KR20230062278A - Novel strain of Lacrimispora algidixylanolytica producing carbohydrase and use thereof - Google Patents

Abstract

The present invention relates to Lacrimispora algidixylanolytica which produces carbohydrate-decomposing enzymes, and a use thereof. More specifically, the present invention provides microbial preparations, feed additives, and food compositions using Laclimispora algicillanolytica CM01 strain (accession number KCTC 14734BP) derived from the digestive tract of cattle and the carbohydrate-decomposing enzyme production ability, wherein the strain produces and secrets carbohydrate-decomposing enzymes such as β-mannanase, amylase, and cellulase.

Description

Lacrimispora algidixylanolytica producing carbohydrase and its use {Novel strain of Lacrimispora algidixylanolytica producing carbohydrase and use thereof}

The present invention relates to Lacrimispora algidixylanolytica and its use, which produce carbohydrate digesting enzymes, and more specifically to β-mannanase, amylase and cellulase ( cellulase), which produces and secretes carbohydrate-degrading enzymes, derived from the digestive system of cattle, Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP) and microbial preparations and feed additives using the ability to produce carbohydrate-degrading enzymes and food compositions.

Indigestible carbohydrates have low nutritional value and are less effective as feed for ruminants and give a physically foreign feeling. Therefore, decomposition of indigestible carbohydrates present in agricultural by-products can greatly increase the value as feed. For example, in the case of cattle, rice straw is not digested directly, but digested with the help of microorganisms present in the stomach. By applying this action to rice straw, the microorganisms can saccharify the cellulose contained in rice straw, thereby increasing the feed value. In order to saccharify cellulose, which is an indigestible carbohydrate, an enzyme such as cellulase is required, and since it is difficult to saccharify with the enzyme itself, it is difficult to put it into practical use, so a microorganism to provide the enzyme is needed.

As another feed material, palm seed meal containing high fiber content is composed of mannan-based fiber, which is difficult to digest and has relatively low metabolic energy. Since the fiber in palm seed meal is mainly mannan-type hemicellulose (Daud and Jarvis, Phytochemistry, 31 (2): 463-464, 1992), the digestibility can be improved when mannanase, a mannan degrading enzyme, is treated. Mannan fiber of palm seed meal is hydrolyzed to produce various digestible sugars including mannose that can be digested and absorbed by unit animals (Dusterhoft et al., Bioresource Technology, 44, 39-46, 1993; Daud et al. al., Proc. of the ^19th Malaysian society of animal Production Annual Conference.8-10 september 1997, Johor Bahru, Johor, Malaysia). Copra meal is a powder remaining after oil extraction from the copra of a cocos palm, a plant of the Arecaceae family, such as the palm seed meal, and is composed of fibers mainly composed of mannan, such as palm seed meal. Therefore, it is not suitable for unit animal feed.

Conventionally, β-mannanase has been isolated from bacteria such as Bacillus , fungi such as Aspergillus , and higher plants. Japanese Patent Registration No. JP5176767 discloses a method for producing β-1,4-mannase for food by anaerobically culturing Clostridium tertium and Lactobacillus sp.

Under this background, the present inventors improved the value of feed raw materials such as rice straw, palm seed meal, or copra meal, which have indigestible carbohydrates as the main component, and in order to secure grain feed substitution and competitiveness by using agricultural and livestock by-products as feed resources, indigestible carbohydrates Strains producing and/or secreting decomposing enzymes were isolated and identified from intestinal microorganisms of Korean cattle, and were intended to be used as livestock feed additives and health functional foods.

The present invention has been made to solve the above problems, the object of the present invention is to provide strains that produce and / or secrete carbohydrate decomposing enzymes, microbial preparations, feed additives and health functional foods containing the same.

In order to solve the above problems, the present invention provides a Lacrimispora algidixylanolytica strain deposited under accession number KCTC14734BP and producing a carbohydrate decomposing enzyme.

Additionally, the present invention is a Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP), a culture solution thereof or a microbial preparation comprising the strain and its culture solution as active ingredients, which produce the carbohydrate degrading enzyme. provides

Furthermore, the present invention provides a feed additive comprising a Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP), a culture thereof, or the strain and its culture medium as active ingredients, which produce the carbohydrate decomposing enzyme. to provide.

Since the Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP) of the present invention produces and secretes various carbohydrate digesting enzymes such as β-mannase, amylase and cellulase, it develops indigestible carbohydrate processing technology. And by using agricultural and livestock by-products such as rice straw as feed resources, an increase in added value can be expected by substituting grain feed.

Figure 1 shows the results of establishing gel diffusion assay conditions using commercially sold β-mannanase and its substrate, Lost bean gum (LBG), for the discovery of candidate strains for producing β-mannanase enzymes. it is shown
Figure 2 shows the degradation activity for each of the four carbohydrate substrates (starch, CM-cellulose, X-gal and LGB) of the cell lysate (cell lysate) of the β-mannase enzyme production candidate strain (Gel diffusion assay) ) shows the results confirmed by the analysis.
Figure 3 shows the degradation activity for each of the four carbohydrate substrates (starch, CM-cellulose, X-gal and LGB) of the live cell strain identified as Lacrimispora algidixylanolytica by gel diffusion assay analysis. It shows the confirmed result.
Figure 4 shows the degradation activity for each of the four carbohydrate substrates (starch, CM-cellulose, X-gal and LGB) of the strain culture broth identified as Lacrimispora algidixylanolytica by gel diffusion assay analysis. It shows the confirmed result.

As described above, the inventors of the present invention improve the feed value of agricultural and livestock by-products containing indigestible carbohydrates as a main component, and produce and/or As a result of diligent efforts to discover secreting microorganisms, strains that produce and/or secrete carbohydrate degrading enzymes such as β-mannanase, amylase, and cellulase were isolated from the microorganisms derived from the intestine of cattle. , Lacrimispora algidixylanolytica was confirmed through 16S RNA gene analysis and deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC: Korean Collection for Type Cultures) (Accession No. KCTC14734BP).

Since the Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP) of the present invention produces and secretes various carbohydrate degrading enzymes such as β-mannase, amylase and cellulase, it has a degrading activity of indigestible carbohydrates. It can be added to feed or food for the purpose of enhancing it, and it can be used as a variety of health functional foods.

Accordingly, the first aspect of the present invention relates to a Lacrimispora algidixylanolytica strain that produces a carbohydrate digesting enzyme deposited under Accession No. KCTC14734BP.

The Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP) of the present invention is derived from the digestive system of cattle, particularly the intestine.

The carbohydrate decomposing enzyme may be any one or more selected from the group consisting of β-mannanase, amylase and cellulase.

The isolation and identification method of the Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP) of the present invention is as follows.

First, after properly diluting a sample taken from cow feces, it was smeared on a medium and cultured to form a colony, and then a microbial strain was isolated and a cell lysate thereof was prepared. Then, commercial enzyme β-mannanase activity assay conditions were established as shown in FIG. 1 using Locust bean gum substrate, and β-mannanase activity candidate strains from more than 100 small intestine-derived microbial cell lysates One species was excavated as shown in Table 1.

At this time, as the medium, LBG plate medium, Tryptic soy plate medium, Nutrient plate medium, Luria-bertani plate medium, etc. may be used, and Depending on the type of enzyme, the enzyme's substrate can be added as the only carbon source to evaluate the activity of the enzyme.

In a specific embodiment of the present invention, in order to confirm the carbohydrate degradation activity of the candidate strain, using its cell lysate, the degradation activity for four carbohydrate substrates (starch, CM-cellulose, X-gal and LBG) It was confirmed according to the gel diffusion assay method. As a result, as shown in FIG. 2, a transparent decomposition ring was formed by decomposition of carboxylmethylcellulose (CM-cellulose), which is a substrate of cellulase enzyme, and locust bean gum, which is a substrate of β-mannanase enzyme. , the candidate strain was shown to possess cellulase and β-mannase enzyme activities.

Candidate strains confirmed to have the cellulase and β-mannase enzyme activities are 16S RNA gene analysis results, as shown in Table 2, the 16S RNA gene sequence is Lacrimispora algidixylanolytica , Synonym: Clostridium algidixylanolyticum ) and 100% homology, finally Lacrimispora algidixylanolytica (Synonym: Clostridium algidixylanolyticum) algidixylanolyticum )).

In a specific embodiment of the present invention, four carbohydrate substrates (starch, CM- cellulose , X-gal and LBG) was further confirmed according to the gel diffusion assay method. As a result, as shown in FIGS. 3 and 4, the live cells of the strain identified as Lacrimispora algidixylanolytica and their culture medium produce amylase, cellulase and β-mannase enzymes, and culture medium experiments Based on the results, it is assumed that the enzymes are all of the extracellular type.

Therefore, the strain identified as Lacrimispora algidixylanolytica of the present invention has cellulase and β-mannase enzyme activities, and at the same time has amylase, cellulase and β-mannase enzyme production and secretion capabilities have Since these carbohydrate decomposing enzymes are indigestible carbohydrate degrading enzymes, the strain of the present invention can be added to feed or food as a microbial preparation for the purpose of enhancing the decomposition activity of indigestible carbohydrates, and can be utilized as various health functional foods.

Accordingly, the second aspect of the present invention is a microbial preparation comprising the above-described Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP), a cell lysate thereof, a culture medium, or a mixture thereof as an active ingredient. and uses thereof.

In the present invention, the term "cell lysate" is present in cells obtained by simply bursting and extracting the Lacrimispora algidixylanolytica strain according to the present invention by a physical method such as disruption using a bead ruptor It means collectively all water-soluble constituents that

In the present invention, the term "culture medium" can be used interchangeably with "culture supernatant", "conditional culture medium" or "conditioned medium", and Lacrimispora algidixylanolytica is grown and It may mean the entire medium including the strain obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients to survive, metabolites thereof, and extra nutrients. In addition, the culture solution may mean a culture solution obtained by removing the cells from the cell culture solution obtained by culturing the strain. On the other hand, the liquid from which the cells are removed from the culture solution is also called "supernatant", and the culture solution is left still for a certain period of time to take only the liquid of the upper layer except for the part sunk in the lower layer, remove the cells through filtration, or centrifuge the culture solution to It can be obtained by removing the precipitate and taking only the upper liquid. The "cell" refers to the strain itself of the present invention, and includes the strain itself isolated and selected from cow feces or the strain isolated from the culture medium by culturing the strain. The cells can be obtained by centrifuging the culture solution and taking the part that has sunk to the lower layer, or it can be obtained by leaving it for a certain period of time and then removing the upper liquid because it sinks to the lower layer of the culture medium by gravity.

The culture solution may include a culture solution itself obtained by culturing the strain, a concentrate thereof, or a lyophilized product or a culture supernatant obtained by removing the strain from the culture solution, a concentrate thereof, or a lyophilisate.

Microbial preparations according to the present invention are prepared in the form of solutions, powders, suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering materials, or granules, and can be variously applied to feeds, foods, or health functional foods.

Specifically, the Lacrimispora algidixylanolytica strain according to the present invention may be added to feed for the purpose of enhancing the decomposition activity of indigestible carbohydrates.

Therefore, the present invention provides a feed additive comprising the strain, its cell lysate, culture medium or a mixture thereof as an active ingredient.

Feed additives according to the present invention include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate) and the like; Natural antioxidants such as polyphenols, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid; there is.

The feed additive according to the present invention may include auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes and other live microbial preparations; grains such as ground or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugar and dairy products, such as various powdered milk and whey powder; main components such as lipids, for example, animal fats and vegetable fats optionally liquefied by heating; It may further include one or more selected from additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventive agents.

The feed additive according to the present invention may be in the form of a dry or liquid formulation, and may include an excipient for adding feed. Excipients for adding the feed include, for example, zeolite, jade powder or rice bran, but are not limited thereto.

The feed additive according to the present invention may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered as a top dressing or directly mixed with livestock feed, separately from feed, in a separate oral formulation, or in combination with other ingredients.

A single daily intake or divided daily intake may be used, as is commonly known in the art to which the present invention pertains.

Animals in which the feed additive according to the present invention can be used include, for example, livestock such as edible cattle, dairy cows, calves, pigs, sheep, goats, horses, rabbits, dogs, cats, chicks, chickens, domestic chickens, roosters, ducks, It includes poultry such as geese, turkeys, quails, small birds, etc., and in particular, cattle is preferred, but is not limited thereto.

The amount of the Lacrimispora algidixylanolytica strain, its cell lysate, culture medium or a mixture thereof of the present invention included in the feed additive according to the present invention is not particularly limited, but the technical field to which the present invention belongs As is commonly known, it is included in an amount suitable for decomposing carbohydrates such as cellulose and/or hemicellulose while surviving for a long time in the digestive tract of livestock.

In addition, the Lacrimispora algidixylanolytica strain of the present invention may be added to food for the purpose of enhancing the decomposition activity of indigestible carbohydrates.

Therefore, the present invention provides a health functional food containing the strain, its cell lysate, culture medium or a mixture thereof as an active ingredient.

In the present invention, the term "Health functional food (Health functional food or nutraceutical)" is the same term as specific health food, functional food, and health food, in addition to quantity supply, medicine and medical treatment that are processed to efficiently display bioregulatory functions. Means highly effective food, the food can be prepared in various forms such as pills, capsules, powders, granules, liquids, pills, etc. to obtain a useful effect in enhancing the decomposition activity of indigestible carbohydrates.

The health functional food may include a food-acceptable carrier.

As used herein, the term "food acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of strains without stimulating them.

The type of carrier that can be used in the health functional food composition of the present invention is not particularly limited, and any carrier commonly used in the art and acceptable in food science can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The health functional food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of using food as a raw material and not having side effects that may occur when taking the drug for a long time.

Specifically, the food is a food prepared by adding the strain according to the present invention to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulated, powdered, or suspended. For example, various foods, It can be used in beverages, gum, tea, vitamin complexes, and health functional foods.

The food may be prepared in formulations such as tablets, granules, powders, capsules, liquid solutions and pills according to known manufacturing methods, and the content of the strain according to the present invention may be adjusted according to the formulation. There is no particular limitation on other ingredients other than including the strain according to the present invention as an active ingredient, and various common flavoring agents or natural carbohydrates may be included as additional ingredients.

Microbial preparations, feed additives, and health functional foods according to the present invention exhibit enhancement of carbohydrate decomposition activity, particularly, indigestible carbohydrate degradation enhancement efficacy, and the indigestible carbohydrates may be included without limitation as long as they contain cellulose and/or hemicellulose as an active ingredient. there is.

Hereinafter, examples of microbial preparations containing the strain of the present invention as an active ingredient will be described, but the present invention is not intended to limit them, but only to be specifically described.

Formulation Example 1: Preparation of powder

20 mg of the Lacrymispora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or a mixture thereof, 100 mg of lactose hydrate and 10 mg of talc.

A powder was prepared by mixing the above components and filling in airtight bags.

Formulation Example 2: Preparation of tablets

10 mg of the Lacrymisspora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or a mixture thereof, 100 mg of corn starch, 100 mg of lactose hydrate, and 2 mg of magnesium stearate.

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3: Preparation of capsule formulation

10 mg of the Lacrymispora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or a mixture thereof, 3 mg of microcrystalline cellulose, 14.8 mg of lactose hydrate and 0.2 mg of magnesium stearate.

After mixing the above ingredients, capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4: Preparation of injection

10 mg of Lacrymisspora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or a mixture thereof, 180 mg of mannitol, 2974 mg of sterile distilled water for injection and 26 sodium hydrogen phosphate mg.

After mixing the above components, it was prepared with the above component content per 1 ampoule (2 mL) according to a conventional method for preparing injections.

Formulation Example 5: Preparation of liquid formulation

10 mg of the Lacrymisspora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or a mixture thereof, 10 g of isomerized sugar, 5 g of mannitol, an appropriate amount of purified water and an appropriate amount of lemon flavor.

The above components are added to and dissolved in purified water according to a conventional manufacturing method, lemon flavor is added in an appropriate amount, purified water is added to adjust the total volume to 100 mL, and then sterilized and filled in a brown bottle to prepare a liquid formulation.

Formulation Example 6: Manufacture of health functional food

10 mg of the Lacrymispora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or a mixture thereof, an appropriate amount of vitamin mixture, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, and vitamin B1 0.13 mg.

Formulation Example 7: Preparation of feed additive composition

Lacrymispora algidisilanolytica strain according to the present invention (Accession No. KCTC14734BP), its cell lysate, culture medium or mixture thereof 10 g, vitamin B1 0.2 g, vitamin B2 0.2 g, vitamin D 0.2 g, vitamin E A feed additive composition of the present invention was prepared by mixing 0.2 g of vitamin P, 0.2 g of vitamin P, 0.2 g of pantothenic acid, 0.2 g of amino acid, 0.2 g of choline, and 178.4 g of glucose as an excipient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Isolation of β-mannanase producing strains

1-1. Isolation of intestinal-derived microbial strains from Korean cattle

1 g of each fecal sample collected from Korean cattle under breeding in the Korean Cattle Research Institute of the National Institute of Animal Science and Technology located in Pyeongchang was suspended in 9 mL of sterile physiological saline (0.85% NaCl), containing 5% sheep blood. TSA (Tryptic soy agar) medium (hereinafter referred to as TSA blood medium) was smeared with 0.1 mL of the diluted solution diluted with 10 ⁴ to 10 ⁶ and cultured in an anaerobic incubator at 37 ° C for 48 hours. Colonies were re-collected with Platinum, and the process of re-inoculation and culturing in the same medium under anaerobic conditions was performed several times to secure more than 100 strains that were purely isolated.

1-2. Establishment of gel diffusion assay analysis conditions using locust bean gum (LBG) as a target enzyme substrate for discovery of β-mannanase enzyme-producing gut-derived microbial candidate strains

In order to confirm the presence or absence of β-mannanase activity of more than 100 species of intestinal microorganisms isolated from Korean cow feces, a sterile TSA medium containing 0.25% of LBG, which is used as a target substrate for β-mannanase enzyme, was prepared, and then, Commercially sold Helix pomatia ( Helix pomatia ) Derived β- metase (Sigma-Aldrich, M9400) enzyme is diluted in sterile phosphate buffered saline (PBS; Phosphate buffered saline) solution in 2, 5 and 10 mU active units 5 μL each was inoculated and cultured for 48 hours, and at the end of culture, gel diffusion analysis conditions were established according to the size of the transparent ring appearing using the Congo red staining method [Fig. 1].

1-3. Selection of strains producing β-mannase enzyme using microbial strain cell lysate derived from Korean cattle

In order to discover candidate strains of microorganisms derived from the intestines of Korean cattle that produce β-mannase enzymes using the previously established LBG-containing TSA medium analysis conditions, each of the obtained strains of more than 100 species was smeared on TSA blood medium and incubated at 37 ° C for 24 hours. miracle culture was performed. Then, in order to prepare cell lysates of microbial strains, each of the microbial colonies that were plated and proliferated was collected by strain, suspended in a PBS solution, and disrupted in a bead ruptor (including glass beads with a diameter of 0.2 mm) for 5 minutes, at 10000 rpm. The supernatant obtained by centrifugation for 15 minutes was prepared by filtering through a sterile filtration filter (pore size 0.2 μm). After diluting the obtained cell lysate for each strain to a protein concentration of 200 μg/mL, 25 μL (5 μg based on protein concentration) was inoculated in three times in TSA medium containing LBG substrate and incubated at 37°C for 48 hours. And the presence or absence of β-mannase enzyme activity was evaluated through Congo red staining. As a result of the experiment, as shown in Table 1, it was confirmed that one strain from more than 100 kinds of bovine intestine-derived microorganisms had β-mannase enzyme activity.

Result of searching for strains with β-mannase activity selected among more than 100 kinds of Korean cattle gut-derived microorganisms enzyme activity strain number of germs β-mannase activity CM01 One

Evaluation of degradation activity by carbohydrate substrate of β-mannanase production candidate CM01 strain

In order to examine whether CM01, a candidate strain of Korean beef intestine-derived microorganisms with β-mannase activity, has degrading ability for other carbohydrates in addition to the glycolytic activity, 1% starch, 0.2% CM-cellulose, and 0.02% X as substrates. Each TSA medium containing -gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside) was prepared, and cell lysate 5 of β-mannanase production candidate strain CM01 was prepared therein. μg was inoculated and cultured for 48 hours under anaerobic conditions. At the end of the culture, the starch degradation activity of strain CM01 was evaluated by Gram's iodine staining method, and the CM-cellulosic degradation activity was evaluated by using Congo red staining method. It was determined that the substrate produced a green ring. As a control for verification of the experiment, as in the previous experiment of FIG. 1, a TSA medium containing 0.25% LBG was used. As a result of the experiment, as shown in Figure 2, it was possible to re-verify that the CM01 strain had the same β-mannase activity as the result, and at the same time, it also had a strong CM-cellulosic activity, so that the candidate strain had at least β-mannase and cellulase enzymes. On the other hand, starch and X-gal degradation activities were not observed under the present conditions (cell lysate or its inoculum concentration, etc.).

Identification of CM01 strains producing cellulase and β-mannase enzymes

In order to identify the strain of CM01, a candidate strain of Korean cattle gut-derived microorganisms confirmed to produce cellulase and β-mannase enzymes, 16S RNA gene analysis of the microorganism was requested from Macrogen, a professional sequencing company. As a result, as shown in Table 2, the CM01 strain was identified as Lacrimispora algidixylanolytica with 100% homology.

Results of 16S RNA gene analysis of cellulase and β-mannase activity candidate strain CM01 No. Identification result homology gene sequence CM01 Lacrimispora algidixylanolytica
(Synonym: Clostridium algidixylanolyticum) 100% TCTTGCTAAGTCTCCCAGGTGGATTACTTTATTGCGTTAGCGACGGCACCGA
AGAGCTTTTGCTTCCCCAACACCTAGTATTCATCGTTTACGGCGTGGACTACC
AGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTAC
AGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTC
ACCGCTACACTAGGAATTCCACTTACCTCTCCTGCACTCTAGCTCCACAGTTT
CCAAAGCAGTCCCGGGGTTGAGCCCCGGGCTTTCACTCCAGACTTGCAGAGCC
GTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTA
TTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTC
ATTTTCTTCCCTGCTGATAGACCTTTACATACCGAAATACTTCTTCAGTCACG
CGGCGTCGCTGGATCAGGGTTTCCCCCATTGTCCAATATTCCCCACTGCTGCC
TCCCGTAGGAGTTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGGTCACCCTCTC
AGGTCGGCTACTGATCGTCGGCTTGGTGGGCCGTTACCTCACCAACTACCTAA
TCAGACGCGGGTCCATCTCATACCACCGGAGTTTTTCACACTGTGCTATGCAG
CACTGTGCGCTTATGCGGTATTAGCAGTCATTTCTAACTGTTATCCCCCTGTA
TGAGGCAGGTTACCCACGCGTTACTCACCCGTCCGCCGCTAAGTCAATTTTAA
TTCCACCCGAAGGCTTCCTTAAAATCGCTTCGCTCGACTTGCATGTGTTAAGC
ACGCCGCCAGCGTTCATCTGACGGAGGAAGAAACCTTAAAAAAAAAAAACCAA
CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
GGGGGGGGGGGGGGGGGGCGGGGGGGGGGGGGGGGGGGGGGGGGCGGGGGCCCC
CCCCGCGGGGGGGGGGGGGGCCGTCCCCCCCCCCGCCCCCGCGGGGGGGGGGGG
GGGCCCCCCCCCCCCGCCGCGCCCGGGGGTTCTTTTTT (SEQ ID NO: 1)

Lacrymisspora Algidixylanolytica ( Lacrimispora algidixylanolytica ) Evaluation of decomposition activity by carbohydrate substrate of live cells and culture medium of strain CM01

The degrading activity of the cell lysate of the Korean cattle intestine-derived Lacrimispora algidixylanolytica CM01 strain on two carbohydrate substrates, such as CM-cellulose and LBG, was also found in the live cells and culture medium of the strain. In order to confirm that it reproduces, the CM01 strain was smeared on TSA blood medium to perform single colony culture, and then a single colony was inoculated into 10 mL of TS culture medium (Tryptic soy broth) and cultured for 24 hours under anaerobic conditions at 37 ° C. . Thereafter, viable cells and culture medium were separated by centrifugation at 4000 rpm for 20 minutes. In the case of CM01 viable cells, after washing twice with a 1 mL PBS solution under the same centrifugation conditions, they were finally suspended in the same volume of PBS solution, and 10 μL of this was the same 1% used in the previous CM01 strain cell lysate experiment. Each TSA medium containing starch, 0.2% CM-cellulose, 0.02% X-gal, and 0.25% LBG was inoculated and cultured for 48 hours under anaerobic conditions to evaluate the degree of decomposition activity for each carbohydrate substrate. In the case of the CM01 strain culture solution, after filtering with a sterile filtration filter (pore size 0.2 μm), it was frozen, water was completely removed using a lyophilizer, and 2 mL of sterile distilled water was added thereto to concentrate 5 times. The prepared 5-fold concentrated BG03 strain culture solution was inoculated in a total of 100 μL of 25 μL each 4 times in the same 4 types of TSA media for each substrate used for live cell inoculation, and the activity was evaluated by culturing for 48 hours under anaerobic conditions. . As a result, as shown in FIG. 3, unlike the previous cell lysate test results, the Lacrimispora algidixylanolytica CM01 strain had strong degradation activity for CM-cellulose and LBG as well as for starch. It showed strong decomposition activity, and it was observed that X-gal decomposition activity was also weak. In view of these results, at least the live bacteria of the CM01 strain indicated that significant amounts of amylase, cellulase, and β-mannanase were producing three types of carbohydrate degrading enzymes. In addition, as shown in FIG. 4, the CM01 strain culture medium also exhibits decomposition activities of starch, CM-cellulose, and LBG substrates very similar to live bacteria, so that Lacrimispora algidixylanolytica CM01 strain is It was assumed that carbohydrate decomposition enzymes were secreted outside the cell. On the other hand, since the culture medium of the CM01 strain did not degrade X-gal, the role of the Lacrimispora algidixylanolytica CM01 strain in the production of β-galactosidase enzyme was considered to be quite limited.

Lacrimispora algidixylanolytica CM01 confirmed to produce and secrete three types of carbohydrate-degrading enzymes, such as amylase, cellulase and β-mannase, through evaluation of the degradation activity of live cells and culture medium for each carbohydrate substrate The strain was deposited with the Korea Research Institute of Bioscience and Biotechnology and was assigned an accession number as follows.

Korea Research Institute of Bioscience and Biotechnology KCTC14734BP 20211018

SEQUENCE LISTING <110> Korea Atomic Energy Research Institute <120> Novel strain of Lacrimispora algidixylanolytica producing carbohydrase and its use <130> 1069327 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 1098 <212> DNA <213> artificial <220> <223> Lacrimispora algidixylanolytica (Clostridium algidixylanolyticum) 16S RNA <400> 1 tcttggctaa gtctcccagg tggattactt tattgcgtta gcgacggcac cgaagagctt 60 ttgcttcccc aacacctagt attcatcgtt tacggcgtgg actaccaggg tatctaatcc 120 tgtttgctcc ccacgctttc gagcctcaac gtcagttaca gtccagtaag ccgccttcgc 180 cactggtgtt cctcctaata tctacgcatt tcaccgctac actaggaatt ccacttacct 240 ctcctgcact ctagctccac agtttccaaa gcagtcccgg ggttgagccc cgggctttca 300 ctccagactt gcagagccgt ctacgctccc tttacaccca gtaaatccgg ataacgcttg 360 ccccctacgt attaccgcgg ctgctggcac gtagttagcc ggggcttctt agtcaggtac 420 cgtcattttc ttccctgctg atagaccttt acataccgaa atacttcttc agtcacgcgg 480 cgtcgctgga tcagggtttc ccccattgtc caatattccc cactgctgcc tcccgtagga 540 gtttgggccg tgtctcagtc ccaatgtggc cggtcaccct ctcaggtcgg ctactgatcg 600 tcggcttggt gggccgttac ctcaccaact acctaatcag acgcgggtcc atctcatacc 660 accggagttt ttcacactgt gctatgcagc actgtgcgct tatgcggtat tagcagtcat 720 ttctaactgt tatccccctg tatgaggcag gttacccacg cgttactcac ccgtccgccg 780 ctaagtcaat tttaattcca cccgaaggct tccttaaaat cgcttcgctc gacttgcatg 840 tgttaagcac gccgccagcg ttcatctgac ggaggaagaa accttaaaaa aaaaaaacca 900 acgggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 960 gggggggggg ggcggggggg gggggggggg gggggggcgg gggccccccc cgcggggggg 1020 gggggggccg tccccccccc cgcccccgcg gggggggggg gggccccccc cccccgccgc 1080 gcccgggggt tctttttt 1098

Claims (10)

Lacrimispora algidixylanolytica strain (Accession No. KCTC14734BP) producing a carbohydrate degrading enzyme. According to claim 1, which is derived from the digestive system of a cow, Lacrimispora algidixylanolytica strain. The method of claim 1, wherein the carbohydrate degrading enzyme is any one or more selected from the group consisting of β-mannanase, amylase and cellulase, Lacrymisspora algidixylanolytica ( Lacrimispora algidixylanolytica ) strain. The Lacrimispora algidixylanolytica strain according to claim 1, comprising the 16S RNA sequence of SEQ ID NO: 1. A microorganism preparation comprising the strain of any one of claims 1 to 4, a cell lysate thereof, a culture medium, or a mixture thereof as an active ingredient. A feed additive comprising the strain of any one of claims 1 to 4, its cell lysate, culture medium or a mixture thereof as an active ingredient. The feed additive according to claim 6, which is added to enhance carbohydrate decomposition activity. 8. The feed additive according to claim 7, wherein the carbohydrates include indigestible carbohydrates. The feed additive according to claim 8, wherein the indigestible carbohydrates contain cellulose and/or hemicellulose as an active ingredient. The feed additive according to claim 6, which is added to cattle feed.

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