CN105039452A - Method for extracting polysaccharides in solid fermentation broth of cordyceps sinensis - Google Patents
Method for extracting polysaccharides in solid fermentation broth of cordyceps sinensis Download PDFInfo
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Abstract
The invention relates to a method for extracting polysaccharides in solid fermentation broth of cordyceps sinensis. The method includes steps of preliminarily treating samples; preparing solid fermentation media; fermenting, cultivating and preparing strain fermentation broth; carrying out ultrasonic reflux extraction; preparing crude polysaccharides in the samples; preparing fine polysaccharides. The method has the advantages that the samples are preliminarily treated, cultivating conditions such as components of the solid fermentation media, cultivating temperatures, material-to-water ratios and illumination are optimized, accordingly, the method is favorable for growth of mycelia of the cordyceps sinensis, and the content of wheat sterol which is one of main effective constituents in the mycelia can be obviously increased; the solid fermentation broth is decolorized by the aid of boiling water with activated carbon when the polysaccharides are about to be extracted, proteins are removed by the aid of heating and cooling processes, accordingly, operation is easy and convenient, and good effects can be realized without affecting the yield of the polysaccharides; 65-80% ethyl alcohol is used for alcohol precipitation, and accordingly the optimal polysaccharide alcohol precipitation effects can be realized; the integral operation is easy and convenient without special instruments and equipment, and accordingly the method is suitable for large-scale production and application.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid.
Background technology
Cordyceps sinensis is Clavicipitaceae fungi, and it parasitizes on stroma on Hepialus section insect larvae and larva corpse.Cordyceps sinensis head is loaded in new compilation of materia medica, taste is sweet, warm in nature, return lung, kidney channel, has multiple physiologically active, as tonifying deficiency, beneficial vital essence, protect lung, kidney-nourishing, hemostasis and phlegm, strengthening by means of tonics, anti-oxidant, improve immunizing power etc., therefore, the diseases such as treatment phthisical cough phlegm blood, night sweat, impotence, seminal emission, jaundice disease, pulmonary tuberculosis and old age is weak, chronic cough and asthma are commonly used to.All the time, Cordyceps sinensis is regarded as precious Chinese medicinal materials always.
Cordyceps polysaccharide is the biologically active substance that in Chinese caterpillar fungus, content is the highest, have antitumor, anti-oxidant, improve immunizing power, reduce the pharmacological action widely such as blood fat, at present, what play a major role in the product such as " paecilomyces hepiall chen ", " Ningxinbao Capsules ", " Cordyceps capsule ", " Caps Bailing " on the market is exactly Cordyceps polysaccharide.But because Cordyceps sinensis requires higher to growing environment, naturally wild output is quite limited, for this reason, how to carry out high efficiency extraction to the polysaccharide active components in Cordyceps sinensis, is the focus of current Chinese scholars research.Existing Cordyceps Polysaccharide extractive technique mainly comprises supersonic method, water extraction and alcohol precipitation method and supercritical carbon dioxide extraction method, but aforesaid method exists respective defect or deficiency, and such as water extraction and alcohol precipitation method extraction yield is low, and purity of polysaccharide is not enough.For this reason, the invention provides a kind of Cordyceps sinensis intracellular polyse extracting method of improvement.
Summary of the invention
The object of the invention is to solve the above-mentioned problems in the prior art, provide the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid, polysaccharide yield is high, easy and simple to handle.
Technical scheme of the present invention is as follows:
An extracting method for polysaccharide in Cordyceps sinensis solid fermentation liquid, comprises the following steps:
(1) sample pre-treatments: by Cordyceps strain 5-10g good for preservation after clear water flushing, drying, for subsequent use;
(2) solid fermentation substratum is prepared: get glucose 2-5g, Semen Maydis powder 2-4g, nitrogenous source 1-2g, wheat bran 2-4g, KH
2pO
42-4g, MgSO
41-2g, VitaminB
12-4g, VitaminB
23-6g, adds distilled water in substratum, heated and boiled, regulates PH, pours into while hot in container, high pressure steam sterilization, be poured on flat board;
(3) fermentation culture: the sample in step (1) after sample pre-treatments is inoculated in the substratum prepared in step (1), gets strain fermentating liquid after having cultivated for subsequent use;
(4) to get in step (3) obtained strain fermentating liquid to add water and carry out ultrasonic refluxing extraction, through 0.45um filtering with microporous membrane, sample thief filtrate is for subsequent use;
(5) by the sample filtrate evaporation concentration of preparation in step (4) to the 1/3-1/2 of original volume, add ethanol again and carry out alcohol precipitation, after filtering, frozen centrifugation, get supernatant liquor and leave standstill 12h under 4 DEG C of conditions, centrifugal at normal temperatures, must precipitate through 0.45um filtering with microporous membrane, adopt absolute ethanol washing 1-3 time, concentrate and obtain sample Crude polysaccharides;
(6) adopted by sample Crude polysaccharides obtained in step (5) boiling water adding activated carbon to dissolve decolouring, cooling, then add boiling water, cool again, remove albumen, insert in dialysis tubing by the sample solution removing albumen, dialyse 24h in distilled water, the ethanol of 5-10 times of volume is added in the solution after dialysis, precipitates overnight under 4 DEG C of conditions, centrifugal, abandon supernatant, taking precipitate is dry, obtained smart polysaccharide.
Preferably, step (1) and the bake out temperature described in step (6) are 50-80 DEG C.
Preferably, in step (2), the amount of adding distil water is by material-water ratio (1-3): the volume of (1.5-5) adds.
Preferably, regulate PH for regulating PH to 6.5-7.2 in step (2).
Preferably, the high pressure steam sterilization time described in step (2) is 10-20min.
Preferably, the culture temperature described in step (3) is 25-28 DEG C, and cultivate as without illumination cultivation, incubation time is 5-7d.
Preferably, the ultrasonic refluxing extraction temperature described in step (4) is 80-100 DEG C, and extraction time is each 1-2h, and extraction time is 3-5 time.
Preferably, the frozen centrifugation temperature described in step (5) is-10--4 DEG C, and the centrifugal speed in step (5) in two times centrifugal and step (6) is 4000-8000r/min, and centrifugation time is 10-20min.
Preferably, in the boiling water of the interpolation activated carbon described in step (6), the mass concentration of activated carbon is 1-3%.
Preferably, the semi-permeable membranes of to be molecular weight cut-off the be 6000-8000 of the dialysis membrane described in step (6), described alcohol concn is 65-80%.
Compared with prior art, beneficial effect is embodied in the present invention:
1. nitrogenous source is that Cordyceps grows necessary nutritive element, because Cordyceps is long-term parasitic in former host bat moth larvae body, adapt to absorbing animal proteinoid, and poor to plant absorbing proteins, cause the content of its effective constituent and biomass lower than animal nitrogenous source.Therefore, using the nitrogenous source as solid medium such as peptone or dried silkworm chrysalis meal in the present invention, the content of Cordyceps mycelium and principle active component thereof can be improved
2. the present invention has carried out strict optimization to the composition of solid medium, strict control has also been carried out to culture condition such as culture temperature during solid state rheology, material-water ratio and illumination, more favourable to Cordyceps mycelium growth, the content of one of principle active component in mycelium wheat sterol can be significantly improved.
3. the extraction of polyose composition often adopts hot water, solvent (acid, alkali or ethanol) extraction, sample is first carried out pre-treatment by the present invention, is concentrated by sample fermented liquid, adds ethanol and refines, and alcohol concn is restricted to 65-80%, Cordyceps sinensis exocellular polysaccharide alcohol precipitation effect can be made best.
4. what the protein in Cordyceps fermented liquid can affect wherein polysaccharide is rate and assay, therefore, in Cordyceps polysaccharide leaching process, normal needs are taken measures the protein removed wherein, research display, it is best that sevage method removes protein effect, and polysaccharide yield is also the highest, but because chloroform toxicity is larger, therefore, be not suitable for the production application of Cordyceps sinensis, and trichloroacetic acid method polysaccharide yield is low, and deproteinated effect is also not obvious, for this reason, after the present invention adopts and is added with the decolouring of certain density activated carbon boiling water, heating, method of cooling removes albumen, easy and simple to handle, effective, and do not affect many rates of polysaccharide.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail:
Embodiment 1
An extracting method for polysaccharide in Cordyceps sinensis solid fermentation liquid, comprises the following steps:
(1) sample pre-treatments: by Cordyceps strain 5g good for preservation after clear water flushing, drying at 50 DEG C, for subsequent use;
(2) solid fermentation substratum is prepared: get glucose 2g, Semen Maydis powder 2g, nitrogenous source 1g, wheat bran 2g, KH
2pO
42g, MgSO
41g, VitaminB
12g, VitaminB
23g, adds distilled water in substratum, heated and boiled, regulates PH to 6.5, pours into while hot in container, high pressure steam sterilization 10min, be poured on flat board;
(3) fermentation culture: be inoculated in the substratum prepared in step (1) by the sample in step (1) after sample pre-treatments, culture temperature is 25 DEG C, cultivate as without illumination cultivation, incubation time is 5d, gets strain fermentating liquid for subsequent use after having cultivated;
(4) to get in step (3) obtained strain fermentating liquid to add water carry out ultrasonic refluxing extraction 3 times, each 1h at 80 DEG C, through 0.45um filtering with microporous membrane, sample thief filtrate is for subsequent use;
(5) by the sample filtrate evaporation concentration of preparation in step (4) to 1/3 of original volume, add ethanol again and carry out alcohol precipitation, after filtering, at-10 DEG C with 4000r/min frozen centrifugation 10min, get supernatant liquor and leave standstill 12h under 4 DEG C of conditions, at normal temperatures with the centrifugal 10min of 4000r/min, must precipitate through 0.45um filtering with microporous membrane, adopt absolute ethanol washing 1 time, concentrate and obtain sample Crude polysaccharides;
(6) adopted by sample Crude polysaccharides obtained in step (5) boiling water of interpolation 1% activated carbon to dissolve decolouring, cooling, then add boiling water, cool again, remove albumen, insert in dialysis tubing by the sample solution removing albumen, dialyse 24h in distilled water, 65% ethanol of 5 times of volumes is added in the solution after dialysis, precipitates overnight under 4 DEG C of conditions, centrifugal, abandon supernatant, taking precipitate is dry, obtained smart polysaccharide.
In step (2), the amount of adding distil water adds by the volume of material-water ratio 1:1.5.
Dialysis membrane described in step (6) to be molecular weight cut-off be 6000 semi-permeable membranes.
Embodiment 2
An extracting method for polysaccharide in Cordyceps sinensis solid fermentation liquid, comprises the following steps:
(1) sample pre-treatments: by Cordyceps strain 10g good for preservation after clear water flushing, drying at 80 DEG C, for subsequent use;
(2) solid fermentation substratum is prepared: get glucose 5g, Semen Maydis powder 4g, nitrogenous source 2g, wheat bran 4g, KH
2pO
44g, MgSO
42g, VitaminB
14g, VitaminB
26g, adds distilled water in substratum, heated and boiled, regulates PH to 7.2, pours into while hot in container, high pressure steam sterilization 20min, be poured on flat board;
(3) fermentation culture: be inoculated in the substratum prepared in step (1) by the sample in step (1) after sample pre-treatments, culture temperature is 28 DEG C, cultivate as without illumination cultivation, incubation time is 7d, gets strain fermentating liquid for subsequent use after having cultivated;
(4) to get in step (3) obtained strain fermentating liquid to add water carry out ultrasonic refluxing extraction 5 times, each 2h at 100 DEG C, through 0.45um filtering with microporous membrane, sample thief filtrate is for subsequent use;
(5) by the sample filtrate evaporation concentration of preparation in step (4) to 1/2 of original volume, add ethanol again and carry out alcohol precipitation, after filtering, at-4 DEG C with 8000r/min frozen centrifugation 20min, get supernatant liquor and leave standstill 12h under 4 DEG C of conditions, at normal temperatures with the centrifugal 20min of 8000r/min, must precipitate through 0.45um filtering with microporous membrane, adopt absolute ethanol washing 3 times, concentrate and obtain sample Crude polysaccharides;
(6) adopted by sample Crude polysaccharides obtained in step (5) boiling water of interpolation 3% activated carbon to dissolve decolouring, cooling, then add boiling water, cool again, remove albumen, insert in dialysis tubing by the sample solution removing albumen, dialyse 24h in distilled water, 80% ethanol of 10 times of volumes is added in the solution after dialysis, precipitates overnight under 4 DEG C of conditions, centrifugal, abandon supernatant, taking precipitate is dry, obtained smart polysaccharide.
In step (2), the amount of adding distil water adds by the volume of material-water ratio 3:5.
Dialysis membrane described in step (6) to be molecular weight cut-off be 8000 semi-permeable membranes.
Embodiment 3
An extracting method for polysaccharide in Cordyceps sinensis solid fermentation liquid, comprises the following steps:
(1) sample pre-treatments: by Cordyceps strain 8g good for preservation after clear water flushing, drying at 65 DEG C, for subsequent use;
(2) solid fermentation substratum is prepared: get glucose 3g, Semen Maydis powder 3g, nitrogenous source 1.5g, wheat bran 3g, KH
2pO
43g, MgSO
41.5g, VitaminB
13g, VitaminB
24.5g, adds distilled water in substratum, heated and boiled, regulates PH to 6.8, pours into while hot in container, high pressure steam sterilization 15min, be poured on flat board;
(3) fermentation culture: be inoculated in the substratum prepared in step (1) by the sample in step (1) after sample pre-treatments, culture temperature is 26 DEG C, cultivate as without illumination cultivation, incubation time is 6d, gets strain fermentating liquid for subsequent use after having cultivated;
(4) to get in step (3) obtained strain fermentating liquid to add water carry out ultrasonic refluxing extraction 4 times, each 1.5h at 90 DEG C, through 0.45um filtering with microporous membrane, sample thief filtrate is for subsequent use;
(5) by the sample filtrate evaporation concentration of preparation in step (4) to 1/3 of original volume, add ethanol again and carry out alcohol precipitation, after filtering, at-8 DEG C with 6000r/min frozen centrifugation 15min, get supernatant liquor and leave standstill 12h under 4 DEG C of conditions, at normal temperatures with the centrifugal 15min of 6000r/min, must precipitate through 0.45um filtering with microporous membrane, adopt absolute ethanol washing 2 times, concentrate and obtain sample Crude polysaccharides;
(6) adopted by sample Crude polysaccharides obtained in step (5) boiling water of interpolation 2% activated carbon to dissolve decolouring, cooling, then add boiling water, cool again, remove albumen, insert in dialysis tubing by the sample solution removing albumen, dialyse 24h in distilled water, 70% ethanol of 8 times of volumes is added in the solution after dialysis, precipitates overnight under 4 DEG C of conditions, centrifugal, abandon supernatant, taking precipitate is dry, obtained smart polysaccharide.
In step (2), the amount of adding distil water adds by the volume of material-water ratio 2:3.
Dialysis membrane described in step (6) to be molecular weight cut-off be 7000 semi-permeable membranes.
Above-described embodiment is only the preferred embodiment of the present invention, and should not be construed as limitation of the invention, and those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (10)
1. the extracting method of polysaccharide in Cordyceps sinensis solid fermentation liquid, is characterized in that, comprise the following steps:
(1) sample pre-treatments: by Cordyceps strain 5-10g good for preservation after clear water flushing, drying, for subsequent use;
(2) solid fermentation substratum is prepared: get glucose 2-5g, Semen Maydis powder 2-4g, nitrogenous source 1-2g, wheat bran 2-4g, KH
2pO
42-4g, MgSO
41-2g, VitaminB
12-4g, VitaminB
23-6g, adds distilled water in substratum, heated and boiled, regulates PH, pours into while hot in container, high pressure steam sterilization, be poured on flat board;
(3) fermentation culture: the sample in step (1) after sample pre-treatments is inoculated in the substratum prepared in step (1), gets strain fermentating liquid after having cultivated for subsequent use;
(4) to get in step (3) obtained strain fermentating liquid to add water and carry out ultrasonic refluxing extraction, through 0.45um filtering with microporous membrane, sample thief filtrate is for subsequent use;
(5) by the sample filtrate evaporation concentration of preparation in step (4) to the 1/3-1/2 of original volume, add ethanol again and carry out alcohol precipitation, after filtering, frozen centrifugation, get supernatant liquor and leave standstill 12h under 4 DEG C of conditions, centrifugal at normal temperatures, must precipitate through 0.45um filtering with microporous membrane, adopt absolute ethanol washing 1-3 time, concentrate and obtain sample Crude polysaccharides;
(6) adopted by sample Crude polysaccharides obtained in step (5) boiling water adding activated carbon to dissolve decolouring, cooling, then add boiling water, cool again, remove albumen, insert in dialysis tubing by the sample solution removing albumen, dialyse 24h in distilled water, the ethanol of 5-10 times of volume is added in the solution after dialysis, precipitates overnight under 4 DEG C of conditions, centrifugal, abandon supernatant, taking precipitate is dry, obtained smart polysaccharide.
2. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, is characterized in that, step (1) and the bake out temperature described in step (6) are 50-80 DEG C.
3. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, is characterized in that, in step (2), the amount of adding distil water is by material-water ratio (1-3): the volume of (1.5-5) adds.
4. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, is characterized in that, regulates PH for regulating PH to 6.5-7.2 in step (2).
5. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, it is characterized in that, the high pressure steam sterilization time described in step (2) is 10-20min.
6. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, it is characterized in that, the culture temperature described in step (3) is 25-28 DEG C, and cultivate as without illumination cultivation, incubation time is 5-7d.
7. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, it is characterized in that, the ultrasonic refluxing extraction temperature described in step (4) is 80-100 DEG C, and extraction time is each 1-2h, and extraction time is 3-5 time.
8. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, it is characterized in that, frozen centrifugation temperature described in step (5) is-10--4 DEG C, centrifugal speed in step (5) in two times centrifugal and step (6) is 4000-8000r/min, and centrifugation time is 10-20min.
9. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, it is characterized in that, in the boiling water of the interpolation activated carbon described in step (6), the mass concentration of activated carbon is 1-3%.
10. the extracting method of polysaccharide in a kind of Cordyceps sinensis solid fermentation liquid according to claim 1, is characterized in that, the semi-permeable membranes of to be molecular weight cut-off the be 6000-8000 of the dialysis membrane described in step (6), described alcohol concn is 65-80%.
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Application publication date: 20151111 |