CN104293854A - Preparation method of cephalotaxus alkaloid product - Google Patents
Preparation method of cephalotaxus alkaloid product Download PDFInfo
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- CN104293854A CN104293854A CN201410543253.3A CN201410543253A CN104293854A CN 104293854 A CN104293854 A CN 104293854A CN 201410543253 A CN201410543253 A CN 201410543253A CN 104293854 A CN104293854 A CN 104293854A
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Abstract
The invention belongs to the field of plant endophyte separation and microorganism fermentation engineering, and in particular relates to a preparation method of a cephalotaxus alkaloid product. The preparation method comprises the following steps: by adopting roots, stems and leaves of perennial cephalotaxus as explants, cutting into strips, sterilizing, then culturing in a culture medium to grow cephalotaxus symbiotic bacteria, then separating strains capable of metabolizing cephalotaxus alkaloid from the cephalotaxus symbiotic bacteria and performing culture and fermentation, concentrating a fermentation liquid, and then precipitating to prepare a product. According to the preparation method disclosed by the invention, in a preparation process of the fermentation liquid, a second batch of strains are inoculated after the strains generate metabolic organisms, metabolic products of the former batch of the strains are used for providing nutrient substances for the second batch of the strains, so that the growth of the second batch of the strains can be greatly promoted, and the biological activity of the strains also can be improved, and thus a great number of fermentation liquids can be generated, and the yield of cephalotaxus alkaloid can be improved.
Description
Technical field
The invention belongs to endophyte of plant to be separated and microbial fermentation engineering field, be specifically related to a kind of preparation method of cephalotaxus alkaloids product.
Background technology
Caephalotaxus sinensis is the distinctive kind of plant of China, has very high nutritive value and economic worth, and cephalotaxus alkaloids is one of important effective constituent of caephalotaxus sinensis, has the effects such as anti-cancer, is usually used in medicine.Current caephalotaxus sinensis resource scarcity, and this species grow is slow, cephalotaxus alkaloids yields poorly down, is difficult to the life requirement meeting people, also result in simultaneously and relate to the expensive of cephalotaxus alkaloids class medicine, one of output important topic becoming people therefore how improving cephalotaxus alkaloids.
Summary of the invention
The object of this invention is to provide a kind of preparation method of cephalotaxus alkaloids product, cephalotaxus alkaloids can be prepared fast and efficiently.
The technical solution used in the present invention is, a kind of preparation method of cephalotaxus alkaloids product, choose the root of perennial caephalotaxus sinensis, stem, leaf is as explant, through slitting, putting into substratum cultivation after sterilizing makes it grow caephalotaxus sinensis fungal component, then isolating from caephalotaxus sinensis fungal component can the bacterial classification of metabolism cephalotaxus alkaloids cultivation and fermentation, i.e. obtained product is separated out after fermented liquid is concentrated, the concrete operation method of described cultivation and fermentation be can metabolism produce the actication of culture of cephalotaxus alkaloids after to put into temperature be that the environment of 26 DEG C cultivates 48h, again strain inoculation is cultivated to the culture dish that PDA substratum is housed, produce metabolism biological until bacterial classification and access second batch bacterial classification after 1-2 days, to promote the growth of second batch bacterial classification, being inoculated into after the bacterial classification that second batch is inoculated grows is equipped with in the shaking flask of fermention medium, then shaking flask being put into rotating speed is 200r/min, temperature be 280C shaking table on cultivate 5d, collect the fermented liquid of bacterial classification,
The concrete operation method that described fermented liquid concentrates is that fermented liquid to be put into rotating speed be that the high-speed refrigerated centrifuge of 10000r/min is centrifugal, collect supernatant liquor, supernatant liquor is put into the high-speed refrigerated centrifuge recentrifuge that rotating speed is 10000r/min, then the supernatant liquor after recentrifuge is put into the Rotary Evaporators that temperature is 40-50 DEG C, pressure is 0.05Mpa and be concentrated into 1/10th of original volume, then concentrated solution is put into the refrigerator that temperature is 4 DEG C;
The concrete operation method of described precipitation for add NaOH solution until pH=8 ~ 9 in concentrated solution, separate out alkaloid and mixture, mixture being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of mixture and ethanol is 1:10, be cooled to 15-18 DEG C after being heated to 30 DEG C, separate out alkaloid, repeat above-mentioned heating, cooling precipitation process 2-3 time, merge alkaloid, i.e. obtained product.
In described shaking flask, fermention medium is by murphy juice, glucose, peptone, Zulkovsky starch, MgSO
4, KH
2pO
4make with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, Zulkovsky starch 1%, MgSO
40.05%, KH
2pO
40.15%, surplus is the pH=7 of water, substratum;
Described murphy juice is put into after Rotary Evaporators is concentrated into 1/3rd of original volume after being mixed according to the weight ratio of 10:1 by component A and B component and is obtained, described component A puts into the pot boiling 20-30min that temperature is 95-100 DEG C after being shredded by potato, cross 100-200 sieve after taking out, collect filtrate obtaining; Described B component puts into juice extractor after being shredded by potato to squeeze, then 80-100 mesh sieve of squeezing the juice, collect filtrate obtaining.
beneficial effect
One, the present invention produces after metabolism biological until bacterial classification and accesses second batch bacterial classification again in the preparation process of fermented liquid, the meta-bolites that before utilizing, batch bacterial classification produces provides nutritive substance for second batch bacterial classification, greatly facilitate the growth of second batch bacterial classification, improve its biological activity simultaneously, thus produce bulk fermentation liquid, improve the output of cephalotaxus alkaloids.
Two, the present invention is by centrifugal by fermented liquid, and recentrifuge after taking-up supernatant liquor, then concentrates, eliminate the most of impurity in fermented liquid, backward concentrated solution in add NaOH solution, and by repeatedly heating, cool precipitation process, substantially increase alkaloidal purity.
Three, by second batch strain inoculation being cultivated to fermention medium of the present invention, make growth rapid, metabolism is vigorous, substantially increases the output of fermented liquid, thus improves the output of cephalotaxus alkaloids.
Embodiment
A kind of preparation method of cephalotaxus alkaloids product, choose the root of perennial caephalotaxus sinensis, stem, leaf is as explant, through slitting, putting into substratum cultivation after sterilizing makes it grow caephalotaxus sinensis fungal component, then isolating from caephalotaxus sinensis fungal component can the bacterial classification of metabolism cephalotaxus alkaloids cultivation and fermentation, i.e. obtained product is separated out after fermented liquid is concentrated, the concrete operation method of described cultivation and fermentation be can metabolism produce the actication of culture of cephalotaxus alkaloids after to put into temperature be that the environment of 26 DEG C cultivates 48h, again strain inoculation is cultivated to the culture dish that PDA substratum is housed, produce metabolism biological until bacterial classification and access second batch bacterial classification after 1-2 days, to promote the growth of second batch bacterial classification, being inoculated into after the bacterial classification that second batch is inoculated grows is equipped with in the shaking flask of fermention medium, then shaking flask being put into rotating speed is 200r/min, temperature be 280C shaking table on cultivate 5d, collect the fermented liquid of bacterial classification,
The concrete operation method that described fermented liquid concentrates is that fermented liquid to be put into rotating speed be that the high-speed refrigerated centrifuge of 10000r/min is centrifugal, collect supernatant liquor, supernatant liquor is put into the high-speed refrigerated centrifuge recentrifuge that rotating speed is 10000r/min, then the supernatant liquor after recentrifuge is put into the Rotary Evaporators that temperature is 40-50 DEG C, pressure is 0.05Mpa and be concentrated into 1/10th of original volume, then concentrated solution is put into the refrigerator that temperature is 4 DEG C;
The concrete operation method of described precipitation for add NaOH solution until pH=8 ~ 9 in concentrated solution, separate out alkaloid and mixture, mixture being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of mixture and ethanol is 1:10, be cooled to 15-18 DEG C after being heated to 30 DEG C, separate out alkaloid, repeat above-mentioned heating, cooling precipitation process 2-3 time, merge alkaloid, i.e. obtained product.
In described shaking flask, fermention medium is by murphy juice, glucose, peptone, Zulkovsky starch, MgSO
4, KH
2pO
4make with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, Zulkovsky starch 1%, MgSO
40.05%, KH
2pO
40.15%, surplus is the pH=7 of water, substratum;
Described murphy juice is put into after Rotary Evaporators is concentrated into 1/3rd of original volume after being mixed according to the weight ratio of 10:1 by component A and B component and is obtained, described component A puts into the pot boiling 20-30min that temperature is 95-100 DEG C after being shredded by potato, cross 100-200 sieve after taking out, collect filtrate obtaining; Described B component puts into juice extractor after being shredded by potato to squeeze, then 80-100 mesh sieve of squeezing the juice, collect filtrate obtaining.
Embodiment 1
A preparation method for cephalotaxus alkaloids product, comprises the following steps:
The selection of step one, explant and process
Choose the root of perennial caephalotaxus sinensis, stem, leaf as explant, with flowing water, explant is cleaned up, then root is cut the long block of growing into 5-6cm, the Lao Pi on removing stem and branches and leaves, for subsequent use;
The sterilizing of step 2, explant
The explant handled well is put into Bechtop, is first the alcohol sterilizing 5min of 75% by volumetric concentration, then is the mercuric chloride sterilizing 12min of 0.1% by mass concentration, after aseptic washing 4 times, the explant handled well is put into sterilized water, for subsequent use;
The purifies and separates of step 3, caephalotaxus sinensis bacterial classification
Explant after sterilizing is accessed respectively in the culturing bottle that PDA substratum is housed, it is that the culturing rack of 25 DEG C is cultivated that the culturing bottle being connected to explant is placed into temperature, after growing bacterial classification in culturing bottle, therefrom isolate metabolism can produce the bacterial strain of cephalotaxus alkaloids according to the colony characteristics of different strain, for subsequent use;
Step 4, cultivation and fermentation
Can metabolism produce the actication of culture of cephalotaxus alkaloids after to put into temperature be that the environment of 26 DEG C cultivates 48h, again strain inoculation is cultivated to the culture dish that PDA substratum is housed, produce after metabolism biological until bacterial classification and access second batch bacterial classification, to promote the growth of second batch bacterial classification, be inoculated into and be equipped with in the shaking flask of fermention medium after the bacterial classification of second batch inoculation grows 1 day, then shaking flask is put into that rotating speed is 200r/min, temperature is 28
05d cultivated by the shaking table of C, collects the fermented liquid of bacterial classification, for subsequent use;
Step 5, fermented liquid are concentrated
Fermented liquid being put into rotating speed is that the high-speed refrigerated centrifuge of 10000r/min is centrifugal, collect supernatant liquor, supernatant liquor is put into the high-speed refrigerated centrifuge recentrifuge that rotating speed is 10000r/min, supernatant liquor after recentrifuge is put into the Rotary Evaporators that temperature is 40 DEG C, pressure is 0.05Mpa and is concentrated into 1/10th of original volume, concentrated solution is put into the refrigerator that temperature is 4 DEG C, for subsequent use;
Step 6, precipitation
NaOH solution is added until pH=8 in concentrated solution, separate out alkaloid and mixture, mixture being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of mixture and ethanol is 1:10, be cooled to 15 DEG C after being heated to 30 DEG C, separate out alkaloid, repeat above-mentioned heating, cooling precipitation process 2 times, merge alkaloid, i.e. obtained cephalotaxus alkaloids;
Step 7, preservation
Cephalotaxus alkaloids being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of cephalotaxus alkaloids and ethanol is 1:10, and it is in the refrigerator of 4 DEG C that sealing dissolving is placed on temperature.
In shaking flask described in step 4, fermention medium is by murphy juice, glucose, peptone, Zulkovsky starch, MgSO
4, KH
2pO
4make with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, Zulkovsky starch 1%, MgSO
40.05%, KH
2pO
40.15%, surplus is the pH=7 of water, substratum.
Described murphy juice is put into after Rotary Evaporators is concentrated into 1/3rd of original volume after being mixed according to the weight ratio of 10:1 by component A and B component and is obtained, described component A puts into the pot boiling 20min that temperature is 95 DEG C after being shredded by potato, cross 100 mesh sieves after taking out, collect filtrate obtaining; Described B component puts into juice extractor after being shredded by potato to squeeze, then 80 mesh sieves of squeezing the juice, collect filtrate obtaining.
Embodiment 2
A preparation method for cephalotaxus alkaloids product, comprises the following steps:
The selection of step one, explant and process
Choose the root of perennial caephalotaxus sinensis, stem, leaf as explant, with flowing water, explant is cleaned up, then root is cut the long block of growing into 5-6cm, the Lao Pi on removing stem and branches and leaves, for subsequent use;
The sterilizing of step 2, explant
The explant handled well is put into Bechtop, is first the alcohol sterilizing 5min of 75% by volumetric concentration, then is the mercuric chloride sterilizing 12min of 0.1% by mass concentration, after aseptic washing 5 times, the explant handled well is put into sterilized water, for subsequent use;
The purifies and separates of step 3, caephalotaxus sinensis bacterial classification
Explant after sterilizing is accessed respectively in the culturing bottle that PDA substratum is housed, it is that the culturing rack of 25 DEG C is cultivated that the culturing bottle being connected to explant is placed into temperature, after growing bacterial classification in culturing bottle, therefrom isolate metabolism can produce the bacterial strain of cephalotaxus alkaloids according to the colony characteristics of different strain, for subsequent use;
Step 4, cultivation and fermentation
Can metabolism produce the actication of culture of cephalotaxus alkaloids after to put into temperature be that the environment of 26 DEG C cultivates 48h, again strain inoculation is cultivated to the culture dish that PDA substratum is housed, produce metabolism biological until bacterial classification and access second batch bacterial classification after 2 days, to promote the growth of second batch bacterial classification, be inoculated into and be equipped with in the shaking flask of fermention medium after the bacterial classification of second batch inoculation grows, then shaking flask is put into that rotating speed is 200r/min, temperature is 28
05d cultivated by the shaking table of C, collects the fermented liquid of bacterial classification, for subsequent use;
Concentrating of step 5, fermented liquid
Fermented liquid being put into rotating speed is that the high-speed refrigerated centrifuge of 10000r/min is centrifugal, collect supernatant liquor, supernatant liquor is put into the high-speed refrigerated centrifuge recentrifuge that rotating speed is 10000r/min, supernatant liquor after recentrifuge is put into the Rotary Evaporators that temperature is 50 DEG C, pressure is 0.05Mpa and is concentrated into 1/10th of original volume, concentrated solution is put into the refrigerator that temperature is 4 DEG C, for subsequent use;
Step 6, precipitation
NaOH solution is added until pH=9 in concentrated solution, separate out alkaloid and mixture, mixture being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of mixture and ethanol is 1:10, be cooled to 18 DEG C after being heated to 30 DEG C, separate out alkaloid, repeat above-mentioned heating, cooling precipitation process 3 times, merge alkaloid, i.e. obtained cephalotaxus alkaloids;
Step 7, preservation
Cephalotaxus alkaloids being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of cephalotaxus alkaloids and ethanol is 1:10, and it is in the refrigerator of 4 DEG C that sealing dissolving is placed on temperature.
In shaking flask described in step 4, fermention medium is by murphy juice, glucose, peptone, Zulkovsky starch, MgSO
4, KH
2pO
4make with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, Zulkovsky starch 1%, MgSO
40.05%, KH
2pO
40.15%, surplus is the pH=7 of water, substratum.
Described murphy juice is put into after Rotary Evaporators is concentrated into 1/3rd of original volume after being mixed according to the weight ratio of 10:1 by component A and B component and is obtained, described component A puts into the pot boiling 30min that temperature is 100 DEG C after being shredded by potato, cross 200 mesh sieves after taking out, collect filtrate obtaining; Described B component puts into juice extractor after being shredded by potato to squeeze, then 100 mesh sieves of squeezing the juice, collect filtrate obtaining.
Claims (2)
1. the preparation method of a cephalotaxus alkaloids product, choose the root of perennial caephalotaxus sinensis, stem, leaf is as explant, through slitting, putting into substratum cultivation after sterilizing makes it grow caephalotaxus sinensis fungal component, then isolating from caephalotaxus sinensis fungal component can the bacterial classification of metabolism cephalotaxus alkaloids cultivation and fermentation, i.e. obtained product is separated out after fermented liquid is concentrated, it is characterized in that: the concrete operation method of described cultivation and fermentation be can metabolism produce the actication of culture of cephalotaxus alkaloids after to put into temperature be that the environment of 26 DEG C cultivates 48h, again strain inoculation is cultivated to the culture dish that PDA substratum is housed, produce metabolism biological until bacterial classification and access second batch bacterial classification after 1-2 days, to promote the growth of second batch bacterial classification, being inoculated into after the bacterial classification that second batch is inoculated grows is equipped with in the shaking flask of fermention medium, then shaking flask being put into rotating speed is 200r/min, temperature be 280C shaking table on cultivate 5d, collect the fermented liquid of bacterial classification,
The concrete operation method that described fermented liquid concentrates is that fermented liquid to be put into rotating speed be that the high-speed refrigerated centrifuge of 10000r/min is centrifugal, collect supernatant liquor, supernatant liquor is put into the high-speed refrigerated centrifuge recentrifuge that rotating speed is 10000r/min, then the supernatant liquor after recentrifuge is put into the Rotary Evaporators that temperature is 40-50 DEG C, pressure is 0.05Mpa and be concentrated into 1/10th of original volume, then concentrated solution is put into the refrigerator that temperature is 4 DEG C;
The concrete operation method of described precipitation for add NaOH solution until pH=8 ~ 9 in concentrated solution, separate out alkaloid and mixture, mixture being joined volumetric concentration is in the ethanol of 95%, and the volume ratio of mixture and ethanol is 1:10, be cooled to 15-18 DEG C after being heated to 30 DEG C, separate out alkaloid, repeat above-mentioned heating, cooling precipitation process 2-3 time, merge alkaloid, i.e. obtained product.
2. a kind of isolating from caephalotaxus sinensis fungal component according to claim 1 can the method for metabolism cephalotaxus alkaloids bacterial classification, it is characterized in that: in described shaking flask, fermention medium is by murphy juice, glucose, peptone, Zulkovsky starch, MgSO
4, KH
2pO
4make with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, Zulkovsky starch 1%, MgSO
40.05%, KH
2pO
40.15%, surplus is the pH=7 of water, substratum;
Described murphy juice is put into after Rotary Evaporators is concentrated into 1/3rd of original volume after being mixed according to the weight ratio of 10:1 by component A and B component and is obtained, described component A puts into the pot boiling 20-30min that temperature is 95-100 DEG C after being shredded by potato, cross 100-200 sieve after taking out, collect filtrate obtaining; Described B component puts into juice extractor after being shredded by potato to squeeze, then 80-100 mesh sieve of squeezing the juice, collect filtrate obtaining.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108496992A (en) * | 2018-05-02 | 2018-09-07 | 张友兰 | A kind of composition pesticide of prevention Laphygma exigua Hubner |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108651487A (en) * | 2018-05-02 | 2018-10-16 | 赖振明 | A kind of composition pesticide containing cephalotaxus alkaloids and four chlorantraniliproles |
Citations (2)
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| CN1545888A (en) * | 2003-12-03 | 2004-11-17 | 华南农业大学 | Method for extracting Cephalotaxus fortunei genus plant nematode-killing alkaloid and application thereof |
| US20100240887A1 (en) * | 2009-03-23 | 2010-09-23 | Yaguang Liu | New methods of producing HHT |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1545888A (en) * | 2003-12-03 | 2004-11-17 | 华南农业大学 | Method for extracting Cephalotaxus fortunei genus plant nematode-killing alkaloid and application thereof |
| US20100240887A1 (en) * | 2009-03-23 | 2010-09-23 | Yaguang Liu | New methods of producing HHT |
Non-Patent Citations (1)
| Title |
|---|
| 陈传文: "抗肿瘤植物内生菌的筛选及菌株PF09005的分类鉴定与发酵优化", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108496992A (en) * | 2018-05-02 | 2018-09-07 | 张友兰 | A kind of composition pesticide of prevention Laphygma exigua Hubner |
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