CN109161571A - A kind of culture medium and preparation method thereof for producing Sodium Hyaluronate - Google Patents
A kind of culture medium and preparation method thereof for producing Sodium Hyaluronate Download PDFInfo
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- CN109161571A CN109161571A CN201811190519.5A CN201811190519A CN109161571A CN 109161571 A CN109161571 A CN 109161571A CN 201811190519 A CN201811190519 A CN 201811190519A CN 109161571 A CN109161571 A CN 109161571A
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Abstract
The present invention provides a kind of culture medium and preparation method thereof for producing Sodium Hyaluronate, culture medium described in every 100kg includes following components: 28~35kg of white granulated sugar, yeast extract 8~12kg, MgSO4·7H2O 0.8~1.1kg, NaH2PO4·2H2O 1.2~1.4kg, K2SO40.5~1.0kg, 15~25kg of L-arginine, 15~30mL of defoaming agent, being mended with water to gross mass is 100kg；Culture medium provided by the present invention simplifies the complexity of formula, impurity is brought in reduction into, when being fermented using streptococcus zooepidemicus H23, the yield of Sodium Hyaluronate can reach 7.5g/L~8.5g/L, compared to the culture medium disclosed in existing method, fermentation yield is higher under identical condition, is conducive to industrialized production, improves the efficiency of production.
Description
Technical field
The invention belongs to field of biotechnology, it is related to a kind of culture medium for producing Sodium Hyaluronate and its preparation side
Method.
Background technique
Hyaluronic acid is mucopolysaccharide necessary to a kind of Human Physiology, is widely present in the various tissues of animal and human body
In.Based on hyaluronic acid high viscosity, lubricity, water-retaining property, good biocompatibility and its special physiological property etc.,
Field of medicaments has been widely used, and carries in the world in orthopaedics, ophthalmology, surgery, wound, sustained release preparation, induction target administration at present
Body, oncotherapy, dermal filler etc. are being applied.
In general, preparing sodium hyaluronate raw material, traditional cockscomb extraction method prepares sodium hyaluronate, but cockscomb extraction method is deposited
The development of extraction method is constrained in the presence of many defects, these defects, in long-term research and exploration, people are tried always
Figure finds the new preparation method of one kind and cockscomb extraction method is replaced to prepare sodium hyaluronate.Biofermentation rule compensates for cockscomb extraction method
Deficiency, be current research hotspot.
CN1786180 discloses a kind of method for improving research on producing hyaluronic acid by fermentation method yield and molecular weight, using epizootic disease
Streptococcus H23 produces hyaluronic acid as starting strain, through liquid fermentation, and fermentation medium selects 8.0~12.0g/L yeast powder
Use speed of agitator for 180~220rpm for single nitrogen source, fermentation process, to improve the yield and molecular weight of hyaluronic acid.Hair
Ferment culture medium composition are as follows: glucose 65.8g/L, yeast powder 8.0~12.0g/L, Na2HPO4·12H2O 6.2g/L, K2SO4
1.3g/L, MgSO4·7H2O 2.0g/L, FeSO4·7H2O 0.005g/L；Process conditions are as follows: speed of agitator 200rpm, temperature
37 DEG C, ventilatory capacity 1.2vvm.Hyaluronic acid volume of production reaches 6.0g/L, and molecular weight reaches 2.00 × 106Da。
CN101041843 discloses a kind of method with microbial fermentation technology production hyaluronic acid, and this method is to utilize
The one plant of streptococcus zooepidemicus voluntarily separated is fermented, using the suitable voluntarily optimal carbon of isolated strains, nitrogen and inorganic salts ratio
Fermentation method, control fermentation process in time, cultivation temperature, pH value, oxyty, the fermentation liquid of acquisition is removed with physical method
Bacterium is then precipitated with Ethanol Method, then with digesting in conjunction with isoamyl alcohol come removing protein, finally obtains hyalomitome with vacuum freeze-drying method
Acid.The group of this method culture medium become concentration of glucose be 42g/L, peptone concentration 22g/L, dipotassium hydrogen phosphate 2g/L,
Magnesium sulfate is 2.4g/L.But the yield of the method production is lower, productivity effect is not high.
CN101643760 discloses a kind of method for improving hyaluronic acid volume of production, transparent with streptococcus zooepidemicus fermenting and producing
Matter is sour, in fermentation process, after 32-36 DEG C of fermentation 8-16h, temperature is adjusted to 36-40 DEG C and continues to ferment.The training that fermentation uses
Support base are as follows: glucose 2% (mass percentage), peptone 1% (mass percentage), (quality percentage contains yeast extract 0.5%
Amount), KH2PO40.2% (mass percentage), MgSO4·7H2O 0.05% (mass percentage), pH 7.5.Yield can
To reach 89.7~105.3 (mg/100mL).
It in summary it can be seen, the yield of Sodium Hyaluronate need to be improved, and how to develop a kind of new for producing
The culture medium of bright matter acid sodium is the key that improve Sodium Hyaluronate yield.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide one kind
To achieve this purpose, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of for producing the culture medium of Sodium Hyaluronate, culture medium packet described in every 100kg
Include following components: 28~35kg of white granulated sugar, yeast extract 8~12kg, MgSO4·7H2O 0.8~1.1kg, NaH2PO4·2H2O
1.2~1.4kg, K2SO40.5~1.0kg, 15~25kg of L-arginine, 15~30mL of defoaming agent, surplus are water.
Provided by the present invention for producing the culture medium of Sodium Hyaluronate, enabling to strain in this recipe has very well
Stability, while simplifying the complexity of formula, impurity is brought in reduction into, when being fermented using streptococcus zooepidemicus H23, thoroughly
The yield of bright matter acid sodium can reach 7.5g/L~8.5g/L, compared to the culture medium disclosed in existing method, in same item
Fermentation yield is higher under part, is conducive to industrialized production, improves the efficiency of production.
In the present invention, each component in culture medium is matched according to gross mass 100kg benchmark.
In the present invention, the quality of the white granulated sugar be 28~35kg, such as can be 28kg, 29kg, 30kg, 31kg,
32kg, 33kg, 34kg or 35kg etc..
In the present invention, the quality of the yeast extract be 8~12kg, such as can be 8kg, 8.5kg, 9kg, 9.5kg,
10kg, 10.5kg, 11kg, 11.5kg or 12kg etc..
In culture medium provided by the invention, white granulated sugar and yeast extract provide most basic energy source for thallus, are
Nutriment necessary to thalli growth.
In the present invention, the MgSO4·7H2The quality of O be 0.8~1.1kg, such as can be 0.8kg, 0.85kg,
0.9kg, 0.95kg, 1kg, 1.05kg, 1.1kg etc..
In the present invention, the NaH2PO4·2H2The quality of O be 1.2~1.4kg, such as can be 1.2kg, 1.25kg,
1.3kg, 1.35kg or 1.4kg etc..
In the present invention, the K2SO4Quality be 0.5~1.0kg, such as can be 0.5kg, 0.6kg, 0.65kg,
0.7kg, 0.8kg, 0.83kg, 0.9kg, 0.95kg or 1kg etc..
In the present invention, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4Three kinds of inorganic salts, can balance when the cell grows
Osmotic pressure, and participate in a certain amount of bacterial metabolism process.But content is unsuitable too high or too low, otherwise will affect thallus
Steady production influences the yield of thallus normal expression production Sodium Hyaluronate.
In the present invention, the quality of the L-arginine be 15~25kg, such as can be 15kg, 16kg, 17kg,
18kg, 19kg, 20kg, 21kg, 22kg, 23kg, 24kg or 25kg etc..
L-arginine is the basic composition of various protein, and there are very extensive, and it is the centre of ornithine circulation
Metabolin can promote ammonia to be transformed into urea.L-arginine in culture medium can promote thallus in the mistake of production Sodium Hyaluronate
The expression of associated metabolic access in journey, promotes the synthesis of Sodium Hyaluronate.
In the present invention, the volume of the defoaming agent be 15~30mL, such as can be 15mL, 16mL, 17mL, 18mL,
19mL, 20mL, 21mL, 22mL, 24mL, 25mL, 28mL, 29mL or 30mL etc..
When calculating culture medium gross mass, defoaming agent is converted into quality and is calculated.
Preferably, the defoaming agent is oiliness defoaming agent and/or polyether antifoam agent, preferably polyether antifoam agent.
In the present invention, polyethet surfactant exists in water with molecular state, and stability is high, not toxic, with it
His the surfactant intermiscibility of type is good, and has good foam inhibition effect, emulsifying capacity and cleaning function.Polyethers surface
Active agent molecule amount usually change it is bigger, composition and structure it is all more complicated, and these differences all can significantly change it is poly-
Close the physico-chemical property of object.Polyether antifoam agent effectively can inhibit bubble to generate during the fermentation, be conducive to growth.But
The content of defoaming agent is unsuitable too high or too low, and the yield that otherwise will cause Sodium Hyaluronate reduces.
Preferably, the culture medium further includes 0.2~0.8kg of inorganic salts containing microelement, for example, can be 0.2kg,
0.3kg, 0.4kg, 0.5kg, 0.6kg, 0.7kg or 0.8kg etc..
Preferably, the inorganic salts containing microelement include manganese nitrate, cobalt nitrate, zinc nitrate, ferrous sulfate, lanthanum nitrate
In rubidium nitrate any one or at least two combination.
In the present invention, by the addition of the inorganic salts containing microelement, the metabolism of thallus can be promoted, compared to lacking
The yield of the culture medium of microelement, Sodium Hyaluronate is promoted.
Preferably, the K2SO4Mass ratio with L-arginine is 1:20~23, such as can be 1:20,1:21,1:22
Or 1:23 etc..
In the present invention, K2SO4It is preferably controlled within the scope of aforementioned proportion with the mass ratio of L-arginine, this is because K+
For the balance in culture medium, L-arginine will affect for the facilitation effect of thalli growth, if be not able to maintain in culture medium
Balance, then L-arginine cannot be by the good Absorption And Metabolism of thallus, and the associated metabolic so as to cause production Sodium Hyaluronate is logical
Road is obstructed, and the yield of Sodium Hyaluronate is reduced.
As optimal technical scheme, culture medium provided by the invention includes following components, is calculated according to every 100kg: white sand
Sugar 30~32kg, yeast extract 10~11kg, MgSO4·7H2O 0.9~1.0kg, NaH2PO4·2H21.25~1.3kg of O,
K2SO40.7~0.8kg, 16~20kg of L-arginine, 20~25mL of polyether antifoam agent, the inorganic salts 0.2 containing microelement~
0.8kg, surplus are water.
On the other hand, the present invention provides a kind of preparation method of culture medium as described above, the preparation method includes
Following steps: white granulated sugar, yeast extract and L-arginine being added to the water together, are uniformly mixing to obtain mixed liquor for the first time,
Then by defoaming agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4Optionally the inorganic salts containing microelement are slowly added to
Into mixed liquor, second of stirring is heated and carried out, the culture medium is obtained.
Preferably, the temperature of first time stirring is 30~50 DEG C, for example, can be 30 DEG C, 35 DEG C, 38 DEG C, 40 DEG C,
42 DEG C, 43 DEG C, 45 DEG C, 47 DEG C, 49 DEG C or 50 DEG C etc..
Preferably, the time of first time stirring is 30~180min, for example, can be 30min, 40min, 50min,
60min、70min、80min、90min、100min、110min、120min、130min、140min、150min、160min、
170min or 180min etc..
Preferably, 55~70 DEG C of the temperature of the heating, such as can be 55 DEG C, 58 DEG C, 60 DEG C, 63 DEG C, 65 DEG C, 67
DEG C, 69 DEG C or 70 DEG C etc..
Preferably, the time of second stirring is 120~360min, for example, can be 120min, 140min,
150min、170min、180min、190min、200min、240min、250min、280min、300min、320min、340min
Or 360min etc..
Preferably, the revolving speed of second stirring is 500~1000rpm, for example, can be 500rpm, 600rpm,
700rpm, 800rpm, 900rpm or 1000rpm etc..
Preferably, further include filtering mixed liquor after second of stirring, remove undissolved impurity, obtain the culture
Base.
Preferably, the preparation method of culture medium provided by the invention includes the following steps: white granulated sugar, yeast extract and L-
Arginine is added to the water together, stirs 30~180min for the first time until uniformly obtaining mixed liquor at 30~50 DEG C, then will
Defoaming agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4Optionally the inorganic salts containing microelement are slowly added into mixing
In liquid, 55~70 DEG C are heated to, second of 120~360min of stirring is carried out in the case where revolving speed is 500~1000rpm, by mixed liquor
Filtering, removes undissolved impurity, obtains the culture medium.
Compared with the existing technology, the invention has the following advantages:
(1) provided by the present invention for the culture medium of production Sodium Hyaluronate, strain is enabled to have in this recipe
Good stability, while the complexity of formula is simplified, impurity is brought in reduction into, is fermented using streptococcus zooepidemicus H23
When, the yield of Sodium Hyaluronate can reach 7.5g/L~8.5g/L, compared to the culture medium disclosed in existing method, same
Under conditions of fermentation yield it is higher, be conducive to industrialized production, improve the efficiency of production.
(2) provided by the present invention for the culture medium of production Sodium Hyaluronate, by being added in the medium containing micro
The inorganic salts of element may advantageously facilitate intake of the thallus for nutriment, promote the steady production of thallus, promotion and hyalomitome
The relevant metabolic capability of sour sodium, finally improves the yield of Sodium Hyaluronate.
(3) culture medium provided by the invention not only breaches the bottleneck of Sodium Hyaluronate industry technology development, creates life
The internal and external environment for producing more quality product, meets the multiple needs of social development more to a certain extent, reduces bad anti-
The probability that should occur, reduces production cost.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
The present embodiment provides a kind of for producing the culture medium of Sodium Hyaluronate
100kg culture medium is composed of the following components: white granulated sugar 31kg, yeast extract 10kg, MgSO4·7H2O 0.9kg,
NaH2PO4·2H2O 1.25kg, K2SO40.8kg, L-arginine 16kg, polyether antifoam agent 23mL, manganese nitrate 0.3kg, surplus are
Water.
Preparation method: white granulated sugar, yeast extract and L-arginine are added to the water together, are stirred for the first time at 40 DEG C
90min is until uniformly obtain mixed liquor, then by polyether antifoam agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4And nitric acid
Manganese is slowly added into mixed liquor, is heated to 60 DEG C, second of stirring 150min is carried out in the case where revolving speed is 700rpm, by mixed liquor
Filtering, removes undissolved impurity, obtains culture medium.
Embodiment 2
The present embodiment provides a kind of for producing the culture medium of Sodium Hyaluronate
100kg culture medium is composed of the following components: white granulated sugar 30kg, yeast extract 11kg, MgSO4·7H2O 1.0kg,
NaH2PO4·2H2O 1.3kg, K2SO40.8kg, L-arginine 20kg, polyether antifoam agent 25mL, lanthanum nitrate 0.8kg, surplus are
Water.
Preparation method: white granulated sugar, yeast extract and L-arginine are added to the water together, are stirred for the first time at 30 DEG C
30min is until uniformly obtain mixed liquor, then by polyether antifoam agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4And nitric acid
Lanthanum is slowly added into mixed liquor, is heated to 70 DEG C, and second of stirring 360min is carried out in the case where revolving speed is 1000rpm, will be mixed
Liquid filtering, removes undissolved impurity, obtains culture medium.
Embodiment 3
The present embodiment provides a kind of for producing the culture medium of Sodium Hyaluronate
100kg culture medium is composed of the following components: white granulated sugar 32kg, yeast extract 10kg, MgSO4·7H2O 0.9kg,
NaH2PO4·2H2O 1.25kg, K2SO40.77kg, L-arginine 21kg, polyether antifoam agent 20mL, ferrous sulfate 0.2kg are remaining
Amount is water.
Preparation method: white granulated sugar, yeast extract and L-arginine are added to the water together, are stirred for the first time at 50 DEG C
180min is until uniformly obtain mixed liquor, then by polyether antifoam agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4And sulfuric acid
Ferrous iron is slowly added into mixed liquor, is heated to 55 DEG C, and second of stirring 120min is carried out in the case where revolving speed is 500rpm, will be mixed
Liquid filtering, removes undissolved impurity, obtains culture medium.
Embodiment 4
The present embodiment provides a kind of for producing the culture medium of Sodium Hyaluronate
100kg culture medium is composed of the following components: white granulated sugar 28kg, yeast extract 8kg, MgSO4·7H2O 0.8kg,
NaH2PO4·2H2O 1.2kg, K2SO40.5kg, L-arginine 15kg, oiliness defoaming agent 15mL, surplus is water.
Preparation method: white granulated sugar, yeast extract and L-arginine are added to the water together, are stirred for the first time at 50 DEG C
180min is until uniformly obtain mixed liquor, then by oiliness defoaming agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4Slowly add
Enter into mixed liquor, be heated to 55 DEG C, second of stirring 120min is carried out in the case where revolving speed is 500rpm, mixed liquor is filtered, is removed
Undissolved impurity is removed, culture medium is obtained.
Embodiment 5
The present embodiment provides a kind of for producing the culture medium of Sodium Hyaluronate
100kg culture medium is composed of the following components: white granulated sugar 35kg, yeast extract 12kg, MgSO4·7H2O 1.1kg,
NaH2PO4·2H2O 1.4kg, K2SO41.0kg, L-arginine 25kg, defoaming agent 30mL, surplus are water.
Preparation method: preparation method: white granulated sugar, yeast extract and L-arginine are added to the water together, at 40 DEG C
Primary stirring 90min is until uniformly obtain mixed liquor, then by polyether antifoam agent, MgSO4·7H2O、NaH2PO4·2H2O、
K2SO4It is slowly added into mixed liquor, is heated to 60 DEG C, second of stirring 150min is carried out in the case where revolving speed is 700rpm, will be mixed
Liquid filtering, removes undissolved impurity, obtains culture medium.
Comparative example 1
The difference of this comparative example and embodiment 1 is only that, does not include K in this comparative example2SO4, remaining component and preparation method
It is same as Example 1, culture medium is prepared.
Comparative example 2
The difference of this comparative example and embodiment 1 is only that, does not include L-arginine in this comparative example, remaining component and preparation
Method is same as Example 1, and culture medium is prepared.
Comparative example 3
The difference of this comparative example and embodiment 1 is only that, does not include L-arginine and K in this comparative example2SO4, remaining component
It is same as Example 1 with preparation method, culture medium is prepared.
Comparative example 4
The difference of this comparative example and embodiment 1 is only that, does not include polyether antifoam agent in this comparative example, remaining component and system
Preparation Method is same as Example 1, and culture medium is prepared.
Comparative example 5
The difference of this comparative example and embodiment 1 is only that the volume of polyether antifoam agent is 34mL, remaining group in this comparative example
It is point same as Example 1 with preparation method, culture medium is prepared.
Comparative example 6
The difference of this comparative example and embodiment 1 is only that the volume of polyether antifoam agent is 10mL, remaining group in this comparative example
It is point same as Example 1 with preparation method, culture medium is prepared.
By the culture medium of above-described embodiment 1-5 and comparative example 1-6 preparation, it is tested for the property.Test method is as follows: by beast
Epidemic disease streptococcus H23 seed is seeded in the fermentation medium of the above-mentioned preparation of 5L, is 360rpm in speed of agitator, and temperature is 35 DEG C,
PH value is 7.3, and ventilatory capacity is cultivated 18 hours under the conditions of being 1vvm, obtains sodium hyaluronate fermentation liquor, adjusts pH in fermentation process
Value, makes pH value be maintained at 7.5 ± 0.5.The yield (g/L) of 11 kinds of preparation-obtained sodium hyaluronates of culture medium is measured,
Shown in obtained yield result table 1 specific as follows:
Table 1
| Sample | Yield (g/L) |
| Embodiment 1 | 8.5 |
| Embodiment 2 | 8.1 |
| Embodiment 3 | 8.2 |
| Embodiment 4 | 7.8 |
| Embodiment 5 | 7.6 |
| Comparative example 1 | 6.7 |
| Comparative example 2 | 6.6 |
| Comparative example 3 | 5.9 |
| Comparative example 4 | 5.4 |
| Comparative example 5 | 6.9 |
| Comparative example 6 | 6.8 |
By the comparison of embodiment 1 and embodiment 2 it is found that working as K2SO4Mass ratio with L-arginine is not in preferred range
When interior, the yield of Sodium Hyaluronate can be declined；By embodiment 4 and embodiment 5 it is found that when micro- without containing containing in culture medium
The yield of the inorganic salts of secondary element, Sodium Hyaluronate is also declined.
By the comparison of comparative example 1-6 and embodiment 1 it is found that K2SO4And/or L-arginine lacks, and will cause hyalomitome
The yield of sour sodium declines to a great extent；And when not containing defoaming agent in culture medium, fermentation efficiency is equally largely effected on, Sodium Hyaluronate
Yield only have 5.4g/L；And when the content of defoaming agent is not in the framework of the present definition, the yield of Sodium Hyaluronate
There is a degree of decline.
To sum up, the culture medium of production Sodium Hyaluronate provided by the invention breaches the development of Sodium Hyaluronate industry technology
Bottleneck creates the internal and external environment of production more quality product, reduces the probability of adverse reaction generation, reduces and be produced into
This, is suitable for large-scale industrial production.
The Applicant declares that the present invention is explained by the above embodiments is of the invention for producing the culture of Sodium Hyaluronate
Base and preparation method thereof, but the invention is not limited to above-mentioned method detaileds, that is, it is above-mentioned detailed not mean that the present invention must rely on
Thin method could be implemented.It should be clear to those skilled in the art, any improvement in the present invention, to product of the present invention
The equivalence replacement of each raw material and addition, the selection of concrete mode of auxiliary element etc., all fall within protection scope of the present invention and public affairs
Within the scope of opening.
Claims (10)
1. a kind of for producing the culture medium of Sodium Hyaluronate, which is characterized in that culture medium described in every 100kg includes with the following group
Point: 28~35kg of white granulated sugar, yeast extract 8~12kg, MgSO4·7H2O 0.8~1.1kg, NaH2PO4·2H2O 1.2~
1.4kg, K2SO40.5~1.0kg, 15~25kg of L-arginine, 15~30mL of defoaming agent, surplus are water.
2. culture medium according to claim 1, which is characterized in that the defoaming agent is that oiliness defoaming agent and/or polyethers disappear
Infusion, preferably polyether antifoam agent.
3. culture medium according to claim 1 or 2, which is characterized in that the culture medium further includes the nothing containing microelement
0.2~0.8kg of machine salt；
Preferably, the inorganic salts containing microelement include manganese nitrate, cobalt nitrate, zinc nitrate, ferrous sulfate, lanthanum nitrate or nitre
In sour rubidium any one or at least two combination.
4. culture medium according to any one of claim 1-3, which is characterized in that the K2SO4With the quality of L-arginine
Than for 1:20~23.
5. culture medium described in any one of -4 according to claim 1, which is characterized in that culture medium described in every 100kg include with
Lower component: 30~32kg of white granulated sugar, yeast extract 10~11kg, MgSO4·7H2O 0.9~1.0kg, NaH2PO4·2H2O
1.25~1.3kg, K2SO40.7~0.8kg, 20~22kg of L-arginine, 20~25mL of polyether antifoam agent, containing microelement
0.2~0.8kg of inorganic salts, surplus are water.
6. the preparation method of culture medium according to any one of claims 1-5, which is characterized in that the preparation method packet
It includes following steps: white granulated sugar, yeast extract and L-arginine is added to the water together, be uniformly mixing to obtain mixing for the first time
Liquid, then by defoaming agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4Optionally the inorganic salts containing microelement slowly add
Enter into mixed liquor, heats and carry out second of stirring, obtain the culture medium.
7. preparation method according to claim 6, which is characterized in that the temperature of the first time stirring is 30~50 DEG C；
Preferably, the time of the first time stirring is 30~180min.
8. preparation method according to claim 6 or 7, which is characterized in that 55~70 DEG C of the temperature of the heating；
Preferably, the time of second of stirring is 120~360min；
Preferably, the revolving speed of second of stirring is 500~1000rpm.
9. preparation method a method according to any one of claims 6-8, which is characterized in that further include after second of stirring
Mixed liquor is filtered, undissolved impurity is removed, obtains the culture medium.
10. the preparation method of culture medium according to claim 1 to 9, which is characterized in that the preparation method
Include the following steps: for white granulated sugar, yeast extract and L-arginine to be added to the water together, in 30~50 DEG C of stirrings 30 for the first time
~180min is until uniformly obtain mixed liquor, then by defoaming agent, MgSO4·7H2O、NaH2PO4·2H2O、K2SO4Optionally
Inorganic salts containing microelement are slowly added into mixed liquor, are heated to 55~70 DEG C, are 500~1000rpm progress in revolving speed
Second of 120~360min of stirring, mixed liquor is filtered, undissolved impurity is removed, obtains the culture medium.
Priority Applications (1)
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| CN110511972A (en) * | 2019-09-22 | 2019-11-29 | 山东众山生物科技有限公司 | A kind of streptococcic culture medium of Sodium Hyaluronate and cultural method |
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