KR20140114524A - Novel microorganism producing butyric acid - Google Patents

Abstract

The present invention relates to a novel strain having excellent productivity of butyric acid and belonging to C lostridium butyricum , a method for producing butyl acid using the strain, and a method for producing And to a pharmaceutical composition for preventing or treating such diseases. The novel Clostridium sp. Strain Clostridium butyricum DKU-01 can be used for economical production of biobutanol produced using butyric acid or the above-mentioned butyric acid, since butyl acid can be produced more effectively than conventional strains.

Description

Novel microorganism producing butyric acid < RTI ID = 0.0 >

The present invention relates to a new butyric acid-producing strain, more specifically, the present invention is excellent in the productivity of butyric acid, and Clostridium beauty rikum (C lostridium butyricum , a method for producing butyl acid using the strain, and a pharmaceutical composition for preventing or treating intestinal diseases containing the strain as an active ingredient.

Currently, most of the petrochemicals are producing butanol, but in the early 20th century before the petrochemical industry was developed, butanol was produced by cultivating an anaerobic microorganism, Clostridium acetobutylicum , which produces butanol, Butanol production The fermentation process is one of the oldest and most traditional biological processes, such as the ethanol fermentation process. Such a biological process is expected to replace the chemical production method of butanol through the hydrogenation reaction of butyraldehyde produced by reacting propylene generated from conventional fossil fuels with carbon monoxide and hydrogen.

Recently, interest in biofuels such as biodiesel and bioethanol has been rising, especially in developed countries, due to an increase in concern about resource exhaustion and environmental pollution due to excessive use of fossil fuels. In particular, , Ease of separation, and the like, and has recently attracted attention as a biofuel. Biobutanol is safer than ethanol-gasoline blends because it reduces vapor pressure when mixed with gasoline. It can be blended at a higher rate when mixed with gasoline compared to ethanol, and biobutanol-gasoline blends can be supplied via existing fuel supply infrastructure. .

However, when the butanol-producing microorganisms are cultured to produce biobutanol, when the concentration of butanol in the culture broth is more than 13.5 g / L, the microbial activity is inhibited, resulting in a problem that the productivity and economical efficiency of biobutanol is deteriorated. In order to solve these problems, various studies have been conducted and a method for converting butyrate to butanol through a bioconversion process has been developed. Generally, butyl acid is chemically produced from propylene by the synthesis of oxalic acid of butylaldehyde, but recently, it has been produced by inoculating an appropriate microorganism into a biomass and fermenting it. In this case, the microorganism is a microorganism of the genus Clostridium are generally used, for example, C. tyrobutyricum, C. butyricum, C. beijerinckii etc. are used. However, since these microorganisms have had a problem that the productivity of the low-butyric acid, butyl Studies to improve acid productivity have continued.

Under these circumstances, the inventors of the present invention have made efforts to improve the productivity of butyric acid, and as a result, they have found that Clostridium butyricum was superior in the productivity of butyric acid , and the present invention was completed.

One object of the present invention is to provide a novel Clostridium butyricum strains.

Another object of the present invention is to provide a method for producing butyl acid using the strain.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating intestinal diseases comprising the above strain as an active ingredient.

In one embodiment for achieving the above object, the present invention deposit number of novel strains of the Clostridium genus Clostridium KCTC 12367BP butyricum DKU-01.

The term " Clostridium " butyricum DKU-01 "is a novel Clostridium sp. strain newly isolated and identified by the present inventors from the feces of newborn infants and can be used in combination with" DKU-01 strain "or" DKU-01. The strain was confirmed to be a strain having excellent ability to produce butyl acid through characterization, and thus the strain can be used for the production of butyric acid.

According to one embodiment of the present invention, the inventors of the present invention found that strains newly isolated and identified from the feces of newborn infants were cultured in a solid plate culture medium and the shape of the colonies formed was analyzed. As a result, the edges of the colonies formed an irregular shape (Fig. 1), and the Clostridium The nucleotide sequence of the 16s rDNA region extracted from butyricum DKU-01 was composed of 1381 bp of polynucleotide (SEQ ID NO: 1), and the nucleotide sequence was applied to ez-Taxon serve to compare homology. Clostridium butyricum strains showed the highest similarity (Table 1). Phylogenetic tree analysis was performed according to the Neighbor-joining method using the mega blast model. As a result, the conventional Clostridium Various strains belonging to butyricum ( Clostridium butyricum DG831126, Clostridium butyricum X68178, Clostridium butyricum AB687551, Clostridium butyricum AB595129, etc.) (Fig. 2). As a result of measuring the growth rate over time, the growth rate reached a maximum after 8 hours from the culture, and the growth rate (Fig. 3).

Thus, the inventors of the present invention found that the strain " Clostridium butyricum DKU-01 ", deposited with the Korean Collection for Type Culture (KCTC) on Feb. 18, 2013, and granted the accession number KCTC 12367BP, which was deposited under the Budapest Treaty It has been deposited internationally.

As another embodiment for achieving the above object, the present invention provides a method for producing butyric acid, comprising: (a) culturing the strain to obtain a culture; and (b) recovering butyric acid from the culture ≪ / RTI >

The term "culture product " of the present invention means a cultured strain obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the DKU-01 strain of the present invention can grow and survive in vitro, its metabolites, Extra nutrients, and the like, and includes cultures from which strains have been removed after culturing the strains. Since the strain DKU-01 is a strain capable of producing butyric acid, butyric acid can be produced by recovering butyric acid from the culture.

In the above method, the step of culturing the strain is not particularly limited, but is preferably carried out by a known batch culture method, a continuous culture method, a fed-batch culture method and the like, and the culturing conditions include, but are not limited to, basic (PH 5 to 9, preferably pH 6 to 8, most preferably pH 6.8) is controlled using a compound such as sodium hydroxide, potassium hydroxide or ammonia or an acidic compound such as phosphoric acid or sulfuric acid The oxygen or oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions and the incubation temperature can be maintained at 20 to 45 캜, preferably 25 to 40 캜, and most preferably at 37 캜 And it is preferable to incubate for about 10 to 160 hours. The butyric acid produced by the culture may be secreted into the medium or may remain in the cells.

In addition, the culture medium used may be a carbon source such as sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower seeds Alcohols such as glycerol and ethanol, and organic acids such as acetic acid, etc. may be used individually or in combination with one or more of the following: ; Examples of nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium nitrate) may be used individually or in combination; As the phosphorus source, potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salt may be used individually or in combination; Other metal salts such as magnesium sulfate or iron sulfate, amino acids and vitamins.

In addition, the step of recovering the butyric acid from the culture can be carried out by methods known in the art. Specifically, the known butyric acid recovery method includes, but is not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation) For example, ion exchange, affinity, hydrophobicity and size exclusion).

According to one embodiment of the present invention, when the DKU-01 strain was cultured, it was confirmed that the production amount of butyric acid was increased with the lapse of the incubation time, and the production amount of such butyric acid was superior to the similar strain 4), the DKU-01 strain can be used for effective production of butyric acid over conventional strains.

As another embodiment for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating intestinal diseases comprising the strain as an active ingredient.

The strain provided in the present invention is a strain naturally distributed in the intestines of a newborn baby and exhibits antimicrobial activity against harmful bacteria which can cause intestinal diseases in the intestines. Therefore, the pharmaceutical composition comprising the strain as a prod Prophylactic or therapeutic effect.

Meanwhile, the pharmaceutical composition for preventing or treating intestinal diseases containing the strain of the present invention in the form of a prodrug may further comprise an appropriate carrier, excipient or diluent commonly used in the production of a pharmaceutical composition. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. It is preferable to be formulated as a preparation for oral administration rather than a preparation for parenteral administration because it is used for prevention or treatment of bacterial intestinal diseases. Examples of the agent for oral administration include suspensions, solutions, emulsions and syrups In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, preservatives and the like may be included.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times.

The use of Clostridium butyricum DKU-01, which is a novel Clostridium spp. Strain provided by the present invention, can more effectively produce butylic acid than conventional strains. Therefore, the economical It will be widely used in production.

1 is a Clostridium provided by the present invention butyricum DKU-01 strain was cultured on a plate medium.
Fig. 2 is a graph showing the relationship between Clostridium butyricum DKU-01 strain of the present invention.
FIG. 3 is a graph showing the relationship between Clostridium butyricum DKU-01 strain after the incubation time.
FIG. 4 is a graph showing the relationship between Clostridium butyricum DKU-01 strain of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Clostridium Beauty Richem DKU -01 Isolation and identification of bacteria

Example  1-1: Newborn Derived from feces  Isolation of strain

A fecal sample from 1g odor minutes newborn beauty rikum basal medium (medium _{(NaCl 0.9g / 875㎖, CaCl 2} 0.028g / 875㎖, (NH 4) 2 SO 4 0.9g / 875㎖, MgCl 2 · 6H 2 O 0.066 g / 875 ml MnCl ₂揃 4H ₂ O 0.020 g / 875 ml CoCl ₂揃 6H ₂ O 0.002 g / 875 ml ZnCl ₂ 0.009 g / 875 ml Resazurin 0.002 g / 875 ml) Phosphate solution KH ₂ PO ₄ 22.5 g / l, Na ₂ CO ₃ 80 g / l, cysteine hydrochloride 25 g / l, EtOH 500 ml / l, Na acetate 2.5 M / l, yeast extract 100 g / And enriched for 48 hours at 37 占 폚.

Subsequently, diluting with 1 ml of sterilized distilled water to 1 ml of the enrichment culture was repeated four times to obtain a dilution diluted to 1 / 10,000 (v / v). 0.1 ml of the diluted solution was mixed with reinforced clostridial L-cysteine, 10 g / l of beef extract, 3 g / l of teast extract, 5 g / l of Dextrose, 5 g / l of NaCl, 3 g / l of Na acetate, soluble Starch 1 g / l, casein enzymic hydrolysate 10 g / , Polymyxin B sulphate 20 mg / l, Resazurin 1 mg / l), and then cultured for 48 hours in a 37 ° C incubator.

The shape of the colony formed after culturing was confirmed by a microscope, and the colonies were inoculated again into the RCB (reinforced clostridial broth) medium and the RCA medium, cultured, and the colonies were again inoculated repeatedly To obtain a colony containing a single strain (Fig. 1). Brief Description of the Drawings Figure 1 is a photomicrograph showing colonies produced from strains isolated from the feces of newborn infants. As shown in Fig. 1, the colonies formed in the RCA medium showed irregular edges.

Example  1-2: 16s rDNA  Identification by base sequence analysis

The strain isolated in Example 1-1 was further subcultured three times, and the resulting cells were inoculated on RCB medium and cultured at 37 DEG C for 24 hours to obtain cells. Genomic DNA was extracted from the obtained microbial cells, and PCR was carried out using the extracted primers (27 forward and 1492 reverse) using the extracted genomic DNA as a template to obtain polynucleotide fragments of the 16S region.

27 forward: 5'-agagtttgatcmtggctcag-3 '(SEQ ID NO: 2)

1492 reverse: 5'-tacggytaccttgttacgactt-3 '(SEQ ID NO: 3)

The nucleotide sequence (SEQ ID NO: 1) of the polynucleotide of the 16S rDNA region obtained was analyzed and subjected to ez-Taxon server for homology analysis (Table 1).

Homology analysis of 16S rDNA region Rank Name strain Accession Pairwise similarity (%) Diff / Total nt Completeness (%) One Clostridium butyricum ATCC 19398 (T) AB075768 99.84 2/1288 100 2 Clostridium saccharoperbutylacetonicum N1-4 (T) U16122 98.14 24/1293 96.1 3 Clostridium beijerinckii NCIMB 8052 (T) CP000721 97.83 28/1292 100 4 Clostridium diolis DSM 5431 (T) AJ458418 97.83 28/1292 100 5 Clostridium puniceum DSM 2619 (T) X71857 97.68 30/1293 100

As shown in Table 1, it was found that the isolated strain exhibited the highest homology with Clostridium butyricum

Example  1-3: Phonetic Analysis

A phylogenetic tree analysis was performed according to the Neighbor-joining method using the mega blast model of the nucleotide sequence of the polynucleotide of the 16S rDNA region obtained in Example 1-2 (FIG. 2). Fig. 2 is a schematic diagram showing phylogenetic analysis results of a strain derived from feces of a neonate. As shown in FIG. 2, the strain was found to be a novel strain belonging to Clostridium butyricum .

The results of Examples 1-1 to 1-3 indicate that the strain derived from neonatal feces was a novel strain belonging to Clostridium butyricum . Therefore, the strain was named "Clostridium Beauty Clostridium butyricum DKU-01 ", and deposited with KCTC12367BP on Feb. 18, 2013 to the Korea Research Institute of Bioscience & Biotechnology.

Example  2: DKU -01 Culture of strain

The strain DKU-01 identified in Example 1 was cultured and the growth rate of the cells was measured with the passage of time. Specifically, the DKU-01 strain was inoculated in 5 ml of a liquid RCA medium prepared by dissolving and sterilizing the RCA medium in distilled water, and incubated at 37 ° C for 18 hours in an anaerobic state. Then, 0.1 ml of the culture was transferred to a new liquid phase Samples were taken at 2-hour intervals while inoculating 5 ml of RCA medium and culturing. Each of the collected samples was applied to a spectrophotometer to measure the growth rate of the cells (FIG. 3). At this time, as the control group, Clostridium butyricum strain (KCTC 1871) was used. FIG. 3 is a graph showing the relationship between Clostridium butyricum DKU-01 strain after the incubation time. As shown in FIG. 3, the maximum OD value of the DKU-01 strain reached 8 hours after the start of the culture and was superior to that of the control group.

Example  3: DKU Butyl acid productivity of strain -01

The cumulative amount of butyric acid produced in the strain cultured by the method of Example 2 was measured with the passage of time. Specifically, each sample collected in Example 2 was centrifuged to obtain a culture supernatant, and 0.1 ml of a saturated HgCl 2 solution and 0.2 ml of a 25% HPO 3 solution were added thereto, and the mixture was allowed to stand at room temperature for 30 minutes . Then, each of the above samples was centrifuged at 13,000 rpm for 10 minutes at 4 ° C to obtain respective supernatant, which was applied to gas chromatography (GC 2010, Shimazhu, Japan) to measure the content of butyl acid contained in each sample (Fig. 4). FIG. 4 is a graph showing changes in the cumulative amount of butyric acid production over time of Clostridium butyricum DKU-01 strain provided in the present invention. FIG. As shown in FIG. 4, cumulative production of butyric acid was found to increase in proportion to the incubation time of DKU-01, and it was confirmed that cumulative production of butyric acid was superior to that of the control.

Korea Research Institute of Bioscience and Biotechnology KCTC12367BP 20130218

<110> Industry-Academic Cooperation Foundation, Dankook University <120> Novel microorganism producing butyric acid <130> PA130275 / KR <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1381 <212> DNA <213> 16S rDNA of Clostridium butyricum DKU-01 <400> 1 agtcgagcga tgaagctcct tcgggagtgg attagcggcg gacgggtgag taacacgtgg 60 gtaacctgcc tcatagaggg gaatagcctt tcgaaaggaa gattaatacc gcataagatt 120 gtagtaccgc atggtacagc aattaaagga gtaatccgct atgagatgga cccgcgtcgc 180 attagctagt tggtgaggta acggctcacc aaggcgacga tgcgtagccg acctgagagg 240 gtgatcggcc acattgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300 aatattgcac aatgggggaa accctgatgc agcaacgccg cgtgagtgat gacggtcttc 360 ggattgtaaa gctctgtctt tagggacgat aatgacggta cctaaggagg aagccacggc 420 taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gatttactgg 480 gcgtaaaggg agcgtaggtg gatatttaag tgggatgtga aatacccggg cttaacctgg 540 gtgctgcatt ccaaactgga tatctagagt gcaggagagg aaaggagaat tcctagtgta 600 gcggtgaaat gcgtagagat taggaagaat accagtggcg aaggcgcctt tctggactgt 660 aactgacact gaggctcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 720 cgccgtaaac gatgaatact aggtgtaggg gttgtcatga cctctgtgcc gccgctaacg 780 cattaagtat tccgcctggg gagtacggtc gcaagattaa aactcaaagg aattgacggg 840 gaggcccgcac aagcagcgga gcatgtggtt taattcgaag caccgcgaag aaccttacct 900 agacttgaca tctcctgaat tactctgtaa tggaggaagc cacttcggtg gcaggaagac 960 aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020 gcgcaaccct tattgttagt tgctaccatt tagttgagca ctctagcgag actgcccggg 1080 ttaaccggga ggaaggtggg gatgacgtca aatcatcatg ccccttatgt ctagggctac 1140 acacgtgcta caatggtcgg tacaatgaga tgcaacctcg cgagagtgag caaaactata 1200 aaaccgatct cagttcggat tgtaggctga aactcgccta catgaagctg gagttgctag 1260 taatcgcgaa tcagaatgtc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1320 acaccatgag agttggcaat acccaaagtt cgtgagctaa ccgcaaggag gcagcgacct 1380 a 1381 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27 forward primers <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1492 reverse primer <400> 3 tacggytacc ttgttacgac tt 22

Claims (7)

Clostridium butyricum DKU-01 strain, a novel Clostridium sp. Strain of accession number KCTC 12367BP.
The method according to claim 1,
Butyric acid.
(a) culturing the strain of claim 1 or 2 to obtain a culture; And
(b) recovering the butyric acid from the culture.
The method of claim 3,
Wherein the culture of the strain is carried out by a method selected from the group consisting of a batch culture method, a continuous culture method, a fed-batch culture method and a combination thereof.
The method of claim 3,
Wherein the butyric acid produced from the strain is secreted into the medium.
The method of claim 3,
Wherein the recovery of the butyric acid is carried out by a method selected from the group consisting of centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution, chromatography and combinations thereof.
A pharmaceutical composition for preventing or treating intestinal diseases comprising the strain of claim 1 or 2 as an active ingredient.

Images