KR20170080033A - Pediococcus pentosaceus KA94 comprising the highly efficient bioconversion activity of arginine into ornithine and its effective bioconversion process condition - Google Patents

Abstract

The present invention relates to a strain Pediococcus pentosaceus KAYE 94 having an ability to biologically convert arginine into ornithine, and a low-cost and high-efficiency bioprocessing method capable of enhancing ornithine productivity using the same.
That is, the present invention relates to a complex medium and a minimum medium composition and culture conditions used in the strain and an efficient bioprocessing method using the same.

Description

 The present invention relates to a Pediococcus pentosaceus KAYE 94 strain having an ability to biologically convert arginine into ornithine, and a method for efficiently producing ornithine using the same, which comprises the steps of: Pediococcus pentosaceus KA94 comprising the highly efficient bioconversion activity of arginine into ornithine and its effective bioconversion process condition}

 The present invention provides a strain Pediococcus pentosaceus KAYE 94 having an excellent ability to biologically convert arginine into ornithine, and provides a biological processing method capable of producing ornithine at a lower cost by using the same.

 The present invention provides the minimum medium composition and culture conditions used during the biological process using the strain.

 Ornithine is a precursor used for the biosynthesis of arginine, proline, and polyamines, and is widely found in plants, animals and microorganisms. It is an essential amino acid and is not found as a protein component. It is present in antibiotic peptides such as tyrosidine and gramicidine. Ornithine plays an important role in the pathway of in vivo metabolism of higher animals to produce elements from amino acids or ammonia via the ornithine circuit and excretion into the body. L-ornithine is widely used in the United States as a food material that secretes growth hormone, increases muscle synthesis, promotes basic metabolism, and prevents obesity. Recently, it has been found that it has new functionalities such as a skin cosmetic effect such as improvement of wrinkles.

 Ornithine is used as a nutritional supplement because it has effects on muscle formation and body fat reduction. Ornithine-alpha-keto glutarylate (OKG), which contains ornithine and alpha-ketoglutarylate in a ratio of 2: Is used as an immunostimulating substance.

 In Europe, L-ornithine-L-aspartate is used as a medicine for improving hepatic disorder. In Japan, it can be used as a food material in the form of L-ornithine hydrochloride, and it can be used as a dietary supplement in the United States. (Korean Patent Publication No. 10-2009-0080797)

 Ornithine is also used as a drug to improve cirrhosis and hepatic dysfunction because it helps remove harmful ammonia from the liver.

 Examples of methods for producing ornithine include a method of treating milk casein with digestive enzymes and a method of producing Corynebacterium, which is an industrial microorganism widely used in the production of transformed Escherichia coli or amino acids, nucleic acids, enzymes and antibiotic- A method using a strain is known (Korean Patent Publication No. 10-2012-0064045)

 In Corynebacterium spp., Arginine (L-arginine) is synthesized from an enzyme expressed from a gene of arginine operon structure consisting of argCJBDFRGH form from glutamate. Arginine operon genes, which play an important role in arginine biosynthesis, are known to synthesize arginine using glutamate (L-glutamate) synthesized in cells as a substrate, and to produce ornithine as an intermediate in the synthesis of arginine . Specifically, in the synthesis route of arginine from glutamate in Corynebacterium sp. Strain, argJ converts glutamate to N-acetylglutamate and argB converts N-acetylglutamate to N-acetylglutamate (N -acetylglutamylphosphate, argC converts N-acetylglutamate to N-acetylglutamate semialdehyde, argD converts N-acetylglutamate semialdehyde to N-acetylornithine, , ArgJ converts N-acetyl ornithine to ornithine, argF converts ornithine to L-citrulline, argG converts citrulline to arginosuccinate, argH is known to encode an enzyme that converts arginylosuccinate to arginine, and arginine It is known that ornithine include the step of generating a route to the production of tin.

 Conventionally known arginine producing strains have been developed by introducing a mutation into arginine operon or by increasing the expression amount of an enzyme involved in arginine biosynthesis through mutation such as a promoter. Of these, argR, which regulates and inhibits the expression of the arginine operon gene, and argB, which is inhibited by the arginine concentration, have been extensively studied as a target for increasing the production of arginine (Korean Patent Publication No. 2010-0060909).

 As a method for improving the productivity of ornithine, a microorganism belonging to the genus Corynebacterium is cultivated in a medium supplemented with proline, and the effect of ornithine cyclodeaminase (ocd) A method of increasing the production amount of the microorganism, a method of improving the ornithine productivity by modifying the impeller and the feeding condition when the microorganism is cultured, and the like. In the case of using the transformed E. coli, argF and argR are deleted A method of enhancing the productivity of ornithine by adding glutamate to the medium when culturing a strain of the present invention is involved in the first step of the pathway for producing proline from glutamate in addition to the pathway from glutamate to ornithine

A method of improving the productivity of ornithine by using a transformant strain in which the gene proB encoding gamma glutamylkinase is deleted is known.

 In addition, studies on high yield production of ornithine precursor, glutamate, have been conducted for a long time in Corynebacterium glutamicum. Among them, glutamate excretion ability of Corynebacterium glutamicum is known to be increased in the case of biotin deficiency condition or treatment with penicillin G or fatty acid ester surfactant, It is known that glutamate activates secretion through damaged cell walls (Korean Patent Publication No. 2010-0017581).

 Kimchi is Korea 's representative fermented food with a long history and obtained the Codex international food standard in July, 2001, and it is a natural fermented food which is different from the pickles of the world. In addition, Kimchi is rich in vitamin A, vitamin B, and vitamin C, and is known to have a number of healthy bacteria, lactic acid bacteria, which help digestion and prevent the growth of cancer cells. The world's healthiest foods Foods) Kimchi was selected as one of the five

 Kimchi is rich in various fermented products and ingredients such as dietary components, vitamins, carotene, lactic acid, lactic acid bacteria, acetylcholine, dextran and acetate, It is known as a functional food having a function of preventing the generation of carcinogens and mutagenic substances. In particular, dietary fiber rich in vegetables prevents constipation, and the rich lactic acid bacteria have a probiotic effect of inhibiting the growth of harmful microorganisms in the intestines and improving intestinal microflora. In fact, it is known that kimchi has a strong effect on the growth of harmful bacteria, because it increases the lactic acid bacteria in the large intestine about 100 times as compared with those who do not eat 300g / day per day.

Kimchi contains major strains such as Lactobacillus , Leuconostoc , Pediococcus , and Weissella , and these strains are known to play an important role in kimchi fermentation have. The abundant lactic acid bacteria in Kimchi produce various substances such as lactate, γ-aminobutyric acid GABA and bacteriocin, and play various roles such as food preservation, acidity and flavor, and inhibition of food poisoning bacteria . Particularly Leuconostoc citreum , Weissella < RTI ID = 0.0 > koreensis ) are known to be the dominant species in determining the taste of Korean kimchi. In addition, the lactic acid bacteria producing materials of Kimchi are used as medicaments for medicines and cosmetics because they have effects of improving lipids in the body, improving immunity and avian influenza (AI) efficacy.

 L-ornithine is an amino acid normally contained in food, though it is not an amino acid constituting a protein. In particular, the content of L-ornithine in freshwater bivalve clams and mussels is relatively high. It is known that the free ornithine content in the 100 g of mulberry extract is 159.9 mg, and the free L-ornithine content is increased when the mulberry is treated at low temperature (Korean Patent Publication No. 10-2011-0081672).

 Therefore, the present invention is to study the isolation, identification, and characteristics of ornithine-producing strains from Korean mushroom, which is one of the unique foods of Korea, to understand the ability of the mushroom strains to produce bioactive substances, . In addition, based on the results of the present invention, an ornithine-producing microorganism derived from fermented food in Korea was utilized to improve the low-cost, high-efficiency production rate.

 That is, a method for producing ornithine biotransformation using an ornithine high productivity strain was proposed.

 Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a novel strain having ornithine hardness.

 It is another object of the present invention to provide a more efficient ornithine production process medium and culture conditions using the strains.

 In order to accomplish the above object, the present invention provides Pediococcus pentosaceus KAYE 94 strain having an excellent ability to biologically convert arginine into ornithine.

 The present invention provides a more efficient ornithine production process and culture conditions using Pediococcus pentosaceus KAYA 94 strain, which is excellent in the ability to biologically convert arginine into ornithine.

 According to the present invention, it is possible to provide Pediococcus pentosaceus KAYA 94 strain having an ability to biologically convert arginine into ornithine, and to provide a more efficient process for producing ornithine using the same. In addition, by applying the minimum medium used in the production process, ornithine can be produced at a lower cost and more efficiently than in the conventional composite medium.

Brief Description of the Drawings Figure 1 is a graphical representation of the Pediococcus pentosaceus ) of KAYA 94 strain,
FIG. 2 is a photograph of the Pediococcus of the present invention pentosaceus ) Photo showing the antifungal activity of KAYA 94 strain
Figure 3 is a graphical representation of the Pediococcus < RTI ID = 0.0 > pentosaceus ) KAYA 94 strain in the phylogenetic tree
Figure 4 is a graphical representation of the Pediococcus < RTI ID = 0.0 > pentosaceus ). The results of TLC analysis of the free amino acids of the strain KAYA 94 showed that the Pediococcus ( Pediococcus) pentosaceus) K A is the result of Figure 94 isolates showing a broad ability lifetime ornithine with arginine tin.
Figure 5 is a graphical representation of the Pediococcus < RTI ID = 0.0 > pentosaceus ). The results of TLC analysis of free amino acids of the KAYA 94 strain show that the ability of 94 strains of Pediococcus pentosaceus KAYA to activate arginine as ornithine in the minimal medium was demonstrated to be.

 One aspect of the present invention relates to Pediococcus pentosaceus KAYE 94 strain, which is superior in the biotransformation of ornithine to arginine.

 The strain Pediococcus pentosaceus KAYE 94 according to the present invention is one of the strains isolated from mukjji as a part of the development of food having enhanced functionality and nutritional value and is a strain excellent in the ability to biologically convert arginine into ornithine.

The strain according to the present invention is a kind of lactic acid bacteria, and its biochemical characteristics were investigated through an API test (API test). As a result, it was found that the strain was a Pediococcus strain, Physiological characteristics and nutritional requirements. In addition, 16S rDNA sequencing was performed for more accurate identification of this strain, and it was found to be Pediococcus pentosaceus .

 Another aspect of the present invention is a method for producing ornithine using the strain. Generally, in order to produce ornithine by inoculating a strain into a complex medium, when the minimum medium of the present invention is used, not only the cost of the culture medium but also the yield of ornithine production is increased, and the ornithine separation becomes very easy I could.

 The biotransformation capacity of ornithine was verified by TLC (Thin Layer Chromatography) analysis and analyzed.

Selection and characterization of strains

 In order to select a strain from the mukjji, it was cultured on MRS medium and Triton yeast (TYE) medium, and then primary sorting was carried out.

 The physiological activity of the microorganisms isolated from the silkworm was determined by measuring the extracellular enzyme activity on the solid medium. The extracellular enzyme activities in solid medium were measured by paper disk method and patching method using media supplemented with various substrates. The medium used in this study is as follows.

  (A) Lipolytic enzyme: A modified version of Sierra's method. The strain was inoculated into a solid medium containing tween 80 (peptone 10 g, CaCl 2 0.1 g, Bacto-agar, Tween 80 10 ml / (Sierra, 1957). After the incubation for 3 days, opaque crystals were observed around the cells.

  (B) Amylase: Amylase was added at 25 ° C in a medium containing Soluble starch (starch 5g, peptone 10g, CaCl2 0.5g, MgCl2 5g, MgSO4 2g, KCl 1g, FeSO4 0.001g, Bacto-agar 20g / After 3 days of incubation, Gram's Iodine solution was added to the cells to observe whether they formed a clear zone around the cells.

  (C) Cellulolytic enzyme: Cellulolytic enzyme: 5 days at 25 ° C in Carboxymethylcelluose (CMC) medium (5 g of CMC, 2 g of MgSO 4, 0.5 g of CaCl 2, 1.0 g of KCl, 0.001 g of FeSO 4, 5 g of peptone and 20 g / After incubation, 0.5% Congo-red solution was applied to the cells to observe clear zone formation.

  (D) Proteolytic enzyme: 1% tannic acid solution was added to the cells grown on gelatin-supplemented medium (gelatin 5 g, beef extract 3 g, peptone 5 g, Bacto-agar 15 g / Were measured.

  (E) β-Glucosidase activity test: Aesculin (6,7-dihydroxycoumarin 6-glucoside) medium [Aesulin 3g, Czapek soln. (50 g of NaNO3, 10 g of KCl, 200 mg / L of MgSO47H2O), 50 ml of 2% K2HPO4, 1 ml of 1% ZnSO47H2O, 1 ml of 0.5% CuSO4 5H2O, 0.2 g of ferric citrate and 12 g / (Skerman, 1969). The cells were incubated for 5 days.

  (F) Ligninase activity test: Medium containing poly R-478 dye (Sigma) (0.6 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.4 g of K2HPO4, 0.22 g of (NH4) 2 tartrate, 4 g of sorbose, 0.2 g of Poly R- agar 15 g, pH 4.5], 25 g of mineral soln. (CaCl 2 2H 2 O 7.4 g, ferric citrate 1.2 g, ZnSO 4 7H 2 O 0.7 g, MnSO 4 5 H 2 O 0.5 g, CoCl 2 6H 2 O 0.1 g, Thiamine HCl 10 mg / (Glenn and Gold, 1983). It was observed that the clear zone was formed around the cells after 5 days incubation.

  (10 g of glucose, 0.2 g of MgSO 4 7 H 2 O, 1 g of NaCl, 0.2 g of CaCL 22 H 2 O, 1.5 g of yeast extract, 0.5 g of yeast extract, 0.5 g of Bacto- Agar was added to 4 g of insoluble phosphate (Tri-Mg 4 g or Tri-Ca) / L] for 5 days at 25 ° C to observe clear zone formation around the cells

 As a result, the general physiological biochemical and morphological characteristics of the selected strains are as follows.

General characteristics of selected strains Characteristics KA 94 Gram's staining + Catalase - Oxidase - Optimum growth temperature (캜) 37 ℃ Growth pH 2.5-9.0 Growth NaCl (%) 3-10% Arabinose + Cellobiose - Fructose + Galactose - Glucose ± Lactose + Mannose + Mannitol - Raffinose + Sucrose - Xylose -

+: Positive reaction, ±: doubtful reaction, -: negative reaction

 The physiological activity of the pure cultured strain was measured by the method of measuring the extracellular enzyme activity on the solid medium. As a result, the activity was exhibited in all of the enzymatic activity measurement media except the β-Glucosidase active medium, and the solubilization ability of the insoluble phosphate was very high , Table 2).

Extracellular enzyme activities of isolated microorganisms Strains CA AA PA LA Tri -Mg Tri -Ca League β-G KA94 O O O O O
1.5 (5) O
0.8 (1) O X X: no growth, no activity
O: growth, weak activity
O (mm): growth, strong activity ex)
3 (5): transparent ring · active ring diameter 3 cm = 30 mm
(Colony tip to active ring edge distance of 5mm)

The separated microorganisms were inoculated with a cell culture medium pre-cultured in a nutrient medium using a paper disk (diameter: 6 mm) in a PDA (Difco Co.) medium coated with plant pathogenic fungi and cultured at 30 ° C for 2 to 3 days, The antifungal activity against pathogenic fungi was investigated. As a result, antifungal activity against various phytopathogenic fungi was shown (Fig. 2)

The genetic similarity between the isolated strains and the existing strains

 MRS, TYE medium, and physiological activity confirmation medium were selected and classified by 16S rDNA analysis and API kit analysis.

 PCR primers were prepared for 16S rDNA analysis of microorganisms showing excellent physiological activity in MRS, TYE medium and physiological activity confirmation medium.

The host strain used in the 16S rDNA analysis, the plasmid DNA and the primer Name Phenotype Plasmids pBluescript II pBluescriptⅡ SK (+/-) phagemid, Amp r, lacz gene, MCS Stratagen E. coli DH5α F ^- Φ80d lac Z Δ ( lac ZYA-argF) U160 end A1 rec A1 hsd R17 (r _k -m _k +) deo R thi -1 sup E44 λ- gyr A96 relA1 Primer 16s 63F 5'-CCCAAGCTTAACTGCAGCAGGCCTAACACATGCAAGTC-3 ' 16s 1406R 5'-CGGAATTCGGGATCCACGGWGTRCAAG-3 '

 The isolated microorganism was inoculated in 5 ml of NB broth and incubated at 37 ° C for 12 to 16 hours. The strain cultured in 50 ml of NB broth was inoculated again and cultured for 12 to 16 hours. The culture was centrifuged at 4,000 rpm for 10 minutes at 13,000 rpm, and the supernatant was discarded to obtain cells. To the obtained cells were added 567 μl of TE buffer, 30 μl of 10% SDS, 3 μl of proteinase K (20 mg / ml), homogenized by vortexing, and incubated at 37 ° C. for 1 hour. 100 μl of 5M NaCl was added and homogenized. After adding 80 μl of CTAB / NaCl solution, vortexing was performed for 10 min at 65 ° C. The same volume of chloroform: isoamyl-alcohol (24: 1) was added to the mixed solution, vortexed, and centrifuged for 5 min. The supernatant was transferred to a new tube. To the supernatant, an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added, vortexed and centrifuged for 10 min to transfer the supernatant to a new tube. To the supernatant obtained, 0.6-fold volume of isopropanol was added, and the resulting solution was inverted slowly to allow genomic DNA to be released. When the white genomic DNA strand was visually observed, it was taken out to a glass rod and 1 ml of 70% ethanol was added, followed by centrifugation at 4 ° C and 13,000 rpm for 10 minutes to discard the supernatant. The cells were suspended in sterile distilled water containing RNase and reacted at 37 ° C for 30 min to remove the RNA and stored at -20 ° C.

Genomic DNA extracted from the strain was used as template DNA and PCR amplification was performed using Gene Cycle ^™ (BIO-RAD Co.). Add 3 μl of the prepared primer (10 pmol), add 5 μl of template DNA (10 ng Genomic DNA) and 39 μl of sterilized distilled water to PCR Mixer (Bioneer Co. Korea), mix with mineral oil Respectively. The PCR reaction conditions were 1 cycle denaturation at 94 ℃ for 4 min, denaturation at 94 ℃ for 1 min, annealing at 60 ℃ for 1min, elongation at 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 10min. The amplified DNA was confirmed by electrophoresis on 0.8% agarose gel.

E. coli strain DH5α was plated on TYE plate medium and cultured at 37 ° C for 12-16 hours to obtain a single colony. Single colony of 5ml SOB (2% tryptone, 0.5 % yeast extract, 0.058% NaCl, 0.019% KCl, pH 7.0) to (about 3 hours) when inoculated with 1/100 volume, and yi be the A ₆₀₀ value of 0.4 to 0.6 Lt; / RTI > The culture was centrifuged at 4 ° C (4,000 rpm, 10 min), and the cells were washed three times with 10% glycerol. The competent cells thus prepared were dispensed in a volume of 80 μl each and stored at -70 ° C. for electroporation. By enzyme digestion with Eco RⅠ p BluescriptⅡ KS vector for self-ligation as a by using an electroporator (BioRad) to a competent cell prepared a p BluescriptⅡ vector 1㎕ each perform a transformation (transformation) by electric shock TYE-Amp solid Transformation rate was confirmed in the medium.

DNA cloning was performed using an amplified PCR product The 16S DNA in Bam HⅠ and Hind Ⅲ double digestion and p BluescriptⅡ KS vector was also digestion with Bam HⅠ and Hind Ⅲ. The cleaved PCR product and cloning vector were mixed and ligation was performed with T4 ligase (Elpis co.) At 16 ° C for 2 hours. Ligation reaction solution was subjected to drop dialysis for 20-30 minutes to remove salts. E. coli DH5α and ligation sample were mixed in a cuvette, and left in ice for 30 minutes, followed by eletroporation. The cuvette sample was transferred to 1 ml of SOC (980 μl SOB, ² μM Mg ^{2 +} 10 μl, 40% glucose 10 μl) and incubated in a shaking incubator at 37 ° C for 1 to 2 hours. The recombinant cultured in SOC was plated on TYE Amp solid medium in which 80 μl of X-gal (20 mg / DMF 1 ml) was plated and cultured at 37 ° C. for 16 hours. The colony selected through Blue-White screen was PCR The plasmid was isolated and cultured to confirm the presence of the product in the vector. The isolated plasmids were digested with Bam HI, Sal I, Eco RI and Sal I for 2 hours at 37 ° C, and then the insert DNA was detected on 0.8% agarose gel.

5 ml of TYE broth supplemented with antibiotic (Amp) was inoculated and cultured at 37 ° C for 12-16 hours. Cells were obtained by centrifugation at 4 ° C and 12,000 rpm for 5 minutes. 100 μl of solution I (0.9% glucose, 10 mM EDTA, 25 mM Tris-HCl pH 8.0, 4 mg / ml) was added to the obtained cell and mixed and homogenized. Then, 200 μl of solution II (0.2 N NaOH, 1% SDS) And allowed to react on ice for 5 minutes. 150 μl of solution III (60 ml of 5M potassium acetate, 11.5 ml of glacial acetic acid, 28.5 ml of dH ₂ O) was added to the reaction mixture for 10 minutes, and the mixture was reacted on ice for 10 minutes. The separated supernatant was transferred to a new tube and equilibrated with the same amount of phenol: chloroform: isoamylalcohol (25: 24: 1) and centrifuged at room temperature for 5 minutes. The resulting supernatant is transferred to a new tube and 1 ml of 100% ethanol (-20 ° C) is added to remove the salt. Centrifuge at 4 ° C for 5 minutes to discard the supernatant. Subsequently, 1 ml of 70% ethanol (-20 ° C) was added to precipitate, followed by centrifugation at 4 ° C for 5 minutes. The supernatant was discarded, dried and dissolved with sterilized distilled water. RNA was digested with RNase (10 mg / ≪ / RTI > For plasmid DNA isolation, Promega plasmid purification kit was used. Nucleotide sequencing was performed by Cosmojin Tech and NCBI's BLAST research program was used for homology analysis.

 The determined 16S rDNA nucleotide sequence was compared with the strains registered in the NCBI gene bank to investigate the phylogeny and genetic similarity, and the results are shown in FIG.

The strain selected as the following base sequence was Pediococcus pentosaceus ) .

tcccttcggggacagaatgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattactagttgccagcattcagttgggcactctagtgagactgccggtgacaaaccggaggaaggtggggacgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagttgcgaaaccgcgaggtttagctaatctcttaaaaccattctcagttcggactgtaggctgcaactcgcctacacgaagtcggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtactcaccgcccgt

The present inventors designated the selected strain as Pediococcus pentosaceus KA94 and deposited it on the 17th of September, 2014 with the Korea Biotechnology Research Center (Accession No .: KCTC 12680BP)

Pediococcus The experiment for measuring the bioconversion efficiency of arginine into ornithine in pentosaceus KA94 strain was analyzed by TLC method.

 In other words, in order to confirm the productivity of ornithine, a functional substance, the experimental condition medium was composed of a combination medium (MRS) and a minimum medium (M) without arginine and arginine added (0.1%).

Each selected microorganism was pre-cultured for 2 days in a liquid medium of MRS ( Lactobacillus genus) and TYE ( Bacillus genus), and 100 μl of each microorganism was inoculated into the following experimental condition medium and cultured at 28 ° C for 50 hours. Were prepared.

Basic complex medium and minimal medium composition Liquid medium Furtherance / L ( only, The solid medium  agar 20 g / L added) MRS Proteose Peptone No.3
Beef Extract
Yeast Extract
Dextrose
Polysorbate 80 10.0 g
10.0 g
5.0g
20.0 g
1.0 g Ammonium Citrate
Sodium Acetate
Magnesium Sulfate
Manganese Sulrate
Dipotassium Phosphate 2.0 g
5.0g
0.1 g
0.05 g
2.0 g TYE Tryptone
Yeast Extract 15.0 g
5.0g Sodium chloride
8.0 g
Minimal Medium
(M) Glucose
KH ₂ PO ₄
Ca (NO _{3) 2} 15.0 g
0.25 g
1.0 g MgSO ₄
KCl
FeCl ₃ 0.25 g
0.12 g
trace

Cultivation conditions for each strain for TLC analysis samples (experimental condition medium and culture conditions) Inoculum Configure Badge Remarks Lactic acid bacteria - MRS, MRS + Arg (0.1%), MRS + Glu (0.1%),

M, M + Arg (0.1%), M + Glu (0.1%), 28 ° C / 50h culture
Arg: Arginine
Glu: Glutamic acid
MRS: Complex medium for lactic acid bacteria
TYE: Bacillus, etc.
Complex medium for general strains
M: Minimum media
(Minimal medium) Bacillus  genus - TYE, TYE + Arg (0.1%), TYE + Glu (0.1%)

M, M + Arg (0.1%), M + Glu (0.1%),

 The supernatant (sup + cell / 500ul) itself and the culture solution (500ul) were centrifuged to prepare supernatant and cells, and the supernatant (sup + cell ), Supernatant (sup) and cell samples were treated with 5 ml of methanol (chloroform: DW = 12: 5: 3) for free amino acid extraction. Samples were mixed well with a vortex mixer. After centrifugation (3000 rpm, 5 min), the supernatant was applied to the amino acid analysis TLC plate (silical G) in an amount of 100 ul, and developed using a developing solvent (1-butanol: acetic acid: DW = 80: 20: Respectively. After development, the cells were dried at room temperature. Then, 1% ninhydrin / ethanol solution was prepared for coloring of amino acids, followed by spraying and reaction at 110 ° C for 5 to 10 minutes.

MRS (lane1), MRS + Arg (lane2), a strain (Pediococcus starter on complex media such as MRS + Glu (lane3) pentosaceus KA94) was inoculated with Arg as a result of lane4 (MRS), lane5 (MRS + Arg) and lane6 (MRS + Glu) -lane5).

 It was investigated whether ornally transformed Orn was released into the extracellular medium or accumulated in the cells.

 Lane 7 (supernatant of LKS 49 inoculation culture supernatant of MRS), lane 8 (supernatant of LKS 49 inoculation culture medium in MRS + Arg addition medium) and lane 9 (LRS 49 culture supernatant in MRS + Glu supplementation medium) Lane 10 (KA94 strain cells that have been precipitated after MRS culturing), lane11 (KA94 strain cells that have been precipitated after MRS + Arg culture), lane12 (MRS + Glu addition medium, (A-lane 11) as shown in FIG. 4), as shown in FIG. It can be seen that almost 100% of the bioconversion occurs in the minimal medium as well as in the complex medium (B-lane 5) (FIG. 5).

 This result is a landmark result

 (1) A medium for the production of arginine (Arg) to ornithine can be prepared as a low-cost minimal medium (less than 100 times cheaper) instead of a complex medium.

 ② It also provides low cost and high production process because it shows 100% biodegradation efficiency even at minimal medium, as well as the efficiency of the biotransformation (Arg → Orn).

③ Therefore, the strain ( Pediococcus pentosaceus KA94) has various functions such as biodegradability (Arg → Orn), insoluble salt availability, extracellular enzyme activity, antibacterial activity, etc., Food, medicines, environmental preservation, etc.).

 In other words, when the strain was applied at low cost and minimum medium for real low cost and high efficiency biodegradation (Arg → Orn) process, very high productivity and high efficiency were also very important in application aspect.

 ④ It is also possible to isolate, collect and purify highly functional ornithine (Orn), which is the final target biotransformation product, because the conversion (Arg → Orn) is 100% in the minimum medium.

    It is yet another advantage of the present invention that the end product as well as the temporal and economic aspects can be obtained easily.

 4 and 5 (TLC sample culture conditions for analysis of free amino acids) drawing Lane No. Medium composition Culture conditions Remarks A Lane 1 MRS badge Before vaccination Ship Support Lane 2 MRS + Arg (0.1%) supplemented medium Lane 3 MRS + Glu (0.1%) supplemented medium Lane 4 MRS badge After inoculation
(Strain KA 94
28 h < / RTI > for 50 h) sup (culture supernatant)
+ Cell Lane 5 MRS + Arg (0.1%) supplemented medium Lane 6 MRS + Glu (0.1%) supplemented medium Lane 7 MRS badge After inoculation
(Strain KA 94
28 h < / RTI > for 50 h) sup (culture supernatant) Lane 8 MRS + Arg (0.1%) supplemented medium Lane 9 MRS + Glu (0.1%) supplemented medium Lane 10 MRS badge After inoculation
(Strain KA 94
28 h < / RTI > for 50 h) cell Lane 11 MRS + Arg (0.1%) supplemented medium Lane 12 MRS + Glu (0.1%) supplemented medium B Lane 1 M medium Before vaccination Ship Support Lane 2 M + Arg (0.1%) supplemented medium Lane 3 M + Glu (0.1%) supplemented medium Lane 4 M medium After inoculation
(Strain KA 94
28 h < / RTI > for 50 h) sup (culture supernatant)
+ Cell Lane 5 M + Arg (0.1%) supplemented medium Lane 6 M + Glu (0.1%) supplemented medium Lane 7 M medium After inoculation
(Strain KA 94
28 ° C for 50 h)) sup (culture supernatant) Lane 8 M + Arg (0.1%) supplemented medium Lane 9 M + Glu (0.1%) supplemented medium Lane 10 M medium After inoculation
(Strain KA 94
28 h < / RTI > for 50 h) cell Lane 11 M + Arg (0.1%) supplemented medium Lane 12 M + Glu (0.1%) supplemented medium

Claims (1)

94 strain of Pediococcus pentosaceus KAE which transforms arginine into ornithine (Accession No .: KCTC 12680BP).

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