CN1900299A - Process for preparing novel fungus extracellular amylose with anti-tumor and immunity regulating activity - Google Patents
Process for preparing novel fungus extracellular amylose with anti-tumor and immunity regulating activity Download PDFInfo
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- CN1900299A CN1900299A CN 200610047234 CN200610047234A CN1900299A CN 1900299 A CN1900299 A CN 1900299A CN 200610047234 CN200610047234 CN 200610047234 CN 200610047234 A CN200610047234 A CN 200610047234A CN 1900299 A CN1900299 A CN 1900299A
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Abstract
The present invention is process of preparing fungus extracellular polysaccharide with antitumor and immunity regulating activity, and relates to bioengineering. The liquid culture medium for Agaricus blazei murrill includes cane sugar 2-6 wt%, bean dregs 0.5-1.5 wt%, wheat bran 0.1-0.5 wt% and growth factor 0.1 wt%. The culture conditions include pH 5.0-6.5, temperature of 25-32 deg.c, rotation speed of the shaking table 100-160 rpm, liquid amount of 25-100 ml/250ml, and culture period of 5-7 days. Through liquid culturing of Agaricus blazei murrill in the liquid culture medium, separating extracellular polysaccharide, purifying polysaccharide and detecting activity, fermented liquid with great amount of active matters and rich polysaccharides is obtained. The fermented liquid with great amount of active matters and rich polysaccharides may be used in producing medicines and functional foods and in preparing immunoregulating injection and orally taken preparation.
Description
Technical field
The present invention relates to a kind of biotechnology, particularly relate to preparation method antitumor outside a kind of fungi born of the same parents, the immunoregulatory activity polysaccharide.
Background technology
Agaricus blazei Murrill (Agaricus blazei Murrill) is a kind of food medicine dual-purpose basidiomycetes, belongs to Agaricus edibilis, and Agaricus contains biologically active substances such as polysaccharide, phytohemagglutinin, nucleic acid-albumen composition, sterols material in its sporophore and the mycelium.At present, the mycelium polysaccharides that the research of Agaricus Blazei Murrill polysaccharide is cultivated based on fruitbody polysaccharide or liquid state mostly, and less to its polysaccharide of fermentation broth research.
The main biological activity of Agaricus Blazei Murrill polysaccharide is immunomodulatory, anti-tumor activity, is ideal pharmaceutical raw material and functional foodstuff.But because cultivation condition requires high factor, its sporophore supply can not be met the need of market far away.Because but liquid submerged fermentation has industrial and annual production with short production cycle, is convenient to advantage such as management, for Agaricus blazei Murrill can also overcome culture cycle reach about three months, comparatively harsh and solid state cultivation biology efficient is low to cultivation condition requirements such as humitures, the output shakiness, unfavorable factors such as more take place in disease and pest.Therefore, adopting the method production Agaricus Blazei Murrill polysaccharide of liquid submerged fermentation is solution.This method also can obtain to contain the fermented liquid of a large amount of active substances except that can producing the mycelium similar to Agaricus blazei Murrill sporophore nutritive value, wherein Feng Fu polysaccharose substance is fully studied at present as yet.With bioactive polysaccharide in the fermented liquid serves as that medicine, the functional foodstuff that the basis produces will have huge market potential.
Summary of the invention
The object of the present invention is to provide preparation method antitumor outside a kind of novel fungitype born of the same parents, the immunoregulatory activity polysaccharide, utilize the liquid state fermentation substratum that the Agaricus blazei Murrill bacterial strain is carried out liquid culture, exocellular polysaccharide separation, polysaccharide purification, the active detection.Can be used as injection or oral immunity and regulate the raw material of preparation.
The objective of the invention is to be achieved through the following technical solutions:
A kind of novel fungitype born of the same parents are antitumor outward, the preparation method of immunoregulatory activity polysaccharide, and its Agaricus blazei Murrill liquid culture based formulas and culture condition are:
Culture medium prescription: sucrose 2-6%, dregs of beans 0.5-1.5%, wheat bran 0.1-0.5%, somatomedin 0.1%, pH5.0-6.5;
Culture condition is: culture temperature 25-32 ℃, shaking speed 100-160rpm, liquid amount are 25-100ml/250ml, incubation time 5-7d.
Aforesaid a kind of novel fungitype born of the same parents are antitumor outward, the preparation method of immunoregulatory activity polysaccharide, when utilizing fermentor tank to carry out submerged fermentation, help the production of exocellular polysaccharide with pH control fermentation; At tank pressure 0.3 ~ 0.5Mpa, stirring velocity 60-100rpm, air flow quantity 0.4-1.0v/vmin, pH is controlled at 5.0-6.5.
Aforesaid a kind of novel fungitype born of the same parents are antitumor outward, the preparation method of immunoregulatory activity polysaccharide, according to the outer structure antitumor, the immunoregulatory activity polysaccharide of the fungi born of the same parents of method for preparing be: the ratio of glucose and seminose is 3: 1 in the molecule, its main chain is connected together by β-(1 → 6) glycosidic link by 3 glucose molecules, be connected with seminose with α-(1 → 6) or β-(1 → 6) and constitute, its structure is as follows:
Advantage of the present invention and effect are:
Fungi born of the same parents of the present invention are antitumor outward, the preparation method of immunoregulatory activity polysaccharide, utilize the liquid state fermentation substratum that the Agaricus blazei Murrill bacterial strain is carried out liquid culture, exocellular polysaccharide separation, polysaccharide purification, the active detection.Can obtain to contain the fermented liquid of a large amount of active substances, abundant polysaccharose substance.With bioactive polysaccharide in the fermented liquid serves as that medicine, the functional foodstuff that the basis produces will have huge market potential.Can be used as injection or oral immunity and regulate the raw material of preparation.
Description of drawings:
Fig. 1 is the high-efficient liquid phase chromatogram of polysaccharide hydrolysis liquid of the present invention;
Fig. 2 is the infrared spectra of polysaccharide of the present invention;
Fig. 3 is the 13C-NMR spectrogram of polysaccharide of the present invention;
Fig. 4 is the 1H-NMR spectrogram of polysaccharide of the present invention.
Embodiment
Agaricus blazei Murrill liquid culture or fermentation
Culture medium prescription: sucrose 2-6%, dregs of beans 0.5-1.5%, wheat bran 0.1-0.5%, somatomedin 0.1%, pH5.0-6.5; Optimal culture conditions is: culture temperature 25-32 ℃, shaking speed 100-160rpm, liquid amount are 25-100ml/250ml, incubation time 5-7d.With this understanding, mycelial biomass of Agaricus blazei Murrill and exopolysaccharides can reach 14.2g/L, 1.35g/L respectively.
When carrying out the fermentor tank submerged fermentation, help the production of exocellular polysaccharide with pH control fermentation.At tank pressure 0.3 ~ 0.5Mpa, stirring velocity 60-100rpm, air flow quantity 0.4-1.0v/vmin, pH are controlled under the condition of 5.0-6.5, and biomass and exopolysaccharides can reach 23.9g/L, 2.4g/L respectively.
Exocellular polysaccharide separates
Mycelium is removed in centrifugal or filtration with the Agaricus blazei Murrill fermented liquid, and the fermented liquid concentrating under reduced pressure adds cold ethanol precipitation, draws Crude polysaccharides.
Polysaccharide purification
After Crude polysaccharides process Sevag method deproteinated, H2O2 decolouring, dialysis, DEAE-cellulose ion-exchange chromatography, the Sephadex G-200 gel-filtration, obtain holosaccharide.
As shown in the figure
Fig. 1: by the peak area of this polysaccharide complete hydrolysis liquid as can be known, the ratio of glucose and seminose is about 3: 1 in this polysaccharide molecule.
Fig. 2: this polysaccharide has absorption peak at the 3406.75cm-1 place, is the stretching vibration of O-H key, shows that polysaccharide exists intramolecularly and intermolecular hydrogen bond; Strong absorption peak at the 2929.50cm-1 place shows the stretching vibration that the carbohydrate c h bond is arranged in the molecule; 1647.38cm-1 and the 1422.66cm-1 place is the charateristic avsorption band of polyose; Absorption peak between 1149cm-1～1017cm-1 is the absorption peak of carbohydrate C-O key; The charateristic avsorption band that seminose is arranged at the 810cm-1 place; The charateristic avsorption band that glucose is arranged at the 749.99cm-1 place; 573.70cm-1 the strong absorption peak at the absorption peak the between～416.56cm-1, especially 416.56cm-1 place is the absorption peak of pyranoid ring carbon skeleton.Do not have absorption between 930cm-1～950cm-1, there is absorption at the 890cm-1 place, and illustrating has α type and β type glycosidic link in the molecule.
Fig. 3: the main chemical shift of resonance signal is less than 5.0ppm, and chemical shift also has absorption peak greater than 5.0 places, shows in the molecule also to contain α type glycosidic link based on β type glycosidic link.The δ value at the 4.95ppm place, there is absorption peak at 5.2～5.4ppm place, showing has glucose and seminose in the molecule.
Fig. 4: the δ value is that 100.3ppm, 101.9ppm place have absorption peak, shows and contains glucose and seminose in the molecule; The δ value is not have near the 86.52ppm to absorb, and shows that no C3 replaces; δ has absorption in 70～78ppm scope, show that no C4 replaces; δ has absorption near 60.6ppm, 60.0ppm, show not exist C6 to replace; And the displacement that the absorption peak at 68.8ppm place is C6 after replacing shows that part C6 is substituted in the molecule, and another part is not substituted.
Comprehensive above monosaccharide component HPLC analysis, Infrared spectroscopy and nuclear magnetic resonance spectroscopy result, the ratio of glucose and seminose is 3: 1 in this polysaccharide molecule as can be known, its main chain is connected together by β-(1 → 6) glycosidic link by 3 glucose molecules, is connected with seminose with α-(1 → 6) or β-(1 → 6) and constitutes.
Claims (3)
1. the preparation method of outer antitumor, the immunoregulatory activity polysaccharide of novel fungitype born of the same parents is characterized in that Agaricus blazei Murrill liquid culture based formulas and culture condition are:
Culture medium prescription: sucrose 2-6%, dregs of beans 0.5-1.5%, wheat bran 0.1-0.5%, somatomedin 0.1%, pH5.0-6.5;
Culture condition is: culture temperature 25-32 ℃, shaking speed 100-160rpm, liquid amount are 25-100ml/250ml, incubation time 5-7d.
2. a kind of novel fungitype born of the same parents according to claim 1 are antitumor outward, the preparation method of immunoregulatory activity polysaccharide, it is characterized in that when utilizing fermentor tank to carry out submerged fermentation, help the production of exocellular polysaccharide with pH control fermentation; At tank pressure 0.3 ~ 0.5Mpa, stirring velocity 60-100rpm, air flow quantity 0.4-1.0v/vmin, pH is controlled at 5.0-6.5.
3. a kind of novel fungitype born of the same parents according to claim 1 are antitumor outward, the preparation method of immunoregulatory activity polysaccharide, it is characterized in that according to the outer structure antitumor, the immunoregulatory activity polysaccharide of the fungi born of the same parents of method for preparing being: the ratio of glucose and seminose is 3: 1 in the molecule, its main chain is connected together by β-(1 → 6) glycosidic link by 3 glucose molecules, be connected with seminose with α-(1 → 6) or β-(1 → 6) and constitute, its structure is as follows:
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831471B (en) * | 2010-05-12 | 2012-10-17 | 浙江省农业科学院 | Method for producing polysaccharides by fermenting yellow serofluid with edible and medicinal fungi |
| CN102786339A (en) * | 2011-05-17 | 2012-11-21 | 吉林农业大学 | Liquid culture medium suitable for tricholoma matsutake fermentation |
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2006
- 2006-07-19 CN CN 200610047234 patent/CN1900299A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831471B (en) * | 2010-05-12 | 2012-10-17 | 浙江省农业科学院 | Method for producing polysaccharides by fermenting yellow serofluid with edible and medicinal fungi |
| CN102786339A (en) * | 2011-05-17 | 2012-11-21 | 吉林农业大学 | Liquid culture medium suitable for tricholoma matsutake fermentation |
| CN102786339B (en) * | 2011-05-17 | 2014-04-09 | 吉林农业大学 | Liquid culture medium suitable for tricholoma matsutake fermentation |
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