CN101112396A - Extract of sparassis crispa MH-3 bacterial strain and method for preparing the same and use thereof - Google Patents
Extract of sparassis crispa MH-3 bacterial strain and method for preparing the same and use thereof Download PDFInfo
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Abstract
The present invention discloses an extract of Sparassis Crispa MH-3 strain NIBH FERM P-17221, which takes the Beta-1, 3-glucan as the main ingredient. The present invention also discloses a preparation method of the extract of sparassis crispa MH-3 strain. In addition, the present invention also discloses the application of the extract of sparassis crispa MH-3 strain in the preparation of foods, beverages and health care products and the application of which in the preparation of anti-tumor drugs.
Description
Technical field
The present invention relates to the extract of a kind of sparassis crispa MH-3-3 bacterial strain (Sparassis Crispa) NIBH FERMP-17221; In addition, the invention still further relates to this sparassis crispa MH-3-3 bacterial strain preparation method of extract and purposes.
Background technology
Had in the past and much utilized nature various mushrooms wild or cultivation, and extracted various active component, and make functional food, beverage and medicine.Though it is ripe to extract the technology of bioactive substance in the present various mushroom, because strain is numerous, composition is extremely complicated, is difficult to understand their biological activitys separately.
For many years, people have carried out a large amount of research and exploration to the immunocompetence and the antitumor action of mushroom polysaccharide.Up to now, developed many mushroom polysaccharide goods,, and obtained certain social benefit and economic benefit as JIGU, dance young pilose antler, Ganoderma etc.But the strain that nature exists is countless, and the composition that contains in the bacterium is also very complicated, still has a lot of active substances not to be found, and whether material also has identical or stronger biological activity still need explore.
Sparassia crispa (Wulf.) Fr. now confirms to exist in the world 1 section 1 to belong to 2 kinds, Japan has only a genus at present, be distributed in geographic alpine belt from Hokkaido to the Northeast, it is a kind of very rare famous and precious edible and medical fungi, summer and autumn Dan Shengzhi all living creatures, be born on the ground or tree root of PiceameyeriRehd. Et Wils., fir or pine forest, stem base portion RUGEN shape links to each other with tree root.Sparassia crispa (Wulf.) Fr. is extremely rare class mushroom, and its mouthfeel is fine, but poor growth, the artificial culture difficulty.The fundamental characteristics of clear and definite Sparassia crispa (Wulf.) Fr., and how to make full use of its still worth discussion, effective bioactive substance of particularly researching and developing in the Sparassia crispa (Wulf.) Fr. is a wide and far-reaching problem.
Summary of the invention
One of the technical problem to be solved in the present invention provides the extract of a kind of sparassis crispa MH-3-3 bacterial strain.
Two of the technical problem to be solved in the present invention provides above-mentioned sparassis crispa MH-3-3 bacterial strain preparation method of extract.
Three of the technical problem to be solved in the present invention provides the application of extract in preparation food, beverage and health product of above-mentioned sparassis crispa MH-3-3 bacterial strain.
Four of the technical problem to be solved in the present invention provides the application of above-mentioned sparassis crispa MH-3-3 bacterial strain extract in the preparation antitumor drug.
For solving the problems of the technologies described above, the invention provides the extract of a kind of sparassis crispa MH-3-3 bacterial strain (SparassisCrispa) NIBH FERM P-17221, it is main component with the glucosan, especially with β-1, the 3-glucosan is an active component.
The present invention use microorganism--sparassis crispa MH-3-3 bacterial strain is the strain that belongs to the silk ball Pseudomonas, its on February 17th, 1999 in life engineering Industrial Technology Research Institute of Japanese industry technology institute (being called for short NIBH) preservation, preservation registration number is FERM P-17221.To contain fallen leaves pinaster bits, fractionlet or the heating of its mixture water proof, remove the solvable composition of hot water after, add nutritional labeling, make artificial culture bacterium bed, carry out the artificial culture of Sparassia crispa (Wulf.) Fr..According to said method, approximately can about two first quarter moons, obtain sparassis crispa MH-3 required for the present invention-3 bacterial strain.
Described sparassis crispa MH-3-3 bacterial strain has following Microbiological Characteristics:
1. biological characteristics:
Sparassis crispa MH-3-3 bacterial strain belongs to the monospore daughter bacteria, and the color of its sporophore is white in color to faint yellow, by sending a lot of branches on the sturdy handle, each intensive one-tenth one ball, likeness in form laurustinus.
2. component analysis
Following table is the component analysis table of sparassis crispa MH-3-3, is the analysis result of juridical person's japanese food analysis center, the content of the various nutrients among the xeraphium 100g of sparassis crispa MH-3-3 strain.(annotate:
Sparassis crispa MH-3-3 component analysis result | ||||
The analytical test project | The result | Detection limit | Annotate | Analytical method |
Moisture | 8.8g/100g | Normal pressure heat drying method | ||
Protein | 6.9g/ |
1 | The nitrogen analysis method | |
Lipid | 0.8g/100g | Acid decomposition | ||
Fiber | 6.3g/100g | |||
Ash | 3.1g/100g | Direct ashing method | ||
Sugar | 74.1g/ |
2 | ||
Ferrum | 2.28mg/100g | Atomic absorption spectrophotometry | ||
Ca | 24.7mg/100g | The potassinm permanganate volumetric method | ||
VitB1 | 1.04mg/100g | 3 | High-pressure liquid phase method | |
VitB2 | 1.63mg/100g | High-pressure liquid phase method | ||
VitD | 3, 200IU/100g | High-pressure liquid phase method | ||
Beta glucan | 43.6g/100g | The ferment method | ||
Annotate 1) ferment, protein conversion coefficient: 6.25 | ||||
Annotate 2) calculating formula: 100-(moisture+protein+lipid+fiber+ash) | ||||
Annotate 3) thiamine hydrochloride |
This table beta glucan amount is the amount of total beta glucan, the amount of non-β-1.3 glucosan.)
3. condition of culture:
At first, mix Fructus Musae powder 6g, beer yeast 45g, peptone 1g, calcium 0.6g, 0.5 in magnesium chloride, Mel powder 2g, wheat flour 100g, secondly add the pinaster wood flour 1kg that contains 20% moisture, add 1.5L water again, sterilization is 5 hours after mixing, and forms culture medium.The optimum temperature that sparassis crispa MH-3-3 is grown on culture medium is 20-25 ℃, and pH value is 6.5, and humidity is 80%.
The present invention also provides the preparation method of extract of described sparassis crispa MH-3-3 bacterial strain, and it adopts thermokalite water extraction process, cold aqueous alkali extraction process or hot-water extraction method.
This extracting method may further comprise the steps: preferably in advance sparassis crispa MH-3-3 bacterial strain is done into fritter or powder.In addition, as required, can before extracting, carry out ungrease treatment earlier: sparassis crispa MH-3-3 bacterial strain is soaked in organic solvent, or in the mixed liquor of organic solvent and water, carries out ungrease treatment.Preferably utilize halogenation hydrocarbon material, the ethanol of methyl chloroform etc., the mixed liquor of water to carry out ungrease treatment.
Use solvent to obtain the extracting solution of sparassis crispa MH-3-3 bacterial strain.Solvent can be water miscible or non-water-soluble, preferred water dissolubility solvent.Described water solublity solvent refers to water or is the water-soluble solvent of main component with water.For example, water, added alkali or other alkali materials aqueous alkali, added organic solvents such as ethanol aqueous solution, added the acidic aqueous solution of acid or acidic materials.Preferred water or aqueous alkali.
Water-insoluble solvent, with dimethyl sulfoxine (DMSO), dimethyl formamide (DMF) etc. has polar organic solvent the most commonly used.When extracting as solvent with water, better with hot water, the temperature of water adopts 80 ℃ to 125 ℃ and is advisable, and is controlled at 90 ℃ to 120 ℃ better.As use aqueous alkali, generally use the cold aqueous alkali about 0 to 20 ℃, perhaps the thermokalite water about 50 to 75 ℃.
Extraction can be carried out stage by stage.In this process, also can adopt different aqueous solvent amounts.Such as employing:
1) hot water extraction repeatedly
2) potass extraction repeatedly
3) once or behind the hot water extraction repeatedly, the further extraction of residue part is adopted once or extracting method such as aqueous alkali solvent extraction repeatedly.
To the use amount of water solublity solvent, there is no particular limitation, and generally according to the amount of sparassis crispa MH-3-3 bacterial strain, the difficulty of residue after perhaps extracting or sedimentary weight, refining/clean post processing etc. decides the consumption of water solublity solvent.In general, the use amount of water solublity solvent is sparassis crispa MH-3-3 bacterial strain or precipitate or extracts 5~50 times of residue weight.
As adopt potass extraction, and can adopt 2~20% caustic alkali such as NaOH, KOH, perhaps adopt the solvent adjuvant added equivalent, denaturant alkaline solution as 1~10% carbamide.In the actual extracting processing procedure, can suitably change step or condition with reference to following examples.
Extract the extract that obtains according to the present invention, main component is the polysaccharide glucosan.Say that in further detail extract of the present invention is to contain β-1, the 3-glucosan is a feature.The glucosan of being introduced in following examples is the β-1 of the 6th base, the 3-glucosan, and molecular weight is about 2,000,000.Glucosan, particularly β-1, the effect in vivo of 3-glucosan is subjected to people's attention in recent years always.The extract of sparassis crispa MH-3 of the present invention-3 bacterial strain, its main component are exactly β-1, the 3-glucosan, thereby extracting solution is extremely valuable.
β-1, the molecular structure of 3-glucosan is as follows:
The biological activity determination appraisement system of beta glucan is as follows:
In the body: antitumor action (solid cancer, ehrlich ascites carcinoma, inbred animal model); Cancerometastasis inhibitory action (leukopenia model); The effect of protecting from infection; Cell is mobilized, activation (phagocyte, cytokine); Leukocyte function enhancement effect (phagocyte, NK cell); Antibody produces potentiation; Cytokine generation effect (in the blood, in the internal organs);
External: complement activity turns usefulness into, the modification of coagulase, cytophagous activation (migration, engulf, cytokine produces); Kill production of cytokines (non-activity oxygen); Lymphocytic activation; Hematoblastic activation; The production of cytokines effect; Receptor is resolved, and the cell internal information is passed on.
The people is to the responsiveness (oral administration) of beta glucan: be difficult to detect beta glucan in the human body blood, except deep fungal infection disease.But can detect the antibody of beta glucan in the human body, can think and once to infect this bioactive substance in the human body.The oral administration beta glucan can cause intravital immunoreation.
The invention provides the application of extract in preparation food, beverage and health product of sparassis crispa MH-3-3 bacterial strain.The extract of sparassis crispa MH-3-3 bacterial strain by adding common additives as constituent, can be made food, beverage and health product liquid, various forms such as solid, powdered.
The present invention also provides the application of sparassis crispa MH-3-3 bacterial strain extract in the preparation antitumor drug.The extract of sparassis crispa MH-3 of the present invention-3 bacterial strain has anti-tumor activity, and this extract can be made the medicine of various forms such as tablet, powder, pill, liquor, injection by adding various adjuvants.
Description of drawings
Fig. 1 is the NMR spectrum analysis figure of sparassis crispa MH-3-3 extract;
Fig. 2 is the β-1 in sparassis crispa MH-3-3 extract, and the 3-glucosan influences sketch map one to quantity of leucocyte in the mice tip blood;
Fig. 3 is the β-1 in sparassis crispa MH-3-3 extract, and the 3-glucosan influences sketch map two to quantity of leucocyte in the mice tip blood.
The specific embodiment
The invention will be further elaborated by the following examples:
Sparassis crispa MH-3-3 bacterial strain is ground into powder, configuration chloroform: methanol: the weight ratio mixed liquor of water=200: 200: 60, at room temperature the dry powder of 25g with above-mentioned pulverizing carries out 2 days ungrease treatment.
After embodiment 1 obtains extract (test sample 2), carry out embodiment 2.With having added the aqueous alkali 500ml (4 degree) of 10% NaOH of 5% carbamide, join in the embodiment 1 resulting residue, the extraction of carrying out 2 days is handled, and obtains extract (test sample 3).After the dialysis, carry out ethanol precipitation, dehydration again, and after the dried, obtain the cold alkaline extraction thing B of 4.93g.
Embodiment 3
After embodiment 2 obtains obtaining extract (test sample 3), carry out embodiment 3.With having added the thermokalite water 500ml (65 degree) of 10% NaOH of 5% carbamide, join in the embodiment 2 resulting residues, the extraction of carrying out 1 hour is handled, and obtains extract (test sample 4).After the dialysis, carry out ethanol precipitation, dehydration again, and after the dried, obtain the thermokalite extract C of 1g.
With the solid matter after the ungrease treatment, carry out air drying, put into autoclave, add the hot water of 500ml, carry out 2 hours hot water extraction, obtain extract (test specimen 1).Residue after will extracting is again put into autoclave, adds the water of 500ml, further carries out 2 hours hot water extraction, obtains extract (test sample 2).The resulting extract of hot water extraction (test sample 1 and 2) is mixed, the ethanol (200ml) that adds volume, with 5000rpm centrifugal 5 minutes, make its precipitation, precipitate is added in the water of 200ml, decompose, add the ethanol (4ml) of equivalent again by dispersion machine, obtain fibrous precipitate, and the clear liquid on upper strata.
Precipitate by behind the ethanol, acetone treatment, is carried out dehydrate, obtain the hot water extract A-1 of 41mg.Supernatant is concentrated, after the lyophilization, obtains the hot water extract A-2 of 415mg.
Main component glucosan to the extract of embodiment 1~3 gained carries out component analysis.Fig. 1 has shown above-mentioned hot water extract A-1 and A-2, cold potass extraction thing B, and thermokalite water extract C is dissolved in the spectrum analysis result of the C-NMR among the 0.6mlDMSO-d.In Fig. 1, SCHWE1v represents that hot water ethanol 1 times of extraction, SCCA1 represent that cold potass extraction 1, SCCA2 represent that cold potass extraction 2, SCHA represent the thermokalite water extraction.As shown in Figure 1, near 85ppm, 3 distinctive divisions are arranged, the feature of Here it is the 6th basic glucosan.From above-mentioned analysis as can be known, the glucosan in sparassis crispa MH-3-3 extract is β-1,3-glucosan (the 6th base).
Below by test the example beneficial effect of the present invention is described in further detail:
The anticancer test of test example 1 mice
Injection S-180 tumor cell (pernicious 180 solid shape hepatoma carcinoma cell, cell concentration 5 * 10 in male (ICR) mice body in 6 weeks (body weight 30g)
6/ only), 10 mices of matched group, 120 mices of test group are one group with 10 and test.To each mouse transplant 20,100 respectively, the tumor tissues of 500m μ g, respectively the 7th, 9, mice was carried out in 11 days extract (the hot water extract A-1 of embodiment 1-3 of lumbar injection variable concentrations, cold alkaline extraction thing B, and thermokalite extract C), observes the tumor growth situation then at the 35th day, and measure the size and the weight of mouse tumor.
The result is as shown in table 1 below, the extract that the result shows sparassis crispa MH-3-3 to mice fixedly Subcutaneous tumor the obvious suppression effect is arranged.Can reach 100% in 4 times of groups of heat extraction and thermokalite water extraction group.
Table 1 sparassis crispa MH-3-3 extracting solution is to the inhibitory action of tumor
Extracting solution | Input amount (μ g*3) | Shrink back fully and count (only) | Suppression ratio (%) |
Matched group | ?0 | ?0 | ?- |
?500 | ?8 | ?99.5 | |
Heat is extracted 1 times | ?100 | ?6 | ?94.4 |
?200 | ?5 | ?83.6 | |
?500 | ?4 | ?91.2 | |
Heat is extracted 4 times | ?100 | ?0 | ?0 |
?20 | ?0 | ?0 | |
?500 | ?5 | ?90 | |
Cold alkaline extraction | ?100 | ?6 | ?95.7 |
?20 | ?3 | ?62.1 | |
?500 | ?6 | ?99 | |
Thermokalite extracts | ?100 | ?10 | ?100 |
?20 | ?4 | ?54.9 |
Test example 2
If 3 test group and 1 matched group, each test group and matched group (control) are 10 mices, totally 40 of 4 groups.Control group mice is irritated the stomach normal saline, and the test group mice is irritated the β-1 in stomach 50 μ g, 100 μ g, 200 μ g sparassis crispa MH-3-3 extracts, 3-glucosan respectively.At first, irritate stomach anticarcinogen-cyclophosphamide (CY) 200mg/kg, cause the leukopenia of mice, and then irritate stomach β-1, the 3-glucosan is observed quantity of leucocyte in the tip blood.As shown in Figure 2, the leukocyte of comparing each test group with matched group obviously increases, this evidence β-1, the 3-glucosan also can strengthen its immune effect according to immunoreation in digestive tube without the absorption of digestive tube.And β-1, the leukopenia phenomenon that the 3-glucosan causes the anticarcinogen effect that improves significantly.
Test example 3
Test is divided into three groups, and experimental subject is a male white mouse, and each organizes 10.As shown in Figure 3, contrast (normal saline) group, anticarcinogen CY (Cyclophosphamide, cyclophosphamide) group and CY+CA (extract of cyclophosphamide+sparassis crispa MH-3-3 bacterial strain) group, ip represents mice is adopted lumbar injection.Matched group, injecting normal saline; Give injected in mice 200mg/KgCY, will begin to reduce as the leukocyte of side effect mice: the CY group after the CY of injected in mice 200mg/Kg, began to occur leukocytic minimizing, leukocyte bottom out again in the 7th day in second day; Injection CY+CA group, the beginning leukocyte increased to some extent in the 5th day, and the 10th day begins leukocytic increase and tends towards stability the 21st day value near matched group.This result shows that the extract of sparassis crispa MH-3-3 bacterial strain can improve the leukopenia phenomenon that anticarcinogen causes.
The test of test example 4 clinical medicine
To the dried powder (300mg/ people's every day) that 14 routine cancer patients carry out NK cell amplification therapy and oral sparassis crispa MH-3-3 strain, the effect that is used for the treatment of various cancer patients sees Table 2 (executions of Jitian hospital).
Table 2
Numbering | Age | Sex | The kind of cancer | The pathological changes phase of cancer | Metastasis site | Viewing duration (moon) | Judge |
?1 | ?67 | The man | Pulmonary carcinoma | IV | Lymph node, bone | ?4 | ?PR |
?2 | ?57 | The man | Pulmonary carcinoma | IV | Brain | ?3 | ?PR |
?3 | ?44 | The woman | Pulmonary carcinoma | IV | ?9 | ?PR | |
?4 | ?66 | The woman | Gastric cancer | III | ?11 | ?CR | |
?5 | ?63 | The man | Gastric cancer | IV | ?7 | ?NC |
?6 | ?59 | The woman | Colorectal cancer | IV | Lung, bone | ?12 | ?PR |
?7 | ?69 | The woman | Colorectal cancer | IV | Liver | ?7 | ?PR |
?8 | ?66 | The woman | Colorectal cancer | III | ?9 | ?CR | |
?9 | ?66 | The woman | Breast carcinoma | IV | Liver | ?12 | ?CR |
?10 | ?50 | The woman | Ovarian cancer | III | ?3 | ?NC | |
?11 | ?74 | The woman | Uterus carcinoma | IV | ?3 | ?NC | |
?12 | ?75 | The man | Carcinoma of prostate | III | ?12 | ?CR | |
?13 | ?62 | The woman | Pancreatic cancer | IV | ?3 | ?NC | |
?14 | ?71 | The woman | Hepatocarcinoma | IV | ?4 | ?NC |
Wherein, PR representative obviously improves (tumor is dwindled, cancer antigen reduces or normal in the blood), and (tumor disappears, blood examination index normal) cured in the CR representative fully, and NC represent no change (cancer antigen no change in the big or small and blood of tumor).The Sparassia crispa (Wulf.) Fr. powder is to carry out after self cell NK cell amplification therapy in that patient is carried out, and NK cell amplification therapy is with patient's NK cell of asking for, and behind amplification in vitro, fails back the intravital a kind of immunotherapy of patient again.
As shown in table 2, after 14 routine cancer patients carry out NK cell amplification therapy and oral sparassis crispa MH-3-3, obvious to the treatment for cancer effect.
Claims (5)
1. the extract of sparassis crispa MH-3-3 bacterial strain (Sparassis Crispa) NIBH FERM P-17221 is characterized in that, it is main component with the glucosan.
2. the extract of sparassis crispa MH-3-3 bacterial strain as claimed in claim 1 is characterized in that, described glucosan is β-1, the 3-glucosan.
3. the preparation method of extract of claim 1 or 2 described sparassis crispa MH-3-3 bacterial strains is characterized in that, adopts thermokalite water extraction process, cold aqueous alkali extraction process or hot-water extraction method.
4. the application of the extract of claim 1 or 2 described sparassis crispa MH-3-3 bacterial strains in preparation food, beverage and health product.
5. the application of the extract of claim 1 or 2 described sparassis crispa MH-3-3 bacterial strains in the preparation antitumor drug.
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CN102277400A (en) * | 2011-08-15 | 2011-12-14 | 上海科爱生物技术有限公司 | Extract containing beta-1,3-D-glucan and use thereof |
CN103570839A (en) * | 2012-07-18 | 2014-02-12 | 上海荷仙菇生物科技股份有限公司 | Extraction method for Sparassia crispa polysaccharide |
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CN115141864A (en) * | 2022-09-06 | 2022-10-04 | 山东久茸生物科技有限公司 | Processing method of selenium-rich sparassis crispa polysaccharide |
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CN102277400A (en) * | 2011-08-15 | 2011-12-14 | 上海科爱生物技术有限公司 | Extract containing beta-1,3-D-glucan and use thereof |
CN103570839A (en) * | 2012-07-18 | 2014-02-12 | 上海荷仙菇生物科技股份有限公司 | Extraction method for Sparassia crispa polysaccharide |
CN105707762A (en) * | 2016-02-02 | 2016-06-29 | 上海荷仙菇生物科技股份有限公司 | Health-care product administrated in female menstrual period |
CN111685234A (en) * | 2020-07-10 | 2020-09-22 | 融和梦(福建)生物科技有限公司 | Pig feed and method for enhancing immunity by feeding pig feed |
CN111718857A (en) * | 2020-07-10 | 2020-09-29 | 融和梦(福建)生物科技有限公司 | Culture method of sparassis crispa, sparassis crispa dry powder, solvent extract and application thereof |
CN111718857B (en) * | 2020-07-10 | 2023-01-06 | 融和梦(福建)生物科技有限公司 | Culture method of sparassis crispa, sparassis crispa dry powder, solvent extract and application thereof |
CN113846021A (en) * | 2021-09-06 | 2021-12-28 | 融和梦(福建)生物科技有限公司 | Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder |
CN115336743A (en) * | 2022-08-19 | 2022-11-15 | 拜泉香虎贸易有限公司 | Application of Ha Louba dew oral liquid in preparation of anti-alcohol product |
CN115141864A (en) * | 2022-09-06 | 2022-10-04 | 山东久茸生物科技有限公司 | Processing method of selenium-rich sparassis crispa polysaccharide |
CN115141864B (en) * | 2022-09-06 | 2022-11-11 | 山东久茸生物科技有限公司 | Processing method of selenium-rich sparassis crispa polysaccharide |
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