CN103570842A - Extracting method of pullulan - Google Patents
Extracting method of pullulan Download PDFInfo
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- CN103570842A CN103570842A CN201310545611.XA CN201310545611A CN103570842A CN 103570842 A CN103570842 A CN 103570842A CN 201310545611 A CN201310545611 A CN 201310545611A CN 103570842 A CN103570842 A CN 103570842A
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Abstract
The invention relates to an extracting method of pullulan. The method comprises the following steps: fermentation; thermal treatment; flocculation and degerming; flocculation and centrifugalization; salting out and deproteinization; salting out and centrifugalization; ultrafiltration and separation; decoloration of macromolecular polysaccharide; decoloration of micromolecular polysaccharide by active carbon; mixing; fractional precipitation; vacuum drying; and crushing. The macromolecular polysaccharide is decolored by hydrogen peroxide while the micromolecular polysaccharide is decolored by active carbon. Impurities such as thallus, proteins, micromolecular salt and the like in fermenting liquid are removed, and molecules with overlarge molecular weight and small molecular weight in pullulan are removed by methods of ultrafiltration and fractional precipitation of alcohol, so that the molecular weight of the product is relatively uniform, and the product is white and stable in property, thereby facilitating application of pullulan.
Description
Technical field
The present invention relates to a kind of extracting method of pulullan polysaccharide, belong to microbial polysaccharide processing technique field.
Background technology
Pulullan polysaccharide, has another name called Pul, pullulan, is a kind of fungus polysaccharide, be Aureobasidium pullulans (
aureobasidium pullulans) synthetic a kind of extracellular water-soluble macromolecule neutral polysaccharide.Its chemical structure is with α-1, the poly-trisaccharide maltose that 6-glycosidic link connects, and glucose is combined into trisaccharide maltose by α-Isosorbide-5-Nitrae-glycosidic link, and two ends are again with α-1, and 6-glycosidic link is with other trisaccharide maltose combination, the macromolecule polysaccharide being so repeatedly formed by connecting.Dry pulullan polysaccharide powder is white, and no hygroscopicity is soluble in cold water and hot water.Pulullan polysaccharide has unique physics and chemistry and biological characteristicses such as splendid film-forming properties, one-tenth fibering, oxygen barrier, plasticity-, cohesiveness and easy natural degradation, it is nontoxic, without any side effects to human body, be a kind of Multifucntional biological products that have very big exploitation value and prospect.
Pulullan polysaccharide has the performance of many uniquenesses, and it is approved as foodstuff additive new variety in the 2006 Nian Bei Ministry of Health.China is the demand rapid growth to it in recent years, particularly in medical auxiliary materials field, has caused people's extensive attention and extensive application.Yet pulullan polysaccharide product is mainly supplied by Japan at present, the production of domestic pulullan polysaccharide does not also have mass-producing, and product price is high, and this is mainly because rear extraction process is immature.Therefore, simple and effective separating and extracting method has great importance.
Aureobasidium pullulans when fermentation the pigment that produces and protein etc. be unfavorable for its application in every respect.Mainly from two aspects, address this problem both at home and abroad at present: the one, by Optimal Medium or mutagenesis or the low Aureobasidium pullulans bacterial strain of the genetic modification acquisition sugared high yield pigment of product; The one, by optimizing rear extraction process, carry out separation and purification pulullan polysaccharide.
The extraction of the Microbial exopolysaccharides of applying in industry is reclaimed generally the precipitator method and direct drying method.The most ripe in the precipitator method is the heavy method of alcohol deposition method and salt, and conventional alcohol precipitation agent has methyl alcohol, ethanol, Virahol etc., and the heavy agent of conventional salt mainly contains quaternary amine, calcium chloride, calcium hydroxide and aluminum chloride etc.Salt precipitation method is mainly applicable to polyanion polysaccharide, and under alkaline condition, high valent cationic and organic cation (as quaternary ammonium salt) can form precipitation with polyanion polysaccharide.The advantage of the heavy method of salt is by alcohol amount than the few 2-3 of alcohol deposition method doubly, but quality product is slightly poor.Direct drying method is by fermented liquid transpiring moisture and convection drying becomes solid phase prod.In laboratory, lyophilize is the best method that reaches this purpose, the product that the methods such as industrial conventional rotating cylinder baking, forced air drying, spraying are dried obtain is all raw product, wherein contain the impurity such as a large amount of thalline, inorganic salt, organic residue, therefore the poorly water-soluble of product, color and luster is dark, rheological is also bad, range of application is restricted.
The extraction step of microbial polysaccharide mainly contains pre-treatment, decolouring, deproteinated and desalination etc.The decoloring method of polysaccharide mainly contains active carbon adsorption, ion exchange method, oxidative decoloration method and metal complex method.Activated carbon decolorizing is thoroughly but because glycocalix charcoal absorption meeting causes the loss of polysaccharide.Ion exchange method is to utilize weakly base resin to carry out adsorpting pigment, and this method is effective to free negativity ion pigment, to the poor effect of the pigment with sugared combination.Oxidative decoloration method is mainly under proper condition pigment oxidation to be removed with oxygenant.The Deproteinated method of polysaccharide mainly contains Sevage method, trichloroacetic acid method, tannic acid method, Freon 113 method and protease method etc.Through the Deproteinated polysaccharide soln of decolouring, can use ethanol precipitation to carry out separating polyose and use membrane technique to remove salt and small molecules.
Pulullan polysaccharide at present can large-scale production only have Japanese Lin Yuan company, but due to reasons such as blockades on new techniques, the domestic rear extraction process to pulullan polysaccharide rarely has report.The Sun Wanru that only has at present the Chinese Academy of Sciences aspect small-scale extraction application a patent of patent and Su Li, remaining extracting method only limits to laboratory.In the leaching process of pulullan polysaccharide, main difficult point is the removal of protein.Su Li adopts membrane filtration technology to the fermented liquid except thalline directly decolour deproteinated and desalination, and the ethanol precipitation of abandoning tradition, uses fluid-bed drying to be dried product, and production process can realize automatization and serialization.Membrane filtration technology in this patent mainly adopts tubular fibre or flat sheet membrane to carry out ultrafiltration, ceaselessly adds deionized water in basin, until colourity and salinity touch the mark.The method that Sun Wanru adopt to add flocculation agent removes thalline, then uses membrane technique to carry out separation and concentrated to polysaccharide soln, through the film of different molecular weight by polysaccharide classification, the rear spray-dried object that reaches removal moisture.In above two kinds of methods, to the removal of protein, be all to use membrane separation technique, yet membrane sepn removal protein also has very large defect, products obtained therefrom protein content is higher, and the pollution of film and obstruction are also comparatively serious.Because pulullan polysaccharide is a kind of water miscible exocellular polysaccharide, the Method and process of its extraction can be with reference to other similar polysaccharide, as xanthan gum, gelling gum and heat setting glue etc.Yet, because the pulullan polysaccharide molecular weight ranges producing in Aureobasidium pullulans fermenting process differs greatly, according to surveying and determination greatly between 6000-1000000, and the pulullan polysaccharide of different molecular weight exists very large difference in properties such as viscosity, film-forming properties, solubleness, make to reach through the difficult quality of the pulullan polysaccharide of simple extraction the requirement of application.In general the larger film-forming properties of molecular weight is better, but the larger viscosity of molecular weight solubleness larger, polysaccharide powder is poorer.If the molecular weight product obtaining differs greatly, lack of homogeneity, can cause unstable product quality.
Patent of the present invention is by adopting the techniques such as thermal treatment, flocculation to remove after the impurity such as thalline in fermented liquid and protein, adopt the method for ultra-filtration and separation that the macromolecular polysaccharide in fermented liquid and micromolecular polysaccharide are separated and processed respectively, make the molecular weight of the pulullan polysaccharide that finally obtains between 10000-100000, the homogeneity of product is greatly improved, and quality product is obviously improved.But also can be according to practical application need to change by adjusting process parameter the molecular weight ranges of product.According to the different decoloring method of the different employings of the molecular size range of pulullan polysaccharide.First be macromolecular polysaccharide and the micromolecular polysaccharide adopting in ultra-filtration and separation fermented liquid, then adopt respectively hydrogen peroxide decolouring or activated carbon decolorizing.It is the ultra-filtration membrane of 0.005 μ m that ultrafiltration for the first time adopts aperture, obtain the pulullan polysaccharide that molecular weight is greater than 100000, remaining ultrafiltrated carries out ultrafiltration for the second time with the ultra-filtration membrane that aperture is 0.001 μ m again, obtaining molecular weight is the pulullan polysaccharide between 10000-100000, and it is discarded or for extracting small molecules oligose that molecular weight is less than 10000 pulullan polysaccharide solution.The pulullan polysaccharide that is greater than 100000 for molecular weight, because molecular weight is too large, after being dried, often solvability is poor, affect its effect, therefore, patent of the present invention adopts the hydrogen peroxide processing of decolour, when decolouring, pulullan polysaccharide is carried out to degraded to a certain degree.For molecular weight, being less than 100000 pulullan polysaccharide directly decolours with gac.
Summary of the invention
Goal of the invention: the object of this invention is to provide a kind of can extract molecular weight ratio evenly, the extracting method of the high-quality pulullan polysaccharide of pure white, the stable in properties of color.
Technical scheme: a kind of extracting method of pulullan polysaccharide, comprises the following steps:
(a) fermentation: Aureobasidium pullulans is through conventional seed culture (slant culture, shake-flask culture), top fermentation tank fermentation culture more than 4 days then;
(b) thermal treatment: fermented liquid is heated to 20-30 minute at 60-80 ℃, with the activity of the relevant degrading enzyme of passivation, avoid Propiram to be degraded in follow-up treating processes;
(c) Flocculation: the inorganic flocculating agent that the mass concentration that adds 1%-2% in heat treated fermented liquid is 10%, standing 8-10h, described inorganic flocculating agent is aluminum chloride;
(d) flocculate centrifugal: the fermented liquid flocculating is placed in to whizzer centrifugal under 3000-4000 r/min, in low-speed centrifugal flocculation process, when making bacterial sediment, a part of protein also can be settled down;
(e) saltout except albumen: clear liquid after centrifugal is regulated to pH to 9.0-10.0, and heating and temperature control is at 85-90 ℃, and adding saturation ratio is the sodium-chlor of 60% left and right, more than heat-up time 1h, the protein of sex change can precipitate by coprecipitated gathering;
(f) saltout centrifugal: with whizzer, the liquid of saltouing is carried out centrifugally, removes precipitation wherein;
(g) ultra-filtration and separation: the clear liquid after centrifugal adds appropriate distilled water, the solution that to be mixed with containing polysaccharide mass concentration be 5%, is then placed in ultrafiltration apparatus ultrafiltration under the pressure of 0.5Mpa.The ultra-filtration membrane of selecting is 0.001 μ m and 0.005 μ m, and molecular weight cut-off is respectively: 10000 and 100000; After ultrafiltration, obtain the polysaccharide of three kinds of different molecular weights: molecular weight is greater than 100000 pulullan polysaccharide, the polysaccharide that pulullan polysaccharide, the molecular weight of molecular weight between 10000-100000 is less than 10000; It is discarded or for the production of the raw material of oligose that molecular weight is less than 10000 pulullan polysaccharide;
(h) macromolecular polysaccharide decolouring: the pulullan polysaccharide employing hydrogen peroxide oxidation process decolouring that is greater than 100000 for molecular weight;
(i) micromolecular polysaccharide activated carbon decolorizing: adopt absorption method decolouring for the pulullan polysaccharide between molecular weight 10000-100000;
(j) mix: two kinds of destainers in step h and step I are mixed;
(k) fractionation precipitation: add 95% alcohol in mixed destainer, make the ethanol content in total solution reach 20%, under this ethanol concn, the pulullan polysaccharide of ultra-high molecular weight can form precipitation, uses centrifugal removal; Supernatant liquor adds alcohol to make the ethanol content of solution reach 70% again, the Propiram precipitation of centrifugal collection molecular weight between 10000-100000, and molecular weight is less than 10000 Propiram oligose and can not precipitates under this ethanol concn, adopt alcohol according to molecular weight, to carry out separation to pulullan polysaccharide;
(l) vacuum-drying: precipitation obtained above is placed in to vacuum drying oven, dry 24h under 0.1MPa;
(m) pulverize: dried Propiram is pulverized with pulverizer.
Wherein, in described step h macromolecular polysaccharide bleaching process, oxidative decoloration method is under alkaline condition, utilize the strong oxidation oxidative degradation colors of edible hydrogen peroxide and decolour, oxidative decoloration method can not cause the loss of polysaccharide, the colourless oxidative breakdown product of coloring matter can be dissolved in water, after ethanol precipitation, can stay in ethanol/water system and remove; During decolouring, regulate between fermented liquid pH to 7.0-9.0, hydrogen peroxide addition is 2.0%-3.0%, and Heating temperature is 40-50 ℃, and be 40-50 minute heat-up time; The carrying out that rising temperature, increase concentration of hydrogen peroxide can be accelerated decolouring; Centrifugal after decolouring, then solution is heated to 90 ℃, keep 10 minutes; Under alkaline condition, heat and unnecessary hydrogen peroxide can be removed; The decolouring of employing hydrogen peroxide oxidation process, makes Propiram have degraded to a certain degree in decolouring, and under above-mentioned oxidizing intensity, wherein the Propiram degradable of most of high molecular is less than 100000 to molecular weight.
Wherein, in described step I micromolecular polysaccharide bleaching process, in fermented liquid, add granular active carbon or powdered active carbon, at 60-80 ℃, keep 30 minutes, after decolouring, filter; Absorption method decolouring can not cause Propiram degraded.
Beneficial effect: the present invention through Overheating Treatment, inorganic salt flocculation sediment thalline, filter, saltout except the operations such as albumen, centrifugal, ultra-filtration and separation, decolouring (macromolecular polysaccharide adopts hydrogen peroxide treatment decolouring, micromolecular polysaccharide activated carbon decolorizing), centrifugal, mixing, alcohol fraction precipitation, centrifugal or filtration, vacuum-drying, pulverizing, the impurity such as the thalline in removal fermented liquid, protein, small molecule salt.And remove excessive molecule and the too small molecule of molecular weight of molecular weight in pulullan polysaccharide by the method for ultrafiltration and alcohol fraction precipitation, and make molecular weight product more even, color is pure white, stable in properties, thus be conducive to the application of pulullan polysaccharide.
Accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
Embodiment
Embodiment 1:
Aureobasidium pullulans is through conventional seed culture (slant culture, shake-flask culture), top fermentation tank fermentation culture more than 4 days then.Fermented liquid is heated 30 minutes at 60 ℃.Then adding 1% concentration is 10%(w/v) inorganic flocculating agent (aluminum chloride), standing 8-10h.Centrifugal under 4000 r/ minutes.Supernatant liquor regulates pH to 9.0, and heating and temperature control is at 90 ℃, and adding saturation ratio is the sodium-chlor of 60% left and right, more than heat-up time 1h.Again with whizzer, the liquid of saltouing is carried out centrifugally, remove precipitation wherein.Clear liquid after filtration adds appropriate distilled water, is mixed with containing polysaccharide 5%(w/v) solution, be then placed in ultrafiltration apparatus ultrafiltration under the pressure of 0.5MPa.The ultra-filtration membrane of selecting is 0.001 μ m and 0.005 μ m, and molecular weight cut-off is respectively: 10000 and 100000.After ultrafiltration, obtain the polysaccharide of three kinds of different molecular weights, molecular weight is greater than the polysaccharide that 100000 pulullan polysaccharide, molecular weight are less than 10000 at pulullan polysaccharide, the molecular weight of 10000-100000.Molecular weight is less than 10000 pulullan polysaccharide discarded (raw material that also can be used for producing oligose fully utilizes).The ultrafiltrated that is greater than 100000 for molecular weight, regulates pH to 9.0, and hydrogen peroxide addition is 2.0%, and Heating temperature is 50 ℃, and be 50 minutes heat-up time.The issuable precipitation of centrifugal removal after decolouring.And the ultrafiltrated that is less than 100000 for molecular weight adds granular active carbon or the powdered active carbon of 10g/L, at 80 ℃, keep 30 minutes, after decolouring, filter.Then above-mentioned two kinds of destainers are mixed.Add 95% alcohol, make the ethanol content in total solution reach 20%, the polymer Propiram that centrifugal removal molecular weight is greater than 100000; And then add alcohol to make the ethanol content of solution reach 70%, the precipitation of centrifugal collection molecular weight between 10000-100000.Be placed in vacuum drying oven, dry 24h under 0.1MPa.With pulverizer, pulverize.
Embodiment 2:
Aureobasidium pullulans is through conventional seed culture (slant culture, shake-flask culture), top fermentation tank fermentation culture more than 4 days then.Fermented liquid is heated 20 minutes at 80 ℃.Then adding 2% concentration is 10%(w/v) inorganic flocculating agent (aluminum chloride), standing 8-10h.Centrifugal under 3000r/ minute.Supernatant liquor regulates pH to 10.0, and heating and temperature control is at 85 ℃, and adding saturation ratio is the sodium-chlor of 60% left and right, more than heat-up time 1h.Again with whizzer, the liquid of saltouing is carried out centrifugally, remove precipitation wherein.Clear liquid after filtration adds appropriate distilled water, is mixed with containing polysaccharide 5%(w/v) solution, be then placed in ultrafiltration apparatus ultrafiltration under the pressure of 0.5MPa.The ultra-filtration membrane of selecting is 0.001 μ m and 0.005 μ m, and molecular weight cut-off is respectively: 10000 and 100000.After ultrafiltration, obtain the polysaccharide of three kinds of different molecular weights, the pulullan polysaccharide that molecular weight is greater than 100000; Molecular weight is at the pulullan polysaccharide of 10000-100000, the polysaccharide that molecular weight is less than 10000.Molecular weight is less than 10000 pulullan polysaccharide discarded (raw material that also can be used for producing oligose fully utilizes).The ultrafiltrated that is greater than 100000 for molecular weight, regulates pH to 7.0, and hydrogen peroxide addition is 3.0%, and Heating temperature is 40 ℃, and be 40 minutes heat-up time.The issuable precipitation of centrifugal removal after decolouring.And the ultrafiltrated that is less than 100000 for molecular weight adds granular active carbon or the powdered active carbon of 10g/L, at 60 ℃, keep 30 minutes, after decolouring, filter.Then above-mentioned two kinds of destainers are mixed.Add 95% alcohol, make the ethanol content in total solution reach 20%, the polymer Propiram that centrifugal removal molecular weight is greater than 100000; And then add alcohol to make the ethanol content of solution reach 70%, the precipitation of centrifugal collection molecular weight between 10000-100000.Be placed in vacuum drying oven, dry 24h under 0.1MPa.With pulverizer, pulverize.
Embodiment 3:
Aureobasidium pullulans is through conventional seed culture (slant culture, shake-flask culture), top fermentation tank fermentation culture more than 4 days then.Fermented liquid is heated 25 minutes at 70 ℃.Then adding 1.5% concentration is 10%(w/v) inorganic flocculating agent (aluminum chloride), standing 9h.Centrifugal under 3500 r/ minutes.Supernatant liquor regulates pH to 9.5, and heating and temperature control is at 87 ℃, and adding saturation ratio is the sodium-chlor of 60% left and right, more than heat-up time 1h.Again with whizzer, the liquid of saltouing is carried out centrifugally, remove precipitation wherein.Clear liquid after filtration adds appropriate distilled water, is mixed with containing polysaccharide 5%(w/v) solution, be then placed in ultrafiltration apparatus ultrafiltration under the pressure of 0.5MPa.The ultra-filtration membrane of selecting is 0.001 μ m and 0.005 μ m, and molecular weight cut-off is respectively: 10000 and 100000.After ultrafiltration, obtain the polysaccharide of three kinds of different molecular weights, the pulullan polysaccharide that molecular weight is greater than 100000; Molecular weight is at the pulullan polysaccharide of 10000-100000, the polysaccharide that molecular weight is less than 10000.Molecular weight is less than 10000 pulullan polysaccharide discarded (raw material that also can be used for producing oligose fully utilizes).The ultrafiltrated that is greater than 100000 for molecular weight, regulates pH to 8.0, and hydrogen peroxide addition is 2.5%, and Heating temperature is 45 ℃, and be 45 minutes heat-up time.The issuable precipitation of centrifugal removal after decolouring.And the ultrafiltrated that is less than 100000 for molecular weight adds granular active carbon or the powdered active carbon of 10g/L, at 70 ℃, keep 30 minutes, after decolouring, filter.Then above-mentioned two kinds of destainers are mixed.Add 95% alcohol, make the ethanol content in total solution reach 20%, the polymer Propiram that centrifugal removal molecular weight is greater than 100000; And then add alcohol to make the ethanol content of solution reach 70%, the precipitation of centrifugal collection molecular weight between 10000-100000.Be placed in vacuum drying oven, dry 24h under 0.1MPa.With pulverizer, pulverize.
Claims (3)
1. an extracting method for pulullan polysaccharide, is characterized in that, comprises the following steps:
(a) fermentation: Aureobasidium pullulans is through seed culture, and then top fermentation tank fermentation culture is more than 4 days;
(b) thermal treatment: fermented liquid is heated to 20-30 minute at 60-80 ℃;
(c) Flocculation: the inorganic flocculating agent that the mass concentration that adds 1%-2% in heat treated fermented liquid is 10%, standing 8-10h;
(d) flocculate centrifugal: it is centrifugal under 3000-4000 r/min that the fermented liquid that Flocculation is crossed is placed in whizzer;
(e) saltout except albumen: the clear liquid by flocculation after centrifugal regulates pH to 9.0-10.0, and heating and temperature control is at 85-90 ℃, and adding saturation ratio is the sodium-chlor of 60% left and right, more than heat-up time 1h;
(f) saltout centrifugal: with whizzer, the liquid of saltouing is carried out centrifugally, removes precipitation wherein;
(g) ultra-filtration and separation: the clear liquid after centrifugal adds distilled water, the solution that to be mixed with containing polysaccharide mass concentration be 5%, is then placed in ultrafiltration apparatus ultrafiltration under the pressure of 0.5Mpa; The ultra-filtration membrane of selecting is 0.001 μ m and 0.005 μ m, and molecular weight cut-off is respectively: 10000 and 100000; After ultrafiltration, obtain the polysaccharide of three kinds of different molecular weights: molecular weight is greater than 100000 pulullan polysaccharide, the polysaccharide that pulullan polysaccharide, the molecular weight of molecular weight between 10000-100000 is less than 10000; It is discarded or for the production of the raw material of oligose that molecular weight is less than 10000 pulullan polysaccharide;
(h) macromolecular polysaccharide decolouring: the pulullan polysaccharide employing hydrogen peroxide oxidation process decolouring that is greater than 100000 for molecular weight;
(i) micromolecular polysaccharide decolouring: the pulullan polysaccharide for molecular weight between 10000-100000 adopts absorption method decolouring;
(j) mix: two kinds of destainers in step h and step I are mixed;
(k) fractionation precipitation: add 95% alcohol in mixed destainer, make the ethanol content in total solution reach 20%, under this ethanol concn, the pulullan polysaccharide of ultra-high molecular weight can form precipitation, uses centrifugal removal; Supernatant liquor adds alcohol to make the ethanol content of solution reach 70% again, the Propiram precipitation of centrifugal collection molecular weight between 10000-100000, and molecular weight is less than 10000 Propiram oligose and can not precipitates under this ethanol concn, adopt alcohol according to molecular weight, to carry out separation to pulullan polysaccharide;
(l) vacuum-drying: precipitation obtained above is placed in to vacuum drying oven, dry 24h under 0.1MPa;
(m) pulverize: dried Propiram is pulverized with pulverizer.
2. the extracting method of pulullan polysaccharide according to claim 1, it is characterized in that: while decolouring in described step h macromolecular polysaccharide bleaching process, regulate between fermented liquid pH to 7.0-9.0, hydrogen peroxide addition is 2.0%-3.0%, Heating temperature is 40-50 ℃, and be 40-50 minute heat-up time; The carrying out that rising temperature, increase concentration of hydrogen peroxide can be accelerated decolouring; Centrifugal after decolouring, then solution is heated to 90 ℃, keep 10 minutes; Under above-mentioned oxidizing intensity, wherein the Propiram degradable of most of high molecular is less than 100000 to molecular weight.
3. the extracting method of pulullan polysaccharide according to claim 1 and 2, is characterized in that: in described step I micromolecular polysaccharide bleaching process, add gac in fermented liquid, at 60-80 ℃, keep 30 minutes, after decolouring, filter.
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| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
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| CN112409506A (en) * | 2020-11-25 | 2021-02-26 | 山东福瑞达生物科技有限公司 | Method for preparing pullulan polysaccharides with different uniform molecular weights |
| CN114034595A (en) * | 2021-11-08 | 2022-02-11 | 江苏力凡胶囊有限公司 | Method for determining content of pullulan |
| CN115521959A (en) * | 2022-10-12 | 2022-12-27 | 江苏力凡胶囊有限公司 | Extraction method of pullulan |
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