together with the sprayed chitooligosaccharide. Therefore, the crops grown using the fermentation broth composition of the present invention contain a large amount of chitooligosaccharide and are detected.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. These examples and experimental examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

< Experimental Example  1>

Selection of chitosan digesting bacteria

In order to select the most suitable microorganism for producing low molecular weight chitooligosaccharides by decomposing chitosan, the following experiment was conducted.

Microorganism strains include Rhizopus ( Rhizopus) oligosporus , Rhizopus oryzae ) and Rhizopus sp. ( Rhizopus sp.) was used.

These strains were inoculated into PDB (Potato dextrose broth), nutrient broth (MRS broth) and MRS broth, respectively. After incubation at 30 ± 1 ° C and 60% humidity, Respectively. The observed growth conditions of mycelium are shown in Table 1 below.

Types of microorganisms Mycelial growth state Raisopus oligosporus +++++ Rijopus Oridge ++ Rizofus sp. +++

As shown in the above Table 1, the mycelial growth state of rizopus oligoporus was the most excellent among the three strains. Therefore, Rizofocus oligoporus was used as a published strain for the production of chito oligosaccharide.

< Experimental Example  2>

Rizofus Oligo spores Measurement of chitosan resolution

1 liter of PDB (Potato dextrose broth, Difco) was prepared and sterilized at 121 ° C for 20 minutes using a high pressure sterilizer. The sterilized medium was inoculated with 1 ml of a strain of Rizofocus oligosporus strain and cultured with shaking for 48 hours at an ambient temperature of 30 1 캜 and a humidity of 60%.

1 kg of a chitosan powder having a molecular weight of 300,000 and the culture of the above-mentioned razorupozolosporus were mixed at a ratio (w / v) of 1: 1, respectively, at 28 to 30 DEG C to confirm the decomposing ability of the polymer chitosan by Rizofusu oligosporus The change in molecular weight over time was measured while incubating for 48 hours.

The molecular weight of chitosan was measured using a Ubbelohde viscometer (SI Analytics GmbH, Ubbelohde Viscometer M), and a solution of 4 M urea, 0.2 M acetic acid and 0.1 M sodium chloride in a volume ratio of 1: 1: 1 The cultured chitosan fermented by the above-mentioned Rizofusu oligosporus strain was added to 0.05%, 0.10%, 0.15% and 0.20%, respectively. The measurement was repeated eight times to obtain an average value. The results are shown in Table 2 below.

Fermentation time (h) Molecular weight (Mw) 6 274,000 12 254,000 18 192,000 24 128,000 30 64,000 36 30,500 42 14,200 48 Not measurable

From the results in Table 2, it was confirmed that chitosan was decomposed by chitinolytic enzyme contained in the cultured solution of Rizofocus oligosporus over time, resulting in a reduced molecular weight and decomposed to such an extent that measurement of molecular weight was impossible at 48 hours after fermentation .

< Experimental Example  3>

Rizofus Oligosporus  Chitinolytic enzyme of  Identification of substrate specificity

For chitosan decomposition rayijo crispus oligonucleotide production sports Russ coenzyme (chitin-degrading enzymes), chitin medium is 15g / ℓ in the production medium 100㎖, yeast extract _{0.5g / ℓ, (NH 4)} 2 SO 4 1.0g / ℓ , and MgSO ₄ 1.36g / l. &lt; / RTI &gt; The production medium was supplemented with 1 mL of a strain of Lysophospholysporus strain and cultured in a 500 mL Erlenmeyer flask at 30 ° C. for 7 days to use as a chitinolytic enzyme coenzyme.

The chitinase enzyme activity was calculated by measuring the absorbance at 540 nm using a microplate reader (Microplate Reader, Anthos Laboratories, Anthos htIII) according to the colorimetric method of Ressig et al. One unit of enzyme was the amount of enzyme capable of hydrolyzing 1 μ mole of N-Acetyl-D-Glucosamine (GlcNAc) for 1 hour.

Substrates specific for the substrate specificity of chitinolytic enzymes were 0.6% colloidal chitin, glycol chitin, purified powder chitin, CM-chitin, A powder chitin (practical grade) solution was used.

Successively chitin-decomposing enzyme (12units / ㎖) as the number of μ mole of the oligosaccharide produced in accordance with the time by reacting said substrate at 37 ℃ was measured according to the analytical method, such as Ressig. That is, N-acetylchitooligosaccharides (N-acetylchitooligosaccharides) produced by degradation products of each substrate by chitinolytic enzymes were prepared by using (GlcNAc) _n (n = 1-6) as the reference solution and μ Bondapak NH ₂ ㎜) column, and detected with a UV detector (Shimadzu, SPD-20A) at 210 nm. The flow rate was 1.0 ml / min and the mobile phase was analyzed using an acetonitrile-water solution at 70:30.

As can be seen from FIG. 1, the chitinolytic enzyme was hydrolyzed to the highest amount of oligosaccharide at a reaction time of 2 hours when colloidal chitin was used as a substrate.

The degree of hydrolysis was in the order of colloidal chitin, glycol chitin, purified powder chitin, practical grade, and CM-quinine, with the amounts of oligosaccharide being 4.55, 1.93, 1.23, 0.82 and 0.06 μmole / ㎖, respectively.

The relative hydrolysis rates according to the substrate were 100% of colloidal chitin, 43% of glycol chitin, 25% of purified chitin, 19% of practical grade and 1.5% of CM-chitin as shown in Table 3 below.

temperament Relative hydrolysis rate (%) Colloidal chitin 100 Glycol chitin 52 Refined powder chitin 28 Powdered chitin (practical grade) 21 CM-chitin 3

As a result of hydrolysis of the chitinolytic enzyme to the colloidal chitin having the highest hydrolysis rate, the oligosaccharide was rapidly hydrolyzed for 20 minutes in the initial stage of the hydrolysis reaction as shown in FIG. 2, Hydrolysis of the substrate.

< Example  1>

Chitooligosaccharide  Preparation of primary fermentation broth containing

1 L of PDB was prepared and sterilized at 121 ° C for 20 minutes using a high pressure sterilizer. On the sterilized medium, Rizofocus oligosporus 1 ml of the strain was inoculated and cultured in a shaking culture for 48 hours at an ambient temperature of 30 ± 1 ° C and a humidity of 60% to obtain a liquid seed culture.

(V / w, 50) was added to 1,500 g of a mixture obtained by mixing 50 ml of the culture of the above-mentioned raiogwuso oligosporus cultured in liquid culture (liquid seed culture) in a mixture of rice bran: zeolite: sterilized water at a weight ratio of 3: 7: Ml / 1,500 g), and the cells were further cultured for 48 hours or more at 30 ± 1 ° C and 60% humidity. The solid culture which had been sufficiently cultured was air-dried and then pulverized to a size of 60 mesh using a pulverizer to obtain a powdery culture.

Separately, chitosan powder was mixed with sterilized water at a ratio of 1: 2 (w / v, 15 kg of chitosan: 30 L of sterilized water) and stirred at 3,000 rpm for 2 hours. 5 L of acetic acid was added thereto, and the mixture was stirred at 3,000 rpm for 1 hour to completely dissolve chitosan in a gel form to obtain a high concentration chitosan solution.

The chitosan solution was loaded with a culture of Lyoporozolosporus liquid phase, followed by stirring at 30 ± 1 ° C and 3,000 rpm for 2 hours. 300 g of the seed culture was added again, sterilized water was added thereto, and the final volume was adjusted to 500 L. The mixture was stirred at 30 ± 1 ° C for 2 days at 3,000 rpm. The supernatant was filtered to use as the primary fermentation broth.

The production process of the fermentation broth of the present embodiment as described above is shown in Fig.

< Experimental Example  4>

Rizofus Oligosporus  Of the primary fermentation broth Chitooligosaccharide  analysis

Preparation of sample

(1) Preparation of D-glucosamine and N-acetyl-D-glucosamine standard solution

D-Glucosamine hydrochloride (≥75% crystalline, Sigma), N-acetyl-D-glucosamine (≥99%, Sigma) was dissolved in water to give 2.5 mg / And 10 mg / ml, and used as a standard solution.

(2) Chitooligosaccharide  Preparation of standard solution

The deacetylated chitosan (≥75% deacetylated chitosan, Sigma) was dissolved in a 1 M acetic acid solution to prepare a 1% (w / v) solution. Hydrochloric acid (35%) was mixed with the chitosan acetic acid solution at a ratio of 1: 1 (v / v) and then hydrolyzed at 96 ° C for 10 hours. The hydrolyzate was again mixed with 10 volumes of acetone, refrigerated for 1 hour, and then centrifuged at 15,000 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the precipitated chitosan degradation product (chito oligosaccharide) was dried at room temperature for 1 hour or more. The dried precipitate was dissolved in an appropriate amount of 1 M acetic acid solution and used as a standard solution of chitooligosaccharide.

(3) Preparation of 5X fermentation broth samples

The fermented culture was concentrated 5 times with a centrifugal vacuum concentrator (Labconco, model-7810015) and used as an analytical sample (5X fermentation broth).

(4) Acid-decomposed  Preparation of 5X fermentation broth samples

For deacetylation of N-acetyl-D-glucosamine residues in the chitooligosaccharide contained in the fermentation broth, hydrochloric acid was added to the 5X fermentation broth to a final concentration of 1 N and hydrolyzed at 96 ° C for 1 hour to obtain 5X fermentation A culture sample was prepared.

One. Thin layer chromatography ( TLC ) analysis

(GlcN) standard solution, N-acetyl-D-glucosamine (GlcNAc) standard solution, chito oligosaccharide standard solution, 5X fermentation broth and acid-decomposed 5X fermentation broth at a concentration of 10 mg / , Silica gel 60), respectively.

A solution of n-butanol, methanol, 25% ammonia solution and water in a volume ratio of 5: 4: 2: 1 was used as a TLC developing solvent.

After the development, aniline-diphenylamine reagent (400 μl of aniline, 400 mg of diphenylamine, 20 ml of acetone, and 3 ml of 85% phosphoric acid) was sprayed, and heated at 180 ° C. or higher to develop color, Respectively. The results are shown in Fig.

In FIG. 4, the lane 1 represents N-acetyl-D-glucosamine (GlcNAc), the lane 2 represents D-glucosamine (GlcN), the lane 3 represents the chitooligosaccharide standard solution, the lane 4 represents the 5X fermentation culture, Acid-degraded 5X fermentation broth. In the chitooligosaccharide standard solution of the third lane, the chitooligosaccharide spot of D-glucosamine polymerization degree of 1 to 6 was confirmed. In the case of the 5X fermentation broth of the fourth lane, D-glucosamine (GlcNAc) - Glucosamine was found to have a degree of polymerization of 1 ~ 6.

To confirm the more accurate sugar composition and polymerization degree of the 5X fermentation broth, hydrolysis and removal of acetyl (N-acetyl) residue from N-acetyl-D-glucosamine in a chitooligosaccharide contained in a 5X fermentation broth with a weak acid, , Lane 5, it was confirmed that the N-acetyl-D-glucosamine spot was almost completely disappeared due to the hydrolysis of the acetyl group, and the D-glucosamine spot increased instead.

It was also confirmed that the 5X fermentation broth mainly contains chitooligosaccharide having a degree of polymerization of 1 to 6 of D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc).

2. HPLC  analysis

The chitooligosaccharide composition of the fermentation broth was separated by HPLC with Asahipak NH2P-50 4E column (4.6 x 250 mm) and then detected by RI detector (Agilent Technologies, G1362A). Column oven temperature was 30 ℃, A flow rate of 0.5 ml / min and a mobile phase of 70:30 (v / v) acetonitrile-water solution were used.

In order to confirm the interference of other compounds other than sugar, it was separated by HPLC with Asahipak NH2P-50 4E column and detected with UV detector (Shimadzu, SPD-20A) at double wavelength (205/254 nm) Was analyzed using an acetonitrile-water solution at 30 캜, a flow rate of 0.5 ml / min, and a mobile phase of 70:30 (v / v). The results are shown in Fig.

(C) is a standard solution of chitooligosaccharide, (D) is a 5X fermentation broth, (E) is an acid-decomposed 5X fermentation broth Lt; / RTI &gt;

First, in each of the standard solutions, the N-acetyl-D-glucosamine (GlcNAc) peak showed retention time (Rt) at 9.94 min (A in FIG. 5) and D-glucosamine (FIG. 5B). The chitooligosaccharide standard solution was prepared according to the polymerization degree (DP) of D-glucosamine according to the degree of polymerization (DP) of DP2 11.51 min, DP3 13.16 min, DP4 15.25 min, DP5 17.79 min, DP6 20.87 min, DP7 24.59 min, DP8 29.55 min (Fig. 5C). &Lt; tb &gt; &lt; TABLE &gt;

The results of the 5X fermentation broth (FIG. 5D) were analyzed along with the results of the acid-degraded 5X fermentation broth (E in FIG. 5). As a result, in the case of the first peak (peak 1), N-acetyl-D-glucosamine Acetylglucosamine dimer (GlcNAc ₂ ), N-acetyl-D-glucosamine-D-glucosamine (GlcNAc-GlcN) as the chitosan dimer in the case of the second peak (peak 2) ) And D-glucosamine-N-acetyl-D-glucosamine (GlcN-GlcNAc). The third peak was judged to be a D-glucosamine dimer (GlcN-GlcN, DP2) Were sequentially judged as D-glucosamine trimers or Hexamers (DP3 to DP6).

Peaks 3 to 6 in red letters were judged to be chitooligosaccharides having a degree of polymerization higher than that of nonamer containing a large number of N-acetyl-D-glucosamine. The remaining shoulder peak was detected with a UV detector 205/245 double detector Was found to be an intermediate product that was not a sugar compound or less deacetylated by N-acetyl-D-glucosamine.

The composition of chitooligosaccharides in the fermentation broth is shown in Table 4 below.

Peak No. Rt Ingredients constitutional formula Content (Area%) One 9.95 N-acetyl-D-glucosamine
(N-Acetyl-D-glucosamine)
(GlcNAc)

15.63 2 10.97 Chitosan dimer
(Chitosan dimer)
(GlcNAc ₂ or GlcNAc-GlcN or GlcN-GlcNAc)

6.20 DP2 11.67 D-glucosamine dimer
(D-Glucosamine dimer)
(GlcN ₂₎

6.59 DP3 12.99 D-glucosamine trimer
(D-Glucosamine trimer)
(GlcN ₃₎

19.48 DP4 15.20 D-glucosamine tetramer
(D-Glucosamine tetramer)
(GlcN ₄₎

10.10 DP5 17.90 D-glucosamine dimer
(D-Glucosamine pentamer)
(GlcN ₅₎

5.15 3 18.67 Chitosan nonamer or more
(GlcNAc or GlcN) _{? 9}

11.02 DP6 20.64 D-glucosamine carrageenan
(D-Glucosamine hexamer)
(GlcN ₆₎

1.08 4 22.28 Chitosan nonamer or more
(GlcNAc or GlcN) _{? 9}

12.12 5 27.24 Chitosan
(GlcNAc or GlcN) _{? 9}

8.09 6 33.32 Chitosan
(GlcNAc or GlcN) _{? 9}

4.55

From the results of Table 4, it was confirmed that the low molecular weight chitooligosaccharides identified in the fermentation broth were mainly composed of chitooligosaccharides having DP2 to DP6.

The content of N-acetyl-D-glucosamine in the fermentation broth was 6.80 mg / ml (0.134%), and the content was calculated as the relative area (Area%) based on the polymerization degree DP2 to DP6 chitooligosaccharide Results The content of main low molecular weight chitooligosaccharide in the fermentation broth was 0.369%.

< Example  2>

Chitooligosaccharide  contain Fermentation  Produce

In order to prevent the contamination of germs in the primary fermentation broth and to prevent the production of chitooligosaccharides and the further decomposition of the resulting low molecular weight chitooligosaccharides by using the residual enzyme activity of the chitinolytic enzyme contained in the culture broth, The final chitosan concentration was adjusted to about 10%, and the mixture was allowed to stand at room temperature for one month to prepare a final fermentation broth ratio.

< Experimental Example  5>

Fermentation Chitooligosaccharide  Character analysis

Pullulan standard set (Mp 342, 1,080, 6,100, 9,600, 21,100, 47,100, 107,000, Sigma) was dissolved in purified water to give each 10 mg / ml and used as a standard solution.

The fermentation broth prepared in Example 2 was concentrated 5 times with a centrifugal vacuum concentrator (model: Lab-7810015) and used as an assay sample.

Each standard and non-fermented broth samples were detected on a UV detector (Shimadzu, SPD-20A) dual wavelength (205/254 nm) by HPLC with a Polysep-GFC-P3000 column (4.6 × 250 mm, Phenomenex) 0.5 ml / min, and water was used as a mobile phase.

The molecular weight of the chitooligosaccharide contained in the final fermentation broth, which was left at room temperature for one month after addition of the final chitosan of the present invention at a final concentration of 10%, was confirmed in comparison with the pullulan standard solution.

The peak retention time (Rt) versus the maximum peak molecular weight (Mp) of the pullulan standard solution is shown in Table 5 below, and a standard curve was prepared based on the peak retention time. A standard curve of the pullulan standard solution is shown in Fig. 6 (A).

Pullulan standard solution (Mp) Rt (min) 107,000 11.247 47,100 12.180 21,100 13.077 9,600 14.043 6,100 14.602 1,080 16.610 342 17.871

Next, the results of analyzing the fermentation broth ratio of the present invention under the same conditions are shown in FIG. 6 (B). As a result of confirming the expected molecular weight by substituting the main peak confirmed in the analysis into the Pullulan standard curve formula, it was confirmed that chitooligosaccharide (DP2 to DP6) having a relatively low molecular degree of polymerization of 6.5 to 18.7 minutes and Rt of 14.4 to 15.6 minutes And the chitooligosaccharides (DP11 to DP38) were two types of chitooligosaccharides.

The expected molecular weight and polymerization degree versus the retention time of the main peak detected in the fermentation broth are shown in Table 6 below.

Rt (min) Expected molecular weight
(Mw, dalton) Degree of polymerization (DP) ^* DP of Mp Remarks 7.50 2,670,906 13,354 Chitosan raw material 10.02 720,877 3,604 3,600 11.00 131,156 655 14.30 7,650 38 Polymerization degree
Chitooligosaccharide 14.44 6,770 30 to 37 34 14.78 5,047 22-28 25 15.30 3,236 14-18 16 15.64 2,413 11-13 12 15.80 2,102 10 16.49 1,160 2 to 6 Low molecular weight polymerization degree
Chitooligosaccharide 18.46 212 N-acetyl-D-glucosamine One 18.67 177 D-glucosamine One * The average molecular weight of each monomer of chitooligosaccharide is calculated as 200
Mp: Molar mass at the peak maximum

< Experimental Example  6>

Sprouts packaging test

The bean sprouts were cultivated according to the usual cultivation method. That is, first of all, it was washed three times for dehydration for a certain time. The dehydrated soybeans were rinsed three times after 30 minutes and the defective soybeans were selectively removed. The selected healthy beans were divided into 5 cultivation cans after 3 hours. At this time, the soybeans were laid on the net laid on the bottom of the cultivation pail to a certain thickness, and the fermentation broth containing chitooligosaccharide was diluted to 10,000 ppm for 3 days at intervals of about 8 to 12 hours while covering the soybean One liquid was developed by watering (primary development stage). In the first stage of development, the voiced vegetation covered on the soybeans was harvested and laid over the beans so that the soybeans could be slightly buried with cleanly washed gypsum. At this stage, the sweet potatoes are introduced into the gaps between the beans so that the distance between the beans is maintained. The vodka was covered with the vodo soap again, and water was supplied 2-3 times a day. The diluted solution was 10,000 ppm in the same manner as described above for about 7 days, and was cultivated to grow the matured soybean sprout.

After cultivated soybean sprouts were randomly collected, the content of chitooligosaccharide contained in bean sprouts was confirmed by Korean Food Research Institute. The resulting test report is shown in Table 7 and FIG.

Specimen name Bean sprouts Test Items Chitooligosaccharide sexual Exterior Yellow solid Chito oligosaccharide (mg / g) 0.3

As shown in the results of Table 7, 0.3 mg / g of chito oligosaccharide was detected in bean sprouts.

< Experimental Example  7>

Tomato Pavement Test

A packed test was carried out by choosing a farmhouse of ripe tomatoes (Super Taotang / Korengon jongmyo) in Icheon-ri, Namseon-myeon, Andong-si, Gyeongbuk province. After the fertilization, the plants were treated 3 times before harvesting. The treatments were 3 times a total of 7 times every 7 days before harvesting, and treated with 200 pyeong of the fermentation broth diluted to 100ppm in water.

After the processed tomatoes were randomly collected, the content of the chitooligosaccharides contained in the tomatoes was confirmed by the Korea Food Research Institute. The resulting test report is shown in Table 8 and FIG.

Specimen name tomato Test Items Chitooligosaccharide sexual Exterior Tomato is a unique form Chito oligosaccharide (mg / g) 0.63

As shown in the results of Table 8, 0.63 mg / g of chito oligosaccharide was detected in tomato.

< Experimental Example  8>

Apple wrapping test

Apple (adverb) farmers in Punggi-eup, Yeongju, Gyeongsangbuk-do were selected and packaged. Treatment method Fertilization of the fermentation broth diluted to 100 ppm in water three times in total every 15 days before harvest was applied to 200 pyeong.

After the processed apple was randomly collected, the content of the chitooligosaccharide contained in the apple was confirmed by the Korea Food Research Institute. The resulting test report is shown in Table 9 and Figure 9 below.

Specimen name Apple (adverb) Test Items Chitooligosaccharide sexual Exterior Red solids Chito oligosaccharide (mg / g) 0.65

As can be seen from the results in Table 9, 0.65 mg / g of chito oligosaccharide was detected in the apple.

Claims (8)

A fermentation broth composition comprising a culture solution containing chitooligosaccharide having a molecular weight of 10,000 or less obtained by decomposing chitosan with a chitosanase-degrading enzyme culture, and chitosan added in a proportion of 5 to 15 wt% in the total weight of the composition. The method according to claim 1,
Wherein the chitosanase is a microorganism of the genus Lyapofus. The method according to claim 1,
Wherein the chitosanase is a lysozyme oligosporus. Culturing the chitosanase in a liquid medium for 40 to 56 hours to obtain a liquid seed culture;
Culturing the liquid seed culture on a solid medium and culturing the solid medium for at least 40 hours to obtain a solid culture;
Drying and pulverizing the solid culture to obtain a powdery culture;
Chitosan powder and sterilized water are mixed at a ratio (w / v) of 1: 1 ~ 3 and stirred at 2,500 ~ 3,500 rpm for 1 ~ 3 hours. Acetic acid is added to the above- And stirring the mixture at 2,500 to 3,500 rpm for 0.5 to 2 hours to obtain a chitosan solution;
Mixing the chitosan solution with the liquid seed culture and stirring at 2,500 to 3,500 rpm for 1 to 3 hours;
Culturing the agar-cultured product by adding the above-mentioned culture medium and sterilized water, stirring the mixture at 2,500 to 3,500 rpm for 40 to 56 hours, and filtering the supernatant to obtain a primary fermentation broth;
Adding the chitosan to the primary fermentation broth so that the final chitosan concentration is 5 to 15% by weight, and then allowing the fermentation broth to remain at room temperature for 20 to 50 days. 5. The method of claim 4,
Wherein the chitosanase is selected from the group consisting of chitosan and chitosan. 5. The method of claim 4,
Wherein the chitosan is added to the primary fermentation broth so that the final chitosan concentration is 10 +/- 1 wt%. 5. The method of claim 4,
Wherein the solid medium is a mixture of rice bran, zeolite, and sterilized water. 5. The method of claim 4,
Wherein the culture of the pulverized seed culture is pulverized to a size of 50 to 70 mesh.

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