CN1880444A - Grifola frondosa strain, culture method and application thereof - Google Patents
Grifola frondosa strain, culture method and application thereof Download PDFInfo
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- CN1880444A CN1880444A CN 200610043944 CN200610043944A CN1880444A CN 1880444 A CN1880444 A CN 1880444A CN 200610043944 CN200610043944 CN 200610043944 CN 200610043944 A CN200610043944 A CN 200610043944A CN 1880444 A CN1880444 A CN 1880444A
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Abstract
The invention discloses a drug and edible two-use fungus grey ramalic GF-932[Grifola frondosa (Dichs.ex Fr.) S.F.Gray] and producing method of grey ramalic beta-dextran, which is characterized by the following: applying in the tablet, soft capsule and hard capsule, liquid drugs injection and freeze dried; reserving in the China biological bacterial reserving management committee common microbe center with CGMCC No.1709 as reserving number.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to a kind of biomaterial---Grifola frondosa strain, its tunning and extract thereof have the microorganism of medicinal function, and at grifolan, the application of injection production aspects such as capsules such as especially grifola frondosa beta-glucose production, tablet, soft capsule and hard capsule, liquid drugs injection and lyophilized injectable powder.
Background technology
After Japanese scientist thousand former Wu Lang in 1969 find that lentinan has anti-tumor activity, the various countries scientist is devoted to the research of fungus polysaccharide in antitumor field one after another, started tumor biotherapy, the frontier of immunotherapy especially, and obtain outstanding progress.In fungus polysaccharide, emerge large quantities of polyose anti-tumor agents evident in efficacy.
Sharp husband of Miyazaki and big wild Shang Ren (1984) have delivered the Grifola Frondosa sporophore polysaccharide to have after the report of anti-tumor activity, and Japan is progressively goed deep into the research of grifolan.Big wild Shang Ren (1986) has compared the mycelium polysaccharides of liquid culture and the anti-tumor activity of fruitbody polysaccharide, finds that the two is quite similar.The processing condition of pointing out to produce polysaccharide by the liquid culture mycelium are suitable for scale operation from sporophore extraction polysaccharide gentleness and simple.Aspect pharmacology, big wild benevolence has still reported that the anti-tumor activity of grifolan is subjected to the influence of the regulating effect of macrophage system, and is except that multiple solid tumor is had the restraining effect, effective too to ascitic tumor.
Takahiro.T etc. (1987) have disclosed the antitumor mechanism of grifolan.Grifolan does not have direct cytotoxicity to the tumour cell of cultivating, but can increase the quantity that tumour cell is had the peritoneal exudate cells and the endothelium skein cell of cytotoxicity, increases the effect of the T cell that cytotoxicity is arranged.The anti-tumor activity that has disclosed grifolan is that the acting in conjunction by intermediary's reaction of host and scavenger cell and T cell realizes.
Iwao.S etc. (1989) have reported that the liquid culture mycelium polysaccharides is having similarity with fruitbody polysaccharide aspect anti-tumor activity and the immunoregulatory activity, can promote the activity of natural killer cell (NK cell) and scavenger cell, enhancing antibody reaction simultaneously, peritoneal exudate cells (PEC) quantity, interleukin 1 (IL-1), acid phosphatase, the phagocytic activity of attached cell all improves greatly among the PEC.Simultaneously, the carbon clearance ability also increases.Theoretical basis has been established in this fermentative production and application thereof that is found to be grifolan.
The wild Zhuo of water (1991) has been reported the anti-tumor activity of Grifola frondosa glycoprotein, and it has, and the tumour of inhibition takes place and the inhibition metastasis effect, and not only injection is effective, oral effective too, and its onset dosage is lower than holosaccharide.Nanba.H etc. (1995) report, grifolan be except that having immunomodulatory and suppressing the function of tumour two aspects, also has anti HIV-1 virus simultaneously, brings high blood pressure down, the effect of aspects such as blood fat reducing, lowering blood glucose.
1986, the tame success of Grifola frondosa was for the research of grifolan provides certain basic substance.Subsequently, discoveries such as big wild Shang Ren, grifolan is one of fungus polysaccharide with the strongest anti-tumor activity of being found.
The production of grifolan is many to be that raw material extracts acquisition with the sporophore.Because Grifola Frondosa sporophore cultivation difficulty, biological yield is low, and difficult quality control makes the Development and Production of grifolan be subjected to very big restriction.Constantly perfect along with the Grifola frondosa Study on Fermentation makes the extensive acquisition of maitake mushroom mycelia become possibility, the pharmacologically active of grifolan and also constantly obtain disclosing in the value aspect oncotherapy and the immunomodulatory.But because the production cost height, the issues of purification of extracting grifolan can not get solving, and the Grifola frondosa strain that especially is fit to large scale fermentation production is difficult to obtain all the time, and the practical application of grifolan is made slow progress.
Grifola frondosa strain involved in the present invention, by wild bacterium separation and purification, through long-term fermentation culture domestication, poor growth, substratum bioavailability and the low shortcoming of biological transformation ratio have been overcome, possessed the characteristic of suitability for industrialized production with bacterial strain, for the application of the application of grifolan, particularly grifola frondosa beta-glucose, established solid basis.
Summary of the invention
First purpose of the present invention is to provide a kind of biomaterial---Grifola frondosa strain, can obtain grifolan, especially grifola frondosa beta-glucose from its tunning.
Grifola frondosa strain of the present invention, called after Grifola frondosa Grifola frondosa (Dichs.ex Fr) S.F.Gray, GF-932, different name Polyporus frondosus (diks.) Fr. is that separation screening obtains from the natural bacterial strain of wild environment.This biological material specimens is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the depositary institution address is No. 13 Institute of Microorganism, Academia Sinica in Zhongguancun N 1st Lane, Beijing City, deposit number is CGMCC No.1709, and preservation date is on May 11st, 2005.
The microbial taxonomy status of above-mentioned Grifola frondosa Grifola frondosa (Dichs.ex Fr.) S.F.Gray belongs to Mycophyta, Basidiomycotina, Hymenomycetes, Aphyllophorales, polyporaceae, tree Pseudomonas.
The cultural characteristic of bacterial strain of the present invention:
The slant strains substratum adopts the potato agar enriched medium, and composition is as follows: potato: 200g; Glucose: 20g; Potassium primary phosphate: 1g; Sal epsom: 0.5g; Agar: 20g; Water: 1000mL; PH=7.0
On the PDA plate culture medium, cultivate 5～6d for 27 ℃, bacteria colony white, therefrom mind-set is radial outward, and the mycelia adhesion is in media surface, and the middle part is sparse, and is outwards intensive, and powdery is to cotton-shaped, and diameter reaches 8cm, back side canescence.
On the test tube slant substratum, to cultivate 5～7d for 25 ℃ and can cover with the inclined-plane, mycelia is white and sturdy, and is intensive, thick along the bevel edge mycelia on top, be cotton-shaped.
The morphological specificity of bacterial strain of the present invention:
At microscopically, morphological structure shows as hyphae colorless, thin, level and smooth, the tool barrier film of wall, branch, no clamp connexion, wide 2～3.2 μ m.Get together in the liquid nutrient medium agglomerating, light yellow sphere or cotton-shaped, the edge is the fine hair shape, diameter 1～4mm.
The sporophore meat, short handle is the coralliform branch, and is terminal living fan-shaped to the cochlear cap, is overlapped into clump; Bacteria cover diameter 2～7cm, grey is to light brown.There is fine, soft fur on the surface, and old back is smooth, the reflectivity striped is arranged, thin edge, curls inward.Bacterial context is thick white, thick 2～7mm.Long 1～the 4mm of tube, pore prolongs life, and hole colourless look extremely faint yellow, mouth of pipe polygon, average 1～3 every millimeter.Spore is colourless, and is smooth, and oval is to oval.The mycelia wall is thin, and branch has tabula.
Second purpose of the present invention provides the fermentation culture method of described Grifola frondosa strain and the composition of substratum.
Liquid culture substratum: potato 1～3%, wheat bran extract 1～5%, glucose 1～5%, Semen Maydis powder 0.3～2%, peptone 0.1～3%, yeast powder 0.1～3%, potassium primary phosphate 0.01～1%, sal epsom 0.05～1%.
The liquid fermentation and culture feature of bacterial strain of the present invention:
Liquid culture 1d in the fermentor tank, promptly visible a large amount of tiny mycelium pellets produce.Cultivate 2d, the mycelia increment has a large amount of mycelium pellets in irregular shape to form rapidly in the substratum, and the mycelium pellet suspension growth is in liquid nutrient medium, and bacterium liquid is sticky, and leaving standstill only has a small amount of layering.Cultivate 3d, mycelium almost is state of saturation, leaves standstill not stratifiedly, and mycelium morphology has barbed mycelium pellet, wadding bunch mycelium pellet and particle mycelium pellet.3～5d can finish a fermentation culture cycle.
After this bacterial strain surpasses the level that goes down to posterity (6～9 generation) that is used to produce, the slant culture speed of growth does not have considerable change, mycelial growth speed does not have considerable change yet in the liquid culture process, mycelial yield and polysaccharide yield do not have obvious downward trend yet, illustrate that this bacterial strain limits the interior stable performance of going down to posterity in production.
The fermentation condition parameter of Grifola frondosa strain is:
Three grade fermemtation, fermentor tank magnification ratio are 1: 10.The coefficient of seeding tank and fermentor tank is 50～80%, and the liquid seeds inoculum size is 5～10% (V/V).Culture condition: culture medium C/N is 20～30: 1, pH value 4～7, dissolved oxygen content 10～100%, 24～29 ℃ of temperature, jar internal pressure 0.5～1.5Mp, mixing speed 100～600r/min.
Above-mentioned feature shows that other fungies of Grifola frondosa strain of the present invention and Ramalina are very different, and also there were significant differences with other Grifola frondosa strains that are used for edible fungus culturing.The main difference point is, no matter on solid medium, or in the liquid medium within, its speed of growth is rapid unusually.This bacterial strain is suitable for liquid submerged fermentation to be cultivated, the mycelium production height, and to the utilization ratio height of substratum, the output height of secondary meta-bolites such as polysaccharide is fit to large-scale industrialization production.
The 3rd purpose of the present invention provides the grifolan in its tunning of described Grifola frondosa strain, and the application approach of grifola frondosa beta-glucose aspect productions such as tablet, capsule, injection especially is provided.
Utilize strain fermentation of the present invention to obtain the technical process of grifolan and grifola frondosa beta-glucose:
During fermentation termination, fermented liquid is carried out solid-liquid separation, reclaim fermented liquid and separate exocellular polysaccharide.The mycelium vacuum-drying and the micronizing that leach are used hot water extraction after the degreasing, residue extracts respectively with weak bronsted lowry acids and bases bronsted lowry.Respectively extract is concentrated alcohol precipitation, vacuum-drying promptly gets grifolan and glycoprotein.Get the extract that wherein contains beta-glucan, remove behind the albumen with isolating grifola frondosa beta-glucose.
Grifolan, glycoprotein and grifola frondosa beta-glucose by above-mentioned technical process acquisition, remove and have the general pharmacology activity of fungus polysaccharide, outside immunomodulatory, inhibition tumour and efficacy enhancing and toxicity reducing effect to chemicotherapy, also has the distinctive pharmacologically active of grifolan, as antiviral, anti tumor activity in vitro, apoptosis of tumor cells induced activity, anti-inflammatory and antalgic activity etc.
Another important characteristic is, Grifola frondosa strain of the present invention, and its meta-bolites grifolan is not only injected effectively, and oral also effective.
The grifolan and the grifola frondosa beta-glucose that utilize strain fermentation of the present invention to obtain can be applicable to production fields such as protective foods, makeup and medicine, can be applicable to the production of capsules such as tablet, hard capsule and soft capsule, also can be used for the production of injections such as liquid drugs injection and freeze-dried powder.
Description of drawings
Accompanying drawing 1 is the mycelium and the bacterium ball microscopic morphology figure (Olympus microscope, 100X, 40X) of bacterial strain of the present invention.
The infrared absorpting light spectra of the grifola frondosa beta-glucose that accompanying drawing 2 obtains for bacterial strain of the present invention.
The chemical structural drawing of the grifola frondosa beta-glucose that accompanying drawing 3 obtains for bacterial strain of the present invention.
Embodiment
The cultural method that goes down to posterity adopts inclined-plane cultivations of going down to posterity, slant medium employing potato agar enriched medium, and composition is as follows: potato 200g; Glucose 20g; Potassium primary phosphate 1g; Sal epsom 0.5g; Agar 20g; Water 1000mL; VB
1In right amount, pH nature.
The slant strains substratum is made according to a conventional method; Inoculation culture by the aseptic technique requirement, on the sterilisable chamber super clean bench, adopts the inoculation of double inclined plane inoculation method.After the inoculation, put 27 ± 1 ℃ and cultivate 1d, can see around moving the tissue block that connects growing crawl shape mycelia and substrate mycelium earlier, extend into aerial hyphae by the shape mycelia that crawls then, also have and directly extend into aerial hyphae.Behind 4～7d, the tender mycelia of children is covered with the inclined-plane, the mycelium fine hair shape that is white in color, and sturdy prosperity, the later stage is felted.
Embodiment 2 shake-flask culture
Liquid culture substratum: potato 1%, wheat bran 1%, glucose 2%, Semen Maydis powder 0.7%, peptone 0.3%, yeast powder 0.14%, potassium primary phosphate 0.05%, sal epsom 0.1%.
The preparation substratum is packed in the Erlenmeyer flask, and coefficient is 0.2～0.4, and tampon seals, sterilization.By 5～10% (V/V) the inoculation Grifola frondosa bacterial classification of charge amount, put shaking table and cultivate.25～30 ℃ of temperature, rotating speed 90～150r/min.Cultivate the visible a large amount of newborn mycelium pellets of 2d and produce, promptly have a large amount of mycelia to form behind 3～5d in the liquid nutrient medium, the mycelia suspension growth is in liquid nutrient medium, and form has branch mycelia, wadding bunch mycelia thing, particle mycelia thing.
Cultivate 3～5d as stated above, maitake mushroom mycelia output is 9～15g/100mL.Intracellular polyse productive rate 8～12% (W/W), exocellular polysaccharide productive rate 0.5～1% (W/V).
Embodiment 3 fermentation culture
Three grade fermemtation, fermentor tank magnification ratio are 1: 10.The coefficient of seeding tank and fermentor tank is 50～80%, and the liquid seeds inoculum size is 5～10% (V/V).
Culture condition: culture medium C/N is 20～30: 1, pH value 4～7,24～29 ℃ of temperature, jar internal pressure 0.5～1.5Mp, mixing speed 100～600r/min, dissolved oxygen content 10～100%.
Fermentor tank amplifies step by step, and in containing the synthetic or semisynthetic medium of sufficient nitrogenous source and carbon source, maitake mushroom mycelia can make the constantly accumulation of mycelia amount in raised growth breeding under the aerobic conditions.Seeding tank growth cycle 24～48h, fermentor tank growth cycle 36～72h.Mode with continuous amplification can make its biomass become hundred times growth in several days, can finish a growth cycle in the time of 3～5d.
During fermentation termination, mycelia yield 1～2%, polysaccharide yield 5～8%, beta-glucan yield 1～3%.
Claims (7)
1, a kind of Grifola frondosa strain, it is characterized in that: called after Grifola frondosa Grifola frondosa (Dichs.ex Fr.) S.F.Gray, bacterium numbering GF-932, different name Polyporus frondosus (diks.) Fr., now be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.1709.
2, according to the described Grifola frondosa strain GF-932 of claim 1, it is characterized in that: on the PDA substratum, cultivate 6d for 27 ℃, bacteria colony white, therefrom mind-set is radial outward, the mycelia adhesion is in media surface, and the middle part is sparse, and is outwards intensive, powdery is to cotton-shaped, and diameter reaches 8cm, back side canescence.On the test tube slant substratum, to cultivate 7d for 25 ℃ and can cover with the inclined-plane, mycelia is white and sturdy, and is intensive, thick along the bevel edge mycelia on top, be cotton-shaped.
3, according to the cultural method of the described Grifola frondosa strain GF-932 of claim 1, it is characterized in that: training method is a liquid submerged fermentation, in 500～1000ml Erlenmeyer flask, shake a bottle production of hybrid seeds, 25～30 ℃ of temperature, rotating speed 90～150r/min, culture cycle 72～96h.In the three grade fermemtation jar, cultivate step by step and amplify, liquid seeds inoculum size 5～10%, aeration-agitation, dissolved oxygen amount is controlled between 10～100%, one-level jar and secondary jar fermentation period 20～30h, three grades of jar fermentation period 36～72h.
4, according to the cultural method of the described Grifola frondosa strain GF-932 of claim 1, it is characterized in that: Grifola frondosa strain GF-932 grows in potato glucose wheat bran juice substratum, described potato glucose wheat bran juice substratum contains potato 1～3%, wheat bran extract 1～5%, glucose 1～5%, Semen Maydis powder 0.3～2%, peptone 0.1～3%, yeast powder 0.1～3%, potassium primary phosphate 0.01～1%, sal epsom 0.05～1%.
5, the grifola frondosa beta-glucose that is produced according to the described Grifola frondosa strain GF-932 of claim 1, it is characterized in that: the beta-glucan that GF-932 Grifola frondosa bacterial classification obtains after liquid culture mostly is β (1-3) ramose β (1-6) dextran (seeing accompanying drawing 3), molecular weight 50 000～1 000 000Dalton, molecular weight distribution width 1.5～2.5, and contain a spot of conjugated protein.Have draw moist; Easily dissolving in water, solution becomes is sticky when solubleness reaches 30～40mg/mL, maximum dissolvable 140～160mg/mL; Be not dissolved in acetone, chloroform, ether.No definite melting point, temperature reach 250 ℃～begin charing more than 260 ℃ the time.The identification of sulfuric acid-phynol feature is yellowish brown, at the 490nm place maximum absorption band is arranged, and sulfuric acid-anthrone feature identification is blue-greenish colour, maximum absorption band is arranged near 620nm; The no obvious characteristics absorption peak of uv-absorbing scanning, in the infrared spectrogram at 840cm
-1There is not absorption about wave number, at 895cm
-1Weak charateristic avsorption band (seeing accompanying drawing 2) is arranged, nucleus magnetic resonance near the wave number
1In the H-NMR collection of illustrative plates, the H1 signal is all less than 4.80ppm.Combine with the Congo red solution of 50mg/L, obvious red shift takes place in maximum absorption wavelength.This beta-glucan novel structure has good stability, still can keep its biological activity behind autoclaving, also has good stability in highly basic.
6, according to described grifolan of claim 5 and grifola frondosa beta-glucose, it is characterized in that: except that having immunomodulatory, function of tumor inhibition, also has the distinctive pharmacologically active of grifolan, as to the efficacy enhancing and toxicity reducing effect of chemicotherapy, antiviral, anti tumor activity in vitro, apoptosis of tumor cells induced activity, anti-inflammatory and antalgic activity etc.
7, according to the range of application of described grifolan of claim 5 and grifola frondosa beta-glucose, it is characterized in that: can be used for the production of capsules such as tablet, hard capsule and soft capsule, also can be used for the production of injections such as liquid drugs injection and freeze-dried powder.
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