CN112592849A - Culture medium and culture method for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus - Google Patents
Culture medium and culture method for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus Download PDFInfo
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Abstract
The invention relates to a culture medium and a culture method for producing high molecular weight hyaluronic acid by using streptococcus zooepidemicus fermentation.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a culture medium and a culture method for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus.
Background
Hyaluronic acid (Hyaluronic acid) is a high-molecular mucopolysaccharide with D-N-acetylglucosamine and D-glucuronic acid as structural units, and the commercial Hyaluronic acid is generally Sodium salt thereof, namely Sodium Hyaluronate (Sodium Hyaluronate), which is abbreviated as HA. Still traditionally referred to as hyaluronic acid. Hyaluronic acid has a special water retention effect, the weight of hyaluronic acid is 100 times higher than that of hyaluronic acid, and the hyaluronic acid is a substance which is found to have the best moisture retention in nature at present and is called as an ideal natural moisture retention factor. It can improve skin nutrition metabolism, make skin tender and smooth, remove wrinkle, increase elasticity, and prevent aging, and is good transdermal absorption promoter while keeping moisture. The nutrient can be used together with other nutrient components to achieve the more ideal effect of promoting nutrient absorption.
At present, domestic hyaluronic acid fermentation production usually adopts a three-stage fermentation mode taking streptococcus zooepidemicus as a production strain, and the method has the following problems:
1. the domestic medical hyaluronic acid has low molecular weight, and most of the medical hyaluronic acid still needs to be imported. According to the physicochemical property of the hyaluronic acid, the higher the molecular weight of the hyaluronic acid is, the better the absorption effect is, particularly in the medical aspect, the molecular weight of the hyaluronic acid reaches more than 4000kDa, and the better the treatment effect is. However, the molecular weight of the medical hyaluronic acid produced in China is only 2000-3000kDa, and the molecular weight of the hyaluronic acid produced by only a few enterprises reaches above 2000 kDa.
2. The fermentation technology level of the hyaluronic acid in China is relatively low and is generally below 11 g/L.
3. The domestic hyaluronic acid fermentation production cost is high, and the main reason is that beef extract or peptone and other animal proteins are added into the culture medium, and the dosage is large, so that the culture medium cost is high. Compared with foreign production enterprises, the international market competitiveness is weaker.
4. In the domestic hyaluronic acid fermentation technology, glucose is usually used as a quick-acting carbon source of a culture medium, and the fermentation titer is influenced because the glucose is easy to generate Maillard reaction in the sterilization process, namely toxic substances inhibiting thalli are generated.
5. In the domestic hyaluronic acid fermentation technology, animal peptone is added into the composition of a culture medium, part of animal protein is remained in the fermentation broth after the fermentation culture is finished, the viscosity of the fermentation broth is increased, and certain influence is exerted on the extraction and purification of the fermentation broth.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the culture medium for producing the hyaluronic acid by fermenting the streptomyces veterinariae, which can improve the molecular weight of the hyaluronic acid, effectively improve the fermentation unit, reduce the production cost and reduce the extraction and purification difficulty.
Another object of the present invention is to provide a culture method for producing hyaluronic acid using the above culture medium.
The technical scheme adopted for realizing the purpose is as follows:
a culture medium for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus is characterized by comprising a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein the culture medium comprises a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium
The first-stage seed culture medium comprises the following components: 25-30 g/L of high fructose corn syrup, 35-40 g/L of degreased silkworm chrysalis powder, 25-30 g/L of fish melt pulp protein powder, 45-50 g/L of corn steep liquor dry powder, 0.5-0.9 g/L of ammonium sulfate and 1-5 g/L of light calcium carbonate;
the secondary seed culture medium comprises the following components: 35-40 g/L of high fructose corn syrup, 35-40 g/L of degreased silkworm chrysalis powder, 25-30 g/L of fish melt pulp protein powder, 45-50 g/L of corn steep liquor dry powder, 0.5-0.9 g/L of ammonium sulfate and 1-5 g/L of light calcium carbonate;
the fermentation medium comprises the following components: 55-60 g/L of high fructose corn syrup, 35-40 g/L of degreased silkworm chrysalis powder, 25-30 g/L of fish molten pulp protein powder, 45-50 g/L of corn steep liquor dry powder, 26-30 g/L of beer yeast protein powder, 0.5-0.9 g/L of ammonium sulfate, 1-5 g/L of light calcium carbonate, 0.1-0.5 g/L of monopotassium phosphate, 0.002-0.006 g/L of sodium chloride, 0.03-0.07 g/L of magnesium sulfate, 0.1-0.5 g/L of ferrous sulfate, 0.003-0.007 g/L of manganese chloride, 800.1-0.5 g/L of span and 0.2-0.4 ml/L of polyether modified silicone oil.
A culture method for producing hyaluronic acid by fermentation by using the culture medium is characterized by comprising the following process steps:
1) first-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium under the protection of flame for seed culture, and transplanting seeds with OD being more than or equal to 2.0;
2) secondary seed culture: sterilizing a secondary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured primary seed culture medium into a secondary seed culture medium for secondary seed culture, wherein OD is more than or equal to 2.0;
3) fermentation culture: sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture, and stopping fermentation until the content of hyaluronic acid is more than 12g/L and the molecular weight of hyaluronic acid is more than 4500 KDa.
The quality requirement of the cultured hyaluronic acid shake flask fermentation liquid is that the thallus concentration is 20-30%; the microscopic examination is free from foreign bacteria.
The first-stage seed culture conditions are as follows: the tank pressure is 0.03-0.05 MPa; the temperature of the tank is 35-36 ℃; air flow rate of 20-40 m3The stirring speed is 60-80 r/min;
the secondary seed culture conditions are as follows: the tank pressure is 0.03-0.05 MPa; the temperature of the tank is 35-36 ℃; air flow rate of 40-60 m3H; stirring at a rotating speed of 60-80 r/min;
the fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 35-36 ℃;
c, stirring speed: the rotating speed is controlled to be 80-100 r/min;
d, pressure control: the tank pressure is 0.05-0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 150 to 200m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
10 h-ending fermentation: the dissolved oxygen is controlled to be more than 40 percent.
Feeding materials by fed-batch method in the fermentation process, wherein the feeding materials comprise uridine diphosphate glucose supplementation, water supplementation and fructose-glucose syrup supplementation, and the feeding materials comprise uridine diphosphate glucose supplementation, water supplementation and fructose-glucose syrup supplementation, wherein
a process control of uridine diphosphate glucose supplementation:
when fermenting for 21h, supplementing adenosine diphosphate glucose, and controlling the concentration of the adenosine diphosphate glucose in the fermentation liquor to be 0.1-0.3 g/L;
b, water supplementing process control:
when the fermentation is carried out for 9-21h, when OD is more than or equal to 2.5, adding sterilized water, controlling OD to be 2-2.5 in the fermentation process, and detecting the OD of the fermentation liquor every 3 h;
after fermentation is finished for 21h to the end of fermentation, adding sterilized water, and controlling the volume of fermentation liquor to be 75-80% of that of the fermentation tank;
c, supplementing high fructose corn syrup:
when the fermentation is carried out for 9-21h, when the total sugar content of the fermentation liquor is less than 5mg/100ml, the sterilized high fructose corn syrup solution is supplemented, so that the total sugar content is controlled to be 10-15 mg/100 ml.
The technical advantages of the invention are embodied in that:
the invention confirms a seed culture medium for producing high molecular weight hyaluronic acid by fermenting streptomyces zooepidemicus, a carbon source, a nitrogen source and an optimal compatibility proportion in a fermentation culture medium.
As is well known, since carbon sources and nitrogen sources have very important influence in the biological growth process, enterprise technicians make a great deal of research on the influence of carbon-nitrogen ratio and the concentrations of the carbon sources and the nitrogen sources on the fermentation process when analyzing the influence of the nutrient sources on the growth of the streptomyces zooepidemicus. The discovery that the carbon-nitrogen ratio is too high and too low, which is not beneficial to cell growth and foreign protein expression and accumulation, and the too low leads to early autolysis of thalli; too high results in an imbalance in bacterial metabolism and ultimately adversely affects product accumulation. Even if the carbon-nitrogen ratio is in a proper level, too high and too low concentrations of a carbon source and a nitrogen source are not beneficial to cell growth and exogenous protein expression and accumulation, too high concentration causes slow cell growth in the later stage of the fermentation process and more metabolic wastes, and finally, the thallus metabolism is abnormal to influence exogenous protein synthesis; too low a concentration, the nutrient substances provided by the culture medium are limited, and the propagation of cells is influenced.
According to the invention, a series of researches are carried out, and finally the seed culture medium for producing the high molecular weight hyaluronic acid by fermenting streptomyces zooepidemicus, a carbon source, a nitrogen source and an optimal compatibility proportion in the fermentation culture medium are confirmed, on the basis, the fermentation technical level of large-scale production is that the HA content is more than 12g/L, HA, the molecular weight is more than 4500KDa, and the fermentation technical level is at the domestic leading level.
2 the culture medium of the invention is introduced with degreased silkworm chrysalis meal, fish melt pulp protein powder and yeast protein powder, and the three raw materials contain complete amino acid groups. The degreased silkworm chrysalis powder has high protein content, good amino acid composition, high content of methionine, lysine and tryptophan, and is rich in leucine, isoleucine and B vitamins; the fish soluble protein powder participates in a large amount of free amino acids formed in the hyaluronic acid biochemical synthesis process, such as glutamic acid and proline, a large amount of unknown growth factors in the fish soluble protein powder, and a plurality of oligopeptides and polypeptides play an important role in protein metabolism; the beer yeast protein powder contains rich protein, polysaccharide, vitamins, minerals and other nutritious components.
According to the biochemical synthesis principle of hyaluronic acid, three key organic nitrogen sources are introduced into the culture medium formula, and can provide key substances and nutrient substances required in the HA biosynthesis process, so that the technical effect of HA fermentation is ensured.
3 the invention elaborates the seed culture and fermentation culture process in detail, and confirms the optimal fermentation process and control parameters of HA. HA is produced by fermentation by adopting the process, and the fermentation unit reaches more than 12 g/L; the molecular weight of the hyaluronic acid reaches above 4500KDa, and the fermentation technical level is at the leading level in China.
3, the high fructose corn syrup is used for replacing glucose in the culture medium, so that the Maillard reaction is avoided.
Compared with the domestic conventional process level, the HA fermentation production level of the invention is improved by about 10 percent, the production cost is reduced, and the market competitiveness of the product is improved.
5, the key organic nitrogen source material used in the formula of the culture medium has lower fat content, wherein the fat content of the degreased silkworm chrysalis powder is less than 3.5 percent; the fish melt pulp protein powder has the fat content of less than 10 percent and the beer yeast protein powder has the fat content of less than 5 percent, and in the culture process of the strains, the utilization rate of key material protein is higher, the fermentation culture is finished, the amino nitrogen content is lower, and the extraction and the purification of the product are facilitated.
Detailed description of the invention
The invention is illustrated below by way of examples, which are to be understood as being illustrative and not limiting. The scope and core content of the invention are to be determined by the claims.
Streptomyces zooepidemicus is selected as the strain of the following examples, and the strain source used for production is shake flask fermentation liquor. The shake flask fermentation liquor quality requirement is as follows: the pH is 6-7; the concentration of the thalli is 25-30%; the microscopic examination is free from foreign bacteria.
Example 1
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-order seed culture medium comprises: 25kg of high fructose corn syrup, 36kg of degreased silkworm chrysalis powder, 28kg of fish melt pulp protein powder, 46kg of corn steep liquor dry powder, 0.5kg of ammonium sulfate and 1kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to the inoculum size of 40L under the protection of flame for seed culture, wherein the primary seed culture conditions are as follows: the tank pressure is 0.03 MPa; the temperature of the tank is 36 ℃; air flow 20m3H, stirring speed is 60 r/min; OD was 2.0 translocated.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The composition of the secondary culture medium is as follows: 350kg of high fructose corn syrup, 360kg of degreased silkworm chrysalis powder, 280kg of fish melt pulp protein powder, 490kg of corn steep liquor dry powder, 5kg of ammonium sulfate and 10kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.03 MPa; the temperature of the tank is 36 ℃; air flow rate of 40m3H; stirring at a rotation speed of 60 r/min; OD 2.0 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 5500kg of high fructose corn syrup, 3900kg of degreased silkworm chrysalis powder, 2800kg of fish melt pulp protein powder, 4600kg of corn steep liquor dry powder, 2600kg of beer yeast protein powder, 50kg of ammonium sulfate, 100kg of light calcium carbonate, 10kg of monopotassium phosphate, 0.2kg of sodium chloride, 5kg of magnesium sulfate, 40kg of ferrous sulfate, 0.5kg of manganese chloride, 8010 kg of span and 20L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 36 ℃;
c, stirring speed: the rotating speed is controlled at 80 r/min;
d, pressure control: the tank pressure is 0.05 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 150m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.3g/L, and the molecular weight of the hyaluronic acid is 4580 KDa.
Example 2
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-order seed culture medium comprises: 30kg of high fructose corn syrup, 36kg of degreased silkworm chrysalis powder, 26kg of fish melt pulp protein powder, 47kg of corn steep liquor dry powder, 0.7kg of ammonium sulfate and 2kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to an inoculation amount of 41L for seed culture under the protection of flame, wherein the primary seed culture conditions are as follows: the tank pressure is 0.03 MPa; the temperature of the tank is 35 ℃; air flow 30m3H, stirring speed is 70 r/min; OD was 2.3 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 400kg of high fructose corn syrup, 360kg of degreased silkworm chrysalis powder, 300kg of fish melt pulp protein powder, 500kg of corn steep liquor dry powder, 6kg of ammonium sulfate and 40kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.03 MPa; the temperature of the tank is 35 ℃; air flow 50m3/h；Stirring at a rotating speed of 70 r/min; OD 2.1 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 6000kg of fructose-glucose syrup, 3700kg of degreased silkworm chrysalis powder, 2600kg of fish melt pulp protein powder, 5000kg of corn steep liquor dry powder, 2600kg of beer yeast protein powder, 60kg of ammonium sulfate, 150kg of light calcium carbonate, 30kg of monopotassium phosphate, 0.4kg of sodium chloride, 3kg of magnesium sulfate, 50kg of ferrous sulfate, 0.7kg of manganese chloride, 8050 kg of span and 30L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 36 ℃;
c, stirring speed: the rotating speed is controlled at 100 r/min;
d, pressure control: the tank pressure is 0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 160m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.3g/L, and the molecular weight of the hyaluronic acid is 4550 KDa.
Example 3
First-order seed culture: the volume of the primary seed culture medium is 1m3。
Primary seed culture medium: 26kg of high fructose corn syrup, 40kg of degreased silkworm chrysalis powder, 26kg of fish melt pulp protein powder, 45kg of corn steep liquor dry powder, 0.6kg of ammonium sulfate and 2kg of light calcium carbonate.
First-order seed culture: sterilizing first-class seed culture medium, cooling to room temperature, maintaining pressure with sterile air, and shaking flask fermentation liquid of cultured sodium hyaluronate under flame protectionInoculating the seeds into a first-level seed culture medium according to the inoculation amount of 43L to perform seed culture, wherein the first-level seed culture conditions are as follows: the tank pressure is 0.05 MPa; the temperature of the tank is 35 ℃; air flow rate 25m3H, stirring speed is 65 r/min; OD was 2.1 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 360kg of high fructose corn syrup, 350kg of degreased silkworm chrysalis powder, 250kg of fish melt pulp protein powder, 460kg of corn steep liquor dry powder, 6kg of ammonium sulfate and 20kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.05 MPa; the temperature of the tank is 35 ℃; air flow rate of 45m3H; stirring speed is 65 r/min; OD 2.1 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 5600kg of high fructose corn syrup, 3500kg of degreased silkworm chrysalis powder, 2600kg of fish melt pulp protein powder, 4600kg of corn steep liquor dry powder, 2700kg of beer yeast protein powder, 60kg of ammonium sulfate, 200kg of light calcium carbonate, 20kg of monopotassium phosphate, 0.3kg of sodium chloride, 4kg of magnesium sulfate, 20kg of ferrous sulfate, 0.4kg of manganese chloride, 8020 kg of span and 25L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 35 ℃;
c, stirring speed: the rotating speed is controlled at 85 r/min;
d, pressure control: the tank pressure is 0.05-0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 160m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.5g/L, and the molecular weight of the hyaluronic acid is 4600 KDa.
Example 4
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-stage culture medium comprises the following components: 27kg of high fructose corn syrup, 37kg of degreased silkworm chrysalis powder, 25kg of fish melt pulp protein powder, 48kg of corn steep liquor dry powder, 0.9kg of ammonium sulfate and 3kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to an inoculation amount of 42L under the protection of flame for seed culture, wherein the primary seed culture conditions are as follows: the tank pressure is 0.04 MPa; the temperature of the tank is 35 ℃; air flow 30m3H, stirring speed is 70 r/min; OD was 2.3 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The composition of the secondary culture medium is as follows: 370kg of high fructose corn syrup, 370kg of degreased silkworm chrysalis powder, 250kg of fish melt pulp protein powder, 470kg of corn steep liquor dry powder, 7kg of ammonium sulfate and 30kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.04 MPa; the temperature of the tank is 35 ℃; air flow 50m3H; stirring at a rotating speed of 70 r/min; OD 2.3 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
Fermentation medium: 5700kg of fructose-glucose syrup, 3800kg of degreased silkworm chrysalis powder, 2500kg of fish melt pulp protein powder, 4700kg of corn steep liquor dry powder, 2800kg of beer yeast protein powder, 70kg of ammonium sulfate, 300kg of light calcium carbonate, 30kg of monopotassium phosphate, 0.4kg of sodium chloride, 3g of magnesium sulfate, 30kg of ferrous sulfate, 0.5kg of manganese chloride, 8030 kg of span and 30L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 35 ℃;
c, stirring speed: the rotating speed is controlled at 90 r/min;
d, pressure control: the tank pressure is 0.05 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 180m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 13.2g/L, and the molecular weight of the hyaluronic acid is 4800 KDa.
Example 5
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-order seed culture medium comprises: 29kg of high fructose corn syrup, 38kg of degreased silkworm chrysalis powder, 30kg of fish melt pulp protein powder, 49kg of corn steep liquor dry powder, 0.8kg of ammonium sulfate and 4kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to an inoculation amount of 45L under the protection of flame for seed culture, wherein the primary seed culture conditions are as follows: the tank pressure is 0.04 MPa; the temperature of the tank is 35 ℃; air flow rate 35m3H, stirring speed is 75 r/min; OD was 2.4 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 390kg of high fructose corn syrup, 390kg of degreased silkworm chrysalis powder, 300kg of fish melt pulp protein powder, 450kg of corn steep liquor dry powder, 8kg of ammonium sulfate and 40kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.04 MPa; the temperature of the tank is 36 ℃; air flow rate 55m3H; stirring at 75 r/min; OD 2.4 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 5900kg of fructose-glucose syrup, 4000kg of degreased silkworm chrysalis powder, 2900kg of fish fused protein powder, 4900kg of corn steep liquor dry powder, 2900kg of beer yeast protein powder, 80kg of ammonium sulfate, 400kg of light calcium carbonate, 40kg of monopotassium phosphate, 0.5kg of sodium chloride, 6kg of magnesium sulfate, 10kg of ferrous sulfate, 0.6kg of manganese chloride, 8040 kg of span and 35L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 35 ℃;
c, stirring speed: the rotating speed is controlled at 95 r/min;
d, pressure control: the tank pressure is 0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 190m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 13.0g/L, and the molecular weight of the hyaluronic acid is 4700 KDa.
Example 6
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-order seed culture medium comprises: 27kg of high fructose corn syrup, 37kg of degreased silkworm chrysalis powder, 28kg of fish melt pulp protein powder, 50kg of corn steep liquor dry powder, 0.5kg of ammonium sulfate and 2kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to an inoculum size of 44L under the protection of flame for seed culture, wherein the primary seed culture conditions are as follows: the tank pressure is 0.04 MPa; the temperature of the tank is 35.5 ℃; air flow rate 25m3H, stirring speed is 65 r/min; OD was 2.1 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 380kg of high fructose corn syrup, 400kg of degreased silkworm chrysalis powder, 290kg of fish melt pulp protein powder, 500kg of corn steep liquor dry powder, 5kg of ammonium sulfate and 30kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.05 MPa; the temperature of the tank is 36 ℃; air flow rate of 45m3H; stirring speed is 65 r/min; OD 2.4 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 5800kg of high fructose corn syrup, 4000kg of degreased silkworm chrysalis powder, 3000kg of fish melt pulp protein powder, 5000kg of corn steep liquor dry powder, 3000kg of beer yeast protein powder, 70kg of ammonium sulfate, 100kg of light calcium carbonate, 20kg of monopotassium phosphate, 0.3kg of sodium chloride, 4kg of magnesium sulfate, 30kg of ferrous sulfate, 0.7kg of manganese chloride, 8040 kg of span and 25L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 36 ℃;
c, stirring speed: the rotating speed is controlled at 100 r/min;
d, pressure control: the tank pressure is 0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 170m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
10 h-ending fermentation: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.2g/L, and the molecular weight of the hyaluronic acid is 4530 KDa.
Example 7
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-stage culture medium comprises the following components: 30kg of high fructose corn syrup, 40kg of degreased silkworm chrysalis powder, 30kg of fish melt pulp protein powder, 46kg of corn steep liquor dry powder, 0.9kg of ammonium sulfate and 5kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to the inoculation amount of 43L under the protection of flame for seed culture, wherein the primary seed culture conditions are as follows: the tank pressure is 0.05 MPa; the temperature of the tank is 35 ℃; air flow rate of 40m3H, stirring speed is 80 r/min; OD was 2.5 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 400kg of high fructose corn syrup, 400kg of degreased silkworm chrysalis powder, 260kg of fish melt pulp protein powder, 470kg of corn steep liquor dry powder, 9kg of ammonium sulfate and 50kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.03-0.05 MPa; the temperature of the tank is 35-36 ℃; air flow 60m3H; stirring at a rotating speed of 80 r/min;OD 2.5 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 6000kg of high fructose corn syrup, 3700kg of degreased silkworm chrysalis powder, 3000kg of fish melt pulp protein powder, 4700kg of corn steep liquor dry powder, 2700kg of beer yeast protein powder, 80kg of ammonium sulfate, 500kg of light calcium carbonate, 50kg of monopotassium phosphate, 0.6kg of sodium chloride, 7kg of magnesium sulfate, 50kg of ferrous sulfate, 0.7kg of manganese chloride, 8050 kg of span and 40L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 36 ℃;
c, stirring speed: the rotating speed is controlled at 100 r/min;
d, pressure control: the tank pressure is 0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 200m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.4g/L, and the molecular weight of the hyaluronic acid is 4600 KDa.
Example 8
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-stage culture medium comprises the following components: 25kg of high fructose corn syrup, 38kg of degreased silkworm chrysalis powder, 28kg of fish melt pulp protein powder, 50kg of corn steep liquor dry powder, 0.7kg of ammonium sulfate and 4kg of light calcium carbonate.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure with sterile air, and then under the protection of flame, shaking a flask of a fermentation broth of cultured sodium hyaluronate according to an inoculum size of 41LInoculating the mixture into a first-level seed culture medium for seed culture, wherein the first-level seed culture conditions are as follows: the tank pressure is 0.03 MPa; the temperature of the tank is 36 ℃; air flow 30m3H, stirring speed is 75 r/min; OD was 2.3 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The composition of the secondary culture medium is as follows: 350kg of high fructose corn syrup, 400kg of degreased silkworm chrysalis powder, 260kg of fish melt pulp protein powder, 460kg of corn steep liquor dry powder, 9kg of ammonium sulfate and 40kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.04 MPa; the temperature of the tank is 35 ℃; air flow rate 55m3H; stirring at 75 r/min; OD 2.2 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 5500kg of high fructose corn syrup, 4000kg of degreased silkworm chrysalis powder, 3000kg of fish melt pulp protein powder, 5000kg of corn steep liquor dry powder, 2900kg of beer yeast protein powder, 90kg of ammonium sulfate, 500kg of light calcium carbonate, 50kg of monopotassium phosphate, 0.6kg of sodium chloride, 7kg of magnesium sulfate, 50kg of ferrous sulfate, 0.3kg of manganese chloride, 8040 kg of span and 40L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 36 ℃;
c, stirring speed: the rotating speed is controlled at 100 r/min;
d, pressure control: the tank pressure is 0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 180m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.1g/L, and the molecular weight of the hyaluronic acid is 4520 KDa.
Example 9
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-order seed culture medium comprises: high fructose corn syrup 30kg, degreased silkworm chrysalis powder 35kg, fish melt pulp protein powder 25kg, corn steep liquor dry powder 46kg, ammonium sulfate 0.5kg, and light calcium carbonate 3 kg.
First-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium according to an inoculation amount of 42L under the protection of flame for seed culture, wherein the primary seed culture conditions are as follows: the tank pressure is 0.05 MPa; the temperature of the tank is 36 ℃; air flow rate of 40m3H, stirring speed is 75 r/min; OD was 2.2 transplants.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 400kg of high fructose corn syrup, 350kg of degreased silkworm chrysalis powder, 290kg of fish melt pulp protein powder, 450kg of corn steep liquor dry powder, 5kg of ammonium sulfate and 20kg of light calcium carbonate.
Firstly, sterilizing a second-level seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured first-level seed culture medium into a second-level seed culture medium for second-level seed culture, wherein the second-level seed culture conditions are as follows: the tank pressure is 0.05 MPa; the temperature of the tank is 36 ℃; air flow 50m3H; stirring at a rotating speed of 70 r/min; OD 2.3 was transferred to the fermentation medium.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 6000kg of fructose-glucose syrup, 3500kg of degreased silkworm chrysalis powder, 2500kg of fish molten pulp protein powder, 4500kg of corn steep liquor dry powder, 2600kg of beer yeast protein powder, 50kg of ammonium sulfate, 200kg of light calcium carbonate, 40kg of monopotassium phosphate, 0.5kg of sodium chloride, 6kg of magnesium sulfate, 40kg of ferrous sulfate, 0.6kg of manganese chloride, 8035 kg of span and 30L of polyether modified silicone oil.
Sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, and transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture.
The fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 36 ℃;
c, stirring speed: the rotating speed is controlled at 100 r/min;
d, pressure control: the tank pressure is 0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 190m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
fermenting for 10 h-finishing: the dissolved oxygen is controlled to be more than 40 percent.
After the fermentation is finished, the content of the hyaluronic acid is 12.6g/L, and the molecular weight of the hyaluronic acid is 4590 KDa.
In the fermentation process of the above examples 1-9, feeding materials including uridine diphosphate, glucose and fructose syrup according to the detection conditions
a process control of uridine diphosphate glucose supplementation:
during 21h of fermentation, adenosine diphosphate glucose was added, and the concentration of the adenosine diphosphate glucose in the fermentation broth was controlled to 0.2 g/L.
b, water supplementing process control:
and (3) when the fermentation time is 9-21h and the OD is more than or equal to 2.5, adding sterilized water, controlling the OD of the fermentation liquor to be 2-2.5, and detecting the OD of the fermentation liquor every 3 h.
After fermentation is finished for 21h, adding sterilized water, and controlling the volume of the fermentation liquor to be 80% of the volume of the fermentation tank.
c, supplementing high fructose corn syrup:
after fermentation for 9-21h, when the total sugar content of the fermentation liquor is less than 5mg/100ml, the sterilized high fructose syrup solution is supplemented to control the total sugar content to be 15mg/100 ml.
Comparative example 1
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-order seed culture medium comprises: 30kg of glucose, 34kg of beef extract, 24kg of yeast extract, 50kg of corn steep liquor, 0.5kg of ammonium sulfate and 2kg of light calcium carbonate.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The secondary seed culture medium comprises: 400kg of glucose, 360kg of beef extract, 260kg of yeast extract, 470kg of corn steep liquor, 6kg of ammonium sulfate and 30kg of light calcium carbonate.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 6000kg of glucose, 3700kg of beef extract, 2600kg of yeast extract, 4600kg of corn steep liquor, 2200kg of peptone, 50kg of ammonium sulfate, 200kg of light calcium carbonate, 30kg of monopotassium phosphate, 0.4kg of sodium chloride, 3kg of magnesium sulfate, 50kg of ferrous sulfate, 0.7kg of manganese chloride, 8050 kg of span and 30L of polyether modified silicone oil.
The fermentation production is carried out according to the process provided in example 1, the fermentation is finished, the hyaluronic acid content is 9.1g/L, and the hyaluronic acid molecular weight is 1850 KDa.
Comparative example 2
First-order seed culture: the volume of the primary seed culture medium is 1m3。
The first-stage culture medium comprises the following components: 10kg of dextrin, 25kg of glucose, 34kg of beef extract, 30kg of yeast powder, 50kg of corn steep liquor, 0.5kg of ammonium sulfate and 2kg of light calcium carbonate.
Secondary seed culture: the volume of the secondary seed culture medium is 10m3。
The composition of the secondary culture medium is as follows: 150kg of dextrin, 270kg of glucose, 350kg of beef extract, 270kg of yeast powder, 460kg of corn steep liquor, 6kg of ammonium sulfate and 30kg of light calcium carbonate.
Fermentation culture: the volume of the fermentation medium is 100m3。
The fermentation medium comprises the following components: 160kg of dextrin, 280kg of glucose, 3800kg of beef extract, 2700kg of yeast powder, 4500kg of corn steep liquor, 1200kg of cholesterol, 1100kg of peptone, 40kg of ammonium sulfate, 250kg of light calcium carbonate, 40kg of monopotassium phosphate, 0.5kg of sodium chloride, 2kg of magnesium sulfate, 40kg of ferrous sulfate, 0.6kg of manganese chloride, 8040 kg of span and 40L of polyether modified silicone oil.
The fermentation was carried out according to the procedure provided in example 1, the fermentation was completed, the hyaluronic acid content was 9.5g/L, and the hyaluronic acid molecular weight was 1900 KDa.
Claims (5)
1. A culture medium for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus is characterized by comprising a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein the culture medium comprises a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium
The first-stage seed culture medium comprises the following components: 25-30 g/L of high fructose corn syrup, 35-40 g/L of degreased silkworm chrysalis powder, 25-30 g/L of fish melt pulp protein powder, 45-50 g/L of corn steep liquor dry powder, 0.5-0.9 g/L of ammonium sulfate and 1-5 g/L of light calcium carbonate;
the secondary seed culture medium comprises the following components: 35-40 g/L of high fructose corn syrup, 35-40 g/L of degreased silkworm chrysalis powder, 25-30 g/L of fish melt pulp protein powder, 45-50 g/L of corn steep liquor dry powder, 0.5-0.9 g/L of ammonium sulfate and 1-5 g/L of light calcium carbonate;
the fermentation medium comprises the following components: 55-60 g/L of high fructose corn syrup, 35-40 g/L of degreased silkworm chrysalis powder, 25-30 g/L of fish molten pulp protein powder, 45-50 g/L of corn steep liquor dry powder, 26-30 g/L of beer yeast protein powder, 0.5-0.9 g/L of ammonium sulfate, 1-5 g/L of light calcium carbonate, 0.1-0.5 g/L of monopotassium phosphate, 0.002-0.006 g/L of sodium chloride, 0.03-0.07 g/L of magnesium sulfate, 0.1-0.5 g/L of ferrous sulfate, 0.003-0.007 g/L of manganese chloride, 800.1-0.5 g/L of span and 0.2-0.4 ml/L of polyether modified silicone oil.
2. The method for producing hyaluronic acid by fermentation of culture medium according to claim 1, characterized in that the process steps are:
1) first-order seed culture: sterilizing a primary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, inoculating a cultured sodium hyaluronate shake flask fermentation broth into the primary seed culture medium under the protection of flame for seed culture, and transplanting seeds with OD being more than or equal to 2.0;
2) secondary seed culture: sterilizing a secondary seed culture medium, cooling to room temperature, maintaining the pressure by using sterile air, transferring the cultured primary seed culture medium into a secondary seed culture medium for secondary seed culture, wherein OD is more than or equal to 2.0;
3) fermentation culture: sterilizing the fermentation culture medium, cooling to room temperature, maintaining the pressure with sterile air, transferring the cultured secondary seed liquid into the fermentation culture medium for fermentation culture, and stopping fermentation until the content of hyaluronic acid is more than 12g/L and the molecular weight of hyaluronic acid is more than 4500 KDa.
3. The culture method according to claim 2, wherein the cultured hyaluronic acid shake flask fermentation liquid has a thallus concentration of 20-30% by mass; the microscopic examination is free from foreign bacteria.
4. The method according to claim 2, wherein the culture medium is a culture medium
The first-stage seed culture conditions are as follows: the tank pressure is 0.03-0.05 MPa; the temperature of the tank is 35-36 ℃; air flow rate of 20-40 m3The stirring speed is 60-80 r/min;
the secondary seed culture conditions are as follows: the tank pressure is 0.03-0.05 MPa; the temperature of the tank is 35-36 ℃; air flow rate of 40-60 m3H; stirring at a rotating speed of 60-80 r/min;
the fermentation culture conditions are as follows:
a initial pH of the medium: the pH value of the fermentation medium is controlled to be 7.0-7.2 after sterilization;
b, temperature: 35-36 ℃;
c, stirring speed: the rotating speed is controlled to be 80-100 r/min;
d, pressure control: the tank pressure is 0.05-0.06 MPa;
e, pH control: controlling the pH value to be 6.9-7.2 in the fermentation process;
f air flow rate: 150 to 200m3/h；
h dissolved oxygen control:
dissolved oxygen is not controlled for 9 hours before fermentation;
10 h-ending fermentation: the dissolved oxygen is controlled to be more than 40 percent.
5. The culture method according to claim 2, wherein the fermentation process is supplemented by feeding a uridine diphosphate-glucose-supplementing solution, a fructose-glucose-supplementing solution and a glucose-fructose-supplementing solution
a process control of uridine diphosphate glucose supplementation:
when fermenting for 21h, supplementing adenosine diphosphate glucose, and controlling the concentration of the adenosine diphosphate glucose in the fermentation liquor to be 0.1-0.3 g/L;
b, water supplementing process control:
when the fermentation is carried out for 9-21h, when OD is more than or equal to 2.5, adding sterilized water, controlling OD to be 2-2.5 in the fermentation process, and detecting the OD of the fermentation liquor every 3 h;
after fermentation is finished for 21h to the end of fermentation, adding sterilized water, and controlling the volume of fermentation liquor to be 75-80% of that of the fermentation tank;
c, supplementing high fructose corn syrup:
when the fermentation is carried out for 9-21h, when the total sugar content of the fermentation liquor is less than 5mg/100ml, the sterilized high fructose corn syrup solution is supplemented, so that the total sugar content is controlled to be 10-15 mg/100 ml.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0694616A2 (en) * | 1994-07-26 | 1996-01-31 | POLI INDUSTRIA CHIMICA S.p.A. | Process for the preparation of hyaluronic acid by fermentation with streptococcus |
| KR100258237B1 (en) * | 1993-04-16 | 2000-06-01 | 성재갑 | Medium for culturing microorganisms producing hyaluronic acid |
| WO2008057637A2 (en) * | 2006-07-21 | 2008-05-15 | Novozymes, Inc. | Methods of increasing secretion of polypeptides having biological activity |
| US20080138865A1 (en) * | 2006-07-06 | 2008-06-12 | Reliance Life Sciences Pvt. Ltd. | Optimisation of culture conditions for production and process of purification of high molecular weight hyaluronic acid |
| CN101451114A (en) * | 2008-12-31 | 2009-06-10 | 桐乡市恒基生物科技有限公司 | Method for breeding fermentation high stability hyaluronic acid sodium bacterial |
| CN102242165A (en) * | 2011-05-26 | 2011-11-16 | 上海应用技术学院 | Method for producing high molecular weight sodium hyaluronate through fermentation and culture medium utilized by same |
| CN102559801A (en) * | 2011-12-20 | 2012-07-11 | 宁夏大学 | Fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining |
| CN108795792A (en) * | 2017-05-02 | 2018-11-13 | 浙江京新药业股份有限公司 | A kind of culture medium and fermentation process of bacillus licheniformis |
| CN108813098A (en) * | 2018-06-07 | 2018-11-16 | 吉林仟客莱科技股份有限公司 | The compound nonreactive feed of microbial fermentation containing natural antibacterial components |
| CN109468355A (en) * | 2018-10-16 | 2019-03-15 | 河北科技大学 | A method of improving fermenting and producing hyaluronan molecule amount |
| CN109706203A (en) * | 2018-12-18 | 2019-05-03 | 贾召鹏 | A kind of preparation method of high yield hyaluronic acid |
-
2020
- 2020-12-14 CN CN202011461609.0A patent/CN112592849A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100258237B1 (en) * | 1993-04-16 | 2000-06-01 | 성재갑 | Medium for culturing microorganisms producing hyaluronic acid |
| EP0694616A2 (en) * | 1994-07-26 | 1996-01-31 | POLI INDUSTRIA CHIMICA S.p.A. | Process for the preparation of hyaluronic acid by fermentation with streptococcus |
| US20080138865A1 (en) * | 2006-07-06 | 2008-06-12 | Reliance Life Sciences Pvt. Ltd. | Optimisation of culture conditions for production and process of purification of high molecular weight hyaluronic acid |
| WO2008057637A2 (en) * | 2006-07-21 | 2008-05-15 | Novozymes, Inc. | Methods of increasing secretion of polypeptides having biological activity |
| CN101451114A (en) * | 2008-12-31 | 2009-06-10 | 桐乡市恒基生物科技有限公司 | Method for breeding fermentation high stability hyaluronic acid sodium bacterial |
| CN102242165A (en) * | 2011-05-26 | 2011-11-16 | 上海应用技术学院 | Method for producing high molecular weight sodium hyaluronate through fermentation and culture medium utilized by same |
| CN102559801A (en) * | 2011-12-20 | 2012-07-11 | 宁夏大学 | Fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining |
| CN108795792A (en) * | 2017-05-02 | 2018-11-13 | 浙江京新药业股份有限公司 | A kind of culture medium and fermentation process of bacillus licheniformis |
| CN108813098A (en) * | 2018-06-07 | 2018-11-16 | 吉林仟客莱科技股份有限公司 | The compound nonreactive feed of microbial fermentation containing natural antibacterial components |
| CN109468355A (en) * | 2018-10-16 | 2019-03-15 | 河北科技大学 | A method of improving fermenting and producing hyaluronan molecule amount |
| CN109706203A (en) * | 2018-12-18 | 2019-05-03 | 贾召鹏 | A kind of preparation method of high yield hyaluronic acid |
Non-Patent Citations (7)
| Title |
|---|
| AMADO, IR等: "Microbial production of hyaluronic acid from agro-industrial by-products: Molasses and corn steep liquor", BIOCHEMICAL ENGINEERING JOURNAL, vol. 117, pages 181 - 187, XP029834448, DOI: 10.1016/j.bej.2016.09.017 * |
| 刘丽等: "微生物发酵生产透明质酸研究进展", 食品与发酵工业, vol. 35, no. 02, pages 127 * |
| 成霞;刘登如;陈坚;堵国成;: "高产量、高分子量透明质酸发酵条件优化", 过程工程学报, no. 05 * |
| 李黔蜀;叶华;贺立虎;: "玉米浆作为氮源对透明质酸发酵的影响", 杨凌职业技术学院学报, no. 02 * |
| 田毅红等: "高分子量透明质酸的发酵研究", 中国医药工业杂志, vol. 35, no. 5, pages 2 * |
| 邱平;柯常毅;邓益全;: "恒速补料与pH胁迫相结合提高透明质酸发酵产量的研究", 食品与发酵科技, no. 05 * |
| 马朝梅;: "发酵法生产透明质酸工艺研究", 广西工学院学报, no. 03 * |
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