CN106191173A - A kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin - Google Patents

A kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin Download PDF

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CN106191173A
CN106191173A CN201610578793.4A CN201610578793A CN106191173A CN 106191173 A CN106191173 A CN 106191173A CN 201610578793 A CN201610578793 A CN 201610578793A CN 106191173 A CN106191173 A CN 106191173A
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cordyceps militaris
cordycepin
link
fermentation
culture
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张楠
段辉国
唐正义
黎勇
徐洁
熊茂
雷雨田
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Neijiang Normal University
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Abstract

The invention discloses a kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin, including actication of culture, Cordyceps militaris spawn makes, Cordyceps militaris (L.) Link. is cultivated, and liquid spawn uses spore concentration 3.0 × 10 during making8The female kind made when CFU/mL or 45 truffles cake is as the primary seed solution in Cordyceps militaris spawn amplification culture；The fermentation medium used in Cordyceps militaris (L.) Link. incubation comprises glucose 25g/L, Rhizoma Solani tuber osi 100g/L, fish peptone 18g/L, (NH4)2SO40.8g/L, KH2PO41.0g/L, MgSO4·7H2O 0.5g/L, dried silkworm chrysalis meal 5.0g/L, VB118mg/L, water 1L.Improve 74.51% by cordyceps militaris biomass of the present invention, Cordycepin total output improves 238.52%.The present invention has significant facilitation to cordyceps militaris biomass and produced from Cordyceps militaris L cordycepin ability.

Description

A kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin
Technical field
The present invention relates to technical field of fungal fermentation, specifically a kind of Cordyceps militaris fermenting and producing Cordycepin that improves produces The method of amount.
Background technology
Cordyceps militaris (L.) Link. is Ascomycotina, Ergota Zoopagales, Clavicipitaceae, the type sepecies of Cordyceps.Formal name used at school is Cordycepsmilitaris, has another name called Cordyceps militaris (L.) Link., Cordyceps militaris etc., has some areas also known as to leap up Cordyceps, worldwide distribution Natural resources quantity is little.Cordycepin is (especially ucleosides) main active in Cordyceps and Cordyceps militaris (L.) Link., is also First nucleoside antibiotics separated from fungus.The medicine of a kind of natural origin of cordycepin, has in traditional Chinese medical science medical knowledge The same function mended with two-ways regulation total balance of the body of negative and positive that Cordyceps is the same；It at hepatoprotective, protect in terms of kidney, lung moistening due to composition Purer effect is more preferable, and strongly invigorating QI and blood, the diseases such as present incurable dysmenorrhea, migraine, hyperosteogeny can be eliminated.From west Doctor's medical knowledge angle see cordycepin have antitumor, defying age, antibacterial, antiviral, immunomodulating, improve metabolism, remove from By multiple pharmacological effect such as bases, there is good potential applicability in clinical practice.The at present research of cordycepin is the most just becoming pharmaceutical chemistry, anti-ageing Always, an extremely active field in beauty treatment, field of health care products.The cultivation of Cordyceps militaris (L.) Link. has focused largely on the research of solid culture, For liquid culture relate to fewer, and the liquid culture related to yields poorly, it is impossible to meet the market demand.
Summary of the invention
It is an object of the invention to provide a kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin, to pupa Cordyceps Biomass and produced from Cordyceps militaris L cordycepin ability have significant facilitation.
For achieving the above object, the present invention provides following technical scheme:
A kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin, including actication of culture, Cordyceps militaris spawn Make, Cordyceps militaris (L.) Link. is cultivated, and liquid spawn uses spore concentration 3.0 × 10 during making8During CFU/mL or 4-5 truffles cake The female kind made is as the primary seed solution in Cordyceps militaris spawn amplification culture；The fermentation culture used in Cordyceps militaris (L.) Link. incubation Base comprises glucose sugar, Rhizoma Solani tuber osi, fish peptone, (NH4)2SO4、KH2PO4、MgSO4·7H2O, dried silkworm chrysalis meal, VB1And water.
As the further scheme of the present invention: in fermentation medium, the content of each composition is: glucose 25g/L, Rhizoma Solani tuber osi 100g/L, fish peptone 18g/L, (NH4)2SO40.8g/L, KH2PO41.0g/L, MgSO4·7H2O 0.5g/L, dried silkworm chrysalis meal 5.0g/L, VB118mg/L, water 1L.
As the further scheme of the present invention: Cordyceps militaris spawn uses NS-810 bacterial strain.
Compared with prior art, the invention has the beneficial effects as follows:
After being optimized by the present invention, cordyceps militaris biomass and Cordycepin total output will be more much higher than be not optimised, optimize Rear cordyceps militaris biomass improves 74.51%, and Cordycepin total output improves 238.52%.The present invention is to cordyceps militaris biomass Significant facilitation is had with produced from Cordyceps militaris L cordycepin ability.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, prepare and produce Cordycepin in order to obtain Cordyceps militaris one-level kind with liquid fermentation Good technique, with Cordyceps militaris (L.) Link. Cordyceps militaris (L) link.NS-810 bacterial strain as strain, by inoculum concentration (spore Concentration and bacterium cake quantity) investigation, explore different spore concentration and bacterium cake number make the shadow of effect to Cordyceps militaris one-level kind Ring；By single single-factor and orthogonal test, optimize Cordyceps militaris (L.) Link. submerged fermentation and produce the optimal medium of cordycepin.Specifically describe such as Under:
1 materials and methods
1.1 strains and culture medium
Cordyceps militaris (L.) Link. [Cordyceps militaris (L) link.] is isolatable from Jiulong County watt, Tibetan Autonomous Prefecture of Garze, Sichuan Province The cordyceps fresh specimen that Hui Shan nature reserve area gathers, named Cordyceps militaris (L.) Link. NS-810, it is stored in high in Sichuan Province through domestication School featured agriculture the Study on Resources with utilize key lab.
Strain activation and culture base (PDA): glucose 20g, Rhizoma Solani tuber osi 200g, fish peptone 5g, water 1L；Cordyceps militaris mother's kind Culture medium: glucose 20g, Rhizoma Solani tuber osi 200g, peptone 5g, KH2PO42g, MgSO4.7H2O 1.5g, water 1L, 112 DEG C of sterilizings 30min；Cordyceps militaris (L.) Link. fermentation basal medium: glucose 20g, Rhizoma Solani tuber osi 200g, peptone 10g, KH2PO42g, MgSO4.7H2O 1.5g, water 1L, 112 DEG C of sterilizing 30min.
1.2 reagent and instrument
Cordycepin standard substance are purchased from Sigma Co., USA；Fish peptone (technical grade) is purchased from Shanghai edge peptide biotechnology Company limited；Agilent company of the Agilent 1100 analytical type high performance liquid chromatograph U.S.；50L fermentation tank Jiangsu Feng Ze is biological Engineering equipment Manufacturing Co., Ltd.
1.3 method
1.3.1 the activation of strains tested
Card punch (diameter D=10mm) will be used to be connected to by Cordyceps militaris (L.) Link. fungus cake in PDA culture dish (15mL PDA culture medium) On centre, being inverted in 26 DEG C of growth cabinets cultivation 5d, 4 DEG C of Refrigerator stores are standby.
1.3.2 the optimization that prepared by Cordyceps militaris strain
(1) inoculation of the different Cordyceps militaris (L.) Link. fungus cakes impact on liquid spawn, with card punch outside activation Cordyceps militaris (L.) Link. thalline Edge takes bacterium cake, in the pure culture biscuits involvng inoculation of varying number to Cordyceps militaris kind culture medium: 2 pieces, 3 pieces, 4 pieces, 5 pieces, 6 pieces, 7 pieces；(2) NS-810 bacterial strain spore suspension is diluted, is counted by blood counting chamber under the microscope, and make a series of different Spore concentration gradient.Then Cordyceps militaris kind culture medium is transferred to following spore concentration: 2.0 × 105、2.0×106、2.0× 107、2×108、2.5×108、3×108、3.5×108、4×108CFU/mL；150r/min, 21 DEG C of shaken cultivation, from the 2nd day The shape of form, size, quantity and the fermentation liquid of liquid first class inoculum mycelium pellet after Continuous Observation varying number truffle inoculation in 5 days State changes, each process 3 repetition.
1.3.3 prepared by seed liquor
NS-810 bacterial strain spore suspension is diluted, is then 3 × 10 by spore concentration8CFU/mL or use card punch Obtain in Cordyceps militaris (L.) Link. fungus cake 4-5 block inoculation liquid mother culture media (liquid amount is 50mL/250mL), 140r/min, 21 DEG C of vibrations Cultivate 4d, then seed liquor accessed in fermentation shake flask (liquid amount is 50mL/250mL) by 10% (v/v) inoculum concentration, 140r/min, 21 DEG C of shaken cultivation 12d, each process 3 repetition.
13.4 inoculation submerged fermentationes
With forwarding in 500mL triangular flask with the inoculum concentration of 10%, 140r/min, 21 DEG C of shaken cultivation 4-5d are as fermentation tank Inoculum.Culture volume 40L in 50L fermentation tank, inoculum concentration 5%, fermentation temperature 24 DEG C, ventilation: 1.25m3/ h, stirring training Supporting 10d rotating speed is 140r/min, quiescent culture 2 days the most again.
1.4 assay method
Cordyceps militaris culture is centrifuged 10min in low speed Large Copacity Multi-pipe centrifugal machine 4000r/min and obtains mycelium, With distilled water flushing mycelium 3 times, dry to constant weight for 60 DEG C in drying baker, weigh；Cordycepin in mycelium and fermentation liquid Mensuration use HPLC chromatogram method, with reference in the progress of cordycepin in the Cordyceps militaris (L.) Link. that Liu Guijun etc. delivers in Food Science Method and be optimized, HPLC chromatogram condition: Extend-C18 (250mm × 4.6mm, 5 μm), flow phase: methanol: water (82: 18), flow velocity: 1.0mL/min, detects wavelength: 260nm, column temperature: 30 DEG C.Computing formula: Cordycepin total output (mg/L) =[outer Cordycepin quality (mg)+intracellular Cordycepin quality (mg) of born of the same parents]/fermentating liquid volume (L).Each process 3 repetition, Take its meansigma methods.
The optimization of 1.5 liquid fermentation medium formula
The following test is all with mycelial Biomass and the yield from cordycepin as inspection target, during each is tested Each process 3 repetition.
1.5.1 quick-acting carbon sources select
Glucose, sucrose, maltose, lactose, mannitol it is respectively adopted as quick-acting carbon sources in liquid fermentation medium (concentration of quick-acting carbon sources is 20g/L), other components are identical, to be not added with quick-acting carbon source with Cordyceps militaris (L.) Link. fermentation basal medium Cordyceps militaris (L.) Link. fermentation basal medium is as comparison.
1.5.2 slow carbon source selects
Liquid fermentation medium is respectively adopted Rhizoma Solani tuber osi (mass concentration is respectively 100g/L, 200g/L and 300g/L), (mass concentration is respectively 10g/L, 20g/ for Semen Maydis powder (mass concentration is respectively 10g/L, 20g/L, 30g/L) and soluble starch L, 30g/L) as slow carbon source, the quick-acting carbon sources in culture medium select the optimal quick-acting carbon sources filtered out, other components and pupa Chinese caterpillar fungus fermentation basal medium is identical, separately to be not added with the fluid medium of slow carbon source as comparison.Wherein, potato decortication is cut into Thin slice, boils 20min, in leaching filtrate adds culture medium.
1.5.3 organic nitrogen source selects
Cultivating, concentration is respectively adopted fish peptone, yeast leaching powder, beef extract, (mass concentration is respectively soybean cake powder 10g/L, 20g/L, 10g/L and 20g/L, nitrogen content is essentially identical) as organic nitrogen source, carbon source selects the optimal speed filtered out Effect and slow carbon source, other components are identical, to be not added with the culture medium of organic nitrogen source as comparison with fermentation basal medium.
The most inorganic nitrogen-sourced selection
It is respectively adopted NH in the fermentation medium4NO3、NH4Cl、(NH4)2SO4、NaNO3(mass concentration is respectively 0.6g/ L, 0.8g/L, 1.0g/L and 1.25g/L, nitrogen content is essentially identical) as inorganic nitrogen-sourced, alternative is the most organic with filter out Nitrogen source, the most quick-acting and slow carbon source, other components are identical with fluid medium, to be not added with inorganic nitrogen-sourced fluid medium work For comparison.
1.5.5 inorganic salt selects
It is separately added into 3 kinds of inorganic salt 2.0g/LKH in the fermentation medium2PO4、1.5g/LMgSO4.7H2O、2.0g/ LNaCl also carries out the nutrition limit assay of inorganic salt, inquires into the different inorganic salt and combinations thereof the impact on cordyceps militaris biomass. Combining the carbon source as fluid medium and nitrogen source using the optimal carbon and nitrogen sources filtered out, other components are identical with fluid medium, To be not added with the fluid medium of inorganic salt as comparison.
1.5.6 somatomedin selects
The most respectively add 2g/L dried silkworm chrysalis meal, 2g/L Semen Maydis starch, 2g/L L-Glu, 2g/L L-Cys, 0.5g/L adenine 20mg/L VB6、20mg/LVB1, 3mg/L NAA carry out the screening of somatomedin, other components are for filtering out Optimum carbon source, nitrogen source and inorganic salt, to be not added with the fluid medium of somatomedin as comparison.Wherein, dried silkworm chrysalis meal uses reel silk from cocoons Silk Pupa bombycis, through 60 DEG C of drying, pulverizer is pulverized, is boiled 30min, adds through leaching filtrate.
1.5.7 the orthogonal optimization of fermentation medium
On the basis of optimum carbon source, nitrogen source, inorganic salt and the somatomedin that above-mentioned assay optimization obtains, use L27 (313) Orthogonal Experiment and Design (table 1), to obtain the optium concentration of each composition in fermentative medium formula and they are to Cordyceps militaris (L.) Link. Bacteria biomass and the influence degree of product Cordycepin total output.
Table 1 factor and level design
1.5.8 the checking of liquid culture based formulas is optimized
Fermentation medium after optimization carries out submerged fermentation test with fermentation basal medium under identical condition of culture, Measuring yield of Cordycepin and Biomass in fermentation liquid, before and after comparing optimization, fermentation medium is to yield of Cordycepin and Biomass Impact, with checking optimize after fermentative medium formula.
2 results and analysis
2.1 prepared by Cordyceps militaris strain
Different Cordyceps militaris (L.) Link. fungus cake numbers and spore concentration suspension inoculation are shown in Table 2 to the impact of liquid spawn, table can obtain mycelia The quantity of ball increases with bacterium cake number and the increase of spore concentration, but the size of mycelium pellet is reducing, and seed liquor is by clarifying change Obtain muddy and start to be a bit sticky.Result shows: inoculation 4-5 truffles cake and spore concentration 3.0 × 108Mycelia during CFU/mL Ball particle size is uniform, and diameter is about 1.2mm, and quantity is about 850, and mycelium pellet color is faint yellow, is uniformly covered with triangular flask, Seed liquor clarification, transparent.From the side such as the quantity of mycelium pellet, size, uniformity coefficient, the clarity of seed liquor and the volume that can inoculate Face considers, selects 4-5 truffles cake or spore concentration 3.0 × 108The optimal inoculum density that CFU/mL plants as fermentation mother.
Table 2 inoculates different truffle number and the spore concentration impact on Cordyceps militaris strain
2.2 liquid fermentations produce the optimised process of Cordycepin
2.2.1 the quick-acting carbon sources in fermentation medium
Respectively using glucose, sucrose, maltose, lactose, mannitol as the quick-acting carbon sources of fermentation medium.Can from table 3 See that fermentation medium is all little without carbon source growth quantity of mycelium and Cordycepin total output, the carbon source life to Cordyceps militaris (L.) Link. is described Length plays very important effect, maximum with sucrose and the Cordycepin total output that glucose is quick-acting carbon source, respectively reaches 548.93g/L and 547.44g/L, from the standpoint of cost and fermenting and producing, selects 20g/L glucose sugar as fermentation medium Quick-acting carbon sources.
In table 3 fermentation medium, different quick-acting carbon sources are on Cordycepin total output and the impact of Biomass
Note: in table, different lower cases represent significant difference in the level of P≤0.05, and capitalization represents P≤0.01 level Upper significant difference.
2.2.2 the slow carbon source in fermentation medium
Fermentation medium adds the Cordycepin total output that 100g/L Rhizoma Solani tuber osi is quick-acting carbon source maximum, reach 644.08g/L.Therefore, select 100g/L Rhizoma Solani tuber osi as the slow carbon source of fermentation medium, select glucose and Rhizoma Solani tuber osi as multiple Close carbon source.
In table 4 fermentation medium, different slow carbon sources are on Cordycepin total output and the impact of Biomass
2.2.3 the organic nitrogen source in fermentation medium
Fermentation medium adds fish peptone, yeast powder, beef extract and soybean cake powder respectively as organic nitrogen source, right The impact of yield of Cordycepin and Biomass is shown in Table 5.When adding fish peptone in culture medium, Cordycepin total output is maximum, Reach 634.36g/L, select 10g/L fish peptone as the organic nitrogen source in fermentation medium.
In table 5 fermentation medium, different organic nitrogen sources are on Cordycepin total output and the impact of Biomass
Note: in table, different lower cases represent significant difference in the level of P≤0.05, and capitalization represents P≤0.01 level Upper significant difference.
2.2.4 inorganic nitrogen-sourced in fermentation medium
Fermentation medium adds NH respectively4NO3、NH4Cl、(NH4)2SO4、NaNO3As inorganic nitrogen-sourced, to Cordycepin The impact of yield and Biomass is shown in Table 6, adds (NH4)2SO4Time yield of Cordycepin and mycelial increment be above comparison. Therefore, 1.0g/L (NH is selected4)2SO4Inorganic nitrogen-sourced as fermentation medium.
In table 6 fermentation medium different inorganic nitrogen-sourced on Cordycepin total output and the impact of Biomass
Note: in table, different lower cases represent significant difference in the level of P≤0.05, and capitalization represents P≤0.01 level Upper significant difference.
2.2.5 the inorganic salt during base is supported in fermentation
Fermentation medium adds KH respectively2PO4、MgSO4·7H2O, NaCl as inorganic salt, selected by inorganic salt and Nutrition limit assay, can search out the optimal inorganic salts combination of yield of Cordycepin and Biomass.As can be seen from Table 7, 2.0g/L KH2PO4+1.5g/L MgSO4·7H2Cordycepin total output and the Biomass of O combination are the highest, are respectively 709.54mg/L and 22.37g/L.Therefore, 2.0g/L KH is selected2PO4+1.5g/L MgSO4·7H2O is as fermentation medium Component of inorganic salts.
Different inorganic salts in table 7 fermentation medium combine Cordycepin total output and the impact of Biomass
Note: in table, different lower cases represent significant difference in the level of P≤0.05, and capitalization represents P≤0.01 level Upper significant difference.
2.2.6 the somatomedin in fermentation medium
Fermentation medium adds different somatomedin respectively the impact of yield of Cordycepin and Biomass is shown in Table 8, Dried silkworm chrysalis meal, VB6, VB as can be seen from Table 51, L-Glu, NAA, adenine to Cordyceps militaris (L.) Link. NS-810 bacterial strain produce cordycepin ability There is certain facilitation.Wherein, dried silkworm chrysalis meal produces the strongest (the Cordycepin total output of Cordycepin ability to Cordyceps militaris (L.) Link. 907.64mg/L), VB1Take second place, reach 863.10mg/L, appropriate dried silkworm chrysalis meal and VB are described1The long-pending of Cordycepin can be advantageously promoted Tired.
Different somatomedin in table 8 fermentation medium are on Cordycepin total output and the impact of Biomass
Note: in table, different lower cases represent significant difference in the level of P≤0.05, and capitalization represents P≤0.01 level Upper significant difference.
2.2.7 the optimization of fermentative medium formula
Orthogonal experiments is shown in Table 9, uses SAS9.0 data analysis software to carry out variance analysis, and analysis result is shown in Table 10. From table 9-10 it can be seen that glucose, fish peptone, VB1, dried silkworm chrysalis meal and KH2PO4Impact on fermenting and producing Fungal biodiversity Reaching significant level, other 3 factors impact is the most notable.As can be drawn from Table 9, optimal level is A3B2C2D1E2F2G1H1, i.e. Portugal Grape sugar 25g/L, Rhizoma Solani tuber osi 100g/L, fish peptone 20g/L, (NH4)2SO40.6g/L, KH2PO41.5g/L, MgSO4· 7H2O1.0g/L, dried silkworm chrysalis meal 3.0g/L, VB118mg/L, water 1L；And the impact on fermenting and producing Cordycepin total output reaches aobvious The factor of work level is glucose, KH2PO4And dried silkworm chrysalis meal, optimal level is A3B2C1D2E1F1G2H1, i.e. glucose 25g/L, soil Bean 100g/L, fish peptone 18g/L, (NH4)2SO40.8g/L, KH2PO41.0g/L, MgSO4·7H2O 0.5g/L, dried silkworm chrysalis meal 5.0g/L, VB118mg/L, water 1L.
The orthogonal experiments that each component proportion of table 9 fermentation medium optimizes
Note: A: glucose/(g/L), B: Rhizoma Solani tuber osi/(g/L), C: fish peptone/(g/L), D:(NH4)2SO4/ (g/L), E: KH2PO4/ (g/L), F:MgSO4·7H2O/ (g/L), G: dried silkworm chrysalis meal/(g/L), H:VB1/(mg/L)。
The SAS that mycelium dry weight is affected by each component of table 10 fluid medium analyzes
2.2.8 the liquid culture checking optimizing fermentative medium formula to Cordyceps militaris (L.) Link. is used
Under Fermentations conditions, using the fermentative medium formula after optimizing, cordyceps militaris biomass and Cordycepin are total Yield will be more much higher than be not optimised, and after optimization, Biomass improves 74.51%, and Cordycepin total output improves 238.52%.Illustrate that the culture medium prescription after optimizing has significantly promotion to cordyceps militaris biomass and produced from Cordyceps militaris L cordycepin ability Effect.
Table 11 ferment basal medium with optimize after culture medium the result compare
3 discuss
During higher fungus submerged fermentation, the preparation of seed, culture medium and fermentation operation condition are three main shadows The factor of sound, wherein the preparation of seed is generation and the basis of secondary metabolite accumulation of Biomass.At present, liquid spawn is at pupa Being widely used in Cordyceps artificial culture, compared with traditional solid vaccination, the advantage of liquid spawn is to have production automatically Changing and intensive degree is high, shorten and send out the bacterium time, reduce strain pollution rate, strain production cost is low, and strain quality has and carries more greatly High.In the manufacturing process of liquid fungus strain especially one-level kind, the concentration of inoculating spores or the block number of bacterium cake are to seed The state of liquid has directly impact, and the state of seed liquor is the key factor determining fermenting and producing success or not.This Bright discovery is inoculating spores concentration 3.0 × 10 in 50mL first order seed culture fluid8When CFU/ml or bacterium cake are 4-5 block, mycelia The quantity of ball, size, uniformity coefficient, the clarity of seed liquor are all in optimal state, and have effective inoculum of maximum Long-pending, therefore, the female kind made during both inoculum densities is most suitable as the primary seed solution in Cordyceps militaris spawn amplification culture.
Cordyceps militaris (L.) Link. can well utilize monosaccharide, disaccharide, result of the present invention to also demonstrate that this point, the product cordycepin filtered out The optimal quick-acting carbon sources of fermentation medium are dextrose plus saccharose.Glucose is replaced by amylum hydrolysate of the sugar in the industrial production, One of raw material that starchy material is commonly used as microbiological industry, is amylum hydrolysate of the sugar through the sugar liquid obtained by saccharifying, its institute It is exactly glucose containing main component, from the standpoint of later industrialized production, selects glucose as submerged fermentation culture medium Carbon source.Additionally, added in fermentation medium training as slow carbon source by Rhizoma Solani tuber osi, the Biomass of Cordyceps militaris (L.) Link. can be effectively improved The yield of cordycepin outer with born of the same parents, makes cordycepin total output improve nearly 20%.Rhizoma Solani tuber osi leaching juice is readily available, and low cost and nutrition are richer Richness, can after fermentation the phase be that Cordyceps militaris (L.) Link. NS-810 bacterial strain provides sufficient carbon source, extend the process producing cordycepin.Therefore, Rhizoma Solani tuber osi, as the slow carbon source in this bacterial strain submerged fermentation culture medium, has reached reduce cost and improve the mesh of cordycepin output 's.
Fish peptone is fish protein enzymolysis or the short peptide chain of microbe fermentation method, micromolecule polypeptide (peptone nitrogen content is high) (small-molecular peptides content is higher than bone protein peptone) and various aminoacid etc., and the powdery product of amino nitrogen, wherein small-molecular peptides contains Amount, higher than bone protein peptone, has preferable water solublity, it is easy to microorganism absorbs, and can be that fermentable provides high-load high-quality organic Nitrogen source.Cordyceps militaris (L.) Link. is as entomogenous fungi, and animal proteinum is to its growth promoter advantageously.So the present invention selects fish peptone to make For nitrogen source, finding to be effectively improved the yield of cordycepin, fish peptone inexpensive and wide material sources, as fermentation medium Nitrogen source can be substantially reduced production cost, and this has important meaning for bacterial strain NS-810 at cordycepin industrialization producer mask Justice.It has also been found that, add inorganic nitrogen-sourced NH4 +Generation to cordycepin plays a driving role.Ammonium salt is a kind of cheap, effect The most inorganic nitrogen-sourced, NH4 +Being used for synthesizing glutamine by Cordyceps militaris (L.) Link. cell, glutamine serves as nitrogen in biosynthesis reacts Donor, be also the indirect precursor of nucleoside, and generate nucleoside, and cordycepin is the derivant of nucleoside, this is probably fermentation culture Middle interpolation NH4 +The reason of cordycepin content can be improved.
Present invention demonstrates that inorganic salt plays very important work during the growth of Cordyceps militaris (L.) Link. and the generation of Cordycepin With, add KH2PO4、MgSO4·7H2O all can be bigger raising Fungal biodiversity and yield of Cordycepin, wherein, KH2PO4To worm Lavendustin yield effect is notable, the KH of low concentration2PO4It is better than the KH of high concentration2PO4, this is probably high concentration KH2PO4Can cause Cytoplasmic osmotic pressure increases, thus suppresses Cordyceps militaris (L.) Link. secretion to produce Cordycepin；Magnesium is the activator of many enzymes and it can Affecting the oxidation of substrate and the synthesis of protein, this is probably MgSO4·7H2O promotes thalli growth and produces the former of cordycepin Cause.Add NaCl as then suppressing the growth of thalline and the generation of cordycepin during inorganic salt.
Pupa bombycis is the Main By product of Silk Industry, great nutritive value, containing rich in protein, fatty acid, vitamin (including vitamin A, vitamin B2, vitamin D and ergosterol etc.) and trace element etc..This test adds in the fermentation medium Adding dried silkworm chrysalis meal as somatomedin, result shows that dried silkworm chrysalis meal shows clear superiority in terms of Cordycepin synthesis accumulation, special The yield effect not being Cordycepin outer to born of the same parents is the most notable.It addition, the fat content in Pupa bombycis is the highest, account for dried silkworm chrysalis quality 25%-30%, the lixiviating solution surface flotation of dried silkworm chrysalis meal by a small amount of silkworm chrysalis oil, it both can promote cordyceps mycelium growth and The secretion of cordycepin, can play again the effect of froth breaking in submerged fermentation.VB1 as somatomedin to Cordyceps in fermentation liquid Element synthesis accumulation also has preferable facilitation.
The optimal deep layer that the present invention determines cordyceps militaris link bacterial strain NS-810 product cordycepin by single factor test and orthogonal test is sent out Ferment culture medium: glucose 25g/L, Rhizoma Solani tuber osi 100g/L, fish peptone 18g/L, (NH4)2SO40.8g/L, KH2PO41.0g/L, MgSO4·7H2O 0.5g/L, dried silkworm chrysalis meal 5.0g/L, VB118mg/L, water 1L.Using this culture medium, Cordyceps militaris (L.) Link. hypha biomass is about For 31.93g/L, the total output of pupa worm element is up to 1597.11mg/L, fermentation raw material wide material sources, low price, and fermentation liquid is clear Clear, steady quality, this produces Cordycepin and high-quality mycelium to industrialization large scale fermentation, has increased economic efficiency important Meaning.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie In the case of the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter From the point of view of which point, all should regard embodiment as exemplary, and be nonrestrictive, the scope of the present invention is by appended power Profit requires rather than described above limits, it is intended that all by fall in the implication of equivalency and scope of claim Change is included in the present invention.
Although moreover, it will be appreciated that this specification is been described by according to embodiment, but the most each embodiment only wraps Containing an independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should Description can also be formed those skilled in the art through appropriately combined as an entirety, the technical scheme in each embodiment May be appreciated other embodiments.

Claims (3)

1. the method improving Cordyceps militaris fermenting and producing yield of Cordycepin, including actication of culture, Cordyceps militaris spawn system Make, Cordyceps militaris (L.) Link. is cultivated, it is characterised in that liquid spawn uses spore concentration 3.0 × 10 during making8CFU/mL or 4- The female kind made during 5 truffles cake is as the primary seed solution in Cordyceps militaris spawn amplification culture；Cordyceps militaris (L.) Link. incubation uses Fermentation medium comprise glucose sugar, Rhizoma Solani tuber osi, fish peptone, (NH4)2SO4、KH2PO4、MgSO4·7H2O, dried silkworm chrysalis meal, VB1With Water.
The method of raising Cordyceps militaris fermenting and producing yield of Cordycepin the most according to claim 1, it is characterised in that In fermentation medium, the content of each composition is: glucose 25g/L, Rhizoma Solani tuber osi 100g/L, fish peptone 18g/L, (NH4)2SO4 0.8g/L, KH2PO41.0g/L, MgSO4·7H2O 0.5g/L, dried silkworm chrysalis meal 5.0g/L, VB118mg/L, water 1L.
3., according to the arbitrary described method improving Cordyceps militaris fermenting and producing yield of Cordycepin of claim 1-2, it is special Levying and be, Cordyceps militaris spawn uses NS-810 bacterial strain.
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CN106810366A (en) * 2017-01-24 2017-06-09 北京新创青龙湖种植专业合作社 A kind of cultural method for improving cordycepin content in Cordyceps militaris mycelia and silkworm chrysalis Cordyceps sinensis
CN108624510A (en) * 2017-03-22 2018-10-09 中国科学院微生物研究所 Cordyceps militaris single cross distribution type bacterial strain 2461 and its application
CN108624510B (en) * 2017-03-22 2020-10-27 中国科学院微生物研究所 Cordyceps militaris single-mating strain 2461 and application thereof
CN107586811A (en) * 2017-10-13 2018-01-16 贵阳中医学院 Improve Cordyceps militaris bark of eucommia liquid fermentation method and the application of cordycepin content
CN107586726A (en) * 2017-10-13 2018-01-16 贵阳中医学院 Improve Cordyceps militaris rhizoma Gastrodiae liquid fermentation method and the application of cordycepin content
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CN108865904A (en) * 2018-08-30 2018-11-23 云南大学 A kind of Hirsutella sinensis expands the Hirsutella sinensis and cultural method of culture medium and its culture
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CN108849257A (en) * 2018-09-26 2018-11-23 张家港市藏联生物研究所有限公司 A kind of Cordyceps militaris science cultural method
CN109971805A (en) * 2019-04-11 2019-07-05 承德森源绿色食品有限公司 Fermentation liquid, drinks and preparation method containing cordycepin and Cordyceps sinensis polysaccharide
CN113151007A (en) * 2021-03-30 2021-07-23 浙江工业大学 Liquid fermentation medium for increasing cordycepin content in cordyceps militaris
CN114736811A (en) * 2022-05-10 2022-07-12 江苏康能生物工程股份有限公司 Culture medium for high-yield cordycepin by submerged fermentation of cordyceps militaris and application of culture medium

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Application publication date: 20161207