KR20130113036A - Novel bacillus subtilis - Google Patents

Abstract

PURPOSE: A Bacillus subtilis CJMPB150 (KCCM11268P) strain is provided to effectively decompose indigestible carbohydrate and to have high endosporulation rate under a high temperature condition with superior thermal resistance, acid resistance, and bile resistance, thereby enhancing survival rate of the strain in digestive organs of livestock. CONSTITUTION: A novel Bacillus subtilis CJMPB150 (KCCM11268P) strain produces complex digestive enzymes and has acid resistance and bile resistance. A culture of the strain is provided. A probiotic formulation contains the strain or the culture thereof. A feed additive contains the probiotic formulation. A feed contains the feed additives.

Description

New Bacillus subtilis {Novel Bacillus subtilis}

The present invention relates to a probiotic preparation containing subtilis (Bacillus subtilis) CJMPB150 (KCCM11268P) and this new strain Bacillus subtilis.

In recent years, international grain prices have been on the rise due to increased demand for grains due to biofuel production and economic growth in emerging economies, reduced agricultural land, and soaring crude oil prices. The rise in international grain prices has led to a rise in domestic blended feed prices, which has been a major blow to the management of livestock farmers. For this reason, studies on probiotics, prebiotics, enzymes, etc., are being actively conducted to promote the growth of livestock and the efficient use of feed grains.

In general, feed grains contain a large number of indigestible carbohydrates, namely cellulose (Cellulose), hemicelluloses (Lignin) and lignin (Lignin). Indigestible carbohydrates inhibit digestion by altering the functioning of the digestive organs, causing stool or reddening in livestock. In order to solve this problem, various feed enzymes for the purpose of digesting indigestible carbohydrates have been widely used.

Cellulase, which can hydrolyze cellulose, Xylan, which is a major component of hemicellulose, and Xylanase, which decompose lignin associated with it, are widely used as an enzyme for feed addition, and livestock of grain feed Increasing the intestinal availability of rats is not only improving feed efficiency (Beguin and Aubert, FEMS Microbiol. Rev. 13: 25-58, 1994), but also contributing to promoting composting of livestock manure (Kim Tae-il et al. 35 (4): 277-282, 1999).

Mannanase is an enzyme that breaks down mannan-containing substances, which are the major components of hemicellulose. Soy protein is used as feed for livestock such as pigs and chickens, and the metase breaks down the galactan (Galactan) in carbohydrates, an energy source for soybean meal, so that it can be easily metabolized by unit animals.

Recently, efforts to use as a probiotic utilizing a strain that produces such enzymes are in progress.

Probiotics is a concept that includes microorganisms that help balance intestinal microflora, microorganisms with antimicrobial and / or enzymatic activity and the products they produce (Fuller, R. J Appl Bacteriol. 66 (5): 365-378, 1989).

Prior arts related to such probiotics include the following.

Korean Patent No. 10-0318554 discloses Bacillus sp . 79-23 strain that produces neutral cellulase, and Korean Patent No. 10-1062309 discloses Bacillus richeni secreting cellulase and xylanase. Bacillus licheniformis DK42 strain KACC 91410P is disclosed. In addition, Korean Patent No. 10-0426930 discloses Bacillus amyloliquefaciens B4 strain KCTC 18083P having amylase, protease, cellulase, and lipase enzyme activity.

However, such prior art only discloses Bacillus species that secrete cellulase and / or xylanase, and relates to strains that secrete all three species of cellulase, xylanase and mannanase, and to probiotic preparations using the same. No technology has been reported.

Republic of Korea Patent Publication B1 No. 10-0318554 (August 25, 2002) Republic of Korea Patent Publication B1 No. 10-1062309 (August 06, 2011) Republic of Korea Patent Publication B1 No. 10-0426930 (August 13, 2004)

Beguin and Aubert, FEMS Microbiol. Rev. 13: 25-58, 1994 Kim Tae-il et al., Korean Journal of Microbiology. 35 (4): 277-282, 1999 Fuller, R. J Appl Bacteriol. 66 (5): 365-378, 1989

An object of the present invention is to provide a Bacillus subtilis CJMPB150 (KCCM11268P) strain excellent in complex digestive enzyme production capacity, acid resistance and bile resistance.

It is also an object of the present invention to provide a culture of Bacillus subtilis CJMPB150 (KCCM11268P) strain.

In addition, an object of the present invention is to provide a probiotic formulation, feed additives and feed comprising the Bacillus subtilis CJMPB150 (KCCM11268P) strain or its culture.

One aspect of the present invention provides a complex of digestive enzymes producing ability, acid resistance and resistance to biliary excellent novel Bacillus subtilis (Bacillus subtilis) CJMPB150 (KCCM11268P) strain.

According to another aspect of the present invention, it provides a culture of the Bacillus subtilis CJMPB150 (KCCM11268P) strain.

According to another aspect of the present invention, there is provided a Probiotics formulation comprising the Bacillus subtilis CJMPB150 (KCCM11268P) strain or a culture of the strain.

According to another aspect of the present invention, there is provided a feed additive comprising the probiotic formulation.

According to another aspect of the present invention, there is provided a feed comprising the feed additive.

The present invention improves the digestibility of livestock by providing probiotics, feed additives and feed comprising a new Bacillus subtilis CJMPB150 (KCCM11268P) strain or its culture having excellent complex digestive enzyme production ability, acid resistance and bile resistance. In addition to improving the efficiency of the feed, it has the effect of contributing to the development of livestock industry by maintaining the intestinal microbial balance and / or antimicrobial activity.

1 is a Bacillus subtilis of the present invention ( Bacillus) subtillis ) CJMPB150 (KCCM11268P) An electron micrograph of the strain.
Figure 2 is a graph showing the growth of the candidate strains of Example 1 over time.
Figure 3 is a photograph showing the digestive enzyme activity of Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention.
Figure 4 is a graph showing the survival rate when the Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention treated with artificial gastric juice or artificial bile.
5 is a graph showing the digestibility of the feed according to Example 6.
Figure 6 is a photograph showing the presence or absence of hemolytic Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention.
Figure 7 shows the 16s rDNA base sequence of Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention.

Hereinafter, the present invention will be described in more detail. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

One aspect of the present invention provides a novel separating a Bacillus subtilis (Bacillus subtilis) CJMPB150 (KCCM11268P) ( hereinafter, 'CJMPB150') strain.

The CJMPB150 morphologically corresponds to Gram-positive bacilli (see FIG. 1), and as a result of analysis of 16s rDNA base sequence (SEQ ID NO: 1, FIG. 7), is a strain having 99% homology with Bacillus subtilis.

The CJMPB150 was deposited on March 22, 2012 at the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under the accession number KCCM11268P.

The CJMPB150 has a complex digestive enzyme production capacity.

Digestive enzyme having the production capacity of the CJMPB150 includes cellulase (Cellulase), xylanase (Xylanase) and mannanase (Mannanase), selected from the group consisting of protease (Protease), amylase (Lipase) and Lipase (Lipase) It may further comprise one or more.

The CJMPB150 may more preferably produce all of cellulase, xylanase, mannanase, protease, amylase and lipase.

The CJMPB150 has excellent heat resistance.

More specifically, the CJMPB150 may be incubated at 37 ° C. and 200 rpm for 24 hours, and then endogenous spore formation rate after heat treatment at 95 ° C. for 10 minutes may be 80% or more, and more preferably 85% or more. Can be.

In addition, the endogenous spore formation rate after incubation for 48 hours at 37 ℃, 200 rpm condition, heat treatment at 95 ℃ 10 minutes may be preferably 100%.

The endogenous spores are resistant to extreme environments such as UV, low temperature, drying and / or high pressure, as well as high temperature conditions, and the higher the endogenous spore formation rate, the higher the survival rate of the strain.

The CJMPB150 has excellent acid resistance.

More specifically, the CJMPB150 has a survival rate after incubation for 3 hours in a medium containing artificial gastric juice prepared by adding 1% (w / v) of pepsin (Pepsin) to a solution adjusted to pH 2.5. It may be more than%.

The CJMPB150 has excellent bile resistance.

More specifically, the CJMPB150 may have a survival rate of preferably 80% or more after incubation for 3 hours in a medium containing artificial bile containing 1% (w / v) pancreatin.

The novel isolated strain CJMPB150 of the present invention can be cultured through a conventional method for culturing Bacillus strains. The CJMPB150 is preferably obtained by incubating for 12 hours to 4 days at a culture temperature in the range of 20 to 40 ℃.

According to another aspect of the present invention, it provides a culture of the CJMPB150.

The culture is a concept including a culture medium or culture medium in which the CJMPB150 strain is cultured, and the resultant cultured in the culture medium or culture solution, and the culture may or may not include the CJMPB150 strain.

The culture is not particularly limited in its dosage form, and may be a dosage form commonly used in the art. For example, the culture may be liquid or solid, and may be in a circular state of the culture, or in its concentrated form or dried form.

Culture medium

As a medium for culturing the CJMPB150 of the present invention, a natural medium or a synthetic medium can be used.

The carbon source of the medium is not particularly limited, and any known in the art may be used. Non-limiting examples of the carbon source include glucose, sucrose, dextrin, glycerol or starch. These may be used alone or in combination of two or more.

The nitrogen source of the medium is not particularly limited, and any known in the art may be used. Non-limiting examples of such nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate and other organic or inorganic nitrogen containing compounds. These may be used alone or in combination of two or more.

The inorganic salt contained in the medium is not particularly limited and may be any known in the art. Non-limiting examples of the inorganic salts include magnesium, manganese, calcium, iron, potassium and the like. These may be used alone or in combination of two or more.

In addition to the carbon source, nitrogen source, and inorganic salt components, the medium for culturing CJMPB150 of the present invention may be added with amino acids, vitamins, nucleic acids, and / or other components that may be contained in the culture medium.

Culture solution

The culture solution of the CJMPB150 strain of the present invention may be a culture stock solution, and may be a solution from which the culture supernatant is removed from the culture stock solution and / or a solution thereof. The culture solution may contain the CJMPB150 strain.

The composition of the culture solution is not particularly limited, and may include not only components known to be necessary for culturing Bacillus strains, but also components that synergistically act on the growth of Bacillus, and the composition thereof is conventional in the art. It can be easily chosen by one with knowledge.

The culture solution may be in a liquid state or a dry state.

The method of drying the culture solution is not particularly limited and may be a method commonly used in the art. Non-limiting examples of the drying method include ventilation drying, natural drying, spray drying or freeze drying. These may be used alone or in combination of two or more methods.

According to another aspect of the present invention, there is provided a probiotic formulation comprising the CJMPB150 strain or a culture of the strain.

Probiotics (Probiotics) means a microorganism or a component that exhibits a beneficial effect on the health of the host, such as humans and animals, and settles on the gut wall of the host intestine and serves to inhibit the settlement of other harmful bacteria and the growth of pathogens. The beneficial digestive enzymes produced by probiotics are known to facilitate the absorption and utilization of nutrients.

The probiotic formulation of the present invention may be one comprising the CJMPB150 strain and / or a culture of the strain.

Probiotics of the present invention may preferably comprise the CJMPB150 strain 5 x 10 ⁴ to 5 x 10 ¹⁰ CFU / ㎖, more preferably 1 x 10 ⁶ to 1 x 10 ⁹ CFU / ㎖ have.

The probiotic formulation may further include a pharmaceutically acceptable carrier, and may be provided in combination with the carrier.

The pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound.

In the carrier, carriers usable in probiotic formulations that are formulated as liquid solutions are suitable for sterilization and living bodies, such as saline solution, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution or glycerol. Can be. These can be used individually or in mixture of 2 or more types, and can add other conventional additives, such as antioxidant, buffer, and / or bacteriostatic agent, as needed.

Diluents, dispersants, surfactants, binders and / or lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Formulations for oral administration comprising the probiotic formulations of the present invention as an active ingredient are not particularly limited and may be used as formulations known in the art. Non-limiting examples of such oral dosage forms include tablets, troches, lozenges, aqueous or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like.

For formulation into such tablets or capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax and the like, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

According to another aspect of the present invention, there is provided a feed additive comprising the probiotic formulation.

Probiotic formulations comprising the CJMPB150 strain of the present invention and / or cultures thereof may be separately prepared in the form of feed additives and mixed into the livestock feed, or may be mixed in such a manner as to add the probiotic preparations directly during feed preparation.

The form of the feed additive is not particularly limited and may be preferably in the liquid or dry state, more preferably in the form of dry powder.

The drying method of the dry powdered probiotic formulation is not particularly limited and may be a method known in the art. Non-limiting examples of the drying method include ventilation drying, natural drying, spray drying or freeze drying. These may be used alone or in combination of two or more methods.

The feed additive may further include other additives that can increase the shelf life of the feed in addition to the probiotic formulations comprising the CJMPB150 of the present invention and / or its culture.

Additives that may be additionally included in the feed additives of the present invention are not particularly limited and may be those known in the art. Non-limiting examples of the additives include, but are not limited to, binders, emulsifiers, preservatives, and the like, which prevent degradation of feed additives; Amino acids, vitamins, enzymes, flavors, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc., which increase their utility. It may include. These may be used alone or in combination of two or more.

The probiotic formulations or feed additives of the present invention may be administered to an animal alone or in combination with other feed additives in an edible carrier. It may also be administered directly to top dressings, livestock feed, oral formulations separate from the feed, or in combination with other ingredients.

According to another aspect of the present invention, there is provided a feed comprising the feed additive.

The feed of the present invention may include the feed additive preferably 0.05 to 10 parts by weight with respect to 100 parts by weight of feed, more preferably 0.1 to 1 part by weight. Within this range, it is possible to effectively increase the digestibility of the livestock, thereby increasing the efficiency of the feed.

The components of the feed of the present invention are not particularly limited and may be known in the art. Non-limiting examples of the feed ingredients include, but are not limited to, cereals, fruits, food processing by-products, algae, fibres, oils, starches, gourds, grain by-products, etc .; Animals include proteins, inorganic substances, fats and oils, minerals, fats and oils, single cell proteins, zooplankton, fish meal and the like. These may be used alone or in combination of two or more.

Livestocks which can use the probiotic formulations, feed additives or feed comprising the feed additives of the present invention include, for example, edible cows, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats and the like. Such as, but not limited to livestock and poultry such as chicks, chickens, domestic chickens, roosters, ducks, geese, turkeys, quails and the like.

Hereinafter, the present invention will be described in more detail by describing Examples. However, the following embodiments are only examples of the present invention and should not be construed as limiting the scope of the present invention.

Example  One

Bacillus Subtilis ( Bacillus subtilis ) CJMBP150  Isolation of strain

(1) Sample acquisition and strain isolation

Meju and various enteric samples, which are traditional fermented foods, were obtained, and the obtained samples were gradually diluted and incubated in BHI solid medium (Difco, USA) added with 3% sodium chloride (Difco, USA) for 24 hours at 37 ° C.

Strains isolated from each sample were grouped according to colony observation to isolate strains. Selected colonies were purely separated by culturing in a new medium over three times, and the pure cultured cells were stored in a medium added with 20% glycerol (glycerol) and stored below minus 70 ° C.

(2) Selection of strains with excellent enzyme activity

In order to select strains having a complex digestive enzyme activity, the enzyme activity evaluation of the three protease, cellulase, and amylase was performed on the isolated enteric-derived bacteria. Enzyme activity evaluation was carried out using the medium containing the substrate for each enzyme was measured the enzyme activity capacity according to the degree of clear zone (clear zone) formation.

Based on the digestive enzyme activity of the enteric bacteria in the culture medium, about 200 species of excellent strains were selected first, and the digestive enzyme secretion activity of the culture supernatant and the culture supernatant were analyzed.

1) Collection of crude enzyme solution

The culture supernatant of the first selected strain was cultured in BHI liquid medium for 24 hours and 48 hours, and centrifuged at 4 ° C. and 13,000 rpm for 5 minutes, and the final supernatant was used as a crude enzyme solution for enzyme activity analysis. As described below, the degree of substrate degradation was determined using a medium containing respective substrates for each enzyme.

2) protease activity

YM (Yeast extract 3g / L, Malt extract 3g / L, Peptone 5g / L, Dextrose 10g / L, Agar 20g / L) with 2% skim milk (Skim milk, Sigma, USA); Difco, USA, 'YM medium') media were prepared. 1.5 μl of the crude enzyme solution collected above was dispensed into the substrate medium, and then reacted at 30 ° C. for 15 hours, and then enzyme activity was measured according to the degree of formation of the transparent ring.

3) cellulase activity

YM medium to which 1% CMC (carboxyl methyl cellulose) substrate was added was prepared. 1.5 μl of the coenzyme solution collected above was dispensed into the substrate medium, reacted at 37 ° C. for 15 hours, and then stained with 0.2% Congo red aqueous solution for 30 minutes, and decolorized with 1M NaCl aqueous solution. The activity of the enzyme was measured according to the degree of formation of the clear ring formed by decomposition of the substrate around the crude enzyme solution.

4) amylase activity

YM medium was prepared to which 1% soluble starch substrate was added. 1.5 μl of the obtained crude enzyme solution was dispensed into the substrate medium, and then reacted at 37 ° C. for 15 hours. After dyeing with an aqueous solution containing 0.1% and 2% of I ₂ and KI, respectively, the activity of the enzyme was measured according to the degree of formation of a transparent ring formed by decomposition of the substrate around the crude enzyme solution.

Based on the secondary evaluation results, 19 strains having excellent digestive enzyme activity were selected as shown in Table 1 below. Among them, 150 and 470 having excellent complex digestive enzyme activity in culture and supernatant were finally selected as candidate strains.

Table 1 below shows the digestive enzyme productivity of the secondary selection strain.

(3) growth evaluation

The two candidate strains excellent in complex digestive enzyme productivity were evaluated for growth. Each strain was incubated at 37 ° C., 200 rpm in BHI liquid medium for 15 hours. 0.1% of the culture was inoculated in 100mL of BHI liquid medium, and cultured at 37 ° C for 10 hours and 24 hours, and each culture was plated in BHI solid medium to measure the number of bacteria.

The results are shown in Figure 2, it was confirmed that the growth of 150 of the two candidate strains more excellent in culture for 24 hours.

Accordingly, 150 strains having excellent complex digestive enzyme production ability and growth ability were finally selected and named as CJMPB150.

Example  2

Selection strain CJMPB150 Digestive Enzyme Activity

In order to examine the digestive enzyme secretion activity of CJMPB150 selected as an excellent digestive enzyme activity strain, the activity of xylanase, mannanase, and lipase in addition to protease, cellulase and amylase was evaluated.

1) Collection of crude enzyme solution

CJMPB150 strain was cultured in BHI liquid medium for 24 hours, and the culture supernatant was collected, centrifuged at 13,000 rpm for 5 minutes at 4 ° C, and the final supernatant was used as crude enzyme solution for enzyme activity analysis. The medium containing each substrate was used to determine the degree of substrate degradation.

2) xylanase activity

YM medium was prepared with 1% xylan added. 1.5 μl of the crude enzyme solution was dispensed into the substrate medium, reacted at 37 ° C. for 15 hours, and then stained with 0.2% aqueous solution of Congo Red for 30 minutes, and decolorized with 1M aqueous NaCl solution. Then, the enzyme activity was measured according to the degree of transparent ring formation.

3) lipase activity

YM medium was prepared to which 1% tricaprylin was added. 1.5 μl of the crude enzyme solution was dispensed into the substrate medium, and then reacted at 37 ° C. for 15 hours, and then enzyme activity was measured according to the degree of formation of the transparent ring.

4) metase activity

Substrate medium (Yeast extract 3g / l, Peptone 5g / l, KH ₂ PO ₄ 1g / l, Agar 20g / l, pH 5) added with 1% mannan (logust bean gum, sigma, USA) was prepared. 1.5 μl of the crude enzyme solution was dispensed into the substrate medium, and then reacted at 37 ° C. for 24 hours, and then enzyme activity was measured according to the degree of transparent ring formation.

Digestive enzyme activity of the CJMPB150 is shown in Table 2 and FIG.

Protease Cellulase Amylase Xylanase Mannanase Lipase CJMPB150 +++ ++++ ++++ ++ ++++ +

Table 2 As shown in FIG. 3, CJMPB150 secreted all six digestive enzymes such as xylanase, mannanase, and lipase in addition to protease, cellulase, and amylase to excellent levels.

Example  3

CJMPB150  Investigation and Identification of Morphological and Biochemical Properties of Strains

(1) Morphological and biochemical characterization

In order to identify the CJMPB150 strain finally selected as an excellent strain of complex digestive enzyme activity, morphological and biochemical investigations were performed primarily. The morphological characteristics were confirmed to be Gram-positive bacillus (FIG. 1), and the sugar fermentation pattern of CJMPB150 strain was analyzed by API 50 CHB system (biomerieux, France) to analyze biochemical properties.

Table 3 below shows the results of analyzing the sugar release pattern of the CJMPB150 strain.

In Table 3, '+' is positive, '-' is negative, and the control is a test group without a substrate.

Based on these results of Table 3, CJMPB150 strain was confirmed that the Bacillus subtilis (Bacillus subtilis) and a homology of 97.8%.

(2) Identification of CJMPB150 Strains

For more accurate identification of CJMPB150 strains, molecular phylogenetic methods based on DNA sequences were performed.

Sequencing was performed to amplify the gene of 16s rDNA using PCR premix (Bionia, South Korea) and Universal Primer 27F (5'AGAGTTTGATCCTGGCTCAG3 ': SEQ ID NO: 2) and 1492R (5'GGTTACCTTGTTACGACTT 3': SEQ ID NO: 3). In the amplification of the gene, the total reaction solution was adjusted to 20 μl, and a total of 30 times of 94 ° C 1 minute, 56 ° C 1 minute, and 72 ° C 1 minute was repeated, and the amplified DNA sequences were analyzed. The base sequence of the analyzed 16s rDNA of the CJMPB150 strain is shown in SEQ ID NO: 1 (FIG. 7).

As a result, it was identified as a microorganism having 99.9% homology with Bacillus subtilis.

The novel microorganism Bacillus subtilis (Bacillus subtilis) CJMPB150 of the present invention are identified in the same way as the Korea Culture Center of Microorganisms on March 22, 2012 (Korean Culture Center of Microorganisms, Seodaemun-gu, Seoul 361-221 Hongje 1 East Deposited as KCCM11268P.

Example  4

CJMPB150  Endogenous Spores of Strains Formability

Bacillus forms endogenous spores to survive when stressed, such as depletion of one or more of the essential nutrients. Endogenous spores are resistant to extreme environments such as ultraviolet light, high temperature, low temperature drying and high pressure, so formation of endogenous spores is very important in maintaining the viability of Bacillus. For this reason, Bacillus subtilis CJMPB150 was cultured for a long time to confirm the endogenous spore formation ability.

0.1% of the bacteria was inoculated into the BHI liquid medium, and cultured at 37 ° C. and 200 rpm for 24 hours and 48 hours. The total bacterial count was measured by smearing the culture medium in each time zone in BHI solid medium, and the culture solution heat-treated at 95 ° C. for 10 minutes was smeared in BHI Agar medium to determine the number of endogenous spores.

Table 4 below shows the measurement results of the endogenous spores.

Incubation time (h) gun Number of bacteria ( CFU / Ml) Endogenous Spore Count ( CFU / Ml) Spores Formation rate (%) 24 1.6 × 10 ⁹ 1.3 × 10 ⁹ 87.8 48 1.3 × 10 ⁹ 1.3 × 10 ⁹ 100

As shown in Table 4, when the CJMPB150 was incubated for 24 hours, about 88% formed endogenous spores, and when incubated for 48 hours, 100% endogenous spores were formed.

Therefore, Bacillus subtilis CJMPB150 of the present invention has a high endogenous spore forming ability when cultured for more than 24 hours, it is determined that it can maintain a high survival rate in the digestive organs of animals when used as probiotics.

Example  5

CJMPB150  Strain Acid resistance  And My bile

Probiotic strains must be resistant to strongly acidic gastric juice and bile fluid in order to survive and reach the intestine, the final target site. For this reason, in order to confirm the application of the Bacillus subtilis CJMPB150 of the present invention as a probiotic strain, acid resistance and bile resistance were confirmed.

(1) acid and artificial gastric juice resistance

Artificial gastric juice was prepared by adding 1% (w / v) of pepsin (Pepsin; Sigma, USA) to a 0.05 M sodium phosphate solution adjusted to pH 2.5 using HCl.

After incubating the CJMPB150 strain for 24 hours at 37 ° C. and 200 rpm in a BHI liquid medium, the supernatant was discarded by centrifugation at 13,000 rpm for 5 minutes, and the cells were recovered. Incubated. After incubation, the BHI medium was plated to measure the number of bacteria to determine acid resistance and resistance to artificial gastric juice.

(2) artificial bile resistance

Artificial bile was sterilized by adding 1% (w / v) Pancreatin (Sigma, USA) to 0.05M sodium phosphate solution, and then sterilized with 10% (w / v) oxagall (Difco Co.) solution. Prepared by adding% (v / v) and adjusting the pH to 6.8.

CJMPB150 strain incubated for 3 hours in the artificial gastric juice of (1) was centrifuged at 13,000 rpm for 10 minutes to discard the supernatant, and the cells were recovered. . After incubation, the plate was plated on BHI medium to measure the number of bacteria to determine acid resistance and resistance to artificial bile.

The results of measuring the acid resistance and resistance to artificial gastric juice and artificial bile are shown in FIG. 4.

As shown in FIG. 4, CJMPB150 was treated with artificial gastric juice at pH 2.5 for 2 hours, and the survival rate was about 83%, and 83% survival rate was maintained even after 24 hours of artificial bile treatment. .

Therefore, Bacillus subtilis CJMPB150 of the present invention showed a high survival rate even after a chain treatment of artificial gastric juice and artificial bile was confirmed that it can be usefully used as a probiotic strain.

Example  6

CJMPB150 In vitro ( in vitro Feed digestibility

The digestibility of feed was investigated to determine whether Bacillus subtilis CJMPB150, which has high ability to produce complex digestive enzymes, could increase digestibility under the same conditions as intestinal intestines.

Standard feed (50% corn, 30% soybean meal, 10% wheat, 5% rice bran and 5% brew Bacillus subtilis CJMPB150 was mixed with 0.1 g of 10 g of the feed prepared by mixing, added to 125 ml of phosphate buffer having a pH of 6.0, and titrated to pH 2 using 3M HCl. 2 ml of pepsin (20 mg / ml) was added thereto and reacted at 200 rpm and 38 ° C. for 2 hours.

After 2 hours reaction, 50 ml of phosphate buffer (pH 6.8) was added and titrated to pH 6.8 using 3M NaOH. Then 2 ml of pancreatin (50 mg / ml) solution is added and incubated at 200 rpm, 38 ° C. for 18 hours.

After incubation, the digested and leftover feed was filtered out using a manure paper (Whatman No. 541) and dried in a 135 ° C. oven. Its dry weight was measured to calculate the digestibility of the feed according to Equation 1 below.

[Formula 1]

Feed digestibility (%) = {(dry weight before reaction-dry weight after reaction) / dry weight before reaction} X 100

The measured feed digestibility is shown in Table 5 and FIG.

sample digestibility(%) Control 22.22 CJMPB150 24.48 enzyme 23.85

When CJMPB150 was added to the feed as shown in Table 5 and FIG. 5, the digestibility was increased by about 2.2% compared to the control group (the test group without adding CJMPB150 or an enzyme to the standard feed), and as a feed additive in the market. When 1% of the used enzyme (Mannanase) was added, the digestibility was increased about 1.6% compared to the control. Therefore, Bacillus subtilis CJMPB150 of the present invention can increase the digestibility of the feed, it was confirmed that can replace the enzyme feed additive.

Example  7

CJMPB150  Strain Stability

To determine the stability of CJMPB150, hemolysis (β-hemolysis) was investigated. β-hemolysis is the action of producing phospholipid enzymes in harmful bacteria to hydrolyze erythrocytes by hydrolyzing phospholipids supplied by red blood cells.

To determine the hemolytic properties of Bacillus subtilis CJMPB150, TSA (Difco, USA) containing 5% sheep blood (sheep blood, Kisan Biotech, Korea) was prepared. After linear inoculation (streaking) in the prepared blood agar medium, and cultured at 37 ℃ for 24 hours to confirm the hemolysis, it was confirmed that the hemolysis does not show as shown in FIG.

Korea Microorganism Conservation Center (overseas) KCCM11268P 20120322

<110> CJ CheilJedang Corporation <120> Novel Bacillus subtilis <130> P11-1057 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 975 <212> DNA <213> 16s rDNA of Bacillus subtilis CJMPB150 <400> 1 gtcacttcgg cggctggctc ctaaaaggtt acctcaccga cttcgggtgt tacaaactct 60 cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc 120 cgcgattact agcgattcca gcttcacgca gtcgagttgc agactgcgat ccgaactgag 180 aacagatttg tgggattggc ttaacctcgc ggtttcgctg ccctttgttc tgtccattgt 240 agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct 300 ccggtttgtc accggcagtc accttagagt gcccaactga atgctggcaa ctaagatcaa 360 gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat 420 gcaccacctg tcactctgcc cccgaagggg acgtcctatc tctaggattg tcagaggatg 480 tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt 540 gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg 600 cttaatgcgt tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt 660 ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 720 cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat 780 ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc cagtttccaa 840 tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg cctgcgagcc 900 ctttacgccc aataattccg gacaacgctt gccacctacg tattaccgcg gctgctggca 960 cgtagttagc cgtgg 975 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 27F <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 1492R <400> 3 ggttaccttg ttacgactt 19

Claims (5)

Composite digestive enzyme producing ability, the acid resistance and having in biliary, novel Bacillus subtilis (Bacillus subtilis) CJMPB150 (KCCM11268P) strain. Culture of the Bacillus subtilis CJMPB150 (KCCM11268P) strain according to claim 1. A probiotics preparation comprising the Bacillus subtilis CJMPB150 (KCCM11268P) strain of claim 1 or a culture of the strain of claim 2. A feed additive comprising the probiotic formulation of claim 3. A feed comprising the feed additive of claim 4.

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