CN1854288A - Lucid ganoderma fungus with high glycopeptide composite yield, its mutagen breeding method and use - Google Patents

Lucid ganoderma fungus with high glycopeptide composite yield, its mutagen breeding method and use Download PDF

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CN1854288A
CN1854288A CN 200510066114 CN200510066114A CN1854288A CN 1854288 A CN1854288 A CN 1854288A CN 200510066114 CN200510066114 CN 200510066114 CN 200510066114 A CN200510066114 A CN 200510066114A CN 1854288 A CN1854288 A CN 1854288A
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peptide complex
polysaccharide
ganoderma
strain
high yield
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CN100387704C (en
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张日俊
鉏晓艳
王铮
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China Agricultural University
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China Agricultural University
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Abstract

The invention involves a kind of Ganoderma Lucidum that can produce saccha-peptide complexes in high yield, the method of mutagenesis and selection, the purpose of it. The name of the fungi is Ganoderma Lucidum according to Systematic nomenclature, held by 2004-11-22 in <the general microbiology center of china culture collection managerial committee >, and the holding number is CGMCC No.1251. The first step of mutagenesis and selection is to culture the Ganoderma Lucidum CAU55 which is bought from <the general microbiology center of china culture collection managerial committee > as the initial strain and the holding number of which is CGMCC 5.533 in solid medium and fluid medium in turn and get the mycelia solution of the initial strain CAU55. The second step is to treat the mycelia solution with enzymolysis and filtration and get the protoplast deposit of the initial strain CAU55. The last step is to treat the protoplast with ultraviolet mutagenesis and regenerative cultivation, in the end getting the Ganoderma Lucidum CAU5501 that can produce saccha-peptide complexes in high yield. This kind of Ganoderma Lucidum is useful for preparing the feed additive of saccha-peptide complexes from liquid fermentation and some microbiological pharmaceutics like immunopotentiator and beneficial biotin.

Description

Lucid ganoderma fungus with high glycopeptide composite yield, its mutagen breeding method and purposes
Invention field
The invention belongs to biological technical field, but specifically relate to a kind of Ganoderma of high yield Polysaccharide-Peptide complex, and mutagenic breeding method and purposes.
Background technology
Since for many years in animal rearing and feed extensively and promotes growth health-care agents such as life-time service microbiotic, chemical synthetic drug, hormone, cause livestock and poultry and aquatic products product Chinese traditional medicine residual serious, the animal products quality descends, bacterial drug resistance strengthens, Resistant strain increases, thereby threatening human health and ecotope greatly.Because antibiotic above-mentioned many side effects, the human consumer is to the concern of food-safety problem in recent years, antibiotic use is being forbidden or limited in countries in the world all one after another, and accelerate development, popularization new type of safe green feed additive efficiently, to reduce or alternative antibiotic etc additive.
Glossy ganoderma and lucidum glycopeptide mixture become the research focus of novel green feed additive field because of its unique physicochemical property and magical biological function.The growth of Ganoderma is the process of spore → mycelium → sporophore.Since glossy ganoderma artificial culture growth cycle long (greater than 2-3 month), and Ganoderma sporophore Polysaccharide-Peptide complex abstraction process complexity, so cost is too high, is difficult to satisfy the needs of large-scale commercial production, thus limited its application in livestock industry; On the other hand, discover, the mycelium of (strain) of the same race Ganoderma is similar to molecular weight, composition, structure, ratio, chemical property and the biologic activity of the Polysaccharide-Peptide complex of sporophore, therefore adopt biofermentation method directly to cultivate the Ganoderma filament, extract in the born of the same parents more respectively, the outer Polysaccharide-Peptide complex of born of the same parents has many advantages.Compare with the former, biological fermentation process is only cultivated the mycelium stage with Ganoderma, and abstraction process is simple behind its Polysaccharide-Peptide complex, and the production cycle shortens, and production efficiency is improved.
In the process that biological fermentation is produced, the principal element of decision production level and production cost height has three: produce bacterial classification, zymotechnique and back extraction process or after-processing technology, wherein, the most important thing is to produce the characteristic of bacterial classification.From the isolated bacterial classification of nature,, often can not satisfy industrial needs because throughput is low.Because under normal physiological conditions, microorganism relies on its metabolism regulation system, and thalline trends towards growth fast and breeding.But the expectation of fermentation industry or needs need the meta-bolites of microorganism in contrast, as polysaccharide or other activeconstituents etc., can accumulate in a large number.For this reason, adopt various methodologies to break the eubolism of bacterial classification, make it to lose the adjusting control of self-conservative property, thereby accumulate our needed target meta-bolites in a large number, as the lucidum glycopeptide mixture.
Summary of the invention
The defective that the objective of the invention is to overcome the cost height of prior art for preparing lucidum glycopeptide mixture fodder additives and be not easy to promote the use of in the animal cultivation industry, but Ganoderma by protoplastis ultraviolet mutagenesis technology seed selection one strain high yield Polysaccharide-Peptide complex, it is low to make it go out in feed addition through fermentative production, definite functions, has the animal body of raising immunizing power, disease resistance, promote immunity system to grow, improve its milk, meat, egg output and throughput, improve milk, meat, the quality of egg and local flavor, improve the speed of growth and the survival rate of aquatic animal, can be widely used in fowl, ox, sheep, pig, aquatic products and economic animal can be used as the lucidum glycopeptide mixture fodder additives of advantages such as antibiotic substitute.
But another object of the present invention is to provide a kind of mutagenic breeding method of Ganoderma of high yield Polysaccharide-Peptide complex.
But a further object of the present invention is the Ganoderma of this high yield Polysaccharide-Peptide complex and is used for the application that liquid fermenting prepares Polysaccharide-Peptide complex fodder additives or immunologic facilitation fodder additives etc.
The objective of the invention is to realize by the following technical solutions:
But the Ganoderma of high yield Polysaccharide-Peptide complex provided by the invention, its called after of classifying: Ganoderma Ganoderma Lucidum, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on November 22nd, 2004, its preserving number is CGMCC No.1251.
This Ganoderma mycelial growth rate is fast, and mycelium is a pure white, produces spore; Need lucifuge to cultivate, it is cultivated needs Mg 2+, K +Deng trace element and vitamin B group; Biologically active substances such as high yield Polysaccharide-Peptide complex.
But the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex provided by the invention, its mutagenic and breeding step is as follows:
1) the mycelial cultivation of starting strain CAU55
In 4 ℃ of preservations, available from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", culture presevation number is that the Ganoderma CAU55 of CGMCC 5.533 is as starting strain with this laboratory;
Starting strain CAU55 is inoculated on the sterilization solid medium 25～30 ℃ of solid culture 5～10 days; With cultured inoculation in the sterilising liq substratum, 28～32 ℃ of shake-flask culture 3～5 days are inoculated in cultured seed liquid in the sterilising liq substratum again, and 28～32 ℃ of shake flask fermentations were cultivated 3～5 days, obtain starting strain CAU55 hypha fluid, standby;
Described solid medium is to prepare in following ratio: potato extracting solution 1000ml, glucose 20～30g, KH 2PO 41.5～3g, MgSO 47H 2O0.75～2.5g, agar 15～20g;
Described liquid nutrient medium is to prepare in following ratio: glucose 20～40g, peptone 2～4g, KH 2PO 40.5～2g, MgSO 47H 2O 0.5～2g, distilled water 1000ml;
2) preparation of starting strain CAU55 protoplastis
Enzymolysis: with the starting strain CAU55 hypha fluid cultivated in the step 1) in the centrifugal 1～3min of 2000～4000r/min; Remove supernatant, add sterilization homeo-osmosis agent 1～3ml, the centrifugal 1～3min of slight vibration back 2000～4000r/min, this step repetitive operation 2～3 times; After removing supernatant, in enzymolysis solution (ml): the ratio of mycelia body weight (g) 1～4: 1, the enzymolysis solution after in the mycelium precipitation, adding bacteriological filtration, fully vibration was cultivated 1～3 hour for 25～30 ℃, and is standby;
Filter: the hypha fluid that enzymolysis is good filters, and with a small amount of homeo-osmosis agent flushing filter membrane, the centrifugal 1～5min of 1000～3000r/min obtains starting strain CAU55 protoplastis precipitation again;
Described homeo-osmosis agent is to prepare in following ratio: 80～130g glucose is dissolved in the 1000ml distilled water, and it is standby to sterilize;
3) ultraviolet mutagenesis of protoplastis and regeneration are cultivated
With step 2) gained starting strain CAU55 protoplastis precipitation makes suspension, and to transfer to the protoplastis number be 10 4～10 6Individual/ml, liquid layer thickness 0.2～0.4cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis is 15～18W, and irradiation time is 0.5～5min, irradiation distance 25～30cm; Dilution immediately is coated on the protoplast regeneration substratum after the irradiation, cultivated 6～10 days for 27～32 ℃, carry out primary dcreening operation, select 10～40 single faster bacterium colonies of mycelial growth speed, carry out shake flask fermentation and sieve again, measure its Polysaccharide-Peptide complex content with the phenolsulfuric acid method, select 6～10 strain Polysaccharide-Peptide complex superior strain CAU550N, do mitotic stability experiment more respectively, it is the fastest to select a strain mycelial growth rate, the bacterial strain CAU5501 of the highest and good stability of Polysaccharide-Peptide complex output;
Described protoplast regeneration substratum is to prepare in following ratio: D-cellobiose 10～15g, peptone 2～3.5g, yeast soak powder 1.5～4g, KH 2PO 42～3g, MgSO 47H 2O 1.0～2.5g, VB 120～50 μ g, homeo-osmosis agent 1000ml;
The step of described mitotic stability experiment is: 6～10 strain Polysaccharide-Peptide complex superior strain CAU550N that will select, be inoculated into respectively on the sterilization solid medium, 25～30 ℃ of solid culture 5～8 days, inoculate after cultivating well on the sterilization solid medium, 25～30 ℃ of solid culture 5～8 days, repeat this step 5～10 time, observe the speed of growth of CAU550N; The CAU550N in 5～10 generations of propagating is inoculated in the liquid nutrient medium, 28～32 ℃ of shake-flask culture 3～5 days, again cultured seed liquid is inoculated in the sterilising liq substratum, carry out shake flask fermentation, cultivated 3～5 days for 28～32 ℃, get hypha fluid, measure their Polysaccharide-Peptide complex output respectively, reject spawn degeneration, the bacterial strain that the speed of growth descends or production performance descends, keep fast growth, production performance is good, stable bacterial strain, but the Ganoderma CAU5501 of high yield Polysaccharide-Peptide complex obtained at last, its called after of classifying: Ganoderma Ganoderma Lucidum, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on November 22nd, 2004, preserving number is CGMCC No.1251.
But the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex provided by the invention comprises that also the superior strain CAU5501 that will obtain is kept on the solid medium II, 4 ℃ of preservations, and switching in per 3 months is once.Switching is used for normally going down to posterity of this bacterial strain, degenerates to prevent bacterial strain.
Described solid medium II prepares in following ratio: potato extracting solution 1000ml, glucose 20～30g, KH 2PO 41.5～3g, MgSO 47H 2O0.75～2.5g, agar 15～20g.
But the invention provides the purposes of the Ganoderma of above-mentioned high yield Polysaccharide-Peptide complex, but the Ganoderma that is somebody's turn to do the high yield Polysaccharide-Peptide complex can be used for liquid fermenting and prepares the Polysaccharide-Peptide complex green feed additive, or is used to prepare microbial preparations such as microorganism feed addictive, immunostimulant.
Compared with prior art, but the advantage of Ganoderma of high yield Polysaccharide-Peptide complex provided by the invention and preparation method thereof be:
1. Protoplast Technique and ultraviolet mutagenesis method are combined and handle, but multi-angle changes the heritability of microorganism, guarantees the stability of dissociant, and improve efficiency of inducing mutation, be easy to set up good regeneration asexual variation strain and be;
2. mutafacient system is uncomplicated, and is easy to operate, and effect is obvious;
3. can obviously shorten the production cycle with its fermentative preparation lucidum glycopeptide mixture fodder additives and reach 50%, reduce fermentation costs, make the application of lucidum glycopeptide mixture in livestock industry become possibility;
4. the superior strain of mutagenic and breeding can be used for preparing microorganism feed addictive;
5. the superior strain of mutagenic and breeding can be used for preparing feeding immunostimulant (reinforcement) agent.
Embodiment
But the Ganoderma of embodiment 1, mutagenic and breeding high yield Polysaccharide-Peptide complex of the present invention
According to mutagenic breeding method provided by the invention, but the Ganoderma of mutagenic and breeding high yield Polysaccharide-Peptide complex, and its step is as follows:
1) the mycelial cultivation of starting strain CAU55
In 4 ℃ of preservations, available from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", culture presevation number is that the Ganoderma CAU55 of CGMCC 5.533 is as starting strain with this laboratory;
Starting strain CAU55 is inoculated on the sterilization solid medium A 27 ℃ of solid culture 10 days; In sterilising liq substratum B, 28 ℃ of shake-flask culture 4 days are inoculated in cultured seed liquid in the sterilising liq culture medium C again with cultured inoculation, and 32 ℃ of shake flask fermentations were cultivated 3 days, obtained starting strain CAU55 hypha fluid, and are standby;
Described solid medium A prepares in following ratio: potato extracting solution 1000ml, glucose 25g, KH 2PO 42g, MgSO 47H 2O 1.5g, agar 15g;
Described liquid nutrient medium B, C prepare in following ratio: glucose 20g, peptone 3g, KH 2PO 41.5g, MgSO 47H 2O 1.5g, distilled water 1000ml;
2) preparation of starting strain CAU55 protoplastis
Enzymolysis: with the starting strain CAU55 hypha fluid cultivated in the step 1) in the sterilization centrifuge tube, the centrifugal 2min of 4000r/min; Remove supernatant, add sterilization homeo-osmosis agent 3ml, the centrifugal 2min of slight vibration back 3000r/min, this step repetitive operation 2 times; After removing supernatant, the gained precipitation is moved in the sterilization Eppendorf pipe, the difference weighting method is weighed, the record mycelium weight; In enzymolysis solution (ml): 1: 1 ratio of mycelia body weight (g), the enzymolysis solution after in the mycelium precipitation, adding bacteriological filtration, fully vibration was cultivated 1 hour for 25 ℃, and is standby;
Filter: be filtered in another sterilization Eppendorf pipe with the sterilization filter hypha fluid that enzymolysis is good, with a small amount of homeo-osmosis agent flushing filter membrane, the centrifugal 3min of 3000r/min obtains starting strain CAU55 protoplastis precipitation again;
Described homeo-osmosis agent is to prepare in following ratio: 100g glucose is dissolved in the 1000ml distilled water, and it is standby to sterilize;
3) ultraviolet mutagenesis of protoplastis and regeneration are cultivated
With step 2) gained starting strain CAU55 protoplastis precipitation makes suspension, and to transfer to the protoplastis number be 10 6Individual/ml, liquid layer thickness 0.4cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis is 15W, and irradiation time is 3min, irradiation distance 30cm; Dilution immediately is coated on the protoplast regeneration substratum D after the irradiation, cultivated 9 days for 27 ℃, carry out primary dcreening operation, select 20 single faster bacterium colonies of mycelial growth speed, carry out shake flask fermentation and sieve again, measure its Polysaccharide-Peptide complex content with the phenolsulfuric acid method, select 10 strain Polysaccharide-Peptide complex superior strain CAU550N, do mitotic stability experiment more respectively, it is the fastest to select a strain mycelial growth rate, the bacterial strain CAU5501 of the highest and good stability of Polysaccharide-Peptide complex output;
Described protoplast regeneration substratum D prepares in following ratio: D-cellobiose 10g, peptone 3g, yeast soak powder 2g, KH 2PO 42g, MgSO 47H 2O 1.5g, VB 120 μ g, homeo-osmosis agent 1000ml;
The step of described mitotic stability experiment is: the 10 strain Polysaccharide-Peptide complex superior strain CAU550N that will select, be inoculated into respectively on the sterilization solid medium A, 25 ℃ of solid culture 8 days, inoculate after cultivating well on the sterilization solid medium A, 25 ℃ of solid culture 8 days, repeat this step 8 time, observe the speed of growth of CAU550N; The CAU550N in 8 generations of propagating is inoculated among the liquid nutrient medium B, 28 ℃ of shake-flask culture 5 days are inoculated in cultured seed liquid among the sterilising liq substratum B again, carry out shake flask fermentation, cultivated 3 days for 30 ℃, get hypha fluid, measure their Polysaccharide-Peptide complex output respectively, reject spawn degeneration, the bacterial strain that the speed of growth descends or production performance descends, keep fast growth, the bacterial strain that production performance is good, stable, but obtain the Ganoderma CAU5501 of high yield Polysaccharide-Peptide complex at last.
The superior strain CAU5501 that obtains is kept on the solid medium II, 4 ℃ of preservations, switching in per 3 months is once.Switching is used for normally going down to posterity of this bacterial strain, degenerates to prevent bacterial strain.
Described solid medium II prepares in following ratio: potato extracting solution 1000ml, glucose 30g, KH 2PO 43g, MgSO 47H 2O 2g, agar 20g.
But the Ganoderma of embodiment 2, mutagenic and breeding high yield Polysaccharide-Peptide complex of the present invention
According to mutagenic breeding method provided by the invention, but the Ganoderma of mutagenic and breeding high yield Polysaccharide-Peptide complex, and its step is as follows:
1) the mycelial cultivation of starting strain CAU55
In 4 ℃ of preservations, available from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", culture presevation number is that the Ganoderma CAU55 of CGMCC 5.533 is as starting strain with this laboratory;
Starting strain CAU55 is inoculated on the sterilization solid medium A 25 ℃ of solid culture 10 days; In sterilising liq substratum B, 30 ℃ of shake-flask culture 3 days are inoculated in cultured seed liquid in the sterilising liq culture medium C again with cultured inoculation, and 30 ℃ of shake flask fermentations were cultivated 3 days, obtained starting strain CAU55 hypha fluid, and are standby;
Described solid medium A prepares in following ratio: potato extracting solution 1000ml, glucose 20g, KH 2PO 41.5g, MgSO 47H 2O 1.5g, agar 20g;
Described liquid nutrient medium B, C prepare in following ratio: glucose 20g, peptone 2.5g, KH 2PO 42g, MgSO 47H 2O 1.5g, distilled water 1000ml;
2) preparation of starting strain CAU55 protoplastis
Enzymolysis: with the starting strain CAU55 hypha fluid cultivated in the step 1) in the sterilization centrifuge tube, the centrifugal 3min of 2000r/min; Remove supernatant, add sterilization homeo-osmosis agent 2ml, the centrifugal 3min of slight vibration back 2000r/min, this step repetitive operation 2 times; After removing supernatant, the gained precipitation is moved in the sterilization Eppendorf pipe, the difference weighting method is weighed, the record mycelium weight; In enzymolysis solution (ml): 2: 1 ratio of mycelia body weight (g), the enzymolysis solution after in the mycelium precipitation, adding bacteriological filtration, fully vibration was cultivated 1 hour for 30 ℃, and is standby;
Filter: be filtered in another sterilization Eppendorf pipe with the sterilization filter hypha fluid that enzymolysis is good, with a small amount of homeo-osmosis agent flushing filter membrane, the centrifugal 2min of 3000r/min obtains starting strain CAU55 protoplastis precipitation again;
Described homeo-osmosis agent is to prepare in following ratio: 130g glucose is dissolved in the 1000ml distilled water, and it is standby to sterilize;
3) ultraviolet mutagenesis of protoplastis and regeneration are cultivated
With step 2) gained starting strain CAU55 protoplastis precipitation makes suspension, and to transfer to the protoplastis number be 10 5Individual/ml, liquid layer thickness 0.2cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis is 15W, and irradiation time is 2.5min, irradiation distance 25cm; Dilution immediately is coated on the protoplast regeneration substratum D after the irradiation, cultivated 6 days for 30 ℃, carry out primary dcreening operation, select 40 single faster bacterium colonies of mycelial growth speed, carrying out shake flask fermentation sieves again, measure its Polysaccharide-Peptide complex content with the phenolsulfuric acid method, select 6 strain Polysaccharide-Peptide complex superior strain CAU55N1, CAU55N2, CAU55N3, CAU55N4, CAU55N5, CAU5501, do the mitotic stability experiment more respectively, it is the fastest to select a strain mycelial growth rate, the bacterial strain CAU5501 of the highest and good stability of Polysaccharide-Peptide complex output;
Described protoplast regeneration substratum D prepares in following ratio: D-cellobiose 12g, peptone 2.0g, yeast soak powder 2.5g, KH 2PO 42.0g, MgSO 47H 2O1.5g, VB 150 μ g, homeo-osmosis agent 1000ml;
The step of described mitotic stability experiment is: 6 strain Polysaccharide-Peptide complex superior strain CAU55N1, CAU55N2, CAU55N3, CAU55N4, CAU55N5, CAU5501 that above-mentioned steps is selected, be inoculated into respectively on the sterilization solid medium A, 26 ℃ of solid culture 6 days, inoculate after cultivating well on the sterilization solid medium A, 26 ℃ of solid culture 6 days, repeat this step 10 time, observe the speed of growth of CAU55N1, CAU55N2, CAU55N3, CAU55N4, CAU55N5, CAU5501; CAU55N1 with 10 generations of propagating, CAU55N2, CAU55N3, CAU55N4, CAU55N5, CAU5501 is inoculated into respectively among the liquid nutrient medium B, 30 ℃ of shake-flask culture 4 days, cultured seed liquid is inoculated among the sterilising liq substratum B again, carry out shake flask fermentation, cultivated 5 days for 30 ℃, get hypha fluid, measure their Polysaccharide-Peptide complex output respectively, reject spawn degeneration, the bacterial strain that the speed of growth descends or production performance descends, keep fast growth, production performance is good, stable bacterial strain, but the Ganoderma CAU5501 of high yield Polysaccharide-Peptide complex obtained at last.
This superior strain CAU5501 that obtains is kept on the solid medium II, 4 ℃ of preservations, switching in per 3 months is once.Switching is used for normally going down to posterity of this bacterial strain, degenerates to prevent bacterial strain.
Described solid medium II prepares in following ratio: potato extracting solution 1000ml, glucose 25g, KH 2PO 42g, MgSO 47H 2O 0.75g, agar 17g.
But the Ganoderma of embodiment 3, mutagenic and breeding high yield Polysaccharide-Peptide complex of the present invention
According to mutagenic breeding method provided by the invention, but the Ganoderma of mutagenic and breeding high yield Polysaccharide-Peptide complex, and its step is as follows:
1) the mycelial cultivation of starting strain CAU55
In 4 ℃ of preservations, available from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", culture presevation number is that the Ganoderma CAU55 of CGMCC 5.533 is as starting strain with this laboratory;
Starting strain CAU55 is inoculated on the sterilization solid medium A 30 ℃ of solid culture 10 days; In sterilising liq substratum B, 32 ℃ of shake-flask culture 4 days are inoculated in cultured seed liquid in the sterilising liq culture medium C again with cultured inoculation, and 32 ℃ of shake flask fermentations were cultivated 5 days, obtained starting strain CAU55 hypha fluid, and are standby;
Described solid medium A prepares in following ratio: potato extracting solution 1000ml, glucose 30g, KH 2PO 43g, MgSO 47H 2O 2.5g, agar 20g;
Described liquid nutrient medium B, C prepare in following ratio: glucose 40g, peptone 4g, KH 2PO 42g, MgSO 47H 2O 1.5g, distilled water 1000ml;
2) preparation of starting strain CAU55 protoplastis
Enzymolysis: with the starting strain CAU55 hypha fluid cultivated in the step 1) in the sterilization centrifuge tube, the centrifugal 1min of 3000r/min; Remove supernatant, add sterilization homeo-osmosis agent 1ml, the centrifugal 1min of slight vibration back 4000r/min, this step repetitive operation 3 times; After removing supernatant, the gained precipitation is moved in the sterilization Eppendorf pipe, the difference weighting method is weighed, the record mycelium weight; In enzymolysis solution (ml): 4: 1 ratio of mycelia body weight (g), the enzymolysis solution after in the mycelium precipitation, adding bacteriological filtration, fully vibration was cultivated 3 hours for 28 ℃, and is standby;
Filter: be filtered in another sterilization Eppendorf pipe with the sterilization filter hypha fluid that enzymolysis is good, with a small amount of homeo-osmosis agent flushing filter membrane, the centrifugal 5min of 1000r/min obtains starting strain CAU55 protoplastis precipitation again;
Described homeo-osmosis agent is to prepare in following ratio: 80g glucose is dissolved in the 1000ml distilled water, and it is standby to sterilize;
3) ultraviolet mutagenesis of protoplastis and regeneration are cultivated
With step 2) gained starting strain CAU55 protoplastis precipitation makes suspension, and to transfer to the protoplastis number be 10 4Individual/ml, liquid layer thickness 0.3cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis is 18W, and irradiation time is 1min, irradiation distance 25cm; Dilution immediately is coated on the protoplast regeneration substratum D after the irradiation, cultivated 10 days for 32 ℃, carry out primary dcreening operation, select 10 single faster bacterium colonies of mycelial growth speed, carry out shake flask fermentation and sieve again, measure its Polysaccharide-Peptide complex content with the phenolsulfuric acid method, select 6 strain Polysaccharide-Peptide complex superior strain CAU550N, do mitotic stability experiment more respectively, it is the fastest to select a strain mycelial growth rate, the bacterial strain CAU5501 of the highest and good stability of Polysaccharide-Peptide complex output;
Described protoplast regeneration substratum D prepares in following ratio: D-cellobiose 15g, peptone 3.5g, yeast soak powder 4g, KH 2PO 42g, MgSO 47H 2O 1g, VB 140 μ g, homeo-osmosis agent 1000ml;
The step of described mitotic stability experiment is: the 6 strain Polysaccharide-Peptide complex superior strain CAU550N that will select, be inoculated into respectively on the sterilization solid medium A, 30 ℃ of solid culture 5 days, inoculate after cultivating well on the sterilization solid medium A, 30 ℃ of solid culture 5 days, repeat this step 5 time, observe the speed of growth of CAU550N; The CAU550N in 5 generations of propagating is inoculated among the liquid nutrient medium B, 32 ℃ of shake-flask culture 3 days are inoculated in cultured seed liquid among the sterilising liq substratum B again, carry out shake flask fermentation, cultivated 5 days for 32 ℃, get hypha fluid, measure their Polysaccharide-Peptide complex output respectively, reject spawn degeneration, the bacterial strain that the speed of growth descends or production performance descends, keep fast growth, the bacterial strain that production performance is good, stable, but obtain the Ganoderma CAU5501 of high yield Polysaccharide-Peptide complex at last.
This superior strain CAU5501 that obtains is kept on the solid medium II, 4 ℃ of preservations, switching in per 3 months is once.Switching is used for normally going down to posterity of this bacterial strain, degenerates to prevent bacterial strain.
Described solid medium II prepares in following ratio: potato extracting solution 1000ml, glucose 20g, KH 2PO 41.5g, MgSO 47H 2O 0.75g, agar 15g.
But to the mycelial growth rate of the Ganoderma CAU5501 of above-mentioned three embodiment gained high yield Polysaccharide-Peptide complex, produce the Polysaccharide-Peptide complex ability and test, but the Ganoderma mycelial growth rate of embodiment 2 gained high yield Polysaccharide-Peptide complex is the fastest, be 7.34cm/7d, the outer Polysaccharide-Peptide complex ability of its product born of the same parents is also the strongest, is 2.45mg/ml.But the Ganoderma CAU5501 of embodiment 2 gained high yield Polysaccharide-Peptide complex is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; the classification called after: glossy ganoderma Ganoderma Lucidum, its preserving number are CGMCC No.1251 on November 22nd, 2004.
Mycelial growth rate is a level of signification of weighing Ganoderma strain growth performance, also is the key that shortens the production cycle.Mycelial growth rate=slant medium mycelial growth length/fate (described fate is 7 days).The mycelial growth rate of each step mutant strain and maternal plant is listed in table 1 among the embodiment 2.
The comparison of the outer Polysaccharide-Peptide complex output of the mycelial growth rate of table 1, starting strain and mutagenic fungi and born of the same parents
Strain number Mycelial growth rate (cm/7d) The outer Polysaccharide-Peptide complex output (mg/ml) of born of the same parents
CAU55 5.23 1.67
CAU55N1 6.30 1.86
CAU55N2 5.60 2.10
CAU55N3 6.82 2.24
CAU55N4 6.51 1.80
CAU55N5 6.05 2.20
CAU5501 7.34 2.45
The outer Polysaccharide-Peptide complex output (mg/ml) of glossy ganoderma born of the same parents is proportionate with lucidum glycopeptide mixture ultimate production, is the important indicator of weighing the Ganoderma production performance, also is the key index of weighing lucidum glycopeptide mixture ultimate production.The principle of utilizing polysaccharide compound to be insoluble to organic solvent (ethanol) during mensuration earlier precipitates it, utilize phenolsulfuric acid reagent can play the principle of color reaction again with the hexose in polysaccharide or the glycopeptide, it is fully reacted, because hexose has maximum absorption at the 490nm place, absorption value and sugared content are linear, therefore, can get the output of the outer Polysaccharide-Peptide complex of born of the same parents according to its absorption value.
The method of the outer Polysaccharide-Peptide complex output of phenolsulfuric acid determination of color glossy ganoderma born of the same parents is as follows:
The drafting of typical curve: at first prepare standard glucose solution, its concentration is 3mg/ml, shakes up standby; Strict then according to the form below 2 listed application of sample amount order application of samples.
The application of sample amount of table 2, glucose, 5% phenol, the vitriol oil
The test tube numbering 1 2 3 4 5 6 7 8 9 10
Glucose concn (mg/ml) 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7
Glucose (ml) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Distilled water (ml) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
5% phenol (ml) 1 1 1 1 1 1 1 1 1 1
The vitriol oil (ml) 4 4 4 4 4 4 4 4 4 4
Press table 2 and add glucose solution and distilled water in the numbering test tube, add 5% phenol solution 1ml again, slowly drip vitriol oil 4ml behind the mixing, the vibration mixing is cooled to room temperature, places 20 minutes, with the first pipe zeroising, measures optical density at 490 wavelength places.With the glucose concn is X-coordinate, is ordinate zou with absorbance (OD value), the drawing standard curve.
The outer Polysaccharide-Peptide complex Determination on content of sample born of the same parents: take the good sample liquid 3ml of Ethanol Treatment, under the 4000r/m centrifugal 20 minutes, precipitation 1ml dissolved in distilled water, add 5% phenol solution 1ml again, slowly drip vitriol oil 4ml, vibration mixing behind the mixing, be cooled to room temperature, placed 20 minutes, 490 wavelength places survey the OD value, get the outer Polysaccharide-Peptide complex content of its born of the same parents according to typical curve.The ability of the outer Polysaccharide-Peptide complex of the product born of the same parents of each step mutant strain and maternal plant is relatively listed in table 1 among the embodiment 2.
But embodiment 4, utilize the Ganoderma of high yield Polysaccharide-Peptide complex of the present invention to prepare the Polysaccharide-Peptide complex fodder additives as liquid fermenting
But utilize the Ganoderma of high yield Polysaccharide-Peptide complex of the present invention as follows as the detailed directions that liquid fermenting prepares the Polysaccharide-Peptide complex fodder additives:
1) preparation seed liquor
But the Ganoderma (Ganoderma Lucidum) of the high yield Polysaccharide-Peptide complex that embodiments of the invention 2 are made inserts in the liquid nutrient medium after the sterilization, cultivates 3～5 days in 28～32 ℃, obtains seed liquor;
Described liquid nutrient medium is prepared in following ratio: glucose 20～40g, peptone 2～4g, KH 2PO 40.5～2g, MgSO 47H 2O 0.5～2g, distilled water 1000ml.
2) fermentative preparation Polysaccharide-Peptide complex fodder additives
Put into fermentor tank after fermentation substrate is crushed to 40～120 orders, add entry and carbon source successively, add the weight of entry and the weight part proportioning of fermentation substrate is 100: 3～20, add the nitrogen content of the carbon content of carbon source and fermentation substrate the weight part proportioning be 10～80: 1, with fermented liquid at 0.1～0.13MPa, sterilized 10～30 minutes for 115～125 ℃, be cooled to 25 ℃, the seed liquor that adds the above-mentioned steps preparation again, the volume ratio of seed liquor and fermented liquid is 0.3～15: 100, mix the back at 25～32 ℃, speed with 0.5～2.5vvm feeds sterile air, stirs fermentation 3～7 days with 80～200rpm speed, obtains the Polysaccharide-Peptide complex fermented liquid, in 140～145 ℃ of sterilizations 2～8 seconds, obtain liquid glycopeptide class fodder additives; Also this Polysaccharide-Peptide complex fodder additives can be carried out ultrafiltration, concentrate, drying etc., obtain the powdered sugar peptide feedstuff additive; In addition,, fermented liquid and mycelium can be extracted respectively that born of the same parents are outer, the intracellular glycopeptide mixture, obtain the refined sugar peptide feedstuff additive by the back extraction process (lixiviate, alcohol precipitation etc.) of fermentation industry---the Polysaccharide-Peptide complex fodder additives.
Described fermentation substrate (being carbon source) is for being selected from one or more the mixture in Semen Maydis powder, vinasse, wheat bran, brown sugar, edible white sugar, maize treacle, starch, cane molasses, the beet sirup;
Described nitrogenous source is one or more the mixture that is selected from soybean protein, the gluten;
Described soybean protein comprises soybean protein isolate, dregs of beans, soya-bean cake, soybean;
Described gluten comprises zein, Zein powder and wheat protein.
The glycopeptide class fodder additives of preparation serves many purposes:
1) this glycopeptide class fodder additives can improve antibody horizontal and the cellular immune function of livestock and poultry, and strengthening immunity and disease resistance reduce case fatality rate.
2) growing of this glycopeptide class fodder additives energy obvious stimulation animal immune organ improved the health level of animal.
3) nontoxic, the noresidue of this glycopeptide class fodder additives has the effect that suppresses harmful bacterium and anti-inflammatory, can be used as the microbiotic substitute products, is used to regulate domestic animal, poultry intestinal microflora balance.
4) this glycopeptide class fodder additives has the adjusting nutrient metabolism, reducing blood-fat, cholesterol, and hypoglycemic effect improves the livestock product quality.
5) this glycopeptide class fodder additives can improve the production performance of animal, as improving the speed of growth, save feed, reducing production costs etc.
The Polysaccharide-Peptide complex fodder additives that embodiment 5, embodiment 4 make is to the influence of broiler chicken production performance.
With 500 of the healthy love of 1 age in days male and female blended denapon fryer, be divided into 5 processing at random, each handles 100, and each processing is divided into 5 repetitions, and each repeats 20 chickens.5 processing are respectively:
The A group, blank group, the broiler chicken basal diet of only feeding;
The B group, the microbiotic group, except that the broiler chicken basal diet of feeding, feed per ton adds 3.2g enramycin (usual amounts);
The C group, lucidum glycopeptide mixture fodder additives group, except that the broiler chicken basal diet of feeding, feed per ton adds the Polysaccharide-Peptide complex fodder additives that 1kg embodiment 4 makes, wherein effective constituent 2.5g/t feed;
The D group, lucidum glycopeptide mixture fodder additives group, except that the broiler chicken basal diet of feeding, feed per ton adds the Polysaccharide-Peptide complex fodder additives that 3kg embodiment 4 makes, wherein effective constituent 7.5g/t feed;
The E group, lucidum glycopeptide mixture fodder additives group, except that the broiler chicken basal diet of feeding, feed per ton adds the Polysaccharide-Peptide complex fodder additives that 5kg embodiment 4 makes, wherein effective constituent 12.5g/t feed.
Polysaccharide-Peptide complex the results are shown in table 3 to the influence of broiler chicken production performance.
Table 3, Polysaccharide-Peptide complex fodder additives are in the influence of broiler chicken production performance
Annotate: the right shoulder of colleague's numeral indicates different capitalizations (A, B, C) person and reaches extremely significantly (P＜0.01) for difference, the right shoulder of colleague's numeral indicates different lowercase person (a, b is significant difference (P＜0.05) c), and same letter person being arranged or do not have alphabetical mark person is difference not remarkable (p＞0.05).A, B, C, a, b, c are the method for expressing of test of significance on the statistics.
As can be seen from Table 3, in 1～4 week and 1～7 week, the day weight gain effect of D group is best, and there are utmost point significant difference (P＜0.01) in day weight gain and other group of organizing at 1～4 all D.In whole breeding cycle (1～7 week), along with the increase of Polysaccharide-Peptide complex addition, the broiler chicken day weight gain rises earlier and afterwards descends, and the day weight gain of D group is peak value and other group significant difference (P＜0.05); And along with the increase of Polysaccharide-Peptide complex addition, the feedstuff-meat ratio of broiler growth descends earlier and afterwards rises, the feedstuff-meat ratio minimum that D organizes, but compare difference not remarkable (p＞0.05) with other group.D group (7.5g/t Polysaccharide-Peptide complex interpolation group) is compared with B group (microbiotic group) as can be seen from the table, and day weight gain is similar, but feedstuff-meat ratio is lower, illustrates that D group effect is better than the B group.In a word, this test shows, adds the Polysaccharide-Peptide complex fodder additives and can improve the poultry day weight gain in the broiler chicken feed, reduces feedstuff-meat ratio, improves production performance, reduces production costs.
The Polysaccharide-Peptide complex fodder additives that embodiment 6, embodiment 4 make is used in Swine Production
Be the effect of checking product of the present invention and the effect on pig industry, and replace antibiotic feasibility.Test is chosen body weight and is divided into 5 processing near 180 healthy of 35 age in days weanling pigs (male and female half and half), 36 pigs of each processing (group), and each handles 6 repetitions, each repeats 6 pigs, be 3 public 3 mothers, in 8 weeks of trial period (2 months), test-results is listed in table 4.5 processing are as follows:
Blank group: do not add any additives, the pig basal diet of only feeding;
The microbiotic control group: pulvis duomycin 100mg/kg, uniform mixing is in basal diet;
1 group of lucidum glycopeptide mixture: the Polysaccharide-Peptide complex fodder additives that 5mg/kg embodiment 4 makes, uniform mixing is in basal diet;
2 groups of lucidum glycopeptide mixtures: the Polysaccharide-Peptide complex fodder additives that 10mg/kg embodiment 4 makes, uniform mixing is in basal diet;
3 groups of lucidum glycopeptide mixtures: the Polysaccharide-Peptide complex fodder additives that 15mg/kg embodiment 4 makes, uniform mixing is in basal diet.
Table 4, the result of use in Swine Production
Processing/repetition 29～50 age in days average daily gains (Kg) 29～50 age in days material anharmonic ratioes
The blank group 0.2707±0.01 2.23±0.09
Microbiotic control group (duomycin 100mg/kg) 0.3261±0.02 1.68±0.08
1 group of lucidum glycopeptide mixture (5mg/kg) 0.3049±0.01 1.76±0.07
2 groups of lucidum glycopeptide mixtures (10mg/kg) 0.3364±0.02 1.68±0.07
3 groups of lucidum glycopeptide mixtures (15mg/kg) 0.3171±0.01 1.84±0.17
As can be seen from Table 5, the average daily gain and the feed efficiency of having added 3 groups of the Polysaccharide-Peptide complex fodder additives that embodiment 4 makes all are better than the blank group, can obviously improve pig production performance only, i.e. day weight gain and feed efficiency.Wherein the average daily gain of lucidum glycopeptide mixture 2 groups (effective constituent 120mg/kg feeds) and feed efficiency significantly are better than blank group and microbiotic group, can substitute microbiotic fully.
The Polysaccharide-Peptide complex fodder additives that embodiment 7, use embodiment 4 make is as the influence (see Table 5) of immunologic facilitation fodder additives to fryer meat immune organ.
The influence that table 5, lucidum glycopeptide mixture additive grow to the fryer immune organ
Figure A20051006611400181
Annotate: 1) immune organ index: the ratio that refers to immune organ (thymus gland, spleen, the fabricius bursa etc.) weight and animal body weight; Last table BW refers to broiler weight.2) the right shoulder of colleague numeral indicates different capitalizations (A, B, C) person and reaches extremely remarkable (P＜0.01) for difference, the right shoulder of colleague's numeral indicates different lowercase person (a, b is significant difference (P＜0.05) c), and same letter person being arranged or do not have alphabetical mark person is difference not remarkable (p＞0.05).A, B, C, a, b, c are the method for expressing of test of significance on the statistics.
As can be seen from Table 5, along with the increase of lucidum glycopeptide mixture addition, fryer immune organ thymus gland, spleen weightening finish are risen, but are not formed significant difference (p＞0.05).Along with the increase of lucidum glycopeptide mixture addition, the weightening finish of the fryer fabricius bursa is risen, and significant difference (P＜0.05); The fabricius bursa between Polysaccharide-Peptide complex group and microbiotic group weightening finish difference is significantly (p＞0.05) not, but the difference of each Polysaccharide-Peptide complex interpolation group and blank group all extremely remarkable (P＜0.01).In a word, this test shows, adds the Polysaccharide-Peptide complex fodder additives that embodiment 4 makes in the fowl feed, can impel the weightening finish of poultry immunity organ, improve antibody titers, strengthens its immunizing power; And can substitute microbiotic fully, to eliminate antibiotic drug residue and toxic side effect.
The Polysaccharide-Peptide complex fodder additives that embodiment 8, embodiment 4 make is to the influence of bovine serum albumin (BSA) antibody titer of chicken
For detecting Polysaccharide-Peptide complex fodder additives that the embodiment of the invention 4 makes to the influence of fryer to bovine serum albumin (BSA) specific antibody titres, broiler chicken is divided into 5 groups, each component becomes 5 repetitions, and each repeats 20 chickens, and trial period was 7 weeks.Test design sees Table 6, and basal diet proportioning and trophic level see Table 7.Give each injection of bovine serum albumin 0.5ml of fryer left and right sides leg flesh (bovine serum albumin concentration is 2.5mg/kg) respectively at 35 ages in days, 42 ages in days.
Table 6, feeding of broiler test design
Group Treatment group Addition (/ ton feed) Effective constituent (g/ ton feed)
A Blank 0 0
B Microbiotic contrast (Xin Meisu+duomycin) 20+100g 20+100
C 0.1% Polysaccharide-Peptide complex additive 1kg 40.0
D 0.3% Polysaccharide-Peptide complex additive 3kg 120.0
E 0.5% Polysaccharide-Peptide complex additive 5kg 200.0
Table 7, broiler chicken daily ration prescription and trophic level
Feed Proportioning (%) Nutrient substance Trophic level
0-3 week 4-7 week 0-3 week 4-7 week
Corn 55.8 59.8 Metabolizable energy (MJ/kg) 2900.11 3000.55
Dregs of beans 37.6 32.2 Crude protein (%) 21.03 19.00
Soya-bean oil 2.3 3.36 Calcium (%) 1.01 0.91
Stone flour 1.3 1.2 Total phosphorus (%) 0.70 0.64
Secondary calcium phosphate 1.92 1.7 Available phosphorus (%) 0.45 0.41
Zeolite powder - 0.66 Methionin (%) 1.10 0.96
Methionin 0.02 - Methionine(Met) (%) 0.45 0.36
Methionine(Met) 0.14 0.07 Egg+Guang (%) 0.79 0.67
Salt 0.3 0.3 Salt (%) 0.3 0.3
Preblend 1 1
Annotate: Preblend provides to every kilogram of complete diet pellet: Cu 6mg, Fe 75mg, Zn 75mg, Mn 80mg, Se 0.2mg, 10.4mg, zeolite powder 1mg, choline 3mg.Vitamin A 6000IU, Vitamin D3 500,000 I.U/GM 2000IU, vitamin-E 12IU, vitamin K3 2mg, vitamin H 0.05mg, folic acid 0.4mg, nicotinic acid 32mg, pantothenic acid 7mg, VITMAIN B1 1mg, Wei ShengsuB2 5mg, vitamin B6 1mg, vitamin B12 10ug.
Get 12 dirty blood samplings of heart in 28 ages in days, every group of group of 49 ages in days, the centrifugal 10min separation of serum of 3000rpm is frozen in-20 ℃ of refrigerators then, detects the BSA antibody titer with enzyme-linked immunoassay method (ELISA), and the result sees Table 8 with the OD value representation.
Table 8, add the influence of Polysaccharide-Peptide complex to 28 age in days broiler chicken BSA antibody titers
Group BSA antibody titer (with the OD value representation) Mortality ratio (%)
A (blank group, no medicine) 0.5537±0.04 cB 6.8
B (microbiotic control group, Xin Meisu 20+ duomycin 100) 2.0123±0.40 bAB 3.1
C (add glycopeptide 0.1%, contain glycopeptide 2.5g/t feed) 2.5330±0.35 bcBA 3.4
D (add peptide 0.3%,, contain glycopeptide 7.5g/t feed) 2.4183±0.48 bcBA 2.1
E (add peptide 0.5%,, contain glycopeptide 12.5g/t feed) 4.1038±0.66 aA 1.8
Annotate: the right shoulder of colleague's numeral indicates different capitalizations (A, B, C) person and reaches extremely significantly (P＜0.01) for difference, the right shoulder of colleague's numeral indicates different lowercase person (a, b is significant difference (P＜0.05) c), and same letter person being arranged or do not have alphabetical mark person is difference not remarkable (p＞0.05).A, B, C, a, b, c are the method for expressing of test of significance on the statistics.
As can be seen from Table 8, the increase of the Polysaccharide-Peptide complex fodder additives addition that makes along with embodiment 4, the bovine serum albumin antibody titer rises in the fryer body, and mortality ratio descends, and E group and other each groups are compared significant difference (P＜0.05).In addition, the antibody titer of the broiler chicken of interpolation saccharide complex group is apparently higher than blank group and microbiotic group, and particularly the E group is more remarkable.Illustrate that the Polysaccharide-Peptide complex fodder additives that embodiment 4 makes can obviously improve the antibody titer (antibody titer is an important indicator of weighing body opposing cause of disease ability, and the antibody titer height shows that promptly the disease resistance of body is strong) of broiler chicken, and obviously reduce mortality ratio.

Claims (10)

  1. But 1, a kind of Ganoderma of high yield Polysaccharide-Peptide complex, its called after of classifying: Ganoderma GanodermaLucidum, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on November 22nd, 2004, its preserving number is CGMCC No.1251.
  2. But 2, the mutagenic breeding method of the Ganoderma of the described high yield Polysaccharide-Peptide complex of a kind of claim 1, its mutagenic and breeding step is as follows:
    1) the mycelial cultivation of starting strain CAU55
    In 4 ℃ of preservations, available from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", culture presevation number is that the Ganoderma CAU55 of CGMCC 5.533 is as starting strain with this laboratory;
    Starting strain CAU55 is inoculated on the sterilization solid medium 25～30 ℃ of solid culture 5～10 days; With cultured inoculation in the sterilising liq substratum, 28～32 ℃ of shake-flask culture 3～5 days are inoculated in cultured seed liquid in the sterilising liq substratum again, and 28～32 ℃ of shake flask fermentations were cultivated 3～5 days, obtain starting strain CAU55 hypha fluid, standby;
    2) preparation of starting strain CAU55 protoplastis
    Enzymolysis: the starting strain CAU55 hypha fluid of cultivating in the step 1) is centrifugal, remove supernatant, add sterilization homeo-osmosis agent 1～3ml, the vibration back is centrifugal, this step repetitive operation 2～3 times; After removing supernatant, in enzymolysis solution: mycelia body weight 1～4ml: the ratio of 1g, the enzymolysis solution after in the mycelium precipitation, adding bacteriological filtration, fully vibration was cultivated 1～3 hour for 25～30 ℃, and is standby;
    Filter: the hypha fluid that enzymolysis is good filters, and obtains starting strain CAU55 protoplastis precipitation;
    3) ultraviolet mutagenesis of protoplastis and regeneration are cultivated
    With step 2) gained starting strain CAU55 protoplastis precipitation makes suspension, and to transfer to the protoplastis number be 10 4～10 6Individual/ml, liquid layer thickness 0.2～0.4cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis is 15～18W, and irradiation time is 0.5～5min, irradiation distance 25～30cm; Dilution immediately is coated on the protoplast regeneration substratum after the irradiation, cultivated 6～10 days for 27～32 ℃, carry out primary dcreening operation, select 10～40 single faster bacterium colonies of mycelial growth speed, carrying out shake flask fermentation sieves again, measure its Polysaccharide-Peptide complex content with the phenolsulfuric acid method, select 6～10 strain Polysaccharide-Peptide complex superior strain CAU550N, do the mitotic stability experiment more respectively, it is the fastest to select a strain mycelial growth rate, the bacterial strain of the highest and good stability of Polysaccharide-Peptide complex output, but be the Ganoderma CAU5501 of high yield Polysaccharide-Peptide complex of the present invention, its called after of classifying: Ganoderma Ganoderma Lucidum, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on November 22nd, 2004, preserving number is CGMCC No.1251.
  3. But 3, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 2 is characterized in that: the solid medium of described step 1) is to prepare in following ratio: potato extracting solution 1000ml, glucose 20～30g, KH 2PO 41.5～3g, MgSO 47H 2O 0.75～2.5g, agar 15～20g.
  4. But 4, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 2 is characterized in that: the liquid nutrient medium of described step 1) is to prepare in following ratio: glucose 20～40g, peptone 2～4g, KH 2PO 40.5～2g, MgSO 47H 2O 0.5～2g, distilled water 1000ml.
  5. But 5, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 2, it is characterized in that: homeo-osmosis agent described step 2) is to prepare in following ratio: 80～130g glucose is dissolved in the 1000ml distilled water, and it is standby to sterilize.
  6. But 6, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 2, it is characterized in that: the protoplast regeneration substratum of described step 3) is to prepare in following ratio: D-cellobiose 10～15g, peptone 2～3.5g, yeast soak powder 1.5～4g, KH 2PO 42～3g, MgSO 47H 2O 1.0～2.5g, VB 120～50 μ g, homeo-osmosis agent 1000ml.
  7. But 7, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 2, it is characterized in that: the step of the mitotic stability experiment of described step 3) is: 6～10 strain Polysaccharide-Peptide complex superior strain CAU550N that will select, be inoculated into respectively on the sterilization solid medium, 25～30 ℃ of solid culture 5～8 days, inoculate after cultivating well on the sterilization solid medium, 25～30 ℃ of solid culture 5～8 days repeat this step 5～10 time, the speed of growth of observing CAU550N; The CAU550N in 5～10 generations of propagating is inoculated in the liquid nutrient medium, 28～32 ℃ of shake-flask culture 3～5 days, again cultured seed liquid is inoculated in the sterilising liq substratum, carry out shake flask fermentation, cultivated 3～5 days for 28～32 ℃, get hypha fluid, measure their Polysaccharide-Peptide complex output respectively, reject spawn degeneration, the bacterial strain that the speed of growth descends or production performance descends, keep fast growth, the bacterial strain that production performance is good, stable, but obtain the Ganoderma CAU5501 of high yield Polysaccharide-Peptide complex at last.
  8. But 8, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 2 is characterized in that: this method comprises that also the superior strain CAU5501 that will obtain is kept on the solid medium II, 4 ℃ of preservations, and switching in per 3 months is once.
  9. But 9, the mutagenic breeding method of the Ganoderma of high yield Polysaccharide-Peptide complex as claimed in claim 8 is characterized in that: described solid medium II prepares in following ratio: potato extracting solution 1000ml, glucose 20～30g, KH 2PO 41.5～3g, MgSO 47H 2O 0.75～2.5g, agar 15～20g.
  10. But 10, the purposes of the Ganoderma of the described high yield Polysaccharide-Peptide complex of a kind of claim 1, but the Ganoderma that is somebody's turn to do the high yield Polysaccharide-Peptide complex can be used for liquid fermenting and prepares the Polysaccharide-Peptide complex fodder additives, or is used to prepare microorganism feed addictive and immunostimulant.
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CN106754432A (en) * 2017-02-16 2017-05-31 江苏中祥高科技实业有限公司 A kind of production method of ganoderma lucidum complex immunity agent
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US10028480B2 (en) 2015-03-18 2018-07-24 Yunnan Mingshida-Science-Tech Co., Ltd. Ganoderma lucidum strain suitable for large-scale liquid fermentation culture, method of mutation breeding the same, and use of the strain
US10721903B2 (en) 2015-03-18 2020-07-28 Yunnan Mingshida-Science-Tech Co., Ltd. Ganoderma lucidum strain suitable for large-scale liquid fermentation culture and method of using the strain
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CN109943488B (en) * 2019-02-19 2022-05-03 中国科学院合肥物质科学研究院 Ganoderma lucidum polysaccharide high-yield strain RWHBW-1 and application thereof
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