CN103146592B - Microzyme converting ginsenoside Rb1 to generate Rd and application thereof - Google Patents
Microzyme converting ginsenoside Rb1 to generate Rd and application thereof Download PDFInfo
- Publication number
- CN103146592B CN103146592B CN201310038798.4A CN201310038798A CN103146592B CN 103146592 B CN103146592 B CN 103146592B CN 201310038798 A CN201310038798 A CN 201310038798A CN 103146592 B CN103146592 B CN 103146592B
- Authority
- CN
- China
- Prior art keywords
- ginsenoside
- generate
- saccharomycetes
- conversion
- microzyme
- 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
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses microzyme converting ginsenoside Rb1 to generate Rd and application of the microzyme converting the ginsenoside Rb1 to generate the Rd. The microzyme is saccharomyces cerevisiae GWYS01. The preservation unit is China representative culture collection center. The preservation address is Wuhan University in Wuhan, China. The preservation number is CCTCC NO: M2013038. The preservation data is January, 22ed, 2013. The invention further discloses the application of the microzyme converting the ginsenoside Rb1 to generate the Rd. According to the microzyme converting the ginsenoside Rb1 to generate the Rd and the application of the microzyme converting the ginsenoside Rb1 to generate the Rd, glycosidase which is a microzyme metabolic product is used for converting the ginsenoside Rb1 to the Rd, bioavailability of effective ingredients in ginseng is improved, a conversion rate of the Rb1 in 24 hours reaches 97.5%, and a rate of generation of the Rd through conversion is 62%. The microzyme converting the ginsenoside Rb1 to generate the Rd and the application of the microzyme converting the ginsenoside Rb1 to generate the Rd are low in cost, simple in operation, and short in conversion period, and promote development of modernization and industrialization of traditional Chinese medicine ginsenoside.
Description
Technical field
The present invention relates to a kind of ginsenoside Rb that transforms
1the yeast that generates Rd also relates to this saccharomycetic application simultaneously, belongs to technical field of traditional Chinese medicines.
Background technology
Ginseng (Panax ginseng G.A.Meyer) is the traditional Chinese medicine of China's preciousness, is applied to the tcm clinical practice history of existing more than 2,000 year.The main active ingredient of ginseng is ginsenoside (ginsenoside), and wherein, protopanoxadiol saponins belongs to dammarane type four-ring triterpenoid saponin, mainly comprises Rb
1, Rc, Rd, Rg
3and Rh
2deng.The textural difference of above monomer is only that C3 is different with the glycosyl side chain on C20 position, but the pharmacologically active demonstrating has difference.As Rb
1show good anti-arrhythmia and anti-aging effects (ginsenoside Rb
1pharmacology activity research progress, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008,33 (12): 1371-1377), Rc can weaken pain (Effects of ginsenosides on vanilloid receptor (VR1) the channels expressed in Xenopus oocytes by capsicine and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 induction, Mol Cells, 2001,12:342-346, Antinociceptive effects ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model, Planta Med, 2003,69:1001-1004) and extend haemolysis occur (Can ginsenosides protect human erythrocytes against free-radical-induced hemolysis retardation time, Biochim Biophys Acta, 2002,1572 (1): 58-66), Rd has the neural stem cell of promotion differentiation (Ginsenoside-Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells, Life Sci, 2005, 76 (9): 983-995), lethality excitotoxicity (the Effects of ginsenoside Rd and decursinol on the neurotoxic responses induced by kainic acid in mice that the acid of selectivity antagonism cacaine causes, Planta Med, 2003, 69 (3): 230-234), weaken pain (Effects of ginsenosides on vanilloid receptor (VR1) the channels expressed in Xenopus oocytes by capsicine and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 induction, Mol Cells, 2001, 12:342-346, Antinociceptive effects ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model, Planta Med, 2003,69:1001-1004) and anti-ageing (Ginsenoside-Rd attenuates oxidative damage related to aging in senescence-accelerated mice, J Pharm Pharmacol, 2004,56 (1): 107-113) effect such as, Rg
3there is significant effect (ginsenoside Rg at aspects such as inducing apoptosis of tumour cell, inhibition tumor cell propagation, inhibition tumor cell invasion and attack and transfer and enhancing body immunizing power
3advances in studies on antitumor activity, modern tumour medical science, 2008,16 (4): 648-652), Rh
2kinds of tumors is all had to certain restraining effect, there is stronger antitumor synergism (Rh
2synergistically enhances paclitaxel or mitoxantrone in prostate cancer models, J Urol, 2006,175 (5): 1926-1931), in addition, also there is hypoglycemic effect (Mediation of β-endorphin by ginsenoside Rh2to lower plasma glucose in streptozotocin-induced diabetic rats, Planta Med, 2006,72 (1): 9-13).In above-mentioned saponin(e, Rb
1, Rc and other three kinds of saponin monomer Rb
2, Re and Rg
1content in ginseng exceedes 80%, and Rd, Rg
3and Rh
2content is but extremely low, but they have stronger pharmacologically active (Microbial conversion of major ginsenoside Rb
1to pharmaceutically active minor ginsenoside Rd, Journal of Microbiology, 2005,43 (5): 456-462).In addition, natural high-content methods of glycosides is difficult to absorb at enteron aisle, and molecular structure is not optimum activity state, is difficult to directly bring into play drug effect, and therefore, rare saponin(e becomes main research and development object both at home and abroad.
The mechanism of ginsenoside in ginseng metabolic process is also indefinite, cannot in process of growth, be controlled with orientation accumulation rare ginsenoside.Investigator often passes through more much higher content glycosyl ginsenoside as Rb
1and Rc is hydrolyzed to obtain Rd, the Rg of low glycosyl
3or Rh
2etc. rare saponin(e.The method of ginsenoside glycosyl transformation has chemical method, enzyme process and microbe transformation method; wherein enzyme process and microbe transformation method are simple to operate; can protect to greatest extent chemical composition of Chinese materia medica to exempt from destruction; be conducive to environment protection simultaneously; reducing production costs, is the desirable mode of production of producing rare saponin(e take Radix Ginseng total saponins as feedstock conversion.At present, the existing glycosyl hydrolase that utilizes institute's production capacity hydrolysis ginsenoside glycosidic link in microorganism or animals and plants metabolic process is catalyzer, the directed technological method that generates rare saponin(e that transforms.The microorganism type relating in research is mainly bacterium and mould (screening of high yield ginsenoside beta-glucosidase bacterial classification, Journal of Dalian Institute of Light, 2000,19 (3): 195-198; From a kind of bacterium of new screening, find out ginsenosidases, Journal of Dalian Institute of Light, 2003,22 (3): 164-166; Microbial conversion of major ginsenoside Rb
1to pharmaceutically active minor ginsenoside Rd, Journal of Microbiology, 2005,43 (5): 456-462; Panoxadiol saponins is converted into Rg
3the bacteria screening that generates of extraordinary ginsenoside Glycosylase, Dalian Polytechnic University's journal, 2008,27 (2): 97-101), also there is report (the Purification and characterization of ginsenoside-β-glucosidase from ginseng that isolates ginsenoside lytic enzyme and use animal digestion enzyme to study from ginseng, Chem.Pharm.Bull, 2001,49 (7): 795-798; Purification and characterization of ginsenoside-α-arabinofuranase hydrolyzing ginsenoside Rc into Rd from the fresh root of Panax ginseng, Process Biochemistry, 2002,37:793-798; A kind of ginsenoside Rb in helicase
1the separation and purification of enzyme, biotechnology journal, 2005,11:929-933), in addition, (fungi is to ginseng soap former times Rb to use in addition fungi
1and the metabolism of panaxadiol saponins, Acta Pharmaceutica Sinica, 2001,3 (18): 603-605) or large-scale medicinal fungi adopt solid fermentation to carry out the research report (research of Microbiological Transformation of Ginsenoside of microbial transformation, Jilin: Jilin Agriculture University, 2004).In research, obtaining more enzyme is ginsenoside Rb
1lytic enzyme, its hydrolysate is mainly Rd, in addition in addition can be by Rg
3hydrolysis generates Rh
2ginsenoside Glycosylase, and separation and purification goes out the ginsenoside-α-arabinofuranosidase/xylosidase that Rc hydrolysis can be generated to Rd from ginseng.
In these method for transformation, mostly use inclined-plane seed activation, prepare liquid seeds and carry out microbial transformation, in the time carrying out bio-transformation, except ginsenoside raw material, also add other auxiliary material as glucose simultaneously, inorganic salt etc., and transformation efficiency is low, is mostly about 15%～20%.Also utilize active dry yeast to transform ginsenoside Rb
1report, transformation efficiency is only 30%～36%.
Summary of the invention
The object of this invention is to provide a kind of ginsenoside Rb that transforms
1generate the yeast of Rd.
In order to realize above object, the technical solution adopted in the present invention is to provide a kind of ginsenoside Rb that transforms
1generate the yeast of Rd, described yeast is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GWYS01, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preserving number: CCTCC NO:M2013038, preservation date: on January 22nd, 2013.
Yeast is a kind of unicellular fungi, there is the enzyme system that various active is powerful, as amylase, saccharifying enzyme, Glycosylase, lipase, proteolytic enzyme, cellulase etc., particularly glycosidase activity is strong, and ginsenoside is made up of aglycon and sugar chain, utilize Glycosylase that yeast produces, the glycosyl on degraded ginsenoside molecule, make it be converted into saponin(e aglycon, can improve the bioavailability of ginsenoside.
In metabolism of yeasts product of the present invention, containing can intense hydrolysis ginsenoside Rb
1ginsenoside Glycosylase, 24 hours Rb
1transformation efficiency reach 97.5%, transforming and forming the ratio of Rd is 62%.
The present invention also aims to provide a Yeasts transforming ginsenoside Rb
1generate the application of Rd aspect.
The technical solution adopted in the present invention is also to provide a Yeasts transforming ginsenoside Rb
1generate the application of Rd aspect.
The present invention also aims to provide a kind of conversion of saccharomycetes ginsenoside Rb
1generate the method for Rd.
The technical solution adopted in the present invention is also to provide a kind of conversion of saccharomycetes ginsenoside Rb
1the method that generates Rd, comprises the following steps:
1) preparation of enzyme liquid:
Saccharomycetes to make fermentation is cultivated, then by filtering fermentation liquor, centrifugal for the first time, remove bacterial sediment, collect supernatant liquor; Under 20-30 ℃ of condition, add ammonium sulfate to 60-80% saturation ratio, 4-6 ℃ of hold over night; Then centrifugal for the second time, abandon supernatant liquor, collect protein precipitation; After being dissolved, the sodium-acetate buffer 6-10mL of 0.02mol/L, pH4.5-5.5 for protein precipitation packs dialysis tubing into, with identical sodium-acetate buffer dialysis 22-26h; After dialysis, centrifugal for the third time, supernatant liquor is enzyme liquid;
2) conversion reaction of enzyme liquid:
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5-5.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:5-9 with enzyme liquid, under 35-45 ℃ of condition, reacts, and obtains Ginsenoside Rd.
The concrete grammar that described saccharomycetes to make fermentation is cultivated is: yeast is inoculated on PDA substratum, under 28-32 ℃ of condition, cultivate 35-45h, then yeast is inoculated in YPD substratum, under 34-38 ℃ of condition, shaking culture 45-51h, obtains saccharomycetes to make fermentation liquid.
Described PDA substratum comprises following component: potato 200g, and glucose 20g, agar 15-20g, water is settled to 1000mL, natural pH.
Described YPD substratum comprises the component of following massfraction: 1% yeast extract, 2% peptone, 2% glucose.
Described centrifugal condition is for the first time: centrifuging temperature 15-17 ℃, rotating speed 10000-12000r/min, centrifugation time 15-20min.
Described centrifugal condition is for the second time: centrifuging temperature 4-6 ℃, rotating speed 12000-14000r/min, centrifugation time 15-20min.
Described centrifugal condition is for the third time: centrifuging temperature 15-17 ℃, rotating speed 12000-14000r/min, centrifugation time 8-12min.
The present invention utilizes metabolism of yeasts product Glycosylase by ginsenoside Rb
1be converted into Rd, improved the bioavailability of effective constituent in ginseng.Cost of the present invention is lower, simple to operate, and the transformation period is short, has promoted the development of the modernization of Chinese medicine ginsenoside and industrialization.
Embodiment
The preparation of substratum:
PDA substratum: potato 200g, glucose 20g, agar 15-20g, water is settled to 1000mL, natural pH, 115 ℃, 0.1MPa autoclaving 20min.
YPD substratum: 1% yeast extract, 2% peptone, 2% glucose, 115 ℃, 0.1MPa autoclaving 20min.YPD Agar substratum i.e. 2% agar in YPD substratum.
Malt juice liquid medium: by kilned malt pulverize, a Fructus Hordei Germinatus adds four parts of water, saccharification 3-4h in 65 ℃ of water-baths, by saccharified liquid boil rear with filtered through gauze to clarification, be diluted to 5-6 degree Beaume, pH6.4,121 ℃, 0.1MPa, autoclaving 20min.
Glucose fermentation substratum: soybean sprout 125g, yeast extract paste 6g, glucose 20g, water 1000mL, 115 ℃ of sterilizings, 20min.
Nitrogenous source assimilation substratum: glucose 20g, KH
2pO
41g, MgSO
47H
2o0.5g, yeast extract paste 0.2g, agar 20g, water 1000mL, packing test tube after filtering, 115 ℃ of sterilizing 20min.
Embodiment 1,
1, saccharomycetic screening
Brewery is stored to mud to be inoculated in and in malt juice liquid medium, to increase bacterium and cultivate, then adopt YPD substratum, with plate dilution method isolated strains, cultivate 2d respectively under 25 ℃ and 37 ℃ of two temperature condition, take bacterium colony mode of appearance as main, coordinate microscopy to observe thalli morphology, it is some that acquisition meets the bacterial strain of yeast colony characteristics, then carry out repeatedly purifying and obtain 5 bacterial strains, access respectively YPD Agar medium slant, be placed in 4 ℃, refrigerator and save backup.5 bacterial strains that separation and purification obtains called after GWYS01, GWYS02, GWYS03, GWYS04, GWYS05 respectively.
2, the preparation of enzyme liquid
The corresponding enzyme liquid of 5 bacterial strains that Preparation Example 1 is screened respectively, concrete steps are as follows:
1) cultivation of bacterial strain and fermentation
The inoculation of embodiment 1 separation and purification, in PDA substratum, is cultivated after 40h for 30 ℃, then proceed to the fermentation of YPD substratum according to the ratio of volume fraction 5%, under 36 ℃, 160r/min condition, shaking culture 48h, obtains fermented liquid;
2) preparation of enzyme liquid:
By filtering fermentation liquor, under 15 ℃, 10000r/min condition, centrifugal 20min, removes bacterial sediment, collects supernatant liquor; Under 25 ℃ of conditions, add ammonium sulfate to 70% saturation ratio, 4 ℃ of hold over night; Then under 4 ℃, 13000r/min condition, centrifugal 20min, abandons supernatant liquor, collects protein precipitation; After being dissolved, the sodium-acetate buffer 8mL of 0.02mol/L, pH5.0 for protein precipitation packs dialysis tubing into, with identical sodium-acetate buffer dialysis 24h; After dialysis, at 15 ℃, under 13000r/min condition, centrifugal 10min, supernatant liquor is enzyme liquid.
3, conversion reaction
By 5 corresponding enzyme liquid of bacterial strain under the same conditions with ginsenoside Rb
1reaction, screening obtains 1 and can effectively transform ginsenoside Rb
1the bacterial strain that generates Rd is GWYS01.
GWYS01 streak inoculation, in YPD substratum, is cultivated to 2d at 37 ℃, its colony morphology characteristic of visual inspection, microscopy observation of cell form.Select glucose fermentation substratum, inoculate rear 37 ℃ of cultivations, observe fermentation situation every 12h, select nitrogenous source assimilation substratum simultaneously, inoculate latter 37 ℃ and cultivate 2d observation.Observe colonial morphology, the cellular form of bacterial strain GWYS01, and Physiology and biochemistry cultural characters, as shown in table 1.
Table 1 morphologic observation and physio-biochemical characteristics test result
18S rDNA fragment amplification and order-checking:
Extract its genome, adopt EF3, EF4 universal primer to carry out 18S rDNA fragment amplification and order-checking, wherein,
EF3：5'-TCCTCTAAATGACCAAGTTTG-3'（SEQ?ID?NO:1）
EF4：5'-GGAAGGGRTGTATTTATTAG-3'（SEQ?ID?NO:2）
Wherein, R represents A or G.
18S rDNA amplified fragments sequencing result:
gatggagttg?cccccttctc?taagcagatc?ctgaggcctc?actaagccat?tcaatcggta?ctagcgacgg?gcggtgtgta
caaagggcag?ggacgtaatc?aacgcaagct?gatgacttgc?gcttactagg?aattcctcgt?tgaagagcaa?taattacaat?gctctatccc
cagcacgacg?gagtttcaca?agattaccaa?gacctctcgg?ccaaggttag?actcgctggc?tccgtcagtg?tagcgcgcgt
gcggcccaga?acgtctaagg?gcatcacaga?cctgttattg?cctcaaactt?ccatcggctt?gaaaccgata?gtccctctaa?gaagtggata
accagcaaat?gctagcacca?ctatttagta?ggttaaggtc?tcgttcgtta?tcgcaattaa?gcagacaaat?cactccacca?actaagaacg
gccatgcacc?accacccaca?aaatcaagaa?agagctctca?atctgtcaat?ccttattgtg?tctggacctg?gtgagtttcc?ccgtgttgag
tcaaattaag?ccgcaggctc?cactcctggt?ggtgcccttc?cgtcaattcc?tttaagtttc?agccttgcga?ccatactccc?cccagaaccc
aaagactttg?atttctcgta?aggtgccgag?tgggtcatta?aaaaaacacc?acccgatccc?tagtcggcat?agtttatggt?taagactacg
acggtatctg?atcatcttcg?atcccctaac?tttcgttctt?gattaatgaa?aacgtccttg?gcaaatgctt?tcgcagtagt?tagtcttcaa
taaatccaag?aatttcacct?ctgacaattg?aatactgatg?cccccgaccg?tccctattaa?tcattacgat?ggtcctagaa?accaacaaaa
tagaaccaaa?cgtcctattc?tattattcca?tgctaatata?ttcgagcaat?acgcctgctt?tgaacactct?aattttttca?aagtaaaagt
cctggttcgc?caagagccac?aaggactcaa?ggttagccag?aaggaaaggc?cccgttggaa?atccagtaca?cgaaaaaatc
ggaccggcca?accgggccca?aagttcaact?acgagctttt?taactgcaac?aactttaata?tacgctattg?gagctggaat?taccgcggct
gctggcacca?gacttgccct?ccaattgttc?ctcgttaagg?tatttacatt?gtactcattc?caattacaag?acccgaatgg?gccctgtatc
gttatttatt?gtcactacct?ccctgaatta?ggattgggta?atttgcgcgc?ctgctgcctt?ccttggatgt?ggtagccgtt?tctcaggctc
cctctccgga?atcgaaccct?tattccccgt?tacccgttga?aaccatggta?ggccactatc?ctaccatcga?aagttgatag?ggcagaaatt
tgaatgaacc?atcgccagca?caaggccatg?cgattcgaaa?agttattatg?aatcatcaaa?gagtccga
Sequencing result carries out homology sequence compare of analysis in GenBank database, and result shows that GWYS01 and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genes involved fragment have 100% similar value in sequence.Combining form observation, physio-biochemical characteristics and Molecular Identification, identify that GWYS01 is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
Embodiment 2
1) saccharomycetic cultivation and fermentation
GWYS01 is inoculated in PDA substratum, cultivates after 35h for 32 ℃, then proceed to the fermentation of YPD substratum according to the ratio of volume fraction 5%, under 38 ℃, 160r/min condition, shaking culture 45h, obtains fermented liquid;
2) preparation of enzyme liquid:
By filtering fermentation liquor, under 16 ℃, 12000r/min condition, centrifugal 17min, removes bacterial sediment, collects supernatant liquor; Under 30 ℃ of conditions, add ammonium sulfate to 80% saturation ratio, 5 ℃ of hold over night; Then under 5 ℃, 12000r/min condition, centrifugal 15min, abandons supernatant liquor, collects protein precipitation; After being dissolved, the sodium-acetate buffer 6mL of 0.02mol/L, pH5.5 for protein precipitation packs dialysis tubing into, with identical sodium-acetate buffer dialysis 22h; After dialysis, at 16 ℃, under 12000r/min condition, centrifugal 12min, supernatant liquor is enzyme liquid.
Embodiment 3
1) saccharomycetic cultivation and fermentation
GWYS01 is inoculated in PDA substratum, cultivates after 45h for 28 ℃, then proceed to the fermentation of YPD substratum according to the ratio of volume fraction 5%, under 34 ℃, 160r/min condition, shaking culture 51h, obtains fermented liquid;
2) preparation of enzyme liquid:
By filtering fermentation liquor, under 17 ℃, 11000r/min condition, centrifugal 15min, removes bacterial sediment, collects supernatant liquor; Under 20 ℃ of conditions, add ammonium sulfate to 60% saturation ratio, 6 ℃ of hold over night; Then under 6 ℃, 14000r/min condition, centrifugal 17min, abandons supernatant liquor, collects protein precipitation; After being dissolved, the sodium-acetate buffer 10mL of 0.02mol/L, pH4.5 for protein precipitation packs dialysis tubing into, with identical sodium-acetate buffer dialysis 26h; After dialysis, at 17 ℃, under 14000r/min condition, centrifugal 8min, supernatant liquor is enzyme liquid.
Embodiment 4
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:5 with the enzyme liquid that GWYS01 in embodiment 1 makes, and under 45 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 5
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 7:50 with the enzyme liquid that GWYS01 in embodiment 1 makes, and under 40 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 6
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH4.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:9 with the enzyme liquid that GWYS01 in embodiment 1 makes, and under 35 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 7
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.0, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 7:50 with the enzyme liquid that GWYS01 in embodiment 1 makes, and under 40 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 8
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.0, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:5 with the enzyme liquid that GWYS01 in embodiment 2 makes, and under 35 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 9
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.0, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:9 with the enzyme liquid that GWYS01 in embodiment 2 makes, and under 45 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 10
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:5 with the enzyme liquid that GWYS01 in embodiment 3 makes, and under 35 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 11
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 7:50 with the enzyme liquid that GWYS01 in embodiment 3 makes, and under 45 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Embodiment 12
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH5.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:9 with the enzyme liquid that GWYS01 in embodiment 3 makes, and under 40 ℃ of conditions, reacts 24h, then in 100 ℃ of water-baths, places 15min, obtains Ginsenoside Rd.
Experimental example, conversion of saccharomycetes ginsenoside Rb
1generate the transformation efficiency analysis of Rd
By the enzyme reaction solution of embodiment 4-12 with do not add the substrate reactions liquid of enzyme liquid to dry at 60 ℃, be then dissolved in respectively in 95% methyl alcohol 700 μ L the centrifugal 10min of 8000rmp.Draw supernatant liquor with syringe, after 0.45 μ m pin type filter filters, be stored in sample bottle to be measured.
Enzyme reaction solution detects analysis by HPLC, HPLC chromatographic condition: detector is UV/VIS, detects wavelength 203nm; Chromatographic column: ODS C18(150mm × 4.6mm, 5 μ are m); Sample size 10 μ L, 35 ℃ of column temperatures, flow velocity 1mL/min, moving phase: 0-23min, acetonitrile: 0.05% phosphoric acid water=32:68; 23-80min, acetonitrile: 0.05% phosphoric acid water=47:53.Record the content of the various compositions in reaction front and back according to typical curve, calculate ginsenoside transformation efficiency according to reaction substrate and product molar ratio relation, result is as shown in table 2.
Table 2 conversion of saccharomycetes ginsenoside Rb
1generate the transformation efficiency analysis of Rd
| ? | Ginsenoside Rb 1Transformation efficiency % | Transform the ratio % that forms Ginsenoside Rd |
| Embodiment 4 | 94 | 51 |
| Embodiment 5 | 95 | 54 |
| Embodiment 6 | 97 | 55 |
| Embodiment 7 | 97.5 | 62 |
| Embodiment 8 | 97 | 60 |
| Embodiment 9 | 98 | 57 |
| Embodiment 10 | 92 | 55 |
| Embodiment 11 | 98 | 58 |
| Embodiment 12 | 96 | 53 |
Claims (9)
1. one kind transforms ginsenoside Rb
1generate the yeast of Rd, it is characterized in that, described yeast be yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) GWYS01, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preserving number: CCTCC NO:M2013038, preservation date: on January 22nd, 2013.
2. a yeast as claimed in claim 1 is transforming ginsenoside Rb
1generate the application of Rd aspect.
3. a conversion of saccharomycetes ginsenoside Rb as claimed in claim 1
1the method that generates Rd, is characterized in that, comprises the following steps:
1) preparation of enzyme liquid:
Saccharomycetes to make fermentation is cultivated, then by filtering fermentation liquor, centrifugal for the first time, remove bacterial sediment, collect supernatant liquor; Under 20-30 ℃ of condition, add ammonium sulfate to 60-80% saturation ratio, 4-6 ℃ of hold over night; Then centrifugal for the second time, abandon supernatant liquor, collect protein precipitation; After being dissolved, the sodium-acetate buffer 6-10mL of 0.02mol/L, pH 4.5-5.5 for protein precipitation packs dialysis tubing into, with identical sodium-acetate buffer dialysis 22-26h; After dialysis, centrifugal for the third time, supernatant liquor is enzyme liquid;
2) conversion reaction of enzyme liquid:
Take ginsenoside Rb
1, by ginsenoside Rb
1mix with the sodium-acetate buffer of 0.02mol/L, pH 4.5-5.5, be mixed with ginsenoside Rb
1concentration is the substrate reactions liquid of 1.70mg/mL, then substrate reactions liquid is mixed according to volume ratio 1:5-9 with enzyme liquid, under 35-45 ℃ of condition, reacts, and obtains Ginsenoside Rd.
4. conversion of saccharomycetes ginsenoside Rb according to claim 3
1generate the method for Rd, it is characterized in that, the concrete grammar that described saccharomycetes to make fermentation is cultivated is: yeast is inoculated on PDA substratum, under 28-32 ℃ of condition, cultivate 35-45h, then yeast is inoculated in YPD substratum, under 34-38 ℃ of condition, shaking culture 45-51h, obtains saccharomycetes to make fermentation liquid.
5. conversion of saccharomycetes ginsenoside Rb according to claim 4
1the method that generates Rd, is characterized in that, described PDA substratum comprises following component: potato 200g, and glucose 20g, agar 15-20g, water is settled to 1000mL, natural pH.
6. conversion of saccharomycetes ginsenoside Rb according to claim 4
1the method that generates Rd, is characterized in that, described YPD substratum comprises the component of following massfraction: 1% yeast extract, 2% peptone, 2% glucose.
7. conversion of saccharomycetes ginsenoside Rb according to claim 3
1generate the method for Rd, it is characterized in that, described centrifugal condition is for the first time: centrifuging temperature 15-17 ℃, rotating speed 10000-12000r/min, centrifugation time 15-20min.
8. conversion of saccharomycetes ginsenoside Rb according to claim 3
1generate the method for Rd, it is characterized in that, described centrifugal condition is for the second time: centrifuging temperature 4-6 ℃, rotating speed 12000-14000r/min, centrifugation time 15-20min.
9. conversion of saccharomycetes ginsenoside Rb1 according to claim 3 generates the method for Rd, it is characterized in that, described centrifugal condition is for the third time: centrifuging temperature 15-17 ℃, rotating speed 12000-14000r/min, centrifugation time 8-12min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310038798.4A CN103146592B (en) | 2013-01-31 | 2013-01-31 | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310038798.4A CN103146592B (en) | 2013-01-31 | 2013-01-31 | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103146592A CN103146592A (en) | 2013-06-12 |
| CN103146592B true CN103146592B (en) | 2014-06-25 |
Family
ID=48544916
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310038798.4A Active CN103146592B (en) | 2013-01-31 | 2013-01-31 | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103146592B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104435032A (en) * | 2013-09-18 | 2015-03-25 | 长春三德天晟科技有限公司 | Method for preparing health-care products from fresh ginseng roots, fruits, stems and leaves |
| KR20170047008A (en) * | 2015-10-22 | 2017-05-04 | (주)아모레퍼시픽 | Method for selectively preparing ginsenoside Rd from saponins of ginseng by enzymatic process |
| CN105533707B (en) * | 2015-12-28 | 2018-06-19 | 江瀚生物科技（上海）有限公司 | A kind of ginseng small molecule nutritional ingredient preparation and preparation method thereof |
| CN106520891B (en) * | 2016-10-27 | 2019-06-04 | 辽宁中医药大学 | A kind of covalent cross-linking method anchoring fiber element enzyme enzymatic hydrolysis ginsenoside-Rb1The method for preparing ginsenoside-Rd |
| CN113249235B (en) * | 2021-04-26 | 2023-03-10 | 安琪纽特股份有限公司 | Pichia pastoris for producing beta-D-glucosidase and application thereof |
| CN114134060B (en) * | 2021-12-09 | 2023-09-19 | 北京理工大学 | Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method |
| CN116396876B (en) * | 2022-11-17 | 2024-06-21 | 云南农业大学 | Saccharomyces cerevisiae engineering bacteria for producing ginsenoside Rd and construction method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1354944B1 (en) * | 2000-12-29 | 2008-11-19 | Fengxie Jin | Ginsenoside glycosidase which hydrolyzes ginsenoside glycosyl and the use thereof |
| KR101098027B1 (en) * | 2009-09-18 | 2011-12-27 | 한국과학기술원 | Ginsenoside glycosidase from Rhodanobacter ginsenosidimutans KCTC22231T and use thereof |
| KR101200571B1 (en) * | 2009-10-23 | 2012-11-13 | 한국과학기술원 | Ginsenoside glycosidase from terrabacter sp and use thereof |
-
2013
- 2013-01-31 CN CN201310038798.4A patent/CN103146592B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103146592A (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103146592B (en) | Microzyme converting ginsenoside Rb1 to generate Rd and application thereof | |
| CN1479787B (en) | Ginsenoside glycosidase of hydrolyze ginseng saponin glycosyl and its application | |
| CN103416223B (en) | Method for improving cordycepin output in cordyceps militaris fermentation broth | |
| CN102559828B (en) | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms | |
| CN107502555A (en) | The fermentation medium and its zymotechnique of a kind of mortierella Diding | |
| CN103966105B (en) | One kind conversion ginsenoside Rg3Produce Rh2Aspergillus oryzae, production method and application | |
| CN102676627B (en) | Microbial fermentation extraction method of total arasaponin | |
| CN111218406B (en) | Mucor circinelloides MF-8 and application thereof in improving content of taxifolin in rhizoma smilacis glabrae | |
| CN104357332B (en) | Aspergillus niger JH-2 and application to biotransformation and synthesis of asiatic acid | |
| CN109749941B (en) | Paecilomyces cicadae fermentation medium and method for preparing ergosterol by fermenting Paecilomyces cicadae | |
| CN103333872B (en) | Method for preparing Beta-glucuronidase crude enzyme preparation | |
| CN113717860B (en) | Application of Huang Lanzhuang bacteria in conversion of total saponins of Notoginseng radix into small-polarity ginsenoside | |
| CN111996223A (en) | Method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing bidirectional fermentation technology | |
| CN104278070A (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| CN116103161A (en) | Plant endophytic fungus for producing eupatorium and application thereof | |
| CN113502230B (en) | Hericium erinaceus strain and culture method thereof, hericium erinaceus-ginseng bidirectional solid fermentation method and method for efficiently converting rare ginsenoside | |
| CN113249235B (en) | Pichia pastoris for producing beta-D-glucosidase and application thereof | |
| Chen et al. | Biotransformation of saponins to astragaloside IV from Radix Astragali by immobilized Aspergillus niger | |
| CN111763705B (en) | Preparation method and application of ginsenoside composition | |
| CN105200102B (en) | Method for extracting glutathione from candida utilis fermentation liquor | |
| CN103865804B (en) | Beta-glucosidase Producing Strain and the application in resveratrol is prepared in conversion thereof | |
| CN102286565A (en) | Preparation method of theaflavin monomer | |
| CN105199966B (en) | A kind of conversion ginsenoside Rb1 produces aspergillus and the application of Rd | |
| CN105087522A (en) | Method for extracting endoenzyme generated by fungal mycelium liquid submerged fermentation | |
| CN1268763C (en) | Method for preparing hypoglycosyl anemonic saponin by encyme method hydrolyzing anemonic saponin glycosyl |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |