CN104177506A - Preparation method of Hirsutella sinensis mycelia polysaccharide - Google Patents
Preparation method of Hirsutella sinensis mycelia polysaccharide Download PDFInfo
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- CN104177506A CN104177506A CN201410341978.4A CN201410341978A CN104177506A CN 104177506 A CN104177506 A CN 104177506A CN 201410341978 A CN201410341978 A CN 201410341978A CN 104177506 A CN104177506 A CN 104177506A
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Abstract
The invention relates to a preparation method of Hirsutella sinensis mycelia polysaccharide, which comprises the following steps: S1: extraction of Hirsutella sinensis mycelia polysaccharide: carrying out superfine pulverization on the raw material Hirsutella sinensis mycelia to obtain superfine powder with the particle size of 7-11 mu m, adding into water, immersing, heating to a boiling state, centrifugating while keeping the micro-boiling state, and taking the supernate to obtain a Hirsutella sinensis mycelia polysaccharide crude extracting solution; S2: concentrating to obtain a concentrated solution; S3: precipitation: adding ethanol while stirring, standing, centrifugating, and collecting the precipitate; and S4: drying: carrying out freeze-drying to obtain the Hirsutella sinensis mycelia polysaccharide. The method is simple and convenient, has the advantages of high efficiency, low cost and high polysaccharide yield and content, and is suitable for large-scale production.
Description
Technical field
The present invention relates to a kind of mycelium polysaccharides in a kind of biological field, be specifically related to a kind of China by the preparation method of hair spore mycelium polysaccharides.
Background technology
China is called again fermented cordyceps hypha Cs-C-Q80 or Hirsutella hepiali Chen et Shen filament by hair spore fermented hypha, is real Cordyceps fungus.The polysaccharide that its mycelium contains high level, this polysaccharide has important effect in protective foods, medicine and other fields, but to this bat moth China, by the extraction method of polysaccharides Shortcomings in hair spore mycelium, is made the extraction yield of polysaccharide lower at present.
Summary of the invention
The object of this invention is to provide a kind of China by a preparation method for hair spore mycelium polysaccharides, the method be a kind of easy, efficient, cost is low, polysaccharide yield and content high, and the method for applicable large-scale production, has overcome some shortcomings of the prior art.
The object of the invention is to be achieved through the following technical solutions:
China is by a preparation method for hair spore mycelium polysaccharides, and described preparation method comprises the following steps:
S1 a: extraction for China's quilt hair spore mycelium polysaccharides: in step S1, China by the concrete grammar of the extraction of hair spore mycelium polysaccharides is: the China's quilt hair spore mycelium of take is raw material, micronizing is powder, join in the water of 15-20 times of raw material weight, soak at room temperature 30-60 minute, reheat to boiling, keep centrifugal after micro-60-120 of boiling minute; Precipitation separation and supernatant liquor, get precipitation and repeat to extract 1～2 time by above-mentioned steps again, gets the supernatant liquor after each centrifugation and merge, and is China by the crude extract of hair spore mycelium polysaccharides;
S2: concentrated: China in step S1 is carried out to underpressure distillation by the crude extract of hair spore mycelium polysaccharides, and the ratio (mass/volume) that is concentrated into liquid and raw material is till 4: 1～2: 1 o'clock, obtains concentrated solution;
S3: precipitation: slowly add ethanol in the concentrated solution described in step S2, limit edged stirs, and described ethanol adds to when ethanol content reaches 70%-80% and stops, and then the standing 12-18 hour of room temperature, centrifugal, abandons supernatant liquor, collecting precipitation;
S4: dry: be dissolved in water in the precipitation described in step S3, add heat extraction ethanol, then carry out lyophilize, obtain a Chinese quilt hair spore mycelium polysaccharides, yield is greater than 20%.
Further preferably, in step S1, by described China, by hair spore mycelium micronizing, to particle diameter, be the ultrafine powder of 10 μ m.
Further preferably, in step S1, after soak at room temperature, reheat to boiling, keep micro-90-105 of boiling minute.
Further preferably, in step S1, the described centrifugal time is 20min.
Further, in step S2, the ratio (mass/volume) that the Chinese crude extract by hair spore mycelium polysaccharides is concentrated into liquid and raw material is till 2: 1 o'clock.
Further, in step S3, described ethanol is dehydrated alcohol.
The invention provides a kind of China by the preparation method of hair spore mycelium polysaccharides, its beneficial effect mainly having is: this preparation method destroys by micronizing the leaching process that cell walls passes through some row again, the Crude polysaccharides yield obtaining is high, and the yield of holosaccharide surpasses 20%; This polysaccharide discharges NO to the strain of RAW264.7 scavenger cell obvious promoter action; In addition, the method be a kind of easy, efficient, cost is low and the method for applicable large-scale production.
Accompanying drawing explanation
With reference to the accompanying drawings the present invention is described in further detail below.
Fig. 1 is that China described in the embodiment of the present invention is by the high-efficient liquid phase chromatogram of hair spore mycelium polysaccharides.
Embodiment
A kind of China described in the embodiment of the present invention is by the preparation method of hair spore mycelium polysaccharides, the specific experiment case of take below illustrates embodiment as example, be to be understood that, specific embodiment described herein is only in order to explain the present invention, be not intended to limit the present invention, in addition, China of the present invention is all referred to that by hair spore mycelium polysaccharides bat moth China is by hair spore mycelium polysaccharides.
Embodiment 1
An extraction for China's quilt hair spore mycelium polysaccharides: the China's quilt hair spore mycelium of take is raw material, by micronizing, particle fineness is pulverized as the ultrafine powder of particle diameter 10 μ m, after micronizing, take the powder after 50g micronizing, add 1000mL distilled water, soak at room temperature 60 minutes, is heated to boiling again, keep micro-and boil 120 minutes, the centrifugal 20min of 8000g; Precipitation separation and supernatant liquor, get precipitation and repeat to extract 1 time by above-mentioned steps again, in this precipitation adding distil water, soak at room temperature, be heated to seethe with excitement, keep centrifugal after micro-boiling, get supernatant liquor, merge the supernatant liquor of two times centrifugal gained, be China by the crude extract of hair spore mycelium polysaccharides.
China is concentrated by hair spore mycelium polysaccharides crude extract: above-mentioned China is evaporated to 100mL by the crude extract of hair spore mycelium polysaccharides in Rotary Evaporators, obtains concentrated solution.
Ethanol precipitation: slowly add dehydrated alcohol in above-mentioned concentrated solution, limit edged stirs, while reaching 70% to ethanol content, standing 12 hours of room temperature, centrifugal ethanol supernatant liquor, the collecting precipitation removed.
Dry: precipitation adds 70mL water dissolution, flings to ethanol on water-bath, put into freeze drier lyophilize after being placed in-20 ℃ of frozen 12h of refrigerator, obtain Chinesely by a hair spore mycelium polysaccharides, this polysaccharide is the Crude polysaccharides that contains trace impurity; China is 20.91% by the yield of hair spore mycelium polysaccharides.
Embodiment 2
An extraction for China's quilt hair spore mycelium polysaccharides: the China's quilt hair spore mycelium of take is raw material, by micronizing, particle fineness is pulverized as the ultrafine powder of particle diameter 9 μ m, take the powder after 50g micronizing, add again 800mL distilled water, soak at room temperature 40 minutes, be heated to boiling, keep micro-and boil 100 minutes, the centrifugal 20min of 8000g; Precipitation separation and supernatant liquor, get precipitation and repeat to extract 1 time by above-mentioned steps again, in this precipitation adding distil water, soak at room temperature, be heated to seethe with excitement, keep centrifugal after micro-boiling, get supernatant liquor, merge the supernatant liquor of two times centrifugal gained, be China by the crude extract of hair spore mycelium polysaccharides.China is concentrated by hair spore mycelium polysaccharides crude extract: above-mentioned China is evaporated to 100mL by the crude extract of hair spore mycelium polysaccharides in Rotary Evaporators, obtains concentrated solution.Ethanol precipitation: slowly add dehydrated alcohol in above-mentioned concentrated solution, limit edged stirs, while reaching 75% to ethanol content, standing 15 hours of room temperature, centrifugal ethanol supernatant liquor, the collecting precipitation removed.Dry: precipitation adds 70mL water dissolution, flings to ethanol on water-bath, put into freeze drier lyophilize after being placed in-20 ℃ of frozen 12h of refrigerator, obtain Chinesely by a hair spore mycelium polysaccharides, this polysaccharide is the Crude polysaccharides that contains trace impurity; China is 20.99% by the yield of hair spore mycelium polysaccharides.
Embodiment 3
In upper table for China of extracting gained by method of the present invention 6 times by the extraction yield of hair spore mycelium polysaccharides and the China by 6 extraction gained of prior art by the extraction yield of hair spore mycelium polysaccharides.
Because the present invention is first by micronizing, China all can be pulverized by the mycelial cell walls of hair spore etc., then by follow-up a series of supplying methods, can more thoroughly more effective China be extracted by the polysaccharide in hair spore mycelium; And prior art is without micronizing, only by ordinary methods such as simple immersions, extract.
Because other impurity of trace is also contained in China after extracting in hair spore mycelium polysaccharides, therefore calculating holosaccharide when (polysaccharide described in the present invention all refers to that China is by hair spore mycelium polysaccharides), to measure absorbancy, again according to typical curve calculation sample concentration, finally release the quality of holosaccharide, finally calculate the content of holosaccharide.
As seen from the above table, the China extracting (is holosaccharide by hair spore mycelium polysaccharides fine work, polysaccharide in correspondence in table) extraction yield is high, all more than 20%, than prior art, improve nearly 10 percentage point, and deviation is smaller in the leaching process of different batches, in actual industrial production, have great importance.
Embodiment 4
Sample preparation: take 5mg according to the prepared China of the present invention by hair spore mycelium polysaccharides, add 1mL HPLC moving phase, fully dissolve, the centrifugal 10min of 13200r/min, supernatant liquor carries out HPLC analysis after the water micro-pore-film filtration of 0.25 μ m.
Chromatographiccondition: analytical column selects TSK PWXL6000 and TSK PWXL3000 gel chromatographic columns series connection post analysis, and moving phase is the NaH containing 0.05mol/L
2pO
4naNO with 0.15mol/L
3solution (pH=7,0.02% sodium azide), flow velocity is 0.5mL/min, chromatographic column temperature is constant in 35 ℃ with column oven; Detector is differential detector; Gained color atlas as shown in Figure 1.
Embodiment 5
Adopt this China by hair spore mycelium polysaccharides stimulated in vitro scavenger cell, to be discharged the determination of activity of nitrogen protoxide (NO), find that this polysaccharide discharges NO to the strain of RAW264.7 scavenger cell and has obvious promoter action, can obviously strengthen the kill capability of engulfing of scavenger cell, thus the anti-tumor capacity of enhancing body.
The present invention is not limited to above-mentioned preferred forms, any modification relevant of the present invention or change that anyone does under enlightenment of the present invention, and every have identical with a application or akin technical scheme, within all dropping on protection scope of the present invention.
Claims (6)
1. China, by a preparation method for hair spore mycelium polysaccharides, is characterized in that: described preparation method comprises the following steps:
S1 a: extraction for China's quilt hair spore mycelium polysaccharides: the China's quilt hair spore mycelium of take is raw material, micronizing be particle diameter in the ultrafine powder of 7～11 μ m, join in the water of 15-20 times of raw material weight soak at room temperature 30-60 minute, reheat to boiling, keep centrifugal after micro-60-120 of boiling minute; Precipitation separation and supernatant liquor, get precipitation and repeat to extract 1～2 time by above-mentioned steps again, gets the supernatant liquor after each centrifugation and merge, and is China by the crude extract of hair spore mycelium polysaccharides;
S2: concentrated: China in step S1 is carried out to underpressure distillation by the crude extract of hair spore mycelium polysaccharides, and the ratio that is concentrated into liquid and raw material is till 4: 1～2: 1 o'clock, obtains concentrated solution;
S3: precipitation: slowly add ethanol in the concentrated solution described in step S2, limit edged stirs, and described ethanol adds to when ethanol content reaches 70%-80% and stops, and then the standing 12-18 hour of room temperature, centrifugal, abandons supernatant liquor, collecting precipitation;
S4: dry: be dissolved in water in the precipitation described in step S3, add heat extraction ethanol, then carry out lyophilize, obtain a Chinese quilt hair spore mycelium polysaccharides, yield is greater than 20%.
2. China according to claim 1, by the preparation method of hair spore mycelium polysaccharides, is characterized in that: in step S1, by described China, by hair spore mycelium micronizing, be the ultrafine powder of 10 μ m to particle diameter.
3. China according to claim 1, by the preparation method of hair spore mycelium polysaccharides, is characterized in that: in step S1, reheat to boiling after soak at room temperature, keep micro-90-105 of boiling minute.
4. China according to claim 1, by the preparation method of hair spore mycelium polysaccharides, is characterized in that: in step S1, the described centrifugal time is 20min.
5. China according to claim 1, by the preparation method of hair spore mycelium polysaccharides, is characterized in that: in step S2, the ratio that the Chinese crude extract by hair spore mycelium polysaccharides is concentrated into liquid and raw material is till 2: 1 o'clock.
6. China according to claim 1, by the preparation method of hair spore mycelium polysaccharides, is characterized in that: in step S3, described ethanol is dehydrated alcohol.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103169065A (en) * | 2013-03-21 | 2013-06-26 | 武汉蜀泰科技有限公司 | Wall breaking method of cordyceps sinensis |
| CN103230419A (en) * | 2012-12-03 | 2013-08-07 | 玉树藏族自治州三江源药业有限公司 | Nitrogen-gas-protected cordyceps sinensis low-temperature ultrafine crushing processing method |
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- 2014-07-18 CN CN201410341978.4A patent/CN104177506B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103230419A (en) * | 2012-12-03 | 2013-08-07 | 玉树藏族自治州三江源药业有限公司 | Nitrogen-gas-protected cordyceps sinensis low-temperature ultrafine crushing processing method |
| CN103169065A (en) * | 2013-03-21 | 2013-06-26 | 武汉蜀泰科技有限公司 | Wall breaking method of cordyceps sinensis |
Non-Patent Citations (4)
| Title |
|---|
| LIANG HE等: "Statistics-based Optimization of Extracellular Polysaccharide Production from Hirsutella sinensis using a Fermentation Process and In vitro Immunomodulatory Activity", 《FOOD SCI. BIOTECHNOL.》, vol. 23, no. 2, 30 April 2014 (2014-04-30) * |
| 孙悦迎等: "冬虫夏草菌丝多糖分离提取特性研究", 《中医药学报》, vol. 31, no. 5, 20 October 2003 (2003-10-20) * |
| 朱西杰等主编: "《中华虫药—宁夏蜥蜴 第一版》", 31 May 2014, article "中药超微粉碎介绍" * |
| 童欣: "中国被毛孢活性物质分离纯化及结构鉴定", 《中国优秀硕士学位论文数据库 农业科技辑》, no. 3, 15 March 2014 (2014-03-15) * |
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Effective date of registration: 20211216 Address after: 212400 Hua Yang Zhen Ji Li Cun Zhu Yao natural village, Jurong City, Zhenjiang City, Jiangsu Province Patentee after: ZHENJIANG GUMANYUAN ECOLOGICAL AGRICULTURE Co.,Ltd. Address before: 201199 room 5, No. 536, Xindong Road, Minhang District, Shanghai Patentee before: SHANGHAI TIAN SONG BIO-TECHNOLOGY Co.,Ltd. |
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