CN104293854B - A kind of preparation method of cephalotaxus alkaloids product - Google Patents
A kind of preparation method of cephalotaxus alkaloids product Download PDFInfo
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- CN104293854B CN104293854B CN201410543253.3A CN201410543253A CN104293854B CN 104293854 B CN104293854 B CN 104293854B CN 201410543253 A CN201410543253 A CN 201410543253A CN 104293854 B CN104293854 B CN 104293854B
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Abstract
The invention belongs to endophyte of plant separation and microbial fermentation engineering field, and in particular to a kind of preparation method of cephalotaxus alkaloids product.Explant is used as by the root, stem, leaf of choosing perennial caephalotaxus sinensis, culture in culture medium is put into after slitting, sterilizing makes it grow caephalotaxus sinensis symbiotic bacteria, then the strain and cultivation and fermentation of metabolizable cephalotaxus sinensis alkaloid are isolated from caephalotaxus sinensis symbiotic bacteria, is separated out after zymotic fluid is concentrated and product is made.The present invention accesses second batch strain again in the preparation process of zymotic fluid after strain produces metabolism biological, the metabolite produced using preceding batch of strain provides nutriment for second batch strain, the growth of second batch strain is greatly facilitated, improve its bioactivity simultaneously, so as to produce bulk fermentation liquid, the yield of cephalotaxus alkaloids is improved.
Description
Technical field
The invention belongs to endophyte of plant separation and microbial fermentation engineering field, and in particular to a kind of cephalotaxus alkaloids production
The preparation method of product.
Background technology
Caephalotaxus sinensis is a kind of distinctive plant of China, and with very high nutritive value and economic value, cephalotaxus alkaloids is thick
One of important active ingredient of Chinese torreya, with anti-cancer the effects such as, be usually used in medicine.Current caephalotaxus sinensis resource scarcity, and seeds life
Long slow, cephalotaxus alkaloids yields poorly down, it is difficult to meet the life requirement of people, is related to cephalotaxus alkaloids class while also result in
Medicine it is expensive, therefore how to improve the yield of cephalotaxus alkaloids and turn into one of important topic of people.
The content of the invention
It is an object of the invention to provide a kind of preparation method of cephalotaxus alkaloids product, can fast and efficiently it prepare thick
Chinese torreya alkaloid.
The technical solution used in the present invention is that a kind of preparation method of cephalotaxus alkaloids product chooses perennial caephalotaxus sinensis
Root, stem, leaf as explant, culture in culture medium is put into after slitting, sterilizing makes it grow caephalotaxus sinensis symbiotic bacteria, then from thick
The strain and cultivation and fermentation of metabolizable cephalotaxus sinensis alkaloid are isolated in Chinese torreya fungal component, is separated out after zymotic fluid is concentrated and production is made
Product, it by temperature is put into after the actication of culture of metabolizable generation cephalotaxus alkaloids is 26 that the concrete operation method of the cultivation and fermentation, which is,
DEG C environment in cultivate 48h, then strain be inoculated on the culture dish equipped with PDA culture medium cultivated, treat that first strain is produced
Metabolism biological accesses second batch caephalotaxus sinensis symbiotic bacteria after 1-2 days, to promote the growth of second batch strain, the strain of second batch inoculation is long
It is inoculated into after going out in the shaking flask equipped with fermentation medium, it is that 200r/min, temperature are shaking for 280C that shaking flask, which is then put into rotating speed,
5d is cultivated on bed, the zymotic fluid of strain is collected；Wherein, in the shaking flask fermentation medium by murphy juice, glucose, albumen
Peptone, soluble starch, MgSO4、KH2PO4It is made with water, and the mass percent of each component is murphy juice 2%, glucose 2%, egg
White peptone 0.12%, soluble starch 1%, MgSO4 0.05%, KH2PO40.15%, surplus is water, pH=7 of culture medium；
The murphy juice is by component A and component B according to 10:1 weight is concentrated into than being put into after mixing in Rotary Evaporators
It is made after 1/3rd of original volume, the component A is that boiling 20- in the pot that temperature is 95-100 DEG C is put into after potato is shredded
30min, crosses 100-200 sieves after taking-up, collect filtrate and be made；The component B is to be put into juice extractor to squeeze after potato is shredded,
Again 80-100 mesh sieves of squeezing the juice, collect filtrate and be made.
The concrete operation method of the zymotic fluid concentration is that zymotic fluid is put into the freezing high speed that rotating speed is 10000r/min
In centrifuge centrifuge, collect supernatant, supernatant be put into rotating speed be 10000r/min high-speed refrigerated centrifuge in again from
The heart, is then put into the supernatant after centrifuging again in the Rotary Evaporators that temperature is 40-50 DEG C, pressure is 0.05Mpa and concentrates
It is put into 1/10th of original volume, then by concentrate in the refrigerator that temperature is 4 DEG C；
The concrete operation method of the precipitation is that NaOH solution is added into concentrate up to pH=8~9, separates out mixture,
Add mixture in the ethanol that volumetric concentration is 95%, and the volume ratio of mixture and ethanol is 1:10, it is heated to after 30 DEG C
15-18 DEG C is cooled to, alkaloid is separated out, above-mentioned heating, cooling precipitation process is repeated 2-3 times, merges alkaloid, that is, production is made
Product.
Beneficial effect
First, the present invention accesses second batch strain again in the preparation process of zymotic fluid after strain produces metabolism biological, profit
The metabolite produced with preceding batch of strain provides nutriment for second batch strain, and the growth of second batch strain is greatly facilitated,
Its bioactivity is improved simultaneously, so as to produce bulk fermentation liquid, the yield of cephalotaxus alkaloids is improved.
2nd, the present invention centrifuges by the way that zymotic fluid is centrifuged, is then concentrated, eliminate hair again after taking out supernatant
Most of impurity in zymotic fluid, backward concentrate in add NaOH solution, and by heating repeatedly, cooling down precipitation process, greatly
The big purity for improving alkaloid.
3rd, cultivated by the way that second batch strain is inoculated on the fermentation medium of the present invention so that growth is fast
Speed, metabolism is vigorous, the yield of zymotic fluid is substantially increased, so as to improve the yield of cephalotaxus alkaloids.
Embodiment
A kind of preparation method of cephalotaxus alkaloids product, chooses the root, stem, leaf of perennial caephalotaxus sinensis as explant, through cutting
Culture in culture medium is put into after bar, sterilizing makes it grow caephalotaxus sinensis symbiotic bacteria, is then isolated from caephalotaxus sinensis symbiotic bacteria metabolizable thick
The strain and cultivation and fermentation of Chinese torreya alkaloid, separate out after zymotic fluid is concentrated and product, the concrete operations of the cultivation and fermentation are made
Method is that will be put into the environment that temperature is 26 DEG C to cultivate 48h after the actication of culture of metabolizable generation cephalotaxus alkaloids, then bacterium
Plant to be inoculated on the culture dish equipped with PDA culture medium and cultivate, second batch is accessed after first strain produces metabolism biological 1-2 days
Caephalotaxus sinensis symbiotic bacteria, to promote the growth of second batch strain, the strain of second batch inoculation to be inoculated into after growing equipped with fermentation medium
Shaking flask in, it is that 200r/min, temperature are that 5d is cultivated on 280C shaking table that shaking flask, which is then put into rotating speed, collects the fermentation of strain
Liquid；
The concrete operation method of the zymotic fluid concentration is that zymotic fluid is put into the freezing high speed that rotating speed is 10000r/min
In centrifuge centrifuge, collect supernatant, supernatant be put into rotating speed be 10000r/min high-speed refrigerated centrifuge in again from
The heart, is then put into the supernatant after centrifuging again in the Rotary Evaporators that temperature is 40-50 DEG C, pressure is 0.05Mpa and concentrates
It is put into 1/10th of original volume, then by concentrate in the refrigerator that temperature is 4 DEG C；
The concrete operation method of the precipitation is to add NaOH solution into concentrate until pH=8~9, separate out alkaloid
And mixture, add mixture in the ethanol that volumetric concentration is 95%, and the volume ratio of mixture and ethanol is 1:10, plus
15-18 DEG C is cooled to after hot to 30 DEG C, alkaloid is separated out, above-mentioned heating, cooling precipitation process is repeated 2-3 times, merges alkaloid,
Product is made.
Fermentation medium is by murphy juice, glucose, peptone, soluble starch, MgSO in the shaking flask4、KH2PO4And water
It is made, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, soluble starch 1%, MgSO4
0.05%, KH2PO40.15%, surplus is water, pH=7 of culture medium；
The murphy juice is by component A and component B according to 10:1 weight is concentrated into than being put into after mixing in Rotary Evaporators
It is made after 1/3rd of original volume, the component A is that boiling 20- in the pot that temperature is 95-100 DEG C is put into after potato is shredded
30min, crosses 100-200 sieves after taking-up, collect filtrate and be made；The component B is to be put into juice extractor to squeeze after potato is shredded,
Again 80-100 mesh sieves of squeezing the juice, collect filtrate and be made.
Embodiment 1
A kind of preparation method of cephalotaxus alkaloids product, comprises the following steps：
Step 1: the selection and processing of explant
The root, stem, leaf of perennial caephalotaxus sinensis are chosen as explant, is cleaned up explant with flowing water, then root is cut into
A length of 5-6cm long block, removes the Lao Pi and branches and leaves on stem, standby；
Step 2: the sterilizing of explant
The explant handled well is put into superclean bench, first with the alcohol sterilizing 5min that volumetric concentration is 75%, then used
Mass concentration is 0.1% mercuric chloride sterilizing 12min, and after sterile washing 4 times, the explant handled well is put into sterilized water, standby
With；
Step 3: the purifies and separates of caephalotaxus sinensis strain
Explant after sterilizing is respectively connected in the blake bottle equipped with PDA culture medium, the blake bottle for being connected to explant
It is placed on the culturing rack that temperature is 25 DEG C and is cultivated, it is special according to the bacterium colony of different strain after strain is grown in blake bottle
The bacterial strain for therefrom isolating metabolizable generation cephalotaxus alkaloids is levied, it is standby；
Step 4: cultivation and fermentation
48h is cultivated by being put into after the actication of culture of metabolizable generation cephalotaxus alkaloids in the environment that temperature is 26 DEG C, then
Strain is inoculated on the culture dish equipped with PDA culture medium and cultivated, and second batch strain is accessed after strain produces metabolism biological, to promote
Enter the growth of second batch strain, the strain of second batch inoculation, which is grown, to be inoculated into after 1 day in the shaking flask equipped with fermentation medium, then
It is that 200r/min, temperature are 28 that shaking flask, which is put into rotating speed,05d is cultivated on C shaking table, the zymotic fluid of strain is collected, it is standby；
Step 5: zymotic fluid is concentrated
Zymotic fluid is put into the high-speed refrigerated centrifuge that rotating speed is 10000r/min and centrifuged, supernatant is collected, supernatant
Liquid is put into the high-speed refrigerated centrifuge that rotating speed is 10000r/min and centrifuged again, and the supernatant after centrifuging again is put into temperature
1/10th of original volume are concentrated into the Rotary Evaporators for being 0.05Mpa for 40 DEG C, pressure, it is 4 that concentrate is put into temperature
DEG C refrigerator in, it is standby；
Step 6: separating out
NaOH solution is added into concentrate until pH=8, separate out alkaloid and mixture, add mixture to volume
Concentration for 95% ethanol in, and mixture and ethanol volume ratio be 1:10, it is heated to after 30 DEG C be cooled to 15 DEG C, separates out life
Alkaloids, repeat above-mentioned heating, cooling precipitation process 2 times, merge alkaloid, that is, cephalotaxus alkaloids is made；
Step 7: preserving
Cephalotaxus alkaloids is added in the ethanol that volumetric concentration is 95%, and the volume ratio of cephalotaxus alkaloids and ethanol is
1:10, it is placed in after sealing dissolving in the refrigerator that temperature is 4 DEG C.
Described in step 4 in shaking flask fermentation medium by murphy juice, glucose, peptone, soluble starch, MgSO4、
KH2PO4It is made with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, soluble starch
1%, MgSO4 0.05%, KH2PO40.15%, surplus is water, pH=7 of culture medium.
The murphy juice is by component A and component B according to 10:1 weight is concentrated into than being put into after mixing in Rotary Evaporators
It is made after 1/3rd of original volume, the component A is that boiling 20min in the pot that temperature is 95 DEG C is put into after potato is shredded,
100 mesh sieves are crossed after taking-up, filtrate is collected and is made；The component B is to be put into juice extractor to squeeze after potato is shredded, then squeezing the juice
80 mesh sieves are crossed, filtrate is collected and is made.
Embodiment 2
A kind of preparation method of cephalotaxus alkaloids product, comprises the following steps：
Step 1: the selection and processing of explant
The root, stem, leaf of perennial caephalotaxus sinensis are chosen as explant, is cleaned up explant with flowing water, then root is cut into
A length of 5-6cm long block, removes the Lao Pi and branches and leaves on stem, standby；
Step 2: the sterilizing of explant
The explant handled well is put into superclean bench, first with the alcohol sterilizing 5min that volumetric concentration is 75%, then used
Mass concentration is 0.1% mercuric chloride sterilizing 12min, and after sterile washing 5 times, the explant handled well is put into sterilized water, standby
With；
Step 3: the purifies and separates of caephalotaxus sinensis strain
Explant after sterilizing is respectively connected in the blake bottle equipped with PDA culture medium, the blake bottle for being connected to explant
It is placed on the culturing rack that temperature is 25 DEG C and is cultivated, it is special according to the bacterium colony of different strain after strain is grown in blake bottle
The bacterial strain for therefrom isolating metabolizable generation cephalotaxus alkaloids is levied, it is standby；
Step 4: cultivation and fermentation
48h is cultivated by being put into after the actication of culture of metabolizable generation cephalotaxus alkaloids in the environment that temperature is 26 DEG C, then
Strain is inoculated on the culture dish equipped with PDA culture medium and cultivated, and second batch strain is accessed after strain produces metabolism biological 2 days,
To promote the growth of second batch strain, the strain of second batch inoculation to be inoculated into after growing in the shaking flask equipped with fermentation medium, so
It is that 200r/min, temperature are 28 that shaking flask, which is put into rotating speed, afterwards05d is cultivated on C shaking table, the zymotic fluid of strain is collected, it is standby；
Step 5: the concentration of zymotic fluid
Zymotic fluid is put into the high-speed refrigerated centrifuge that rotating speed is 10000r/min and centrifuged, supernatant is collected, supernatant
Liquid is put into the high-speed refrigerated centrifuge that rotating speed is 10000r/min and centrifuged again, and the supernatant after centrifuging again is put into temperature
1/10th of original volume are concentrated into the Rotary Evaporators for being 0.05Mpa for 50 DEG C, pressure, it is 4 that concentrate is put into temperature
DEG C refrigerator in, it is standby；
Step 6: separating out
NaOH solution is added into concentrate until pH=9, separate out alkaloid and mixture, add mixture to volume
Concentration for 95% ethanol in, and mixture and ethanol volume ratio be 1:10, it is heated to after 30 DEG C be cooled to 18 DEG C, separates out life
Alkaloids, repeat above-mentioned heating, cooling precipitation process 3 times, merge alkaloid, that is, cephalotaxus alkaloids is made；
Step 7: preserving
Cephalotaxus alkaloids is added in the ethanol that volumetric concentration is 95%, and the volume ratio of cephalotaxus alkaloids and ethanol is
1:10, it is placed in after sealing dissolving in the refrigerator that temperature is 4 DEG C.
Described in step 4 in shaking flask fermentation medium by murphy juice, glucose, peptone, soluble starch, MgSO4、
KH2PO4It is made with water, and the mass percent of each component is murphy juice 2%, glucose 2%, peptone 0.12%, soluble starch
1%, MgSO4 0.05%, KH2PO40.15%, surplus is water, pH=7 of culture medium.
The murphy juice is by component A and component B according to 10:1 weight is concentrated into than being put into after mixing in Rotary Evaporators
It is made after 1/3rd of original volume, the component A is that boiling 30min in the pot that temperature is 100 DEG C is put into after potato is shredded,
200 mesh sieves are crossed after taking-up, filtrate is collected and is made；The component B is to be put into juice extractor to squeeze after potato is shredded, then squeezing the juice
100 mesh sieves are crossed, filtrate is collected and is made.
Claims (1)
1. a kind of preparation method of cephalotaxus alkaloids product, chooses the root, stem, leaf of perennial caephalotaxus sinensis as explant, through cutting,
Culture in culture medium is put into after sterilizing makes it grow caephalotaxus sinensis symbiotic bacteria, and metabolizable caephalotaxus sinensis life is then isolated from caephalotaxus sinensis symbiotic bacteria
The strain and cultivation and fermentation of alkaloids, separate out after zymotic fluid is concentrated and product are made, it is characterised in that：
The concrete operation method of the cultivation and fermentation is that will be put into temperature after the actication of culture of metabolizable generation cephalotaxus alkaloids to be
48h is cultivated in 26 DEG C of environment, then strain is inoculated into culture on the culture dish equipped with PDA culture medium, treats that first strain is produced
Raw metabolism biological accesses second batch caephalotaxus sinensis symbiotic bacteria after 1-2 days, to promote the growth of second batch strain, the strain of second batch inoculation
It is inoculated into after growing in the shaking flask equipped with fermentation medium, it is that 200r/min, temperature are 280C's that shaking flask, which is then put into rotating speed,
5d is cultivated on shaking table, the zymotic fluid of strain is collected；Wherein, in the shaking flask fermentation medium by murphy juice, glucose, egg
White peptone, soluble starch, MgSO4、KH2PO4Be made with water, and each component mass percent be murphy juice 2%, glucose 2%,
Peptone 0.12%, soluble starch 1%, MgSO4 0.05%, KH2PO40.15%, surplus is water, pH=7 of culture medium；It is described
Murphy juice is by component A and component B according to 10:1 weight is concentrated into three points of original volume than being put into after mixing in Rotary Evaporators
One of it is rear be made, the component A is that temperature is put into after potato is shredded is boiling 20-30min in 95-100 DEG C of pot, after taking-up
100-200 sieves are crossed, filtrate is collected and is made；The component B is to be put into juice extractor to squeeze after potato is shredded, then squeezing the juice
80-100 mesh sieves, collect filtrate and are made；
The concrete operation method of the zymotic fluid concentration is that zymotic fluid is put into the freezing high speed centrifugation that rotating speed is 10000r/min
Centrifuged in machine, collect supernatant, supernatant is put into the high-speed refrigerated centrifuge that rotating speed is 10000r/min and centrifuged again, so
The supernatant after centrifuging again is put into the Rotary Evaporators that temperature is 40-50 DEG C, pressure is 0.05Mpa afterwards and is concentrated into substance
Long-pending 1/10th, then concentrate is put into the refrigerator that temperature is 4 DEG C；
The concrete operation method of the precipitation is to add NaOH solution into concentrate until pH=8~9, precipitation mixture will be mixed
Compound is added in the ethanol that volumetric concentration is 95%, and the volume ratio of mixture and ethanol is 1:10, it is heated to cool down after 30 DEG C
To 15-18 DEG C, alkaloid is separated out, above-mentioned heating, cooling precipitation process is repeated 2-3 times, merges alkaloid, that is, product is made.
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Cited By (1)
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| 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 |
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| 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;陈传文;《中国优秀硕士学位论文全文数据库》;20130415;正文第2.1.1-2.2.4、3.1.2、3.2.1、3.3节，表7、10 * |
Cited By (1)
| 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 |
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