CN102936609A - Preparation method of swift moth paecilomyces varioti extracellular acid glycoprotein - Google Patents
Preparation method of swift moth paecilomyces varioti extracellular acid glycoprotein Download PDFInfo
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- CN102936609A CN102936609A CN2012104340925A CN201210434092A CN102936609A CN 102936609 A CN102936609 A CN 102936609A CN 2012104340925 A CN2012104340925 A CN 2012104340925A CN 201210434092 A CN201210434092 A CN 201210434092A CN 102936609 A CN102936609 A CN 102936609A
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Abstract
The invention relates to a preparation method of swift moth paecilomyces varioti extracellular acid glycoprotein. The method includes culturing first-level seeds of swift moth paecilomyces varioti bacterial strains, conducting inoculated fermentation to obtain swift moth paecilomyces varioti fermentation liquid, conducting work procedures including ethanol precipitation, deionized water redissolution, deproteinization, freezing, drying, diethyl-aminoethanol (DEAE)-52 column chromatography and the like on the fermentation liquid to extract the swift moth paecilomyces varioti extracellular acid glycoprotein. The preparation method has the advantages of being simple in process, mild in extraction condition, low in cost, environment-friendly, apt to industrial production and the like. The obtained swift moth paecilomyces varioti extracellular acid glycoprotein can serve as raw materials of medicines and health food.
Description
Technical field
The present invention relates to separate the technical field that obtains acid glycoprotein outside the born of the same parents with bat moth bacterium Paecilomyces varioti bacterial strain liquid fermenting and from its fermented liquid, be specifically related to the preparation method of the outer acid glycoprotein of a kind of peacilomyce hepiahi born of the same parents.
Background technology
Cordyceps sinensis is traditional famous and precious strong, tonic herb of China, and it is that for human being's production and the real huge value of life they can produce the multiple physiologically active substance that important value is arranged in biotechnology and medicines and health protection.Their physiologically active is rich and varied, can be antibiotic, antitumor, antiplatelet condenses, radioprotective, improve memory, regulate body immunity, calcium ion antagonism, brain and heart are had provide protection, calmness and analgesia etc. under normobaric hypoxia.
Because the parasitics that natural cordyceps is strict and special ecotope requirement, its resource is extremely limited, and is expensive.In recent years, along with the Anamorph of Cordyceps Sinensis progress of research, the research of relevant Anamorph of Cordyceps polysaccharide is just carried out.Cordyceps sinensis can carry out fermentative production by bionic method, Cordyceps mycelium to artificial culture, through chemistry, pharmacological research, proof is basically identical with natural cs, through clinical application for many years, be known as the substitute that can be used as Cordyceps sinensis by the world of medicine, the People's Republic of China's existing clear and definite regulation of version pharmacopeia in 2000.Simultaneously, the also various bioactivators such as the trace element such as rich in proteins, VITAMIN, zinc, copper and cordycepic acid (D one N.F,USP MANNITOL), cordycepin (3 '-deoxyadenosine), polyose, alkaloids, cyclic peptide class, SOD enzyme in the Anamorph of Cordyceps fermented liquid.Wherein, polyose is as one of the abundantest most important monoid in the contained physiologically active substance of Cordyceps sinensis, having the different physiological roles such as anti-oxidant, antitumor, hypoglycemic, reducing blood-fat, is an important breakthrough mouth of Cordyceps sinensis development research, has a good application prospect.
Peacilomyce hepiahi (Paecilomyces hepiali) is a kind of of Anamorph of Cordyceps, and this bacterial strain separated in the natural cs sporophore of Qinghai Province's Yushu Regions in 2002, and was identified by DSMZ of the Chinese Academy of Sciences.Because the popularity that Chinese caterpillar fungus distributes and the diversity of anamorphic strain, there is very big-difference in different scientific research institutions to the research of anamorph Chinese caterpillar fungus, and there is not yet report about the research that utilizes this strain liquid fermentation and separate acid glycoprotein outside the born of the same parents from fermented liquid.
Summary of the invention
The object of the invention aims to provide a kind of method for preparing the outer acid glycoprotein of peacilomyce hepiahi born of the same parents, realizes that concrete scheme of the present invention is as follows:
(1) (size is 0.5cm * 0.5cm) to take the mycelium piece the mother on being stored in the potato slope substratum plants, be inoculated in (every bottled seed culture medium 100mL of 250mL triangle) in the seed culture medium, static cultivations are after 12 hours under 17 ℃, shaking culture was cultivated 72 hours under the 120rpm, can obtain seed liquor.Seed culture medium (g/L): sucrose 30.00, yeast extract paste 6.00, potassium primary phosphate 1.50, sal epsom 0.50, wort 10.00, murphy juice 10.00, pH 6.5.
The seed liquor of (2) getting in the step (1) is inoculated into (every bottled fermention medium 200mL of 500mL triangle) in the fermention medium, inoculum size is 1% (V/V), static cultivations are after 12 hours under 17 ℃, shaking culture was cultivated after 120 hours under the 120rpm, tunning centrifugation (centrifugation condition: 4000rmp, 15min) is obtained the peacilomyce hepiahi fermented liquid.Fermentative medium formula (g/L): sucrose 50.86, yeast extract paste 2.00, ammonium sulfate 6.61, potassium primary phosphate 1.36, sal epsom 0.05, murphy juice 15.00, wort 15.00, pH 6.5;
(3) with the peacilomyce hepiahi fermented liquid that obtains in the step (2), 50 ℃ are evaporated to 1/2 of original volume, the dehydrated alcohol that adds 3 times of volumes, precipitate 12 hours, after 80% washing with alcohol 3 times, centrifugation (centrifugation condition: 4000rmp, 15min) obtains crude extracellular polysaccharide.
(4) crude extracellular polysaccharide that step (3) made ventilates and flings to ethanol, after adding a small amount of deionized water and redissolving, with Sevag method (chloroform: propyl carbinol=4: 1) behind the deproteinated 3 times, the aqueous phase solution lyophilize is obtained the Crude polysaccharides freezing dry powder.
(5) with after the dissolving of step (4) gained Crude polysaccharides, be splined on the DEAE-52 cellulose column, and carry out wash-out with deionized water, 0.1mol/LNaCl and 0.3mol/L NaCl respectively, the 0.3mol/L NaCl elution fraction of collecting is carried out concentrating under reduced pressure, deionized water dialysis 72 hours, concentrated postlyophilization namely obtains the outer acid glycoprotein component of born of the same parents.The outer acid glycoprotein of the born of the same parents of preparation is pale powder, and wherein polysaccharide content 80.56%, protein content 18.76%.
(6) the outer acid glycoprotein of step (5) gained born of the same parents is detected its molecular weight distribution situation with molecular-exclusion chromatography, form with the Infrared spectroscopy characteristic group, determine with uv scan whether it has protein component.
2, require 1 described a kind of preparation method with the outer acid glycoprotein of biological activity born of the same parents according to patent, it is characterized in that: the fermentative medium formula (g/L) of described step (2): sucrose 50.86, yeast extract paste 2.00, ammonium sulfate 6.61, potassium primary phosphate 1.36, sal epsom 0.05, murphy juice 15.00, wort 15.00, pH 6.5.
The outer acid glycoprotein of the peacilomyce hepiahi born of the same parents of preparation is pale powder, and wherein polysaccharide content 80.56%, protein content 18.76%, the easily moisture absorption.The infrared absorption spectrum of this acid glycoprotein shows that it has polysaccharide, carboxyl, sulfate and amino charateristic avsorption band.The uv-absorbing scanning of the outer acid glycoprotein of these born of the same parents shows that it contains the absorption peak of albumen.Molecular weight to the outer glycoprotein of these born of the same parents is measured, and shows that its molecular weight distribution is between 20-80kD.
Description of drawings
Fig. 1 substratum forms the impact on the outer acid glycoprotein output of peacilomyce hepiahi born of the same parents
The infrared absorption pattern of the outer acid glycoprotein of Fig. 2 peacilomyce hepiahi born of the same parents
The UV scanning collection of illustrative plates of the outer acid glycoprotein sugar of Fig. 3 peacilomyce hepiahi born of the same parents
Embodiment
Below by embodiment, the invention will be further described.
Embodiment 1:
(1) (size is 0.5cm * 0.5cm) to take the mycelium piece the mother on being stored in the potato slope substratum plants, be inoculated in (every bottled seed culture medium 100mL of 250mL triangle) in the seed culture medium, static cultivations are after 12 hours under 17 ℃, shaking culture was cultivated 72 hours under the 120rpm, can obtain seed liquor.Seed culture medium (g/L): sucrose 30.00, yeast extract paste 6.00, potassium primary phosphate 1.50, sal epsom 0.50, wort 10.00, murphy juice 10.00, pH 6.5.
The seed liquor of (2) getting in the step (1) is inoculated into (every bottled fermention medium 200mL of 500mL triangle) in the fermention medium, inoculum size is 1% (V/V), static cultivations are after 12 hours under 17 ℃, shaking culture was cultivated after 120 hours under the 120rpm, tunning centrifugation (centrifugation condition: 4000rmp, 15min) is obtained the peacilomyce hepiahi fermented liquid.Fermentative medium formula (g/L): sucrose 50.86, yeast extract paste 2.00, ammonium sulfate 6.61, potassium primary phosphate 1.36, sal epsom 0.05, murphy juice 15.00, wort 15.00, pH 6.5;
(3) with the peacilomyce hepiahi fermented liquid that obtains in the step (2), 50 ℃ are evaporated to 1/2 of original volume, the dehydrated alcohol that adds 3 times of volumes, precipitate 12 hours, after 80% washing with alcohol 3 times, centrifugation (centrifugation condition: 4000rmp, 15min) obtains crude extracellular polysaccharide.
(4) crude extracellular polysaccharide that step (3) made ventilates and flings to ethanol, after adding a small amount of deionized water and redissolving, with Sevag method (chloroform: propyl carbinol=4: 1) behind the deproteinated 3 times, the aqueous phase solution lyophilize is obtained the Crude polysaccharides freezing dry powder.
(5) with after the dissolving of step (4) gained Crude polysaccharides, be splined on the DEAE-52 cellulose column, and carry out wash-out with deionized water, 0.1mol/LNaCl and 0.3mol/L NaCl respectively, the 0.3mol/L NaCl elution fraction of collecting is carried out concentrating under reduced pressure, deionized water dialysis 72 hours, concentrated postlyophilization namely obtains the outer acid glycoprotein component of born of the same parents.The outer acid glycoprotein of the born of the same parents of preparation is pale powder, and wherein polysaccharide content 80.56%, protein content 18.76%.
(6) the outer acid glycoprotein of step (5) gained born of the same parents is detected its molecular weight distribution situation with molecular-exclusion chromatography, form with the Infrared spectroscopy characteristic group, determine with uv scan whether it has protein component.
Embodiment 2:
The fermentation condition optimization experiment of the outer acid glycoprotein of peacilomyce hepiahi born of the same parents
(1) the single Factor screening experiment of the fermentation condition optimization of the outer acid glycoprotein of peacilomyce hepiahi born of the same parents
1. the some factors design is to the screening of the remarkable factor of fermentation condition.
The outer acid glycoprotein fermentation condition factor of peacilomyce hepiahi born of the same parents and code are: glucose (X
1), Semen Maydis powder (X
2), yeast extract paste (X
3), ammonium sulfate (X
4), KH
2PO
4(X
5), MgSO
4(X
6), murphy juice (X
7), wort (X
8) central point (0) be respectively (g/L): 30,10,2,6,1,0.5,10,10, step-length is respectively: 10,5,1,1,0.5,0.2,5,5.The output of the outer acid glycoprotein of the peacilomyce hepiahi born of the same parents of 20 groups of tests sees Table 1.
Table 1 fermention medium significance conditional filtering
Carry out variance analysis through Design-Expert 7.0 softwares, obtain table 2:
The variance analysis of table 2 fractional factorial design
As can be seen from Table 2, sucrose (X
1), yeast extract paste (X
3), ammonium sulfate (X
4) and potassium primary phosphate (X5) be the significance influence factors of the outer acid glycoprotein output of peacilomyce hepiahi born of the same parents.
But from table, can't judge sucrose (X
1), yeast extract paste (X
3) and ammonium sulfate (X
4) and potassium primary phosphate (X5) to the remarkable sex concentration central point of the outer acid glycoprotein yield of peacilomyce hepiahi born of the same parents.In order further to optimize sucrose (X
1), yeast extract paste (X
3) and ammonium sulfate (X
4) and potassium primary phosphate (X
5) optimum concn and interaction, the present invention adopts the response surface method further to test sucrose (X
1), yeast extract paste (X
3) and ammonium sulfate (X
4) and potassium primary phosphate (X
5) optimum concn.
2. the response surface method is optimized the outer acid glycoprotein fermentation condition result of peacilomyce hepiahi high yield born of the same parents
Sucrose (X
1), yeast extract paste (X
3) and ammonium sulfate (X
4) and potassium primary phosphate (X
5) central point be respectively (g/L): 50,3,6 and 1.0.Response surface test design and the results are shown in Table 3.According to table 3 result, obtain multiple regression equation with Design EXpert7.0 software analysis and be:
Y＝-37.99+1.84X
1-0.14X
3+1.19X
4+3.53X
5-0.014X
1 2-0.97X
3 2-0.04X
4 2-0.79X
5 2+0.085X
1X
3-0.093X
1X
4-0.018X
1X
5
Obtaining optimal conditions of fermentation according to Equation for Calculating is: the outer acid glycoprotein fermentative medium formula (g/L) of peacilomyce hepiahi high yield born of the same parents: sucrose 50.86, yeast extract paste 2.00, ammonium sulfate 6.61, potassium primary phosphate 1.36, sal epsom 0.05, murphy juice 15.00, wort 15.00, pH 6.5, and the outer acid glycoprotein production peak of born of the same parents can reach 5.33g/L.
The test of table 3 response surface and fermentation condition optimization result
Claims (2)
1. the preparation method of the outer acid glycoprotein of peacilomyce hepiahi born of the same parents is characterized in that comprising following operation steps:
(1) (size is 0.5cm * 0.5cm) to take the mycelium piece from the mother who is stored in the potato slope substratum plants, be inoculated in (every bottled seed culture medium 100mL of 250mL triangle) in the seed culture medium, static cultivations are after 12 hours under 17 ℃, shaking culture was cultivated 72 hours under the 120rpm, can obtain seed liquor.Seed culture medium (g/L): sucrose 30.00, yeast extract paste 6.00, potassium primary phosphate 1.50, sal epsom 0.50, wort 10.00, murphy juice 10.00, pH6.5.
The seed liquor of (2) getting in the step (1) is inoculated into (every bottled fermention medium 200mL of 500mL triangle) in the fermention medium, inoculum size is 1% (V/V), static cultivations are after 12 hours under 17 ℃, shaking culture was cultivated after 120 hours under the 120rpm, tunning centrifugation (centrifugation condition: 4000rmp, 15min) is obtained the peacilomyce hepiahi fermented liquid.Fermentative medium formula (g/L): sucrose 50.86, yeast extract paste 2.00, ammonium sulfate 6.61, potassium primary phosphate 1.36, sal epsom 0.05, murphy juice 15.00, wort 15.00, pH6.5;
(3) with the peacilomyce hepiahi fermented liquid that obtains in the step (2), 50 ℃ are evaporated to 1/2 of original volume, the dehydrated alcohol that adds 3 times of volumes, precipitate 12 hours, after 80% washing with alcohol 3 times, centrifugation (centrifugation condition: 4000rmp, 15min) obtains crude extracellular polysaccharide.
(4) crude extracellular polysaccharide that step (3) made ventilates and flings to ethanol, after adding a small amount of deionized water and redissolving, with Sevag method (chloroform: propyl carbinol=4: 1) behind the deproteinated 3 times, the aqueous phase solution lyophilize is obtained the Crude polysaccharides freezing dry powder.
(5) with after the dissolving of step (4) gained Crude polysaccharides, be splined on the DEAE-52 cellulose column, and carry out wash-out with deionized water, 0.1mol/LNaCl and 0.3mol/L NaCl respectively, the 0.3mol/L NaCl elution fraction of collecting is carried out concentrating under reduced pressure, deionized water dialysis 72 hours, concentrated postlyophilization namely obtains the acid glycoprotein component.The acid glycoprotein of preparation is pale powder, and wherein polysaccharide content 80.56%, protein content 18.76%.
(6) step (5) gained acid glycoprotein is detected its molecular weight distribution situation with molecular-exclusion chromatography, form with the Infrared spectroscopy characteristic group, determine with uv scan whether it has protein component.
2. require the preparation method of the outer acid glycoprotein of 1 described a kind of peacilomyce hepiahi born of the same parents according to patent, it is characterized in that: the fermentative medium formula (g/L) of described step (2): sucrose 50.86, yeast extract paste 2.00, ammonium sulfate 6.61, potassium primary phosphate 1.36, sal epsom 0.05, murphy juice 15.00, wort 15.00, pH6.5.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104762341A (en) * | 2015-04-02 | 2015-07-08 | 河南科技学院 | Preparation method of low-molecular-weight paecilomyces hepialid exopolysaccharides |
| CN104911109A (en) * | 2015-05-28 | 2015-09-16 | 吉林大学 | High-yield paecilomyces hepiali mutant strain and cultivation method |
| CN105147715A (en) * | 2015-06-15 | 2015-12-16 | 河南科技学院 | Novel application of neutral extracellular polysaccharides of paecilomyces hepiali |
| CN105176845A (en) * | 2015-10-26 | 2015-12-23 | 河南科技学院 | Method for preparing paecilomyces hepiali fermentation full-liquid instant particles |
| CN105177050A (en) * | 2015-10-26 | 2015-12-23 | 河南科技学院 | Method for preparing paecilomyces hepiali purpurin |
| CN106008662A (en) * | 2016-05-16 | 2016-10-12 | 南京农业大学 | Method for preparing zinc-rich neutral glycoprotein from bee-collected Chinese wolfberry pollen serving as raw material |
| CN107343889A (en) * | 2016-05-06 | 2017-11-14 | 元龙生技国际股份有限公司 | A kind of peacilomyce hepiahi bacterium fermentation culture medium for being used to treat the 1st type or diabetes B |
| CN111139277A (en) * | 2020-03-03 | 2020-05-12 | 长春工业大学 | Crude polysaccharide and preparation process thereof, health-care oral liquid and preparation method thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762341A (en) * | 2015-04-02 | 2015-07-08 | 河南科技学院 | Preparation method of low-molecular-weight paecilomyces hepialid exopolysaccharides |
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| CN105147715A (en) * | 2015-06-15 | 2015-12-16 | 河南科技学院 | Novel application of neutral extracellular polysaccharides of paecilomyces hepiali |
| CN105147715B (en) * | 2015-06-15 | 2019-01-01 | 河南科技学院 | A kind of new application of Paecilomyces hepiali chen neutrality exocellular polysaccharide |
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| CN107343889A (en) * | 2016-05-06 | 2017-11-14 | 元龙生技国际股份有限公司 | A kind of peacilomyce hepiahi bacterium fermentation culture medium for being used to treat the 1st type or diabetes B |
| CN106008662A (en) * | 2016-05-16 | 2016-10-12 | 南京农业大学 | Method for preparing zinc-rich neutral glycoprotein from bee-collected Chinese wolfberry pollen serving as raw material |
| CN111139277A (en) * | 2020-03-03 | 2020-05-12 | 长春工业大学 | Crude polysaccharide and preparation process thereof, health-care oral liquid and preparation method thereof |
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