CN103621564B - A kind of preparation method of biological antibiosis agent - Google Patents
A kind of preparation method of biological antibiosis agent Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of biological antibiosis agent, to induce source bacterial classification to be inoculated into respectively five five-age living silkworms biological antibody to carry out immunity cultivation, make biologically active non-pathogenic bacteria antibody bacterium liquid, then merged, through super-dry, add bio-hormone, magnetic agitation to cultivate to obtain; Or the bacterial classification that is prepared the mixing source kind bacterium spore suspension of bacterium and fungi by biological antibody induction source bacterial classification is inoculated on five five-age living silkworms, carry out antibacterial cultivation, then make with bio-hormone or the basic element of cell division. Prepared antiseptic product can prevent bacterial wilt and droop to crops, and the prevention incidence of disease reaches 80% ~ 90%; This product has stronger antibacterial action and antibacterial advantage efficient, wide spectrum, has completed pilot scale, and this method technology path is easy, and product is stable, low production cost, and productive rate degree of controllability is high, and pollution-free, achievement easily transforms, is convenient to industrialization and produces.
Description
Technical field
The present invention relates to a kind of antiseptic, specifically a kind of biological antibiosis agent.
Background technology
Antimicrobial (antimicrobialpeptide) or biological antibiosis agent are that the class that Immune System produces is supportedThe activated protein peptide material in anti-extraneous biological infection source extensively exists in insect, plant, animal and human body. It is apart from non-Outside the effect of specific antibacterium, fungi, virus, anti-infective, anti-disease, also has the work of anti-cell variation, antitumor cellWith, and do not damage human normal cell, especially still effective to multiple antibiotic resistant strain. Biological antibiosis agent is to Gram-positive and feminine genderThe endurance strains such as bacterium approximately more than 40 kinds, Pseudomonas aeruginosa and paratyphoid bacterium all have stronger killing action. Effectively biotic resistance medicineEmphasis and the focus of the research of the Wu Shi world today, the tomato being caused by Stalk Rot and pepper ralstonia solanacearum (BacterialThe crushing soil-borne disease of the worldwide bacterium such as the banana blight (Fusariumwilt) Wilt), being caused by Fusarium wiltThrough there being many research reports. But, utilize five age silkworm larva live body, through middle part, 1/2 sericterium place inoculated with subcutaneous injections induction is biologicalThe research that resistance is directly used in agricultural antibiotic prevention has no report.
Since present inventor place group guides from Guangdong Province's emphasis in 2004 subsidy of approving and initiate a project, ten years pairThe regenerated resources of silkworm have carried out going deep into systematic research, evidence silkworm has superpower immune protein synthesis capability, oneSilkworm, in the growth period less than one month, the mulberry leaf of edible 30 ~ 40g can synthesize and secrete and can utilize fresh sericterium 3 ~ 5g or spueThe fibroin of 0.5 ~ 1.5g. Evidence silkworm antimicrobial (antimicrobialpeptide) or biological antibiosis agent are silkwormsThe class that Immune System produces is resisted the activity polypeptid substance in extraneous biological infection source, has antibacterium, fungal infection, anti-Multiple antibiotic resistant strain variation. To Gram-positive, part gramnegative bacterium, to drug resistances such as Pseudomonas aeruginosa and paratyphoid bacteriumBacterial strain all has certain effect. Utilize biological antibiosis agent effectively to prevent the above disease of crops, especially at China coastThe environment of area warm moist reduces the incidence of disease can reach 80 ~ 90%. By the extensive use of biological antibiosis agent, can be in plant rootsIn border, body surface or body and in soil, multiply and surely grow fast, in a large number, effectively repel, stop and disturb plant pathogenic microorganismsTo the infecting and surely grow of plant, thereby reach antibacterial and effect diseases prevention. Not only direct Suppressing phytopathogens, and can lead toThereby the disease-resistant potential of crossing inducing plant self strengthens the disease resistance of plant. Can effectively prevent, alleviate agricultural pest, reduceThe amount of application of agricultural chemicals, reduces agricultural production and drops into, and guarantees agricultural product edible safety, realizes agricultural production and income, thus the thing of playingHalf merit doubly, the effect of environmental protection.
Now existing silkworm correlative study, produces anti-as Chinese patent ZL200910116463.3 discloses a kind of inducing silkwormsThe method of bacterium peptide, described the method is to adopt virus or bacterium injection, or can adopt the method that makes silkworm hunger; Adopt sickWhen poison or bacterium injection, inject micro-bacterium or virus to silkworm, adopt while making the method for silkworm hunger, do not feed mulberry to silkwormLeaf, allows its hungry 48 hours, more normally raises 24 hours, and abductive approach is simple described in this patent, can effectively induceMany antibacterial peptides, greatly improve antibacterial peptide extraction efficiency; Chinese patent ZL200910193620.0 discloses a kind of silkwormAntibacterial peptide BmcecE and cDNA sequence and application, the cultivated silkworm antimicrobial peptide of this invention is to gram-positive bacteria (Su Yun gold gemma barBacterium) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum) all there is antibacterial activity, anti-Bacterium spectrum is wide, and cultivated silkworm antimicrobial peptide BmcecE can be used for medicament research and development, prevention and the treatment of people, animal, fowl by bacillus thuringiensis, largeEnterobacteria, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum infect and cause causing a disease of people, animal, fowl, can also be used for biological productThe preparation of additive, prevents from being caused by bacillus thuringiensis, Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearumBiological product putrid and deteriorated.
Up to now, not yet have about utilizing immune induction technology, taking five age silkworm larva live body as raw material, in silkworm bodyPortion's 1/2 sericterium place inoculated with subcutaneous injections biotic induce bacterial classification is prepared biological antibiosis agent and is directly used in the concrete of agricultural antibiotic preventionReport.
Summary of the invention
The object of the invention is to overcome shortcoming or the deficiency of Modern Agriculture biological antibiosis agent, utilizes silkworm body to have superpower exempting fromEpidemic disease albumen synthesis capability, taking natural five age silkworm as inoculation raw material, by acceptor adaptive immunity and body fluid, cellular immunity approachThe activation signal factor, makes acceptor lymphocyte immune globulin produce the NK of antibody or activation, and preparation has spyThe pathogen of different resistance is lost to sick ability and for the biological antibiosis agent of agricultural production, reaches to alleviate toxic property agricultural chemicals and useAmount, reduces agriculture production cost, progressively rehabilitating soil ecology, make agricultural rationally, the eucyclic order of safety, efficient, high yield.
For addressing the above problem, the technical solution adopted in the present invention is as follows: a kind of preparation method of biological antibiosis agent,Be to induce source bacterial classification to be inoculated into respectively five five-age living silkworms biological antibody to carry out immunity cultivation, prepare each biological antibody induction source bacteriumKind biologically active non-pathogenic bacteria antibody bacterium liquid, then is merged, through super-dry, add bio-hormone, magnetic agitation cultivationObtain, concrete steps are as follows:
(1) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of Ralstonia solanacearum: under normal temperature, humidity 75 ~ 80% conditions, getThe five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections Ralstonia solanacearum bacterium of sterilizing are dense is 108The bacteria suspension 0.01mL of cfu/ml, entersAfter the living silkworm immunity of row 48h is cultivated, by the living silkworm after cultivating to organize pulverizer to organize pulverizing, magnetic under shading conditionPower stir culture 72h, heats to constant temperature 10-15min at 100 DEG C, filters, and filtered fluid leaves standstill 12-14h, obtains the biology of Ralstonia solanacearumActive non-pathogenic bacteria antibody bacterium liquid;
(2) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of phytophthora blight of pepper: in normal temperature, humidity 75 ~ 80% conditionsUnder, cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 106The phytophthora blight of pepper of the cfu/ml spore that moves aboutDaughter bacteria suspension 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to carry outTissue is pulverized, and shading condition lower magnetic force stir culture 72h heats to constant temperature 10-15min at 60 DEG C, filters, and filtered fluid leaves standstill12-14h, obtains the biologically active non-pathogenic bacteria antibody bacterium liquid of phytophthora blight of pepper;
(3) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of cucumber fusarium axysporum: in normal temperature, humidity 75 ~ 80% conditionsUnder, cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 106The cucumber fusarium axysporum spore of cfu/mlBacteria suspension 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to carry out groupKnit pulverizing, shading condition lower magnetic force stir culture 72h, heats to constant temperature 10-15min at 100 DEG C, filters, and filtered fluid leaves standstill 12-14h, obtains the biologically active non-pathogenic bacteria antibody bacterium liquid of cucumber fusarium axysporum;
(4) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of bacillus subtilis: in normal temperature, humidity 75 ~ 80% conditionsUnder, cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 109The bacillus subtilis bacterium of cfu/ml is outstandingLiquid 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to carry out tissue powderBroken, shading condition lower magnetic force stir culture 72h, heats to constant temperature 10-15min at 100 DEG C, filters, and filtered fluid leaves standstill 12-14h,Obtain the biologically active non-pathogenic bacteria antibody bacterium liquid of bacillus subtilis;
(5) by the biology of the above-mentioned Ralstonia solanacearum preparing, phytophthora blight of pepper, cucumber fusarium axysporum, bacillus subtilisActive non-pathogenic bacteria antibody bacterium liquid merges, and under infrared lamp, living silkworm tissue pulverizing liquid is dried to moisture and isAfter 70% ~ 80%, drop in 50kg reactor, and add bio-hormone, magnetic agitation is cultivated 120h, dry under 40 DEG C of infrared lamp illuminationDry, to obtain final product.
A preparation method for biological antibiosis agent, will prepare bacterium and fungi by biological antibody induction source bacterial classificationThe bacterial classification of mixing source kind bacterium spore suspension is inoculated on five five-age living silkworms, carries out antibacterial cultivation cultivation, then with bio-hormone systemStandby obtaining, concrete step is as follows:
(1) for the preparation of the mixing source kind bacterium spore suspension of the bacterium that inoculates and fungi: the green grass or young crops that collection is obtained is witheredThe bacteria suspension of bacterium, bacillus subtilis first uses respectively the 0.5% normal saline dilution bacterial classification prepared by sterilized water under the microscopeObserve a visual field 45-50 spore, then the bacteria suspension after diluting is respectively fitted together, mix, bacterium is dense is104Cfu/ml, is placed in 4 DEG C of refrigerators and preserves; The phytophthora blight of pepper that collection is obtained, the spore suspension of cucumber fusarium axysporum divideDo not use the 0.5% normal saline dilution bacterial classification of being prepared by sterilized water to examining under a microscope a visual field 75-80 spore, thenSpore suspension after diluting is respectively fitted together, mix bacterium dense 108cfu.g-1, protect being placed in 4 DEG C of refrigeratorsDeposit; The plastc ring finally twice assembling being obtained and mixing spore suspension fit together again, mix, and make and connectPlant bacterium, fungi mixing source kind bacterium spore suspension; Inoculum concentration is controlled at 1 × 10 with liquid bacteria seed6cfu.g-1, at 4 DEG C of iceIn case, preserve; Treat inoculation use;
(2) under the condition of normal temperature, humidity 75 ~ 80%, cancel poison after five five-age living silkworms, with skin test needle through five five-age living silkwormsMiddle division of silkgland 1/2 place is dense through inoculated with subcutaneous injections 0.01ml bacterium is 106cfu.g-1The bacterium that makes of step (1) and fungi mixedClose source kind bacterium spore suspension, carry out the antibacterial cultivation of living silkworm of 48h, cultivate and finish, with organizing the silkworm after pulverizer is cultivated to liveBody is pulverized, and pulverize the living silkworm tissue obtaining and pulverize in liquid input 50kg reactor, and add bio-hormone, be 200 at rotating speedUnder～500rp/min stirring condition, cultivate after 10 ~ 20d, heat to 60 DEG C, constant temperature 2 hours under this temperature conditions, filters,Can make.
A preparation method for biological antibiosis agent, will prepare bacterium and fungi by biological antibody induction source bacterial classificationThe bacterial classification of mixing source kind bacterium spore suspension is inoculated on five five-age living silkworms, carries out antibacterial cultivation cultivation, then with the basic element of cell divisionPrepare with clear water, concrete step is as follows:
(1) for the preparation of the mixing source kind bacterium spore suspension of the bacterium that inoculates and fungi: the green grass or young crops that collection is obtained is witheredThe bacteria suspension of bacterium, bacillus subtilis first uses respectively the 0.5% normal saline dilution bacterial classification prepared by sterilized water under the microscopeObserve a visual field 45-50 spore, then the bacteria suspension after diluting is respectively fitted together, mix, bacterium is dense is104Cfu/ml, is placed in 4 DEG C of refrigerators and preserves; The phytophthora blight of pepper that collection is obtained, the spore suspension of cucumber fusarium axysporum divideDo not use the 0.5% normal saline dilution bacterial classification of being prepared by sterilized water to examining under a microscope a visual field 75-80 spore, thenSpore suspension after diluting is respectively fitted together, mix bacterium dense 108cfu.g-1, protect being placed in 4 DEG C of refrigeratorsDeposit; The plastc ring finally twice assembling being obtained and mixing spore suspension fit together again, mix, and make and connectPlant bacterium, fungi mixing source kind bacterium spore suspension; Inoculum concentration is controlled at 1 × 10 with liquid bacteria seed6cfu.g-1, at 4 DEG C of iceIn case, preserve; Treat inoculation use;
(2) under normal temperature, humidity 75 ~ 80%, cancel poison after five age silkworm, skin test needle is through five five-age living silkworm middle division of silkgland 1/2 place's warpsInoculated with subcutaneous injections 0.01ml bacterium is dense is 106cfu.g-1Bacterium and the mixing source kind bacterium spore suspension of fungi, carry out 48h'sThe antibacterial cultivation of living silkworm, cultivates and finishes, and with organizing the living silkworm after pulverizer is cultivated to pulverize, the living silkworm tissue obtaining is pulverizedLiquid; In 50kg reactor, add 20kg clear water, add living silkworm tissue to pulverize liquid 20kg, then add basic element of cell division 500g,Stir culture 10 ~ 20d at normal temperatures, can make.
Bio-hormone of the present invention is dexamethasone sodium phosphate or dexamethasone acetate, bio-hormone and living silkworm groupThe usage ratio of knitting pulverizing liquid quality is 1-1.2:1000.
The basic element of cell division of the present invention is 6-BA, i.e. 6-benzyl aminoadenine.
Biological antibody of the present invention induction source bacterial classification is that Ralstonia solanacearum bacterium liquid, phytophthora blight of pepper zoospore liquid, cucumber are witheredWither germ spore liquid, bacillus subtilis bacterium liquid, its collection method is separately as follows:
(1) collection of biological antibody induction source Ralstonia solanacearum: from the disease serious plants stems body fluid of bacterial wilt, with rotating speed 1500The centrifugal extraction Stalk Rot of ~ 2000rp/min (Pseudomonassolanacearum) supernatant, gets 1mL supernatant,Water on rice medium with line partition method line, in 36 DEG C of winters, under summer room temperature, humidity 80-90%, cultivates after 3 ~ 5d, thenPicking colony central authorities pinkiness, outer rim are white butyrous, and wild type Ralstonia solanacearum colony lift in irregular shape is to sameIn sample culture medium, transfer, under room temperature, in dark, 200 ~ 500rpm/min speed oscillation is cultivated after 30 ~ 48d, and Ralstonia solanacearum short-term can be protectedExist on rice medium; According to turbidity counting method, be diluted to concentration as 10 taking aqua sterilisa8The bacteria suspension of cfu/ml, as connecingPlant body, for subsequent use;
(2) collection of biological antibody induction source phytophthora blight of pepper zoospore liquid: by pathogenic Phytophthora capsici bacteria strain fromIn test tube, be transferred on rice medium, under summer room temperature, in 36 DEG C of winters, humidity 80-90%, secretly cultivates 5-7d, covers with to myceliaMedia surface adds 300mL sterilized water in incubator, places it in illumination box and cultivates 7d, induces sporangiumGeneration; Taking-up is placed on 30 ~ 40min in 4 DEG C of refrigerators, and then room temperature is placed 40 ~ 60min, and ascus release trip is embraced in induction repeatedlyZoospore is cultivated after 3 ~ 5d, then picking bacterium piece is transferred in same culture medium and transfers, under room temperature with 200 ~ 500rpm/min rotating speedShaken cultivation 30 ~ 48d in the dark, is diluted to concentration as 10 taking aqua sterilisa6The bacteria suspension of cfu/ml, as inoculum, for subsequent use;
(3) collection and the sprouting of biological antibody induction source cucumber fusarium axysporum spore liquid: cucumber fusarium axysporum is seeded inOn rice medium, be placed in summer under room temperature, in 36 DEG C of winters, humidity 80-90%, secretly cultivates 6 ~ 8d; Get the newborn bacterium of mycelia extensionSilk, is inoculated in and in rice medium, adds 0.5% physiological saline 500mL, under room temperature with 200 ~ 500rpm/min rotating speed in the darkShaken cultivation 30 ~ 48d, induction Sporulation, nutrient solution, through Filter paper filtering, is removed mycelia, obtains and embraces sub-suspension; With aqua sterilisaBeing diluted to concentration is 106The bacteria suspension of cfu/ml, as inoculum, for subsequent use;
(4) collection method of biological antibody induction source bacillus subtilis bacterium liquid: buy by oneself by biologically pure commercial generation, go down to posterityNumber of times is no more than the bacillus subtilis bacterium liquid in 5 generations, gets bacterium liquid 1mL, is 50 ~ 100cfum, on inoculation rice medium, and winter38 DEG C, under summer normal temperature, humidity 80-90%, cultivates after 3 ~ 5d, is transferred to transferring in same culture medium chamber to picking bacterium piece againUnder temperature, with 200 ~ 500rpm/min rotating speed shaken cultivation cultivation 30 ~ 48d in the dark, add 0.5% physiological saline homogenate, use500ml centrifuge tube is centrifugal with 20000rpm/min rotating speed, gets supernatant, is diluted to concentration as 10 taking aqua sterilisa9cfu.g-1Preserve,For subsequent use.
Of the present invention five age silkworm larva live body acquisition pattern as follows: silkworm seed completes four times and casts off a skin spring from newly-hatched silkwormAbout 25 days, Xia Qiu 23 ~ 25 days, every silkworm is feed 30 ~ 40g mulberry leaf approximately, develop into 5 age silkworm, then after taking food 6-8 days, stop foodWhen mulberry leaf, become matured silkworm, entire body is transparent. Through artificial go-on-go, except the ripe silkworm of end removing, sprayed for five ages with 1/10000 potassium permanganateLiving silkworm, silkworm body liquid natural air drying, for subsequent use under room temperature.
The present invention with respect to the beneficial effect of prior art is:
(1) utilize silkworm body to there is superpower immune protein synthesis capability, taking natural five age silkworm as inoculation raw material, by being subject toBody adaptive immunity and body fluid, cellular immunity pathway activation signal factor, make acceptor lymphocyte immune globulin produce antibodyOr the NK activating, preparation has the biological antibiosis agent of specific resistance, and prepared antiseptic product is to cropsCan prevent bacterial wilt and droop, the prevention incidence of disease reaches 80% ~ 90%;
(2) this product can multiply and surely grow fast, in a large number in plant rhizosphere, body surface or body and in soil, effectivelyRepulsion, prevention and interference plant pathogenic microorganisms infect and surely grow plant, thereby reach antibacterial and effect diseases prevention; Not onlyDirectly Suppressing phytopathogens, thereby and can be by the disease resistance of the disease-resistant potential enhancing plant of inducing plant self;
(2) as agriculture biological and ecological methods to prevent plant disease, pests, and erosion antiseptic, be natural products, have no side effect, pollution-free, do not produce drug-resistant effect, rawProduce with low costly, productive rate is stable, and achievement easily transforms, is convenient to industrialization and produces.
Detailed description of the invention
Below by embodiment, the present invention is described in further details, these embodiment are only used for illustrating the present invention, andDo not limit the scope of the invention.
In following embodiment, five age silkworm larva live body acquisition pattern as follows: silkworm seed completes four times and sloughs off from newly-hatched silkwormPi Xiaqiu 25 days, every silkworm is feed 30 ~ 40g mulberry leaf approximately, develop into 5 age silkworm, then while stopping eating mulberry leaf after taking food 6-8 days, becomeFor matured silkworm, entire body is transparent. Through artificial go-on-go, except the ripe silkworm of end removing, spray five five-age living silkworms, silkworm with 1/10000 potassium permanganateBody liquid natural air drying, for subsequent use under room temperature.
Embodiment 1 adopts following steps to prepare the biological antibiosis agent of the present embodiment:
(1) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of Ralstonia solanacearum: under normal temperature, humidity 75% condition, cancel poisonFive five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections Ralstonia solanacearum bacterium are dense is 108The bacteria suspension 0.01mL of cfu/ml, carries outAfter the living silkworm immunity of 48h is cultivated, by the living silkworm after cultivating to organize pulverizer to organize pulverizing, shading condition lower magnetic forceStir culture 72h, heats to constant temperature 10min at 100 DEG C, filters, and filtered fluid leaves standstill 12h, obtains non-the causing of biologically active of Ralstonia solanacearumCharacteristic of disease bacteria antibody bacterium liquid;
(2) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of phytophthora blight of pepper: under normal temperature, humidity 80% condition, getThe five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium of sterilizing are dense is 106The phytophthora blight of pepper zoospore bacterium of cfu/mlSuspension 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to organizePulverize, shading condition lower magnetic force stir culture 72h, heats to constant temperature 12min at 60 DEG C, filters, and filtered fluid leaves standstill 14h, obtains pepperyThe biologically active non-pathogenic bacteria antibody bacterium liquid of green pepper phytophthora;
(3) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of cucumber fusarium axysporum: under normal temperature, humidity 78% condition,Cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 106The cucumber fusarium axysporum pityrosporion ovale of cfu/mlSuspension 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to organizePulverize, shading condition lower magnetic force stir culture 72h, heats to constant temperature 10min at 100 DEG C, filters, and filtered fluid leaves standstill 13h, obtains HuangThe biologically active non-pathogenic bacteria antibody bacterium liquid of cucurbit wilt bacterium;
(4) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of bacillus subtilis: under normal temperature, humidity 76% condition,Cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 109The bacillus subtilis bacteria suspension of cfu/ml0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to organize pulverizing,Shading condition lower magnetic force stir culture 72h, heats to constant temperature 15min at 100 DEG C, filters, and filtered fluid leaves standstill 12h, obtains withered grass budThe biologically active non-pathogenic bacteria antibody bacterium liquid of spore bacillus;
(5) by the biology of the above-mentioned Ralstonia solanacearum preparing, phytophthora blight of pepper, cucumber fusarium axysporum, bacillus subtilisActive non-pathogenic bacteria antibody bacterium liquid merges, and under infrared lamp, living silkworm tissue pulverizing liquid is dried to moisture and isAfter 75%, drop in 50kg reactor, and add bio-hormone dexamethasone sodium phosphate, bio-hormone and living silkworm tissue are pulverizedThe usage ratio of liquid quality is 1:1000, and magnetic agitation is cultivated 120h, dry under 40 DEG C of infrared lamp illumination, to obtain final product.
The biological antibody induction source bacterial classification of the present embodiment is that Ralstonia solanacearum bacterium liquid, phytophthora blight of pepper zoospore liquid, cucumber are witheredWither germ spore liquid, bacillus subtilis bacterium liquid, its collection method is separately as follows:
(1) collection of biological antibody induction source Ralstonia solanacearum: from the disease serious plants stems body fluid of bacterial wilt, with rotating speedThe centrifugal extraction Stalk Rot of 2000rp/min (Pseudomonassolanacearum) supernatant, gets 1mL supernatant, usesLine partition method line is watered on rice medium, and under summer room temperature, humidity 85%, cultivate after 3d, then picking colony central authorities is powderRed, outer rim is white butyrous, and wild type Ralstonia solanacearum colony lift in irregular shape is transferred to same culture medium, chamberIn the lower dark of temperature, 300rpm/min speed oscillation is cultivated after 40d, and Ralstonia solanacearum short-term can be kept on rice medium; According to turbidDegree counting method, is diluted to concentration as 10 taking aqua sterilisa8The bacteria suspension of cfu/ml, as inoculum, for subsequent use;
(2) collection of biological antibody induction source phytophthora blight of pepper zoospore liquid: by pathogenic Phytophthora capsici bacteria strain fromIn test tube, be transferred on rice medium, under summer room temperature, humidity 90%, secretly cultivates 5d, covers with media surface to mycelia,In incubator, add 300mL sterilized water, place it in illumination box and cultivate 7d, induce sporangial generation; Taking-up is putPut 350min in 4 DEG C of refrigerators, then room temperature is placed 50min, and induction is embraced after ascus release zoospore cultivation 4d repeatedly, then choosesGet bacterium piece and be transferred in same culture medium and transfer, under room temperature with 300rpm/min rotating speed shaken cultivation 48d in the dark, with sterilizingIt is 10 that water is diluted to concentration6The bacteria suspension of cfu/ml, as inoculum, for subsequent use;
(3) collection and the sprouting of biological antibody induction source cucumber fusarium axysporum spore liquid: cucumber fusarium axysporum is seeded inOn rice medium, be placed in summer under room temperature, humidity 80%, secretly cultivates 8d; The newborn mycelia of getting mycelia extension, is inoculated in riceIn culture medium, add 0.5% physiological saline 500mL, under room temperature, with 500rpm/min rotating speed shaken cultivation 30d in the dark, induceSporulation, nutrient solution, through Filter paper filtering, is removed mycelia, obtains and embraces sub-suspension; Be diluted to concentration as 10 taking aqua sterilisa6cfu/The bacteria suspension of ml, as inoculum, for subsequent use;
(4) collection method of biological antibody induction source bacillus subtilis bacterium liquid: buy by oneself by biologically pure commercial generation, go down to posterityNumber of times is no more than the bacillus subtilis bacterium liquid in 5 generations, gets bacterium liquid 1mL, is 100cfum, inoculation rice medium on, summer normal temperatureUnder, humidity 85%, cultivates after 3 ~ 5d, be transferred to transferring in same culture medium to picking bacterium piece again, under room temperature with 200rpm/minRotating speed in the dark shaken cultivation is cultivated 42d, adds 0.5% physiological saline homogenate, uses 500ml centrifuge tube with 20000rpm/minRotating speed is centrifugal, gets supernatant, is diluted to concentration as 10 taking aqua sterilisa9cfu.g-1Preserve, for subsequent use.
Embodiment 2 adopts following steps to prepare the biological antibiosis agent of the present embodiment:
(1) the mixing source kind bacterium spore suspension of bacterium and fungi adopts following steps: Ralstonia solanacearum that collection is obtained, witheredThe bacteria suspension of grass bacillus first uses respectively the 0.5% normal saline dilution bacterial classification of being prepared by sterilized water to examining under a microscope45, a visual field spore, then the bacteria suspension after diluting is respectively fitted together, mixing, bacterium is dense is 104Cfu/ml, putsIn 4 DEG C of refrigerators, preserve; The phytophthora blight of pepper that collection is obtained, the spore suspension of cucumber fusarium axysporum are used respectively by sterilized waterPreparation 0.5% normal saline dilution bacterial classification to examining under a microscope 78, visual field spore, then by respectively dilution after sporeSub-suspension fits together, and mixes bacterium dense 108cfu.g-1, preserve being placed in 4 DEG C of refrigerators; Finally by twice assemblingThe plastc ring obtaining and mixing spore suspension fit together again, mix, and make inoculated bacteria, fungi mixing sourcePlant bacterium spore suspension; Inoculum concentration is controlled at 1 × 10 with liquid bacteria seed6cfu.g-1, in 4 DEG C of refrigerators, preserve, treat inoculation use;(2) under the condition of normal temperature, humidity 78%, cancel poison after five five-age living silkworms, with skin test needle through five five-age living silkworm middles division of silkgland 1/2Locating dense through inoculated with subcutaneous injections 0.01ml bacterium is 106cfu.g-1Bacterium and the mixing source kind bacterium spore suspension of fungi, carry outThe antibacterial cultivation of living silkworm of 48h, cultivates and finishes, and with organizing the living silkworm after pulverizer is cultivated to pulverize, pulverizes the living silkworm obtainingTissue is pulverized liquid and is dropped in 50kg reactor, and adds bio-hormone dexamethasone acetate, bio-hormone and living silkworm tissueThe usage ratio of pulverizing liquid quality is 1.2:1000, is to cultivate after 15d under 400rp/min stirring condition at rotating speed, heats to 60DEG C, constant temperature 2 hours under this temperature conditions, filter, can make, by 500mL/ bottle with bottled.
Prove through test of many times, the product that the present embodiment prepares is when for crops prevention bacterial wilt and droopThe prevention incidence of disease reaches 80% ~ 90%, and this product has stronger antibacterial action and antibacterial advantage efficient, wide spectrum.
Embodiment 3 adopts following steps to prepare the biological antibiosis agent of the present embodiment:
(1) the mixing source kind bacterium spore suspension of bacterium and fungi adopts following steps: Ralstonia solanacearum that collection is obtained, witheredThe bacteria suspension of grass bacillus first uses respectively the 0.5% normal saline dilution bacterial classification of being prepared by sterilized water to examining under a microscope50, a visual field spore, then the bacteria suspension after diluting is respectively fitted together, mixing, bacterium is dense is 104Cfu/ml, putsIn 4 DEG C of refrigerators, preserve; The phytophthora blight of pepper that collection is obtained, the spore suspension of cucumber fusarium axysporum are used respectively by sterilized waterPreparation 0.5% normal saline dilution bacterial classification to examining under a microscope 80, visual field spore, then by respectively dilution after sporeSub-suspension fits together, and mixes bacterium dense 108cfu.g-1, preserve being placed in 4 DEG C of refrigerators; Finally by twice assemblingThe plastc ring obtaining and mixing spore suspension fit together again, mix, and make inoculated bacteria, fungi mixing sourcePlant bacterium spore suspension; Inoculum concentration is controlled at 1 × 10 with liquid bacteria seed6cfu.g-1, in 4 DEG C of refrigerators, preserve; Treat inoculation use;
(2) under normal temperature, humidity 75 ~ 80%, cancel poison after five age silkworm, skin test needle is through five five-age living silkworm middle division of silkgland 1/2 place's warpsInoculated with subcutaneous injections 0.01ml bacterium is dense is 106cfu.g-1Bacterium and the mixing source kind bacterium spore suspension of fungi, carry out 50h'sThe antibacterial cultivation of living silkworm, cultivates and finishes, and with organizing the living silkworm after pulverizer is cultivated to pulverize, the living silkworm tissue obtaining is pulverizedLiquid; In 50kg reactor, add 20kg clear water, add living silkworm tissue to pulverize liquid 20kg, then add basic element of cell division 6-benzyl ammoniaBase adenine 500g, stir culture 20d at normal temperatures, can make.
Prove through test of many times, the product that the present embodiment prepares is when for crops prevention bacterial wilt and droopThe prevention incidence of disease reaches 80% ~ 90%, and this product has stronger antibacterial action and antibacterial advantage efficient, wide spectrum.
Claims (1)
1. a preparation method for biological antibiosis agent, is characterized in that: induce source bacterial classification to be inoculated into respectively for five ages biological antibodyLiving silkworm carries out immunity to be cultivated, and prepares the biologically active non-pathogenic bacteria antibody bacterium liquid of each biological antibody induction source bacterial classification, then willIt merges, and through super-dry, add bio-hormone, magnetic agitation to cultivate to obtain, concrete steps are as follows:
(1) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of Ralstonia solanacearum: under normal temperature, humidity 75 ~ 80% conditions, cancel poisonFive five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium are dense is 108The Ralstonia solanacearum bacteria suspension 0.01mL of cfu/ml, carries outAfter the living silkworm immunity of 48h is cultivated, by the living silkworm after cultivating to organize pulverizer to organize pulverizing, shading condition lower magnetic forceStir culture 72h, heats to constant temperature 10-15min at 100 DEG C, filters, and filtered fluid leaves standstill 12-14h, the biology of acquisition Ralstonia solanacearumActive non-pathogenic bacteria antibody bacterium liquid;
(2) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of phytophthora blight of pepper: under normal temperature, humidity 75 ~ 80% conditions, getThe five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium of sterilizing are dense is 106The phytophthora blight of pepper zoospore bacterium of cfu/mlSuspension 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to organizePulverize, shading condition lower magnetic force stir culture 72h, heats to constant temperature 10-15min at 60 DEG C, filters, and filtered fluid leaves standstill 12-14h, the biologically active non-pathogenic bacteria antibody bacterium liquid of acquisition phytophthora blight of pepper;
(3) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of cucumber fusarium axysporum: under normal temperature, humidity 75 ~ 80% conditions,Cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 106The cucumber fusarium axysporum pityrosporion ovale of cfu/mlSuspension 0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to organizePulverize, shading condition lower magnetic force stir culture 72h, heats to constant temperature 10-15min at 100 DEG C, filters, and filtered fluid leaves standstill 12-14h, the biologically active non-pathogenic bacteria antibody bacterium liquid of acquisition cucumber fusarium axysporum;
(4) prepare the biologically active non-pathogenic bacteria antibody bacterium liquid of bacillus subtilis: under normal temperature, humidity 75 ~ 80% conditions,Cancelling malicious five five-age living silkworm middle part 1/2 sericterium place inoculated with subcutaneous injections bacterium dense is 109The bacillus subtilis bacteria suspension of cfu/ml0.01mL, after the living silkworm immunity of carrying out 48 hours is cultivated, by the living silkworm after cultivating to organize pulverizer to organize pulverizing,Shading condition lower magnetic force stir culture 72h, heats to constant temperature 10-15min at 100 DEG C, filters, and filtered fluid leaves standstill 12-14h, obtainsObtain the biologically active non-pathogenic bacteria antibody bacterium liquid of bacillus subtilis;
(5) by the biologically active of the above-mentioned Ralstonia solanacearum preparing, phytophthora blight of pepper, cucumber fusarium axysporum, bacillus subtilisNon-pathogenic bacteria antibody bacterium liquid merges, living silkworm tissue is pulverized to liquid under infrared lamp to be dried to moisture and to be 70% ~After 80%, drop in 50kg reactor, and add bio-hormone, magnetic agitation is cultivated 120h, dry under 40 DEG C of infrared lamp illumination,Obtain;
Wherein, described bio-hormone is dexamethasone sodium phosphate or dexamethasone acetate, bio-hormone and living silkworm tissue powderThe usage ratio of broken liquid quality is 1-1.2:1000.
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| CN102533678A (en) * | 2012-01-10 | 2012-07-04 | 特菲(天津)生物医药科技有限公司 | Vivax malaria erythrocytic stage vaccine and preparation method thereof |
| CN102584452A (en) * | 2012-02-22 | 2012-07-18 | 湛江师范学院 | Method for preparing biological antibacterial organic fertilizer by using immune induced biological technology |
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| CN102533678A (en) * | 2012-01-10 | 2012-07-04 | 特菲(天津)生物医药科技有限公司 | Vivax malaria erythrocytic stage vaccine and preparation method thereof |
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