CN1475101A - Agaric pilose mixed fungus fermentation culturing method and its culturing medium and product - Google Patents
Agaric pilose mixed fungus fermentation culturing method and its culturing medium and product Download PDFInfo
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Abstract
A culture medium for the pine mushroom cultured by fungus-yeast mixed fermenting method contains Co (0.01-50 mg/kg). Said fungus-yeast mixed fermenting method includes respectively preparing the fermenting liquid and seed liquid from yeast and pine mushrom, inoculating them in fermentor containing said culture medium, and deep culturing. Its products include pine mushroom, active matrix (such as polyose) and the composition containing the active matrix.
Description
Technical field
The present invention relates to the mixed fungus fermentation field, is to be total to the method that yeast-like fungi changes product biologically active and sensory properties with matsutake especially.
Background technology
Matsutake [Tricholoma matsutake (s Ito.et Imai) sing] is called the rare large-scale wild edible fungi of ' king in the mushroom ', and the mouthfeel uniqueness gives off a strong fragrance.Though, once having its polysaccharose substance of research report can reach 91.8% to the tumour inhibiting rate of small white mouse sarcoma S-180, is 70% to the EC tumour inhibiting rate, has extremely effectively antitumaous effect, in 27 kinds of basidiomycetes with antitumaous effect, occupy second, be important screening anticancer medicine object, can improve nospecific immunity and specific immune function and anti-stress effect (Fu Weijie, Xu Guangbo, permitted perigene etc., Shandong Agricultural University's journal, 1999,30 (increasing): 230-233).
Realize on the artificial cultivation technique that the research report of relevant its bioactive functions material (polysaccharide, glycoprotein etc.) structure and function is very few but at present its research is mainly concentrated on.
Because worldwide matsutake is that minimum number, trees live body the rarest, that distribution is the narrowest are total to ectotrophic mycorrhiza, the artificial cultivation of over half a century research fruit body still is unrealized so far, along with the international market demand amount constantly increases, because excessive gathering causes the natural matsutake output in the world to descend year by year, frequently face extinction, cause costing an arm and a leg, the Japanese market retail price is up to 1275 dollars/Kg.
Discover fungi mixed fungus fermentation with two or more, kind, structure, the biologically active of the mixed fungus polysaccharide that produces, for example anticancer cancer suppressing action improves immunologic function etc., with single fungi difference to some extent, be not equal to the simple superposition of two kinds of independent tunnings of fungi yet.Reason is owing to working in coordination with between active substance, synergy, and complementation, and produced new bioactivator, its physiological function effect is better than original independent product.At present, still few to the mixed fungus fermentation research of fungi, be not to be total to yeast-like fungi with matsutake, utilize the mixed fungus fermentation technology to change the report of fungi activity composition.
Therefore, must go by the suitability for industrialized production power of microbial engineering acquisition matsutake product.To using matsutake as being total to yeast-like fungi, the product that acquisition improves and change biologically active and sensory properties presses for.
Summary of the invention
Therefore, an object of the present invention is to provide a kind of method of mixed fungus fermentation, wherein, improved the sensory properties of its biologically active and product by having changed the independent products of two kinds of single bacterium with agaric pilose mixed fungus fermentation.
Another object of the present invention provides a kind of medium that is exclusively used in above-mentioned agaric pilose mixed fungus fermentation method.
Another object of the present invention provides the tunning that is obtained by above-mentioned fermentation process, the polysaccharide that wherein contains high branch degree with have the polysaccharide of polyalcohol group with β-structure that (1 → 3) main chain links to each other.
Another object of the present invention provides above-mentioned tunning and prepares purposes in biological active base material and medicine, health products, the food.
In one aspect of the invention, provide a kind of medium that is used for the matsutake fermentation, this medium contains 0.01-50mg/kg, preferred 0.1-5mg/kg cobalt element.In a preference, also contain the fumaric acid that concentration is 0.005-0.5wt% in the medium of the present invention.
In another aspect of the present invention, a kind of matsutake fermentation method is provided, the method comprising the steps of:
(a) preparation matsutake fermentation seed liquid; With
(b) the matsutake fermentation seed liquid that makes in the step (a) is inoculated in fermentation tank, under the condition of 0.01-50mg/kg cobalt element, cultivates matsutake.
In another aspect of the present invention, a kind of mixed fungus fermentation method is provided, the method comprising the steps of:
(a) prepare matsutake and the fermentation seed liquid that is total to the yeast-like fungi strain respectively; With
(b) fermentation seed liquid with the matsutake that makes in the step (a) and yeast-like fungi strain altogether is inoculated in fermentation tank, under the condition of the mixed bacteria growing of 0.01-50mg/kg cobalt element, mixes the bacterium deep layer and cultivates matsutake and yeast-like fungi strain altogether.
In a preference, contain the 0.1-5mg/kg cobalt element in this medium, and the described suitable bacteria growing condition of mixing comprises that also concentration is the fumaric acid of 0.005-0.5wt%.
In another preference, another kind of yeast-like fungi is altogether selected good strains in the field for seed from rainbow conk, glossy ganoderma or its combination.
In also having a preference, fermentation temperature is 25-37 ℃, and pH is 4.5-5.5, and incubation time is 12 hours-10 days.
In another preference, this method also comprises step:
(c) intracellular polyse in the extraction mycelia and the exocellular polysaccharide in the zymotic fluid.
Another aspect of the present invention provides a kind of polysaccharide, this polysaccharide is the intracellular polyse and/or the exocellular polysaccharide of agaric pilose mixed fungus fermentation, and has content and account for the fermentation exocellular polysaccharide and the polysaccharide of the high branch degree of intracellular polyse total amount more than 20% with polyalcohol group and β-structure that (1 → 3) main chain links to each other that obtain; This polysaccharide prepares by following steps:
(a) prepare matsutake and the fermentation seed liquid that is total to the yeast-like fungi strain respectively;
(b) matsutake that makes in the step (a) and the fermentation seed liquid that is total to the yeast-like fungi strain are inoculated in fermentation tank, under the condition that is fit to mixed bacteria growing of 0.01-50ug/kg cobalt element, mix the bacterium deep layer and cultivate matsutake and yeast-like fungi strain altogether;
(c) intracellular polyse in the extraction mycelia and the exocellular polysaccharide in the zymotic fluid.
Another aspect of the present invention also provides a kind of composition, it is characterized in that, said composition contains carrier and is selected from down the active base-material of group: the mixed bacterium mycelium of agaric pilose mixed fungus fermentation, mixed fungus fermentation liquid, pure mixed fungus polysaccharide, pure mixed fungus glycoprotein, or its mixture.
The accompanying drawing summary
Fig. 1 is a flow chart, has shown embodiment 1 described glossy ganoderma/rainbow conk, matsutake bacterial classification fermentation and mixed bacterium ferment altogether separately, with and the flow chart that experimentizes of each product preparing product.
Embodiment
Fermentation medium technology, prescription are the key points of agaric pilose mixed fungus fermentation production technology.According to the inventor's field study for many years,, find all reductions greatly of the content of cobalt ions before and after the growth in the environment of growth matsutake from the research of sampling of the environment of various places growths matsutake.After exploring, find that cobalt ions is the essential factor of tricholoma matsutake mycelium growth through the laboratory cultures condition.By adding certain density cobalt ions, can make matsutake breed growth fast in fermentation or the mixed culture fermentation separately, the accumulation polysaccharide.
Matsutake medium of the present invention (as submerged fermentation culture medium) outside the conventional ingredients such as nitrogenous source and trace element, also contains cobalt element except containing carbon source, and its content is about 0.01-50mg/kg, preferably about 0.1-5mg/kg.Matsutake seed (tricholoma matsutake mycelium) and common ferment fungi can breed growth fast under this concentration.Cobalt ions can various forms adds, and preferably adds in the medium with the cobalt salt form, for example cabaltous nitrate hexahydrate etc.
In medium, can also contain fumaric acid.Fumaric acid can play growth-promoting effect in the growth of matsutake, its concentration range is generally about 0.005-0.5wt%, preferably is 0.01-0.1wt%, by the medium gross weight.
As for other conventional ingredient that contains in the medium of the present invention, carbon source for example, nitrogenous source and trace element are not particularly limited.Can adopt corresponding composition and consumption in the conventional medium, 3% maltose for example, 0.2%KH
2PO
4, 0.1%Mg
2SO
47H
2O and trace element.These compositions can substitute with other composition well known to those skilled in the art.Those skilled in the art is not difficult to determine its content.
Mixed fungus fermentation method of the present invention is not particularly limited for the matsutake bacterial strain that uses, and available any commercially available matsutake bacterial strain (can from for example academy of agricultural sciences, Shanghai etc. buy) perhaps separates the bacterial strain of the matsutake that obtains with conventional method.
Also be not particularly limited available for example rainbow conk, glossy ganoderma etc., or the combination of the fungi of other tool same functions with the fungi of its mixed culture fermentation.The bacterial strain of glossy ganoderma, rainbow conk can be any commercially available bacterial strain (can from for example China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) etc. buy), perhaps separates the bacterial strain that obtains with conventional method.
In mixed fungus fermentation method of the present invention, add the cobalt element except essential, as broad as long with the fermentation process of routine.Fermentation can be batch (-type) or continous way fermentation.In batch (-type) mixed fungus fermentation production process, can adopt common two step method, promptly cultivate each bacterial classification earlier and obtain seed liquor, then seed liquor is seeded to big fermentation tank, carry out large scale fermentation.
Fermentation temperature can be about 25-37 ℃, and pH is 4.5-5.5, and incubation time is 12 hours-10 days.Preferred fermentation temperature is about 30 ℃, and pH5.0, incubation time are 48 hours-5 days.
Behind large scale fermentation, obtain mycelium and zymotic fluid according to a conventional method, and process processing.For example, first filtering and concentrating zymotic fluid obtains sediment with 95% alcohol chromatography, and with 75% washing with alcohol for several times, freeze-drying obtains exocellular polysaccharide.By pulverizing mycelium, the poach extracting concentrates then, same with alcohol precipitation and dialysis,, can obtain intracellular polyse.
In the tunning except matsutake with altogether the polysaccharide of yeast-like fungi itself, the polysaccharide that has also produced high branch degree with have the polysaccharide of polyalcohol group with β-structure that (1 → 3) main chain links to each other, its content accounts for the about more than 20% of exocellular polysaccharide that fermentation obtains and intracellular polyse total amount.Illustrate that this mixed fungus fermentation method has changed the The Nomenclature Composition and Structure of Complexes of polysaccharide in various degree.The pharmacologically active that proves polysaccharide by experiment is relevant with the glycosidic bond combination between monose, and is relevant with the branch degree of polysaccharide.
With the product of this mixed fungus fermentation method preparation, comprise and mix bacterium mycelium, mixed fungus fermentation liquid, pure mixed fungus polysaccharide and pure mixed fungus glycoprotein.They all can be used as biological active base material and are used to prepare products such as medicine, food, health products.Product can be a various forms, and for example medicine is with forms such as tablet, elixir, syrup, and health products are with mycelium lozenge form, forms such as oral liquid, and food is as mixing forms such as bacterium mycelium bread, biscuit, cake, and mixed fungus fermentation liquid exists with drink form.
Fermentation process of the present invention and products thereof mainly contains following advantage:
(1) method of the present invention has realized the mixed fungus fermentation cultivation of tricholoma matsutake mycelium first, has realized the large-scale production tricholoma matsutake mycelium and has obtained its fermented product;
(2) cost is low: carry out with conventional fermentation process, reduce greatly than the matsutake price of spontaneous growth;
(3) culture medium prescription is simple, and is easy to use.In medium of the present invention, use be the conventional ingredient that is easy to obtain.Preparation is convenient, does not need through specially treated.And usually during the fermentation, needn't regulate pH.
(4) kind of tunning is many, and structure is than single fungi complexity, and its biologically active the experiment proved that also and improves greatly than single fungi.Owing to there is the composition of matsutake, with the sense organ impression of the product of its preparation, fragrance for example, mouthfeels etc. all improve greatly.
Embodiment hereinafter further illustrates the present invention, but should be noted that they are not for special qualification.
Embodiment
Embodiment 1
The mixed fungus fermentation of matsutake and rainbow conk, glossy ganoderma is cultivated
According to the flow process of Fig. 1, with identical medium and condition carry out matsutake and rainbow conk/glossy ganoderma separately, the cultivation of matsutake and rainbow conk/glossy ganoderma mixed fungus fermentation.
The mixed fungus fermentation of embodiment 1a glossy ganoderma and matsutake CS992 bacterial strain is cultivated
1. bacterial classification:
(1), glossy ganoderma [Ganoderma lucidum (Fr.) Karst]:
60Co-gamma-rays and ultraviolet alternately accumulated mutagenic strain CJL990 (1999 through Beijing institute of microbiology of the Chinese Academy of Sciences identify) were than present lucidum strain fast growth 2-4 days.
(2), rainbow conk [Polyporus versicolorL.:Fr] (available from China Committee for Culture Collection of Microorganisms common micro-organisms center)
(3), matsutake [Tricholoma matsutake (s Ito.et Imai) sing]: Jilin is prolonged sky, limit Buddhist and is referred to that the living Tricholoma matsutake (lto et lmai) Singer sporophore of hill separates the matsutake bacterial strain CS992 that obtains through routine.
This bacterial strain is the proof of true tricholoma matsutake mycelium:
1, by the breeding of this bacterial strain being discovered self growing of having only mycelia, do not observe sexual and vegetative propagation (mycelia does not have clamp connection) feature, prove that this bacterial strain can not produce fruit body through pure culture;
2, with randomly amplified polymorphic DNA (RAPD) technical Analysis, the RAPD finger-print similarity factor of this bacterial strain RAPD finger-print and Tricholoma matsutake (lto et lmai) Singer sporophore is 0.932, illustrates that this bacterial strain and matsutake have very high close bacterium (edge) relation.
3, the mycelium to Tricholoma matsutake (lto et lmai) Singer sporophore and this bacterial strain CS992 carries out isoenzyme analysis: the isodynamic enzyme zymogram mainly is that the gene institute by each bar enzyme band of coding directly determines, Isozyme Analysis is from one of important means of the biological population genetic differentiation of protein molecule level research, has been applied to the research that multiple large edible bacteria strain is differentiated at present.Zymoprotein is first product of gene expression, and the isozymogram of edible mushroom different strain is generally different.
The mycelium of Tricholoma matsutake (lto et lmai) Singer sporophore and this bacterial strain CS992 is carried out esterase isozyme and peroxidase isozyme to be analyzed its isodynamic enzyme similar value (S) and is respectively 0.908 and 0.887, the two isozymogram very close (because Tricholoma matsutake (lto et lmai) Singer sporophore is not to be the fruit body of setting out of bacterial strain CS992 separate tissue, otherwise similar value (S) can be more close).
Prove all that below bacterial strain CS992 is true matsutake bacterial strain.
Two, mix bacterium deep layer ferment medium altogether
(wt%) maltose 3, bean cake powder 0.5, CaCO
30.05, KH
2PO
40.2, MgSO
47H
2O0.1, Na
2CO
30.056, tartaric acid 0.1, fumaric acid 0.066, K
2HPO
40.36;
(mg/Kg) Fe
SO
47H
2O 495, CuSO
45H
2O 8, V
c6.2, MnSO
4H
2O 440, Co (NO
3)
2.6H
2O 5 (by cobalt ions, concentration is 1.066), growth factor VB
16
Three, zymotechnique (2T fermentation tank)
Inoculum concentration: seed liquor, proportioning are 2: 1, amount to 5%; Be total to ferment medium acid-base value: PH4.5-5.5; Temperature: 30 ℃; Throughput: 1:0.5-0.8 (V/V/min); Time: 36h.
Four, biological accumulation amount (36h, dried g/100ml) (2T fermentation tank)
Glossy ganoderma: 4.22; Matsutake: 6.15; Mix bacterium: 5.87
Five. the influence of cobalt element concentration and fumaric acid concentration
Repeat above-mentioned cultivation, difference is to replace ganoderma strain capable with rainbow conk, 0,0.01,0.5, the 2mg/kg cobalt element concentration cobalt element concentration of replacing 1.066mg/kg, and 0.01,0.1, the fumaric acid of 0.5wt% concentration replaces the fumaric acid of 0.066wt%.
The result shows, the deep layer mixed fungus fermentation that carries out matsutake and glossy ganoderma or rainbow conk of equal energy success under above-mentioned concentration of cobalt ions, and under the condition that does not have cobalt ions, the independent cultivation of matsutake almost can not obtain product, and the effect of mixed bacteria growing also reduces greatly.In addition, the fumaric acid of above-mentioned concentration all has facilitation to matsutake and glossy ganoderma or the growth of rainbow conk under the deep layer mixed fungus fermentation.
Embodiment 1b
Use the matsutake bacterial strain available from academy of agricultural sciences, Shanghai edible mushroom research institute to substitute matsutake CS992 bacterial strain, cultivate under identical condition, the results obtained are as follows:
Biological accumulation amount (36h, dried g/100ml) (2T fermentation tank)
Glossy ganoderma: 4.3; Matsutake: 6.0; Mix bacterium: 5.6
Repeat above-mentioned cultivation, difference is to replace ganoderma strain capable with rainbow conk, 0,0.01,0.5, the 2mg/kg cobalt element concentration cobalt element concentration of replacing 1.066mg/kg, and 0.01,0.1, the fumaric acid of 0.5wt% concentration replaces the fumaric acid of 0.066wt%.
The result shows, the deep layer mixed fungus fermentation that carries out matsutake and glossy ganoderma or rainbow conk of equal energy success under above-mentioned concentration of cobalt ions, and under the condition that does not have cobalt ions, the independent cultivation of matsutake almost can not obtain product, and the effect of mixed bacteria growing also reduces greatly.In addition, the fumaric acid of above-mentioned concentration all has facilitation to matsutake and glossy ganoderma or the growth of rainbow conk under the deep layer mixed fungus fermentation.
The extraction of embodiment 2 tunnings
1. the extraction of exocellular polysaccharide
With 100 milliliters of zymotic fluids through filtered through gauze, with filtrate at 70 ℃ of vacuum concentration to 1/4 of original volume, add isopyknic 95% ethanol, alcohol was analysed 20 hours.With 75% washing with alcohol sediment 5-6 time, to there not being reducing sugar, use absolute ethanol washing 5-6 time again, vacuum cooling drying obtains thick exocellular polysaccharide.Be placed in the test tube, add the 5ml hot water dissolving, constant volume to 10 milliliter.Get 1 milliliter in another test tube, add 2N HCl, water-bath heating hydrolysis 5 hours obtains the outer holosaccharide of born of the same parents.
2. the extraction of intracellular polyse:
Pulverize mycelium, sieve (80-90 order), poach extracts 3-4 time, merging extract, 70 ℃ of vacuum concentration be to original volume 1/4, below identical with the extraction exocellular polysaccharide, obtain pure intracellular polyse.
3. active component initial analysis (with the active polysaccharide is example, Biostat-MD type 10L automatic fermenter)
(1), polysaccharide accumulation
Table 1 ferments separately and the polysaccharide of mixed fungus fermentation is accumulated
Intracellular polyse (%)
Prepared from coriolus versicolor mycelium (fermenting 3 days) 14.90
Tricholoma matsutake mycelium (fermenting 3 days) 4.95
Mix bacterium mycelium (fermenting 3 days) 14.90
Exocellular polysaccharide (g/l)
Prepared from coriolus versicolor mycelium (fermenting 3 days) 13.15
Tricholoma matsutake mycelium (fermenting 3 days) 5.15
Mix bacterium mycelium (fermenting 3 days) 12.18
(2), the initial analysis of active polysaccharide kind and structure
1) analytical method of exocellular polysaccharide in the zymotic fluid: carry out purifying by ethanol precipitation deproteinization, degreasing, decolouring, desalination, separate with the DEAE cellulose column, obtain exocellular polysaccharide, adopt gel chromatography further to carry out fractionated, measure relative molecular weight, functional group, various glucosides and the glycosidic bond of polysaccharide with high-pressure liquid phase, infrared chromatography and nuclear magnetic resonance apparatus.
2) analytical method of intracellular polyse in the mycelium: mycelium hot water extracting, alcohol are analysed, dialysis, SephadexG-75 column chromatography purification, still adopt gel chromatography further to carry out fractionated, measure relative molecular weight, functional group, various glucosides and the glycosidic bond of polysaccharide with high-pressure liquid phase, infrared chromatography and nuclear magnetic resonance apparatus.
3) analysis result:
The initial analysis of rainbow conk [Polyporus versicolorL.:Fr] active polysaccharide kind and structure:
Mainly be the compound of polysaccharide-protein and polysaccharide with β-(1 → 3), β-(1 → 4), β-(1 → 6) glycosidic bond and branch.
The initial analysis of matsutake [Tricholoma matsutake (s Ito.et Imai) sing] active polysaccharide kind and structure:
Mainly be GLSP
2GLSP
3The isoreactivity polysaccharide, molecular weight 12790-14200 contains β-(1 → 3) β-(1 → 6) and β-(1 → 4) β-(1 → 6) glycosidic bond
Mix the initial analysis of bacterium (rainbow conk [Polyporus versicolorL.:Fr] matsutake [Tricholoma matsutake (s Ito.etImai) sing]) active polysaccharide kind and structure:
Except that the part active polysaccharide of rainbow conk, matsutake, the polysaccharide of finding to exist high branch degree with have polyalcohol group and the structure that β-(1 → 3) main chain links to each other, its content is greater than about 20%.
Analysis result is tentatively thought:
1) active polysaccharide in all kinds of mycelium and the zymotic fluid is basic identical on the structure, just the part difference of relative molecular weight and glucosides.
2) change the The Nomenclature Composition and Structure of Complexes of polysaccharide with matsutake in various degree for the mixed fungus fermentation of yeast-like fungi altogether.
3) combination of glycosidic bond is relevant between the pharmacologically active of polysaccharide and monose.
4) pharmacologically active of polysaccharide is relevant with the branch degree of polysaccharide.
Embodiment 3-5 has illustrated the biologically active of mixed fungus fermentation product and the reinforced effects of immunization.
Embodiment 3
The cancer resistant effect that mixes the outer active polysaccharide of born of the same parents
By being suppressed experimental result, culture in vitro people liver cancer SMMC-7721 cell proves that mix exocellular polysaccharide, cancer suppressing ratio (95.17%) compares P＜0.05 with single bacterium exopolysaccharide cancer suppressing ratio (rainbow conk 80.32%, matsutake 80.91).
(1), method
With the RPMI-1640 culture fluid of people's liver cancer SMMC-7721 cell → 0.25% trypsinization → 10% calf serum (FCS) of logarithmic phase growth suspend, counting → cell concentration 1-5 * 10
5/ ml → inoculation 24 well culture plates (every hole 2ml) → 37 ℃ of 5%CO
2Culture fluid → RPMI-1640 washing amount → (every kind of exocellular polysaccharide is established four holes to add exocellular polysaccharide (300 μ g/ml) respectively to cultivate 24h → abandon, if there are not four holes of RPMI-1640 culture fluid of exocellular polysaccharide, four holes of no exocellular polysaccharide 5%FCS RPMI-1640 culture fluid are contrast) → cultivate 24h → every hole to add
3Stop to cultivate → remove supernatant → 0.25% trypsinization → cell behind H-TdR74KBq → 4h to collect respectively on the 49 type filter paper → trichloroacetic acid, the absolute ethyl alcohol processing → scraps of paper dry → go in the scintillating disc → add scintillation solution → two pass liquid scintillation counter count pulse number/min (cpm value).
(2), result
1, three kinds of exocellular polysaccharide (rainbow conk-agaric pilose mixed fungus, rainbow conk, matsutake) cpm values and control group RPMI-1640 culture fluid cpm value all have utmost point significant difference P＜0.01.
Table 2 exocellular polysaccharide is to the SMMC-7721 cell
3The influence of H-TdR74KBq incorporation
Handle 5%FCS RPMI-300 μ g and mix 300 μ g clouds, 300 μ g matsutakes
The outer many exocellular polysaccharides of the outer many sesames born of the same parents of 1640 mycetocytes
Sugar sugar
CPM 3304±209 4316±398 2021?± 2345?± 2302±39
**
43
** 56
**
Significance (p) P＜0.01 P＜0.01 P＜0.01
2, rainbow conk-agaric pilose mixed fungus exocellular polysaccharide is remarkable difference P＜0.05 with rainbow conk exocellular polysaccharide, matsutake exocellular polysaccharide cpm value respectively.
(3) conclusion
1. explanation rainbow conk-agaric pilose mixed fungus, rainbow conk, three kinds of exocellular polysaccharides of matsutake all have obvious inhibition culture in vitro people liver cancer SMMC-7721 cell
3H-TdR mixes, the tool antitumaous effect.
2. illustrate simultaneously that the cancer resistant effect remarkable (P＜0.05) of rainbow conk-agaric pilose mixed fungus exocellular polysaccharide is higher than rainbow conk exocellular polysaccharide, the matsutake exocellular polysaccharide of single bacterium.
3. to the analytical test of the kind of polysaccharide, structure, ratio, disclose with matsutake for the polysaccharide of the new high branch degree that produces the rainbow conk-agaric pilose mixed fungus exocellular polysaccharide of yeast-like fungi altogether with to have the polysaccharide of polyalcohol group with β-structure that (1 → 3) main chain links to each other be one of reason of cancer resistant effect enhancing from molecular level.
Cancer resistant effect strengthen former two be synergy between active substance, complementation, synergistic result therefore.
5. illustrate with matsutake to being total to the mechanism that yeast-like fungi changes product bioactive functions and sensory properties from the cell biology and the molecular biological degree of depth.
Embodiment 4 glossy ganodermas-agaric pilose mixed fungus is the health drink immunoloregulation function of ferment altogether
Glossy ganoderma-agaric pilose mixed fungus altogether health drink the mouthfeel of ferment is pure and fresh, the pleasant aroma, and scalable crowd drinks quantity-unlimitingly, possesses the standard and the feature of food fully.Testing result by Shanghai Medical Univ's food toxicity and health food Function detection center proves:
1) influence and the control group to mouse internal organs/body weight ratio compares P＜0.05.
2) influence and control group comparison P＜0.05 that transform of the mouse lymphocyte that ConA is induced.
3) influence and the control group to the NK cytoactive compares P＜0.05.
4) influence and the control group to serum hemolysin compares P＜0.05.
5) Turnover of Mouse Peritoneal Macrophages is engulfed relatively P＜0.05 of chicken red blood cell experimental and control group.
Experimental result show glossy ganoderma-agaric pilose mixed fungus altogether the health drink of ferment to experimental mice lymphocyte transformation ability, NK cytoactive, serum hemolysin level, and peritoneal macrophage is engulfed percentage and phagocytic index all obviously raises.According to the evaluation criterion glossy ganoderma-agaric pilose mixed fungus of defined in " the health food function assessment assessment process and the method for inspection " altogether the health drink of ferment have extraordinary immunoregulation effect.
(1), method
Use the function assessment assessment process and the method for inspection of health food
1, internal organs/weight ratio pH-value determination pH: when experiment finishes, slaughter animal, take out internal organs, weigh behind the rejecting connective tissue, calculate internal organs/weight ratio.
2, the ConA mouse spleen lymphocyte conversion test of inducing: select the mtt assay in " health food function assessment assessment process and method " for use.
3, the NK cytoactive is measured: select isotope in " health food function assessment assessment process and method " for use
3The HTdR determination method.
4, the mensuration of serum hemolysin: select the blood clotting method in " health food function assessment assessment process and method " for use.
5, Turnover of Mouse Peritoneal Macrophages is engulfed the chicken red blood cell test: by the method for regulation in " health food function assessment assessment process and method ".
(2), result
1, glossy ganoderma--matsutake drinks is to the influence of mouse internal organs/body weight ratio
Table 3 glossy ganoderma-matsutake drinks is to the influence of mouse spleen, thymus gland/body weight ratio (X ± SD)
Dosage, (ml/Kg.bw) thing number, dosage group 83.3 10 7.87 ± 2.55 1.93 ± 0.07 high dose group 250.0 10 12.12 ± 3.82 in (only) spleen/weight ratio thymus gland/weight ratio control group 10 7.85 ± 2.20 1.93 ± 0.09 low dose group 25.0 10 5.45 ± 1.28 1.96 ± 0.07
*2.03 ± 0.13
*Compare p＜0.05 with control group
Through variance analysis and statistical procedures,, obvious significant difference p＜0.05 is arranged by the visible high dose group spleen/weight ratio of table 1.
2. the influence that the mouse spleen lymphocyte that glossy ganoderma--matsutake drinks is induced ConA transforms
The influence that the mouse spleen lymphocyte that table 4 glossy ganoderma-matsutake drinks is induced ConA transforms (X ± SD)
Dosage group 83.3 10 0.325 ± 0.013 in dosage (ml/Kg.bw) number of animals (only) lymphopoiesis ability (OD) control group 10 0.295 ± 0.018 low dose group 25.0 10 0.296 ± 0.036
*High dose group 250.0 10 0.429 ± 0.036
*
*Compare p＜0.05 with control group.
Each treated animal represents with optical density value that through the lymphproliferation response that ConA induces the result shows glossy ganoderma-matsutake function drink p＜0.05.Illustrate that glossy ganoderma-matsutake drinks has the promotion function to the lymphopoiesis of mouse.
3, glossy ganoderma--the matsutake drinks is to the influence of NK cytoactive
Table 5 glossy ganoderma-matsutake drinks is to the influence of NK cytoactive (dosage group 83.3 10 66.7 ± 8.1 in dosage (ml/Kg.bw) number of animals (only) NK cytoactive (0/0) control group 10 48.2 ± 15.1 low dose group 25.0 10 53.6 ± 9.0 of X ± SD)
*High dose group 250.0 10 68.2 ± 10.7
*
*Compare p＜0.05 with control group
The NK cytoactive of the visible height of table 5, middle dosage group mouse is than control group height, and there was a significant difference, illustrates that glossy ganoderma-matsutake drinks has the function that improves the NK cells in mice activity.
4, glossy ganoderma--the matsutake drinks is to the influence of serum hemolysin
Table 6 glossy ganoderma-matsutake drinks is to the influence of serum hemolysin (dosage group 83.3 10 111.4 ± 22.3 high dose group 250.0 10 126.4 ± 14.5 in dosage (ml/Kg.bw) number of animals (only) antibody product control group 10 94.8 ± 9.2 low dose group 25.0 10 103.4 ± 20.1 of X ± SD)
*
*Compare p＜0.05 with control group
The level of mouse serum hemolysin after sensitization can be checked by sheep cell condensation degree, and the height of its antibody titer can partly reflect the humoral immunity of organism ability.To finding in each dosage group mice serum hemolysin mensuration, the serum hemolysin of each dosage group all is higher than control group and increases and rising gradually with dosage, through variance analysis and statistical procedures, the antibody product of high dose group illustrates the obviously humoral immune function of enhancing body of glossy ganoderma-matsutake drinks apparently higher than control group, p＜0.05.
5, Turnover of Mouse Peritoneal Macrophages is engulfed the chicken red blood cell experiment
What table 7 was respectively organized Turnover of Mouse Peritoneal Macrophages engulfs percentage and phagocytic index (dosage (ml/Kg.bw) number of animals (only) phagocytic rate (%) phagocytic index control group 10 16.9 ± 2.0 0.25 ± 0.03 low dose group 25.0 10 32.1 ± 2.9 of X ± SD)
*0.51 ± 0.05
*Middle dosage group 83.3 10 49.3 ± 9.4
*0.66 ± 0.10
*High dose group 250.0 10 66.8 ± 11.7
*0.79 ± 0.108
*
*Compare p＜0.05 with control group.
The visible glossy ganoderma of the table 7-basic, normal, high dosage group of matsutake drinks, Turnover of Mouse Peritoneal Macrophages engulf percentage and phagocytic index all apparently higher than control group P＜0.05.Drinks has improved the phagocytic activity of Turnover of Mouse Peritoneal Macrophages significantly, but glossy ganoderma-matsutake drinks enhancing body non-specific immune function is described.
(3) conclusion
1, the decision principle as a result of basis " the function assessment assessment process and the method for inspection of health food ": in cellular immunity, humoral immunity, monokaryon-macrophage and NK cell function detect, if any the Function detection that (contains two) more than two positive as a result, promptly decidable this tried thing and had immunoregulation effect.Glossy ganoderma--the matsutake drinks is studied at this, in the Function detection, more than four all be positive, so can think to have immunoregulation effect.
2, the phagocytic function of macrophage not only has non-specific immune function, and can also engulf, old and feeble in the cracking body, dead cell and abnormal cell, promptly have rubbish, delaying senility function in the body of removing.The basic, normal, high dosage group of glossy ganoderma-matsutake drinks, Turnover of Mouse Peritoneal Macrophages engulf percentage and phagocytic index all apparently higher than control group P＜0.05, and increase and phagocytic activity increases gradually with dosage.As long as glossy ganoderma-matsutake drinks is described to be drunk low dosage and promptly can obviously improve the macrophage phagocytic ability.
3, the NK cell claims that natural killer cell is directly from marrow and lymphocyte with NK cell, not only have immunologic function but also energy killing tumor cell, play important immunosurveillance in vivo, so can think tentatively that glossy ganoderma-matsutake drinks has the prevention function of tumor.
Embodiment 5: mix bacterium hypha powder buccal tablet antifatigue effect
It is tasty and refreshing to mix bacterium hypha powder buccal tablet, and delicate fragrance is old, few all suitable.
To mix the bacterium hypha powder is the buccal tablet of active function composition, carries out anoxia enduring, antifatigue effect mensuration.The result shows, but mixes bacterium hypha powder buccal tablet significant prolongation mouse swimming time and the time-to-live under anoxia condition.Mouse hypoxia endurance test death time and control group be P＜0.01 relatively, and difference is extremely remarkable; Mouse swimming test death time and control group compare, P＜0.01, and difference is extremely remarkable; Improve the aerobic capacity of body, reduce blood lactic acid after the mouse strenuous exercise, compare P＜0.01 with control group, difference is extremely remarkable; Blood urea nitrogen increment and control group compare, P＜0.01, and difference is extremely remarkable; Reduce the consumption of hepatic glycogen, compare P＜0.01 with control group, difference is extremely remarkable.Illustrate that mixing bacterium hypha powder buccal tablet has the body of raising function, the effect of enhancing body anoxia enduring, antifatigue.
(1), method
1. animal subject
Cleaning level SD mouse inbred lines, 18-22g, male, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides
2, for test agent
Only contain this product major ingredient part, do not add other carbohydrates as yet, fungi polysaccharide content reaches 878.7mg/L.
3, basal feed
Provide by Chinese Academy of Sciences's Shanghai Experimental Animal Center
4, experimental animal grouping
Mouse was divided into 4 groups at random after 2 day laundering period, 10 every group, wherein two groups is control group, and basal feed is fed, free drinking pure water, and two groups is experimental group in addition, mixes bacterium hypha powder buccal tablet and feeds free drinking pure water.Experiment periods is 30 days, carries out the test of anoxia enduring, antifatigue.
5, hypoxia endurance test: the method for regulation in " the health food function assessment assessment process and the method for inspection "
6, anti-fatigue test: the method for regulation in " the health food function assessment assessment process and the method for inspection "
7, blood lactic acid is measured: the parazon colorimetric method
8, serum urea nitrogen determination: DAM method
9, hepatic glycogen is measured: anthrone method
(2), result
1, the edible comparison that mixes bacterium hypha powder buccal tablet and basal feed amount of mouse
Table 8 mouse is edible to mix comparison (group number of animals (only) time of x ± s) (my god) daily mean intake control group 10 30 32.9 ± 1.32 experimental group 10 30 37.4 ± 2.01 of bacterium hypha powder buccal tablet and basal feed amount
*
*P＜0.05 significant difference.
Table 8 as seen, mouse is edible to mix bacterium hypha powder buccal tablet and the basal feed amount has notable difference.This may be that the food back is comfortable because mixed bacterium hypha powder buccal tablet mouthfeel is suitable.
2, mix of the influence of bacterium hypha powder buccal tablet to the mouse body weight
Table 9 mixes bacterium hypha powder buccal tablet to the influence of mouse body weight (group number of animals (only) experimental period of x ± s) (day) the last weight of starting weight (gram) (gram) average weight gain (gram) control group 10 30 18.24 ± 0.61 28.7 ± 0.88 10.47 ± 0.69 experimental group 10 30 18.33 ± 0.65 30.03 ± 0.3 11.70 ± 0.83
It is more variant that as seen table 9 drinks the mouse weight increase and the control group of L-S drinks, but no significant difference.
3, mix of the influence of bacterium hypha powder buccal tablet to the mouse hypoxia-bearing capability
The table 10 mouse hypoxia endurance test death time other number of animals of comparative group (only) experimental period (my god) death time (second) control group 10 30 16.33 ± 1.46 experimental group 10 30 22.83 ± 1.10
*
*P＜0.01, difference are extremely remarkable
Anoxic is a kind of pessimal stimulation to body, influences the various metabolism of body, table 10 as seen, the experimental mice hypoxia-bearing capability is compared with control group, difference is (P＜0.01) extremely significantly.Show that mixing bacterium hypha powder buccal tablet can improve the hypoxia-bearing capability of mouse.
4, mix of the influence of bacterium hypha powder buccal tablet to the mouse anti-reflecting fatigue ability
1) swimming of duration
The table 11 mouse swimming test death time other number of animals of comparative group (only) experimental period (my god) death time (branch) control group 10 30 115.00 ± 9.04 experimental group 10 30 126.60 ± 4.99
*
*P＜0.01, difference are extremely remarkable.
Mix bacterium hypha powder buccal tablet and can prolong the swimming death time of mouse, illustrate that body is had the anti-fatigue ability function.
5, mix of the influence of bacterium hypha powder buccal tablet to mouse blood lactic acid
The other number of animals of comparative group (only) experimental period of table 12 mouse blood lactic acid (my god) blood lactic acid (mg/100ml) control group 10 30 222.52 ± 13.43 experimental group 10 30 196.67 ± 12.16
*
*P＜0.01, difference are extremely remarkable.
Content explanation glucolytic degree in the oxygen free condition lower body of blood lactic acid is to estimate antifatigue biochemical indicator commonly used.By table 12 as seen, experimental mice is after swimming, and the accumulation of blood lactic acid is starkly lower than control group in the body.P＜0.01, difference are extremely remarkable.This explanation mixes the ability that bacterium hypha powder buccal tablet can increase the body aerobic metabolism.
6, mix of the influence of bacterium hypha powder buccal tablet to the mice serum urea nitrogen
The other number of animals of the comparative group of table 13 mice serum urea nitrogen (only) experimental period (my god) serum urea nitrogen (mg/100ml) control group 10 30 84.70 ± 4.46 experimental group 10 30 73.78 ± 4.73
*
*P＜0.01, difference are extremely remarkable.
The content of serum urea nitrogen can illustrate nitrogen substance katabolism situation in the body, also is sensitive index estimating body physical work load ability to bear under specific condition.This experimental result, the experimental group serum urea nitrogen is starkly lower than control group.(P＜0.01) illustrates and mixes the decomposition that bacterium hypha powder buccal tablet can slow down nitrogen substance after the special manual labor.
7, mix of the influence of bacterium hypha powder buccal tablet to the Mouse Liver glycogen
The other number of animals of the comparative group of table 14 Mouse Liver glycogen (only) experimental period (my god) hepatic glycogen (mg/100g) control group 10 30 177.42 ± 8.85 experimental group 10 30 205.27 ± 8.51
*
*P＜0.01, difference are extremely remarkable.
As seen from Table 14, experimental mice is after swimming, and hepatic glycogen is apparently higher than control group (P＜0.01) in the body, prompting mixes the storage level that bacterium hypha powder buccal tablet can increase hepatic glycogen, reduce the consumption of glycogen after the violent manual labor, thereby have the enhancement physical efficiency, strengthen the effect of endurance, antifatigue.
(3), conclusion
1, anoxic is a kind of pessimal stimulation to body, influences the various metabolism of body, influences the oxidation energy supply of body especially, finally can cause the major organs such as the heart, brain of body to lack, the oxygen energy supply is not enough and dead.By table 10 as seen, the experimental mice hypoxia-bearing capability is compared with control group, and death time difference is (P＜0.01) extremely significantly.Illustrate that the L-S drinks can improve the hypoxia-bearing capability of mouse, the energy enhancing body is resisted the function of this poor environment of anoxic.
2, physical efficiency is the adaptive capacity of body to physical exertion and motion.Physical exertion influences key reaction in two basic physiological, biochemical pathway to body, i.e. the adjusting and the adaptation of each system's physiological function of energy metabolic exhaustion and body.With regard to energy, mainly decompose rapidly during muscle activity energy is provided by the ATP among the myocyte, and the additional main of ATP leans on the anerobic glycolysis of glycogen, glucose to generate the interior sugar of mitochondria under release energy in the lactic acid process synthetic ATP and the aerobic situation, fatty acid and the synthetic ATP of amino-acid oxidase decomposition produce power, and CP decomposes and synthesizes ATP in the muscle in addition.Because mainly obtain energy during physical exertion by glycolysis.When glycogen was consumed in a large number, the body mobility just reduced, and the accumulation of glucolytic product one lactic acid can cause fatigue.Absorb blood sugar when consuming muscle glycogen from blood, the latter can be decomposed by hepatic glycogen, is replenished and keeps blood sugar level.In case flesh, hepatic glycogen are consumed in a large number, then blood sugar decline can cause central nervous system energy supply deficiency, thereby causes the generation of whole body fatigue, and physical efficiency descends.
By table 11～table 14 as seen, in the experiment of a series of antifatigues, the result is all the positive, and difference extremely significantly (P＜0.01).By table 12 as seen, experimental mice is after swimming, and the accumulation of blood lactic acid is starkly lower than control group in the body.The results of analysis of variance P＜0.01, difference are extremely remarkable.Illustrate that the L-S drinks can increase aerobic capacity, promote lactic acid metabolism.As seen from Table 14, experimental mice is after swimming, hepatic glycogen is apparently higher than control group (P＜0.01) in the body, prompting mixes the storage level that bacterium hypha powder buccal tablet can increase hepatic glycogen, the increase that hepatic glycogen is stocked can be kept the blood sugar level of long period, for body provides more energy, reduces the consumption of glycogen after the violent manual labor, thereby have the enhancement physical efficiency, strengthen the effect of endurance, antifatigue.
3, the function assessment assessment process and the method for inspection of experimental result basis＜health food) decision principle as a result: this is tried the positive explanation of anoxia enduring experiment thing and is had resisting oxygen lack, in the antifatigue effect, if the exercise test that (contains) more than and the biochemical indicator that (contains two) more than two positive, can judge that this is tried thing and has antifatigue effect.In this research Function detection, it is extremely remarkable to mix bacterium hypha powder buccal tablet anoxia enduring experiment (P＜0.01) difference that is positive, two exercise tests and three biochemical indicators all are positive in the anti-fatigue test, have anoxia enduring, anti-fatigue effect so mix bacterium hypha powder buccal tablet.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a medium that is used for the matsutake fermentation is characterized in that this medium contains the 0.01-50mg/kg cobalt element.
2. medium as claimed in claim 1 is characterized in that, contains the 0.1-5mg/kg cobalt element in this medium.
3. medium as claimed in claim 1 is characterized in that, also contains the fumaric acid that concentration is 0.005-0.5wt% in this medium.
4. mixed fungus fermentation method is characterized in that the method comprising the steps of:
(a) prepare matsutake and the fermentation seed liquid that is total to the yeast-like fungi strain respectively; With
(b) matsutake that makes in the step (a) and the fermentation seed liquid that is total to the yeast-like fungi strain are inoculated in fermentation tank, under the condition that is fit to mixed bacteria growing of 0.01-50mg/kg cobalt element, mix the bacterium deep layer and cultivate matsutake and yeast-like fungi strain altogether.
5. method as claimed in claim 4 is characterized in that, contains the 0.1-5mg/kg cobalt element in the described medium, and the described suitable bacteria growing condition of mixing comprises that also concentration is the fumaric acid of 0.005-0.5wt%.
6. method as claimed in claim 4 is characterized in that, described another kind yeast-like fungi is altogether selected good strains in the field for seed from rainbow conk, glossy ganoderma or its combination.
7. method as claimed in claim 4 is characterized in that, described fermentation temperature is 25-37 ℃, and pH is 4.5-5.5, and incubation time is 12 hours-10 days.
8. method as claimed in claim 4 is characterized in that, this method also comprises step:
(c) intracellular polyse in the extraction mycelia and the exocellular polysaccharide in the zymotic fluid.
9. polysaccharide, it is characterized in that, this polysaccharide is the intracellular polyse and/or the exocellular polysaccharide of agaric pilose mixed fungus fermentation, and has content and account for the fermentation exocellular polysaccharide and the polysaccharide of the high branch degree of intracellular polyse total amount more than 20% with polyalcohol group and β-structure that (1 → 3) main chain links to each other that obtain; Described polysaccharide is to prepare by following steps:
(a) prepare matsutake and the fermentation seed liquid that is total to the yeast-like fungi strain respectively;
(b) matsutake that makes in the step (a) and the fermentation seed liquid that is total to the yeast-like fungi strain are inoculated in fermentation tank, under the condition that is fit to mixed bacteria growing of 0.01-50mg/kg cobalt element, mix the bacterium deep layer and cultivate matsutake and yeast-like fungi strain altogether;
(c) intracellular polyse in the extraction mycelia and the exocellular polysaccharide in the zymotic fluid.
10. a composition is characterized in that, said composition contains carrier and is selected from down the active base-material of group: the mixed bacterium mycelium of agaric pilose mixed fungus fermentation, mixed fungus fermentation liquid, pure mixed fungus polysaccharide, pure mixed fungus glycoprotein, or its mixture.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191297A (en) * | 2011-04-06 | 2011-09-21 | 黑龙江省轻工科学研究院 | Method for making tricholoma matsutake polysaccharides |
| CN102771857A (en) * | 2012-07-20 | 2012-11-14 | 黄晓青 | Truffle health-care drink prepared through liquid submerged fermentation and preparation method of truffle health-care drink prepared through liquid submerged fermentation |
| CN104025904A (en) * | 2014-05-27 | 2014-09-10 | 中国林业科学研究院森林生态环境与保护研究所 | Culturing method for tricholoma matsutake mycelia and special device thereof |
| CN110353261A (en) * | 2019-08-29 | 2019-10-22 | 浙江省医学科学院 | A kind of sesame young pilose antler hypha powder and its preparation method and application |
| CN115786129A (en) * | 2022-06-16 | 2023-03-14 | 中国医学科学院药用植物研究所 | Fermentation method for medicinal planting of space No.1 ganoderma lucidum new strain |
-
2002
- 2002-08-14 CN CN 02136484 patent/CN1217570C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191297A (en) * | 2011-04-06 | 2011-09-21 | 黑龙江省轻工科学研究院 | Method for making tricholoma matsutake polysaccharides |
| CN102771857A (en) * | 2012-07-20 | 2012-11-14 | 黄晓青 | Truffle health-care drink prepared through liquid submerged fermentation and preparation method of truffle health-care drink prepared through liquid submerged fermentation |
| CN104025904A (en) * | 2014-05-27 | 2014-09-10 | 中国林业科学研究院森林生态环境与保护研究所 | Culturing method for tricholoma matsutake mycelia and special device thereof |
| CN110353261A (en) * | 2019-08-29 | 2019-10-22 | 浙江省医学科学院 | A kind of sesame young pilose antler hypha powder and its preparation method and application |
| CN115786129A (en) * | 2022-06-16 | 2023-03-14 | 中国医学科学院药用植物研究所 | Fermentation method for medicinal planting of space No.1 ganoderma lucidum new strain |
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