CN107177662A - The fermentation preparation of Chinese yam saponin - Google Patents
The fermentation preparation of Chinese yam saponin Download PDFInfo
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- CN107177662A CN107177662A CN201710565754.5A CN201710565754A CN107177662A CN 107177662 A CN107177662 A CN 107177662A CN 201710565754 A CN201710565754 A CN 201710565754A CN 107177662 A CN107177662 A CN 107177662A
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Abstract
The invention belongs to Secondary Metabolites of Microorganisms technical field, a kind of fermentation preparation of Chinese yam saponin is disclosed, is fermented using Chinese yam endophyte, fermentation process comprises the steps：1) Chinese yam endophyte is accessed in seed culture medium and carries out seed culture, obtain fermented bacterium；2) defoamer being added into fermentation medium, by step 1) fermented bacterium access is loaded with the fermentation tank of fermentation medium and fermented, open and stir, set fermentation condition as 28 DEG C of fermentation temperature, the 5.5L/min of throughput 1.0；3) the isolated Chinese yam saponin from tunning.The present invention establishes the basic technology of canister fermenting and producing Chinese yam saponin, significantly improves the yield of Chinese yam saponin, overcomes the technological deficiency that existing Chinese yam saponin production yields is low, power consumption is high, and the exploitation for peltate yam endophytic bacterium provide Research foundation.
Description
Technical field
The invention belongs to Secondary Metabolites of Microorganisms technical field, it is related to the optimum preparation condition of raw material medicine saponin,
Particularly relate to the method that peltate yam endophytic bacterium fermentation prepares Chinese yam saponin.
Background technology
Dioscorea zingiberensis wright (Dioscorea zingiberensis C.H.Wright) is the distinctive Chinese yam kind of China, also known as
It is the main plant raw material for producing saponin for yellow ginger.Chinese yam saponin is referred to as " medicinal gold " by the world of medicine, and it can synthesize cortex
The thousands of kinds of steroid hormone class medicines such as hormone, protein anabolic hormone, sex hormone.Dioscorea zingiberensis wright is the important original for obtaining Chinese yam saponin
One of material.At present, the whole world has found 138 kinds of yams containing Chinese yam saponin, but only 10% or so Dioscorea
Plant has industrial utility value.With the continuous increase of Chinese yam saponin market demand breach, dioscorea zingiberensis wright suffers from predation formula
Excavation so that this precious wild resource is petered out.Long-term artificial growth dioscorea zingiberensis wright has resulted in Chinese yam germ plasm resource
Degenerate, its saponin yield is constantly reduced, and the ecological environment fragile to mountain area causes serious destruction.
In traditional saponin producing process, a large amount of waste water can be produced.These Organic Pollutants in Wastewater concentration are high, colourity
High, acidity is high, therefore intractability is very big.The main producing region of dioscorea zingiberensis wright includes Hubei, Shaanxi, Henan etc. and saved.Shaanxi Province is long-term
Large area plants dioscorea zingiberensis wright, there is 80 Yu Jia production of saponin enterprises, and processing capacity accounts for the 50% of the whole nation, wherein, Hanjiang River
Upstream city Hanzhong City just has 38 production of saponin enterprises, and annual sewage effluent is ten thousand tons about more than 100.Due to the soap of many enterprises
Element production waste water is directly discharged into Han River, extremely serious to Middle Line Project For Transferring Water From South To North watershed water pollution.According to《Project of South-to-North water diversion
Overall planning》With the severe situation of current Huang-Huai-Hai shortage of water resources, Shaanxi Province part saponin producer stops production in succession.In order to
Environmental protection, the whole nation saponin of more than half extracts enterprise and stopped production.This is also to cause one of the reason for saponin price rises steadily.
Production-demand gap is big, wild dioscorea zingiberensis wright resource is increasingly reduced, artificial growth Quality Down and environment are destroyed
Deng, it has also become numerous problems in Chinese yam saponin production process, China's medical industry has been had a strong impact on to steroid hormone class medicine
Research and development and application.In order to seek the solution of above mentioned problem, it is necessary to change the existing production model of Chinese yam saponin, this respect
Change be mainly reflected in：The Chinese yam new varieties of one side breeding high-yield, stable yields, using science, scale implantation methods；
On the other hand environmentally friendly, efficient Chinese yam saponin preparation approach is explored.
The problems such as pollution for puzzlement production of saponin enterprise and scarcity of raw material, researcher has carried out substantial amounts of study and should
For putting into practice.Current research and application direction mainly uses isolated strains to ferment or spontaneous fermentation dioscorea zingiberensis wright subterranean stem,
Chinese yam saponin is separated again, compared with conventional acid-hydrolysis method, although the active ingredient of dioscorea zingiberensis wright can be made full use of, hence it is evident that improve
The recovery rate of saponin, but do not solved at all the problem of a large amount of acid waste waters are produced in production process.
The content of the invention
The purpose of the present invention is to explore environmentally friendly, efficient Chinese yam saponin new preparation process, overcomes and is prepared using prior art
Many technical problems of Chinese yam saponin.Endophyte of plant can be produced and the same or analogous physiological activator of its host.Base
In this, peltate yam endophytic bacterium as R＆D direction, is sought the secondary generation from peltate yam endophytic bacterium by the inventor of this patent
Thank to the possibility that Chinese yam saponin is extracted in product.This will be solution industrial pollution and find one of feasible way of new resources.
Specifically, the present invention provides a kind of fermentation preparation of Chinese yam saponin, is fermented using Chinese yam endophyte,
The fermentation preparation comprises the steps：
1) the Chinese yam endophyte is accessed in seed culture medium and carries out seed culture, obtain fermented bacterium.
2) defoamer being added into fermentation medium, by step 1) fermented bacterium access is loaded with fermentation medium
Fermented in fermentation tank, open stirring, set fermentation condition as 28 DEG C of fermentation temperature, throughput 1.0-5.5 L/min.
3) the isolated Chinese yam saponin from tunning.
Further, the Chinese yam endophyte that uses of the present invention is bacillus licheniformis Syb06.11.1, and the bacterial strain is from shield leaf
It is isolated in Chinese yam, CGMCC is stored in, the preservation time is on January 10th, 2008, deposit number 2334.
The inventor of this patent filtered out one plant of peltate yam endophytic bacterium in 2006 from the root-like stock of dioscorea zingiberensis wright.
Identify that the bacterial strain belongs to bacillus by colony characteristicses, cellular morphology, physio-biochemical characteristics.Then with molecular biology
Method, ITS sequence, 16S rDNA sequences to the bacterial strain are measured, and are compared with blast, find its with
Bacillus licheniformis ASC585 16S rDNA homologys are 99%, so that it is determined that it belongs to lichens gemma bar
Bacterium, and it is named as Endophytic Bacillus licheniformis Syb06.11.1.About the screening technique of the bacterial strain
And identification and the preservation information of bacterial strain, it has been expressly recited in patent ZL200810150274.3, therefore not to repeat here.
In this patent, above-mentioned bacillus licheniformis Endophytic Bacillus licheniformis Syb06.11.1 are abbreviated as
SYt1 in SYt1, this patent is identical with the bacterial strain being expressly recited in patent ZL200810150274.3.Pass through the hair to SYt1
Ferment product carries out column chromatography and crystallization, obtains crystalline solid, determines that its structure is steroid sapogenines by NMR, and to the sapogenin
Fusing point and optical activity be determined, determine that SYt1 bacterial strains can be with fermenting and producing Chinese yam saponin.
Based on the studies above, embodiments of the invention pass through to SYt1 seed culture medium, fermentation medium, fermentation temperature
The factors such as degree, shaking speed are optimized, and optimize dividing for Chinese yam saponin using Orthogonal Experiment and Design and Responds Surface Methodology
Separating process.
Embodiments of the invention establish the basic technology of canister fermenting and producing saponin, it is preferable that the appearance of this fermentation tank
Product is 5L or 3L.In order to eliminate the bubble caused by the factors such as stirring, ventilation in fermentation process, one is added in the fermentation medium
Quantitative defoamer.As the fermentation process of an optimization, the amount of being included in of the defoamer is fermentation cylinder for fermentation culture volume
One thousandth.In a further embodiment, those skilled in the art, can also be to froth breaking under the technical inspiration of the present invention
The amount of being included in of agent is in optimized selection.
As the fermentation process of an optimization, the fermentation time of the fermentation preparation of the Chinese yam saponin is 100-180h.
It is further preferred that fermentation time is set as 168h.
Throughput into fermentation tank has direct influence to the yield of the Chinese yam saponin.It is used as the fermentation of an optimization
Method, the fermentation throughput of the Chinese yam saponin is set as 5.5L/min.
The present invention gives the processing method of tunning, specifically, concentrated hydrochloric acid is added into zymotic fluid, in boiling water bath
Under the conditions of sour water hydrolysis and fermentation liquid, after hydrolyzate is cooled to room temperature, into hydrolyzate add aqueous slkali adjust hydrolyzate pH into
Property, filtering obtains filtrate and filter residue, the Chinese yam saponin is isolated from filtrate and/or filter residue.
As the separation of the Chinese yam saponin of an optimization, the Chinese yam saponin dichloromethane is separated from filtrate to filter
Liquid is extracted；The Chinese yam saponin is separated from filter residue to extract filter residue with petroleum ether.It is further preferred that the extraction
Take methylene chloride and the filtrate of filtrate isometric；The petroleum ether boiling range for extracting filter residue is 60-90 DEG C.
Composition suitable for the seed culture medium of the fermentation process of the present invention is beef extract 3g, peptone 10g, chlorination
Sodium 5g, water 1000ml, the pH7.0-7.2 of seed culture medium.
Composition suitable for the fermentation medium of the fermentation process of the present invention is sucrose 100g, peptone 50g, yeast
Powder 15g, sodium chloride 7g, dipotassium hydrogen phosphate 1g, corn steep liquor 6g, dextrin 14g, calcium carbonate 10g, water 1000ml.
The fermentation preparation of Chinese yam saponin of the present invention, at least with following beneficial effect or advantage.
The present invention bacterial strain that using peltate yam endophytic bacterium SYt1 as Chinese yam saponin prepared by extension, establishes canister fermentation
Produce the basic technology of Chinese yam saponin.By to SYt1 seed culture medium, fermentation medium, fermentation temperature, shaking speed etc.
Single factor test is optimized so that Chinese yam saponin yield is significantly improved, and fermentation period is greatly shortened.Chinese yam of the present invention
Production of saponin technique, substantially increases the yield of Chinese yam saponin, overcomes that the yield of existing Chinese yam saponin production method is small, power consumption is high
Technological deficiency.The present invention lays a good foundation for the early stage application study that SYt1 bacterial strains are developed.
Brief description of the drawings
Fig. 1 is influence of the different throughputs to Chinese yam saponin yield.
Fig. 2 is the yield comparison figure of Chinese yam saponin in filter cake slag and filtrate.
Embodiment
For the ease of understanding the purpose of the present invention, technical scheme and its effect, now the present invention will be done in conjunction with the embodiments into
One step is elaborated.
Embodiment 1
The present embodiment provides a kind of fermentation preparation of Chinese yam saponin.This method for preparing Chinese yam saponin is different from existing
Have and the preparation method for obtaining Chinese yam saponin is isolated and purified from Chinese yam, it leads to using peltate yam endophytic bacterium SYt1 as starting strain
Cross specific zymotechnique and separated from its zymotic fluid and obtained.The peltate yam endophytic bacterium SYt1 that the present embodiment is used is lichens bud
Born of the same parents bacillus Syb06.11.1, bacterial strain is stored in CGMCC, and the preservation time is on January 10th, 2008, deposit number 2334.It is relevant to be somebody's turn to do
Bacterial strain separation identification, and its metabolite dioscorea zingiberensis wright sapogenin separation referring to patent ZL200810150274.3.
The method that fermentation described in the present embodiment prepares Chinese yam saponin, comprises the steps.
The first step, peltate yam endophytic bacterium SYt1 expansion culture.
Concrete operations are described as：Seed training will be carried out in the peltate yam endophytic bacterium SYt1 access seed culture mediums of purifying
Support, after its species community expands to certain amount, you can be used as the fermented bacterium of next step.
The seed culture medium being related to, consisting of beef extract 3g, peptone 10g, sodium chloride 5g, water 1000ml, seed training
Support the pH7.0-7.2 of base.
Second step, inoculation fermentation.
Defoamer is added into fermentation medium, as the preferred of embodiment, the amount of being included in of the defoamer is fermentation tank
The one thousandth of middle fermentation medium volume.The fermented bacterium access that the first step is obtained is loaded with the fermentation tank of fermentation medium
In, stirring is opened, setting fermentation condition carries out fermenting and producing.
On stirring and its mixing speed, those skilled in the art can be using artificial stirring or motor-driven side
Formula, is preferred with motor driving stirring, mixing speed is set as 100-500rpm, it is further preferred that being tasted through multiple practice
Examination, with 400rpm mixing speed, can obtain ideal ferment effect.Other fermentation conditions also include fermentation temperature
28 DEG C, throughput 1.0-5.5L/min.For fermentation time, it should have a rational period, but in produce reality, to
The beginning that fermented bacterium is explanation fermentation is accessed in fermentation tank, also just there is the generation of target secondary metabolite, only in hair
Ferment initial stage, the amount of target secondary metabolite is less, after fermentation is carried out to a certain extent, the target secondary metabolism in zymotic fluid
The amount region of product is steady.In the present embodiment, in theory, fermentation time can be set as 0-180h, or can be by
Fermentation time further extends to 210h, and this is also that those skilled in the art can directly be known by the present embodiment.Enter one
Preferably, fermentation time can be using initial setting as 100-180h for step.
The fermentation medium being related to, consisting of sucrose 100g, peptone 50g, dusty yeast 15g, sodium chloride 7g, phosphoric acid hydrogen
Dipotassium 1g, corn steep liquor 6g, dextrin 14g, calcium carbonate 10g, water 1000ml.
3rd step, acidolysis zymotic fluid.
Separation obtains Chinese yam saponin from the zymotic fluid of peltate yam endophytic bacterium, in addition it is also necessary to acidolysis is carried out to zymotic fluid, then
Zymotic fluid after acidolysis is neutralized.The concrete operations of acidolysis zymotic fluid are that concentrated hydrochloric acid is added into zymotic fluid, in boiling water bath
Under the conditions of sour water hydrolysis and fermentation liquid, after hydrolyzate is cooled to room temperature, into hydrolyzate add aqueous slkali adjust hydrolyzate pH into
Property, filtering obtains filtrate and filter residue, the Chinese yam saponin is isolated from filtrate and/or filter residue.
In order to further verify that peltate yam endophytic bacterium fermentation prepares Chinese yam saponin, the present embodiment also further provides Chinese yam
The detection method and quantitative approach of saponin.
Concentrated hydrochloric acid is added into sample zymotic fluid, concentration of hydrochloric acid in zymotic fluid is reached 2mol/L.Under the conditions of boiling water bath
Sour water solution 4h, is cooled to room temperature, and NaOH solution is added into hydrolyzate and adjusts pH to neutrality, filter residue is stayed after filtering.In 90 DEG C of water
Under the conditions of bath, surname extraction 4h is carried out to filter residue for 60-90 DEG C of petroleum ether using 250mL boiling ranges, obtained saponin is extracted thick
Product are used for saponin concentration mensuration in zymotic fluid.
Dissolved with chloroform by dry Chinese yam saponin crude extract is evaporated into culture dish, lysate is transferred to 10mL grounds tool plug
In test tube, then with a small amount of chloroform by culture dish rinse twice, washed down completely with the Chinese yam saponin ensured in crude extract, flushing liquor
Equally it is transferred in ground tool plug test tube, 10mL is settled to chloroform, takes the saponin crude product solution of 4mL constant volumes, then add 4mL
Liebermann-Barcharad reagents, scale are added dropwise to chloroform, beyond the Great Wall grinding port plug, shaken up, in water bath with thermostatic control in 50 DEG C
60min, taking-up be cooled to room temperature, with spectrophotometer wavelength be 460nm at determine its light absorption value.
Embodiment 2
The present embodiment establishes the basic technology of canister fermenting and producing saponin, i.e., with volume be 5L or 3L fermentation tank is carried out
Peltate yam endophytic bacterium is fermented.Above-mentioned fermentation condition is optimized the present embodiment, seeks fermentation method production Chinese yam saponin
Process conditions.
(1) throughput optimizes
The optimization of throughput is carried out based on fermentation medium, the fermentation medium composition being related to is sucrose 100g, egg
White peptone 50g, dusty yeast 15g, sodium chloride 7g, dipotassium hydrogen phosphate 1g, corn steep liquor 6g, dextrin 14g, calcium carbonate 10g, water 1000ml.
Fig. 1 shows influence of the different throughputs to Chinese yam saponin yield.As shown in Figure 1, throughput is set to be 1L/min, 3L/ respectively
Min, 4.5L/min, 5.5L/min, are carried out after 5L canister fermenting experiments, fermentation ends, determine the yield of Chinese yam saponin.Throughput
The fermentation condition of Optimal Experimental is set as 28 DEG C of fermentation temperature, mixing speed 400rpm, fermentation time 168h.Different throughputs
Corresponding Chinese yam saponin yield is shown in Fig. 1, from figure 1 it appears that the increase of throughput can significantly improve the production of Chinese yam saponin
Amount, optimal throughput is 5.5L/min, and corresponding Chinese yam saponin yield is 437.36mg/L, is control group (1L/min) yield
69.59mg/L 6.28 times.
(2) in filter cake slag and filtrate Chinese yam saponin yield
Peltate yam endophytic bacterium SYt1 is accessed canister fermentation is carried out in basal medium, condition of culture is set as throughput
5.5L/min, 28 DEG C of fermentation temperature, mixing speed 400rpm, fermentation time 168h.After fermentation ends, fermented to 50mL samples
Concentrated hydrochloric acid is added in liquid, concentration of hydrochloric acid in zymotic fluid is reached 2mol/L.Sour water solution 4h, is cooled to room under the conditions of boiling water bath
Temperature, NaOH solution is added into hydrolyzate and adjusts pH to neutrality, filter residue and filtrate are obtained after filtering.Using different Chinese yam saponins
Extraction process, respectively from filter residue and filtrate separation Chinese yam saponin.The use of 250mL boiling ranges it is 60 DEG C -90 under 90 DEG C of water bath conditions
DEG C petroleum ether surname extraction 4h is carried out to filter residue, extracting obtained saponin crude product is used for saponin concentration mensuration in zymotic fluid.With
Isometric dichloromethane is extracted twice to filtrate, and extraction conditions is：In equal volume, 6 hours, 200rmp measured saponin after concentration
Yield.
Fig. 2 gives the yield comparison of Chinese yam saponin in filter cake slag and filtrate.From figure 2 it can be seen that Chinese yam saponin master
It is present in filtrate, is fermented during through 168h, the yield of Chinese yam saponin reaches up to 716.31mg/L, is Chinese yam soap in filter residue
1.64 times of cellulose content.
Further narration has been done to the present invention above in conjunction with embodiment, but the present invention is not limited to above-mentioned embodiment,
In the knowledge that one skilled in the relevant art possesses, it can also be made on the premise of present inventive concept is not departed from
Various change.
Claims (10)
1. the fermentation preparation of Chinese yam saponin, is fermented using Chinese yam endophyte, the fermentation preparation includes following
Step：
1) the Chinese yam endophyte is accessed in seed culture medium and carries out seed culture, obtain fermented bacterium；
2) defoamer being added into fermentation medium, by step 1) fermented bacterium access is loaded with the fermentation of fermentation medium
Fermented in tank, open stirring, set fermentation condition as 28 DEG C of fermentation temperature, throughput 1.0-5.5L/min；
3) the isolated Chinese yam saponin from tunning.
2. the fermentation preparation of Chinese yam saponin according to claim 1, it is characterised in that the Chinese yam endophyte is ground
Clothing bacillus Syb06.11.1, the bacterial strain is isolated from dioscorea zingiberensis wright, is stored in CGMCC, and the preservation time is 2008 1
The moon 10, deposit number 2334.
3. the fermentation preparation of Chinese yam saponin according to claim 1, it is characterised in that the volume of the fermentation tank is
5L or 3L, the amount of being included in of the defoamer is the one thousandth of fermentation cylinder for fermentation culture volume.
4. the fermentation preparation of Chinese yam saponin according to claim 1, it is characterised in that the fermentation time is 100-
180h。
5. the fermentation preparation of Chinese yam saponin according to claim 4, it is characterised in that the fermentation time is
168h。
6. the fermentation preparation of Chinese yam saponin according to claim 1, it is characterised in that the fermentation throughput is
5.5L/min。
7. the fermentation preparation of Chinese yam saponin according to claim 1, it is characterised in that the processing of the tunning
Method is：Concentrated hydrochloric acid is added into zymotic fluid, the sour water hydrolysis and fermentation liquid under the conditions of boiling water bath, after hydrolyzate is cooled to room temperature,
Aqueous slkali is added into hydrolyzate and adjusts hydrolyzate pH to neutrality, filtering obtains filtrate and filter residue, the Chinese yam saponin is from filtrate
And/or it is isolated in filter residue.
8. the fermentation preparation of Chinese yam saponin according to claim 7, it is characterised in that the potato is separated from filtrate
Chinese yam saponin is extracted with dichloromethane to filtrate；The Chinese yam saponin is separated from filter residue to carry filter residue with petroleum ether
Take.
9. the fermentation preparation of Chinese yam saponin according to claim 8, it is characterised in that the dichloro of the extraction filtrate
Methane consumption and filtrate are isometric；The petroleum ether boiling range for extracting filter residue is 60-90 DEG C.
10. the method that peltate yam endophytic bacterium fermentation according to claim 1 prepares Chinese yam saponin, it is characterised in that institute
The composition for stating seed culture medium is beef extract 3g, peptone 10g, sodium chloride 5g, water 1000ml, the pH7.0- of seed culture medium
7.2；The composition of the fermentation medium be sucrose 100g, peptone 50g, dusty yeast 15g, sodium chloride 7g, dipotassium hydrogen phosphate 1g,
Corn steep liquor 6g, dextrin 14g, calcium carbonate 10g, water 1000ml.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808116A (en) * | 2020-07-24 | 2020-10-23 | 西北农林科技大学 | Fluorescent compound and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932030A (en) * | 2006-09-14 | 2007-03-21 | 中国科学院武汉植物园 | Process of preparing dioscin with dioscin penicillium notatum |
| CN101619294A (en) * | 2008-07-04 | 2010-01-06 | 西北农林科技大学 | Screening method of dioscorea zingiberensis endophyte for producing sapogenin |
| CN103146795A (en) * | 2012-12-27 | 2013-06-12 | 西安绿泉生物技术有限公司 | Method for producing diosgenin through microbial fermentation of peltate yam rhizome-yellow ginger |
-
2017
- 2017-07-12 CN CN201710565754.5A patent/CN107177662A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932030A (en) * | 2006-09-14 | 2007-03-21 | 中国科学院武汉植物园 | Process of preparing dioscin with dioscin penicillium notatum |
| CN101619294A (en) * | 2008-07-04 | 2010-01-06 | 西北农林科技大学 | Screening method of dioscorea zingiberensis endophyte for producing sapogenin |
| CN103146795A (en) * | 2012-12-27 | 2013-06-12 | 西安绿泉生物技术有限公司 | Method for producing diosgenin through microbial fermentation of peltate yam rhizome-yellow ginger |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808116A (en) * | 2020-07-24 | 2020-10-23 | 西北农林科技大学 | Fluorescent compound and preparation method and application thereof |
| CN111808116B (en) * | 2020-07-24 | 2023-04-07 | 西北农林科技大学 | Fluorescent compound and preparation method and application thereof |
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