CN109486879A - A kind of Sodium Hyaluronate and its fermentation process - Google Patents
A kind of Sodium Hyaluronate and its fermentation process Download PDFInfo
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Abstract
The present invention provides a kind of fermentation process of Sodium Hyaluronate, include the following steps: that strain is cultivated activation 12-24h by (A) on plating medium；(B) strain after activation is seeded on fermentation medium according to the inoculum concentration of 5-15wt%, ventilatory capacity control between 1-2vvm, fermented and cultured for 24 hours more than.The Sodium Hyaluronate that fermentation process of the invention is prepared itself is with high purity, belong to the product of high molecular weight range, and the acquisition further expansion of the product application range of Sodium Hyaluronate can form good economic benefit.
Description
Technical field
The present invention relates to Sodium Hyaluronate manufacture fields, in particular to a kind of Sodium Hyaluronate and its fermentation process.
Background technique
Hyaluronic acid (hyaluronic acid, HA) also known as Hyaluronic Acid, water conservation, moisturizing and antibacterial invasion with height
Protective effect and be widely used as the additives of cosmetics.HA is mainly used for treatment of arthritis, ophthalmology as a kind of biochemical drug
Viscosurgery, soft tissue repair and sticky inhibitor of surgical site infections etc..HA is by D- glucuronic acid and acetylaminohydroxyphenylarsonic acid D-
The acid mucopolysaccharide that glucose is formed by the molar ratio of 1:1.In cytoplasma membrane inner wall, in the case where HA synthesizes enzyme effect, HA is by uridine
Extract synthesizes before diphosphate-N-acetyl aminoglucose (UDP-GlcNAc) and uridine 5'-diphosphate-glucuronic acid (UDP-GlcA) two.
HA without species difference, no matter which kind of source, structure and composition is the same, and there is only the difference of relative molecular mass.
Currently, the production of HA is still mainly animal tissue's extraction method and microbe fermentation method.Animal tissue's extraction method
Raw material is limited, low output, isolates and purifies process complexity (the polysaccharide impurity such as sulfur acid chondroitin, sulfuric acid Portugal amine glycan), is produced into
This height, HA molecular weight obtained is also smaller, is not able to satisfy the market demand.And microbe fermentation method has production scale not by dynamic
Raw material limits, and HA exists in fermentation liquid with free state, is easily isolated purifying, at low cost, and it is raw to be easily formed large-scale industrial
The advantages that production, the danger of the Causative virus pollution of animal origin-free.But due between bacterium own metabolism and product expression
Imbalance seriously constrains the yield of hyaluronic acid and the change of molecular weight.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of fermentation process of Sodium Hyaluronate, which passes through to fermentation
Technique is made rational planning for, and to the Optimization of fermentation medium after, improve the yield of Sodium Hyaluronate, and realize
The molecular weight of Sodium Hyaluronate is controlled, obtains possessing the Sodium Hyaluronate between ideal molecular weight area, expands hyalomitome
The application field of sour sodium is related in this method with seeking the balance in fermentation process between microorganism own growth and product expression
And numerous parameters be that inventor have passed through a large amount of practices and obtain, by the way that parameters to be adjusted to suitable range
It is interior, it is able to produce out the Sodium Hyaluronate of excellent quality.
The second object of the present invention is to provide the Sodium Hyaluronate that above-mentioned fermentation process is prepared, the Sodium Hyaluronate
Purity is high itself, belongs to the product of high molecular weight range, the acquisitions further expansion of the product application model of Sodium Hyaluronate
It encloses, good economic benefit can be formed.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention provides a kind of fermentation process of Sodium Hyaluronate, include the following steps:
(A) strain is cultivated to activation 12-24h on plating medium；
(B) strain after activation is seeded on fermentation medium according to 5-15wt% inoculum concentration, ventilatory capacity is controlled in 1-
Between 2vvm, fermented and cultured for 24 hours more than；
Wherein, glucose is added in the fermentation medium stage by stage, glucose adds 2-3kg at interval of 2-3h.
The fermentation process of Sodium Hyaluronate provided by the present invention, by making rational planning for zymotechnique, and to hair
After the Optimization of ferment culture medium, improve the yield of Sodium Hyaluronate, and realize to the molecular weight of Sodium Hyaluronate into
Row control, obtains possessing the Sodium Hyaluronate between ideal molecular weight area, expands the application field of Sodium Hyaluronate, to seek to send out
Balance during ferment between microorganism own growth and product expression, numerous parameters involved in this method are inventor's warps
Cross what a large amount of practices obtained, by being able to produce out the saturating of excellent quality in range that parameters are adjusted to suitable
Bright matter acid sodium.
It should be noted that vvm:air volume/culture volume/min (ventilation ratio) is: minute ventilation volume
With the ratio of the practical material liquid volume of tank body.Ventilatory capacity in fermentor is generally in terms of vvm, and gas volume therein is with standard state
Meter.
In fact, the production of early stage hyaluronic acid is mainly extracted from rooster comb, mainly uses microbial fermentation at present
Method production, streptococcus is widely used in the industrial production as main production bacterial strain, main reason is that fermentation
Method HA production have it is at low cost, and because in fermentation HA be in free state and be easy to purify this point feature.In pharmaceutical grade Hyaluronic Acid
In the application of sodium, there is the demand of high molecular weight to sodium hyaluronate bulk pharmaceutical chemicals, meanwhile, for the needs of industrialized production and application,
Also there is certain requirement to high yield.
Preferably as further enforceable scheme, in the step (A), the composition of plating medium are as follows: brain heart leaching
Powder 30-40g/L, yeast extract 5-15g/L, glucose 0.5-2.0g/L, agar 10-15g/L.
Preferably as further enforceable scheme, in the step (A), the cultivation temperature control on plating medium
System is between 37-39 DEG C.
Plating medium culture activated spawn, and the strain more purified is obtained, strain of the present invention is common
Streptococcus, there is no big improvement in the selection of strain.
Culture is carried out as follows when practical culture: preparing plating medium, (the brain heart soaks powder: 30-40g/L；Yeast
Soak powder: 5-15g/L；Glucose: 0.5-2.0g/L；Agar: 10-15g/L), work seed is seeded in flat by inverted plate after sterilizing
On plate, cultivated 12-24 hours in 37 ± 2 DEG C of constant incubators.
Preferably as further enforceable scheme, in the step (B), the every 400L of fermentation medium includes
Composition are as follows: glucose 13-16kg, yeast extract 6-9kg, magnesium sulfate 0.6-1.3kg, sodium dihydrogen phosphate 1.0-1.3kg, sulfuric acid
Potassium 0.3-0.6kg, L-arginine 16-23g, trace element solution 0.9-1.3L, defoaming agent 15-23ml.
Preferably as further enforceable scheme, in the step (B), the pH of fermentation medium is controlled in 6-14
Between, temperature is between 37-39 DEG C, and revolving speed is in 60rpm or more.
Preferably as further enforceable scheme, in the step (A), the hydrogen that is supplemented in the fermentation medium
The mass ratio of sodium oxide molybdena and glucose is (1.2-1.5): 1；
Preferably, sodium hydroxide is made into the sodium hydroxide solution that concentration is 3-5mol/L, and more preferably concentration is 4mol/L.
The quality of the sodium hydroxide of above-mentioned supplement is the mass ratio between the glucose of supplement, rather than is fermented with preparing
The mass ratio of glucose used in culture medium.
The process of fermented and cultured determines between the yield and molecular weight area obtained of subsequent clear matter acid sodium, therefore its
The preparation of culture medium and the mode of benefit sugar, benefit alkali are required to scheme operation according to the invention, wherein the process for mending alkali is to hold
Continuous progress, because hyaluronic acid itself is to will affect the pH of system in acidity, so needing lasting benefit alkali, supplementing
It prevents alkalinity too strong in the process, is generically configured to aqueous slkali, the concentration general control of aqueous slkali, should not mistake between 3-5mol/L
Height prevents the damage for having certain to strain, influences its fermentating breeding.
The process for mending sugar is to add 2-3kg at interval of 2-3h, by carrying out supplement lasting stage by stage on the one hand to energy
The enriched of nutrition is avoided, strain on the other hand can be promoted to continue normally to grow, optimal operation is supplemented every 2h
The glucose of 2kg, general fermented and cultured more than for 24 hours, then can respectively 2 hours, 4 hours, 6 hours, 8 hours, 10 hours,
12 hours, 14 hours, 16 hours, be separately added into 2kg glucose on 20 hours time points.
It further include expanding between the step (A) and the step (B) preferably as further enforceable scheme
The step of culture: after the strain after activation is cultivated in shaking flask, then continue to expand culture on seed culture medium.
Preferably as further enforceable scheme, in the step (A), the every 3.5L of culture medium in shaking flask includes
Composition are as follows: glucose 55-70g, magnesium sulfate 4.5-7.3g, yeast extract 43-63g, potassium dihydrogen phosphate 4.6-6.7g, bicarbonate
Sodium 0.20-0.31g, phosphate buffer 1 15-123ml, trace element solution 2.4-3.3ml, defoaming agent 0.7-1.4ml；
Preferably, the composition that the every 25L of the seed culture medium includes are as follows: glucose 434-477g, magnesium sulfate 42-49g, ferment
Mother soaks powder 431-485g, potassium dihydrogen phosphate 41-47g, sodium bicarbonate 2.0-2.9g, phosphate buffer 910-930ml, micro member
Plain solution 17-30ml, defoaming agent 0.8-1.2ml.
The present invention expand culture method be the two-stage of shaking flask culture and seed culture expand culture by way of, it
So need expand culture, be because inoculum concentration needs and and tamed strain, simultaneously for shorten fermentation time, guarantee
Production level also has positive help.
The present invention also provides the Sodium Hyaluronates being prepared using above-mentioned fermentation process, using above-mentioned fermentation process system
The yield of standby obtained Sodium Hyaluronate is 11g/L or more, and molecular weight is between 130-200Mw.
Compared with prior art, the invention has the benefit that
(1) fermentation process of Sodium Hyaluronate of the invention is trained by making rational planning for zymotechnique, and to fermentation
After the Optimization for supporting base, the yield of Sodium Hyaluronate is improved, and realize and control to the molecular weight of Sodium Hyaluronate
System, obtains possessing the Sodium Hyaluronate between ideal molecular weight area, expands the application field of Sodium Hyaluronate, to seek to ferment
Balance in journey between microorganism own growth and product expression, numerous parameters involved in this method are that inventor have passed through
What a large amount of practices obtained, by being able to produce out the hyalomitome of excellent quality in range that parameters are adjusted to suitable
Sour sodium.
(2) Sodium Hyaluronate itself that the present invention is obtained using above-mentioned fermentation process is with high purity, belongs to high molecular weight range
Product, the acquisition further expansion of the product application range of Sodium Hyaluronate can form good economic benefit.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The fermentation process of Sodium Hyaluronate specifically comprises the following steps:
1) plate culture:
Preparing plating medium, (the brain heart soaks powder: 30g/L；Yeast extract: 15g/L；Glucose: 0.5g/L；Agar: 15g/
L), work seed is seeded on plate, cultivates 24 hours in 39 DEG C of constant incubators by inverted plate after sterilizing.
2) shaking flask culture:
Prepare Shake flask medium 3.5L [glucose: 70g, magnesium sulfate (seven water): 7.3g, yeast extract: 43-63g, phosphoric acid
Potassium dihydrogen: 4.6-6.7g, sodium bicarbonate: 0.31g, phosphate buffer: 115ml (0.1mol/L sodium dihydrogen phosphate (two water) and
0.1mol/L disodium hydrogen phosphate dodecahydrate mixes in equal volume), trace element solution: 3.3ml, defoaming agent: 0.7ml], use NaOH
PH to 7.2 is adjusted, culture medium is sub-packed in 10 conical flasks by 350ml/ bottles, is sterilized after cold, respectively from plate culture
Strain access is scraped on base, is placed in constant-temperature table, and setting revolving speed is not less than 100rpm, 37 DEG C of cultures to net added value >=1.0 OD.
3) seed culture:
Seed culture medium 25L [glucose: 434g, magnesium sulfate (seven water): 49g, yeast extract: 431g, biphosphate is added
Potassium: 47g, sodium bicarbonate: 2.0g, phosphate buffer: 910ml, trace element solution: 17ml, defoaming agent: 0.8ml]；Sterilizing
5% inoculum concentration is pressed afterwards, is seeded in seeding tank, is not less than 70rpm, temperature: 37 DEG C, ventilatory capacity: the condition of 1.5vvm in revolving speed
Cultivate 6h.
4) fermented and cultured:
Prepare fermentation medium 400L [glucose: 16kg, yeast extract: 9kg, magnesium sulfate (seven water): 0.6kg, di(2-ethylhexyl)phosphate
Hydrogen sodium (two water): 1.0kg, potassium sulfate: 0.3kg, L-arginine: 16g, trace element solution: 0.9L, Polyalcoxyether
(defoaming agent): 15ml], culture transferring after sterilizing is controlled revolving speed and is not less than 60rpm by 15wt% inoculum concentration；Temperature: 37 DEG C；PH:
6.0；Ventilatory capacity: 2vvm；
It is separately added into 2kg glucose in 2h, 4h, 6h, 8h of fermented and cultured, 10h, 12h, 14h, 16h, 20h, is controlled
The sodium hydroxide and glucose weight ratio of 3mol/L is 1.2:1.
Embodiment 2
The fermentation process of Sodium Hyaluronate specifically comprises the following steps:
1) plate culture:
Preparing plating medium, (the brain heart soaks powder: 40g/L；Yeast extract: 5g/L；Glucose: 2.0g/L；Agar: 10g/
L), work seed is seeded on plate, cultivates 12 hours in 37 DEG C of constant incubators by inverted plate after sterilizing.
2) shaking flask culture:
Prepare Shake flask medium 3.5L [glucose: 55g, magnesium sulfate (seven water): 4.5g, yeast extract: 63g, biphosphate
Potassium: 4.6g, sodium bicarbonate: 0.20g, phosphate buffer: 123ml (0.1mol/L sodium dihydrogen phosphate (two water) and 0.1mol/L
Disodium hydrogen phosphate dodecahydrate mixes in equal volume), trace element solution: 2.4ml, defoaming agent: 1.4ml], with NaOH adjust pH to
7.3, culture medium is sub-packed in 10 conical flasks by 350ml/ bottles, sterilizes after cold, is scraped from plating medium respectively
Strain access, is placed in constant-temperature table, and setting revolving speed is not less than 100rpm, 39 DEG C of cultures to net added value >=1.0 OD.
3) seed culture:
Seed culture medium 25L [glucose: 477g, magnesium sulfate (seven water): 42g, yeast extract: 485g, biphosphate is added
Potassium: 41g, sodium bicarbonate: 2.9g, phosphate buffer: 930ml, trace element solution: 30ml, defoaming agent: 1.2ml]；Sterilizing
15% inoculum concentration is pressed afterwards, is seeded in seeding tank, is not less than the condition training of 70rpm, temperature: 39 DEG C, ventilatory capacity: 1vvm in revolving speed
Support 3h.
4) fermented and cultured:
Prepare fermentation medium 400L [glucose: 13kg, yeast extract: 6kg, magnesium sulfate (seven water): 1.3kg, di(2-ethylhexyl)phosphate
Hydrogen sodium (two water): 1.3kg, potassium sulfate: 0.6kg, L-arginine: 23g, trace element solution: 1.3L, Polyalcoxyether
(defoaming agent): 23ml], culture transferring after sterilizing is controlled revolving speed and is not less than 60rpm by 5wt% inoculum concentration；Temperature: 39 DEG C；PH:
14.0；Ventilatory capacity: 1.5vvm；
It is separately added into 3kg glucose in 2h, 4h, 6h, 8h of fermented and cultured, 10h, 12h, 14h, 16h, 20h, is controlled
The sodium hydroxide and glucose weight ratio of 5mol/L is 1.5:1.
Embodiment 3
The fermentation process of Sodium Hyaluronate specifically comprises the following steps:
1) plate culture:
Preparing plating medium, (the brain heart soaks powder: 35g/L；Yeast extract: 10g/L；Glucose: 1.5g/L；Agar: 12g/
L), work seed is seeded on plate, cultivates 20 hours in 38 DEG C of constant incubators by inverted plate after sterilizing.
2) shaking flask culture:
Prepare Shake flask medium 3.5L [glucose: 65g, magnesium sulfate (seven water): 6g, yeast extract: 50g, biphosphate
Potassium: 5g, sodium bicarbonate: 0.25g, phosphate buffer: 120ml (0.1mol/L sodium dihydrogen phosphate (two water) and 0.1mol/L ten
Two hypophosphite monohydrate disodium hydrogens mix in equal volume), trace element solution: 3.0ml, defoaming agent: 1.0ml], with NaOH adjust pH to
7.3, culture medium is sub-packed in 10 conical flasks by 350ml/ bottles, sterilizes after cold, is scraped from plating medium respectively
Strain access, is placed in constant-temperature table, and setting revolving speed is not less than 100rpm, 37 DEG C of cultures to net added value >=1.0 OD.
3) seed culture:
Seed culture medium 25L [glucose: 450g, magnesium sulfate (seven water): 45g, yeast extract: 450g, biphosphate is added
Potassium: 43g, sodium bicarbonate: 2.1g, phosphate buffer: 920ml, trace element solution: 20ml, defoaming agent: 1.0ml]；Sterilizing
5wt% inoculum concentration is pressed afterwards, is seeded in seeding tank, is not less than 70rpm, temperature: 39 DEG C, ventilatory capacity: the condition of 1vvm in revolving speed
Cultivate 3h.
4) fermented and cultured:
Prepare fermentation medium 400L [glucose: 13kg, yeast extract: 6kg, magnesium sulfate (seven water): 1.3kg, di(2-ethylhexyl)phosphate
Hydrogen sodium (two water): 1.3kg, potassium sulfate: 0.6kg, L-arginine: 23g, trace element solution: 1.3L, Polyalcoxyether
(defoaming agent): 23ml], culture transferring after sterilizing is controlled revolving speed and is not less than 60rpm by 10wt% inoculum concentration；Temperature: 39 DEG C；PH:
14.0；Ventilatory capacity: 1.5vvm；
It is separately added into 2kg glucose in 3h, 6h, 9h, 12h, 15h, 18h, 21h of fermented and cultured, controls the hydrogen of 3mol/L
Sodium oxide molybdena and glucose weight ratio are 1.4:1.
Comparative example 1
Concrete operation step and embodiment 3 are consistent, and only there is no supplement glucose stage by stage, but disposably supplement
Glucose 20kg.
Comparative example 2
Concrete operation step and embodiment 3 are consistent, only fermentation medium 400L [glucose: 30kg, yeast extract:
4kg, magnesium sulfate (seven water): 1.3kg, sodium dihydrogen phosphate (two water): 1.3kg, potassium sulfate: 0.6kg, L-arginine: 23g, it is micro
Element Solution: 1.3L, Polyalcoxyether (defoaming agent): 23ml].
Experimental example 1
The content for the Sodium Hyaluronate that the fermentation process of above-described embodiment 1-3 and comparative example 1-2 are prepared and
Relative molecular weight is measured, and concrete outcome is as follows:
1 measurement result of table
| Group | Yield (g/L) | Weight average molecular weight |
| Embodiment 1 | 15 | 150-200Mw |
| Embodiment 2 | 14 | 150-200Mw |
| Embodiment 3 | 14 | 150-200Mw |
| Comparative example 1 | 11 | 130-150Mw |
| Comparative example 2 | 11 | 130-150Mw |
Yield as can be seen from the above table using fermentation process of the invention not only Sodium Hyaluronate is high, but also can do
It is controllable to molecular weight, within the scope of the molecular weight of Sodium Hyaluronate is adjusted to controllable, obtain such high molecular weight range
Interior Sodium Hyaluronate, its application of further expansion.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of fermentation process of Sodium Hyaluronate, which comprises the steps of:
(A) strain is cultivated to activation 12-24h on plating medium；
(B) strain after activation is seeded on fermentation medium according to 5-15wt% inoculum concentration, ventilatory capacity is controlled in 1-2vvm
Between, fermented and cultured for 24 hours more than；
Wherein, glucose is added in the fermentation medium stage by stage, glucose adds 2-3kg at interval of 2-3h.
2. fermentation process according to claim 1, which is characterized in that in the step (A), the composition of plating medium
Are as follows: the brain heart soaks powder 30-40g/L, yeast extract 5-15g/L, glucose 0.5-2.0g/L, agar 10-15g/L.
3. fermentation process according to claim 1, which is characterized in that in the step (A), the culture on plating medium
Temperature controls between 37-39 DEG C.
4. fermentation process according to claim 1, which is characterized in that in the step (B), the fermentation medium is every
The composition that 400L includes are as follows: glucose 13-16kg, yeast extract 6-9kg, magnesium sulfate 0.6-1.3kg, sodium dihydrogen phosphate 1.0-
1.3kg, potassium sulfate 0.3-0.6kg, L-arginine 16-23g, trace element solution 0.9-1.3L, defoaming agent 15-23ml.
5. fermentation process according to claim 4, which is characterized in that in the step (B), the pH of fermentation medium is controlled
Between 6-14, temperature is between 37-39 DEG C, and revolving speed is in 60rpm or more.
6. fermentation process according to claim 4, which is characterized in that in the step (A), mended in the fermentation medium
The mass ratio of the sodium hydroxide and glucose that fill is (1.2-1.5): 1.
7. fermentation process according to claim 4, which is characterized in that in the step (A), sodium hydroxide is made into concentration and is
The sodium hydroxide solution of 3-5mol/L, more preferably concentration are 4mol/L.
8. fermentation process according to claim 1, which is characterized in that between the step (A) and the step (B), also
Include the steps that expanding culture: after the strain after activation is cultivated in shaking flask, then continuing to expand culture on seed culture medium.
9. fermentation process according to claim 8, which is characterized in that in the step (A), the culture medium in shaking flask is every
The composition that 3.5L includes are as follows: glucose 55-70g, magnesium sulfate 4.5-7.3g, yeast extract 43-63g, potassium dihydrogen phosphate 4.6-
6.7g, sodium bicarbonate 0.20-0.31g, phosphate buffer 1 15-123ml, trace element solution 2.4-3.3ml, defoaming agent
0.7-1.4ml；
Preferably, the composition that the every 25L of the seed culture medium includes are as follows: glucose 434-477g, magnesium sulfate 42-49g, yeast leaching
Powder 431-485g, potassium dihydrogen phosphate 41-47g, sodium bicarbonate 2.0-2.9g, phosphate buffer 910-930ml, microelement are molten
Liquid 17-30ml, defoaming agent 0.8-1.2ml.
10. the Sodium Hyaluronate that the described in any item fermentation process of claim 1-9 ferment；
Preferably, the yield of the Sodium Hyaluronate is 11g/L or more, and molecular weight is between 130-200Mw.
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