CN113652456A - Method for producing pullulan polysaccharide by cell recycling and application - Google Patents
Method for producing pullulan polysaccharide by cell recycling and application Download PDFInfo
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- CN113652456A CN113652456A CN202110880337.6A CN202110880337A CN113652456A CN 113652456 A CN113652456 A CN 113652456A CN 202110880337 A CN202110880337 A CN 202110880337A CN 113652456 A CN113652456 A CN 113652456A
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- 229920001218 Pullulan Polymers 0.000 title claims abstract description 52
- 239000004373 Pullulan Substances 0.000 title claims abstract description 52
- 235000019423 pullulan Nutrition 0.000 title claims abstract description 52
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- 238000004064 recycling Methods 0.000 title claims abstract description 17
- 150000004676 glycans Chemical class 0.000 title claims abstract description 12
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 12
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 74
- 230000004151 fermentation Effects 0.000 claims abstract description 74
- 239000007788 liquid Substances 0.000 claims abstract description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 241000223678 Aureobasidium pullulans Species 0.000 claims abstract description 13
- 230000016615 flocculation Effects 0.000 claims abstract description 13
- 238000005189 flocculation Methods 0.000 claims abstract description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000000701 coagulant Substances 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims abstract description 6
- 239000008394 flocculating agent Substances 0.000 claims abstract description 5
- 238000011081 inoculation Methods 0.000 claims abstract description 4
- 230000014759 maintenance of location Effects 0.000 claims description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 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 5
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 2
- 229920001661 Chitosan Polymers 0.000 claims description 2
- 235000010413 sodium alginate Nutrition 0.000 claims description 2
- 239000000661 sodium alginate Substances 0.000 claims description 2
- 229940005550 sodium alginate Drugs 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 9
- 238000011218 seed culture Methods 0.000 abstract description 8
- 238000011177 media preparation Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 5
- 238000011049 filling Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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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
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P19/10—Pullulan
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The invention provides a method for producing pullulan polysaccharide by cell recycling fermentation and application thereof, comprising the following steps: inoculating the aureobasidium pullulans seed liquid to a fermentation culture medium for fermentation; after fermentation culture for 7 days, adjusting the pH value in the fermentation liquor to be stable by using hydrochloric acid or sodium hydroxide, dropwise adding a flocculating agent and a coagulant aid, shaking, uniformly mixing, standing, layering, separating bacterial liquid to obtain a fermentation liquor clear liquid containing pullulan polysaccharide and flocculation-precipitated Aureobasidium pullulan thallus, collecting the fermentation liquor clear liquid containing pullulan polysaccharide, resuspending the Aureobasidium pullulan thallus by using a fresh fermentation liquor, and continuing fermentation. According to the invention, the fermentation production of the pullulan is carried out by adopting a flocculation method to recover and repeatedly utilize a cell fermentation mode, so that the yield of the pullulan is improved; the links of seed culture medium preparation, seed culture, inoculation and the like in the fermentation production process are reduced, the fermentation time is saved, the raw material cost is reduced, and the production efficiency is further improved.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to a method for producing pullulan polysaccharide by cell recycling and application of the pullulan polysaccharide.
Background
The pullulan is a colorless and tasteless dextro-glucoside polymer, is powdery, can give off slight sweet taste when dissolved in water, has the obvious advantages of no toxicity, biological safety, easy degradability, good edibility, good film coating property and the like, and has huge application potential in the fields of medicines, cosmetics, environment, food and the like. The microbial fermentation method has the advantages of mild reaction conditions, high purity of target products and little pollution, shows greater production advantages and has huge development prospect. Aureobasidium pullulans: (Aureobasidium pullulans) Is a yeast-like fungus and is a main pullulan production strain. The current strategies for efficiently producing the pullulan by using a microbial fermentation method mainly comprise breeding high-yield strains, optimizing culture conditions, controlling metabolic processes and the like, however, the future demand space of the pullulan is large, and the strategies cannot realize large-scale production of the pullulan.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for producing pullulan by cell recycling and application thereof, wherein the method can maintain the continuous fermentation performance of cells, reduce the product inhibition, save the fermentation time, reduce the production cost and improve the total yield of fermentation products.
The invention is realized by the following technical scheme:
the method for producing the pullulan polysaccharide by cell recycling fermentation is characterized by comprising the following steps:
1) inoculating a prepared aureobasidium pullulans seed solution into a fermentation culture medium for fermentation culture;
2) after fermentation culture for one week, adjusting the pH value in the fermentation liquor by using hydrochloric acid or sodium hydroxide;
3) after the pH value in the fermentation liquor is stable, adding a flocculating agent and a coagulant aid dropwise into the fermentation liquor, and uniformly mixing by oscillation;
4) standing the fermentation liquor after oscillation and uniform mixing until the bacterial liquor is layered, pouring to separate the bacterial liquor to obtain a fermentation liquor clear liquid containing pullulan and flocculently precipitated Aureobasidium pullulan thallus, collecting the fermentation liquor clear liquid containing pullulan to extract pullulan, suspending the flocculently obtained Aureobasidium pullulan with a fresh culture medium again, continuing to ferment, and continuing to prepare the pullulan.
Preferably, the inoculation amount of the aureobasidium pullulans seed solution in the step 1) is 6%, the culture time of the seed solution is 2-3 days, and the culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.
Preferably, the culture medium for preparing the seed liquid in the step 1) comprises the following components in percentage by weight: glucose 5%, peptone 0.1%, yeast extract 0.1%, KH2PO4 0.5%，MgSO4•7H20.04% of O, 0.1% of NaCl and the balance of water.
Preferably, the fermentation medium in step 1) comprises the following components in percentage by weight: glucose 12%, NH4NO30.1%，KH2PO4 0.01%，KCl 0.05%，MgSO4•7H20.02% of O, the balance of water, and the fermentation culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.
Preferably, the concentration of the hydrochloric acid or the sodium hydroxide in the step 2) is 2mol/L, and the pH value of the fermentation liquor is adjusted to 5.0-5.5.
Preferably, the flocculating agent in the step 3) is 10g/L chitosan solution prepared by 1% acetic acid solution and autoclaved, and the dropping amount is 0.03-0.04 mL/mL; the coagulant aid is 10g/L sodium alginate solution prepared by ultrapure water and autoclaved, and the dropping amount is 0.02-0.04 mL/mL.
Preferably, the condition of shaking and mixing the fermentation liquor in the step 3) is 28 ℃, 180r/min, and the time is 20-30 min.
Preferably, the calculation formula of the cell flocculation rate after the flocculation treatment of the fermentation liquor is as follows:
(ii) a General purposeThe formula for calculating the retention rate of the luran polysaccharide is as follows: retention rate
。
The method for producing the pullulan by repeatedly utilizing the cells through fermentation should be used in the production of the pullulan.
According to the method, a flocculation method is adopted, the fermentation production of the pullulan is carried out in a cell fermentation mode, after a fermentation period is finished, the bacterial cells are recovered through flocculation treatment, a fresh culture medium is supplemented for resuspension and continuous fermentation, products are discharged in time, the product inhibition is reduced, meanwhile, metabolic waste is continuously transferred out, the toxic hazard to the cell growth is avoided, and the yield of the pullulan is effectively improved; the method for producing the pullulan polysaccharide by cell recycling fermentation reduces the links of seed culture medium preparation, seed culture, inoculation and the like in the fermentation production process, saves the fermentation time, reduces the raw material cost and further improves the production efficiency. After the fermentation liquor is flocculated, the cell flocculation rate is over 80 percent, the retention rate of the pullulan is over 95 percent, and the yield of the pullulan is improved by over 45 percent.
Detailed Description
The present invention will be described in further detail with reference to specific examples to better understand the technical solution.
Example 1
The method for producing the pullulan polysaccharide by cell recycling fermentation comprises the following steps:
1) selecting a well-cultured slant strain, selecting 2 rings, inoculating the strain into a seed culture medium, filling 50mL of the seed culture medium into a triangular flask with the liquid filling amount of 250mL, and culturing for 2d at the temperature of 180r/min in a shaking table;
the seed culture medium comprises: glucose 5%, peptone 0.1%, yeast extract 0.1%, KH2PO4 0.5%，MgSO4•7H20.04% of O, 0.1% of NaCl and the balance of water;
2) fermentation culture: inoculating the prepared seed liquid into a fermentation culture medium according to the proportion of 6 percent, filling 50mL of the seed culture medium into a triangular flask with the liquid filling amount of 250mL, and culturing for 7d at the temperature of 180r/min by a shaking table;
3) dropwise adding HCl into the fermentation liquor fermented in the step 2), adjusting the pH of the fermentation liquor to 5.1, respectively dropwise adding 2mL of flocculant and 2mL of coagulant aid, and then uniformly mixing for 20 min;
4) standing the fermentation liquid treated in the step 3) for 2 hours, pouring out the supernatant, adding a fresh fermentation medium with the same volume again for suspension, and continuing fermentation culture for 7 days;
through determination, the cell flocculation rate is 84.8%, the pullulan retention rate is 96.1%, and the pullulan yield is improved by 46.4%.
Wherein, the calculation formula of the cell flocculation rate is as follows:
(ii) a The calculation formula of the pullulan retention rate is as follows: retention rate
。
Example 2
This example is the same as the process step of example 1, except that the amount of flocculant added dropwise in step 3) was 1.5 mL.
The final determination in the example shows that the cell flocculation rate is 80.3%, the pullulan retention rate is 95.1%, and the yield of the pullulan is improved by 45.1%.
Example 3
This example is the same as the procedure of example 1 except that the coagulant aid was added in an amount of 1.5mL in step 3).
The final determination in the example shows that the cell flocculation rate is 81.4%, the pullulan retention rate is 95.3%, and the yield of the pullulan is improved by 45.9%.
Example 4
This example is identical to the procedure of example 1, except that in step 3) the shaking and homogenisation time is 30 min.
The final determination in the example shows that the cell flocculation rate is 83.7%, the pullulan retention rate is 95.6%, and the yield of the pullulan is improved by 46.2%.
Claims (9)
1. The method for producing the pullulan polysaccharide by cell recycling fermentation is characterized by comprising the following steps:
1) inoculating a prepared aureobasidium pullulans seed solution into a fermentation culture medium for fermentation culture;
2) after fermentation culture for one week, adjusting the pH value in the fermentation liquor by using hydrochloric acid or sodium hydroxide;
3) after the pH value in the fermentation liquor is stable, adding a flocculating agent and a coagulant aid dropwise into the fermentation liquor, and uniformly mixing by oscillation;
4) standing the fermentation liquor after oscillation and uniform mixing until the bacterial liquor is layered, pouring to separate the bacterial liquor to obtain a fermentation liquor clear liquid containing pullulan and flocculently precipitated Aureobasidium pullulan thallus, collecting the fermentation liquor clear liquid containing pullulan to extract pullulan, suspending the flocculently obtained Aureobasidium pullulan with a fresh culture medium again, continuing to ferment, and continuing to prepare the pullulan.
2. The method for producing pullulan through cell recycling and fermentation according to claim 1, wherein the inoculation amount of the aureobasidium pullulans seed solution in step 1) is 6%, the time for culturing the seed solution is 2-3 days, and the culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.
3. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the culture medium for preparing the seed liquid in step 1) comprises the following components in percentage by weight: glucose 5%, peptone 0.1%, yeast extract 0.1%, KH2PO4 0.5%，MgSO4•7H20.04% of O, 0.1% of NaCl and the balance of water.
4. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the fermentation medium in the step 1) comprises the following components in percentage by weight: glucose 12%, NH4NO3 0.1%，KH2PO4 0.01%，KCl 0.05%，MgSO4•7H20.02% of O, the balance of water, and the fermentation culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.
5. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the concentration of hydrochloric acid or sodium hydroxide in step 2) is 2mol/L, and the pH value of the fermentation broth is adjusted to 5.0 to 5.5.
6. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the flocculating agent in step 3) is 10g/L chitosan solution prepared from 1% acetic acid solution and autoclaved, and the dropping amount is 0.03-0.04 mL/mL; the coagulant aid is 10g/L sodium alginate solution prepared by ultrapure water and autoclaved, and the dropping amount is 0.02-0.04 mL/mL.
7. The method for producing pullulan through cell recycling and fermentation according to claim 1, wherein the oscillating and mixing condition of the fermentation liquid in the step 3) is 28 ℃, 180r/min and the time is 20-30 min.
8. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the calculation formula of the flocculation rate of the cells after the flocculation treatment of the fermentation liquid is as follows:
(ii) a The calculation formula of the pullulan retention rate is as follows: retention rate
。
9. The method for producing pullulan by cell recycling fermentation according to claims 1 to 8 should be used in the production of pullulan.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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