CN103255193A - Ginsenoside conversion method by use of ginseng endophytic Paenibacillus polymyxa - Google Patents
Ginsenoside conversion method by use of ginseng endophytic Paenibacillus polymyxa Download PDFInfo
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Abstract
The invention relates to the field of biotechnology and specifically discloses a ginsenoside conversion method by the use of ginseng endophytic Paenibacillus polymyxa. The endophytic Paenibacillus polymyxa was preserved in the China General Microbiological Culture Collection Center on February, 5th, 2013. The address is No.3, Yard 1, West Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The collection number is CGMCC No. 7250. The ginseng endophytic Paenibacillus polymyxa has high conversion activity. By the adoption of the method, contents of ginsenoside monomer Rg1, Re, Rf, Rg2, Rb1, Rb2, Rb3 and Rd are raised or new compounds are induced. According to the strain, a raw material ginseng powder can be directly used as a medium for conversion of saponins. The method is simple, requires low cost, and is suitable for industrial large-scale production.
Description
Technical field
The present invention relates in the medicinal plants such as genseng a kind of genseng endogenetic polymexa bacillus (
Paenibacillus polymyxa) transform the method for ginsenoside.
Background technology
Ginsenoside is the effective component of genseng, but genseng content is lower, and particularly rare ginsenoside content is lower.After the ginsenoside oral administration, almost can not be decomposed by human intestines and stomach's digestive ferment in addition, but depend on the utilization that just can be absorbed by the body after the peculiar microbial metabolism in the enteron aisle, and play pharmacological action.But mainly being the enzyme by microorganisms, the transformation of energy of panoxadiol saponins come β-(1-6)-glycosidic link on the hydrolysis C20 and C3 to go up β-(1-2)-glucoside bond, then generate Rd, the further hydrolysis of Rd generates F2 or Rg3, F2 then is hydrolyzed into C-K, Rg3 can be converted into Rh2, finally changes into the diol type ginsengenin.Twentieth century Japan's seventies Gang Ji etc., fermented liquid conversion ginsenoside with soil bacteria has obtained C-K, nineties Japan foot bridges etc. are to having found ginsenoside Rb1 and ginsenoside Rg1's metabolic mechanism: transform back absorption, Rb1 → Rd → F2 → C-K → aglycon through enteric microorganism; Su Jin-Huan in 2006 etc. isolate bacterial strain from soil
Fusarium proliferatumECU2042, this bacterium can become Rh2 by hydrolysis panaxoside Rg 3, and transformation efficiency is 60.3%.Utilizations such as Yousef
Pytium irregulaeThe bacterium fermentation transforms glycols ginsenoside Rb1, Rb2, Rc, Rd etc. and is converted into F2.2007, doctor Cheng Leqin screened a strain new bacteria from soil
Microbacterium esteraromatiumGS514, it is Rd, Rg3, F2 and C-K that enzymatic production transforms Rb1.Tong Xin etc. have invented with moral formula Bacterium lacticum Bulgaria subspecies Radix Ginseng total saponins to be transformed and have obtained the rare saponin(e Rh2 of genseng.Yang Yuan in 2011 is superfine screen a strain Fusarium moniliforme (
Fuasarium moniliforme) to transform notoginseng stem and leaf total saponin Rb1, Rd be C-K.As seen can produce rare saponin(e or improve its output by microbial transformation.But do not see the report that transforms ginsenoside with the genseng endogenetic polymexa bacillus.
Summary of the invention
In view of the low problem of ginsenoside monomer content in the genseng, the object of the present invention is to provide a kind of genseng endogenetic polymexa bacillus to transform the method for ginsenoside, producing rare saponin(e or to improve its output, and then improve pharmaceutical use and the bioavailability of genseng.
1 one kinds of genseng endogenetic polymexa bacillus of example transform the method for ginsenoside
The present invention relates to biological technical field, is the method that a kind of genseng endogenetic polymexa bacillus transforms ginsenoside.Endogenetic polymexa bacillus of the present invention is kept at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC 7250.Endogenetic polymexa bacillus of the present invention has higher activity of conversion.This method has improved ginsenoside monomer Rg1, Re, Rf, Rg2, Rb1, Rb2, Rb3, Rd content.This bacterial strain can directly utilize conversion saponin(e raw material ginseng powder to be substratum, and method is easy, and cost is low, is conducive to industrialization scale operation.Concrete scheme is as follows:
1, the method for living bacillus polymyxa conversion ginsenoside in a kind of genseng, the used genseng endogenetic polymexa bacillus of the present invention (Paenibacillus polymyxa), be kept at China Committee for Culture Collection of Microorganisms common micro-organisms center on February 5th, 2013, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, this bacterium of deposit number CGMCC No.7250. is activated to be inoculated into fermentation culture in the genseng substratum.Substratum is: water: ginseng powder's's (60-100 order) ratio is 100ml:3-10 g, 121 ℃, cools off behind the sterilization 10-30min.Culture condition: liquid amount is 80-120ml/250ml, and living bacillus polymyxa seed liquor (OD600=0.5) inoculum size is 0.5-4% (V/V) in the genseng, and rotating speed is that the 120-200r/min concussion is cultivated, and culture temperature is 25 ℃-35 ℃, and incubation time is 1d-16d.
2, be to be substratum with genseng and distilled water only by 1 described this method, carry out the method that liquid state fermentation transforms ginsenoside.
3, transform the method for ginsenoside by 1 described a kind of genseng endogenetic polymexa bacillus, it is characterized in that this method has improved ginsenoside monomer Rg1, Re, Rf, Rg2
,Rb1, Rb2, Rb3, Rd content.
4, by 1 described optimal medium: water 100ml, 80 order ginseng powder 3g, cool off behind the sterilization 20min by 121 ℃.
5, by 1 described its actication of culture condition: adopt PDB substratum (potato 20g, glucose 2g, distilled water 100ml), the 120r/min shaking culture is spent the night in 25 ℃ of following thermostat containers, when measuring its OD600=0.5 as fermentation seed liquid.
6, by 1 described optimal culture condition: liquid amount is 100ml/250ml, and genseng endogenetic polymexa bacillus seed liquor (OD600=0.5) inoculum size is 1% (V/V), and rotating speed is that the 150r/min concussion is cultivated, and culture temperature is 25 ℃, and incubation time is 12d.
Test example 1 different fermentations temperature transforms the influence of ginsenoside to the genseng endogenetic polymexa bacillus
1 materials and methods
1.1 genseng endogenetic polymexa bacillus actication of culture
Insert under the inclined-plane genseng endogenetic polymexa bacillus bacterial classification aseptic technique with 4 degree preservations in the liquid PDB substratum, the 150r/min shaking culture is spent the night in 25 ℃ of following thermostat containers, measures its OD
600=0.5 o'clock as fermentation seed liquid.
The genseng endogenetic polymexa bacillus transforms ginsenoside
In 12 bottles of 250ml triangular flasks, add 100ml water and 3g ginseng powder respectively, 121 ℃, the 20min cooling of sterilizing, under aseptic condition, respectively connect 1ml genseng endogenetic polymexa bacillus seed liquor (OD600=0.5) with liquid-transfering gun, cultivate (4 repetitions of each temperature) 25 ℃, 30 ℃, 35 ℃ 150r/min concussions respectively, behind the cultivation 8d, get genseng endogenetic polymexa bacillus converted product.
Ginseng saponins is measured in the genseng endogenetic polymexa bacillus converted product
After 50-60 ℃ of water-bath oven dry of genseng endogenetic polymexa bacillus converted product, soxhlet extraction extracts saponin(e, measures ginseng saponins Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd content in the converted product with high-efficient liquid phase technique.
Data processing
Spss17.0 software carries out statistical study to data.
, result and analysis
Temperature is an important factor that influences bacteria growing, can influence the activity of enzyme, so also directly influence saponin content in the genseng endogenetic polymexa bacillus converted product.As shown in table 1, except ginseng saponins Rd, 25 ℃ when fermentation ginseng saponins content and 9 kinds of ginseng saponinses add and value reaches maximum.30 ℃ when fermentation ginseng saponins Rd value the highest, ginseng saponins Re, Rf, Rb1, Rb2, Rb3 content and 9 kinds of ginseng saponinses add and be worth and 25 ℃ of differences when fermenting not remarkable.
Table 1 different fermentations temperature transforms the influence (%) of ginsenoside to the genseng endogenetic polymexa bacillus
| Handle | Rg1 | Re | Rf | Rb1 | Rg2 | Rc | Rb2 | Rb3 | Rd | Rg1＋Re＋Rf＋Rb1＋Rg2＋Rc＋Rb2＋Rb3＋Rd |
| 25℃ | 0.59 a | 0.34 a | 0.13 a | 0.57 a | 0.04 a | 0.16 a | 0.23 a | 0.04 a | 0.53 b | 2.61 a |
| 30℃ | 0.44 b | 0.33 a | 0.14a | 0.58 a | 0.04 b | 0.12 b | 0.20 a | 0.04 a | 0.65 a | 2.53 a |
| 35℃ | 0.31 c | 0.21 b | 0.09 b | 0.38 b | 0.03 c | 0.05 c | 0.21 a | 0.01 b | 0.37 c | 1.65 b |
Annotate: same column different rows subscript letter identical table differential different not significantly (P〉0.05), same column different rows subscript letter different table differential different significantly (P＜0.05).
The test 2 different fermentations times of example transform the influence of ginsenoside to the genseng endogenetic polymexa bacillus
1 materials and methods
1.1 genseng endogenetic polymexa bacillus actication of culture
With test example 1.
The genseng endogenetic polymexa bacillus transforms ginsenoside
In 24 bottles of 250ml triangular flasks, add 100ml water and 3g ginseng powder respectively, 121 ℃, the 20min cooling of sterilizing, under aseptic condition, respectively connect 1ml genseng endogenetic polymexa bacillus seed liquor (OD600=0.5) with liquid-transfering gun, at 25 ℃, 150r/min shakes cultivation, cultivate 1d, 2 d, 4 d, 8 d, 12 d, 16d(each time 4 repetitions respectively) after, genseng endogenetic polymexa bacillus converted product got.
Ginseng saponins is measured in the genseng endogenetic polymexa bacillus converted product
With test example 1.
, data processing
With test example 1.
, result and analysis
Microorganism has growth cycle separately, and the length of fermentation time not only influences the microbial growth state, also influences microorganism to the conversion of ginsenoside, sees Table 2.Except ginseng saponins Rc and Rb2, add and be worth all with ginseng saponins content in the prolongation genseng endogenetic polymexa bacillus converted product of fermentation time and 9 kinds of ginseng saponinses and improve, ginseng saponins Rg1, Re, Rg2, Rc, Rb3 content and 9 kinds of ginseng saponinses add and be worth 12 days and during fermentation in 16 days difference not remarkable.From fermentation period and the fermentation of 9 kinds of saponin content angle analysis in the time of 12 days effect better.
The table 2 different fermentations time transforms the influence of ginsenoside to the genseng endogenetic polymexa bacillus(%)
| Handle (my god) | Rg1 | Re | Rf | Rb1 | Rg2 | Rc | Rb2 | Rb3 | Rd | Rg1＋Re＋Rf＋Rb1＋Rg2＋Rc＋Rb2＋Rb3＋Rd |
| 1 | 0.31 c | 0.17 e | 0.08 f | 0.31 e | 0.02 c | 0.22 b | 0.15 b | 0.03 d | 0.19 f | 1.47 e |
| 2 | 0.48 b | 0.28 d | 0.10 e | 0.45 d | 0.03 b | 0.26 a | 0.22 a | 0.04 c | 0.31 e | 2.16 d |
| 4 | 0.54 a | 0.31 c | 0.11 d | 0.51 c d | 0.03 b | 0.26 a | 0.22 a | 0.04 c | 0.37 d | 2.38 c |
| 8 | 0.59 a | 0.34 b | 0.13 c | 0.56 c | 0.04 a | 0.16 c | 0.23 a | 0.04 b | 0.53 c | 2.61 b |
| 12 | 0.56 a | 0.40 a | 0.16 b | 0.69 b | 0.04 a | 0.15 c | 0.11 c | 0.05 | 0.86 b | 3.02 a |
| 16 | 0.59 a | 0.41 a | 0.17 a | 0.77 a | 0.05 a | 0.16 c | 0.08 d | 0.05 a | 0.94 a | 3.22 a |
Annotate: same column different rows subscript letter identical table differential different not significantly (P〉0.05), same column different rows subscript letter different table differential different significantly (P＜0.05).
Test example 3 differences connect the bacterium amount transforms ginsenoside to the genseng endogenetic polymexa bacillus influence
1 materials and methods
1.1 genseng endogenetic polymexa bacillus actication of culture
With test example 1.
The genseng endogenetic polymexa bacillus transforms ginsenoside
In 16 bottles of 250ml triangular flasks, add 100ml water and 3g ginseng powder respectively, 121 ℃, the 20min cooling of sterilizing, under aseptic condition, respectively connect living bacillus polymyxa seed liquor (OD600=0.5) in 0.5 ml, 1ml, 2 ml, the 4 ml gensengs (4 repetitions of each inoculum size) with liquid-transfering gun, at 25 ℃, 150r/min shakes cultivation, after cultivating 12 d, get living bacillus polymyxa converted product in the genseng.
Ginseng saponins is measured in the genseng endogenetic polymexa bacillus converted product
With test example 1.
Data processing
With test example 1.
, result and analysis
Connecing the bacterium amount in the fermenting process also is the important factor that influences microorganism growth, and it is as shown in table 3 to the influence that the genseng endogenetic polymexa bacillus transforms ginsenoside that difference connects the bacterium amount.With 9 kinds of ginseng saponins content in the increase genseng endogenetic polymexa bacillus converted product of inoculum size and and add and be worth all raisings, but 2ml and 4ml treatment group differences are not remarkable.Effect is better when connecing the bacterium amount for 2ml from fermentation costs and 9 kinds of saponin content angle analysis.
Table 3 difference connects the bacterium amount transforms ginsenoside to the genseng endogenetic polymexa bacillus influence (%)
| Handle | Rg1 | Re | Rf | Rb1 | Rg2 | Rc | Rb2 | Rb3 | Rd | Rg1＋Re＋Rf＋Rb1＋Rg2＋Rc＋Rb2＋Rb3＋Rd |
| 0.5ml | 0.50 c | 0.39 a | 0.17 a | 0.71 a | 0.04 c | 0.16 a | 0.30 a | 0.06 a | 0.66 a | 3.00 b |
| 1ml | 0.59 bc | 0.40 a | 0.16 a | 0.74 a | 0.04 bc | 0.16 a | 0.30 a | 0.06 a | 0.67 a | 3.12 ab |
| 2ml | 0.77 a | 0.41 a | 0.16 a | 0.77 a | 0.05 ab | 0.17 a | 0.29 a | 0.07 a | 0.68 a | 3.36 a |
| 4ml | 0.75 ab | 0.41 a | 0.15 a | 0.77 a | 0.05 a | 0.15 a | 0.30 a | 0.08 a | 0.72 a | 3.38 a |
Annotate: same column different rows subscript letter identical table differential different not significantly (P〉0.05), same column different rows subscript letter different table differential different significantly (P＜0.05).
Claims (9)
- A genseng endogenetic polymexa bacillus ( Paenibacillus polymyxa) transform the method for ginsenoside, it is characterized in that with the genseng endogenetic polymexa bacillus be bacterial classification, be kept at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC 7250 activatedly is inoculated into fermentation culture in the genseng substratum.
- 2. by the described substratum of claim 1 be: water 100ml, 60-100 order ginseng powder 3-10 g cools off behind 121 ℃ of sterilization 10-30min.
- 3. by the described culture condition of claim 1: liquid amount is 80-120ml/250ml, genseng endogenetic polymexa bacillus seed liquor (OD600=0.5) inoculum size is 0.5-4% (V/V), rotating speed is that the 120-200r/min concussion is cultivated, and culture temperature is 25 ℃-35 ℃, and incubation time is 1d-16d.
- 4. be to be substratum with genseng and distilled water only by described this method of claim 1, carry out the method that liquid state fermentation transforms ginsenoside.
- 5. transform the method for ginsenoside by the described a kind of genseng endogenetic polymexa bacillus of claim 1, it is characterized in that this method has improved ginsenoside monomer Rg1, Re, Rf, Rg2 ,Rb1, Rb2, Rb3, Rd content.
- 6. the method optimised process that transforms ginsenoside by the described a kind of genseng endogenetic polymexa bacillus of claim 1 is substratum: water 100ml, 80 order ginseng powder 3g cool off behind 121 ℃ of sterilization 20min.
- 7. by claim 1 optimal culture condition: liquid amount is 100ml/250ml, and genseng endogenetic polymexa bacillus seed liquor (OD600=0.5) inoculum size is 2% (V/V), and rotating speed is that the 150r/min concussion is cultivated, and culture temperature is 25 ℃, and incubation time is 12d.
- 8. transform the method for ginsenoside by the described a kind of genseng endogenetic polymexa bacillus of claim 1, its actication of culture adopts PDB substratum (potato 20g, glucose 2g, distilled water 100ml), the 120r/min shaking culture is spent the night in 25 ℃ of following thermostat containers, when measuring its OD600=0.5 as fermentation seed liquid.
- 9. be applicable to terpenoids such as transforming ginsenoside with the genseng endogenetic polymexa bacillus or induce new compound by described this method feature of claim 1.
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| CN106929465A (en) * | 2017-04-10 | 2017-07-07 | 吉林农业大学 | A kind of Cu2+Ion promotes Paenibacillus polymyxa propagation and its application process |
| CN107312720A (en) * | 2017-03-01 | 2017-11-03 | 东北林业大学 | One plant of Efficient Conversion ginsenoside Rb1 is Rd pigeonpeas endogenetic fungus and application |
| CN107663528A (en) * | 2017-04-10 | 2018-02-06 | 吉林农业大学 | A kind of Cu2+The method that ion promotes Paenibacillus polymyxa conversion ginsenoside |
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| CN104784230A (en) * | 2014-01-17 | 2015-07-22 | 百济红蔘株式会社 | Fermented red ginseng and manufacturing method thereof |
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| CN106367371A (en) * | 2016-09-18 | 2017-02-01 | 中南民族大学 | Enterobacter cloacae clx-14 strain for generating polyphyllin I and application thereof |
| CN107312720A (en) * | 2017-03-01 | 2017-11-03 | 东北林业大学 | One plant of Efficient Conversion ginsenoside Rb1 is Rd pigeonpeas endogenetic fungus and application |
| CN107312720B (en) * | 2017-03-01 | 2020-02-04 | 东北林业大学 | Cochinchinensis endophytic fungus for efficiently converting ginsenoside Rb1 into Rd and application thereof |
| CN106929465A (en) * | 2017-04-10 | 2017-07-07 | 吉林农业大学 | A kind of Cu2+Ion promotes Paenibacillus polymyxa propagation and its application process |
| CN107663528A (en) * | 2017-04-10 | 2018-02-06 | 吉林农业大学 | A kind of Cu2+The method that ion promotes Paenibacillus polymyxa conversion ginsenoside |
| CN108034614A (en) * | 2018-01-19 | 2018-05-15 | 吉林农业大学 | A kind of Paenibacillus polymyxa while 5 kinds of pesticide residue methods of degrading |
| CN108034614B (en) * | 2018-01-19 | 2022-05-27 | 吉林农业大学 | Method for simultaneously degrading 5 pesticide residues by using Paenibacillus polymyxa |
| CN108753900A (en) * | 2018-06-13 | 2018-11-06 | 延边大学 | A kind of method that the fermentation of ginseng endophyte prepares rare ginsenoside |
| CN111979139A (en) * | 2020-05-30 | 2020-11-24 | 郑州大学 | Bacillus belgii G9y and application thereof in ginsenoside biotransformation |
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