CN104805170B - Ganodenic acid acid submerged fermentation process for extracting - Google Patents
Ganodenic acid acid submerged fermentation process for extracting Download PDFInfo
- Publication number
- CN104805170B CN104805170B CN201510147874.4A CN201510147874A CN104805170B CN 104805170 B CN104805170 B CN 104805170B CN 201510147874 A CN201510147874 A CN 201510147874A CN 104805170 B CN104805170 B CN 104805170B
- Authority
- CN
- China
- Prior art keywords
- acid
- extracting
- extract
- afterwards
- fermentation process
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of ganodenic acid acid fermentation process for extracting, and in particular to a kind of ganodenic acid acid submerged fermentation process for extracting that can effectively improve yield belongs to technical field of biological fermentation.A kind of ganodenic acid acid submerged fermentation process for extracting, includes the following steps：(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：4.9 6.9g/L of 25.0 27.0g/L of glucose, 10.2 12.2g/L of corn flour, 9.8 11.8g/L of wheatfeed and peptone；(2) fermented and cultured；(3) extraction separation.Ganodenic acid acid submerged fermentation process for extracting provided by the present invention can effectively solve the ganoderma lucidum fruitbody cultivation wasting of resources in the prior art, and triterpenic acid culture extraction time is long, and effective recovery rate and purity is not high, the problem of being not easy to carry out large-scale industrial production.
Description
Technical field
The present invention relates to a kind of ganodenic acid acid fermentation process for extracting, and in particular to a kind of to effectively improve yield
Ganodenic acid acid submerged fermentation process for extracting, belongs to technical field of biological fermentation.
Background technology
Ganodenic acid acid belongs to the secondary metabolism object of ganoderma lucidum, be the key drug effect of another kind in ganoderma lucidum in addition to polysaccharide into
Point, with the works such as analgesic, calm inhibition histamine release, removing toxic substances, liver protection, poisoning tumour cell, raising immunity of organisms, hypoglycemic
With.
Horizontal not high, the relatively low hair for limiting its industrialized production of yield of fermentation extraction of ganodenic acid acid in the prior art
Exhibition, it is impossible to meet the needs of modern industrialization fermenting and producing.
Solution fermentation, which is proposed, in the Chinese patent of application number 00111953.2 produces ganoderma lucidum polysaccharide and ganoderma lucidum simultaneously
The technology of triterpenic acid.The shortcomings that technology is that the fermentation period of ganodenic acid is too long, and liquid aerobic fermentation is plus quiescent culture
Total fermentation time is up to 2-3 weeks, and the production cycle is long, and therefore, whole production efficiency is relatively low.
At present, the organic solvent method used in the prior art usually requires longer extraction time and consumes substantial amounts of extraction
Solvent, while the use of a large amount of organic solvents can bring detrimental effect to human body and environment.Additionally, due to ganoderma lucidum fruitbody by
The main components such as cellulose, hemicellulose and lignin are formed, close structure, and traditional extraction time is long, consume energy high, activity
Component extract yield is low.
The content of the invention
It is provided by the present invention it is an object of the invention to provide a kind of ganodenic acid acid submerged fermentation process for extracting
Ganodenic acid acid submerged fermentation process for extracting can effectively solve the ganoderma lucidum fruitbody cultivation wasting of resources in the prior art, triterpene
Acid culture extraction time is long, and effective recovery rate and purity is not high, the problem of being not easy to carry out large-scale industrial production.
Technical scheme is as follows：
A kind of ganodenic acid acid submerged fermentation process for extracting, the ganodenic acid acid submerged fermentation process for extracting
Include the following steps：
(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：Glucose 25.0-27.0g/
L, corn flour 10.2-12.2g/L, wheatfeed 9.8-11.8g/L and peptone 4.9-6.9g/L；
(2) fermented and cultured：The campanulaceae water extract of 400-500mg/L or the gold and silver of 180-220mg/L are added in the medium
Flower and matrimony vine alcohol extract access ganoderma lucidum inoculum and carry out fermented and cultured afterwards, and fermentation culture conditions are as follows：Fermentation starting pH
It is worth for 5.5-6.5, temperature is 28-32 DEG C, shaking speed 150-170r/min, fermentation time 166-170h；Fermentation process
After by obtained glossy ganoderma fermentation thalline, drying to constant weight；The ganoderma lucidum inoculum is real for ganoderma lucidum mycelium or ganoderma lucidum
Body；
(3) extraction separation：Comprise the following steps：
1. being extracted three to four times after the dry glossy ganoderma fermentation thalline to constant weight is crushed, the extracting process is：First by spirit
The crushed material of sesame fermentation thalli presses 0.8-1.2 with chloroform extracted solution:14(W:V) ratio mixes, then by the mixed liquor and concentration
For 4.8-5.2% (W:V NaHCO)3Solution is according to 2.8-3.2:1(V:V) ratio mixes, after impregnating 22-26h, extraction extraction
Liquid is extracted surplus materials according to this method next time；The extract liquor for extracting acquisition several times is merged afterwards, utilizes ultrasound
Ancillary equipment under 380-420W ultrasound output powers, using the supersonic frequency of 50-60kHz, is ultrasonically treated 28-32min, stops
Stagnant 8-12min, is ultrasonically treated 28-32min again；Then it is separated by filtration；
2. filtrate α and filter residue are obtained after being separated by filtration；Filter residue is first pressed into 0.8-1.2:8(W:V) ratio addition concentration is
4.8-5.2% (W:V)NaHCO3Solution, using ultrasonic wave added equipment, under 380-420W ultrasound output powers, using 50-
The supersonic frequency of 60kHz is ultrasonically treated 28-32min, stagnates 8-12min, is ultrasonically treated 28-32min again, utilizes hole afterwards
Footpath is that 0.4-0.5 μm of filtering with microporous membrane obtains filtrate β；Then filtrate α and filtrate β is merged, opens dispersion machine, adjustment turns
Speed disperses 18-22min, obtains supernatant to 4800-5200r/min；Afterwards under 3.8-4.2 DEG C of condition of ice bath, supernatant is used
The dilute hydrochloric acid of 5.8-6.2mol/L carries out being acidified to pH=2.8-3.2, obtains acidifying solution；
3. after acidifying solution is extracted 3-5 times with isometric chloroform extracted solution, simultaneously combining extraction liquid is collected, extract liquor is quiet
35-45min is put, with 0.4-0.5 μm of the filtering with microporous membrane in aperture, obtains yellow clear liquid；
4. evaporated under reduced pressure obtains yellow viscous liquid under the conditions of 44-46 DEG C by yellow clear liquid, yellow viscous liquid is existed
It is dry under 58-62 DEG C of water bath condition that drying to constant weight；
1. 3. the chloroform extracted solution is according to 95 by chloroform and absolute ethyl alcohol to step with step:5-98:2 volume is matched somebody with somebody
Than being formulated.
Further, the extraction process of the campanulaceae water extract is as follows：Campanulaceae 90-110g is taken, boils 25- with decocting system
35min pours out the dregs of a decoction, adds appropriate cold water in the dregs of a decoction, then boils 18-22min, pours out concoction, adds again afterwards appropriate cold
Water boils 18-22min in the dregs of a decoction, pours out concoction, merges concoction, concoction is filtered, by the concoction after filtering at 55-65 DEG C
It is dry, obtain campanulaceae water extract.
The extraction process of the honeysuckle and matrimony vine alcohol extract is as follows：By honeysuckle and matrimony vine according to 0.8-1.2：1 matter
After amount proportioning mixing, 30-50 mesh crushes, and binds up with gauze tighten afterwards, be put into reflow of alcohol extraction apparatus, with 2.5-3.5 times
Ethyl alcohol extraction 2.5-3.5h, obtain extract liquor, by surplus materials add 2.5-3.5 times ethyl alcohol extraction 1.5-2.5h, then
Secondary acquisition extract liquor, merges the extract liquor obtained twice afterwards, and revolving is concentrated under reduced pressure to give honeysuckle and matrimony vine alcohol extracting thick liquid；
The concentration expressed in percentage by volume of the ethyl alcohol is 93-97%.
Further, extract liquor of the of step (3) 3. described in step first added in before standing cellulose complex enzyme into
Row enzymolysis, 45-55 DEG C, pH=5.5-6.5, enzymolysis time 18-22min of hydrolysis temperature, the addition of cellulose complex enzyme is extraction
Take the 1.5-2.5% (W of liquid:V)；The cellulose complex enzyme presses Rate activity 4.8-5.2 by cellulase, protease, pectase:
2.8-3.2:1.8-2.2 it mixes.
The ganodenic acid acid submerged fermentation process for extracting comparison prior art provided by the present invention has the following advantages：
1) cultivation cycle of strain is short；
2) raw material are easy to get, and production cost is low；
3) it is energy-saving；
4) production efficiency of ganodenic acid acid and extraction yield, the ganodenic acid acid stable quality of gained are improved；
5) stable yield, from the influence of season and raw material；
6) it is suitble to large-scale industrial production.
Specific embodiment
The present invention will be described in detail with specific embodiment With reference to embodiment.
(1) specific embodiment is as follows：
A kind of ganodenic acid acid submerged fermentation process for extracting, the ganodenic acid acid submerged fermentation process for extracting
Include the following steps：
(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：Glucose 25.0-27.0g/
L, corn flour 10.2-12.2g/L, wheatfeed 9.8-11.8g/L and peptone 4.9-6.9g/L；
(2) fermented and cultured：The campanulaceae water extract of 400-500mg/L or the gold and silver of 180-220mg/L are added in the medium
Flower and matrimony vine alcohol extract access ganoderma lucidum inoculum and carry out fermented and cultured afterwards, and fermentation culture conditions are as follows：Fermentation starting pH
It is worth for 5.5-6.5, temperature is 28-32 DEG C, shaking speed 150-170r/min, fermentation time 166-170h；Fermentation process
After by obtained glossy ganoderma fermentation thalline, drying to constant weight；The ganoderma lucidum inoculum is real for ganoderma lucidum mycelium or ganoderma lucidum
Body；
(3) extraction separation：Comprise the following steps：
1. being extracted three to four times after the dry glossy ganoderma fermentation thalline to constant weight is crushed, the extracting process is：First by spirit
The crushed material of sesame fermentation thalli presses 0.8-1.2 with chloroform extracted solution:14(W:V) ratio mixes, then by the mixed liquor and concentration
For 4.8-5.2% (W:V NaHCO)3Solution is according to 2.8-3.2:1(V:V) ratio mixes, after impregnating 22-26h, extraction extraction
Liquid is extracted surplus materials according to this method next time；The extract liquor for extracting acquisition several times is merged afterwards, utilizes ultrasound
Ancillary equipment under 380-420W ultrasound output powers, using the supersonic frequency of 50-60kHz, is ultrasonically treated 28-32min, stops
Stagnant 8-12min, is ultrasonically treated 28-32min again；Then it is separated by filtration；
2. filtrate α and filter residue are obtained after being separated by filtration；Filter residue is first pressed into 0.8-1.2:8(W:V) ratio addition concentration is
4.8-5.2% (W:V)NaHCO3Solution, using ultrasonic wave added equipment, under 380-420W ultrasound output powers, using 50-
The supersonic frequency of 60kHz is ultrasonically treated 28-32min, stagnates 8-12min, is ultrasonically treated 28-32min again, utilizes hole afterwards
Footpath is that 0.4-0.5 μm of filtering with microporous membrane obtains filtrate β；Then filtrate α and filtrate β is merged, opens dispersion machine, adjustment turns
Speed disperses 18-22min, obtains supernatant to 4800-5200r/min；Afterwards under 3.8-4.2 DEG C of condition of ice bath, supernatant is used
The dilute hydrochloric acid of 5.8-6.2mol/L carries out being acidified to pH=2.8-3.2, obtains acidifying solution；
3. after acidifying solution is extracted 3-5 times with isometric chloroform extracted solution, simultaneously combining extraction liquid is collected, extract liquor is quiet
35-45min is put, with 0.4-0.5 μm of the filtering with microporous membrane in aperture, obtains yellow clear liquid；
4. evaporated under reduced pressure obtains yellow viscous liquid under the conditions of 44-46 DEG C by yellow clear liquid, yellow viscous liquid is existed
It is dry under 58-62 DEG C of water bath condition that drying to constant weight；
1. 3. the chloroform extracted solution is according to 95 by chloroform and absolute ethyl alcohol to step with step:5-98:2 volume is matched somebody with somebody
Than being formulated.
Extract liquor of the of step (3) 3. described in step can first add in cellulose complex enzyme before standing and be digested, enzyme
45-55 DEG C, pH=5.5-6.5, enzymolysis time 18-22min of temperature is solved, the addition of cellulose complex enzyme is the 1.5- of extract liquor
2.5% (W:V)；The cellulose complex enzyme presses Rate activity 4.8-5.2 by cellulase, protease, pectase:2.8-3.2:
1.8-2.2 it mixes.
The extraction process of the campanulaceae water extract is as follows：Campanulaceae 90-110g is taken, 25-35min is boiled with decocting system, pours out
The dregs of a decoction add appropriate cold water in the dregs of a decoction, then boil 18-22min, pour out concoction, add appropriate cold water again afterwards in the dregs of a decoction,
18-22min is boiled, pours out concoction, merges concoction, filters concoction, the concoction after filtering is dry at 55-65 DEG C, obtain tangerine
Obstruct water extract.
The extraction process of the honeysuckle and matrimony vine alcohol extract is as follows：By honeysuckle and matrimony vine according to 0.8-1.2：1 matter
After amount proportioning mixing, 30-50 mesh crushes, and binds up with gauze tighten afterwards, be put into reflow of alcohol extraction apparatus, with 2.5-3.5 times
Ethyl alcohol extraction 2.5-3.5h, obtain extract liquor, by surplus materials add 2.5-3.5 times ethyl alcohol extraction 1.5-2.5h, then
Secondary acquisition extract liquor, merges the extract liquor obtained twice afterwards, and revolving is concentrated under reduced pressure to give honeysuckle and matrimony vine alcohol extracting thick liquid；
The concentration expressed in percentage by volume of the ethyl alcohol is 93-97%.
(2) embodiment is as follows：It is further illustrated the present invention below by embodiment：
Embodiment group 1
Embodiment 1-1
A kind of ganodenic acid acid submerged fermentation process for extracting, the ganodenic acid acid submerged fermentation process for extracting
Include the following steps：
(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：Glucose 26.0g/L, it is beautiful
Rice flour 11.2g/L, wheatfeed 10.8g/L and peptone 5.9g/L；
(2) fermented and cultured：The campanulaceae water extract of 450mg/L is added in the medium, accesses ganoderma lucidum inoculum afterwards
Fermented and cultured is carried out, fermentation culture conditions are as follows：Initial ph value of fermenting is 6, and temperature is 30 DEG C, shaking speed 160r/min,
Fermentation time is 168h；By obtained glossy ganoderma fermentation thalline, drying to constant weight after fermentation process；The ganoderma lucidum seed culture
Object is ganoderma lucidum mycelium or ganoderma lucidum fruitbody；
The extraction process of the campanulaceae water extract is as follows：Campanulaceae 100g is taken, boils 30min with decocting system, pours out the dregs of a decoction, is mended
Add appropriate cold water in the dregs of a decoction, then boil 20min, pour out concoction, boil 20min again afterwards, pour out concoction, merge concoction, mistake
Filter, it is dry at 60 DEG C, obtain campanulaceae water extract；
(3) extraction separation：Comprise the following steps：
1. being extracted three times after the dry glossy ganoderma fermentation thalline to constant weight is crushed, the extracting process is：First ganoderma lucidum is sent out
The crushed material of yeast-like fungi body presses 1 with chloroform extracted solution:14(W:V) ratio mixes, and is then 5% (W by the mixed liquor and concentration:V)
NaHCO3Solution is according to 3:1(V:V) ratio mix, impregnate for 24 hours after, extract extract liquor, by surplus materials according to this method into
Row extracts next time；The extract liquor for extracting acquisition several times is merged afterwards, using ultrasonic wave added equipment, in 400W ultrasound output works
Under rate, using the supersonic frequency of 55kHz, 30min is ultrasonically treated, 10min is stagnated, is ultrasonically treated 30min again；Then filtering point
From；
2. filtrate α and filter residue are obtained after being separated by filtration；Filter residue is first pressed 1:8(W:V it is 5.0% (W that) ratio, which adds in concentration,:
V)NaHCO3Solution, using ultrasonic wave added equipment, under 400W ultrasound output powers, using the supersonic frequency of 55kHz, at ultrasound
30min is managed, 10min is stagnated, is ultrasonically treated 30min again, is filtered afterwards using the filtering with microporous membrane that aperture is 0.45 μm
Liquid β；Then filtrate α and filtrate β is merged, opens dispersion machine, adjustment rotating speed to 5000r/min disperses 20min, obtains supernatant；
Afterwards under 4 DEG C of condition of ice bath, supernatant is carried out being acidified to pH=3 with the dilute hydrochloric acid of 6mol/L, obtains acidifying solution；
3. it after acidifying solution is extracted 4 times with isometric chloroform extracted solution, collects and combining extraction liquid, standing 40min is used
The filtering with microporous membrane that 0.45 μm of aperture obtains yellow clear liquid；
4. by yellow clear liquid, evaporated under reduced pressure obtains yellow viscous liquid under the conditions of 45 DEG C, by yellow viscous liquid at 60 DEG C
It is dry under water bath condition that drying to constant weight；
1. 3. the chloroform extracted solution is according to 18 by chloroform and absolute ethyl alcohol to step with step:1 volume proportion is matched somebody with somebody
It makes.
Embodiment 1-2
Embodiment 1-2 and embodiment 1-1 difference lies in：The campanulaceae water extract added in culture medium is replaced with into 200mg/L
Honeysuckle and matrimony vine alcohol extract.
The extraction process of the honeysuckle and matrimony vine alcohol extract is as follows：By honeysuckle and matrimony vine according to 1：1 quality proportioning
After mixing, 40 mesh crush, and bind up with gauze tighten afterwards, be put into reflow of alcohol extraction apparatus, extract 3h with 3 times of ethyl alcohol, obtain
Extract liquor is obtained, surplus materials is added to 3 times of ethyl alcohol and extracts 2h, extract liquor is obtained again, merges the extraction obtained twice afterwards
Liquid is taken, revolving is concentrated under reduced pressure to give honeysuckle and matrimony vine alcohol extracting thick liquid；The concentration expressed in percentage by volume of the ethyl alcohol is 95%.
Embodiment 1-3
Embodiment 1-3 and embodiment 1-1 difference lies in：The of step (3) 3. the extract liquor described in step before standing first
It adds in cellulose complex enzyme to be digested, 50 DEG C, pH=6.0, enzymolysis time 20min of hydrolysis temperature, cellulose complex enzyme adds
Enter the 2.0% (W that amount is extract liquor:V)；The cellulose complex enzyme presses Rate activity 5 by cellulase, protease, pectase:3:
2 mix.
Embodiment group 2
Embodiment 2-1
A kind of ganodenic acid acid submerged fermentation process for extracting, the ganodenic acid acid submerged fermentation process for extracting
Include the following steps：
(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：Glucose 25.0g/L, it is beautiful
Rice flour 10.2g/L, wheatfeed 9.8g/L and peptone 4.9g/L；
(2) fermented and cultured：The campanulaceae water extract of 400mg/L is added in the medium, accesses ganoderma lucidum inoculum afterwards
Fermented and cultured is carried out, fermentation culture conditions are as follows：Initial ph value of fermenting is 5.5, and temperature is 28 DEG C, shaking speed 160r/
Min, fermentation time 166h；By obtained glossy ganoderma fermentation thalline, drying to constant weight after fermentation process；The ganoderma lucidum seed
Culture is ganoderma lucidum mycelium or ganoderma lucidum fruitbody；
The extraction process of the campanulaceae water extract is as follows：Campanulaceae 100g is taken, boils 30min with decocting system, pours out the dregs of a decoction, is mended
Add appropriate cold water in the dregs of a decoction, then boil 20min, pour out concoction, boil 20min again afterwards, pour out concoction, merge concoction, mistake
Filter, it is dry at 60 DEG C, obtain campanulaceae water extract；
(3) extraction separation：Comprise the following steps：
1. being extracted three times after the dry glossy ganoderma fermentation thalline to constant weight is crushed, the extracting process is：First ganoderma lucidum is sent out
The crushed material of yeast-like fungi body presses 0.8 with chloroform extracted solution:14(W:V) ratio mixes, and is then 4.8% by the mixed liquor and concentration
(W:V NaHCO)3Solution is according to 2.8:1(V:V) ratio mixes, and after impregnating 22h, extract liquor is extracted, by surplus materials according to this
Method is extracted next time；The extract liquor for extracting acquisition several times is merged afterwards, using ultrasonic wave added equipment, in 380W ultrasounds
Under output power, using the supersonic frequency of 50kHz, 28min is ultrasonically treated, 8min is stagnated, is ultrasonically treated 28min again；Then
It is separated by filtration；
2. filtrate α and filter residue are obtained after being separated by filtration；Filter residue is first pressed 0.8:8(W:V it is 4.8% that) ratio, which adds in concentration,
(W:V)NaHCO3Solution using ultrasonic wave added equipment, under 380W ultrasound output powers, using the supersonic frequency of 50kHz, surpasses
Sonication 28min stagnates 8min, is ultrasonically treated 28min again, is obtained afterwards using the filtering with microporous membrane that aperture is 0.4 μm
Filtrate β；Then filtrate α and filtrate β is merged, opens dispersion machine, adjustment rotating speed to 4800r/min disperses 18min, obtains supernatant
Liquid；Afterwards under 3.8 DEG C of condition of ice bath, supernatant is carried out being acidified to pH=2.8 with the dilute hydrochloric acid of 5.8mol/L, is acidified
Liquid；
3. it after acidifying solution is extracted 3 times with isometric chloroform extracted solution, collects and combining extraction liquid, standing 35min is used
The filtering with microporous membrane that 0.5 μm of aperture obtains yellow clear liquid；
4. by yellow clear liquid, evaporated under reduced pressure obtains yellow viscous liquid under the conditions of 44 DEG C, by yellow viscous liquid at 58 DEG C
It is dry under water bath condition that drying to constant weight；
1. 3. the chloroform extracted solution is according to 19 by chloroform and absolute ethyl alcohol to step with step:1 volume proportion is matched somebody with somebody
It makes.
Embodiment 2-2
Embodiment 1-2 and embodiment 1-1 difference lies in：The campanulaceae water extract added in culture medium is replaced with into 180mg/L
Honeysuckle and matrimony vine alcohol extract.
The extraction process of the honeysuckle and matrimony vine alcohol extract is as follows：By honeysuckle and matrimony vine according to 0.8：1 quality is matched somebody with somebody
After mixing, 30 mesh crush, and bind up with gauze tighten afterwards, be put into reflow of alcohol extraction apparatus, are extracted with 2.5 times of ethyl alcohol
2.5h obtains extract liquor, and surplus materials is added to 2.5 times of ethyl alcohol and extracts 1.5h, extract liquor is obtained again, merges two afterwards
The extract liquor of secondary acquisition, revolving are concentrated under reduced pressure to give honeysuckle and matrimony vine alcohol extracting thick liquid；The concentration expressed in percentage by volume of the ethyl alcohol
For 93%.
Embodiment 2-3
Embodiment 1-3 and embodiment 1-1 difference lies in：Extract liquor of the of step (3) 3. described in step can before standing
It first adds in cellulose complex enzyme to be digested, 45 DEG C, pH=5.5, enzymolysis time 18min of hydrolysis temperature, cellulose complex enzyme
Addition is 1.5% (W of extract liquor:V)；The cellulose complex enzyme presses Rate activity by cellulase, protease, pectase
4.8:2.8:1.8 it mixes.
Embodiment group 3
Embodiment 3-1
A kind of ganodenic acid acid submerged fermentation process for extracting, the ganodenic acid acid submerged fermentation process for extracting
Include the following steps：
(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：Glucose 27.0g/L, it is beautiful
Rice flour 12.2g/L, wheatfeed 11.8g/L and peptone 6.9g/L；
(2) fermented and cultured：The campanulaceae water extract of 500mg/L is added in the medium, accesses ganoderma lucidum inoculum afterwards
Fermented and cultured is carried out, fermentation initial ph value is 6.5, and temperature is 32 DEG C, shaking speed 170r/min, fermentation time 170h；
By obtained glossy ganoderma fermentation thalline, drying to constant weight after fermentation process；The ganoderma lucidum inoculum for ganoderma lucidum mycelium or
Ganoderma lucidum fruitbody；
The extraction process of the campanulaceae water extract is as follows：Campanulaceae 110g is taken, boils 35min with decocting system, pours out the dregs of a decoction, is mended
Add appropriate cold water in the dregs of a decoction, then boil 22min, pour out concoction, add appropriate cold water again afterwards in the dregs of a decoction, boil 22min,
Concoction is poured out, merges concoction, filters concoction, the concoction after filtering is dry at 65 DEG C, obtain campanulaceae water extract；
(3) extraction separation：Comprise the following steps：
1. being extracted three to four times after the dry glossy ganoderma fermentation thalline to constant weight is crushed, the extracting process is：First by spirit
The crushed material of sesame fermentation thalli presses 1.2 with chloroform extracted solution:14(W:V) ratio mixes, and is then with concentration by the mixed liquor
5.2% (W:V NaHCO)3Solution is according to 3.2:1(V:V) ratio mixes, and after impregnating 26h, extract liquor is extracted, by surplus materials
It is extracted next time according to this method；The extract liquor that acquisition will be extracted several times afterwards merges, using ultrasonic wave added equipment,
Under 420W ultrasound output powers, using the supersonic frequency of 60kHz, 32min is ultrasonically treated, 12min is stagnated, is ultrasonically treated again
32min；Then it is separated by filtration；
2. filtrate α and filter residue are obtained after being separated by filtration；Filter residue is first pressed 1.2:8(W:V it is 5.2% that) ratio, which adds in concentration,
(W:V)NaHCO3Solution using ultrasonic wave added equipment, under 420W ultrasound output powers, using the supersonic frequency of 60kHz, surpasses
Sonication 32min stagnates 12min, is ultrasonically treated 32min again, is obtained afterwards using the filtering with microporous membrane that aperture is 0.5 μm
Filtrate β；Then filtrate α and filtrate β is merged, opens dispersion machine, adjustment rotating speed to 5200r/min disperses 22min, obtains supernatant
Liquid；Afterwards under 4.2 DEG C of condition of ice bath, supernatant is carried out being acidified to pH=3.2 with the dilute hydrochloric acid of 6.2mol/L, is acidified
Liquid；
3. after acidifying solution is extracted 5 times with isometric chloroform extracted solution, simultaneously combining extraction liquid is collected, extract liquor is stood
45min with 0.5 μm of the filtering with microporous membrane in aperture, obtains yellow clear liquid；
4. by yellow clear liquid, evaporated under reduced pressure obtains yellow viscous liquid under the conditions of 46 DEG C, by yellow viscous liquid at 62 DEG C
It is dry under water bath condition that drying to constant weight；
1. 3. the chloroform extracted solution is according to 49 by chloroform and absolute ethyl alcohol to step with step:1 volume proportion is matched somebody with somebody
It makes.
Embodiment 3-2
Embodiment 1-2 and embodiment 1-1 difference lies in：The campanulaceae water extract added in culture medium is replaced with into 220mg/L
Honeysuckle and matrimony vine alcohol extract.
The extraction process of the honeysuckle and matrimony vine alcohol extract is as follows：By honeysuckle and matrimony vine according to 1.2：1 quality is matched somebody with somebody
After mixing, 50 mesh crush, and bind up with gauze tighten afterwards, be put into reflow of alcohol extraction apparatus, are extracted with 3.5 times of ethyl alcohol
3.5h obtains extract liquor, and surplus materials is added to 3.5 times of ethyl alcohol and extracts 2.5h, extract liquor is obtained again, merges two afterwards
The extract liquor of secondary acquisition, revolving are concentrated under reduced pressure to give honeysuckle and matrimony vine alcohol extracting thick liquid；The concentration expressed in percentage by volume of the ethyl alcohol
For 97%.
Embodiment 3-3
Embodiment 1-3 and embodiment 1-1 difference lies in：Extract liquor of the of step (3) 3. described in step can before standing
It first adds in cellulose complex enzyme to be digested, 55 DEG C, pH=6.5, enzymolysis time 22min of hydrolysis temperature, cellulose complex enzyme
Addition is 2.5% (W of extract liquor:V)；The cellulose complex enzyme presses Rate activity by cellulase, protease, pectase
5.2:3.2:2.2 it mixes.
In various embodiments above：
By the test to each embodiment, the ganodenic acid acid submerged fermentation process for extracting all technical is as follows
Shown in each table：
Table 1 is culture before each embodiment inoculation of ganodenic acid acid submerged fermentation process for extracting provided by the present invention
The residual sugar amount of culture medium and the experimental data of residual nitrogen concentration when total sugar amount, total nitrogen concentration and the carbon-nitrogen ratio and fermentation ends of base：
| Project | Total sugar amount | Total nitrogen concentration | Carbon-nitrogen ratio | Residual sugar amount | Residual nitrogen concentration |
| Embodiment 1-1 | 3.0% | 0.103% | 29.1:1 | 0.8% | 8.6% |
| Embodiment 1-2 | 2.8% | 0.091% | 30.7:1 | 0.7% | 6.7% |
| Embodiment 1-3 | 3.3% | 0.106% | 31.1:1 | 1% | 9.5% |
| Embodiment 2-1 | 2.8% | 0.104% | 26.8:1 | 0.9% | 7.9% |
| Embodiment 2-2 | 3.5% | 0.115% | 30.2:1 | 1.2% | 10% |
| Embodiment 2-3 | 3.1% | 0.113% | 27.4:1 | 0.9% | 8.8% |
| Embodiment 3-1 | 2.9% | 0.101% | 28.7:1 | 0.7% | 6.7% |
| Embodiment 3-2 | 3.2% | 0.116% | 25.8:1 | 1.1% | 9.6% |
| Embodiment 3-3 | 2.9% | 0.114% | 25.4:1 | 0.9% | 7.6% |
From 1 each embodiment of table inoculation before culture medium total sugar amount, total nitrogen concentration and carbon-nitrogen ratio and fermentation ends when it is residual
Sugar amount and residual nitrogen concentration, comparison is it will be evident that ganodenic acid acid submerged fermentation process for extracting provided by the present invention has
Have suitable Preliminary fermentation environment, the raising for biomass in fermentation process is had laid a good foundation, after fermentation residual sugar amount and
Residual nitrogen concentration is relatively low.
Table 2 is ganoderma lucidum acquired in each embodiment of ganodenic acid acid extraction and separation process method provided by the present invention
The experimental data of fermentation mycelium
| Project | Unit | Embodiment 1-1 | Embodiment 2-1 | Embodiment 3-1 | Common process |
| Biomass (DCW) | g/L | 9.5 | 9.1 | 8.9 | 7.6 |
| Biomass increase rate | - | 25.0% | 19.7% | 17.1% | - |
| Intracellular triterpene acid | mg/L | 229 | 217 | 211 | 192.5 |
| Intracellular triterpene acid increase rate | - | 19.0% | 12.9% | 9.6% | - |
| Extracellular triterpenic acid | mg/L | 289 | 308 | 334 | 230 |
| Extracellular triterpenic acid increase rate | - | 25.7% | 33.9% | 45.2% | - |
| Project | Unit | Embodiment 1-2 | Embodiment 2-2 | Embodiment 3-2 | Common process |
| Biomass (DCW) | g/L | 9.7 | 9.3 | 9.1 | 7.6 |
| Biomass increase rate | - | 27.7% | 22.4% | 19.8% | - |
| Intracellular triterpene acid | mg/L | 249 | 237 | 221 | 192.5 |
| Intracellular triterpene acid increase rate | - | 29.3% | 23.1% | 14.8% | - |
| Extracellular triterpenic acid | mg/L | 310 | 340 | 366 | 230 |
| Extracellular triterpenic acid increase rate | - | 34.7% | 47.8% | 59.1% | - |
| Project | Unit | Embodiment 1-3 | Embodiment 2-3 | Embodiment 3-3 | Common process |
| Biomass (DCW) | g/L | 10.67 | 10.23 | 10.0 | 7.6 |
| Biomass increase rate | - | 30.5% | 24.6% | 21.8% | - |
| Intracellular triterpene acid | mg/L | 274 | 261 | 243 | 192.5 |
| Intracellular triterpene acid increase rate | - | 32.2% | 25.4% | 16.3% | - |
| Extracellular triterpenic acid | mg/L | 341 | 374 | 403 | 230 |
| Extracellular triterpenic acid increase rate | - | 38.2% | 52.6% | 75.2% | - |
As apparent from Table 2, it is acquired in ganodenic acid acid extraction and separation process procedure provided by the present invention
The more conventional technique of biomass of fermented hyphostroma of Ganoderma can improve 17.1%-30.5%, final acquired and extracellular triterpene
The more conventional technique of acid greatly improves, and wherein intracellular triterpene acid increase rate is 9.6%-32.2%, extracellular triterpenic acid increase rate
For 25.7%-75.2%.
Each embodiment organic solvent of 3 ganodenic acid acid submerged fermentation process for extracting provided by the present invention of table returns
The experimental data of receipts ratio and this consumption ratio：
| Project | Organic solvent recycles ratio | Organic solvent this consumption ratio |
| Embodiment 1-1 | 98.46% | 1.54% |
| Embodiment 1-2 | 98.77% | 1.23% |
| Embodiment 1-3 | 99.39% | 0.61% |
| Embodiment 2-1 | 97.31% | 2.69% |
| Embodiment 2-2 | 97.69% | 2.31% |
| Embodiment 2-3 | 96.92% | 3.08% |
| Embodiment 3-1 | 98.67% | 1.33% |
| Embodiment 3-3 | 98.57% | 1.43% |
| Embodiment 3-3 | 99.24% | 0.76% |
From table 3, it is evident that each embodiment of ganodenic acid acid extraction and separation process method provided by the present invention just like
Lower advantage：(1) organic solvent consumption is less, it is not easy to cause secondary pollution to the ganodenic acid acid for extracting isolated；(2) return
Receipts ratio is high, saves cost to a certain extent.
The effective recovery rate of each implementation and DNA purity of 4 Ganoderma total triterpenes acid extraction and separation process provided by the present invention of table
Experimental data：
| Project | Effective recovery rate | Total triterpene purity |
| Embodiment 1-1 | 4.58% | 94.7% |
| Embodiment 1-2 | 4.72% | 95.3% |
| Embodiment 1-3 | 5.38% | 93.6% |
| Embodiment 2-1 | 5.11% | 96.9% |
| Embodiment 2-2 | 4.22% | 95.3% |
| Embodiment 2-3 | 6.12% | 95.6% |
| Embodiment 3-1 | 5.52% | 96.6% |
| Embodiment 3-2 | 5.63% | 94.2% |
| Embodiment 3-3 | 6.11% | 95.9% |
From table 4, it is evident that each embodiment of ganodenic acid acid extraction and separation process method provided by the present invention has
There is higher effective recovery rate, it is high to extract separating obtained ganodenic acid acid purity；Particularly add in the implementation of composite fibre enzyme
Example 1-3, embodiment 2-3, embodiment 3-3 ratios are not plus the embodiment 1-1 of composite fibre enzyme, embodiment 2-1, embodiment 3-1 increase
20% or so the effective recovery rate of total triterpene acid.
Above-mentioned specific embodiment is simply explained in detail technical scheme, the present invention not only only office
It is limited to above-described embodiment, every any improvement or replacement according to the principle of the invention should all be within protection scope of the present invention.
Claims (6)
1. a kind of ganodenic acid acid submerged fermentation process for extracting, it is characterised in that：The ganodenic acid acid submerged fermentation carries
Taking technique method includes the following steps：
(1) culture medium is configured：The culture medium is mainly formulated (W/V) by following components：Glucose 25.0-27.0g/L, it is beautiful
Rice flour 10.2-12.2g/L, wheatfeed 9.8-11.8g/L and peptone 4.9-6.9g/L；
(2) fermented and cultured：In the medium add 400-500mg/L campanulaceae water extract or 180-220mg/L honeysuckle and
Matrimony vine alcohol extract accesses ganoderma lucidum inoculum and carries out fermented and cultured afterwards, and fermentation culture conditions are as follows：Fermentation initial ph value be
5.5-6.5, temperature are 28-32 DEG C, shaking speed 150-170r/min, fermentation time 166-170h；Fermentation process terminates
By obtained glossy ganoderma fermentation thalline, drying to constant weight afterwards；The ganoderma lucidum inoculum is ganoderma lucidum mycelium or ganoderma lucidum fruitbody；
(3) extraction separation：Comprise the following steps：
1. being extracted three to four times after the dry glossy ganoderma fermentation thalline to constant weight is crushed, the extracting process is：First ganoderma lucidum is sent out
The crushed material of yeast-like fungi body presses 0.8-1.2 with chloroform extracted solution:14(W:V) ratio mixes, and is then with concentration by the mixed liquor
4.8-5.2% (W:V NaHCO)3Solution is according to 2.8-3.2:1(V:V) ratio mixes, and after impregnating 22-26h, extracts extract liquor,
Surplus materials is extracted next time according to this method；The extract liquor for extracting acquisition several times is merged afterwards, it is auxiliary using ultrasound
Equipment is helped, under 380-420W ultrasound output powers, using the supersonic frequency of 50-60kHz, 28-32min is ultrasonically treated, stagnates
8-12min is ultrasonically treated 28-32min again；Then it is separated by filtration；
2. filtrate α and filter residue are obtained after being separated by filtration；Filter residue is first pressed into 0.8-1.2:8(W:V it is 4.8- that) ratio, which adds in concentration,
5.2% (W:V)NaHCO3Solution, using ultrasonic wave added equipment, under 380-420W ultrasound output powers, using 50-60kHz's
Supersonic frequency is ultrasonically treated 28-32min, stagnates 8-12min, is ultrasonically treated 28-32min again, is afterwards 0.4- using aperture
0.5 μm of filtering with microporous membrane obtains filtrate β；Then filtrate α and filtrate β is merged, opens dispersion machine, adjustment rotating speed is extremely
4800-5200r/min disperses 18-22min, obtains supernatant；Afterwards under 3.8-4.2 DEG C of condition of ice bath, by supernatant 5.8-
The dilute hydrochloric acid of 6.2mol/L carries out being acidified to pH=2.8-3.2, obtains acidifying solution；
3. after acidifying solution is extracted 3-5 times with isometric chloroform extracted solution, simultaneously combining extraction liquid is collected, extract liquor is stood
35-45min with 0.4-0.5 μm of the filtering with microporous membrane in aperture, obtains yellow clear liquid；
4. by yellow clear liquid, evaporated under reduced pressure obtains yellow viscous liquid under the conditions of 44-46 DEG C, by yellow viscous liquid in 58-62
It is dry under DEG C water bath condition that drying to constant weight；
1. 3. the chloroform extracted solution is according to 95 by chloroform and absolute ethyl alcohol to step with step:5-98:2 volume proportion is matched somebody with somebody
It makes.
2. ganodenic acid acid submerged fermentation process for extracting according to claim 1, it is characterised in that：
Step (1) described culture medium is mainly formulated (W/V) by following components：Glucose 26.0g/L, corn flour 11.2g/L,
Wheatfeed 10.8g/L and peptone 5.9g/L.
3. ganodenic acid acid submerged fermentation process for extracting according to claim 2, it is characterised in that：
450mg/L campanulaceaes water extract or 200mg/L honeysuckles and matrimony vine alcohol extract are added in culture medium described in step (2),
Initial ph value of fermenting is 6.0, and temperature is 30 DEG C, shaking speed 160r/min, fermentation time 168h.
4. the ganodenic acid acid submerged fermentation process for extracting according to claim 1 or 2 or 3, it is characterised in that：It is described
The extraction process of campanulaceae water extract is as follows：Campanulaceae 90-110g is taken, 25-35min is boiled with decocting system, pours out the dregs of a decoction, is added appropriate
Cold water is in the dregs of a decoction, then boils 18-22min, pours out concoction, adds appropriate cold water again afterwards in the dregs of a decoction, boils 18-22min,
Concoction is poured out, merges concoction, filters concoction, it is afterwards that the concoction after filtering is dry at 55-65 DEG C, obtain campanulaceae water extract.
5. the ganodenic acid acid submerged fermentation process for extracting according to claim 1 or 2 or 3, it is characterised in that：It is described
The extraction process of honeysuckle and matrimony vine alcohol extract is as follows：By honeysuckle and matrimony vine according to 0.8-1.2：After 1 quality proportioning mixing,
30-50 mesh crushes, and binds up with gauze tighten afterwards, be put into reflow of alcohol extraction apparatus, and 2.5- is extracted with 2.5-3.5 times of ethyl alcohol
3.5h obtains extract liquor, and surplus materials is added to 2.5-3.5 times of ethyl alcohol and extracts 1.5-2.5h, obtains extract liquor again, it
Merge the extract liquor obtained twice afterwards, be concentrated to give honeysuckle and matrimony vine alcohol extracting thick liquid；The concentration expressed in percentage by volume of the ethyl alcohol
For 93-97%.
6. the ganodenic acid acid submerged fermentation process for extracting according to claim 1 or 2 or 3, it is characterised in that：Step
(3) extract liquor described in first adds in cellulose complex enzyme before standing and is digested, 45-55 DEG C of hydrolysis temperature, pH=5.5-
6.5, enzymolysis time 18-22min, the addition of cellulose complex enzyme are the 1.5-2.5% (W of extract liquor:V)；The cellulose
Complex enzyme presses Rate activity 4.8-5.2 by cellulase, protease, pectase:2.8-3.2:1.8-2.2 it mixes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510147874.4A CN104805170B (en) | 2015-02-03 | 2015-04-01 | Ganodenic acid acid submerged fermentation process for extracting |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015100564742 | 2015-02-03 | ||
| CN201510056474 | 2015-02-03 | ||
| CN201510147874.4A CN104805170B (en) | 2015-02-03 | 2015-04-01 | Ganodenic acid acid submerged fermentation process for extracting |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104805170A CN104805170A (en) | 2015-07-29 |
| CN104805170B true CN104805170B (en) | 2018-06-01 |
Family
ID=53690367
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510147874.4A Active CN104805170B (en) | 2015-02-03 | 2015-04-01 | Ganodenic acid acid submerged fermentation process for extracting |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104805170B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106852496A (en) * | 2015-12-09 | 2017-06-16 | 黄建钧 | The method of the triterpenes synthesis contained by induction mushroom gill fungus |
| CN106722875A (en) * | 2016-12-07 | 2017-05-31 | 盐城工学院 | The extracting method and Ganoderma lucidum of a kind of ganodenic acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348513A (en) * | 2007-07-18 | 2009-01-21 | 澳门药物及健康应用研究所 | Ganoderic acid extraction method and preparation of soft capsule of ganoderic acid |
| CN102488719A (en) * | 2011-12-25 | 2012-06-13 | 南京农业大学 | Method for improving triterpene output of Ganoderma lucidum liquid fermented mycelia |
| CN102772448A (en) * | 2012-08-15 | 2012-11-14 | 苏州菩芸生物科技有限公司 | Lucid ganoderma triterpene extraction method |
-
2015
- 2015-04-01 CN CN201510147874.4A patent/CN104805170B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348513A (en) * | 2007-07-18 | 2009-01-21 | 澳门药物及健康应用研究所 | Ganoderic acid extraction method and preparation of soft capsule of ganoderic acid |
| CN102488719A (en) * | 2011-12-25 | 2012-06-13 | 南京农业大学 | Method for improving triterpene output of Ganoderma lucidum liquid fermented mycelia |
| CN102772448A (en) * | 2012-08-15 | 2012-11-14 | 苏州菩芸生物科技有限公司 | Lucid ganoderma triterpene extraction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104805170A (en) | 2015-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102391387B (en) | Microwave chemical extraction method of active polysaccharide in higher plants or edible and medicinal fungi | |
| CN102942410B (en) | Edible fungi culture medium and preparation method thereof | |
| CN103981054B (en) | A kind of biological enzyme brewages the method for oil tea wine | |
| CN103555556B (en) | Method for preparing ginseng vinegar by virtue of fermentation with corncobs and corn ears as raw materials | |
| CN106581083A (en) | Extraction method for ganoderma lucidum components, biological feed and preparation method thereof | |
| CN101560260A (en) | Technique for extracting polysaccharide in glossy ganoderma mycelium cell and method thereof | |
| CN103589185B (en) | Method for extracting pigment and polyphenol from water chestnut peels | |
| CN103804503A (en) | Method of extracting polysaccharide of enteromorpha by enzymolysis in steps | |
| CN106491662B (en) | A method of polysaccharide and flavones are extracted simultaneously from Phellinus cultivation waste material | |
| CN104710541A (en) | Method for preparing laminarin from kelp | |
| CN101423855A (en) | Method for preparing polysaccharide by using lucidum strain fermented laminaria leftover | |
| CN104961839A (en) | Preparation method of specific pachyman formula granule | |
| CN104805170B (en) | Ganodenic acid acid submerged fermentation process for extracting | |
| WO2014166430A2 (en) | Solid-state alcohol fermentation production method | |
| CN105543289A (en) | Oak acorn tannin extraction and starch thick mash fermentation coupling method | |
| CN102154427A (en) | Method for extracting tea saponin by microbial fermentation | |
| CN101233920A (en) | Method for preparing highland barley embryo oil extracted by ultrasonic method | |
| CN106244390A (en) | A kind of Herba Dendrobii Cordyceps fermentation original plasm wine and preparation method thereof | |
| CN101376669B (en) | Preparation of 6-O-beta-D- glucosyl-3,6,16,25-tetrahydroxy cycloartane | |
| CN104761612B (en) | Ganoderma triterpenoids acid extraction and separation process method | |
| CN110628840B (en) | Method for extracting myricetin by microbial fermentation | |
| CN103923146B (en) | The extracting method of free ergosterol in a kind of Chinese caterpillar fungus | |
| CN102220381B (en) | Technology for producing edible alcohol by using crushed vermicelli and bean dregs | |
| CN104357527A (en) | Method for extracting tea saponin from tea seed meal with microbial fermentation method | |
| CN101828628A (en) | Biological treatment method for effectively extracting rapeseed protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |