KR20110107076A - Enterobacter sp. sd255 and method for preparation of extracellular polysaccharide from the same - Google Patents

Abstract

The present invention relates to a SD255 strain of the genus Enterobacter sp. Secreting extracellular polysaccharides, a method for producing extracellular polysaccharides using the strain, and an extracellular polysaccharide produced by the method. By using the new strain of the present invention, it is possible to mass-produce extracellular polysaccharides of a new composition, and the extracellular polysaccharides obtained by the method of the present invention can be industrially used and developed as a biofunctional biopolymer.

Description

SD 255 strain of Enterobacter and method for producing extracellular polysaccharides from the same {Enterobacter sp. SD255 and Method for Preparation of Extracellular Polysaccharide from the Same}

The present invention relates to a SD255 strain of the genus Enterobacter sp. Secreting extracellular polysaccharides, a method for producing extracellular polysaccharides from the strain, and an extracellular polysaccharide produced by the method.

Polysaccharides secreted out of cells by microorganisms are not used as an energy source for microorganisms, but they protect and defend microorganisms from other organisms, and neutralize toxic substances scattered around, preventing osmotic stress and evaporation of intracellular moisture. It protects itself from (Sutherland, 1983). Extracellular polysaccharides are long-chain polymers that dissolve or disperse in water and play an important role in thickening and gelling in food products.They can form gel, emulsify, surface tension, water absorption, adhesion, Since it has a wide range of functions such as lubricity and film forming ability, it is used as a substitute for food and various industrial materials (Fu and Tseng, 1990; Irene et al . , 1990; Low et al ., 1998; Marra et al ., 1990).

In addition, extracellular polysaccharides are the most polysaccharides produced by microorganisms in practical terms, and can improve productivity by improving production culture conditions, and can mass-produce using fermenters in a short time, and isolate and recover polysaccharides of produced cells. Because of its ease of use, polysaccharides have the highest commercial potential. Therefore, studies on the extracellular polysaccharides of microorganisms or microalgae are actively conducted, and the material structure and physical properties are various, so that the application range of food, medicine, chemistry, etc. using the physical properties in aqueous solution is very wide.

Zooglan produced by Zoogloea ramigera has been applied to the removal of heavy metals in the bioenvironmental industry, Pseudomonas Gellan gum produced by elodea ) has a specific physical property not produced by the existing polysaccharides, and is in the limelight as a food material. In addition, there are various types of extracellular polysaccharides derived from microorganisms such as pollulan, dextran, and curdlan, and their usage and range of use are continuously increasing. As there is much room for development.

Because of the wide variety of microorganisms, research on the extracellular polysaccharides derived from various microorganisms is still insufficient, and the search for new microorganisms and the development of biological materials are needed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have conducted research to develop a microorganism capable of producing a new extracellular polysaccharide different from the conventional extracellular polysaccharides, and as a result, the step of growing shiitake mycelia through sawdust fermentation and pasteurization for the ground culture of shiitake mushrooms We discovered a new microorganism that produces a large amount of extracellular polysaccharides in the biofilm formed in, and finally identified it as a new strain belonging to SD255 in Enterobacter through phylogenetic analysis based on morphological characteristics, substrate availability, and 16S rRNA gene sequence. The present invention was completed.

Accordingly, an object of the present invention is to provide a strain of Enterobacter SD255 (KCTC 11631BP) that secretes extracellular polysaccharides.

Another object of the present invention to provide a method for producing extracellular polysaccharides from the strain.

Still another object of the present invention is to provide an extracellular polysaccharide produced from the strain.

According to one aspect of the present invention, the present invention provides a new strain Enterobacter sp. SD255 strain (KCTC 11631BP) secreting extracellular polysaccharides.

The strain of the present invention is a strain newly identified from microorganisms that secrete a large amount of extracellular polysaccharides in the biofilm formed during the sawdust fermentation and pasteurization for shiitake mushroom ground cultivation. The strains of the present invention were finally identified as belonging to the genus SD255 strain by phylogenetic analysis based on morphological characteristics, substrate availability and 16S rRNA gene sequence.

The strain of the present invention was deposited on February 4, 2010 to the Korea Biotechnology Research Institute Gene Bank and was given accession number KCTC 11631BP.

The strain of the present invention is very excellent in the ability to secrete extracellular polysaccharides.

According to another aspect of the invention, the present invention provides a method for producing extracellular polysaccharide comprising the step of culturing the Enterobacter genus SD255 strain (KCTC 11631BP) and from the culture medium extracellular polysaccharide.

According to one preferred embodiment of the invention, the extracellular polysaccharide is a heteropolysaccharide (heteropolysaccharide) including xylose, glucose (glucose) and lactose (lactose).

According to another preferred embodiment of the present invention, xylose, glucose and lactose in the extracellular polysaccharide is 14-16: 3-5: 0.5-2 by weight.

According to another preferred embodiment of the present invention, the culture of Enterobacter genus SD255 strain (KCTC 11631BP) is carried out in PDB (Potato Dextrose Broth) medium.

The PDB medium used in the method of the present invention includes potato starch and dextrose. Preferably, the potato starch in the PDB medium is included in an amount of 0.1 to 10% by weight, and the dextrose is included in an amount of 0.5 to 10% by weight.

The strain of the present invention is inoculated in the PDB medium of the composition and cultured. After adjusting the pH of the medium in the range of 4 to 7, the strain of the present invention is inoculated. The inoculation amount of the strain is 0.1-10% by weight, preferably 0.5-5% by weight, more preferably 0.7-3% by weight, and most preferably 1% by weight, based on the total weight of the medium. The temperature at which the strain is inoculated and then cultured is not particularly limited, but is preferably cultured at a temperature of 20 to 40 ° C, more preferably at a temperature of 25 to 35 ° C.

According to another aspect of the present invention, the present invention is obtained from a culture medium (a) cultured Enterobacter SD255 strain (KCTC 11631BP) secreting extracellular polysaccharides, (b) xylose, glucose, lactose It provides an extracellular polysaccharide characterized in that the heteropolysaccharide (heteropolysaccharide) present in xylose: glucose: lactose = 14-16: 3-5: 0.5-2 by weight ratio.

According to another preferred embodiment of the present invention, the extracellular polysaccharide comprises a strain of the present invention containing potato starch in an amount of 0.1 to 10% by weight, and the dextrose in an amount of 0.5 to 10% by weight of PDB ( Potato Dextrose Broth) was obtained by culturing in medium.

The extracellular polysaccharide obtained by culturing the genus SD255 strain (KCTC 11631BP) by the method of the present invention was named "Y-3261".

The present invention relates to a SD255 strain of the genus Enterobacter sp. Secreting extracellular polysaccharides, a method for producing extracellular polysaccharides using the strain, and an extracellular polysaccharide produced by the method. By using the new strain of the present invention, it is possible to mass-produce extracellular polysaccharides of a new composition, and the extracellular polysaccharides obtained by the method of the present invention can be industrially used and developed as a biofunctional biopolymer.

Figure 1 is a micrograph observed at 1500 times by Gram staining Enterobacter genus SD255 strain (KCTC 11631BP) of the present invention.
Figure 2 is a colony picture of the Enterobacter genus SD255 strain of the present invention (KCTC 11631BP) when incubated in PDA medium for 24 hours.
Figure 3 is a molecular biological phylogenetic diagram based on 16S rRNA sequencing of Enterobacter genus SD255 strain (KCTC 11631BP) of the present invention.
Figure 4 is a graph of growth curve according to the type and concentration of the Enterobacter genus SD255 strain (KCTC 11631BP) of the present invention.
Figure 5 shows the composition of monosaccharides by thin layer chromatography analysis of extracellular polysaccharides obtained from Enterobacter genus SD255 strain (KCTC 11631BP) of the present invention (1: xylose, 2: galactose, 3: hydrolyzed SD255 strain) Extracellular polysaccharide, 4: lactose, 5: glucose).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  1: Pure separation of strain

Samples were collected after visual observation of the biofilm formed during the sawdust fermentation and pasteurization for the cultivation of shiitake ground. Samples were first plated on potato dextrose solid medium (PDA: potato starch 4 g / L, dextrose 20 g / L, agar 15 g / L) and incubated at 30 ° C for 24 hours to give viscosity. Eggplant strains were isolated.

Example 2 Identification of Production Strains

   2-1. Morphological characteristics

The results of observing the morphological characteristics of the genus Enterobacter strain of the present invention are shown in Figures 1 and 2. This strain is an aerobic Gram-negative bacillus, and the motility strain is a color of colony (colony) was changed from light yellow to dark yellow with the incubation time. The appearance of early colonies changed from circular to mucoid, and the viscosity was very high in potato dextrose solid medium.

   2-2. Substrate

Substrates and metabolism of SD255 strains of Enterobacter were examined for 60 substrates using API 20E and API 50CHE kit, and the results are shown in Table 1. The starting strain was incubated for 24 hours using potato dextrose liquid medium (PDB: potato starch 4 g / L, dextrose 20 g / L), and then inoculated the bacterial suspension in the wells of API 20E and API 50CHE, and then at 37 ° C. The cells were cultured for 24 hours at, and the availability of 60 substrates was determined using the reading table. In the case of sugars, the substrate utilization of SD255 was found in the case of saccharides such as rhamnose, dulcitol, methyl-α, D-glucoside, methyl sucrose, and glucose. ), Galactose, maltose, lactose and the like were used as substrates. In addition, D-arabitol, melibiose, L-fucose, inositol, and the like were not used as substrates.

   2-3. 16S rRNA  Genetic analysis

Amplification of the 16S rRNA gene of the strain SD255 genus Enterobacter after determining the nucleotide sequence is shown in FIG. 3. 16S rRNA gene sequences were analyzed using ABI 3730XL DNA analyser (Applide biosystem, USA). The identified nucleotide sequence is about 1100 bp in length and contains a portion of the 16S rRNA gene (see SEQ ID NO: 1). BLAST search of the Genebank database was used to estimate the approximate taxonomic location of the strain. 16S rRNA gene sequence of the identified strain and the SD255 strain and lineage from the database of Ribosomal database project (RDP; http://rdp.cme.msu.edu) and Genbank (http://ncbi.nlm.nih.gov) The nucleotide sequence of the taxonomically closest group was obtained and sorted using the ClustalX (version 1.83) program. The ordered sequences were inferred by evolutionary distances and schematics between the sequences using the Jukes and Canter distance model and the neighbor-joining method using the PHYLIP package (version 3.6a3). In addition, the bootstrap value was calculated from 1,000 resampled data. Phylogenetic analysis showed that SD255 had a relatively low similarity with other strains of the genus Enterobacter.

Example  3: Extracellular polysaccharide  Production and characteristics

   3-1. Extracellular polysaccharides  To produce Optimal medium  Furtherance

The optimal production medium in which the enteric bacterium SD255 produces extracellular polysaccharides was determined by the following method.

① Preculture

Nutrient solid medium (NB: beef extract 3 g / L, peptone 5 g / L, agar 15 g / L) was incubated for 24 hours at 30 ℃. 1 colony was taken from the grown colonies and inoculated in 5 mL of nutrient liquid medium and shaken at 30 ° C. for 24 hours.

② Bacterial Growth Measurement

Absorbance (OD ₆₀₀ ) was measured using a spectrophotometer (OPTIZEN 3220UV, Korea).

③ Extracellular polysaccharides  Measure

The cells were centrifuged (13000 rpm, 15 minutes) to remove the cells, and 2 times of ethanol was added to the culture medium from which the cells were removed to precipitate polysaccharides for at least 15 hours, followed by centrifugation (13000 rpm, 15 minutes) again. Recovered polysaccharides. The polysaccharide thus recovered was dried and the dry weight was measured.

   3-2. Environmental factor investigation

In order to analyze the environmental factors required for the production of extracellular polysaccharides, the results of the investigation on the type, concentration and incubation time of the medium are as follows.

① Type and concentration of medium

The type and concentration of medium on bacterial growth and the formation of extracellular polysaccharides are shown in Table 2. After inoculating 1% by weight of SD255 strain in 200 mL of medium, bacterial growth and extracellular polysaccharide production during shaking for 25 hours in an incubator at 30 ° C. are shown in FIGS. 4 and 3. Bacterial growth according to the concentration of medium showed high cell growth when both cultures were inoculated at 1.0X concentration. Also, when inoculated in PDB medium, the highest amount of extracellular polysaccharide was formed at 3.02 g / L, and bacterial growth was the highest at 2.08 (OD ₆₀₀ ).

Badge type NB PDB 0.2 X (Medium concentration) Beef extract 0.6 g / L, Peptone 1 g / L Potato starch 0.8 g / L, Dextrose 4 g / L 1.0 X (Medium concentration) Beef extract 3 g / L, Peptone 5 g / L Potato starch 4 g / L, Dextrose 20 g / L

Badge type and
Medium concentration NB PDB 0.2X 1.0X 0.2X 1.0X Extracellular Polysaccharides Dry Weight (g / L) 0.16 0.15 0.14 3.02

② Culture time

Extracellular polysaccharides were formed according to the culture time by incubating 200 mL of 1X PDB medium with the best bacterial growth in a 500 mL Erlenmeyer flask and inoculating 1% by weight of SD255 strain and incubating for 8, 18 and 24 hours in a 30 ° C incubator. The results of the observation are shown in Table 4. The production of extracellular polysaccharides by incubation time was the highest at 2.42 g / L after 18 hours of incubation.

Time (h) 8 18 24 Extracellular Polysaccharides (dry weight g / L) 0.78 2.42 2.08

③ in enterobacter SD255 of Extracellular polysaccharides  Production optimum

Optimal conditions for the production of extracellular polysaccharides of SD255 in Enterobacter are inoculated with 1% by weight of bacteria in 1X PDB medium and incubated for 18 hours at pH 5.1 and 30 ° C for optimal formation of extracellular polysaccharides. It was a condition.

   3-3. Extracellular polysaccharides Monosaccharide composition of ‘Y-3261’

The polysaccharide extracted in order to investigate the constituent sugars of the SD255 extracellular polysaccharide in Enterobacter was analyzed by thin layer chromatography (Thin layer chromatography). As shown in FIG. 5, a small amount of xylose, glucose and It was confirmed that it is composed of lactose, the weight ratio was 14-16: 3-5: 0.5-2 ratio.

As described above, new genus Enterobacter SD255 (KCTC 11631BP) of the present invention is a soil microorganism that secretes viscous polysaccharides, and the secreted extracellular polysaccharides Y-3261 can be used as a new functional biopolymer, When cultured in PDB medium shows a high production of extracellular polysaccharides, the method of producing extracellular polysaccharides using the Enterobacter strain is very industrial value.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

references

Fu, J. F., and Y. H. Teeng. 1990. Construction of the Jactose utilizing Xanthomonas campestris and production of xanthan gum from whey. Appl. Environ. 56: 919-923.

2. Irene, B. M., P. E. Jansson, and B. Lindberg. Structural studied of the capsular polysaccharide from Streptococcus pneumoniae type 7A. Carbohydr Res, 198: 67-77.

3. Low, D., J. A. Ahlgren, D. Horne, D. J. McMahon, C. J. Oberg, and J. R. Boradbert. 1998. Role of Streptococcus thermophillus MR-1C capsular expolysaccharide in cheese moisture retention. Appl. Environ. 64: 2147-2151.

Marra, M. 1990. Structure characterization of the exocellular polysaccharide from Cyanospira capsular. Carbohydr Res, 197: 338-344.

5. Sutherland, IW 1983. Extracellular polysaccharides. In: Biotechnology. Vol. 3. Verlag Chemie. Weinheim .. P 533-574.

Korea Research Institute of Bioscience and Biotechnology KCTC11631 20100204

<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Enterobacter sp. SD255 and Method for Preparation of          Extracellular Polysaccharide from the Same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1080 <212> DNA <213> Enterobacter sp. <400> 1 aactactgga aacggtagct aataccgcat aacgtcgcaa gaccaaagag ggggaccttc 60 gggcctcttg ccatcagatg tgcccagatg ggattagcta gtaggcgggg taacggccca 120 cctaggcgac gatccctagc tggtctgaga ggatgaccag ccacactgga actgagacac 180 ggtccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat 240 gcagccatgc cgcgtgtgtg aagaaggcct tcgggttgta aagcactttc agcggggagg 300 aaggcggtac ggttaataac cgtgctgatt gacgttaccc gcagaagaag cacccggcta 360 actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc 420 gtaaagcgca cgcaggcggt ctgtcaagtc ggatgtgaaa tccccgggct caacctggga 480 actgcattcg aaactggcag gctggagtct cgtagaggga ggtagaattc caggtgtagc 540 ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa ggcggcctcc tggacgaaga 600 ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 660 ccgtaaacga tgtcgatttg gagttgtgcc cttgaggcgt ggcttccgga gctaacgcgt 720 taaatcgacc gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc 780 ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac gcgaagaacc ttacctggtc 840 ttgacatcca cagaacctgg cagagatgcc ggggtgcctt cgggaactgt gagacaggtg 900 ctgcatggct gtcgtcagct cgtgttgtga aatgtggggt ttagtcccgc aaagagcgca 960 acctttttgt gcagcgtcgg cggtcaaacg agactgcagt gataaactga agagtgacgt 1020 caggtcatca tggccgacta ccctaagccc agcactgcga ccagcgtaca ataaggccga 1080                                                                         1080

Claims (5)

SD255 strain of the genus Enterobacter (KCTC 11631BP) secreting extracellular polysaccharides.
A method for producing extracellular polysaccharide, comprising the step of culturing the enteric bacterium SD255 strain (KCTC 11631BP) and then obtaining extracellular polysaccharide from the culture medium.
According to claim 2, wherein the extracellular polysaccharide xylose (xylose), glucose (glucose) and lactose (lactose) is a heteropolysaccharide having a weight ratio of 14-16: 3-5: 0.5-2 heteropolysaccharide).
The method according to claim 2, wherein the culture of Enterobacter SD255 strain (KCTC 11631BP) is performed in a Potato Dextrose Broth (PDB) medium containing potato starch and dextrose.
(a) obtained from a culture medium of an enterobacter SD255 strain (KCTC 11631BP) that secretes extracellular polysaccharides, and (b) xylose: glucose: lactose in weight ratio of xylose, glucose, and lactose. 16: 3-5: extracellular polysaccharide, characterized in that the heteropolysaccharide (heteropolysaccharide) present in 0.5-2.

Images