CN115521959A - Extraction method of pullulan - Google Patents
Extraction method of pullulan Download PDFInfo
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- CN115521959A CN115521959A CN202211248117.2A CN202211248117A CN115521959A CN 115521959 A CN115521959 A CN 115521959A CN 202211248117 A CN202211248117 A CN 202211248117A CN 115521959 A CN115521959 A CN 115521959A
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- 239000004373 Pullulan Substances 0.000 title claims abstract description 28
- 229920001218 Pullulan Polymers 0.000 title claims abstract description 28
- 235000019423 pullulan Nutrition 0.000 title claims abstract description 28
- 238000000605 extraction Methods 0.000 title claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 33
- 230000004151 fermentation Effects 0.000 claims abstract description 33
- 230000001954 sterilising effect Effects 0.000 claims abstract description 30
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 30
- 150000004676 glycans Chemical class 0.000 claims abstract description 27
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 27
- 239000005017 polysaccharide Substances 0.000 claims abstract description 27
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229940072056 alginate Drugs 0.000 claims abstract description 17
- 235000010443 alginic acid Nutrition 0.000 claims abstract description 17
- 229920000615 alginic acid Polymers 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 238000001704 evaporation Methods 0.000 claims abstract description 16
- 238000011049 filling Methods 0.000 claims abstract description 13
- 238000011065 in-situ storage Methods 0.000 claims abstract description 13
- 230000008020 evaporation Effects 0.000 claims abstract description 11
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 239000012263 liquid product Substances 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 239000000706 filtrate Substances 0.000 claims description 25
- 159000000007 calcium salts Chemical class 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 241000223678 Aureobasidium pullulans Species 0.000 claims description 10
- 239000003513 alkali Substances 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 6
- 235000010408 potassium alginate Nutrition 0.000 claims description 6
- 239000000737 potassium alginate Substances 0.000 claims description 6
- MZYRDLHIWXQJCQ-YZOKENDUSA-L potassium alginate Chemical compound [K+].[K+].O1[C@@H](C([O-])=O)[C@@H](OC)[C@H](O)[C@H](O)[C@@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@@H](O)[C@H]1O MZYRDLHIWXQJCQ-YZOKENDUSA-L 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000012266 salt solution Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 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 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 235000011148 calcium chloride Nutrition 0.000 claims description 5
- FNAQSUUGMSOBHW-UHFFFAOYSA-H calcium citrate Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FNAQSUUGMSOBHW-UHFFFAOYSA-H 0.000 claims description 5
- 239000001354 calcium citrate Substances 0.000 claims description 5
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 claims description 5
- 239000001527 calcium lactate Substances 0.000 claims description 5
- 229960002401 calcium lactate Drugs 0.000 claims description 5
- 235000011086 calcium lactate Nutrition 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 239000008213 purified water Substances 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 235000010413 sodium alginate Nutrition 0.000 claims description 5
- 239000000661 sodium alginate Substances 0.000 claims description 5
- 229940005550 sodium alginate Drugs 0.000 claims description 5
- 235000013337 tricalcium citrate Nutrition 0.000 claims description 5
- 239000000728 ammonium alginate Substances 0.000 claims description 4
- 235000010407 ammonium alginate Nutrition 0.000 claims description 4
- KPGABFJTMYCRHJ-YZOKENDUSA-N ammonium alginate Chemical compound [NH4+].[NH4+].O1[C@@H](C([O-])=O)[C@@H](OC)[C@H](O)[C@H](O)[C@@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@@H](O)[C@H]1O KPGABFJTMYCRHJ-YZOKENDUSA-N 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000011033 desalting Methods 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000010612 desalination reaction Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 abstract description 2
- 239000006227 byproduct Substances 0.000 abstract description 2
- 229910001424 calcium ion Inorganic materials 0.000 abstract description 2
- 239000003337 fertilizer Substances 0.000 abstract description 2
- 238000002372 labelling Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 239000012065 filter cake Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 239000002808 molecular sieve Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003860 storage Methods 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0018—Pullulan, i.e. (alpha-1,4)(alpha-1,6)-D-glucan; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention discloses an extraction method of pullulan polysaccharide, which comprises the steps of fermentation, sterilization, protein removal, ultrafiltration desalination, evaporation concentration, in-situ sterilization and filling. According to the invention, the gel formed by the reaction of the natural extract (water-soluble alginate) and calcium ions is used for wrapping thallus impurities in the fermentation liquor, so that solid-liquid separation is realized, the use of a filter aid is reduced, and meanwhile, the value of mushroom dregs can be improved by adding natural components, thereby being beneficial to the development of byproducts such as feed or fertilizer. In addition, the natural extract meets the requirement of organic labeling, and can simultaneously meet the production requirement of organic pullulan. And a concentration mode is adopted, and an in-situ sterilization mode is adopted when the content of the pullulan reaches a certain concentration, so that a liquid product which is high in purity and can be directly used for filling is obtained.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to an extraction method of pullulan.
Background
The pullulan is an extracellular mucopolysaccharide obtained by fermenting aureobasidium pullulans, has the characteristics of excellent film forming property, water solubility, biocompatibility, degradability and the like, and is widely applied to the fields of food additives, cosmetic raw materials and medicines. However, the production level of the product is low in the global scope at present, and the main production technology difficulty is the extraction method of the pullulan, especially the post-treatment extraction of the high-viscosity pullulan fermentation liquor. Therefore, the production cost is high, the additional value is relatively low, and the industrialization process is delayed.
Currently, the research and the industrial production of pullulan in China are relatively lack, and the conventional treatment method of the known fermentation liquor mostly uses an organic solvent or an inorganic flocculant and is added with a filter aid, so that the problems of organic solvent residue, large pollutant discharge, high environmental protection cost and the like are easily caused. Meanwhile, due to high viscosity of the pullulan, the pullulan is usually subjected to low-concentration high-temperature instantaneous sterilization or bactericide addition and high-temperature drying to meet the requirements of microbial control and storage. The sterilization treatment and drying of a large amount of water in the extraction process cause a large amount of energy loss and resource waste.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems, the invention provides the extraction method of the pullulan polysaccharide, which can effectively solve the defects of organic solvent residue, large pollutant discharge, high environmental protection cost, energy consumption, resource waste and the like in the traditional treatment method.
The technical scheme is as follows: a method for extracting pullulan comprises the following steps:
s1, fermentation: culturing the aureobasidium pullulans by seeds, then putting the aureobasidium pullulans into a fermentation tank, and fermenting and culturing for 70 to 120h at the temperature of 27 to 30 ℃ and the ventilation ratio of 0.8 to 1.5 vvm;
s2, degerming: mixing the alginate aqueous solution with the fermentation liquor, and uniformly stirring; then adding calcium salt solution, and stirring again to form floccule; then adjusting the pH value to 5-8 with an alkali solution, heating and stirring, and then keeping the temperature and standing for 0.5-2h at 60-90 ℃; removing thalli and insoluble substances to obtain a first filtrate and recovering bacterial residues;
s3, protein removal: adjusting the pH value of the first filtrate to 5 to 8 by using alkali liquor, and then adding the first filtrate into a plate and frame filter coated with a filter aid for filtering to obtain a second filtrate;
s4, ultrafiltration desalting: filtering the second filtrate by using an ultrafiltration membrane, adding purified water, and circularly filtering to remove salt and other impurities to obtain a polysaccharide solution with the molecular weight of 1w to 50w;
s5, evaporation and concentration: evaporating and concentrating the polysaccharide solution obtained in the step S4 to obtain a high-concentration polysaccharide solution with the mass concentration of 10-60%;
s6, in-situ sterilization: adding the high-concentration polysaccharide solution obtained in the step S5 into a sterilization tank for in-situ sterilization, wherein the sterilization temperature is 115-140 ℃, and the sterilization time is 0.2-30min;
s7, filling: and filling the sterilized high-concentration polysaccharide solution into a sterile container to obtain a liquid product.
Preferably, the concentration of the alginate aqueous solution is 30 to 50g/L, and the volume ratio of the alginate aqueous solution to the fermentation liquid is 1 to 3.
Further, the alginate is one or a mixture of sodium alginate, potassium alginate and ammonium alginate.
Preferably, in the step S2, the concentration of the calcium salt solution is 300 to 400g/L, and the volume ratio of the calcium salt solution to the fermentation liquid is 1 to 3.
Further, the calcium salt is one or more of calcium chloride, calcium lactate and calcium citrate.
Preferably, in step S2, the method for removing the cells and insoluble matter is centrifugation or plate-and-frame filter pressing.
Preferably, in step S5, the condensed water generated during the evaporation and concentration is recycled as the raw material of the water produced by the water purifier.
Has the advantages that: according to the extraction method of the pullulan, the natural extract (water-soluble alginate) reacts with calcium ions to form gel to wrap thallus impurities in fermentation liquor, so that solid-liquid separation is realized, the use of a filter aid is reduced, meanwhile, the value of mushroom dregs can be improved by adding natural ingredients, and the development of byproducts such as feed or fertilizer is facilitated. In addition, the natural extract meets the requirement of organic labeling, and can simultaneously meet the production requirement of organic pullulan. By adopting a concentration mode and adopting an in-situ sterilization mode when the content of the pullulan reaches a certain concentration, a liquid product with higher purity and capable of being directly used for filling is obtained, condensed water is recycled, and meanwhile, the effects of energy conservation and emission reduction are achieved.
Drawings
FIG. 1 is a process flow diagram of a method for extracting pullulan according to the present invention;
FIG. 2 is a photograph showing floc formation by adding a calcium salt to the fermentation broth in step S1 of example 1;
FIG. 3 is a drawing showing the solid-liquid separation by heating and standing after the flocs have been adjusted in pH in step S1 in example 1;
FIG. 4 is a graph of the cake after filtration in step S1 in example 1.
Detailed Description
The invention is described in detail below with reference to the following figures and specific examples:
example 1
As shown in fig. 1, a method for extracting pullulan comprises the following steps:
s1, fermentation: the aureobasidium pullulans SWP35 has a preservation number of CGMCC NO.11602, and the aureobasidium pullulans SWP35 used in the embodiment of the invention is purchased from China general microbiological culture collection center, cultured by seeds, put into a fermentation tank, and fermented and cultured for 70 hours at the temperature of 27 ℃ and the aeration ratio of 0.8vvm to obtain fermentation liquor;
s2, degerming: mixing sodium alginate, potassium alginate and ammonium alginate according to any proportion to obtain mixed alginate, preparing into alginate aqueous solution of 30g/L (w/v), mixing with fermentation liquor according to a ratio of 1; then mixing calcium chloride, calcium lactate and calcium citrate according to any proportion to obtain a mixed calcium salt, preparing a calcium salt aqueous solution with the volume ratio of 300g/L (w/v) to the fermentation liquor of 1 (v/v), and stirring again to form floccule, as shown in figure 2; then adjusting the pH value to 5 by using an alkali solution, heating and stirring, then keeping the temperature and standing for 0.5h at 60 ℃, and layering the solid and liquid of the mixed feed liquid as shown in figure 3; removing thalli and insoluble substances by centrifugation to obtain a first filtrate and recovering bacterial residues;
s3, protein removal: adjusting the pH value of the first filtrate to 5 by using alkali liquor, adding the first filtrate into a plate-and-frame filter coated with filter aid No. 10 diatomite, and filtering to obtain a second filtrate, wherein the generated filter cake is shown in figure 4, the water content of the filter cake is low, and most of feed liquid is collected;
s4, ultrafiltration desalting: filtering the second filtrate by using an ultrafiltration membrane of a 3k molecular sieve, adding purified water, and circularly filtering to remove salt and other impurities to obtain a polysaccharide solution with the weight-average molecular weight of 1 w;
s5, evaporation and concentration: evaporating and concentrating the polysaccharide solution obtained in the step S4 to obtain a polysaccharide solution with the mass concentration of 10%, and recycling condensed water generated in evaporation and concentration as a water raw material of a water purification machine;
s6, in-situ sterilization: adding the polysaccharide solution obtained in the step S5 into a sterilization tank for in-situ sterilization, wherein the sterilization temperature is 115 ℃, and the sterilization time is 30min;
s7, filling: and filling the sterilized high-concentration polysaccharide solution into a sterile container to obtain a liquid product.
Example 2
A method for extracting pullulan comprises the following steps:
s1, fermentation: the aureobasidium pullulans SWP35 has a preservation number of CGMCC NO.11602, and the aureobasidium pullulans SWP35 used in the embodiment of the invention is purchased from China general microbiological culture Collection center, cultured by seeds, put into a fermentation tank, and fermented and cultured for 96 hours under the conditions of 28 ℃ and the aeration ratio of 1.0 vvm to obtain fermentation liquor;
s2, degerming: mixing sodium alginate, potassium alginate and potassium alginate according to any proportion to obtain mixed alginate, preparing into 40g/L (w/v) alginate water solution, mixing with fermentation liquor according to a ratio of 2 (v/v), and stirring and mixing uniformly; then mixing calcium chloride, calcium lactate and calcium citrate according to any proportion to obtain a mixed calcium salt, preparing 360g/L (w/v) of calcium salt aqueous solution, wherein the volume ratio of the calcium salt aqueous solution to fermentation liquor is 1 (v/v), and stirring again to form floccule; then adjusting the pH value to 6.5 by using an alkali solution, heating and stirring, then keeping the temperature and standing for 1h at 80 ℃, and layering the solid and liquid of the mixed feed liquid; removing thalli and insoluble substances through centrifugation to obtain a first filtrate and recovering bacterial residues;
s3, protein removal: adjusting the pH value of the first filtrate to 6.5 by using alkali liquor, adding the first filtrate into a plate-and-frame filter coated with filter aid No. 10 diatomite, and filtering to obtain a second filtrate, wherein the water content of a generated filter cake is low, and most of feed liquid is collected;
s4, ultrafiltration and desalination: filtering the second filtrate by using an ultrafiltration membrane of a 5k molecular sieve, adding purified water, and circularly filtering to remove salt and other impurities to obtain a polysaccharide solution with the weight-average molecular weight of 25 w;
s5, evaporation and concentration: evaporating and concentrating the polysaccharide solution obtained in the step S4 to obtain a polysaccharide solution with the mass concentration of 30%, and recycling condensed water generated in evaporation and concentration as a water raw material of a water purification machine;
s6, in-situ sterilization: adding the polysaccharide solution obtained in the step S5 into a sterilization tank for in-situ sterilization, wherein the sterilization temperature is 121 ℃, and the sterilization time is 20min;
s7, filling: and filling the sterilized high-concentration polysaccharide solution into a sterile container to obtain a liquid product.
Example 3
A method for extracting pullulan comprises the following steps:
s1, fermentation: the aureobasidium pullulans SWP35 has a preservation number of CGMCC NO.11602, the aureobasidium pullulans SWP35 used in the embodiment of the invention is purchased from China general microbiological culture Collection center, cultured by seeds, put into a fermentation tank, fermented and cultured for 120 hours under the conditions of 30 ℃ and the aeration ratio of 1.5vvm to obtain fermentation liquor;
s2, degerming: mixing sodium alginate, potassium alginate and ammonium alginate according to any proportion to obtain mixed alginate, preparing into alginate aqueous solution with the concentration of 50g/L (w/v), mixing the alginate aqueous solution with fermentation liquor according to the volume ratio of 1; then mixing calcium chloride, calcium lactate and calcium citrate according to any proportion to obtain a mixed calcium salt, preparing a 400g/L calcium salt water solution, mixing the calcium salt water solution with a fermentation liquor according to the volume ratio of 1; then adjusting the pH value to 8 by using an alkali solution, heating and stirring, then keeping the temperature and standing for 2 hours at 90 ℃, and layering the solid and liquid of the mixed feed liquid; removing thalli and insoluble substances through plate-and-frame filter pressing to obtain a first filtrate and recovering mushroom dregs;
s3, removing protein: adjusting the pH value of the first filtrate to 8 by using alkali liquor, adding the first filtrate into a plate-and-frame filter coated with 10# diatomite, and filtering to obtain a second filtrate, wherein the water content of a generated filter cake is low, and most of feed liquid is collected;
s4, ultrafiltration and desalination: filtering the second filtrate with ultrafiltration membrane of 10k molecular sieve, adding purified water, circularly filtering to remove salt and other impurities to obtain polysaccharide solution with weight average molecular weight of 50 w;
s5, evaporation and concentration: evaporating and concentrating the polysaccharide solution obtained in the step S4 to obtain a polysaccharide solution with the mass concentration of 60%, and reusing condensed water generated in evaporation and concentration as a water raw material of a water purification machine;
s6, in-situ sterilization: adding the polysaccharide solution obtained in the step S5 into a sterilization tank for in-situ sterilization, wherein the sterilization temperature is 140 ℃, and the sterilization time is 0.2min;
s7, filling: and filling the sterilized high-concentration polysaccharide solution into a sterile container to obtain a liquid product.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (7)
1. The extraction method of the pullulan is characterized by comprising the following steps of:
s1, fermentation: culturing aureobasidium pullulans by seeds, and then fermenting and culturing for 70 to 120h in a fermentation tank under the conditions of the temperature of 27 to 30 ℃ and the ventilation ratio of 0.8 to 1.5vvm to obtain fermentation liquid;
s2, degerming: mixing the alginate water solution with the fermentation liquid, and stirring and mixing uniformly; then adding calcium salt solution, and stirring again to form floccule; then adjusting the pH value to 5-8 with an alkali solution, heating and stirring, and then keeping the temperature and standing for 0.5-2h at 60-90 ℃; removing thallus and insoluble substances to obtain a first filtrate and recovering mushroom dregs;
s3, protein removal: adjusting the pH value of the first filtrate to 5 to 8 by using alkali liquor, and then adding the first filtrate into a plate and frame filter coated with a filter aid for filtering to obtain a second filtrate;
s4, ultrafiltration desalting: filtering the second filtrate by using an ultrafiltration membrane, adding purified water, and circularly filtering to remove salt and other impurities to obtain a polysaccharide solution with the molecular weight of 1w to 50w;
s5, evaporation and concentration: evaporating and concentrating the polysaccharide solution obtained in the step S4 to obtain a high-concentration polysaccharide solution with the mass concentration of 10-60%;
s6, in-situ sterilization: adding the high-concentration polysaccharide solution obtained in the step S5 into a sterilized audience for in-situ sterilization, wherein the sterilization temperature is 115-140 ℃, and the sterilization time is 0.2-30min;
s7, filling: and filling the sterilized high-concentration polysaccharide solution into a sterile container to obtain a liquid product.
2. The extraction method of pullulan according to claim 1, wherein the extraction method comprises the following steps: in the step S2, the concentration of the alginate aqueous solution is 30 to 50g/L, and the volume ratio of the alginate aqueous solution to the fermentation liquid is 1 to 3.
3. The extraction method of pullulan according to claim 2, wherein: the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate.
4. The method for extracting pullulan according to claim 1, wherein the method comprises the following steps: in the step S2, the concentration of the calcium salt solution is 300 to 400g/L, and the volume ratio of the calcium salt solution to the fermentation liquor is 1 to 3.
5. The method for extracting pullulan according to claim 4, wherein the method comprises the following steps: the calcium salt is one or more of calcium chloride, calcium lactate, and calcium citrate.
6. The extraction method of pullulan according to claim 1, wherein the extraction method comprises the following steps: in step S2, the method for removing the thalli and the insoluble substances is centrifugation or plate-and-frame filter pressing.
7. The extraction method of pullulan according to claim 1, wherein the extraction method comprises the following steps: and in the step S5, the condensed water generated in the evaporation concentration is reused as a water raw material of the water purifier.
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