CN102220270B - Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate - Google Patents

Abstract

The invention relates to a screening method for producing chondroitin sulfate bacillus subtilis and application of a bacterial strain fermentation method in the production of chondroitin sulfate, belonging to the technical field of biological engineering. The bacterial strain disclosed by the invention is bacillus subtilis which is obtained by the following steps of: diluting and coating fermented soya beans subjected to a boiling water bath, dyeing and carrying out microscopic examination on an obtained soporiferous strain, then carrying out primary fermentation and shaking culture one by one on screened bacterial strains, adding chondroitin sulfate standard substance of sharks in a formation liquor to be used as an internal standard, carrying out qualitative analysis and screening by adopting a high performance liquid chromatography, and carrying out morphological, physiological and biochemistric and molecular biological identification on the screened bacterial strains to obtain the bacillus subtilis for producing the chondroitin sulfate. Meanwhile, the invention discloses a method for producing the chondroitin sulfate by using the bacterial strain; and the method is used for fermenting the chondroitin sulfate for 24h, wherein the yield of the chondroitin sulfate is 177mg/L.

Description

A kind of screening method that produces the chondroitin sulfate bacterial strain and produce chondroitin sulfate with this strain fermentation method

Technical field

The screening method of a kind of subtilis that produces chondroitin sulfate and use this bacterial strain and produce chondroitin sulfate belongs to bioengineering field, and particularly the subtilis of chondroitin sulfate is produced in a strain.

Background technology

Chondroitin sulfate (chondroitin sulfate, CS) have another name called CS, it is a kind of glycosaminoglycan polysaccharide, its two sugar monomer by glucuronic acid (D-GlcUA) and N-acetylgalactosamine (GalNAc) with β-1,3 keys are connected, sugar chain carries out sulfation by sulfotransferase at 4, carbon or 6, the carbon of GalNAc after generating, as Fig. 1.Can be divided into A, B, C, D, E, F, H etc. according to its chemical composition and structure difference multiple, the chondroitin sulfate that extraction obtains from mammalian tissues usually is take CS-A, CS-C as main.

CS is the natural acid mucopolysaccharide class medicine that extracts from animal cartilage, is mainly used in nervous headache, arthrodynia, neurodynia etc.For many years, chondroitin sulfate is the little kind of pharmaceutical always, and volume of production and marketing is little.But in recent years, chondroitin sulfate is widely used in fields such as pharmaceutical preparation, medical material, daily chemical products, makeup, hair growth promoter, healthcare products as a kind of novel medicinal activeconstituents, more and more is subject to people's attention, and it is prosperous that the market requirement becomes.China produces and export volume increases substantially year by year, has now become the third-largest export varieties of biochemical drug, and development prospect is good.

At present, the domestic traditional technologys of using are extracted the production chondroitin sulfate more from animal cartilage, there are no the report that adopts the Production by Microorganism Fermentation chondroitin sulfate.Can synthesize a kind of capsular polysaccharide as far back as Rodriguez et al. in 1988 the bacterium E.coli O5:K4:H4 that just finds the cause of disease abroad, its repeated monomer connects for be connected a β with fructose by GlcA, GalNAc the disaccharides that the furanose residue of end consists of.Except residue of fructose, it is closely similar with not Sulfated chrondroitin skeleton.DeAngelis et al. proved by structural analysis that F type P.multocida produced not Sulfated chrondroitin in 2002.Jolly in 2010 finds that to the analysis of fermenting of a large amount of microorganisms the microorganism of numerous species can produce chondroitin sulfate or its analogue.

The research of adopting the Production by Microorganism Fermentation chrondroitin is in the preliminary stage, still a kind of method that can utilize microbial fermentation direct production chondroitin sulfate useless.

Summary of the invention

The purpose of this invention is to provide a kind of chondroitin sulfate produces bacterium and screening method thereof and produces chondroitin sulfate with this strain fermentation method.

Technical scheme of the present invention:

1, a kind of screening method that produces the chondroitin sulfate subtilis:

1) the initial gross separation purifying of subtilis

Take approximately the 0.2g fermented soya bean in 20ml 0.85% stroke-physiological saline solution, bacteria suspension is vibrated in boiling water after heating 10min, be cooled to rapidly room temperature.This bacteria suspension is diluted to 10 as mother solution gradient ^-8, get 10 ^-6-10 ^-8Dilution bacterium liquid is coated on the LB flat board, is inverted cultivation 24h for 37 ℃, and single bacterium colony of the different colonial morphologies of picking carries out violet staining, and microscopically is observed gemma, the gemma bacterial strain will be arranged as the doubtful bacterial strain of subtilis that further screens, and numbering is preserved;

2) produce the screening of chondroitin sulfate subtilis

The bacterial classification that above-mentioned separation and purification is obtained is inoculated in nutrient broth medium, centrifugal collection fermented liquid after cultivation 24h, the marker method of fermented liquid in the high performance liquid phase method analyzed, and the bacterial strain that makes chondroitin sulfate standard substance peak height increase is the subtilis that produces chondroitin sulfate.

2, it is as follows that a kind of screening method that produces the chondroitin sulfate subtilis according to claim 1, its screening obtain producing the authentication method of subtilis of chondroitin sulfate:

1) Morphological Identification

Colonial morphology, cellular form, gramstaining, spore staining;

2) Physiology and biochemistry is identified

Catalase test, V.P. and M.R. test, gelatine liquefication and Starch Hydrolysis, salt tolerant acid resistance test, nitrate reduction test;

3) 16S rDNA molecular biology identification

Extract test kit with bacterial genomes and extract bacterial genomes DNA, adopt subtilis 16S rDNA universal primer to carry out pcr amplification, amplified production reclaims test kit with glue and reclaims purifying, and be connected to cloning vector pMD 18 T Vector, be transformed in e. coli jm109, utilize the penicillin resistance of cloning vector to screen positive transformant, and with universal primer, transformant is carried out plasmid PCR and identify.

Nucleotide sequence in positive colony order-checking gained 16S rDNA sequence and GenBank database carries out homology analysis, obtains the nearest bacterial classification Bacillus.subtilis of sibship with it, and homology reaches 99%.

Determine that by morphology, Physiology and biochemistry and 16S rDNA molecular biology identification this bacterial strain is subtilis, and called after Bacillus.subtilis BN.

3, the production method of a kind of chondroitin sulfate disclosed by the invention, it is characterized in that adopting Bacillus.subtilis BN is starting strain, produces chondroitin sulfate with seed culture and liquid fermenting;

1) seed culture:

Seed culture medium is in g/L: extractum carnis 3, peptone 5, initial p H 7.2-7.4;

Culture condition: under 37 ℃ of temperature, shaking speed 200rpm condition, cultivate 12-14h;

2) liquid fermentation and culture:

Fermention medium is in g/L: extractum carnis 3, peptone 5, initial p H 7.2-7.4;

Fermentation condition: inoculum size 10%, under 37 ℃ of temperature, shaking speed 200 conditions, fermentation 20-24h.

4, the mensuration of chondroitin sulfate output

Get the centrifugal 30min of fermented liquid 3000rpm, collect supernatant liquor, and with shark chondroitine as standard substance, the standardized solution of preparation 100,200,300,400,500mg/L.With supernatant liquor and standardized solution after 0.22 μ m filtering with microporous membrane, with the content of high effective liquid chromatography for measuring chondroitin sulfate.

Chromatographic condition:

Chromatographic column: 150mm ZORBAX SB-AQ;

Moving phase: acetonitrile-2.28mmol/L tetramethyl ammonium chloride aqueous solution (volume ratio is 10: 90), with 0.45 μ m membrane filtration;

Column temperature: 25 ℃;

Detect wavelength: 195nm;

Sample size: 10 μ l

Flow velocity: 0.5ml/min.

Typical curve presents good linear relationship (Fig. 2), regression equation: y=0.1469x-1.5009, R between 100-500mg/L ²=0.9990.The output that obtains accordingly the 24h chondroitin sulfate is 177mg/L, as Fig. 3.

Description of drawings

The structural formula of Fig. 1 chondroitin sulfate, R and R ₁Identical or different, represent H or SO ₃Na, but can not be H simultaneously, n=5-50

Fig. 2 shark chondroitine typical curve

Fig. 3 chromatogram detected result, A 500mg/L shark chondroitine, B 24h fermented liquid

Embodiment

Below the embodiment of subtilis (Bacillus.subtilis BN) screening, evaluation and fermentative production chondroitin sulfate.

Embodiment 1

Take approximately the 0.2g fermented soya bean in 20ml 0.85% stroke-physiological saline solution, bacteria suspension is vibrated in boiling water after heating 10min, be cooled to rapidly room temperature.This bacteria suspension is diluted to 10 as mother solution gradient ^-8, get 10 ^-6-10 ^-8Dilution bacterium liquid is coated on the LB flat board, is inverted for 37 ℃ and cultivates 24h, and single bacterium colony of the different colonial morphologies of picking carries out violet staining, and microscopically is observed gemma, selects the further multiple sieve of fermentation of genus bacillus.Centrifugal collection fermented liquid after 37 ℃ of cultivation 24h of nutrient broth medium, the marker method of fermented liquid in the high performance liquid phase method analyzed, and mark in doing with shark chondroitine, the bacterial strain that chondroitin sulfate standard substance peak height is increased are and produce the chondroitin sulfate bacterial strain.

Embodiment 2

The BN bacterial strain of screening is carried out physio-biochemical characteristics by " microbial taxonomy " identify (seeing Table 1,2), and extract the test kit extracting method by bacterial genomes and extract genomic dna, take P1 and P2 as forward and reverse primer:

P1：5’-CAGATGGGAGCTTGCTCCCTG-3’，

P2：5’-CGACTTCACCCCAATCATCTG-3’。

With pcr amplification 16S rDNA gene, entrust the large cara gene of China to carry out 16S rDNA order-checking, obtain this bacterial strain part 16S rDNA sequence (GenBank accession number: JF922967), carry out the sequence analysis analysis with the BLAST gopher on the NCBI website.Identify based on 16S rDNA sequential analysis and physio-biochemical characteristics, in conjunction with growth and the fermentation character research to this bacterium, think subtilis, called after Bacillus.subtilis BN.

Table 1 bacterial strain (BN) and the contrast of subtilis colony morphology characteristic

Table 2 bacterial strain (BN) Physiology and biochemistry qualification result

Embodiment 3

It is in 15% glycerine pipe that bacterial strain is preserved in final concentration, gets in 100 μ l preservation bacterium liquid access 30ml nutrient broth mediums and carries out seed culture, and 37 ℃, after 200rpm cultivated 14h, the inoculum size access 50ml nutrient broth medium with 10% carried out fermentation culture.Nutrient broth consists of extractum carnis 3g/L, peptone 5g/L, the initial pH 7.2-7.4 of substratum.At 37 ℃, 200rpm condition bottom fermentation 24h is with the content of the chondroitin sulfate in HPLC mensuration fermented liquid.Output is 177mg/L.

Claims (2)

1. screening method that produces the chondroitin sulfate subtilis is characterized by:

1) the initial gross separation purifying of subtilis

Take approximately the 0.2g fermented soya bean in the 20ml0.85% stroke-physiological saline solution, bacteria suspension is vibrated in boiling water the heating 10min after, be cooled to rapidly room temperature, the dilution bacteria suspension is coated on the LB flat board, be inverted for 37 ℃ and cultivate 24h, single bacterium colony of the different colonial morphologies of picking carries out violet staining, and microscopically is observed gemma, the gemma bacterial strain will be arranged as the doubtful bacterial strain of subtilis that further screens, numbering is preserved;

2) produce the screening of chondroitin sulfate subtilis

The bacterial classification that above-mentioned separation and purification is obtained is inoculated in nutrient broth medium, centrifugal collection fermented liquid after cultivation 24h, fermented liquid adopts marker method to carry out qualitative analysis through high performance liquid chromatography, obtain the bacterial strain that chondroitin sulfate standard substance peak height increases, the bacterial strain that to make bacterial strain that chondroitin sulfate standard substance peak height increases be subtilis through morphology, Physiology and biochemistry and 16S rDNA molecular biology identification is the subtilis that produces chondroitin sulfate.

2. the production method of a chondroitin sulfate, the product chondroitin sulfate subtilis that it is characterized in that adopting screening method screening according to claim 1 to obtain is starting strain, produces chondroitin sulfate with seed culture and liquid fermenting;

1) seed culture:

Seed culture medium is in g/L: extractum carnis 3, peptone 5, initial p H7.2-7.4;

Culture condition: under 37 ℃ of temperature, shaking speed 200rpm condition, cultivate 12-14h;

2) liquid fermentation and culture:

Fermention medium is in g/L: extractum carnis 3, peptone 5, initial p H7.2-7.4;

Fermentation condition: inoculum size 10%, under 37 ℃ of temperature, shaking speed 200rpm condition, fermentation 20-24h.

Images