KR101170978B1 - Microorganism producing chitosanase, method for producing chitooligosaccharides using the same and use thereof - Google Patents

Abstract

The present invention relates to a strain for producing chitosan degrading enzyme, a method for producing chitosan oligosaccharide using the same, and more particularly, a method for producing chitosan degrading enzyme, and more specifically, komamonas corrensis or chryseobacterium indologenes strain for producing chitosan degrading enzyme, It relates to a chitosan oligosaccharide production method and a feed, antibacterial or food composition containing the strain or culture thereof. The strain of the present invention is effective in producing chitosan oligosaccharides by producing chitosan degrading enzymes. The composition of the present invention contains chitosan oligosaccharides and has antimicrobial activity, and has an effect of enhancing immunity and anti-cholesterol ability, and thus can be effectively used as feed additives or food additives.

Description

Microorganism producing chitosan degrading enzyme, chitosan oligosaccharide manufacturing method using the same and its use {microorganism producing chitosanase, method for producing chitooligosaccharides using the same and use}

The present invention relates to a strain for producing chitosan degrading enzyme, a method for producing chitosan oligosaccharide using the same, and more particularly, a method for producing chitosan degrading enzyme, and more specifically, komamonas corrensis or chryseobacterium indologenes strain for producing chitosan degrading enzyme, It relates to a chitosan oligosaccharide production method and a feed, antibacterial or food composition containing the strain or culture thereof.

An ongoing challenge for the management of the Korean beef industry is to reduce mortality from calf diarrhea. Since calves are not born with immunity at birth, they are already faced with poor conditions at birth, so their defenses against disease are defenseless. In order to solve this problem, high-cost immune substances are directly supplied with antibiotics and colostrum. However, it is possible to prevent calf diarrhea and the like more effectively by feeding microbes with colostrum, which does not feed antibiotics and expensive immune substances, and allows the calf to produce immune substances.

Chitosan (Chitosan) is a polymer obtained from chitin through a deacetylation reaction, the macromolecule of chitin can be said to be a low molecular form. Chitosan is dissolved in dilute acidic solutions of inorganic acids such as hydrochloric acid and nitric acid and organic acids such as acetic acid, lactic acid, ascorbic acid and malic acid.It has a reactive free amino group, which lowers cholesterol, boosts immunity, lowers blood pressure, improves liver function, and lowers blood sugar. It has physiological activities such as antibacterial properties, and is widely applied to food, cosmetics, medicine, agricultural, livestock, and fisheries fields.

Chitosan made in this way is insoluble in water and alcohol solvents, has a characteristic of being soluble in weak acid, and is a pollution-free and non-toxic biodegradable substance. On the other hand, the low molecular weight of the chitosan with molecular weight of 100,000 to 200,000 is once again broken down to 2 to 10 glucosamines called chito-oligosaccharides. Although chitosan is a natural polysaccharide with high physiological activity, it does not dissolve in aqueous solution and is a very high molecular weight polymer, so even if it is ingested, most humans cannot absorb it because there is no enzyme to decompose chitosan in the human body. Rarely used. However, when chitosan is hydrolyzed to oligosaccharides, the molecular weight decreases, solubility and absorption in the body are greatly increased, and the bitter and astringent taste of chitosan is greatly reduced.

Chitosan oligosaccharides have antimicrobial activity against Gram-negative bacteria such as Escherichia coli and Salmonella, which cause diarrhea, because the cationic amine group of chitosan reacts with anionic carboxyl groups of microbial cell walls to inhibit the action of pathogenic microorganisms. Chitosan oligosaccharides also have the effect of enhancing immunity in livestock by activating T-cells, macrophage and natural killer cells in the immune system. In addition, the glucosamine finally produced by the microorganism is used as a precursor of antibiotics, and also widely used in the production of proteins by biotransformation and other food and pharmaceutical fields.

The production of chitosan oligosaccharides generally involves chemical degradation, physical degradation and enzymatic degradation. The acid hydrolysis method can easily decompose chitosan by using hydrochloric acid, hydrogen peroxide, sulfuric acid, etc., but it is difficult to control the molecular weight, the yield of oligosaccharide is notably low, and the distribution of oligosaccharides produced is not constant and most of the monomers are produced. Its disadvantage is its weak biological activity. In addition, the physical decomposition method is a method of decomposing chitosan by using ultrasonic waves, microwaves, ozone, ultraviolet rays, etc., and requires a special facility, and the reaction time is long, resulting in a change in physiological activity according to physical properties of chitosan. Therefore, many methods for producing oligosaccharides using microbial-derived enzymes that can compensate for these shortcomings have been studied, and it is urgent to discover microorganisms producing such chitosan degrading enzymes.

Accordingly, the present inventors completed the present invention by finding that the Komamonas corensis strain and the chrysobacterium indologenes strain derived from calf feces produced chitosan degrading enzymes while studying the microorganisms producing chitosan degrading enzyme. .

It is therefore an object of the present invention to provide a Komamonas corensis strain producing chitosan degrading enzymes.

It is another object of the present invention to provide a chrysobacterium indologenes strain producing chitosan degrading enzyme.

Another object of the present invention is to provide a feed composition containing the strain or its culture.

Another object of the present invention to provide an antimicrobial composition containing the strain or its culture.

Another object of the present invention is to provide a food composition containing the strain or culture thereof.

Another object of the present invention is to provide a chitosan oligosaccharide manufacturing method comprising culturing the strain in a medium containing chitosan.

In order to achieve the above object, the present invention provides a Komamonas corensis strain for producing chitosan degrading enzyme.

In order to achieve another object of the present invention, the present invention provides a chrysobacterium indologenes strain producing chitosan degrading enzyme.

In order to achieve another object of the present invention, the present invention provides a feed composition containing the strain or its culture.

In order to achieve another object of the present invention, the present invention provides a composition for antimicrobial containing the strain or its culture.

In order to achieve another object of the present invention, the present invention provides a food composition containing the strain or culture thereof.

In order to achieve another object of the present invention, the present invention provides a method for producing chitosan oligosaccharide comprising culturing the strain in a medium containing chitosan.

Hereinafter, the content of the present invention will be described in more detail.

The present invention provides a Comamonas corensis strain or a Chryeobacterium indologenes strain producing chitosan degrading enzyme.

The strain Comamonas corensis or chryseobacterium indologenes of the present invention is characterized by producing chitosan degrading enzyme.

Especially comamonas of the present invention isolated from calf feces ( Comamonas) koreensis hanwoo: Accession No. KACC 91496P) or Chryseobacterium indologenes hanyo ( Chryseobacterium) indologenes hanyoo: Accession No. KACC 91497P) produces chitosan-degrading enzymes, which produce chitosan oligosaccharides when cultured in chitosan-containing media. Therefore, the strain comamonas corensis of the present invention is preferably comamonas corensis Hanwoo ( Comamonas) koreensis hanwoo: Accession No. KACC 91496P), the strain of the present invention, Chloriseobacterium indologenes is preferably Chryseobacterium indologenes hanyo ( Chryseobacterium indologenes hanyoo: accession number KACC 91497P).

Chitosan oligosaccharides of the present invention are oligomers in which chitosan, a polymer of β-1,4-D-glucosamine, is partially degraded, and has various physiological functions. In the chitosan oligosaccharide of the present invention, preferably, 2 to 10 glucosamine may be polymerized, and more preferably 4 to 6 polymerized.

Comamonas corensis or chryseobacterium indologenes for producing chitosan degrading enzymes to produce chitosan oligosaccharides is to be provided for the first time in the present invention.

Meanwhile, the present invention provides a method for preparing chitosan oligosaccharide comprising culturing the strain in a medium containing chitosan.

The production method of the present invention is characterized by culturing the strain of the present invention in a medium containing chitosan. If the strain of the present invention is capable of producing chitosan degrading enzyme and thereby to decompose chitosan to produce chitosan oligosaccharide, any culture conditions are included in the culture of the production method of the present invention. In the case of Comamonas Corensis Hanwoo, preferably 0.1 to 10% (w / v) of chitosan, 0.5 to 5% (w / v) of glucose, and 1.0 to 5.0% (w / v) of yeast extract per liter of distilled water , NH ₄ Cl 0.05 to 0.5% (w / v) and NaCl 0.01 to 0.2% (w / v) can be prepared by incubating for 2 to 5 days at 20 ℃ to 40 ℃ in a medium containing, most preferably 1 to 5% (w / v) of chitosan, 0.5 to 1.5% (w / v) of glucose, yeast extract 1.0 to 2.0% (w / v), and NH ₄ Cl 0.05 to 0.15% (1 L of distilled water) w / v) and NaCl 0.05 to 0.1% (w / v) can be prepared by incubating for 3 to 5 days at 25 ℃ to 35 ℃ medium.

In addition, in the case of Cryeobacterium indologenes hanyo preferably 0.1 to 10% (w / v) chitosan per 1 L of distilled water, 0.1 to 5.0% (w / v) corn flour, soybean flour 20 ° C.-40 ° C. in a medium comprising 0.5-5% (w / v), (NH ₄ ) ₂ SO ₄ 0.01-0.5% (w / v) and K ₂ HPO ₄ 0.01-0.2% (w / v) It can be prepared by incubating for 2 to 5 days, most preferably 0.1 to 5% (w / v) chitosan per 1 L of distilled water, 0.1 to 1.5% (w / v) corn flour, soybean powder ( soybean flour) 25 ° C. in a medium containing 0.5-2% (w / v), (NH ₄ ) ₂ SO ₄ 0.01-0.1% (w / v) and K ₂ HPO ₄ 0.05-0.1% (w / v) It can be prepared by incubating at 3 to 5 days at 35 ℃.

In one embodiment of the present invention, microorganisms were isolated from the calf feces and cultured in a medium containing chitosan to find a strain having chitosan degrading activity.

As a result of the identification, it was confirmed that the comamonas corensis and chryseobacterium indologenes.

In another embodiment of the present invention to find the optimum culture conditions for the strain of the present invention and to measure the activity of chitosan degrading enzyme according to the culture days.

As a result, the strain of the present invention showed the highest chitosan degrading enzyme activity on the 4th day of culture, and was confirmed to produce chitosan oligosaccharides most efficiently.

In another embodiment of the present invention was confirmed the type of chitosan oligosaccharide prepared by chitosan degrading enzyme of the strain of the present invention.

As a result, coma Pseudomonas Koren of the present invention cis beef (Comamonas koreensis hanwoo: Accession No. KACC 91496P) was found to produce chitosan oligosaccharide hexamer mainly, Chryseobacterium indologenes hanyo ( Chryseobacterium Indologenes hanyoo: accession number KACC 91497P) was mainly produced chitosan oligosaccharide pentamers.

Therefore, the strain of the present invention or a culture thereof may be used as an antimicrobial or feed composition for animal health promotion. The cationic amine group of the chitosan oligosaccharide prepared using the strain of the present invention reacts with the anionic carboxyl group of the microbial wall to inhibit the activity of the pathogenic microorganism. In addition, chitosan oligosaccharides act to enhance immunity by activating T-cells, macrophages and natural killer cells of the immune system. Therefore, when feeding the animal with a strain or a composition containing the culture of the present invention to feed the animal, even without supplying antibiotics or expensive immune substances, it is possible to prevent diarrhea due to pathogenic microorganisms, and to increase the immunity animal Can reduce mortality.

Feed composition according to the invention can be prepared in the form of fermented feed, compound feed, pellet form and silage (silage) and the like. The fermented feed may be prepared by fermenting an organic material by adding the strain of the present invention and various microorganisms or enzymes, and the blended feed may be prepared by mixing various kinds of general feed and the strain of the present invention. The feed in pellet form may be prepared by formulating the fermented feed or blended feed into a pellet machine, and the silage may be prepared by fermenting the green feed with the strain according to the present invention.

In addition, the strain of the present invention or its culture may be used as a food composition. The food composition of the present invention contains chitosan oligosaccharides, which not only increases the preservation of food but also enhances immunity. In addition, chitosan oligosaccharides have a function of reducing blood cholesterol (Kim Se-jae et al., Korean J. Food Sci . Technol . 1998. 30 (3), 693-696) The composition of the present invention also helps to prevent and alleviate atherosclerosis.

The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the composition of the present invention may be prepared in the form of tea, juice and drink for drinking, or may be ingested by granulation, encapsulation and powdering.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage cornebipe) Breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese), edible vegetable oils, margarine, vegetable protein , Retort foods, frozen foods, various seasonings (e.g., miso, soy sauce, sauce, etc.) may be added to the composition of the present invention.

The preferred content of the culture of the present invention in the food composition of the present invention is not limited to this, but is preferably 0.01 to 50% by weight of the finally prepared food.

In addition, in order to use the composition of the present invention in the form of a food additive, it can be prepared in powder or concentrate form.

As described above, the present invention comprises a method for producing chitosan oligosaccharide, comprising the step of culturing the strain with the comamonas corensis or chryseobacterium indologenes strain producing chitosan degrading enzyme and the strain or the culture thereof To feed, antibacterial, or food composition is provided. The strain of the present invention is effective in producing chitosan oligosaccharides by producing chitosan degrading enzymes. The composition of the present invention contains chitosan oligosaccharides and has antimicrobial activity, and has an effect of enhancing immunity and anti-cholesterol ability, and thus can be effectively used as feed additives or food additives.

Figure 1 is a result of verifying the chitosan decomposing ability of the strain after inoculating calf feces in chitosan as the only carbon source to isolate the strain producing chitosan degrading enzyme. The medium stained with Congo red dye reagent is changing color as the strain degrades chitosan.
2 is a result of similarity analysis of 16S rDNA of the Comamonas koreensis hanwoo (Accession No. KACC 91496P) strain of the present invention.
Figure 3 is a result of the similarity analysis of 16S rDNA of Chryseobacterium indologenes hanyoo strain (KACC 91497P) strain of the present invention.
Figure 4 is a graph of the results of measuring the change of strain growth and chitosan degrading enzyme activity according to the culture time of the comamonas koreensis hanwoo (Accession Number KACC 91496P) strain of the present invention (unit: 1umol N per hour N) The amount of enzyme that produces glucosamin; unit / ml: The amount of enzyme in 1 ml of culture.
5 is a graph showing the results of measuring strain growth and chitosan degrading enzyme activity according to culture time of Chryseobacterium indologenes hanyoo (Accession No. KACC 91497P) strain of the present invention (unit: per hour) The amount of enzyme that produces 1 um of N-glucosamin; unit / ml: The amount of enzyme in 1 ml of culture.
6 is a result of analyzing the components of the chitosan oligosaccharide prepared by chitosan decomposition by the comamonas koreensis hanwoo (Accession Number KACC 91496P) strain of the present invention.
7 is a result of analyzing the components of the chitosan oligosaccharide prepared by chitosan decomposition by Chryseobacterium indologenes hanyoo (Accession No. KACC 91497P) strain of the present invention.

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

Isolate chitosan degrading enzyme

A strain producing chitosan degrading enzyme was isolated from feces of 1 month-old Korean calf and explored culture conditions for identification and bacterial growth.

Put 100ml of sterile water into 500ml Erlenmeyer flask and add 1% chitosan, yeast extract 0.1%, K ₂ HPO ₄ 0.07%, KH ₂ PO ₄ Make culture medium at concentrations of 0.03%, 0.01% MgSO ₄ 7H ₂ O and 0.01% FeSO ₄ 7H ₂ O, and inoculate 1 g of Hanwoo calf feces and incubate at 30 ° C. for 5 days. After completion of shaking culture, the liquid culture solution was continuously diluted by 10-fold dilution with peptone water (peptone 1%, NaCl 0.5%, pH 7.2), and each dilution was smeared on a solid medium containing 1.5% agar in the liquid medium. By culturing at 30 ℃ was isolated strain around the colonies appeared chitosan degradation ring was confirmed by Congo red (Congo red) staining method (see Fig. 1).

< Example  2>

Identification of Selected Chitosan Degrading Enzyme-producing Strains

a. Burgess  Analysis according to classification bacteriology manual

After separating the strains selected in <Example 1> into a single colony, morphological characteristics were investigated in accordance with Burgess's manual of systematic bacteriology, and Gram staining was performed. As a result, it was confirmed that the isolated strain is a Gram-negative strain and has a form of bacilli (see Table 1 and Table 2).

Morphological Properties of the Strains of the Invention division Characteristic Comamonas koreensis hanwoo Chryseobacterium indologenes hanyoo shape Bacillus Bacillus motility none none Sporulation none none Gram Dyeing Gram voice Gram voice

Culture characteristics of the strain of the present invention Factors Characteristics hanwoo hanyoo Colony characteristics when incubated in solid medium
(Colony on the Nutrient agar)
(35 ℃, 1 ~ 2 days) Form Spindle Circular Colony surface shape
(Surface) Smooth Smooth Edge No feature No feature Colony Central
(Elevation) Convex Convex Opacity Opaque Opaque Colony Color Creamy Strong yellow Colony Brilliance Glistening Glistening Culture Characteristics on Nutrient Broth
(Growth on Nutrient broth)
(35 ℃, 1 ~ 2 days) +++ +++ The turbidity of the liquid is thickened without being precipitated
(Turbid without pellicle and not sediment)

b. 16S rDNA  Sequence and G + C mol % analysis

The base sequence and G + C mol% (base composition) of 16S rDNA of the selected strains were analyzed according to conventional methods known in the art.

As a result, Coma Monas Corensis Hanwoo had a G + C mol% of 66.14%, as shown in FIG.Comamonas koreensisIt was confirmed that 99% identical to the nucleotide sequence.

Therefore, the inventors of the strain ' Comamonas koreensis hanwoo 'and it was deposited on September 24, 2009 at KACC Agricultural Research Center (KACC) (accession number: KACC91496P).

In addition, it was confirmed that Chrysobacterium indologenes Hannyo was 42.29% of G + C mol% and 98% identical to Chryseobacterium indologenes as shown in FIG. 3.

Therefore, the present inventors referred to the bacterium ' Chryseobacterium indologenes hanyoo ', which was deposited on September 24, 2009 with the National Institute of Agricultural Science (KACC) (accession number: KACC91497P).

< Example  3>

Confirmation of optimum growth conditions and activity

<3-1> Confirmation of optimum growth conditions and activity

The physicochemical conditions such as culture time, incubation temperature, initial pH, carbon source, nitrogen source, and inorganic salts were examined to determine the optimal growth conditions of each strain producing chitosan degrading enzyme.

After inoculating the strain cultured with platinum in a 100 ml Erlenmeyer flask to which 20 ml of Tryptic soy broth was added once, and then incubating at 35 ° C for 24 hours, the strain was reached. It was used as a seed of. Growth of the strains of the present invention was measured by the spread plate method (Spread plate method). That is, 0.5 ml of each culture solution is added to a sterile test tube containing 4.5 ml of saline solution, followed by stirring using a vortex mixer. This 10-fold dilution was performed continuously, 0.1 ml of the suspension at the appropriate dilution rate was taken, plated in Tryptic soy agar, incubated at 35 ° C for 24 hours, and the colonies appeared to be counted. The growth degree (CFU / ml) of each strain was determined.

As a result of examining the culture conditions for the incubation temperature, initial pH, carbon source effect, nitrogen source effect, and inorganic salts, the optimum conditions for growth of each strain were shown in Table 3 or Table 4.

Results of Optimal Culture Conditions of C. koreensis hanwoo ingredient Concentration (%, w / v) Chitosan 1.0 Glucose 1.0 Yeast extract
(Yeast extract) 1.6 NH ₄ Cl 0.1 K ₂ HPO ₄ 0.07 KH ₂ PO ₄ 0.03 MgSO ₄ ? 7H ₂ O 0.01 FeSO ₄ ˜7H ₂ O 0.01 NaCl 0.08 pH 7.0, 30 ℃

Optimal Culture Conditions Results of C. indologenes hanyoo ingredient Concentration (%, w / v) Chitosan 1.0 Corn flour 1.0 Yeast extract
(Yeast extract) 0.1 Soybean Powder
(Soybean flour) 1.0 (NH ₄ ) ₂ SO ₄ 0.05 K ₂ HPO ₄ 0.07 KH ₂ PO ₄ 0.03 MgSO ₄ ? 7H ₂ O 0.01 FeSO ₄ ? 7H ₂ O 0.01 K ₂ HPO ₄ 0.08 pH 7.0, 30 ℃

The strain was cultured under the optimum culture conditions, and the activity of chitosanase was determined according to the culture time. One unit was defined as the amount of enzyme that produced 1umol of N-glucosamin per hour.

The culture solution was centrifuged at 10,000 rpm and 0.5 ml of 1% swollen chitosan (Swollen chitosan) was added to 0.5 ml of the coenzyme solution from which the supernatant was collected. After completion of the reaction, the reaction was stopped for 3 minutes at 100 ° C. to stop the enzymatic reaction, and the supernatant was taken by centrifugation (4 ° C., 10,000 rpm) to remove the remaining expanded chitosan.

The measurement of the reducing sugar was measured by Schales's method for the reaction mixture produced over time. Trivalent ferricyanide ions (yellow) in Schales' reagent are reduced to tetravalent ferricyanide ions (colorless) by reducing sugars when heated under alkaline conditions.

0.5 g of sodium carbonate solution was added to 0.5 g of potassium ferricyanide (Potassuim ferricyanide) (Anhydrate) to prepare a Schales' reagent.

The enzyme reaction was stopped and 1 ml of Schales' reagent was added to 0.5 ml of the supernatant (sample) from which chitosan was removed by centrifugation. The mixture was mixed well, heated in a boiling water bath for 15 minutes, cooled to room temperature, and then cooled at room temperature at 420 nm with UV-Spectrophotometer. Absorbance was measured. Standard curves were prepared in the same manner using standard reagents of N-acetylglucosamine. If the solution was colorless or brown after the reaction was completed, the sample solution was diluted with too much reducing sugar.

As a result, as shown in FIG. 4 or 5, the strain isolated in the present invention had a life of 5 days, and it was confirmed that the highest productivity of chitosan oligosaccharide was found on the fourth day of culture.

<3-2> Degradation product analysis of the strain of the present invention

In order to confirm the chitosan-degrading enzyme production activity of the strain of the present invention, the culture solution of the best strain of the isolated strain, validated in Example 3-1, was centrifuged at 10,000 rpm for 1% swelling in 0.5 ml of the coenzyme solution. 0.5 ml of Swollen chitosan was added thereto, mixed well with a vortex mixer, and reacted at 37 ° C. for 12 hours. After completion of the reaction, the reaction was stopped for 3 minutes at 100 ° C. to stop the enzymatic reaction, and the supernatant was taken by centrifugation (4 ° C., 10,000 rpm) to remove the remaining expanded chitosan.

The separated supernatant was analyzed by high performance liquid chromatography (HPLC) using 70% acetonitrile and amino column (NH ₂ column).

As a result, in the case of Komamonas corensis Hanwoo strain, it can be seen that the secreting enzyme mainly degrades the substrate to produce (GlcN) ₆ (see FIG. 7).

In the case of Cryeobacterium indologenes Hanuria strain, it was found that the secreting enzyme mainly degrades the substrate to produce (GlcN) ₅ (see FIG. 8).

As a result, it was confirmed that the comamonas corensis and the chrysobacterium indologenes of the present invention decomposed chitosan to prepare chitosan oligosaccharides.

Therefore, the present invention provides a method for producing chitosan oligosaccharide comprising the step of culturing the strain with the Komamonas corensis or chryseobacterium indologenes strain producing chitosan degrading enzyme, and for the feed, antimicrobial containing the strain or its culture Or a food composition. The strain of the present invention is effective in producing chitosan oligosaccharides by producing chitosan degrading enzymes. The composition of the present invention contains chitosan oligosaccharides, has antimicrobial activity, has an effect of enhancing immunity and anti-cholesterol ability, and thus can be effectively used as a feed additive or a food additive, and thus has great industrial applicability.

Agricultural Biotechnology Research Institute KACC91496 20090924 Agricultural Biotechnology Research Institute KACC91497 20090924

Claims (8)

Comamonas koreensis hanwoo (Accession No. KACC 91496P), which produces chitosanase.
Chryseobacterium indologenes hanyoo (accession number KACC 91497P) that produces chitosanase.
delete delete A method for producing chitosan oligosaccharide comprising culturing the strain of claim 1 or 2 in a medium containing chitosan.
A feed composition comprising the strain of claim 1 or 2 or a culture thereof.
An antimicrobial composition comprising the strain of claim 1 or 2 or a culture thereof.
A food composition comprising the strain of claim 1 or 2 or a culture thereof.

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