CN1982438A - Bacillus and production of monodesmosidic panasaponin and aglucon therewith - Google Patents

Bacillus and production of monodesmosidic panasaponin and aglucon therewith Download PDF

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CN1982438A
CN1982438A CN 200610046660 CN200610046660A CN1982438A CN 1982438 A CN1982438 A CN 1982438A CN 200610046660 CN200610046660 CN 200610046660 CN 200610046660 A CN200610046660 A CN 200610046660A CN 1982438 A CN1982438 A CN 1982438A
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ginsenoside
microorganism
ppd
genus bacillus
glycolysis
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赵余庆
姜彬慧
韩颖
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XINZHONG MODERN MEDICAL CO Ltd LIAONING CITY
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XINZHONG MODERN MEDICAL CO Ltd LIAONING CITY
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Abstract

Bacillocin and production of monose-chain ginseng saponin and aglycone are disclosed. The process is carried out by zymolysis acting by bacillocin A8 at 25-45degree and pH value 5.0-7.0 for 2-15hrs, collecting reactant, removing microorganism by 0.2-0.45mum micro-porous filter film, filling it into D101 macro-porous adsorptive resin column, washing by water to be colorless, gradient washing for resin column by alcohol, concentrating for 80-90% alcohol eluent, volatilizing and drying to obtain C-K, PPD and PPT mixture. The converting liquid is aqueous solution and the proportion of ginseng saponin and microbe strain A is 4:1-8:1. Its absorbing capacity reaches to 70-90%.

Description

One bacillus and prepare the method for monose chain ginsenoside and aglycon with it
Technical field:
The present invention relates to prepare the method for monose chain ginsenoside and aglycon by microbial transformation (hydrolysis) ginsenoside.Particularly relate to a bacillus A8[(Bacillus sp.), preservation date: on 04 26th, 2006, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), deposit number: 1703, classification name: bacillus (Bacillus sp.)] transform ginsenoside and prepare monose chain ginsenoside 20 (S) protopanoxadiol-20-O-β-D-glucopyranoside [20 (S)-protopanaxadiol-20-O-β-D-glucopyranoside in a large number, ginsenoside Compound-K is called for short C-K], protopanoxadiol aglycon [20 (S)-protopanaxadiol, be called for short PPD] and the method for Protopanaxatriol's aglycon [20 (S)-protopanaxatriol are called for short PPT].
Background technology:
Ginsenoside is araliaceae ginseng plants' such as genseng, Radix Panacis Quinquefolii, pseudo-ginseng a main active ingredient, the Rh in the ginsenoside 1,-Rh 2, C-K, PPD and PPT be the micro-rare secondary saponin(e that exists among the araliaceae ginseng plants such as genseng, Radix Panacis Quinquefolii, pseudo-ginseng.Studies show that these rare ginsenosides all have notable antitumor activity.
Just find the ginsenoside Rh as far back as people such as Japanese scholar Odashima in 1979 2The propagation that can suppress lung carcinoma cell 3LL (mouse), Mo Lisi liver cell (rabbit), BA-16 malignant tumour (mouse) and HELA cell (people), and the proof ginsenoside can cause that cancer cells transforms to normal cell.1991, reports such as Japanese scholar Kikuchi were given the oral ginsenoside Rh of nude mice 2Can obviously suppress the hyperplasia of human ovarian cancer cell (Human ovarian cancer cells HRA), the survival rate of mouse significantly rises [3]Summer in 1996, the beautiful beautiful experiment in vitro method of utilizing was studied-Rh 2To the differentiation-inducing action of murine melanoma B16 cell, the result shows :-Rh 2Under the concentration of 10 μ g0.mL-L the B16 cell is had stronger differentiation-inducing action, showing as the melanochrome generative capacity obviously increases; Form is broken up to the epithelioid cell; Cell is reticulated structure; Melanin granule increases, and growth slows down slow [20]Kim etc. [21]Research also show the ginsenoside Rh 2With-Rh 3Can induce the HL-60 cell to granulocytic differentiation, can make the blocking-up of HL-60 cell in the G1/S phase.By-Rh 2The differentiation of the HL-60 cell that causes can make the activity of proteinase activated dose of C (PKC) increase.The same year Park etc. [22]Report, the ginsenoside Rh 1With-Rh 2Propagation to the human fibrosarcoma cell has obvious restraining effect, compares-Rh with universally acknowledged the most effective anti-HRA medicine CDDP (Cis-diamminedichloroplatinum) 2The effect that suppresses tumor growth is similar to CDDP, but is free from side effects, and CDDP has significant side effects.-Rh 2The ability that also has stronger increase natural killer tumor cell viability, and energy significant prolongation life, these characteristics are not found when using CDDP.
1995-1996, reports such as Japanese scholar Kitagawa, ginsenoside Rg 3Can suppress effect by rat intestinal cancer intraabdominal metastasis due to the Bomesin.Azuma etc. have reported the ginsenoside Rg 3,-Rb 2In vivo, external all have restraining effect to the B16-BL6 tumour cell in the transfer of lung.The rich power of domestic scholars, Lu Qi have improved and extracted ginsenoside 20 (R)-Rg from red ginsengs by years of researches 3Method, realized suitability for industrialized production first, through taking the lead by Guang-amen Hospital, China Traditional Chinese Medicine Instl, what big unit of 8 families such as Attached Hospital No.1, dalian Medical Univ., tumor center of the entire PLA of Nanjing Aug. 1st hospital met the GCP requirement is the center clinical study, now obtained the first class national new drug certificate, formal medicine ginseng by name one capsule.Research mechanism shows ,-Rg 3In metastases, can suppress five key links such as tumour increment growth, implantation, infiltration, adhesion and new vessel formation.
According to another studies show that, C-K no matter in vivo, externally all have good anticancer growth effect and an anti-metastasis effect, no matter entity or fluid JEG-3 all there is unusual effect, as: the anti-infiltration and transferance, utilize the influence of the research C-K such as easy transfer hypotype B16-F10 cell of multiple inside and outside metastasis model such as lewis lung cancer cell LLC, human fibrosarcoma HT1080, murine melanoma B16 cell to tumor cell invasion and transfer, found that: do not form though C-K does not directly suppress tumor mass, have tangible anti-metastasis effect; The inducing apoptosis of tumour cell effect, C-K promptly appears at behind the 15min in the nuclear of tumour cell when hatching jointly with B16-BL6 and two kinds of tumour cells of LLC, induces within the 24h morphologic apoptosis performance to occur; The newborn effect of antineoplastic vascular, studies show that: propagation and tube chamber that the C-K of non-toxic can suppress the HSE cell due to the CM-L5 form activity, and the cardiovascular formation of tumor inducing is produced inhibition [28]Anti-mutation and prevention canceration effect, C-K has the mutagenesis of anti-benzopyrene inductive, and its effect possesses pharmacological dose-effect relationship.In addition, C-K also can reduce the chromosome aberration that benzopyrene causes Chinese hamster lung fibroblast (Chinesehamster lungfibmbl～tcells CHL), illustrates that C-K can be used as the sudden change preventive.
The Wakabayashi report ,-Rg 1,-Re and have very strong antitumor cell transfer activity after intestinal bacteria metabolite 20 (S)-PPT is oral.
The secondary rare saponin(e-Rg of genseng 3,-Rh 2Have notable antitumor activity with C-K, but as the basic substance of performance such as natural drug genseng, Radix Panacis Quinquefolii, pseudo-ginseng drug activity, their content in natural phant is extremely low.For example :-Rg 3Content at Bai Canzhong is 0.0003% only, and the content in red ginseng is about 0.03%; And-Rh 2In red ginseng content only be 0,001%, in the Stem and leaf of Radix Ginseng-Rh 2Content is high 10 times than red ginseng, separate in the stem and leaf of Radix Panacis Quinquefolii and obtain-Rh 2Content is 0.001%-0.0043%.C-K does not exist in natural ginseng, is the microbial metabolites of ginsenoside, and the content in pseudo-ginseng only is 0.032%.Therefore, how improving the content of these micro-secondary activated saponins, is the research focus in the present natural medicine field.
And with regard to natural plant resource, except that the minority activeconstituents, major part all is the low activity composition in certain kind of plant, but these compositions often all are the close or similar of source of students relation.With the monomer saponin in the genseng is example, as-Rb ,-Re ,-Rg ,-Rh, the parent nucleus of their molecular structures is identical, it all is metastable dammarane type four-ring triterpenoid saponin, different is side chain sugar (glucose, rhamnosyl, the pectinose etc.) difference that they connect on the specific position of parent nucleus, or on the side chain sugar different amts, or hydroxyl is different with the link position of sugar chain, moreover be exactly their space multistory configuration difference (space multistory rotation configuration), in a word, their structure all is very close.But because the difference of side chain, the pharmacologically active that they had is also different fully.-Rb 1,-Rb 2,-Rc ,-Rd and-Re etc. are because the sugar that is connected on the side chain is too many, make molecular weight huge, and pharmacologically active is very low; And-Rg 3,-Rh 2With-Rh 1Deng, the glucose on the side chain is less, but is proved to be the small-molecule active substance with different pharmacologically actives.Therefore,, reduce the number of sugar as far as possible, perhaps change the sterie configuration of saponin(e, the high reactivity saponin(e that can both obtain expecting as long as can take off the syrup on the very big low activity saponin(e side chain of molecular weight.
The method of hydrolyzing saponin commonly used has acid-hydrolysis method, alkali hydrolysis method and biotransformation method at present.The hydrolysis method of discovering saponin(e has two kinds, and a kind of is once thoroughly hydrolysis of saponin(e, generates aglycon and sugar, and another kind is the saponin(e fractional hydrolysis, and promptly part sugar is hydrolyzed earlier, or sugar chain of first hydrolysis forms secondary glycosides or preceding sapogenin in the bisdesmosidic saponins.
The mechanism of acid hydrolytic reaction is: the glycosidic bond atom is at first protonated, the glycosidic bond fracture generates the oxocarbonium ion intermediate of aglycon and sugar then, at the water oxygen carbon ion through solvation, slough hydrogen ion again and form glycan molecule, the difficulty or ease of acid-catalyzed hydrolysis are closely-related with the basicity of glycosides atom, cloud density and its space environment on the glycosides atom.Have data to show because the sugar that is connected on the aglycon C20 tertiary alcohol base is easily obtained secondary glycoside by 50% HAC partial hydrolysis, thus monomer saponin with the HAC partial hydrolysis after, the ginsenoside Ra 1,-Ra 2,-Rb 1,-Rb 2,-Rb 3,-Rc and-Rd all identical secondary glycoside, ginsenoside Re's portion water solves-Rg 1, the ginsenoside Rg 1Solve-Rh through portion water 1There is data to show in addition: can obtain aglycon and sugar through the peracid all-hydrolytic.
When being hydrolyzed with alkali, glycosidic bond should be relatively stable to alkaline reagents, be difficult for by alkali catalyzed hydrolysis, but in some saponin(e because contain ethylene linkage, meet alkali and also can react.Some new compounds of for example finding from genseng in recent years are protopanoxadiol or the derivation of triol type side chain or the acetylize or the malonyl-ization of sugar moieties mostly.
Though these two kinds of methods can make the saponin(e hydrolysis, but their hydrolysising condition is all very violent, wayward, and owing in hydrolytic process, can make variations such as sapogenin dewaters, cyclization, double-bond shift, substituting group displacement, configuration conversion, the by product of hydrolysis is increased, and target product is difficult to obtain [2]The more important thing is that these two kinds of methods expect that from former reaction process all discharged pollutents such as a large amount of organic solvents, bronsted lowry acids and bases bronsted lowry to environment, environment is caused to a great extent harm; And bronsted lowry acids and bases bronsted lowry also can mordantly injure operator itself, so need to seek new transformation technology and method, can be more effective low activity, high-load plant saponin(e be changed into high reactivity, monomer saponin that natural content is low, can accomplish not cause the transformation technology and the method for burden again to environment, this is raising natural drug utilization ratio, carries out cleaner production, Chinese medicine is gone to the world, and sets up the only way of green Chinese medicine industry.
In the various new technologies that latest developments are got up, the most noticeable with conversion technology especially.Bio-transformation (Biotransformation or Bioconversion) is that a certain privileged site of external source organic substrates or the functional group that utilize living things system will join in the reactive system carry out specific structural modification to obtain the technology of valuable different chemical product.What the bio-transformation was here emphasized is to finish by single step reaction between external source substrate and the product, its essence is a kind of catalyzed reaction of enzyme.Because the enzyme of reactions such as vegetable cell contains catalyzed oxidation, reduction, hydroxylation, acetylize, esterification, glucosyl, isomerization, methylates, demethylation, epoxidation, so they have the ability that the external source substrate conversion is become useful compound.The zone and the stereoselectivity of bio-transformation is strong, reaction conditions is gentle, easy and simple to handle, cost is lower, public hazards are few, and can finish some chemosynthesis and is difficult to the reaction carried out, is subjected to organic chemist, Pharmaceutical Chemist and microbiologists' very big attention.The wide range of bio-transformation almost comprises various types of chemical reactions, as hydrolysis, condensation, acyl group transfer, redox or the like.Therefore utilize the biological defective that has both overcome classical acid, basic hydrolysis method that the plant saponin(e is transformed, reduced the generation of pollutent again, meet the novel modern production pattern that transforms to source control from end treatment, thereby more and more come into one's own, biotransformation method has become current research focus.
Because microorganism itself derives from nature, activity of conversion is higher, and low production cost, only be directly to use 1/100 of industrialization enzymatic conversion, in addition lower.In a single day bacterial strain can also forever use behind separation, the purifying, preservation.This method is harmless fully to operator in process of production, whole process is expected product from former, can not pollute environment, and the remaining thalline raffinate in reaction back, through cultivating again, still can be used as reactant and experimentize once more, therefore this have a revolutionary innovation production technique, the novel environment friendly theory that meets 21 century fully is undisputed " friendly process ".
The bioconversion method present Research of ginsenoside
The bioconversion reaction of microorganism, in fact be exactly the single-minded catalysis that utilizes one or more enzymes that one or more microorganisms contain, specific position effect to as the molecular structure of certain organic compound of substrate makes it change into the more valuable new compound of structural similitude.Along with contemporary development of biology, new technologies such as solid phase alcohol (immobilized cell), enzyme mebrane reactor, solvent engineering, protoplastis and fusion, mutagenesis and gene recombination are introduced the enzymic catalytic reaction system, the efficient of microbial transformation is doubled and redoubled, and can make whole process of production continuously, automatization, represented wide prospect for microbial transformation is applied to organic synthesis.
The application of microbial transformation starts from after the clinical application of the steroid drugs fifties, but has just found that in the thirties microorganism has the ability that transforms some compound.In recent years, because the attention of Chinese scholars, microbiological transformation technology has also had new breakthrough, for example: the production of protein fodder, produce Xylitol etc. with the regenerated cellulose for the substrate bio-transformation, all become current focus.
At present, Chinese scholars mainly concentrates on three aspects to the research that the plant saponin(e carries out bio-transformation, and the one, the bacterial strain that utilizes strain library to store carries out microbial transformation to the plant saponin(e; The 2nd, utilize the interior bacterium of intestines that monomer saponin is carried out metabolic conversion; The 3rd, utilize enzyme that the plant saponin(e is transformed.
Humans such as the Ma Jisheng of Changchun Traditional Chinese Medical College available from 10 kinds of fungies at institute of Chinese section microbial strains preservation center right respectively-Rb 1With panoxadiol be that saponin(e (PDS) carries out bio-transformation, the result shows that wherein 6 kinds of fungies have metabolic activity after transforming, and prolongation in time, has four kinds of meta-bolitess to occur in succession, be respectively-Rd, C-K, PPD and-Rg 3People such as the Dong Aling of Peking University once used several single strains of buying to the monomer saponin Rb in the genseng 1,-Rb 3Carry out bio-transformation with Rd etc., discover, conversion has in various degree all taken place in the ginsenoside after bacterial strain transforms.
It is comparatively extensive at present to utilize the interior bacterium of intestines that monomer saponin is carried out metabolic research.It is because saccharide compound is the important carbon source of bacterium in the enteron aisle that intestinal flora is hydrolyzed to the medicine with glycosidic bond.In recent years, China and Japanese scholar studies have shown that bacterium metabolism in the intestines, ginsenoside Rb 1With-Rg 1Under the effect of bacterium in the enteron aisle midgut, can be converted to multiple meta-bolites ,-Rb by people and the oral back of rat 1Metabolic patterns is-Rb under the effect of bacterium in intestines 1→-Rd → F 2→ C-K → PPD ,-Rg 1Metabolic patterns is-Rg under the effect of bacterium in intestines 1→-Rh 1/ F 1→ Protopanaxatriol (PPT).People such as the Chen Xin of Changchun Traditional Chinese Medical College in 1999 just to ginsenoside Rb 1Intestines in the bacterium metabolism study, the result shows-Rb 1By bacterium metabolism in people's intestines of isolated culture, its R 4 kinds of meta-bolitess appear, in prolongation in time in succession fThe value respectively with standard substance-Rd ,-Rg 3,-Rh 2, the PPD value is identical; And-Rb 1By bacterium in people's intestines of meta-bolites after the bacterium metabolism in the rat intestine and isolated culture right-Rb 1Meta-bolites identical, still, in the rat intestine bacterium right-Rb 1In the meta-bolites of metabolism after 48 hours after testing less than-Rd and-Rg 3, have only-Rh 2And PPD.Human To Humans such as Wang Yi joined saponin(e Rg in 2000 1Intestines in the bacterium metabolism study, find in human body-Rg 1By bacterium metabolism in the intestines be-Rh 1And PPT ,-Rh 1Be absorbed into blood, and get rid of external by urine; And-Rg 1By the metabolism of rat intestinal flora be-Rh 1, F 1And PPT.
Enzymatic conversion method plant saponin(e is a new developing technology just in recent years.Humans such as the Zhao Liya of Dalian Light Industry College isolating crude enzyme liquid from store bacterial strain transforms Radix Ginseng total saponins, and the result shows that enzyme process can change the glycosyl of Panaxadiol saponin ,-Rh 2With-Rg 3Hydroxyl on the 20th carbon of saponin(e loses water formation-Rh easily 3With-Rg 5,-Rh 1Be a spot of-Re of being mixed with by the panoxadiol saponins ,-Rg 1Generate, and primary product is the ginsenoside Rh in the reaction product of enzyme process 2, by product is the ginsenoside Rg 3,-Rg 5,-Rh 1,-Rh 3With panoxadiol saponins unit.Gold wind is mediate to wait the people to carry out utilizing ginsenoside glycosyl enzyme to prepare the ginsenoside Rh 2Research, and applied for patent.Liu Ke utilizes enzyme process that Radix Ginseng total saponins is carried out bio-transformation and obtained C-K, and enzymolysis process is as follows: the total saponins → glycol saponins → enzyme-added bio-transformation → C-K that carries out.In addition, people such as Tian Jing find can also change with enzyme process the glycosyl of soybean saponin, and hydrolyzing saponin becomes low sugar saponin(e product and aglycon.People such as the red sudden strain of a muscle of fish also study improving its sugariness with enzyme process change liquirtin alditol acidic group.
The research of ginsenoside antitumor activity component
Ginsenoside is araliaceae ginseng plants' such as genseng, Radix Panacis Quinquefolii, pseudo-ginseng a main active ingredient, the Rh in the ginsenoside 1,-Rh 2, C-K, PPD and PPT be the micro-rare secondary saponin(e that exists among the araliaceae ginseng plants such as genseng, Radix Panacis Quinquefolii, pseudo-ginseng.Studies show that these rare ginsenosides all have notable antitumor activity.
Herba Herminii is the mature fruit of plant genseng, and color and luster is scarlet, in two semicircle seeds are arranged.In gathering the process of genseng, seed and fruit are gathered simultaneously, and seed is used to raise up seed, and pulp is often lost as refuse, have not only polluted environment but also land occupation.After scholar's research confirms, the Herba Herminii glucoside of in the pericarp of genseng fruit and pulp, containing, except that to ginseng in effective constituent-ginsenoside have the similar effect, also have the effect of its uniqueness.Detect through China pharmacy expert, the content of ginsenoside in the genseng pulp is 4 times of ginseng, contains ginsenoside Re's 30 times for ginseng, 20 (R)-Rg 3, 20 (R)-Rh 2Content in red ginseng and Stem and leaf of Radix Ginseng-Rh 2Content.Therefore, the development and utilization of Herba Herminii has been caused the extensive attention of Chinese scholars.At present, from Herba Herminii isolation identification go out ginsenoside Rb 1,-Rb 2,-Rc ,-Rd ,-Re ,-Rg 1, 20 (S)-Rg 2, 20 (R)-Rg 2, 20 (R)-Rg 3, 20 (S)-Rh 1, 20 (R)-Rh 1, 20 (R)-Rh 2With compositions such as 20 (R)-Protopanaxatriols, daucosterol, β-Gu Zaichuns.
We study the Herba Herminii chemical ingredients on the basis of forefathers' research, and have compared the ginsenoside Rg who has anti-tumor activity in Herba Herminii and the Fructus Panacis Quinquefolii saponin extract 3, 20 (R)-ginsenoside Rgs 3And ginsenoside Rh 2Content, for further this cheap medicine resource of development and use Herba Herminii provides theoretical foundation, also lay the first stone simultaneously for the natural PTS of research and development.
Summary of the invention:
The invention provides and a kind ofly from the soil of cultivated ginseng, filter out a bacillus A8, and the glycolysis ginsenoside prepares the method for monose chain ginseng saponin C-K and aglycon PPD and PPT mixture.The present invention can comprise panax species the total saponins that extracts purifying in root, cauline leaf and the fruit of plants such as genseng, pseudo-ginseng, Radix Panacis Quinquefolii, or the monomer saponin after separation and purification, through microbial transformation (hydrolysis) effect, obtain the mixture of monose chain ginseng saponin C-K and aglycon PPD and PPT.The chemical structure of C-K and aglycon PPD and PPT is as follows:
The chemical structure of I ginseng saponin C-K
The chemical structure of II PPD
III PPT (R 1=R 2=OH) chemical structure
III PPT (R 1=R 2=OH) chemical structure
For simple, convenient, low-costly and in high volume preparation monose chain ginseng saponin C-K, and aglycon PPD and PPT mixture, the present invention at first provides a kind of microorganism strains A8 glycolysis ginsenoside to prepare the method for C-K, PPD and PPT mixture.And provide a kind of method of from the soil of cultivated ginseng, screening strains A 8.
The present invention prepares in the method for C-K, PPD and PPT mixture with microbial transformation (hydrolysis) panax species, and used microorganism strains is A8.
A8 glycolysis ginsenoside of the present invention prepares in the method for C-K, PPD and PPT mixture, and the strain culturing condition is slightly spacious, can grow in Ma Dingshi substratum commonly used in the experiment.
A8 glycolysis ginsenoside of the present invention prepares in the method for C-K, PPD and PPT mixture, 25-45 ℃ of glycolysis temperature, and pH value 5.0-7.0, glycolysis time 2h-15d, transforming (hydrolysis) liquid is the aqueous solution.
The present invention prepares in the method for C-K, PPD and PPT mixture with microbial transformation (hydrolysis) ginsenoside, and the ratio of ginsenoside and microorganism strains A8 is 4-8: 1, and the most economical with 6: 1; In general, behind the hydrolysis 15d, prolong hydrolysis time again, the output of mixture increases few, therefore, need not the hydrolysis time that extends.
A8 glycolysis ginsenoside of the present invention prepares in the method for C-K, PPD and PPT mixture, can obtain C-K, PPD and PPT mixture by glycolysis natural ginseng total saponins and each monomer saponin.
The present invention prepares in the method for C-K, PPD and PPT mixture with microbial transformation (hydrolysis) ginsenoside, and collection process is to be injected in the macroporous adsorptive resins after reaction product is removed microorganism, and the water flushing is to colourless, again with ethanol gradient flushing resin column.The 80-90% ethanol eluate is concentrated, evaporate into driedly, obtain C-K, PPD and PPT mixture, yield 70-90%.
Reaction product is injected in the macroporous adsorptive resins in the collection process, and the water flushing is to colourless, and washing lotion and mother liquor merge, and is used for microbial transformation (hydrolysis) once more after concentrating.
Implement above operating process, A8 provided by the present invention can also be used for other glucoside compounds of glycolysis as the root that comprises plants such as genseng, pseudo-ginseng, Radix Panacis Quinquefolii, gynostemma pentaphylla, cauline leaf and fruit extract purifying total saponins (cauline leaf/fruit total saponin), Fructus Momordicae charantiae glucoside, Semen Trigonellae glycosides, peoniflorin, saikoside, baicalin, astragalin, remove hydroxyl glycosides etc., prepare corresponding microorganism glycolysis product.
The used A8 of the present invention changes it in substratum that contains ginsenoside over to, the bacterial strain that can the glycolysis ginsenoside prepares C-K, PPD and PPT mixture that obtains through the cultivation of going down to posterity, domestication, screening.The A8 bacterium colony is smooth, and is moistening; It is round that phylonepionic is, and the single thalline in back of growing up is long ellipse at microscopically, and is bigger than common bacteria bacterium colony.
Advantage of the present invention is: the present invention adopts environmentally friendly technology---conversion technology, provides a kind of convenient sources, cheap microbial bacteria bacillus A8 for utilizing ginsenoside to prepare C-K, PPD and PPT mixture.Prepare with this bacterial strain that the method for antitumor activity component monose chain ginseng saponin C-K, aglycon PPD and PPT is simple and convenient, safe and reliable, cost is low, product rate of recovery height; Converted product is stablized single, is easy to separate; Reduced the discharging of pollutent simultaneously and to the destruction of ecotope.Use C-K, the PPD of present method preparation and the transformation efficiency of PPT mixture to be 20%-60%, yield 70-90%.
Description of drawings:
The correlation curve of accompanying drawing 1 A8 thalline logarithm and absorbancy
The logarithmic growth curve of accompanying drawing 2 A8
Accompanying drawing 3 pH are to the influence of A8 growth
The culture presevation date: on 04 26th, 2006, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), No. 13, one in village north in the Haidian District, Beijing City, address, deposit number: 1703, and classification name: bacillus Bacillus sp.
Embodiment:
One, the isolation and purification process of microorganism strains A8
(1), soil microorganisms concentration method separate microorganism
(1) enrichment of microorganism: utilize sterile vessel to get the soil sample 1g of cultivated ginseng, add the 10mL stroke-physiological saline solution, concussion is made soil supension for a moment, gets the 1mL suspension and puts into the triangular flask that fills 25mL enrichment medium 1 respectively.In the biochemical incubator 30 ℃, 48h.
(2) primary dcreening operation of bacterial strain: be linked into respectively in the primary dcreening operation substratum 2 with the microorganism of dilution plate coating method with growth in the enrichment medium 1, every kind of weaker concn connects 3 parallel samples, and inoculum size is 0.2mL.Postvaccinal plane ware is just being placed earlier 28-30 ℃ incubator 24h, and cultivated 3-4 days in being inverted in incubator the back.
(2), liquid soil dilution method separate microorganism
The primary dcreening operation of bacterial strain: be linked into respectively in the primary dcreening operation substratum 2 with the soil diluent of dilution-plate method with different concns, every kind of weaker concn connects 3 parallel samples, and inoculum size is 1mL.The incubator that postvaccinal plane ware is inverted in 28-30 ℃ was cultivated 3-4 days.
(3), the separation of bacterial classification, purifying are cultivated
With the single bacterium colony on the primary dcreening operation flat board separate, purifying (numbering).With the plate streaking partition method each single bacterium colony is inoculated in respectively in the isolation medium 3, the incubator that is inverted in 28-30 ℃ was cultivated 3-4 days.When treating that strain growth is more vigorous, will separate again, purifying obtains pure bacterial strain and inserts liquid and divide and support in the base 4, cultivated 2-3 days in 30 ℃, 160r/min shaking table.
(4), the multiple sieve of bacterial classification
The thalline of growth in the liquid separation culture medium 4 is transferred to respectively in the multiple sieve substratum 5, cultivated 2-3 days in 30 ℃, 160r/min shaking table, observe the growing state of thalline and the transformation of arasaponin.Select the good and tool of growing state with the bacterial strain of higher transformation as experimental bacteria,, further this pure bacterial strain is identified that tentatively definite A8 is the microorganism of bacillus with its called after A8.
(5), microorganism is to the conversion of saponin of Radix Notoginseng leaf
Microorganism behind the above multiple sieve is inoculated in respectively in the liquid inoculation substratum 6, inoculum size is 2%, and cultivate in 30 ℃, 160r/min shaking table inoculation back, when treating that strain growth is more vigorous, add conversion substratum 8, and the content of saponin of Radix Notoginseng leaf composition is observed in increase in time.
Microbial inoculant behind the part purifying is in solid vaccination substratum 7, and 4 ℃ of refrigerators are preserved.
The substratum that uses when table 1 screening and cultivation A8
The substratum title The composition of substratum The purposes of substratum Remarks
Enrichment medium 1 Glucose 4.0g, notoginseng haulm saponin(e 6.0g, KH 2PO 41.0g, CMC 10.0g, MgSO 47H 2O 0.2g, Benzylpenicillin sodium 0.1g, distilled water 1000mL, sterilization. The enrichment of microorganism 4 ℃ of refrigerators are preserved
Primary dcreening operation substratum 2 Extractum carnis 5.0g, peptone 10.0g, NaCl 5.0g, agar 20.0g, distilled water 1000mL, pH 7.6.After the sterilization, contra bevel or flat board. The preliminary screening microorganism Mixing
Isolation medium 3 Extractum carnis 5.0g, peptone 10.0g, NaCl 5.0g, agar 20.0g, distilled water 1000mL, pH 7.6.After the sterilization, contra bevel or flat board. The separation and purification microorganism Line separates
Liquid separation culture medium 4 Extractum carnis 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH 7.6, sterilization. Increase the separation and purification microbial numbers 4 ℃ of refrigerators are preserved
Sieve substratum 5 again Notoginseng haulm saponin(e 5.0g, peptone 3.0g, KH 2PO 41.0g, NaCL5.0g, MgSO 47H 2O 0.2g, distilled water 1000mL, pH 7.6, sterilization. Further screen microorganism 4 ℃ of refrigerators are preserved
Liquid inoculation substratum 6 Take by weighing 16.5g Martin substratum, add the 1000mL dissolved in distilled water, natural pH, sterilization. The preparation seed liquor Matching while using
Solid (inclined-plane or flat board) inoculation medium 7 The agar that adds 2-3% in the liquid inoculation substratum 10, after the sterilization, contra bevel or flat board. Bacterial classification is preserved Went down to posterity once in 3 months
Liquid transforms substratum 8 Weighing notoginseng stem and leaf total saponin 50g, add distilled water 1000mL, ultrasonic mixing, the concentration that makes notoginseng stem and leaf total saponin is 50mg/mL. Substrate is transformed Matching while using
Two, the A8 optimal culture condition determines
Maximum absorption wavelength with the A8 of RF-5000 type spectrophotometric instrumentation is 384.4mn, and therefore selected maximum absorption wavelength is 380nm, with the mensuration wavelength of this wavelength as A8.At first, be X-coordinate with absorbancy (A), thalline logarithm [logN (individual mL -1)] be ordinate zou, determine the equation of linear regression of thalline logarithm logN and absorbance A, as follows:
logN＝0.8187A+7.2293 r＝0.997 (1)
Then: the thalline number in the stoste is N` (individual mL -1)=n10 LogN(2)
logN`＝logn+logN (3)
Wherein: N` is the thalline sum; N is an extension rate.
With 721 spectrophotometer λ=380nm, measure absorbance at A8 cultivation 0h, 1h, 2h, 4h, 8h, 24h, 36h, 48h, 72h, 96h respectively, by the logarithmic growth value of above 3 Equation for Calculating thalline, the experimental result of different time thalline logarithmic growth is seen Fig. 2 again.
As shown in Figure 2: the process of growth of bacterium A8 has experienced lag phase (in the 8h), increased logarithmic phase (8h-36h), stationary phase three phases such as (36h-96h).According to experimental result, the best incubation time of bacterium A8 is 48h.Change wall scroll spares such as transforming the initial pH of substratum, culture temperature, calculate according to formula (3) again, determine the growing state of thalline.The result shows that the best initial pH of bacterium A8 is 6.0 (the results are shown in Figure 3), and optimum culturing temperature is 30 ℃, and best shaking speed is 160r/min.
Three, optimum conversion condition determines
A8 transforms selection of time 1～15d that ginsenoside generates C-K, PPD and PPT mixture, 15,20,25,30,35,40 ℃ of invert points, shaking speed 120,140,160,180,200 rmin -1, transform pH value 4.0,5.0, natural pH value (pH=5.5), 6.0,7.0,8.0.Detect according to TLC and to show, the optimal conversion time of bacterium A8 is that 13d, optimal conversion temperature are that 30 ℃, best shaking table revolution are 160rmin -1, optimal conversion pH value is nature pH value.Transformation efficiency is 20-60%.
Embodiment 1
A8 is to the conversion of notoginseng haulm saponin(e
The A8 that will cultivate under optimal culture condition is inoculated in the aqueous solution that contains 10g notoginseng haulm saponin(e (content＞80%), is 160rmin at 30 ℃ of temperature, shaking table revolution -1, transform (hydrolysis) under the natural pH value condition.Begin sampling behind the 3d.The result of TLC shows: compare with control group, along with the prolongation of time, C-K, PPD and PPT spot increase gradually, color burn, and the corresponding minimizing of the content of the Rb that the Rf value is bigger in the notoginseng haulm saponin(e, Rd and Rg.Through the transformation of 13d, reaction product after 0.2 μ m-0.45 μ m millipore filtration is removed microorganism, is injected in the D101 macroporous adsorptive resins, the water flushing is to colourless, again with ethanol gradient flushing resin column.The 80-90% ethanol eluate is concentrated, volatilization as for, obtain C-K, PPD and PPT mixture 4.7g, transformation efficiency is 47%.
Embodiment 2
A8 is to Herba Herminii glucoside's conversion
The A8 that will cultivate under optimal culture condition is inoculated in the aqueous solution that contains 1g Herba Herminii glucoside (content＞60%), is 160rmin at 30 ℃ of temperature, shaking table revolution -1, transform (hydrolysis) under the natural pH value condition.Through the transformation of 13d, reaction product after 0.2 μ m millipore filtration is removed microorganism, is injected in the D101 macroporous adsorptive resins, the water flushing is to colourless, again with ethanol gradient flushing resin column.The 80-90% ethanol eluate is concentrated, evaporate into driedly, obtain C-K, PPD and PPT mixture 0.24g, transformation efficiency is 24%.
Embodiment 3
A8 is to the conversion of Radix Panacis Quinquefolii saponin
The A8 that will cultivate under optimal culture condition is inoculated in the aqueous solution that contains 2g Radix Panacis Quinquefolii saponin (content＞90%), is to transform (hydrolysis) under 160rmin-1, the natural pH value condition at 30 ℃ of temperature, shaking table revolution.Through the transformation of 13d, reaction product after 0.2 μ m millipore filtration is removed microorganism, is injected in the D101 macroporous adsorptive resins, the water flushing is to colourless, again with ethanol gradient flushing resin column.The 80-90% ethanol eluate is concentrated, evaporate into driedly, obtain C-K, PPD and PPT mixture 1.16g, transformation efficiency is 58%.
Embodiment 4
The separation of converted product
Silica gel is adorned post in proportion, and to wherein slowly injecting developping agent (chloroform-methanol system), be stirred to no bubble clockwise with glass stick and produce, in the chromatography column that is injected into, sedimentation 12 hours.Stand-by.
With C-K, PPD and PPT mixture 1.0g and the silica gel mixing wet method upper prop of getting well by 1: 2 ratio and activation.Begin calculated flow rate from adding sample, during to elutriant to 30mL, begin to connect flow point.With developping agent [chloroform: methyl alcohol (v/v)] gradient elution, receive 38 flow points altogether.Investigate through thin layer, merge identical flow point.
With 4-14 flow point products therefrom, concentrate the Compound I crude product.Behind preparation Thin-layer separation purifying, get a white powder Compound I, yield 30.74%; TLC calibrating and standard control determine that Compound I is PPD.
With 18-23 flow point products therefrom, concentrate Compound I I crude product.Behind preparation Thin-layer separation purifying, get a unformed white powder Compound I I, yield 20.62%; TLC calibrating and standard control determine that Compound I I is PPT.
With 26-35 flow point products therefrom, concentrate the compound III crude product.Behind preparation Thin-layer separation purifying, get a unformed white powder compound III, yield 41.16%; TLC calibrating and standard control determine that compound III is C-K.

Claims (8)

1. a bacillus, it is characterized in that: by the bacillocin of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC1703, preservation date is on 04 26th, 2006.
2, the method for cultivation of a kind of genus bacillus as claimed in claim 1 is characterized in that:
One, soil microorganisms concentration method separate microorganism
The enrichment of a, microorganism: utilize sterile vessel to get the soil sample 1g of cultivated ginseng, add the 10mL stroke-physiological saline solution, concussion is made soil supension for a moment, gets the 1mL suspension and puts into the triangular flask that fills 25mL enrichment medium 1 respectively.In the biochemical incubator 30 ℃, 48h;
The primary dcreening operation of b, bacterial strain: be linked into respectively in the primary dcreening operation substratum 2 with the microorganism of dilution plate coating method with growth in the enrichment medium 1, every kind of weaker concn connects 3 parallel samples, and inoculum size is 0.2mL.Postvaccinal plane ware is just being placed earlier 28-30 ℃ incubator 24h, and cultivated 3-4 days in being inverted in incubator the back;
Two, liquid soil dilution method separate microorganism
The primary dcreening operation of bacterial strain: be linked into respectively in the primary dcreening operation substratum 2 with the soil diluent of dilution-plate method with different concns, every kind of weaker concn connects 3 parallel samples, and inoculum size is 1mL.The incubator that postvaccinal plane ware is inverted in 28-30 ℃ was cultivated 3-4 days;
Three, the separation of bacterial classification, purifying are cultivated
With the single bacterium colony on the primary dcreening operation flat board separate, purifying (numbering).With the plate streaking partition method each single bacterium colony is inoculated in respectively in the isolation medium 3, the incubator that is inverted in 28-30 ℃ was cultivated 3-4 days.When treating that strain growth is more vigorous, will separate again, purifying obtains pure bacterial strain and inserts in the liquid separation culture medium 4, cultivated 2-3 days in 30 ℃, 160r/min shaking table;
Four, the multiple sieve of bacterial classification
The thalline of growth in the liquid separation culture medium 4 is transferred to respectively in the multiple sieve substratum 5, cultivated 2-3 days in 30 ℃, 160r/min shaking table, observe the growing state of thalline and the transformation of arasaponin.Select the good and tool of growing state with the bacterial strain of higher transformation as experimental bacteria,, further this pure bacterial strain is identified that tentatively definite A8 is the microorganism of bacillus with its called after genus bacillus, genus bacillus falls smooth, and is moistening; It is round that phylonepionic is, and is long ellipse after growing up, bigger than common bacteria bacterium colony.
3, the method for cultivation of genus bacillus according to claim 2 is characterized in that: bacterial classification can grow in Ma Dingshi substratum commonly used in the experiment.
4, transform the method that ginsenoside prepares monose chain ginsenoside and aglycon with genus bacillus, it is characterized in that: prepare with the microbial transformation panax species in the method for C-K, PPD and PPT mixture, used microorganism strains is genus bacillus A8.
5, the method for preparing monose chain ginsenoside and aglycon with genus bacillus conversion ginsenoside according to claim 4, it is characterized in that: A8 glycolysis ginsenoside prepares in the method for C-K, PPD and PPT mixture, 25-45 ℃ of glycolysis temperature, pH value 5.0-7.0, glycolysis time 2h-15d, conversion fluid is the aqueous solution, and the ratio of ginsenoside and microorganism strains A8 is 4: 1-8: 1; Collection process be with reaction product after 0.2-0.45 μ m millipore filtration is removed microorganism, be injected in the D101 macroporous adsorptive resins, the water flushing is to colourless, again with ethanol gradient flushing resin column, the 80-90% ethanol eluate is concentrated, evaporate into driedly, obtain C-K, PPD and PPT mixture, yield 70-90%.
6, according to claim 4 or the 5 described methods that prepare monose chain ginsenoside and aglycon with genus bacillus conversion ginsenoside, it is characterized in that: A8 bacterial classification glycolysis ginsenoside prepares in the method for C-K, PPD and PPT mixture, can obtain C-K, PPD and PPT mixture by glycolysis natural ginseng total saponins and each monomer saponin.
7, according to claim 4 or the 5 described methods that prepare monose chain ginsenoside and aglycon with genus bacillus conversion ginsenoside, it is characterized in that: the A8 bacterial classification that is provided can also be used for the root, cauline leaf of other glucoside compound gensengs of glycolysis, pseudo-ginseng, Radix Panacis Quinquefolii, gynostemma pentaphylla plant and total saponins, Fructus Momordicae charantiae glucoside, Semen Trigonellae glycosides, peoniflorin, saikoside, baicalin, astragalin that fruit extracts purifying, remove hydroxyl glycosides, prepares corresponding microorganism glycolysis product.
8, according to claim 4 or the 5 described methods that prepare monose chain ginsenoside and aglycon with genus bacillus conversion ginsenoside, it is characterized in that: reaction product is injected in the D101 macroporous adsorptive resins in the collection process, the water flushing is to colourless, washing lotion and mother liquor merge, and are used for microbial transformation once more after concentrating.
CN 200610046660 2006-05-24 2006-05-24 Bacillus and production of monodesmosidic panasaponin and aglucon therewith Pending CN1982438A (en)

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