CN106399397A - Method for increasing content of tyrosol in rhodiola rosea by microorganism fermentation - Google Patents
Method for increasing content of tyrosol in rhodiola rosea by microorganism fermentation Download PDFInfo
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- CN106399397A CN106399397A CN201610829202.6A CN201610829202A CN106399397A CN 106399397 A CN106399397 A CN 106399397A CN 201610829202 A CN201610829202 A CN 201610829202A CN 106399397 A CN106399397 A CN 106399397A
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- radix rhodiolae
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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Abstract
The invention provides a method for increasing content of tyrosol in rhodiola rosea by microorganism fermentation. The method comprises the following steps of (1) pretreatment of the rhodiola rosea: slicing, crushing and drying the rhodiola rosea; (2) preparation of rhodiola rosea extracting liquid: mixing the rhodiola rosea powder in step (1) and distilled water, extracting, filtering by eight layers of gauze, mixing the extracting liquid, settling and staying overnight, centrifuging to remove impurities, and using a supernatant to prepare a fermentation culture medium; (3) preparation of a seed culture medium; (4) preparation of the fermentation culture medium: adding the rhodiola rosea extracting liquid obtained in step (2) into 0.5 to 2 percent of glucose; (5) fermentation method: inoculating ring saccharomycetes into the seed culture medium, culturing and staying overnight, and inoculating into the fermentation culture medium to culture, so as to increase the content of tyrosol in the rhodiola rosea by the microorganism fermentation technique. The method has a beneficial effect that the content of the tyrosol is increased.
Description
Technical field
The invention belongs to biological fermentation field, more particularly, to a kind of utilization fermentable raising Radix Rhodiolae butyl alcohol content
Method.
Background technology
Radix Rhodiolae system Crassulaceae (Crassulaceae) rhodiola (Rhodiola) plant, perennial herb or sub- filling
Wood.China's Rhodida plant accounts for the 80% of world's Radix Rhodiolae kind total resources, widely distributed, is typically grown in high and cold, dry
The High aititude area dry, anoxia, strong ultraviolet radiation, day and night temperature are big, has extremely strong environmental suitability and indomitable life
Power.Because its growing environment is badly so as to have antioxidation, anti-hypoxia, resisting fatigue, radioprotective, defying age, antiviral, adjust god
The effects such as through system, cardio-cerebrovascular and enhance immunity.Mainly effective and active component in Radix Rhodiolae is rhodioside
And its tyrosol, the height of Chang Yiqi total content is as the standard weighing Rhodida plant crude drug and its quality of the pharmaceutical preparations.
But both contents in Radix Rhodiolae are not high, using being chemically synthesized low yield, pollute environment, how under mild conditions
Improving effective ingredient is problem in the urgent need to address at present.
Herb fermenting pharmaceutical technology is on the basis of inheriting science of Chinese drug processing fermentation method, has drawn Microecology one-tenth
Really, the high-tech herbal pharmaceutical new technique being formed in conjunction with the fermentation technique of modern biological project.During herb fermenting, micro-
Bioenergy produces multiple secondary metabolites such as cellulase, lignoenzyme, amylase, protease, makes the biochemistry of complexity
Reaction quickly completes at normal temperatures and pressures, more greatly strengthens and adjust the property of medicine than general processing means physically or chemically,
Improve curative effect, reduce toxic and side effects.After by fermentation, thus it is possible to vary its original performance, effective ingredient is sufficiently separated, extracts, has
With more biological activity, strengthen or produce new effect, expand clinical application scope, more adapt to the needs of clinical application.
Content of the invention
This patent provides a kind of new technology, and the butyl alcohol being improved using fermentable pharmaceutical technology in Radix Rhodiolae is contained
Amount, makes effective ingredient increase.
The technical solution adopted in the present invention is：
A kind of method that utilization fermentable improves butyl alcohol content in Radix Rhodiolae, comprises the following steps：
(1) pretreatment of Radix Rhodiolae：Radix Rhodiolae is cut into slices and pulverizes, cross 40-60 mesh sieve；It is dried, described dry temperature again
Spend for 80-100 DEG C, drying time, 2-3 hour, made water content 2%-6%, obtain final product Radix Rhodiolae powder, standby；
(2) preparation of Radix Rhodiolae extracting solution：Step (1) gained Radix Rhodiolae powder and distilled water are pressed material-water ratio 1:25-1:50
100 DEG C of ratio extract twice, mixed extract after filtered through gauze, supply the moisture of loss, extracting solution sunk in 2-8 DEG C of refrigerator
Overnight, 5000r is centrifuged 20-30 minute remove impurity to fall, and supernatant is used for preparing fermentation medium；
(3) preparation of seed culture medium：By 10g yeast powder, 20g peptone, 20g glucose is dissolved in water, is settled to 1L, point
It is contained in the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min；
(4) preparation of fermentation medium：The Radix Rhodiolae extracting solution of step (2) gained is added the glucose of 0.5%-2%,
It is divided in the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min；
(5) fermentation process：Ring yeast CICC1339 is taken to be seeded in seed culture medium, incubated overnight, then press 2%-5%
Inoculum concentration be seeded in fermentation medium, 28 DEG C of 200r cultivate 2-3 days；
(6) butyl alcohol detection method of content：Fermentation liquid 12000r centrifugation 10min is removed thalline, supernatant crosses 0.22 μm of filter,
Using C18 chromatographic column, in 220nm with 25% methanol as mobile phase, sample size 10 μ l flow velocity 1ml/min, 30 DEG C of samples of column temperature divide
Analysis time 20min.
In order to obtain superior technique effect, after the excessively complete 40-60 mesh sieve of Radix Rhodiolae in step (1), unsanctioned coarse powder is again
Pulverize, cross 40-60 mesh sieve to whole.
In order to obtain superior technique effect, when step (2) carries out water extraction to Radix Rhodiolae, carried out once every 5 minutes
Stirring.
In order to obtain superior technique effect, when step (2) carries out water extraction to Radix Rhodiolae, extract one hour every time.
In order to obtain superior technique effect, in step (2) during filtered through gauze, gauze used is eight layers of gauze overlap one
Rise.
Due to employing technique scheme, the present invention compared with prior art has the advantages that：
The present invention using numbering be CICC1339 yeast fermentation Radix Rhodiolae, fermented after, its effective ingredient can be filled
Separate, extract.In the presence of multiple enzymes of yeast secretion, the content of butyl alcohol significantly improves, before butyl alcohol content can be by fermenting
0.6mg/g bring up to 1.8mg/g after fermentation, effective ingredient is improved significantly.This method can than typically more physically or chemically
Processing means more greatly strengthen and adjust the property of medicine, improve curative effect, reduce toxic and side effects.The method is simple to operate, cost
Low, cycle is short, it is the method improving rhodiola active ingredient in a mild condition, is conducive to the comprehensive utilization of Chinese medicine, have wide
Wealthy market prospect.
Specific embodiment
Embodiment 1
(1) pretreatment of Radix Rhodiolae：Radix Rhodiolae section 20g is pulverized, crosses 40-60 mesh sieve, middle powder and fine powder are continued to employ, coarse powder
Pulverize again, to whole mistake 60 mesh sieves；It is dried, described baking temperature is 80 DEG C, 2 hours drying times, makes water content again
6%, obtain final product Radix Rhodiolae powder, standby.
(2) preparation of Radix Rhodiolae extracting solution：Step (1) gained Radix Rhodiolae powder and distilled water are pressed material-water ratio 1:25 ratio
100 DEG C are extracted twice, one hour every time, and mixed extract after eight layers of filtered through gauze supplies moisture.By extracting solution in 4 DEG C of refrigerators
Overnight, 5000r is centrifuged remove impurity in 20 minutes for sedimentation, and supernatant is used for preparing fermentation medium.
(3) preparation of seed culture medium：By 10g yeast powder, 20g peptone, 20g glucose is dissolved in water, is settled to 1L, point
It is contained in the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min.
(4) preparation of fermentation medium：The Radix Rhodiolae extracting solution of step (2) gained is added 2% glucose, be divided in
In the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min.
(5) fermentation process：A ring yeast CICC1339 is taken to be seeded in seed culture medium, incubated overnight, then press 5%
Inoculum concentration is seeded in fermentation medium, and 28 DEG C of 200r cultivate 3 days.
(6) butyl alcohol detection method of content：Fermentation liquid 12000r centrifugation 10min is removed thalline, crosses 0.22 μm of filter, adopt
C18 chromatographic column, in 220nm with 25% methanol as mobile phase, sample size 10 μ l flow velocity 1ml/min, during 30 DEG C of sample analysis of column temperature
Between 20min.Finally give butyl alcohol content 1.5mg/g, improve 150% than before fermentation.
Embodiment 2
(1) pretreatment of Radix Rhodiolae：Radix Rhodiolae is cut into slices and pulverizes 20g, cross 60 mesh sieves, middle powder and fine powder are continued to employ, and coarse powder is again
Pulverize, to whole mistake 60 mesh sieves；It is dried, described baking temperature is 100 DEG C, 3 hours drying times, makes water content 5% again,
Obtain final product Radix Rhodiolae powder, standby.
(2) preparation of Radix Rhodiolae extracting solution：Step (1) gained Radix Rhodiolae powder and distilled water are pressed material-water ratio 1:25 ratio
100 DEG C are extracted twice, one hour every time, and mixed extract after eight layers of filtered through gauze supplies moisture.By extracting solution in 5 DEG C of refrigerators
Overnight, 5000r is centrifuged remove impurity in 30 minutes for sedimentation, and supernatant is used for preparing fermentation medium.
(3) preparation of seed culture medium：By 10g yeast powder, 20g peptone, 20g glucose is dissolved in water, is settled to 1L, point
It is contained in the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min.
(4) preparation of fermentation medium：The Radix Rhodiolae extracting solution of step (2) gained is added 1% glucose, be divided in
In the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min.
(5) fermentation process：A ring yeast CICC1339 is taken to be seeded in seed culture medium, incubated overnight, then press 4%
Inoculum concentration is seeded in fermentation medium, and 28 DEG C of 200r cultivate 3 days.
(6) butyl alcohol detection method of content：Fermentation liquid 12000r centrifugation 10min is removed thalline, crosses 0.22 μm of filter, adopt
C18 chromatographic column, in 220nm with 25% methanol as mobile phase, sample size 10 μ l flow velocity 1ml/min, during 30 DEG C of sample analysis of column temperature
Between 20min, finally give butyl alcohol content 1.6mg/g, than fermentation before improve 166%.
Embodiment 3
(1) pretreatment of Radix Rhodiolae：Radix Rhodiolae section 20g is pulverized, crosses 50 mesh sieves, middle powder and fine powder are continued to employ, and coarse powder is again
Pulverize, to whole mistake 50 mesh sieves；It is dried, described baking temperature is 90 DEG C, 3 hours drying times, makes water content 4% again,
Obtain final product Radix Rhodiolae powder, standby.
(2) preparation of Radix Rhodiolae extracting solution：Step (1) gained Radix Rhodiolae powder and distilled water are pressed material-water ratio 1:25 ratio
100 DEG C are extracted twice, one hour every time, and mixed extract after eight layers of filtered through gauze supplies moisture.By extracting solution in 4 DEG C of refrigerators
Overnight, 5000r is centrifuged remove impurity in 25 minutes for sedimentation, and supernatant is used for preparing fermentation medium.
(3) preparation of seed culture medium：By 10g yeast powder, 20g peptone, 20g glucose is dissolved in water, is settled to 1L, point
It is contained in the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min.
(4) preparation of fermentation medium：The Radix Rhodiolae extracting solution of step (2) gained is added 2% glucose, be divided in
In the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min.
(5) fermentation process：A ring yeast CICC1339 is taken to be seeded in seed culture medium, incubated overnight, then press 5%
Inoculum concentration is seeded in fermentation medium, and 28 DEG C of 200r cultivate 3 days.
(6) butyl alcohol detection method of content：Fermentation liquid 12000r centrifugation 10min is removed thalline, crosses 0.22 μm of filter, adopt
C18 chromatographic column, in 220nm with 25% methanol as mobile phase, sample size 10 μ l flow velocity 1ml/min, during 30 DEG C of sample analysis of column temperature
Between 20min.Finally give butyl alcohol content 1.8mg/g, improve 200% than before fermentation.
The butyl alcohol content of embodiment 1-3 gained Radix Rhodiolae fermentation liquid and increase rate
Above the specific embodiment of the present invention is described in detail, but described content has been only the preferable enforcement of the present invention
Example is it is impossible to be considered the practical range for limiting the present invention.All impartial changes made according to the present patent application scope and improvement
Deng all should still belong within the patent covering scope of the present invention.
Claims (6)
1. a kind of utilization fermentable improve butyl alcohol content in Radix Rhodiolae method it is characterised in that：Comprise the following steps：
（1）The pretreatment of Radix Rhodiolae：Radix Rhodiolae is cut into slices and pulverizes, cross 40-60 mesh sieve；It is dried, described baking temperature is again
80-100 DEG C, drying time, 2-3 hour, made water content 2%-6%, obtained final product Radix Rhodiolae powder, standby；
（2）The preparation of Radix Rhodiolae extracting solution：By step（1）Material-water ratio 1 pressed by gained Radix Rhodiolae powder and distilled water:25-1:50 ratio
100 DEG C of example is extracted twice, and mixed extract after filtered through gauze is supplied the moisture of loss, extracting solution was settled in 2-8 DEG C of refrigerator
At night, 5000r is centrifuged 20-30 minute remove impurity, and supernatant is used for preparing fermentation medium；
（3）The preparation of seed culture medium：By 10g yeast powder, 20g peptone, 20g glucose is dissolved in water, is settled to 1L, is divided in
In the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min；
（4）The preparation of fermentation medium：By step（2）The Radix Rhodiolae extracting solution of gained adds the glucose of 0.5%-2%, is divided in
In the conical flask of 500ml, often bottled liquid measure 100ml, 115 DEG C of sterilizing 20min；
（5）Fermentation process：Ring yeast CICC1339 is taken to be seeded in seed culture medium, incubated overnight, then press the inoculation of 2%-5%
Amount is seeded in fermentation medium, and 28 DEG C of 200r cultivate 2-3 days；
（6）Butyl alcohol detection method of content：Fermentation liquid 12000r centrifugation 10min is removed thalline, supernatant crosses 0.22 μm of filter, adopts
C18 chromatographic column, in 220nm with 25% methanol as mobile phase, sample size 10 μ l flow velocity 1ml/min, 30 DEG C of sample analysis times of column temperature
20min.
2. utilization fermentable as claimed in claim 1 improve butyl alcohol content in Radix Rhodiolae method it is characterised in that：Institute
Step（1）After the excessively complete 40-60 mesh sieve of middle Radix Rhodiolae, unsanctioned coarse powder is pulverized again, crosses 40-60 mesh sieve to whole.
3. utilization fermentable as claimed in claim 1 improve butyl alcohol content in Radix Rhodiolae method it is characterised in that：Step
Suddenly（2）When water extraction is carried out to Radix Rhodiolae, once stirred every 5 minutes.
4. utilization fermentable as claimed in claim 1 improve butyl alcohol content in Radix Rhodiolae method it is characterised in that：Institute
State step（2）When water extraction is carried out to Radix Rhodiolae, extract one hour every time.
5. utilization fermentable as claimed in claim 1 improve butyl alcohol content in Radix Rhodiolae method it is characterised in that：Institute
State step（2）During middle filtered through gauze, gauze used is for eight layers of gauze overlap together.
6. the method that the utilization fermentable described in claim 1 improves butyl alcohol content in Radix Rhodiolae, extracts from Radix Rhodiolae
The butyl alcohol content obtaining is 1.5-1.8mg/g.
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Cited By (2)
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| JP2018193341A (en) * | 2017-05-19 | 2018-12-06 | 共栄化学工業株式会社 | Plant fermentation product |
| CN110624271A (en) * | 2019-10-10 | 2019-12-31 | 安徽德正堂药业有限公司 | Chinese herbal medicine micro-molecule fermentation extraction process |
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| CN110624271A (en) * | 2019-10-10 | 2019-12-31 | 安徽德正堂药业有限公司 | Chinese herbal medicine micro-molecule fermentation extraction process |
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Application publication date: 20170215 |