CN102391978A - Aloe endophytic bacterium with broad-spectrum antibacterial activity and colonization capability in animal intestinal tract - Google Patents

Abstract

An aloe endophytic bacterium with broad-spectrum antibacterial activity and colonization capability in animal intestinal tracts of Paenibacillussp. Aloe-11 is separated from a three-year-old Chinese aloe artificially cultivated by the applicant, and is identified as a paenibacillus ash based on the morphological characteristics, physiology, biochemistry, and molecular biology technology; the optimal carbon source and nitrogen source for fermentation are respectively cane sugar and ammonium nitrate (generally a PDA culture medium is selected); the optimal fermentation condition is 28 DEG C; the pH adaptation range is 4-10; several active substances with a broad-spectrum antibacterial function and a growth promotion function are generated during secondary metabolism; the bacterium also has the functions of nitrogen fixation and colonization in animal intestinal tracts; therefore, the invention is applicable to the development of new antibiotics, growth promoters, animal microbial ecological agents, and the like, and has potential business development and application value.

Description

One strain has the aloe endogenetic bacteria of broad spectrum antibiotic activity and animal intestinal field planting ability

Technical field

The present invention relates to microorganism field, be specifically related to a strain aloe endogenetic bacteria Paenibacillus sp.Aloe -11 and use.

Background technology

Since penicillium mould was found, people hankered after antibiotic research and use always.Antibiotic discovery is undoubtedly the last great transition of physianthropy history, particularly in the control of some bacterial infection diseases.But As time goes on, antibiotic frequent use makes the bacterial drug resistance problem constantly manifest, and has occurred almost anti-all existing antibiotic superbacterias in some areas, so the new antibiotic research and development is extremely urgent.Traditional production of antibiotics derives from the secondary metabolite of some soil microorganisms fermentations mostly, and in recent years, endophyte of plant also can produce multiple material with different anti-microbial activities because of its source is abundant, receives people's attention gradually.

DeBary had proposed endophyte of plant (endophyte) speech at first in 1866, and definition was at that time: within plant tissue the mikrobe of referring to live, come with plant surface mikrobe (epiphyte) difference.By popular generally accepted definition be now: refer to various tissues and inner fungi or the bacterium of organ that those live in health plant; In the certain phase or whole life process of its life history; Host plant does not show external illness; Can isolate through Histological method or from the plant tissue of strict surface sterilization, perhaps the method through DNA cloning proves.Plant and its endophyte generally all are reciprocal symbiosis, and plant is that endophyte provides photosynthate and mineral substance on the one hand; What the metabolite of endophyte can stimulating plant on the other hand grows, and improves host plant biology is coerced the resistivity with abiotic stress.The report that many relevant endophyte of plant can produce antimicrobial substance or other physiologically active substance constantly appearred in recent years; Wherein widespread reports the microbiotic that produces of endophyte of plant not only kind is many; And wherein existence is undeveloped novel substance greatly, is the valuable source of research and development new antibiotic.At present relevant endophyte of plant in animal intestinal field planting and do not appear in the newspapers as yet based on the animal microecological formulation research of endophyte of plant.

Summary of the invention

It is active and have an endophytic bacterium of nitrogen fixation to the purpose of this invention is to provide a strain and have broad spectrum antibiotic activity, actuate plant-growth.According to morphological feature, Physiology and biochemistry and Protocols in Molecular Biology it is accredited as the novel strain that a series bacillus belongs to (Paenibacillus); Its biological characteristics is furtherd investigate, confirmed optimum condition, the antibiotic antimicrobial spectrum of producing and animal intestinal colonization ability that its growth and microbiotic produce.

The present invention realizes through following technical scheme:

Bacterial strain of the present invention comes through the following steps isolation identification:

3 years living the Chinese aloe aloes of applicant's artificial culture; Get its root, stem, leaf three parts to its surface sterilization sterilization back section; Adopt potato sucrose substratum (PDA); Obtain dissimilar bacteriums through cultivation and purifying, be transferred to respectively again and carry out the thalline fermentation on the PDA liquid nutrient medium, confirm to produce the bacterium (Aloe-11) of bacteriostatic activity material at last through the bacteriostatic experiment of fermented liquid.According to " uncle Jie Shi Bacteria Identification handbook is measured and 16SrRNA sequencing analysis (AC:GU481684) through morphological observation, physiological and biochemical property, confirms Aloe-11 type of belonging to bud pole Pseudomonas.

Endophytic bacterium of the present invention belongs to series bacillus and belongs to called after Paenibacillus sp.Aloe-11 is deposited in CCTCC, deposit number No:M 2011237, preservation date 2011.07.12.

Below do Paenibacillus sp. Aloe-11 describes in detail:

Paenibacillus sp. the morphological specificity of Aloe-11 bacterial strain and physiological and biochemical property: On potato sucrose solid medium (PDA), colony growth is fastest, is creamy white, and bacterium colony surface wettability, protuberance become concentric(al) circles to grow towards periphery in media surface, with the prolongation of incubation time, forms bigger bacterium colony.On No. 1 substratum of Gao Shi, Cha Shi substratum, grow better, the flora of going up cultivation with PDA compares, and the form color distortion of its bacterium colony is little, but the bacterium colony of formation protuberance is gone up height than PDA.This bacterial strain Gram-positive or feminine gender, thalline are shaft-like, and size is 0.8 * 3.2 m; Produce gemma, gemma is long oval, and catalase is positive; Amphimicrobian is according to " uncle Jie Shi Bacteria Identification handbook belongs to bacillus, analyzes in conjunction with the 16SrRNA sequencing; This bacterium is that series bacillus belongs to, and the Latin formal name used at school does Paenibacillus sp. Aloe-11Its physiological and biochemical property is as shown in table 1.

Table 1 bacterial strain Paenibacillus sp.The physiological and biochemical property of Aloe-11

Test event	Measure the result	Test event	Measure the result	Test event	Measure the result
						Gramstaining	+/-, shaft-like, 0.8 * 3.2 m	Salt tolerance (Nacl concentration)	0%-4%	SANMALT-S	+, produce acid, aerogenesis
Flagella staining	Peritrichous	Hydrogen sulfide produces	-	N.F,USP MANNITOL	+, produce acid, aerogenesis
						The dyeing of bud robe	+, long oval	Nitrate reduction	+	Art sugar	+
Mobility	+	Rely helium acid	+	Urea	+
						Catalase	+	The starch hydrolysis	+, produce acid, aerogenesis	The acid of egg helium	-
Oxydase	+	Glucose	+, produce acid, aerogenesis	The acid of bird helium	+
						Gelatine liquefication	+	Fructose	+, produce acid, aerogenesis	Phenylalanine(Phe)	+
The V.P test	+	Lactose	+, produce acid, aerogenesis	?	?

"+" represents test result positive, and "-" represents test result negative.

The antimicrobial substance optimal conditions of fermentation:Aloe-11 the righttest carbon source of fermentation and nitrogenous source are respectively sucrose and an ammonium nitrate (generally selecting the PDA substratum for use); The fermentation top condition is 28 ℃ of (10 ℃-45 ℃ of adaptive temperature scopes), 200r/min 72h that ferments; Liquid amount 30%; Inoculum size 5%, original ph are pH 7-8 (accommodation pH 4-10).After adding inorganic nitrogen, organonitrogen or not adding several kinds of processing of nitrogenous source, cultivated 72 hours.Compare with contrast, add the generation that organic nitrogen source can suppress active substance; And that the addition of organic nitrogen source is 2% o'clock an inhibition effect is more obvious than 0.5%.And from inorganic nitrogen-sourced influence, except that Sodium Nitrite was inhibited, other inorganic nitrogen-sourced generation that helps active substance to a certain extent especially when inorganic nitrogen-sourced addition is 0.5%, had strengthened the anti-microbial activity of active substance.Fermentation promotor methyl alcohol commonly used both can't be utilized by Aloe-11 as carbon source, and Aloe-11 being produced active substance does not have any promoter action yet; Tensio-active agent (Tween-80) then plays restraining effect to the generation of antibacterial substance.

Paenibacillus sp. Aloe-11 anti-microbial activity:The secondary metabolite that this bacterial strain produces all shows good broad-spectrum antibacterial action to pathogenetic bacteria and fungi.With pathogenic bacterias such as intestinal bacteria, klebsiella, streptococcus aureus, streptococcus suis 2-type, Bacillus thuringiensis, subtilis, gibberella saubinetii, pink powder pathogenic bacteria, Chinese cabbage dehiscence bacterium, Sclerotinia sclerotiorum, the former bacterium of peach brown rot, oranges and tangerines green mold, grey mold, rotten mould, mycoplasma gallinarums is antibacterial test bacterium; Adopt cup-plate method, suppress methods such as mycelial growth rate method, minimal inhibitory concentration (MIC) mensuration, the antibacterial substance that endophyte Aloe-11 is produced has carried out the antimicrobial spectrum analysis.The result shows: the antibacterial substance that Aloe-11 produces all has very strong restraining effect to gram-positive microorganism such as streptococcus aureus, streptococcus suis 2-type, Bacillus thuringiensis, subtilis and the plant pathogenic fungi tested, and to Gram-negative bacteria and mycoplasma gallinarum DeGrains such as intestinal bacteria, klebsiellas.

Fixed nitrogen and the growth promoting function of Paenibacillus sp. Aloe-11:This bacterium grows on vinelandii isolation medium Ah Xu Bei Shi (Ashby) substratum better.Mix the back to this bacterium with the another kind of bacterium that can decompose agar-agar and on the agar plate that does not have other carbon source (only containing agar) and nitrogenous source, cultivate, it is very good that the result finds that two kinds of bacterium all grow, and in this substratum, do not grow separately.Through to the complete genomic sequencing of this bacterial strain (genome sequence number: AGFI00000000) with the gene compare of analysis, find this bacterial strain exist a series of can coding and the genes that utilize inorganic nitrogen and promoting growth of plants function associated protein.

The field planting of Paenibacillus sp. Aloe-11 in animal intestinal:This bacterium can produce plurality of enzymes such as cellulase, glycase, LSD.PDA fermented liquid through Aloe-11 bacterium that mouse and chick are fed finds, this bacterium has in a large number in its small intestine separately and grows surely, feeds this bacterium and can also detect this bacterium at its enteron aisle after 15 days disconnected, can significantly improve cellulosic utilization in the feed behind this bacterium of feeding.

Description of drawings:

Fig. 1 is the morphological specificity of Aloe-11 thalline (10 * 100)

Fig. 2 is the antibiotic situation of antibacterial substance to the bacillus green mold

Fig. 3 is the antibiotic situation of antibacterial substance to the former bacterium of peach brown rot

Fig. 4 is the antibiotic situation of antibacterial substance to pink powder pathogenic bacteria

Fig. 5 is the antibiotic situation of antibacterial substance to Sclerotinia sclerotiorum

Advantage of the present invention is: from aloe, separate a strain endophyte A11 (Paenibacillus sp. Aloe-11) who obtains; Its secondary metabolism produces some and has broad-spectrum antimicrobial and the active substance that promotes functions such as growth; This bacterium also has the effect of fixed nitrogen and field planting in animal intestinal simultaneously; So the present invention can be used for researching and developing new antibiotic, growth promoter, animal microecological formulation etc., has potential business development and using value.

Embodiment

Below in conjunction with embodiment the present invention is further described

Embodiment 1:

Paenibacillus sp.The separation and purification of Aloe-11 bacterial strain and evaluation

Material: 3 years living aloes of Southwestern University's artificial culture.Substratum: potato sucrose substratum (PDA), by conventional compound method preparation.

The separation of endophyte: get aloetic root, stem, leaf respectively water rinse well, after 75% alcohol-pickled 6 minutes, use aseptic water washing 3-4 time again; Soak after 50 seconds aseptic water washing 5 times with 0.1% mercuric chloride then.Dry the back and be cut into the fritter that 0.5cm grows after therefrom cutting, be cut into the thin slice of the wide 0.5cm of long 0.8cm to blade, insert PDA solid culture primary surface to them with aseptic hilt root, stem; 28 ℃ of constant temperature culture; After treating that its edge grows bacterium, picking colony carries out purifying and cultivates the 4 ℃ of preservations in PDA inclined-plane.

The thalline fermentation: the PDA liquid nutrient medium, 20% loading amount, 16 hours seed culture fluid is cultivated in inoculation 1%, and 28 ℃ of 150r/min shaker fermentations were cultivated 72 hours, handled 15 minutes for 121 ℃, and the centrifuging and taking supernatant is cooked following bacteriostatic experiment.

Bacteriostatic experiment: punch method.Punch tool with diameter 0.5cm punches in the petridish of good substratum; Surface coated indicator suspension to be detected; Add fermented liquid in the hole, lie against in 37 ℃ (bacterium does indicator) or 28 ℃ of (fungi does indicator) constant incubators and cultivate, observe the inhibition zone size.To only occur the corresponding microbionation of the fermented liquid of obvious fungistatic effect again to the PDA solid medium, constant temperature culture makes it form single bacterium colony, and preservation is subsequent use.

The evaluation of bacterial strain: with reference to " uncle Jie Shi Bacteria Identification handbook; Measure through morphological observation and Physiology and biochemistry; This bacterial strain be Gram-positive/negative bacterium, shaft-like, produce gemma, positive, the amphimicrobian of catalase; Analyze in conjunction with the 16SrRNA sequencing, confirm Aloe-11 type of belonging to bud pole Pseudomonas.

Embodiment 2:

Confirming of optimal conditions of fermentation

The activation bacterium: connect bacterium to the PDA inclined-plane from-80 ℃, 28 ℃ cultivate 36h after, enlarge and carry out seed culture, from the inclined-plane picking one ring bacterial classification inoculation in the PDA nutrient solution, 28 ℃, 200r/min shaking culture 15h.

Determination of activity: 121 ℃ of sterilizations of fermented liquid 15min, the centrifuging and taking supernatant is an indicator with the streptococcus aureus, adopts punch method to detect the inhibition zone size of active substance in the supernatant.

(1) liquid amount influence that antimicrobial substance is produced: the PDA liquid nutrient medium of in the 100ml triangular flask, packing into of the liquid amount by 10%, 20%, 30%, 40%, 50%; The seed culture fluid of inoculation 5%; 28 ℃, 200r/min shaking table cultivation 72h; Measure the inhibition zone size through the fermented liquid bacteriostatic experiment, confirm the Aloe-11 best liquid amount that ferments thus.

(2) original ph influence that antimicrobial substance is produced: the pH value of PDA liquid nutrient medium is adjusted to 2,3,4,5,6,7,8,9 respectively; The seed culture fluid of inoculation 5%; 28 ℃, 200r/min shaking table cultivation 72h; Measure the inhibition zone size through the fermented liquid bacteriostatic experiment, confirm the Aloe-11 best original ph of fermenting thus.

(3) inoculum size influence that antimicrobial substance is produced: cultured A11 seed liquor is inserted in the fermention medium by 1%, 5%, 10%, 15%, 20%, 25% inoculum size, and 28 ℃, 200r/min shaking table are cultivated 72h.Observe fermentation broth viscosity, and detect the active size of the antimicrobial substance of its generation, confirm Aloe-11 fermentation optimum inoculation amount thus through the fermented liquid bacteriostatic experiment.

(4) different carbon sources influence that fermented liquid viscosity and antimicrobial substance are produced: with the yam leach liquor is basic medium; Nature pH; Liquid amount branch by 20% installs in the 100ml triangular flask; Add glucose, sucrose, SANMALT-S, lactose, Zulkovsky starch, N.F,USP MANNITOL, dextrin, Hydrocerol A, glycerine respectively by 2% of culture volume again, compare, the seed culture fluid of inoculation 5% not add any carbon source.28 ℃, 200r/min shaking table cultivation 72h.Observe fermentation broth viscosity, and detect the active size of the antimicrobial substance of its generation, confirm Aloe-11 fermentation optimum carbon source thus through the fermented liquid bacteriostatic experiment.

(5) different nitrogen sources influence that fermented liquid viscosity and antimicrobial substance are produced: with the yam leach liquor is basic medium; Nature pH; Liquid amount branch by 20% installs in the 100ml triangular flask; Add an ammonium nitrate, Sodium Nitrite, ammonium chloride, ammonium sulfate, Carnis Bovis seu Bubali cream, peptone, yeast powder, yeast extract paste, fish meal, soya-bean cake respectively by 0.5% and 2% of culture volume again, compare, the seed culture fluid of inoculation 5% not add any nitrogenous source.28 ℃, 200r/min shaking table cultivation 72h.Observe fermentation broth viscosity, and detect the active size of the antimicrobial substance of its generation, confirm Aloe-11 fermentation optimum nitrogen source thus through the fermented liquid bacteriostatic experiment.

(6) methyl alcohol influence that antimicrobial substance is produced: the seed culture fluid of inoculation 5% in the PDA fermentation culture; After 28 ℃, 200r/min shaking table are cultivated 24h; Add 0%, 1%, 1.5%, 2%, 2.5%, 3%, 4% methanol solution respectively; Continue 28 ℃, 200r/min shaking table and be cultured to 72h, measure the inhibition zone size, confirm the influence of methyl alcohol thus the Aloe-11 fermentation through the fermented liquid bacteriostatic experiment.

(7) tensio-active agent influence that antimicrobial substance is produced: the seed culture fluid of inoculation 5% in the PDA fermentation culture; After 28 ℃, 200r/min shaking table are cultivated 24h; Add 0%, 0.01%, 0.05%, 0.1%, 0.15%, 0.2% Tween-80 solution respectively; Continue 28 ℃, 200r/min shaking table and be cultured to 72h, measure the inhibition zone size, confirm to show the influence of promoting agent thus the Aloe-11 fermentation through the fermented liquid bacteriostatic experiment.

Embodiment 3:

The separation and purification of fermented liquid active substance and analysis

(1) preparation of fermentation using bacteria product: with the A11 colony inoculation in the solid PDA substratum (every 100mL triangular flask packing 25mL seed culture medium) in the seed liquor substratum that configures, 28 ℃, 200r/min are cultivated 15h in the constant-temperature shaking culture case.The fermention medium branch that configures is installed in the 500mL triangular flask every bottle of packing 120mL substratum; Under aseptic technique, seed liquor is inserted respectively in each bottle fermention medium, inoculum size is 5%.Fermention medium is cultured continuously 48h under 28 ℃, 200r/min condition.

(2) slightly carrying of antibacterial substance: with the fermented liquid of 48h with the absolute ethyl alcohol of 3 times of volumes precipitate, extracting.After fully stirring, vibrating, through filter paper filtering, gained filtrating is through obtaining crude extract behind the rotary evaporation with extract, and with the amount of methanol dissolving, gained solution is the active substance crude extract to crude extract again.

(3) silica gel separates: with 250mL silica gel (200-300 order) after 110 ℃ of activation, with the methyl alcohol of 1:9: the chromatography column of packing into after the abundant swelling of ETHYLE ACETATE; (methyl alcohol of 1:9: ETHYLE ACETATE) chromatography column is carried out balance, flow velocity is 10mL/min during balance with the initial moving phase that doubles the post appearance; The crude extract of 6mL is splined on the silicagel column after the balance, and uses 1:9,2:8,3:7,4:6; 5:5,6:4,7:3,8:2,9:1; The methyl alcohol of 10:0: ETHYLE ACETATE carries out gradient elution, and each gradient elution volume is 500mL, and elution speed is 5mL/min, and every 50mL collects a component; Each component of collecting is carried out the bacteriostatic activity test with punch method; Have active component and carry out purifying with 300-400 purpose silica gel once more, purified reagent, method are with the silica gel purification first time.

(4) polydextran gel separates: after the isolating active ingredient of silica gel merges for the second time, to dry, add the small amount of methanol dissolving through rotary evaporation in the dry sample, the gained solution example is all article.Polydextran gel Sephadex LH-20 is packed into after with methyl alcohol swelling (swelling coefficient is 4) in the chromatography column; With 2 times methyl alcohol (analytical pure methyl alcohol is through the organic membrane filtration of 0.45um) balanced gel post, flow velocity is 1mL/min during balance; Separate carrying out exclusion chromatography in appearance to the VISOSE chromatography column on the molten sample of above-mentioned methyl alcohol, moving phase is methyl alcohol (analytical pure methyl alcohol is through the organic membrane filtration of 0.45um), and elution speed is 0.3mL/min, and elution volume is 4 times a column volume.Component under the wash-out is collected through full-automatic Fraction Collector, component of collection in per 10 minutes; The component of collecting is carried out the bacteriostatic activity test through punch method, will have active component and merge.

(5) HPLC purifying: active substance is carried out all wave band scanning in the 200-400nm wavelength region, confirm the uv-absorbing optimal wavelength.Adopt gradient method to use half preparative high performance liquid chromatography post to carry out separation and purification: each appearance volume of going up is 1mL, and moving phase is methyl alcohol: zero(ppm) water 1:9,3:7,5:5,7:3,9:1; Elution speed is 1mL/min, each gradient elution 10min; Column temperature is 25 ℃; Collect the corresponding component of each chromatographic peak, and adopt punching to send out each component is carried out the bacteriostatic activity test; Merge and have active component.

(6) mass spectrometric detection: will have active component and carry out 100 times of dilutions, the solution after the dilution through the organic membrane filtration of 0.45um, is guaranteed no any insoluble foreign matter in the solution; The sample of handling well is carried out mass spectroscopy, and ion source is electron spray ionisation source (ESI).

(7) magnetic resonance detection: in 50 ℃ of baking ovens, evaporate into active substance solution dried; The exsiccant material is got the above sample adding 1mL deuterated methanol of 200mg and is fully dissolved; Above-mentioned dissolved sample is carried out nuclear magnetic resonance spectroscopy.

Embodiment 4:

Paenibacillus sp.The antimicrobial spectrum experiment of Aloe-11 antibacterial substance

(1) preparation of active substance: the Aloe-11 bacterial classification growing fine with transfering loop picking one ring on the aseptic technique platform is connected on the solid PDA substratum, and the back is subsequent use about 28 ℃ of cultivation 36h.Preparation seed liquor substratum divides to install in the triangular flask of 100mL, and each liquid amount is 25mL; 121 ℃ of sterilization 20min.One ring of cultured Aloe-11 bacterial classification picking on the PDA substratum is inoculated in the seed liquor substratum, puts into 28 ℃ then, that the shaking table of 200r/min is cultivated 12h is subsequent use.Preparation fermented liquid substratum divides to install in the triangular flask of 500mL, and each liquid amount is 120mL; 121 ℃ of sterilization 20min.Under aseptic condition, cultured seed liquid is transferred in the fermented liquid substratum, inoculum size is 5%, puts into 28 ℃, the shaking table cultured continuously 48h of 200r/min then.After the fermentation ends, add absolute ethyl alcohol by the amount of every bottle of 3 times of volumes, abundant mixing, treat that solid matter fully precipitates for some time in the fermented liquid after, remove with the vacuum pump suction filtration and to precipitate.Filtrating is further removed deposition through centrifugal 10 min of 5000r/min again after rotary evaporation concentrates, use the filter filtration sterilization of 0.22 μ m filter opening at last.

(2) preparation of indicator bacteria suspension: the bacterium bacteria suspension prepare-is got bacterial classification inoculation respectively and in the corresponding liquid substratum, is cultured to logarithmic phase, and colony counting method is measured bacteria suspension concentration, and adjustment bacterium number is 10 ⁵Do about cfu/mL to supply the examination bacterium, packing is subsequent use.The fungi bacteria suspension prepare-is connected to indicator on the PDA flat board, cultivates 4 days for 25 ℃; Each flat board washes lawn or spore with the 10mL sterilized water, counts at microscopically with blood counting chamber, and making its thalline or spore concentration is 10 ⁶About individual/mL, packing is subsequent use.

(3) pipe dish laboratory method: under aseptic condition, draw 100 each bacteria suspension of μ L on corresponding culture medium flat plate, it is coated on planar surface equably, till the liquid on the substratum becomes dry, punch in substratum with punch tool again with spreading rod.With each hole back cover, the bacteriostatic activity material crude extract of drawing 50 μ L adds in the hand-hole (can not overfolw hole outer), puts into constant incubator to petridish and cultivates about 24h after accomplishing fluently.It is generally acknowledged that inhibition zone explains that at 6～10mm this material has bacterinertness; Below the 10mm is slight sensitive; 11～15mm is a medium sensitivity; Then be extremely sensitive more than the 16mm.The result shows: the Aloe-11 antibacterial substance has very strong restraining effect to Gram-positive such as the streptococcus aureus of causing a disease, streptococcus suis 2-type and multiple bacterium plant pathogenic fungi, and Gram-negative bacteria and mycoplasma gallinarum are not had obvious inhibition effect.

(4) suppressing mycelial growth rate measures: under aseptic condition, get the aseptic PDA substratum that 10mL crude extract and 90mL melted and mix, pour into to process in the sterile petri dish and be with the medicine culture medium flat plate.Wait to solidify the confession examination pathogenic fungi bacterium cake of back inoculation diameter 7mm, handle repetition 3 times at every turn, put 28 ℃ of cultivations, be treated to contrast, measure bacterium cake diameter with the right-angled intersection method after 4 days, calculate inhibiting rate with sterilized water.The result shows: in 8 kind of plant pathogenic fungies of test; Gibberella saubinetii, pink powder pathogenic bacteria, Sclerotinia sclerotiorum, the former bacterium of peach brown rot, oranges and tangerines green mold, grey mold, rotten 7 kinds of pathogenic fungies such as mould there is the better inhibited effect; Its inhibiting rate is greater than 90%; Lower to Chinese cabbage dehiscence bacterium inhibiting rate, be 69.2%.

(5) minimal inhibitory concentration (MIC) is measured: will slightly carry active substance and adopt doubling dilution dilution back subsequent use, each weaker concn is respectively stoste, 1:2,1:4,1:8,1:16,1:32,1:64,1:128.Getting different extent of dilution active substance 1mL, indicator bacteria suspension 1mL, aseptic culture medium 2mL respectively in vitro mixes in difference; Not add active substance but the inoculation bacterium test tube as positive control; As negative control, each concentration is done 3 repetitions with the test tube that do not add active substance and do not inoculate bacterium.Place shaking table (37 ℃, 150 r/min) to cultivate about 12h in the test tube of inoculated bacteria, place shaking table (28 ℃, 200 r/min) to cultivate about 20h in the test tube of inoculated fungi, measure the OD630nm value.The result shows; Minimal inhibitory concentration to streptococcus aureus, pink powder pathogenic bacteria, the former bacterium of peach brown rot and oranges and tangerines green mold is extent of dilution 1:64; Minimal inhibitory concentration to Sclerotinia sclerotiorum is extent of dilution 1:32; To gibberella saubinetii and grey mold, rotten mould minimal inhibitory concentration is extent of dilution 1:16 then, and to Gram-negative bacteria intestinal bacteria unrestraint effect.

Claims (1)

1. a strain has the aloe endogenetic bacteria of broad spectrum antibiotic activity and animal intestinal field planting ability Paenibacillus sp.Aloe -11, it is characterized in that the preserving number of this bacterial strain is CCTCC No:M 2011237.

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