CN103173507A - Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium - Google Patents
Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium Download PDFInfo
- Publication number
- CN103173507A CN103173507A CN 201110436265 CN201110436265A CN103173507A CN 103173507 A CN103173507 A CN 103173507A CN 201110436265 CN201110436265 CN 201110436265 CN 201110436265 A CN201110436265 A CN 201110436265A CN 103173507 A CN103173507 A CN 103173507A
- Authority
- CN
- China
- Prior art keywords
- solution
- fermentation
- produce
- bacterium
- hyaluronate sodium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a production technology for fermentatively producing sodium hyaluronate by utilizing bacterium. The production technology comprises the following steps of: using streptococcus equi subsp. zooepidemicus (Streptococcus Zooepidemicus) as an original strain; mutagenizing by virtue of nitrosoguanidine, and also mutagenizing by virtue of a protoplast in the presence of ultraviolet rays; introducing a special culture medium; carrying out submerged fermentation to produce high-output hyaluronic acid; and then carrying out a relative separation and purification technology to obtain a high-purity sodium hyaluronate product. According to the production technology for fermentatively producing the sodium hyaluronate by utilizing the bacterium, under the culture conditions that the culture temperature is 32 to 38 DEG C, the pH (Potential Of Hydrogen) of fermentation liquor is maintained within the range of 5.0 to 9.0, and a fermentation tank is at speed of stirring of 150 to 600rev/min, the output of the hyaluronic acid is greatly increased; and the production technology is suitable for being applied to industrial production.
Description
Technical field
The present invention relates to the microbial fermentation technology field of hyaluronate sodium, especially relate to a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium.
Background technology
Hyaluronic acid is a kind of extensively being present in animal connective tissue and certain micro-organisms pod membrane, macromole acidic mucopolysaccharide with unique molecular structure and physico-chemical property, because of its very high visco-elasticity and extremely strong water-retentivity, can play such as lubricated joint, regulate the permeability of vessel wall, regulate protein, water, electrolyte diffusion and running, promote wound healing and improve the skin-nourishing metabolism, make skin tender, smooth, go wrinkle, increase elasticity, prevent the effects such as old and feeble, be widely used at present clinical medicine, pharmacy and cosmetic industry.
Early stage hyaluronic acid is take animal tissues such as people's umbilical cord, cockscomb, bovine vitreous body etc. as raw material, make after extraction, removal of impurities, precipitation and fractional separation, this method is limited because of starting material sources, and separation and purification is complicated, with high costs and replaced by microbe fermentation method gradually.The latter has output and is not subjected to the animal material resource limit, and cost is lower, and separation purifying technique is simple and direct, is easy to the advantages such as large-scale production.But the general output of wild hyaluronic acid production bacterial strain is lower; the expensive problem that causes thus makes it not be suitable for large-scale production; for this reason; we have comprehensively adopted multiple mutagenic breeding method that one strain wild mushroom has been carried out preferably; finally obtained a strain Producing Strain; its output reaches as high as 10g/L, greatly reduces thus production cost.Follow-up separation purifying technique is greatly simple and direct than the methods such as employing ion-exchange, has improved separation efficiency, and the highest yield can reach 80%.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of fermentative production hyaluronic acid that makes its high expression level amount under the condition after optimization by the transformation to thalline for the defective that overcomes above-mentioned prior art existence, and use filtration, absorption, complexing etc. to separate purification technique with purpose product and separation of fermentative broth, finally make the qualified product of meeting the need of market.Use the present invention to produce hyaluronate sodium and can increase substantially the biological expression amount, effectively reduce production cost, thereby obtain the production technique of utilizing fermentation using bacteria production hyaluronate sodium of market income preferably.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium comprises the following steps:
(1) with streptococcus equi epizootic disease subspecies for the bacterium that sets out, through nitrosoguanidine mutagenesis and Mutagenesis, seed selection obtains not producing beta hemolysin and Unidasa and produces the stable mutant strain of hereditary property that the hyaluronic acid ability improves greatly, with its on the inclined-plane seed culture medium after the temperature of 32～38 ℃ is cultivated 18～24h, be seeded in the first order seed substratum;
(2) cultured first order seed nutrient solution is seeded in the secondary seed nutrient solution, cultivates 6～12h and be in the early stage of exponential phase of growth to the flora in this nutrient solution under 32～38 ℃;
(3) cultured seed liquor is seeded in the higher fermention medium of nutrition degree, make thalline carry out fermentative production under following condition: to adopt fed-batch fermentation, fermented liquid pH is 5.0～9.0, temperature is 32～38 ℃, mixing speed 150～600rev/min, air flow 1.0～15.0vvm, incubation time 18～24h;
(4) finish fermentation, fermented liquid is transferred to extracts in storage tank, use while stirring 20% trichoroacetic acid(TCA) adjust pH to 3.0, hold over night, filter and collect filtrate with sheet frame, then be about 5g/L with hyaluronic content in the deionized water regulator solution, measure the pH value of this solution, with the NaOH solution of 2N with the pH regulator to 6.0 of this solution～9.0;
(5) addition with 10～100g/L adds the diatomite of specific model and stirs 2h, filters with the stainless steel sheet frame, collects filtrate;
(6) diatomite of the another kind of specific model addition according to 10～100g/L is joined in above-mentioned filtrate, stir 2h, filter with the stainless steel sheet frame, and collect filtrate;
(7) gac with 1～10g/L adds in above-mentioned filtrate, stirs after 2h with Plate Filtration and collects filtrate;
(8) then hyaluronic content in the filtrate of sampling and measuring unit volume add certain density cetylpyridinium chloride (CPC) solution according to the ratio of 2: 1 (mass ratio), and the limit edged stirs, and after all adding, continues to stir 2h;
(9) above-mentioned suspension is carried out Plate Filtration and is precipitated;
(10) the NaCl solution of configuration 0.2～1.0mol/L, then dissolve above-mentioned precipitation with this sodium chloride solution, and dissolution process will constantly stir, with accelerate dissolution speed;
(11) solution after above-mentioned dissolving is fully added the gac of 1～10g/L concentration, stir after 2h Plate Filtration and collect filtrate;
(12) with above-mentioned filtrate by the aperture be the membrane filtration degerming of 0.22 μ m, collect filtrate;
(13) add 95% ethanolic soln of 2～3 times of volumes in the above-mentioned filtrate, stirring is precipitated out the hyaluronic acid in original solution, when producing, precipitation carries out Plate Filtration to no longer including, collecting precipitation is to clean container, wash 2～3 times with 95% ethanol, then precipitation is positioned over and carries out drying in vacuum drying oven;
(14) collect dried product and pulverize and namely obtain final product hyaluronate sodium.
Selection in step (1) is: take strain streptococcus equi epizootic disease subspecies as starting strain, the screening means of 1～10% aseptic Sheep Blood or 0.01～0.1% hyaluronate sodium are added in employing in plate culture medium, through nitrosoguanidine mutagenesis and Mutagenesis breeding technique, screening obtain expression amount high, do not produce the stable mutant strain of beta hemolysin and Unidasa and hereditary property.
In step (1), the selection of the mutant strain of streptococcus equi epizootic disease subspecies is:
(1) prepare by the following method the blood agar plate substratum: weighing glucose 5.0g, extractum carnis 10g, yeast powder 5g, MgSO
41.0g agar 20g adds water 1L, after autoclave sterilization, adds the aseptic Sheep Blood of 20ml when being cooled to 45 ℃, mixes, and is positioned in 45 ℃ of water-baths, and is standby.
(2) the bacterium streptococcus equi epizootic disease subspecies of setting out are carried out nitrosoguanidine mutagenesis: the fresh bacterial classification after cultivation 24h on slant medium is seeded in liquid seed culture medium, 12h is cultivated in concussion under 32 ℃, abandon supernatant after this liquid medium is centrifugal, the bacterial sediment thing that obtains suspends with stroke-physiological saline solution, and filter with absorbent cotton, suitably dilution makes 10
8The bacteria suspension of individual/ml, the bacteria suspension that obtains is placed in little Erlenmeyer flask, and add the nitroso guanidine solution of 200 μ g/ml, shake under 32 ℃ after mixing and cultivate 30min, then use the dilution method termination reaction, and add in liquid nutrient medium under 32 ℃ concussion to cultivate 1h the bacterium liquid after mutagenesis, getting 1ml adds in above-mentioned blood agar plate substratum through the middle bacterium liquid of cultivating, pour in plate after mixing, cultivate 48h, the single bacterium colony that does not produce transparent circle on the picking flat board carries out preservation, obtains not producing the mutant strain of beta hemolysin.
(3) the preparation plate culture medium identical with composition described in (1), after autoclave sterilization, adding aseptic hyaluronate sodium to make its concentration is 0.02%, but does not add Sheep Blood, puts into 45 ℃ of water-baths, standby.
(4) getting according to method described in (2) mutant strain that does not produce beta hemolysin prepares bacteria suspension and processes with nitrosoguanidine as the bacterium that sets out, then the bacterium liquid 1ml that cultivated 1h in the middle of learning from else's experience adds in the plate culture medium described in (3), pour in plate after mixing, after cultivating 48h, be poured on uniformly the aseptic cetylpyridinium chloride solution of 1% concentration on flat board, after placing 10min, on the picking flat board, periphery of bacterial colonies has single bacterium colony of turbidity zone to carry out preservation, obtains neither producing beta hemolysin and does not also produce the mutant strain of Unidasa simultaneously.
(5) get the mutant strain that obtains described in (4) as setting out bacterium, carry out the hyaluronic variant of Mutagenesis breeding high-yield.Protoplastis preparation with the top condition of regeneration is: the bacterium that will set out is cultivated the centrifugal thalline that obtains after 11h with liquid seed culture medium, the helicase and the cellulase that add the high osmotic buffer dissolving, process 1h at 35 ℃ of constant temperature, centrifugal, obtain the protoplastis suspension with the high osmotic buffer washing, under the 15w ultraviolet lamp, in the 30cm place, irradiation 300s, the bacterium liquid that then will process is sneaked into the soft agar regeneration culture medium, is poured onto on the regeneration culture medium flat board.After cultivating 48h, to set out bacterium as a control group, grow fine on the picking flat board, the large and bacterium colony of viscosity carries out shake flask fermentation greater than single bacterium colony of control group bacterium to be cultivated, and after 32 ℃ of shaking culture 24h, measures the hyaluronic acid contents in fermented liquid.
The composition of above-mentioned soft agar regeneration culture medium is: glucose 5.0g, extractum carnis 10g, yeast powder 5g, MgSO
41.0g agar 4g adds water 1L; The composition of regeneration culture medium is; Glucose 5.0g, extractum carnis 10g, yeast powder 5g, MgSO
41.0g agar 20g adds water 1L.
Can repeatedly carry out the ultraviolet mutagenesis seed selection, to obtaining the higher gain mutant bacterial strain of output.We have carried out ultraviolet mutagenesis altogether 6 times, finally obtain a strain and neither produce the Producing Strain SY-121 that beta hemolysin does not produce Unidasa simultaneously yet, cultivate by fermentation, and in final fermented liquid, hyaluronic concentration is 10g/L.Through 10 cultivations of going down to posterity, produce the hyaluronic acid ability and do not descend.
The composition of described inclined-plane seed culture medium is: glucose 1.0～10g/L, extractum carnis 10～50g/L, yeast powder 5～15g/L, Na
2HPO
40.5～2.0g/L, MgSO
40.2～1.0g/L.
Described fermentation culture based component is: glucose 10～70g/L, yeast powder 10～50g/L, Na
2HPO
41～5g/L, MgSO
41～5g/L, defoamer 0.5～1.5g/L.
Described fed-batch fermentation is to be 70g/L with total reducing sugar, just sugar is 10g/L, remaining glucose was according to 1: 3: 5: 7 amount is divided four times respectively at the 8h of fermentation, 9h, 10h, 14h add in fermented liquid, continuous adjusting air inflow quantity and mixing speed in fermenting process, the dissolved oxygen of fermented liquid is maintained in 10～70% scope, and mixing speed can not be greater than 600rev/min.
The diatomite of the specific model described in step (5) is that model is the diatomite of CD10.
The diatomite of the specific model described in step (6) is that model is the diatomite of CD3.
Gac described in step (7) is the aciculiform gac.
Certain density cetylpyridinium chloride (CPC) solution described in step (8) is CPC to be dissolved in deionized water obtain the solution that concentration is 100g/L.
Compared with prior art, technological method of the present invention by lab scale, pilot scale, has been set up a ripe production technique of cover, can be used for large-scale production, and the product that makes at last meets general international standard, meets the need of market.Concrete target level of product quality can reach:
| Quality standard | Parameter |
| Glucuronic acid content | ≥44.0％ |
| The hyaluronic acid sodium content | ≥90％ |
| The solution transparence | ≥99.0％ |
| Weight loss on drying | ≤10％ |
| Molecular weight | ≥1.0×10 6 |
| Protein content | ≤0.1％ |
| Heavy metal | ≤10ppm |
| Total plate count | ≤10cfu/g |
| Total number of molds | ≤10cfu/g |
| Streptococcus aureus | Must not detect |
| Pseudomonas aeruginosa | Must not detect |
| Salmonellas | Must not detect |
| Intestinal bacteria | Must not detect |
Beneficial effect of the present invention: adopt the NTG mutagenic and breeding not produce the bacterial classification of beta hemolysin, its efficiency of inducing mutation is high, and only once experiment has namely obtained a satisfactory variant of strain; It is single nitrogenous source that fermention medium adopts yeast powder, has both saved production cost, is conducive to again the separation and purification process, significant to large production; Adopt and add at times, in proportion the feeding strategy of carbon source, cater to the growth characteristics of thalline, can increase substantially the production capacity of bacterial classification, than no-feed supplement or a feed supplement, the production capacity of thalline has improved respectively 32% and 18%; The product separation purge process adopts the diatomite auxiliary agent to filter, and has accelerated filtration velocity, has reduced and has produced power consumption, thereby saved production cost.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Embodiment 1
With mutant strain SY-121 as the bacterium that sets out, get one, its freeze-dried vaccine powder, with 2ml stroke-physiological saline solution dissolving bacterium powder, get the streak inoculation on slant medium of bacterium liquid with transfering loop, after 32 ℃ of cultivation 24h, it is inoculated in 1.6L first order seed substratum, after 32 ℃ of shaking culture 18h, is inoculated in the 80L secondary seed medium, after 32 ℃ of ventilation stir culture 10h, be transferred in the 0.8t fermention medium, before the fermention medium sterilization, composition is: yeast powder 10g/L, Na
2HPO
45g/L, MgSO
45g/L, defoamer 1.5g/L.after sterilization is completed, the aseptic Glucose Liquid 16L that shifts to an earlier date ready 500g/L is transferred in fermentor tank, then add the 80L seed liquor, open automatically and cultivate, with the pH of the NaOH controlled fermentation liquid of 10N in 7.2 left and right, temperature is controlled at 32.5 ℃, mixing speed 0～4h is 150rev/min, 4～6h is 200rev/min, 6～10h is 250rev/min, 10～12h is 300rev/min, 12～18h is 400rev/min, air flow is regulated and is regulated between 3.0～12vvm according to mixing speed, make dissolved oxygen can maintain 10～40%, with the aseptic Glucose Liquid 96L of 500g/L according to 1: 3: 5: 7 amount is divided four times respectively at the 8h of fermentation, 9h, 10h, 14h adds in fermented liquid, cultivate altogether 24h.
Finish fermentation, in the sampling and measuring fermented liquid, hyaluronic concentration is 7.5g/L.Fermented liquid is transferred in extractor, and fermentating liquid volume is 910L, opens tank mixer, transfers fermented liquid pH value to 3.0 with 20% trichoroacetic acid(TCA) solution while stirring, and filtrate is filtered and collected to hold over night with sheet frame.Be 4.55g/L with hyaluronic content in the deionized water regulator solution, amount to 1.5t.With the NaOH solution of the 2N pH regulator to 6.5 with this solution.Addition with 50g/L adds diatomite (CD10) 75Kg and stirs 2h, filters with the stainless steel sheet frame, collects filtrate 1.5t altogether.Be that 37.5Kg joins in above-mentioned filtrate with the diatomite CD3 of another kind of model according to the addition of 25g/L, stir 2h, filter with the stainless steel sheet frame, and collect filtrate 1.52t altogether.The gac 11.4Kg of 7.5g/L is added in above-mentioned filtrate, stir after 2h with Plate Filtration and collect filtrate 1.56t altogether, sampling records hyaluronic concentration 4.0g/L in this filtrate, then add cetylpyridinium chloride (CPC) the solution 31.2L of 10% concentration according to the ratio of 2: 1 (mass ratio), the limit edged stirs, after all adding, continue to stir 2h.Above-mentioned suspension is carried out Plate Filtration and is precipitated.Then the NaCl solution of the 0.2mol/L of configuration 1.12t dissolve above-mentioned precipitation with this sodium chloride solution, dissolution process will constantly stir, with accelerate dissolution speed.Solution after above-mentioned dissolving is fully added the gac 6Kg of 5g/L concentration, stir after 2h Plate Filtration and collect filtrate.Be the membrane filtration degerming of 0.22 μ m by an aperture with above-mentioned filtrate, collect filtrate.The 95% ethanolic soln 3t that adds 2.5 times of volumes in the above-mentioned filtrate, stirring is precipitated out the hyaluronic acid in original solution, when producing, precipitation carries out Plate Filtration to no longer including, in collecting precipitation to a clean container, wash this precipitation 3 times with 95% ethanol, then precipitation is positioned over and carries out drying in vacuum drying oven.
Collect dried product and pulverize to obtain final product, weighing knows that this batch obtains meeting the common 5.4Kg of qualified product hyaluronate sodium of quality standard altogether, and the total recovery of separation purifying technique is 79%.
Embodiment 2
With mutant strain SY-121 as the bacterium that sets out, get one, its freeze-dried vaccine powder, with 2ml stroke-physiological saline solution dissolving bacterium powder, get the streak inoculation on slant medium of bacterium liquid with transfering loop, after 33 ℃ of cultivation 24h, it is inoculated in 1.6L first order seed substratum, after 33 ℃ of shaking culture 12h, is inoculated in the 80L secondary seed medium, after 33 ℃ of ventilation stir culture 10h, be transferred in the 0.8t fermention medium, before the fermention medium sterilization, composition is: yeast powder 25g/L, Na
2HPO
43g/L, MgSO
43g/L, defoamer 1.5g/L.after sterilization is completed, the aseptic Glucose Liquid 16L that shifts to an earlier date ready 500g/L is transferred in fermentor tank, then add the 80L seed liquor, open automatically and cultivate, with the pH of the NaOH controlled fermentation liquid of 10N in 8.0 left and right, temperature is controlled at 33 ℃, mixing speed 0～6h is 150rev/min, 6～8h is 200rev/min, 8～10h is 250rev/min, 10～12h is 300rev/min, 12～18h is 400rev/min, air flow is regulated and is regulated between 3.0～12vvm according to mixing speed, make dissolved oxygen can maintain 10～30%, with the aseptic Glucose Liquid 96L of 500g/L according to 1: 3: 5: 7 amount is divided four times respectively at the 8h of fermentation, 9h, 10h, 14h adds in fermented liquid, cultivate altogether 20h.
Finish fermentation, in the sampling and measuring fermented liquid, hyaluronic concentration is 8.2g/L.Fermented liquid is transferred in extractor, and fermentating liquid volume is 860L, opens tank mixer, transfers fermented liquid pH value to 3.0 with 20% trichoroacetic acid(TCA) solution while stirring, and filtrate is filtered and collected to hold over night with sheet frame.Be 4.7g/L with hyaluronic content in the deionized water regulator solution, amount to 1.5t.With the NaOH solution of the 2N pH regulator to 6.5 with this solution.Addition with 50g/L adds diatomite (CD10) 75Kg and stirs 2h, filters with the stainless steel sheet frame, collects filtrate 1.5t altogether.Be that 37.5Kg joins in above-mentioned filtrate with the diatomite CD3 of another kind of model according to the addition of 25g/L, stir 2h, filter with the stainless steel sheet frame, and collect filtrate 1.51t altogether.The gac 11.33Kg of 7.5g/L is added in above-mentioned filtrate, stir after 2h with Plate Filtration and collect filtrate 1.54t altogether, sampling records hyaluronic concentration 4.3g/L in this filtrate, then add cetylpyridinium chloride (CPC) the solution 33.1L of 10% concentration according to the ratio of 2: 1 (mass ratio), the limit edged stirs, after all adding, continue to stir 2h.Above-mentioned suspension is carried out Plate Filtration and is precipitated.Then the NaCl solution of the 0.2mol/L of configuration 1.14t dissolve above-mentioned precipitation with this sodium chloride solution, dissolution process will constantly stir, with accelerate dissolution speed.Solution after above-mentioned dissolving is fully added the gac 6Kg of 5g/L concentration, stir after 2h Plate Filtration and collect filtrate.Be the membrane filtration degerming of 0.22 μ m with above-mentioned filtrate by an aperture, again collect filtrate.The 95% ethanolic soln 3t that adds 2.5 times of volumes in the above-mentioned filtrate, stirring is precipitated out the hyaluronic acid in original solution, when producing, precipitation carries out Plate Filtration to no longer including, in collecting precipitation to a clean container, wash this precipitation 3 times with 95% ethanol, then precipitation is positioned over and carries out drying in vacuum drying oven.
Collect dried product and pulverize to obtain final product, weighing knows that this batch obtains meeting the common 5.64Kg of qualified product hyaluronate sodium of quality standard altogether, and the total recovery of separation purifying technique is 80.3%.
Claims (10)
1. a production technique of utilizing fermentation using bacteria to produce hyaluronate sodium, is characterized in that, this technique comprises the following steps:
(1) with streptococcus equi epizootic disease subspecies for the bacterium that sets out, through nitrosoguanidine mutagenesis and Mutagenesis, seed selection obtains not producing beta hemolysin and Unidasa and produces the stable mutant strain of hereditary property that the hyaluronic acid ability improves greatly, with its on the inclined-plane seed culture medium after the temperature of 32～38 ℃ is cultivated 18～24h, be seeded in the first order seed substratum;
(2) cultured first order seed nutrient solution is seeded in the secondary seed nutrient solution, cultivates 6～12h and be in the early stage of exponential phase of growth to the flora in this nutrient solution under 32～38 ℃;
(3) cultured seed liquor is seeded in the higher fermention medium of nutrition degree, make thalline carry out fermentative production under following condition: to adopt fed-batch fermentation, fermented liquid pH is 5.0～9.0, temperature is 32～38 ℃, mixing speed 150～600rev/min, air flow 1.0～15.0vvm, incubation time 18～24h;
(4) finish fermentation, fermented liquid is transferred to extracts in storage tank, use while stirring 20% trichoroacetic acid(TCA) adjust pH to 3.0, hold over night, filter and collect filtrate with sheet frame, then be about 5g/L with hyaluronic content in the deionized water regulator solution, measure the pH value of this solution, with the NaOH solution of 2N with the pH regulator to 6.0 of this solution～9.0;
(5) addition with 10～100g/L adds the diatomite of specific model and stirs 2h, filters with the stainless steel sheet frame, collects filtrate;
(6) diatomite of the another kind of specific model addition according to 10～100g/L is joined in above-mentioned filtrate, stir 2h, filter with the stainless steel sheet frame, and collect filtrate;
(7) gac with 1～10g/L adds in above-mentioned filtrate, stirs after 2h with Plate Filtration and collects filtrate;
(8) then hyaluronic content in the filtrate of sampling and measuring unit volume add certain density cetylpyridinium chloride (CPC) solution according to the ratio of 2: 1 (mass ratio), and the limit edged stirs, and after all adding, continues to stir 2h;
(9) above-mentioned suspension is carried out Plate Filtration and is precipitated;
(10) the NaCl solution of configuration 0.2～1.0mol/L, then dissolve above-mentioned precipitation with this sodium chloride solution, and dissolution process will constantly stir, with accelerate dissolution speed;
(11) solution after above-mentioned dissolving is fully added the gac of 1～10g/L concentration, stir after 2h Plate Filtration and collect filtrate;
(12) with above-mentioned filtrate by the aperture be the membrane filtration degerming of 0.22 μ m, collect filtrate;
(13) add 95% ethanolic soln of 2～3 times of volumes in the above-mentioned filtrate, stirring is precipitated out the hyaluronic acid in original solution, when producing, precipitation carries out Plate Filtration to no longer including, collecting precipitation is to clean container, wash 2～3 times with 95% ethanol, then precipitation is positioned over and carries out drying in vacuum drying oven;
(14) collect dried product and pulverize and namely obtain final product hyaluronate sodium.
2. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, it is characterized in that, selection in step (1) is: take strain streptococcus equi epizootic disease subspecies as starting strain, the screening means of 1～10% aseptic Sheep Blood or 0.01～0.1% hyaluronate sodium are added in employing in plate culture medium, through nitrosoguanidine mutagenesis and Mutagenesis breeding technique, screening obtain expression amount high, do not produce the stable mutant strain of beta hemolysin and Unidasa and hereditary property.
3. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 2, it is characterized in that, described nitrosoguanidine mutagenesis method is: take 10mg NTG and pour in aseptic centrifuge tube, add 0.1mol/L, the phosphoric acid buffer 10mL of pH 6.0, dark place vibration dissolving, divide and be filled in aseptic small test tube, every pipe 1mL, getting 1mL bacterium liquid adds in above-mentioned centrifuge tube, fully shake up, be placed in immediately 37 ℃ of water-bath oscillation treatment 30min, after the final concentration of NTG is 500 μ g/mL, centrifugal collection thalline, the supernatant liquor that will contain NTG is poured concentrated NaOH solution into and is discarded, wash thalline 3 times with sterilized water, stop the mutagenesis of NTG with the Macrodilution method, add the 2mL stroke-physiological saline solution in the most backward centrifuge tube, be coated with aseptic SBA dull and stereotyped, the screening periphery of bacterial colonies is not produced the variation bacterium of transparent circle and is differentiated and preservation, finally obtain a strain and do not produce the dissociant of beta hemolysin, it is proceeded NTG mutagenesis as the bacterium that sets out, the ultimate density of mutagenesis NTG used is adjusted to 600 μ g/mL, coating contains the screening flat board of 0.01～0.1% hyaluronate sodium, not producing single bacterium colony of transparent circle around choosing colony differentiates and preservation, can obtain neither producing beta hemolysin, do not produce again the dissociant of Unidasa,
described Mutagenesis method is: with the above-mentioned neither product beta hemolysin that seed selection obtains through nitrosoguanidine mutagenesis, do not produce again the dissociant of Unidasa as setting out bacterium, with the centrifugal thalline that obtains after seed culture medium cultivation 10h, add the sucrose solution of 2mol/L as high osmotic buffer, helicase and the 0.2mg/ml cellulase solution 5ml of the 0.5mg/ml of dissolving, process 1h at 32 ℃ of constant temperature, centrifugal, obtain the protoplastis suspension with the high osmotic buffer washing, under the 15w ultraviolet lamp, in the 30cm place, irradiation 1min, then the bacterium liquid that will process is sneaked in the soft agar seed culture medium, agar content is 0.5% seed culture medium, be poured onto on the seed culture medium flat board.Cultivate 24～48h in 35 ℃, after dull and stereotyped upper bacterium colony grows, picking is compared the larger single bacterium colony of area of colony than the bacterium that sets out and is carried out the shake flask fermentation cultivation, and mensuration hyaluronate sodium output, finally obtain a strain and improve 400% bacterial strain than original bacterium production capacity, be and do not produce beta hemolysin and Unidasa and produce the stable mutant strain of hereditary property that the hyaluronic acid ability improves greatly.
4. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, is characterized in that, the composition of described inclined-plane seed culture medium is: glucose 1.0～10g/L, extractum carnis 10～50g/L, yeast powder 5～15g/L, Na
2HPO
40.5～2.0g/L, MgSO
40.2～1.0g/L.
5. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, is characterized in that, described fermentation culture based component is: glucose 10～70g/L, yeast powder 10～50g/L, Na
2HPO
41～5g/L, MgSO
41～5g/L, defoamer 0.5～1.5g/L.
6. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, it is characterized in that, described fed-batch fermentation is to be 70g/L with total reducing sugar, and just sugar is 10g/L, and remaining glucose was according to 1: 3: 5: 7 amount is divided four times respectively at the 8h of fermentation, 9h, 10h, 14h add in fermented liquid, continuous adjusting air inflow quantity and mixing speed in fermenting process, the dissolved oxygen of fermented liquid is maintained in 10～70% scope, and mixing speed can not be greater than 600rev/min.
7. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, is characterized in that, the diatomite of the specific model described in step (5) is that model is the diatomite of CD10.
8. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, is characterized in that, the diatomite of the specific model described in step (6) is that model is the diatomite of CD3.
9. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, is characterized in that, the gac described in step (7) is the aciculiform gac.
10. a kind of production technique of utilizing fermentation using bacteria to produce hyaluronate sodium according to claim 1, it is characterized in that, certain density cetylpyridinium chloride (CPC) solution described in step (8) is CPC to be dissolved in deionized water obtain the solution that concentration is 100g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110436265 CN103173507A (en) | 2011-12-23 | 2011-12-23 | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110436265 CN103173507A (en) | 2011-12-23 | 2011-12-23 | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103173507A true CN103173507A (en) | 2013-06-26 |
Family
ID=48633695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110436265 Pending CN103173507A (en) | 2011-12-23 | 2011-12-23 | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103173507A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317257A (en) * | 2016-08-30 | 2017-01-11 | 新疆阜丰生物科技有限公司 | Efficient energy-saving method of extracting hyaluronic acid |
| CN106434443A (en) * | 2016-09-23 | 2017-02-22 | 东辰控股集团有限公司 | Production process of sodium hyaluronate |
| CN107338201A (en) * | 2017-06-23 | 2017-11-10 | 浙江工业大学 | Malian drainage SXY36 and the application in fermenting and producing hyaluronic acid |
| CN108852946A (en) * | 2018-09-18 | 2018-11-23 | 广州上官靖文化传播有限公司 | A kind of rapid detumescence, sewing product and preparation method thereof that is antibacterial, repairing skin |
| CN109486879A (en) * | 2018-12-06 | 2019-03-19 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and its fermentation process |
| CN109536550A (en) * | 2018-12-06 | 2019-03-29 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and preparation method thereof |
| CN110423291A (en) * | 2019-09-06 | 2019-11-08 | 焦点生物医药有限公司 | A kind of instant Sodium Hyaluronate preparation method |
| CN116200319A (en) * | 2022-12-27 | 2023-06-02 | 山东丰金美业科技有限公司 | Genetically engineered bacterium for producing low molecular weight hyaluronic acid by one-step fermentation and construction method and application thereof |
-
2011
- 2011-12-23 CN CN 201110436265 patent/CN103173507A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317257A (en) * | 2016-08-30 | 2017-01-11 | 新疆阜丰生物科技有限公司 | Efficient energy-saving method of extracting hyaluronic acid |
| CN106317257B (en) * | 2016-08-30 | 2019-07-16 | 新疆阜丰生物科技有限公司 | A kind of energy-efficient method for extracting hyaluronic acid |
| CN106434443A (en) * | 2016-09-23 | 2017-02-22 | 东辰控股集团有限公司 | Production process of sodium hyaluronate |
| CN107338201A (en) * | 2017-06-23 | 2017-11-10 | 浙江工业大学 | Malian drainage SXY36 and the application in fermenting and producing hyaluronic acid |
| CN107338201B (en) * | 2017-06-23 | 2021-02-02 | 浙江工业大学 | Streptococcus equi subsp zooepidemicus SXY36 and application thereof in fermentation production of hyaluronic acid |
| CN108852946A (en) * | 2018-09-18 | 2018-11-23 | 广州上官靖文化传播有限公司 | A kind of rapid detumescence, sewing product and preparation method thereof that is antibacterial, repairing skin |
| CN109486879A (en) * | 2018-12-06 | 2019-03-19 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and its fermentation process |
| CN109536550A (en) * | 2018-12-06 | 2019-03-29 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and preparation method thereof |
| CN110423291A (en) * | 2019-09-06 | 2019-11-08 | 焦点生物医药有限公司 | A kind of instant Sodium Hyaluronate preparation method |
| CN110423291B (en) * | 2019-09-06 | 2020-06-16 | 焦点生物医药有限公司 | Preparation method of instant sodium hyaluronate |
| CN116200319A (en) * | 2022-12-27 | 2023-06-02 | 山东丰金美业科技有限公司 | Genetically engineered bacterium for producing low molecular weight hyaluronic acid by one-step fermentation and construction method and application thereof |
| CN116200319B (en) * | 2022-12-27 | 2024-01-12 | 山东丰金美业科技有限公司 | Genetically engineered bacterium for producing low molecular weight hyaluronic acid by one-step fermentation and construction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103173507A (en) | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium | |
| CN104312834B (en) | A kind of preparation method of Sugarcane fruit wine | |
| CN101037661A (en) | Pseudoalteromonas and its usage | |
| CN106399422A (en) | Preparation method of bacterial cellulose | |
| CN105255741B (en) | The aspergillus niger mutant strain of one plant of high-yield glucoamylase and its industrial fermentation technology | |
| CN105309750A (en) | Comprehensive utilization and production technology of high-content antrodia camphorate fungal active polysaccharide, triterpene fine powder, and byproducts thereof | |
| CN102584411A (en) | Agar protococcus biologic fertilizer and preparation method thereof | |
| CN105385607A (en) | Lentinus edodes liquid submerged fermentation culture medium formula and fermentation technology | |
| CN101933439A (en) | Method for improving phellinus igniarius hypha amount of submerged culture by utilizing plant oil | |
| CN103074401B (en) | Method for producing neomycin sulfate | |
| CN101993847A (en) | Bacterial cellulose strain | |
| CN102630493B (en) | Method and equipment for preparing liquid strain of edible and medicinal fungi by using conventional drinking water | |
| CN106635919A (en) | Spirulina culture method | |
| CN109504725A (en) | A kind of method and fermentation medium of fermentation Hericium erinaceus preparation high-purity Hericium erinaceus Polysaccharides | |
| CN102002482B (en) | Method for producing PRRS (Porcine Reproductive and Respiratory Syndrome) viruses | |
| CN101492705A (en) | Method of preparing pleurotu ostreatus polysaccharide | |
| CN105176859A (en) | Strain MQO-153 for production of arginine deiminase | |
| CN102002481A (en) | Production method of porcine reproductive and respiratory syndrome virus | |
| CN1766082A (en) | Culturing method for heterotrophic chlorella growth without irradiation | |
| CN101864471A (en) | Microbial fermentation method for producing hyaluronic acid | |
| CN109628509B (en) | Method for producing pyrroloquinoline quinone by semi-continuous fermentation process | |
| CN103146769A (en) | Method for preparating citric acid by fermentation | |
| CN114591878B (en) | Process for preparing mariculture tail water treatment microbial inoculum and tail water treatment method | |
| CN109680025A (en) | Fermentation process for improving production level of recombinant human collagen and reducing protein degradation speed | |
| CN105177075A (en) | Method for preparation of L-citrulline with arginine as raw material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1183508 Country of ref document: HK |
|
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130626 |
|
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1183508 Country of ref document: HK |