CN111321187A - Fermentation production process of hyaluronic acid - Google Patents
Fermentation production process of hyaluronic acid Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 77
- 230000004151 fermentation Effects 0.000 title claims abstract description 77
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 46
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 45
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 45
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 49
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 12
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 13
- 108010080698 Peptones Proteins 0.000 claims description 13
- 235000019319 peptone Nutrition 0.000 claims description 13
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 12
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 238000002525 ultrasonication Methods 0.000 claims 1
- 235000011187 glycerol Nutrition 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 9
- 210000000170 cell membrane Anatomy 0.000 description 7
- 229910052751 metal Inorganic materials 0.000 description 7
- 239000002184 metal Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 5
- 238000002604 ultrasonography Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
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- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- Engineering & Computer Science (AREA)
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- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention belongs to the technical field of biological fermentation, and discloses a fermentation production process of hyaluronic acid, which comprises the following steps: inoculating the streptococcus zooepidemicus seed liquid into a fermentation tank containing a fermentation culture medium for fermentation culture for 6 hours, adding glycerol and acetaldehyde into the fermentation tank, continuing the fermentation culture for 8-10 hours, then carrying out ultrasonic treatment, continuing the fermentation culture for 2 hours, and stopping the fermentation. In the method for preparing hyaluronic acid by fermentation, various factors are optimized and improved, and the yield of hyaluronic acid is improved.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation production process of hyaluronic acid.
Background
Hyaluronic Acid (HA) is named as furfural (hyaluronic acid) acid by chemical name, and is chain-shaped high-molecular acidic mucopolysaccharide formed by repeatedly and alternately connecting D-glucuronic acid and N-acetylglucosamine monomers serving as structural units through β -1, 4 glycosidic bonds.
Hyaluronic acid is a biochemical substance with excellent performance and has extremely wide application. The production method is developed from the initial extraction method to the microbial fermentation method. The production of hyaluronic acid by fermentation has been rapidly developed for decades and is the predominant method for hyaluronic acid production today. At present, there have been some studies to improve fermentation methods, including mixed fermentation techniques, control of nutrient components and fermentation conditions to increase the content of hyaluronic acid metabolites; the applicant's prior patent technology ' CN105368912A, a method for extracting sodium hyaluronate by spray counter-current method ' adopts two strains to mix and ferment, greatly improving the fermentation efficiency, but the method has high requirements on culture conditions, possibly resulting in that dominant strains cannot be formed, and the produced hyaluronic acid comprises hyaluronic acid with small, medium and large molecular weights, cannot form a single product with high purity, and reduces the added value of the product.
The applicant's prior patent technology "a method for producing high molecular weight hyaluronic acid by fermentation", in the method for producing hyaluronic acid by fermentation, optimization and improvement of various factors, including addition of glycerin, can exert influence on cell membrane voids, thereby exerting a positive effect on sugar chain secretion; the cavitation of the ultrasound can cause the non-thermal biological effect of cells, so that the cell membranes are locally ruptured in a short time, thereby changing the permeability of the cell plasma membranes and releasing the substances in the cells out of the cells; the method improves hyaluronic acid yield and molecular weight. On the basis of the above studies, the applicant has continued to clarify the production method of hyaluronic acid.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a fermentation production process of hyaluronic acid.
The invention is realized by the following technical scheme:
a fermentation production process of hyaluronic acid comprises the following steps:
inoculating streptococcus zooepidemicus seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 10% for fermentation culture, performing fermentation culture for 6 hours, adding glycerol and acetaldehyde into the fermentation tank, continuing the fermentation culture for 8-10 hours, performing ultrasonic treatment, continuing the fermentation culture for 2 hours, and stopping the fermentation; during the fermentation culture process, the residual sugar concentration is controlled to be not less than 20g/L by feeding 100g/L glucose solution, the temperature of the fermentation culture is controlled to be 38-40 ℃, and the dissolved oxygen is 2-4L/min.
Preferably, the first and second electrodes are formed of a metal,
the addition amount of the glycerol is 200-300 mg/L.
Preferably, the first and second electrodes are formed of a metal,
the addition amount of the acetaldehyde is 20-30 mg/L.
Preferably, the first and second electrodes are formed of a metal,
the parameters of the ultrasonic treatment are as follows: the ultrasonic power is 200-300w respectively, the action time of the ultrasonic is 3s each time, the interval time is 3s, and the total time of the ultrasonic treatment is 90 s.
Preferably, the first and second electrodes are formed of a metal,
the fermentation medium comprises the following components: 50g/L glucose, 5g/L corn steep liquor, 1g peptone, 1.8 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate and pH 7.1.
Preferably, the first and second electrodes are formed of a metal,
the preparation method of the streptococcus zooepidemicus seed liquid comprises the following steps:
taking out Streptococcus zooepidemicus, inoculating in solid activating culture medium, inoculating in seed culture medium, and culturing to strain concentration of 108-109cfu/ml streptococcus zooepidemicus seed liquor.
Preferably, the first and second electrodes are formed of a metal,
the components of the solid activation medium are as follows:
according to weight percentage, 10 percent of sucrose, 2 percent of peptone, 0.125 percent of sodium chloride, 0.5 percent of magnesium sulfate, 2 percent of dipotassium hydrogen phosphate, 2 percent of agar and the balance of sterile water.
Preferably, the first and second electrodes are formed of a metal,
the seed culture medium comprises the following components: according to weight percentage, 10 percent of sucrose, 2 percent of peptone, 0.125 percent of sodium chloride, 0.5 percent of magnesium sulfate, 2 percent of dipotassium hydrogen phosphate and the balance of sterile water.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
in the method for preparing hyaluronic acid by fermentation, various factors are optimized and improved, and the yield of hyaluronic acid is improved.
After the bacterial strain is proliferated to a certain concentration, the nitrogen source concentration is reduced, the metabolism is not beneficial to the proliferation of the bacterial strain, and at the moment, the glycerol is added, so that the carbon source can be promoted to synthesize the hyaluronic acid, the metabolism is carried out towards the direction beneficial to the synthesis of products, the cell wall secretory pores and permeability can be increased, and the synthesis and the secretion of the hyaluronic acid with large molecular weight are facilitated; addition of glycerol at the initial stage of fermentation does not improve the expression level of hyaluronic acid, and it is likely that the strain rapidly proliferates at the initial stage of fermentation, but the synthesis of hyaluronic acid is small.
The cost can be reduced by replacing part of peptone with corn steep liquor, and the yield of hyaluronic acid is improved.
In large-scale fermentation culture, the situation of insufficient oxygen can occur, acetaldehyde and the like can be generated by using sugar through strains, and the acetaldehyde with proper concentration is added into a culture medium, so that feedback inhibition can be caused on an acetaldehyde metabolic pathway, the flow of a glycolytic pathway can enter a hyaluronic acid synthesis pathway to the maximum extent, and the hyaluronic acid with more and higher polymerization degree can be synthesized.
The cavitation of the ultrasound can cause the non-thermal biological effect of cells, so that the cell membranes are locally ruptured in a short time, thereby changing the permeability of the cell plasma membranes and releasing the substances in the cells out of the cells; the proper ultrasonic time and intensity are selected, so that the production of hyaluronic acid is facilitated.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A fermentation production process of hyaluronic acid comprises the following steps:
streptococcus zooepidemicus ATCC39920 is taken out, inoculated in a solid activated culture medium (10% by weight of sucrose, 2% of peptone, 0.125% of sodium chloride, 0.5% of magnesium sulfate, 2% of dipotassium hydrogen phosphate, 2% of agar, and the balance of sterile water), and then inoculated in a seed culture medium (10% by weight of sucrose, 2% of peptone, 0.125% of sodium chloride, 0.5% of magnesium sulfate, 2% of dipotassium hydrogen phosphate, and the balance of sterile water) to be cultured until the strain concentration is 108cfu/ml of seed solution;
inoculating the seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 10% for fermentation culture, wherein the fermentation culture medium comprises the following components: 50g/L glucose, 5g/L corn steep liquor, 1g peptone, 1.8 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate, pH 7.1, temperature 38 ℃, dissolved oxygen 3.2L/min; fermenting and culturing for 6h, adding glycerol and acetaldehyde into the fermentation tank, and controlling the concentration of the glycerol to be 200mg/L and the concentration of the acetaldehyde to be 30 mg/L; continuing fermentation culture for 10h, and then carrying out ultrasonic treatment, wherein the parameters of the ultrasonic treatment are as follows: the ultrasonic power is 200w respectively, the action time of the ultrasonic is 3s each time, the interval time is 3s, and the total time of the ultrasonic treatment is 90 s; then continuing fermentation culture for 2h, and terminating fermentation; the total time of fermentation culture is 18 h; during the fermentation process, the residual sugar concentration is controlled to be not less than 20g/L by feeding glucose solution with the concentration of 100 g/L.
Example 2
A fermentation production process of hyaluronic acid comprises the following steps:
streptococcus zooepidemicus ATCC39920 is taken out, inoculated in a solid activated culture medium (10% by weight of sucrose, 2% by weight of peptone, 0.125% by weight of sodium chloride, 0.5% by weight of magnesium sulfate, 2% by weight of dipotassium hydrogen phosphate, 2% by weight of agar, and the balance of sterile water), and then inoculated in a seed culture medium (10% by weight of sucrose, 2% by weight of peptone, 0.125% by weight of sodium chloride, 0.5% by weight of magnesium sulfate, 2% by weight of dipotassium hydrogen phosphate, and the balance of sterile water) to be cultured until strains are obtainedConcentration 108cfu/ml of seed solution;
inoculating the seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 10% for fermentation culture, wherein the fermentation culture medium comprises the following components: 50g/L glucose, 5g/L corn steep liquor, 1g peptone, 1.8 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate, pH 7.1, temperature 39 ℃, dissolved oxygen 3.2L/min; fermenting and culturing for 6h, adding glycerol and acetaldehyde into the fermentation tank, and controlling the concentration of the glycerol to be 300mg/L and the concentration of the acetaldehyde to be 20 mg/L; continuing fermentation culture for 8h, and then carrying out ultrasonic treatment, wherein the parameters of the ultrasonic treatment are as follows: the ultrasonic power is 300w respectively, the action time of the ultrasonic is 3s each time, the interval time is 3s, and the total time of the ultrasonic treatment is 90 s; then continuing fermentation culture for 2h, and terminating fermentation; the total time of fermentation culture is 16 h; during the fermentation process, the residual sugar concentration is controlled to be not less than 20g/L by feeding glucose solution with the concentration of 100 g/L.
Example 3
The influence of the addition of glycerol and acetaldehyde on the yield of hyaluronic acid was examined by orthogonal experiments:
taking the fermentation conditions of example 1 as an example, a plurality of glycerol concentration groups are set, and are respectively 0mg/L, 100 mg/L, 200mg/L, 300mg/L, 400 mg/L and 500 mg/L; a plurality of acetaldehyde concentration groups of 0mg/L, 10 mg/L, 20mg/L, 30mg/L, 40 mg/L and 50 mg/L were set as in example 1. The yield of hyaluronic acid in the fermentation broth was determined, and is specifically shown in table 1:
and (4) conclusion: as shown in Table 1, in the middle stage of fermentation, the yield of hyaluronic acid was increased by adding glycerol and acetaldehyde, and it was found that the increase in the yield of hyaluronic acid was maximized by combining glycerol in an amount of 200-300mg/L with acetaldehyde in an amount of 20-30mg/L through an orthogonal test.
The invention also detects the molecular weight of the hyaluronic acid, and the molecular weight is about 240 ten thousand Da; the glycerin can increase the molecular weight of the hyaluronic acid, and the acetaldehyde has no influence on the molecular weight of the hyaluronic acid.
Example 4
The effect of sonication intensity and time on hyaluronic acid production was examined using an orthogonal test:
taking example 1 as an example, a plurality of ultrasonic intensity groups of 0, 100w, 200w, 300w, 400w and 500w are set, and a plurality of ultrasonic intensity times of 30s, 60s, 90s and 120s are set. The yield of hyaluronic acid in the fermentation broth was determined, and is specifically shown in table 2:
TABLE 2
| Factors of the fact | 30s | 60s | 90s | 120s |
| 0w | 3.07 | 3.07 | 3.07 | 3.07 |
| 100w | 3.26 | 3.45 | 3.61 | 3.64 |
| 200w | 3.41 | 3.78 | 3.97 | 3.92 |
| 300w | 3.59 | 3.81 | 3.88 | 3.77 |
| 400w | 3.66 | 3.75 | 3.71 | 3.60 |
| 500w | 3.34 | 3.62 | 3.46 | 3.51 |
And (4) conclusion: the cavitation of the ultrasound can cause the non-thermal biological effect of cells, so that the cell membranes are locally ruptured in a short time, thereby changing the permeability of the cell plasma membranes and releasing the substances in the cells out of the cells; as shown in table 2, the production of hyaluronic acid increased first and then decreased with the increase of intensity and time of ultrasound, and it is likely that ultrasound can change the cell membrane structure, adversely affecting bacteria, resulting in the increase of the production of hyaluronic acid, but damage was caused to the strain with the increase of intensity and time, resulting in the decrease of the production of hyaluronic acid.
The foregoing list is only illustrative of the preferred embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (8)
1. A fermentation production process of hyaluronic acid comprises the following steps:
inoculating streptococcus zooepidemicus seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 10% for fermentation culture, performing fermentation culture for 6 hours, adding glycerol and acetaldehyde into the fermentation tank, continuing the fermentation culture for 8-10 hours, performing ultrasonic treatment, continuing the fermentation culture for 2 hours, and stopping the fermentation; during the fermentation culture process, the residual sugar concentration is controlled to be not less than 20g/L by feeding 100g/L glucose solution, the temperature of the fermentation culture is controlled to be 38-40 ℃, and the dissolved oxygen is 2-4L/min.
2. The process as claimed in claim 1, wherein the amount of glycerol added is 200-300 mg/L.
3. The process according to claim 1, wherein the acetaldehyde is added in an amount of 20 to 30 mg/L.
4. The process according to claim 1, characterized in that the parameters of the ultrasonication are: the ultrasonic power is 200-300w respectively, the action time of the ultrasonic is 3s each time, the interval time is 3s, and the total time of the ultrasonic treatment is 90 s.
5. The process of claim 1, wherein the fermentation medium comprises: 50g/L glucose, 5g/L corn steep liquor, 1g peptone, 1.8 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate and pH 7.1.
6. The process of claim 1, wherein the preparation method of the streptococcus zooepidemicus seed solution comprises the following steps:
taking out Streptococcus zooepidemicus, inoculating in solid activating culture medium, inoculating in seed culture medium, and culturing to strain concentration of 108-109cfu/ml streptococcus zooepidemicus seed liquor.
7. The process according to claim 6, wherein the solid activation medium has the composition:
according to weight percentage, 10 percent of sucrose, 2 percent of peptone, 0.125 percent of sodium chloride, 0.5 percent of magnesium sulfate, 2 percent of dipotassium hydrogen phosphate, 2 percent of agar and the balance of sterile water.
8. The process of claim 6, wherein the seed medium comprises the following components: according to weight percentage, 10 percent of sucrose, 2 percent of peptone, 0.125 percent of sodium chloride, 0.5 percent of magnesium sulfate, 2 percent of dipotassium hydrogen phosphate and the balance of sterile water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811540445.3A CN111321187A (en) | 2018-12-17 | 2018-12-17 | Fermentation production process of hyaluronic acid |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320484A (en) * | 2013-06-28 | 2013-09-25 | 四川柯森油田化学有限公司 | Method for improving the fermentation yield of hyaluronic acid (HA) |
| CN108410926A (en) * | 2017-12-03 | 2018-08-17 | 新疆阜丰生物科技有限公司 | A method of preparing extraction high molecular weight hyaluronic acid |
| CN108410927A (en) * | 2017-12-09 | 2018-08-17 | 新疆阜丰生物科技有限公司 | A kind of method of fermentation method production high molecular weight hyaluronic acid |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320484A (en) * | 2013-06-28 | 2013-09-25 | 四川柯森油田化学有限公司 | Method for improving the fermentation yield of hyaluronic acid (HA) |
| CN108410926A (en) * | 2017-12-03 | 2018-08-17 | 新疆阜丰生物科技有限公司 | A method of preparing extraction high molecular weight hyaluronic acid |
| CN108410927A (en) * | 2017-12-09 | 2018-08-17 | 新疆阜丰生物科技有限公司 | A kind of method of fermentation method production high molecular weight hyaluronic acid |
Non-Patent Citations (1)
| Title |
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| 郑建仙, 中国轻工业出版社 * |
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