CN100420736C - Method for preparing zymophyte powder of caterpillar fungus - Google Patents
Method for preparing zymophyte powder of caterpillar fungus Download PDFInfo
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- CN100420736C CN100420736C CNB2006100502728A CN200610050272A CN100420736C CN 100420736 C CN100420736 C CN 100420736C CN B2006100502728 A CNB2006100502728 A CN B2006100502728A CN 200610050272 A CN200610050272 A CN 200610050272A CN 100420736 C CN100420736 C CN 100420736C
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- caterpillar fungus
- preparation
- cordyceps sinensis
- powder
- solid
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- 239000003206 sterilizing agent Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 229940032362 superoxide dismutase Drugs 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
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- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
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Abstract
The present invention discloses a method for preparing Chinese caterpillar fungus zymocyte powder. The Chinese caterpillar fungus comes from the China center for Culture Collection of Agricultural Microorganisms, and the collection number is ACCC50560. The method comprises the following steps: 1) the preparation of a solid culture medium: first, a solid material is formed from 82 to 88 wt % of rice, 8 to 12 wt% of bran, 3 to 4 wt% of soybean flour and 1 to 2 wt % of ammonium sulfate, and after the solid material is pulverized, 0.9 to 1.1 wt% of water is added, and then the mixture is sterilized and cooled; 2) fermentation: under a sterile condition, cultured Chinese caterpillar fungus liquid seeds are inoculated to the solid medium with the thickness of 4.55cm to 5.5cm according to the proportion by weight of 1.5 to 2.5%, and are cultured for 7 to 10 days under the condition of 20 DEG C to 22 DEG C. Obtained white mycelium is dried under the condition of 40 DEG C to 50 DEG C and pulverized, and Chinese caterpillar fungus zymocyte powder is obtained. The method for preparing Chinese caterpillar fungus zymocyte powder has the advantages of low cost and high yield.
Description
Technical field
The present invention relates to a kind of preparation method of zymophyte powder of caterpillar fungus.
Background technology
Along with the fast development of modern medicine and chemical industry, fodder additives popularization and applications in feed constantly such as microbiotic, somatotropin, wormer, sanitas have been played huge pushing effect undoubtedly to the intensification Developing of Animal Industry.But this also causes the residual of animal products chemicals inevitably simultaneously, seriously jeopardizes human beings'health.Mainly show: the one, in feed, add antibiotic for a long time, can bring out the generation of interior high resistant microorganism group of fowl carcass and superbacteria, because these microorganisms have the ability of antagonism antibiotic, thereby in case infect human body, patient is pasted medical help, thereby the mankind are caused great threat.1992, the U.S. had the death because of the bacterial disease of the uncontrollable infection of microbiotic of people more than 12000 at least; The 2nd, the somatropin of interpolation synthetic in the feed makes the fowl dirty swine long unusual, and product has lost original nutritive value and local flavor, and quality descends, and commodity value reduces; The 3rd, the chemicals of synthetic such as antibiotic, somatotropin, sanitas, antioxidant, sterilizing agent often has toxic side effect, can cause distortion, the sudden change of normal tissue cell even cause canceration, be detrimental to health, these chemical substances are by the inrichment of fowl poultry, in the liver of fowl poultry and muscle, in large quantities under the accumulation, more aggravated hazardness; In addition, these chemicalses also may exist the not clear toxic side effect of some other mechanism, cause potential to threaten.Therefore, since the nineties in 20th century, developed country progressively strengthens the use control of animal breeding antibiotic etc, as U.S. food and FAD (FDA) several microbiotic below the cancellation from agricultural use: penicillin, tsiklomitsin, erythromycin, lincomycin, tylosin and virginiamycin; EU Committee's legislation forbids adding bacitracin, Spiramycin Base, virginiamycin, 4 kinds of microbiotic of tylosin feed from July 1st, 1999; British agriculture portion reports according to Swan, only allows the 4 kinds of antimicrobial drugs such as bacitracin, nitre furan alkene hydrazone with lower concentration to be used for cattle food; Germany forbids adding all microbiotic during raising section from the second half year in 1999; In the meeting of " microbiotic and the growth stimulant " that hold in Copenhagen in December, 1998, the participant thinks, will eliminate Antibiotic Additive gradually in 10 years of future.China is maximum in the world aquaculture big country, also is one of country that chemical for animals is the most general, usage quantity is high that uses at animal farming industry all kinds of non-nutritives such as all kinds of disease-resistant expelling parasites, promotes growth, mildew-resistant.In recent years owing to a large amount of uses of using even abuse, violate a ban, the hygienic quality of animal product is low, arrived startling degree in partial area, not only had a strong impact on human consumer's health, and the case of various in recent years animal product outlet experience importer Green Trade Barrier constantly takes place.Develop green meat, egg, milk deli and just becoming focus and the difficult point that everybody pays close attention to.The key problem that realizes livestock product " greenization " is few usefulness or without antibiotic etc class fodder additives.That development has is pollution-free, low residue, do not produce resistance, can promote growth of animals or poultry simultaneously, improves immunizing power, increase animal has become current animal husbandry development to the novel green additive of the resistibility of disease inexorable trend.The biological species green feed additive of exploitation comprises probiotics, zymin, herbal medicine, oligosaccharides and biologically active peptides and souring agent etc. both at home and abroad at present.
Utilize the immunologic function and the body defense function of the biologically active substance raising cultivated animals of natural origin, reach the purpose of preventing and treating Animal diseases and just paid attention to by more and more researchers.Chinese caterpillar fungus contains the abundant active substance of multiple content, comprises polysaccharide, N.F,USP MANNITOL, cordycepin, adenosine, amino acid and unsaturated fatty acids etc.It has immunomodulatory, anti-oxidant, regulate neuroendocrine, strengthen effects such as metabolism, and can stimulate the secretion of hormone in vivo to discharge, strengthen multiple efficacies such as cardiovascular systems, Digestive tract, hemopoietic system, Skeletal system.Therefore, Cordyceps sinensis can be used as the control that protective foods new resources and source new drugs have been widely used in human multiple disease.It has all obtained curative effect preferably at treatment cardiovascular disorder, respiratory system disease, sexual dysfunction, renal failure, hepatic diseases, tumour and blood, internal secretion, collagen systemic disease etc.But few because of Cordyceps sinensis quantity, cost an arm and a leg, can't in animal and fowl fodder, be used.
Summary of the invention
At the deficiencies in the prior art part, the invention provides the preparation method of the zymophyte powder of caterpillar fungus that a kind of cost is low, output is high.
The present invention is for reaching above purpose, be to realize: the preparation method that a kind of zymophyte powder of caterpillar fungus is provided by such technical scheme, Cordyceps sinensis is ACCC50560 from Chinese agriculture microbial strains preservation administrative center, preserving number, may further comprise the steps:
1), preparation solid medium: be that 82～88% rice, 8～12% wheat bran, 3～4% soyflour and 1～2% ammonium sulfate are formed solid material earlier by weight content; Add entry after then will solid material pulverizing, the weight ratio of solid material and water is 0.9～1.1, is that 0.1～0.15Mpa, temperature are the 40～60min that sterilizes under 121～125 ℃ the condition at pressure; Be cooled to 25～35 ℃ again;
2), fermentation: under aseptic condition, cultured Cordyceps sinensis liquid seeds is seeded on the solid medium that thickness is 4.5～5.5cm, the weight ratio of Cordyceps sinensis liquid seeds and solid medium is 1.5～2.5%, cultivates 7～10 days 20～22 ℃ of conditions; White hypha dry also pulverizing under 40～50 ℃ of conditions of gained promptly got zymophyte powder of caterpillar fungus.
A kind of improvement as the preparation method of zymophyte powder of caterpillar fungus of the present invention: cultured Cordyceps sinensis liquid seeds is seeded on the solid medium that thickness is 5cm, and the weight ratio of Cordyceps sinensis liquid seeds and solid medium is 2%.
Further improvement as the preparation method of zymophyte powder of caterpillar fungus of the present invention: above-mentioned cultured Cordyceps sinensis liquid seeds carries out following steps successively and makes:
1), the preparation first order seed: under aseptic condition, with preserving number be the former bacterial classification inoculation of ACCC50560 to the PDA solid medium, cultivated 5～7 days for 22 ℃;
2), preparation secondary seed: under aseptic condition, above-mentioned first order seed is seeded to the PDA liquid nutrient medium, cultivated 5～7 days for 22 ℃;
3), three grades of seeds of preparation: under aseptic condition, above-mentioned secondary seed is seeded to liquid nutrient medium, cultivated 3～5 days for 22 ℃; The weight composition of this liquid nutrient medium is: 1.5% glucose, 3% bean powder, 0.15% potassium primary phosphate, 0.15% sal epsom, all the other are water.
Use the zymophyte powder of caterpillar fungus of the solid state that preparation method of the present invention obtains, its polysaccharide content reach gross weight 24%, adenosine content reaches 150 μ g/g, cordycepin content reaches 3.0 μ g/g, these active ingredient content approach natural cs.Preparation method's technology of the present invention is simple, cost is low, is suitable for scale operation.Zymophyte powder of caterpillar fungus of the present invention has the effect that improves antioxidant ability of organism, animal immune relevant enzyme vigor and body fluid and cellular immunity.Zymophyte powder of caterpillar fungus of the present invention has the effect that improves livestock and poultry and aquatic animal to grow performance as green feed additive.
In order to prove creativeness of the present invention, the contriver has done following experiment:
Experiment 1, the zymophyte powder of caterpillar fungus of gained of the present invention is carried out measurement of the polysaccharide content
The sample polysaccharide extracts: zymophyte powder of caterpillar fungus is added the water of 5 times of volumes, 90--95 ℃ of digestion 10h, digestion liquid suction filtration, digestion 4-5 time, filtrate merges, and is evaporated to certain volume, the centrifugal filter residue that discards, adding 3 times of volume 95% ethanol sedimentations in supernatant liquor spends the night, centrifugal, collecting precipitation adds acetone, ether repetitive scrubbing, put then and vacuumize drying in the vacuum drier, get yellow powder shape Crude polysaccharides.
Adopt sulfuric acid-phynol method to measure polysaccharide content: accurately take by weighing analytical pure glucose 20mg in the 500mL volumetric flask, add water to scale, the concentration of the glucose reference liquid of this moment is 40 μ g/mL.Draw this solution 0.4,0.6,0.8,1.0,1.2,1.4,1.6 and 1.8ml respectively in the 10ml centrifuge tube, water mends volume to 2.0mL.Add 6% phenol 1.0ml and vitriol oil 5.0mL then, static 10min shakes up, and places 20min under the room temperature, the absorbancy of working sample under the 490nm wavelength (making blank with 2.0mL water).By sample concentration and light absorption value drawing standard curve.Get said extracted Cordyceps polysaccharide a little, measure with the preparation method by typical curve after the 2.0mL water dissolution, and by obtaining polysaccharide content on the typical curve.
After measured, Cordyceps sinensis solid fermentation bacterium powder polysaccharide content reaches 21.0%-26.0%.
Experiment 2, the zymophyte powder of caterpillar fungus of gained of the present invention carried out the mensuration of cordycepin and adenosine content
Sample pre-treatments: take by weighing about zymophyte powder of caterpillar fungus 0.5g, place triangular flask, add 10ml water, the ice-bath ultrasonic ripple is transferred in the centrifuge tube the centrifugal 10min of 2000rpm after handling 20min (ultrasonic 5s is 5s at interval), get supernatant liquor and cross 0.45 μ m membrane filtration, sample introduction 20 μ L during mensuration.
Buffer preparation: take by weighing SODIUM PHOSPHATE, MONOBASIC 3.12g, sodium hydrogen phosphate 1.41g adds water to 1L (pH 1) after water-soluble, shakes up after 0.45 μ m membrane filtration.
The standard substance preparation: accurately take by weighing cordycepin and adenosine standard substance 1.2mg and 8.3mg, be respectively applied in 10mL and the 8.3mL buffered soln, this is a standard reserving solution.Get each storing solution and be prepared into the working fluid that cordycepin and adenosine concentration are respectively 2.4 μ g/mL and 5.15 μ g/mL by three grades of dilutions successively, with cordycepin and adenosine working fluid with 3: 1 mixed, get the hybrid standard liquid that concentration is respectively 1.8 μ g/mL and 1.2875 μ g/mL, under the chromatographic condition of drafting, sample introduction 20 μ L.
Instrument: U.S. HP11OO series of high efficiency liquid chromatograph comprises automatic sampler, UV detector, quaternary gradient pump, online de-gassing vessel and HP chem workstation etc.
Chromatographic condition: mobile phase A: chromatographically pure methyl alcohol; Mobile phase B: phosphate buffer solution (pH, 6.8, take by weighing 3.12 gram SODIUM PHOSPHATE, MONOBASIC and 1.41 gram Sodium phosphate dibasics, be dissolved in the 1.0L water 0.45 μ m membrane filtration), adopt single concentration wash-out during operation, be 15.0min working time.Operation concentration: mobile phase A 8%, Mobile phase B 92%.Flow velocity: 1.0mL/min detects wavelength: 259nm, 30 ℃ of post oven temperature, degree, separator column: Hypersil ODS 5 μ m/100mm * 4.6mm.
After measured, Cordyceps sinensis solid fermentation bacterium powder adenosine content reaches 130-180 μ g/g, cordycepin content reaches 2.0-5.0 μ g/g.
The effect of the zymophyte powder of caterpillar fungus of experiment 3, gained of the present invention
1), the application on weanling pig:
Add 0.1-0.3%MSP and make DLY piglet day weight gain improve 5.44% in feed, feed efficiency and diarrhea rate have reduced by 4.24% and 75.15%, have reached the identical effect of microbiotic.Compare with control group, 0.2%MSP makes lactic acid bacteria number raising 141.67% in the piglet colon, and coliform count reduces by 82.47%; Make piglet jejunum villi height improve 29.45%, the gland nest degree of depth has reduced by 18.56%; Make that IgG and IgA have improved 22.02% and 69.23% respectively in the serum, make the T-lymphocyte transformation rate improve 75.91%.
2), the application on laying hen:
In diet, add 0.1-0.3%MSP and make the laying rate of commodity Dai Luoman laying hen improve 4.29%, egg laying performances such as feedstuff-egg ratio, rate of broken eggs, average daily food consumption are had the trend of improvement, but do not have significant difference; Adding 0.2%MSP can make in the laying hen serum superoxide-dismutase and catalase activity compare respectively according to improving 14.70% and 50.30%; Make in the laying hen small intestine coliform count reduce by 77.39% than control group, lactic acid bacteria number improves 2.32 times than control group; Make that NBT and T-lymphocyte transformation rate have improved 35% and 42.86% respectively in the laying hen whole blood.
3), the application on bolti:
Add 0.1-0.3%MSP in the feed and all can obviously improve the growth of bolti, improve day weight gain, specific growth rate, protein efficiency ratio, PER and the survival rate of bolti, can significantly reduce feed coefficient.Wherein, day weight gain has improved 15.56%; Specific growth rate has improved 10.19%, protein efficiency ratio, PER has improved 23.40%; Survival rate has improved 4.13%; Feed coefficient reduces by 11.59%; And compare no significant difference with the antibiosis group.In addition, add 0.20%MSP in the feed and can improve the digestibility of proteolytic enzyme, amylase and lipase activity and various nutrients in the fish enteron aisle, wherein dry-matter has improved 6.99%, crude protein has improved 3.82%, crude fat has improved 3.71%, improve the enteron aisle structure, improve blood middle leukocytes percentage phagocytosis and phagocytic index and lysozyme activity.
4), the application on prawn:
0.1-0.3%MSP can make the average weight gain of prawn improve 18.49% in feed, make the immunocompetence (hemocyte sum, respiratory burst vigor and hemocyte activate the phagocytic capacity) of prawn and also obviously improve with the Ia enzyme of prawn (blood plasma phenol oxidase, alkaline phosphatase, acid phosphatase and N,O-Diacetylmuramidase) activity, wherein serum neutral and alkali Phosphoric acid esterase, acid phosphatase and antalzyme activity also respectively comparison according to high by 123.61%, 78.14% and 28.30%.In addition, MSP can make superoxide dismutase activity, Total antioxidant capacity, reduced glutathion content and the active o content in the prawn liver pancreas obviously improve, wherein the latter two comparisons you can well imagine high 26.71% and 14.07% according to components, but activity of glutathione peroxidase and mda content do not make significant difference in the prawn liver pancreas.Add MSP and also significantly reduce vibrios sum sum in the prawn enteron aisle.
Embodiment
The preparation method of embodiment 1, a kind of zymophyte powder of caterpillar fungus, this Cordyceps sinensis is ACCC50560 from Chinese agriculture microbial strains preservation administrative center, preserving number, carries out following steps successively:
1), preparation first order seed (test tube slant seed): under aseptic condition, with preserving number be the former bacterial classification inoculation of ACCC50560 to the PDA solid medium, cultivated 5～7 days for 22 ℃; The moiety of this PDA solid medium is 1000 milliliters in peeled potatoes 200 gram, glucose 20 grams, agar 20 grams and a water.
2), preparation secondary seed (test tube liquid seeds): under aseptic condition, above-mentioned first order seed is seeded to the PDA liquid nutrient medium, cultivated 5～7 days for 22 ℃; The moiety of this PDA liquid nutrient medium is 1000 milliliters in peeled potatoes 200 gram, glucose 20 grams and a water.
3), preparation three grades of seeds (triangular flask liquid seeds): under aseptic condition, above-mentioned secondary seed is seeded to liquid nutrient medium, cultivated 3～5 days, and just can obtain the Cordyceps sinensis liquid seeds for 22 ℃; The weight composition of this liquid nutrient medium is: 1.5% glucose, 3% bean powder, 0.15%KH
2PO
4, 0.15%MgSO
4, all the other are water.
4), preparation solid medium: be that 85% rice, 10% wheat bran, 3.5% soyflour and 1.5% ammonium sulfate are formed solid material earlier by weight content; Then solid material being pulverized the water that the back adds identical weight, is that 0.11Mpa, temperature are the 50min that sterilizes under 121～125 ℃ the condition at pressure; Be cooled to 30 ℃ again.
5), fermentation: under aseptic condition, above-mentioned cultured Cordyceps sinensis liquid seeds is seeded on the solid medium that thickness is 5cm, the weight of this Cordyceps sinensis liquid seeds is 2% of solid medium, cultivates 7～10 days 20～22 ℃ of conditions then; The white hypha of gained is promptly got zymophyte powder of caterpillar fungus 40～50 ℃ of dry down also pulverizing.
At last, it is also to be noted that what more than enumerate only is a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. the preparation method of a zymophyte powder of caterpillar fungus, described Cordyceps sinensis is ACCC50560 from Chinese agriculture microbial strains preservation administrative center, preserving number, it is characterized in that may further comprise the steps:
1), preparation solid medium: be that 82～88% rice, 8～12% wheat bran, 3～4% soyflour and 1～2% ammonium sulfate are formed solid material earlier by weight content; Add entry after then will solid material pulverizing, the weight ratio of described solid material and water is 0.9～1.1, is that 0.1～0.15Mpa, temperature are the 40～60min that sterilizes under 121～125 ℃ the condition at pressure; Be cooled to 25～35 ℃ again;
2), fermentation: under aseptic condition, cultured described Cordyceps sinensis liquid seeds is seeded on the solid medium that thickness is 4.5～5.5cm, the weight ratio of described Cordyceps sinensis liquid seeds and solid medium is 1.5～2.5%, cultivates 7～10 days 20～22 ℃ of conditions; White hypha dry also pulverizing under 40～50 ℃ of conditions of gained promptly got zymophyte powder of caterpillar fungus.
2. the preparation method of a kind of zymophyte powder of caterpillar fungus according to claim 1, it is characterized in that: cultured Cordyceps sinensis liquid seeds is seeded on the solid medium that thickness is 5cm, and the weight ratio of described Cordyceps sinensis liquid seeds and solid medium is 2%.
3. the preparation method of a kind of zymophyte powder of caterpillar fungus according to claim 1 and 2, it is characterized in that: described cultured Cordyceps sinensis liquid seeds carries out following steps successively and makes:
1), the preparation first order seed: under aseptic condition, with preserving number be the former bacterial classification inoculation of ACCC50560 to the PDA solid medium, cultivated 5～7 days for 22 ℃;
2), preparation secondary seed: under aseptic condition, above-mentioned first order seed is seeded to the PDA liquid nutrient medium, cultivated 5～7 days for 22 ℃;
3), three grades of seeds of preparation: under aseptic condition, above-mentioned secondary seed is seeded to liquid nutrient medium, cultivated 3～5 days for 22 ℃; The weight composition of this liquid nutrient medium is: 1.5% glucose, 3% bean powder, 0.15% potassium primary phosphate, 0.15% sal epsom, all the other are water.
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|---|---|---|---|---|
| CN1275403A (en) * | 1999-09-01 | 2000-12-06 | 朱宏图 | Method for preparation of fungus powder medicine of artificial cultivated cordyceps |
| CN1372002A (en) * | 2001-02-26 | 2002-10-02 | 孙悦迎 | Method for producing cordycepin powder and active immne cordyceps sinensis polysaccharide frozen dried powder and products thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1275403A (en) * | 1999-09-01 | 2000-12-06 | 朱宏图 | Method for preparation of fungus powder medicine of artificial cultivated cordyceps |
| CN1372002A (en) * | 2001-02-26 | 2002-10-02 | 孙悦迎 | Method for producing cordycepin powder and active immne cordyceps sinensis polysaccharide frozen dried powder and products thereof |
Non-Patent Citations (4)
| Title |
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| 中国农业菌种目录. 姜瑞波 等，128. 2001 |
| 中国农业菌种目录. 姜瑞波 等，128. 2001 * |
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