CN101225430A - Separation screening method for antibiotic antituberculotic plant endophyte - Google Patents
Separation screening method for antibiotic antituberculotic plant endophyte Download PDFInfo
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- CN101225430A CN101225430A CNA2008100205701A CN200810020570A CN101225430A CN 101225430 A CN101225430 A CN 101225430A CN A2008100205701 A CNA2008100205701 A CN A2008100205701A CN 200810020570 A CN200810020570 A CN 200810020570A CN 101225430 A CN101225430 A CN 101225430A
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Abstract
The invention discloses a separation and selection method of antibiotic antitubercular endophyte, which comprises the steps as follows, the preparation of antibiotic antitubercular plant tissue, the surface sterilization and the sterility test, the separation and the purification of the endophyte, the ferment of endogenetic fungi, the ferment of endogenetic bacterium, the process of the zymotic fluid, the process of the thalli, the selection of the effective antibiotic bacterial strains, the selection of the effective antitubercular bacterial strains and the preservation of the strains. The separation and selection method of antibiotic antitubercular endophyte ensures the realization of obtaining the effective antibiotic antitubercular matter from microorganisms, thanks to separation and selection method, a batch of effective bacterial strains are obtained.
Description
Technical field
The present invention relates to medicinal plants triage techniques field, be specifically related to the separating screening method of antibiotic antituberculotic plant endophyte.
Background technology
In recent years, because the scarcity of plant resources, and endophyte of plant can produce some special physiological active substance, therefore domestic and international pharmacy men are to the research extensive concern of endophyte.In the sepn process of endophyte of plant, the sterilization method of using mainly contains physical sterilization and chemical disinfection, has several combinations in the selection of chemostefilant: 75% alcohol; 75% alcohol+mercuric chloride; 75% alcohol+clorox.Because it is not thorough to sterilize, so select physical sterilization for use and select separately alcohol less as sterilizing agent.Both combinations of back are used always, and the foreign scholar adopts this combination of 75% alcohol+clorox more, mainly considers clorox, and is than mercuric chloride, nontoxic.But this combination is thorough not as this combined sterilizing of 75% alcohol+mercuric chloride usually, and longer to the explant treatment time, also causes endophyte death easily.Domestic scholars is normal uses this sterilizing agent combination the carrying out surface sterilization of 75% alcohol+mercuric chloride, but because of investigator's hobby difference, the difference of the kind of host plant and host tissue type, this combination also is not quite similar.Therefore, a chemical disinfection combination fast and effectively is vital to the separation of endophyte.
The antibiotic anti-tumor activity of endophyte of plant, many scientists have carried out deep research, and a series of reactive monomers have been obtained, and the processes for disinfecting surfaces by optimizing, study the antibiotic antituberculotic activity of endophyte of plant such as fragrant camphor tree, narrow leaf Gaulis Mahoniae, obtain people and animals pathogenic bacteria and the inhibited secondary metabolite of human-like mycobacterium tuberculosis by fermentation, thereby obtain the antibiotic antituberculotic active bacterial strain, this does not also receive too much concern in the endophyte research field.
Summary of the invention
The objective of the invention is to propose a kind of chemical process of passing through to optimize from the antibiotic antituberculotic plant tissue, separate secondary metabolite and people and animals pathogenic bacteria and human-like mycobacterium tuberculosis are had the method for obvious inhibiting endophyte, the endophyte of plant resource is used more effectively, thereby realize screening antibiotic antituberculotic bacterial strain from microorganism, from microbial fermentation product, extract the antibiotic antituberculotic material.
For achieving the above object, the present invention is achieved by the following technical programs.
1, a kind of separating screening method of antibiotic antituberculotic plant endophyte is characterized in that step is as follows:
(1) preparation of antibiotic antituberculotic plant tissue: tissues such as the root of herborization, stem, leaf, fruit;
(2) surface sterilization and aseptic detection: in aseptic worktable, step (1) each several part tissue with aseptic water washing 3 times, is adopted 75% alcohol+0.1% mercuric chloride combination carrying out surface sterilization, behind the aseptic water washing 5 times, plant in water agar 27 ℃ of following constant temperature culture.The last flushing of surface sterilization uses sterilized water as blank;
(3) separation and purification of endophyte of plant: with the endophyte that step (2) grows, the bacterium colony of picking different shape, fungi is to the PDA substratum, and bacterium is to the beef extract solid medium, and is streak culture after pure, is stored in the corresponding test tube slant standby;
(4) endogenetic fungus fermentation: the PDA liquid nutrient medium is divided in the triangular flask, and the endogenetic fungus mycelia of using transfering loop a little step of picking (3) purifying is put 27 ℃ of shaking tables in culturing bottle, 150r/min, shaking culture 6-7 days, fermented liquid was got supernatant liquor through the centrifugal 10-15min of 4000r/min;
(5) endogenetic bacteria fermentation: the beef extract liquid nutrient medium is divided in the triangular flask, and the endogenetic bacteria bacterium colony of using transfering loop picking few steps (3) purifying is put 37 ℃ of shaking tables in culturing bottle, 150r/min, shaking culture 2-3 days, fermented liquid was got supernatant liquor through the centrifugal 10-15min of 4000r/min;
(6) processing of fermented liquid: the ethanol of adding 95% in the supernatant liquor of step (4) (5), making alcohol concn after the mixing is 75%, standing over night, the centrifugal 10-15min of treatment solution 4000r/min, get supernatant liquor, 40 ℃ of underpressure distillation, it is standby to be stored in 4 ℃ of refrigerators after the vacuum-drying;
(7) processing of thalline: place at the bottom of the garden flask with the acetone or the ethyl acetate water-bath refluxing extraction 2h of equivalent the thalline of step (4) (5), lixiviate twice, the centrifugal 20min of 4000r/min, 60 ℃ of underpressure distillation, it is standby to be stored in 4 ℃ of refrigerators behind the concentrate drying;
(8) effective antibiotic bacterial strain screening: take by weighing step (6) (7) powder, add certain sterilized water, making liquor strength is 10mg/ml, adopts and improves the K-B paper disk method, by measuring bacterium circle size, judges and suppresses effect;
(9) effectively the tuberculosis bacterial strain screens: after 10mg/ml soup and certain modified Russell medium were mixed into the inclined-plane, inoculum density was the human-like mycobacterium tuberculosis of 103mg/ml, and inoculum size is 0.1ml.37 ℃ of cultivations are observed bacterium colony and situation occurred;
(10) preservation of bacterial classification: the height that step (7) is filtered out suppresses efficient endophyte inoculation in 25% aseptic glycerine, and-80 ℃ of preservations are standby.
Step of the present invention (2) organizes the each several part disinfecting time as follows to antibiotic antituberculotic plant: root, stem 75% alcohol-pickled 1min, aseptic water washing 3 times, 0.1% mercuric chloride rinsing 1.5min; Leaf, fruit 75% alcohol-pickled 1min, sterilized water 3 times, 0.1% mercuric chloride rinsing 0.5-1min.The water agar component is agar 1.5g, distilled water 1000ml, PH nature.
Step of the present invention (3), (4), (5) described PDA substratum, its component and content are: potato 200g, glucose 20g, agar 15g, distilled water 1000ml is about PH7.0.Described its component of beef extract substratum and content are: extractum carnis 3g, and peptone 10g, sodium-chlor 5g, agar 15g, distilled water 1000ml is about PH7.0.
The present invention (8), (9) are described to be streptococcus aureus (ATCC 25923), pseudomonas aeruginosa (ATCC 27853), subtilis (CMCC (B) 63501), intestinal bacteria (CMCC (B) 44103), candida albicans (ATCC 10231), Fei Yanshi suis (ATCC 49619), human-like mycobacterium tuberculosis reference culture (H37Rv), human-like mycobacterium tuberculosis Resistant strain etc. for the examination bacterium, wherein one or more (more than be people and animals are common causes a disease and the epiphytotic pathogen bacterium).
The improvement K-B paper disk method that step of the present invention (8) adopts is that the examination bacterium that supplies that activation is good is diluted to 1.0 * 10 according to the Maxwell than turbid
6CFU mixes with the beef extract solid medium that is cooled to about 50 ℃, and after waiting to solidify, the aseptic scraps of paper that four diameters are 5mm are put on each substratum plane, and the soup for preparing is squeezed into the scraps of paper by the amount of 15ul, puts 37 ℃ of incubators and cultivates.
The described modified Russell medium of step of the present invention (9): monosodium glutamate (Sodium Glutamate is more than 95%) 7.2g, potassium primary phosphate 2.4g, sal epsom 0.24g, magnesium citrate 0.6g, glycerine 12mL, distilled water 600mL, yam starch 30g, whole egg liquid 1000mL, 2% Victoria Green WPB 20mL; Preparation method: behind potassium primary phosphate, sal epsom, magnesium citrate, monosodium glutamate and glycerine adding distil water, put middle heating for dissolving in the boiling water, solution is taken out from boiling water bath, cold slightly back adds yam starch, put and continue heating in the boiling water bath, and constantly stir or shake, make it into even pasty state.Getting new fresh hen egg soaps in clean rearmounted 75% alcohol and soaks 30min, the taking-up back is dried with sterile gauze and is broken up eggshell, yolk and albumen are collected, be collected in the lump in the sterilization beaker, break into even egg liquid with the sterilization glass rod, filter, collect ovum liquid 1000ml with sterile gauze, add the potato that is cooled to below 60 ℃ and stick with paste, and add malachite green solution.Behind the thorough mixing, be sub-packed in the sterilization test tube, in baking oven, put into the inclined-plane, 85 ℃ of twice of 60min tyndallizations (once a day).Pastille is cultivated based on quantitatively adding before the sterilization, after the sterilization not the pastille substratum put and carry out sterility test in 37 ℃ of incubators, prove that asepsis growth can use.
The present invention is the difference according to the antibiotic antituberculotic plant surface sterilization time, to the disinfection of different plant different tissues, determines a cover feasible scheme fast by chemical process, for screening antibiotic antituberculotic plant endophyte provides a kind of method; The 2nd, tracking and measuring of the present invention isolating each endophyte bacterial strain secondary metabolite to restraining effect for examination people and animals pathogenic bacteria and human-like mycobacterium tuberculosis, thereby obtain effective strain, for the research and development of antibiotic antituberculotic medicine provide a collection of starting strain; The 3rd, the present invention's row simple to operate, easy.
Embodiment
Be further described below in conjunction with the separating screening method of example antibiotic antituberculotic plant endophyte of the present invention.
Embodiment 1:
Select for use the narrow leaf Gaulis Mahoniae each several part tissue that picks up from the University Of Chongqing campus as endophyte host sample, carry out surface sterilization by following program:
(1) in the aseptic technique platform, with host's sample aseptic water washing 3 times of endophyte of plant;
(2) adopt the sterilization of 75% alcohol+0.1% mercuric chloride to make up, set and soak 1-5min (1min at interval) in 75% alcohol, aseptic water washing 3 times, rinsing 0.5-2min in 0.1% mercuric chloride (0.5min at interval), aseptic water washing 5 times is collected for the last time with the sterilized water that washes plant tissue in contrast.
The result shows: root, stem 75% alcohol-pickled 1min, aseptic water washing 3 times, 0.1% mercuric chloride rinsing 1.5min, sterilized water 5 times; Leaf, fruit 75% alcohol-pickled 1min, sterilized water 3 times, 0.1% mercuric chloride rinsing 1.5-1min, sterilized water 5 times.Separate the endophyte that obtains under this condition and do not have assorted bacterium interference.
Embodiment 2:
(1) the sterilization scheme by example 1, the endophyte that will from the antibiotic antituberculotic plant tissue, grow, according to different colonial morphologies, the streak inoculation of picking endogenetic bacteria to the beef extract solid medium, 37 ℃ of constant temperature culture; The streak inoculation of picking endogenetic fungus to the PDA substratum, 27 ℃ of constant temperature culture.Purified back inoculation respective ramp is preserved standby;
(2) above-mentioned endophyte bacterial strain is carried out liquid fermentation and culture: the endogenetic fungus substratum is the PDA liquid nutrient medium, endogenetic bacteria is the beef extract liquid nutrient medium, and substratum is divided in the triangular flask, and the endophyte of using a little purifying of transfering loop picking is in culturing bottle, fungi is put 27 ℃ of shaking tables, 150r/min, shaking culture 6-7 days, bacterium was put 37 ℃ of shaking tables, 150r/min, shaking culture 2-3 days, fermented liquid was got supernatant liquor through the centrifugal 10-15min of 4000r/min;
(3) fermentation liquor treatment: add 95% ethanol in supernatant liquor, making alcohol concn after the mixing is 75%, standing over night, and the centrifugal 10-15min of treatment solution 4000r/min gets supernatant liquor, underpressure distillation, after the vacuum-drying, it is standby that powder collection is stored in 4 ℃ of refrigerators;
(4) processing of thalline: place at the bottom of the garden flask with the acetone or the ethyl acetate water-bath refluxing extraction 2h of equivalent thalline, lixiviate twice, the centrifugal 20min of 4000r/min, underpressure distillation, it is standby to be stored in 4 ℃ of refrigerators behind the concentrate drying;
(5) anti-microbial activity is measured: powder is mixed with the soup that concentration is 10mg/ml, with activation good for the examination bacterium according to the Maxwell than turbid, mixes with the beef extract solid medium that is cooled to about 50 ℃, make for trying bacteria concentration to reach 1.0 * 10
6CFU, after waiting to solidify, the aseptic scraps of paper that four diameters are 5mm are put on each substratum plane, and the soup for preparing is squeezed into the scraps of paper by the amount of 15ul, put 37 ℃ of incubators and cultivate after one day, measure bacterium circle size, judge and suppress effect;
(6) result shows: respectively be separated to a strain endogenetic fungus from Phellodendron sachalinense stem (PE-S11), Wild Buckwheat Rhizome root (FE-R9), coltsfoot root (TE-R7), its secondary metabolite is to all having the obvious suppression effect for examination people and animals pathogenic bacteria, and the inhibition zone size is as follows:
| Indicator | Endogenetic fungus inhibition zone (mm) | ||
| FE-R9 | TE-R7 | PE-S11 | |
| Streptococcus aureus subtilis Fei Yanshi suis intestinal bacteria pseudomonas aeruginosa candida albicans | 10.8±0.4 15.3±0.2 19.5±1.1 23.2±1.3 14.9±0.8 15.4±0.3 | 12.7±1.4 18.3±1.8 22.5±0.9 25.3±1.6 23.5±1.7 19.7±0.5 | 21.4±0.9 16.2±1.4 24.2±1.6 15.7±0.5 30.0±0.7 18.5±0.3 |
Embodiment 3:
(1) the sterilization scheme by example 1, the endophyte that will from the medicinal plant tissue, grow, according to different colonial morphologies, the streak inoculation of picking endogenetic bacteria to the beef extract solid medium, 37 ℃ of constant temperature culture; The streak inoculation of picking endogenetic fungus to the PDA substratum, 27 ℃ of constant temperature culture.Purified back inoculation respective ramp is preserved standby;
(2) above-mentioned endophyte bacterial strain is carried out liquid fermentation and culture: the endogenetic fungus substratum is the PDA liquid nutrient medium, endogenetic bacteria is the beef extract liquid nutrient medium, and substratum is divided in the triangular flask, and the endophyte of using a little purifying of transfering loop picking is in culturing bottle, fungi is put 27 ℃ of shaking tables, 150r/min, shaking culture 6-7 days, bacterium was put 37 ℃ of shaking tables, 150r/min, shaking culture 2-3 days, fermented liquid was got supernatant liquor through the centrifugal 10-15min of 4000r/min;
(3) fermentation liquor treatment: add 95% ethanol in supernatant liquor, making alcohol concn after the mixing is 75%, standing over night, and the centrifugal 10-15min of treatment solution 4000r/min gets supernatant liquor, underpressure distillation, after the vacuum-drying, it is standby that powder collection is stored in 4 ℃ of refrigerators;
(4) processing of thalline: place at the bottom of the garden flask with the acetone or the ethyl acetate water-bath refluxing extraction 2h of equivalent thalline, lixiviate twice, the centrifugal 20min of 4000r/min, underpressure distillation, it is standby to be stored in 4 ℃ of refrigerators behind the concentrate drying;
(5) preparation of modified Russell medium: monosodium glutamate (Sodium Glutamate is more than 95%) 7.2g, potassium primary phosphate 2.4g, sal epsom 0.24g, magnesium citrate 0.6g, glycerine 12mL, distilled water 600mL, yam starch 30g, whole egg liquid 1000mL, 2% Victoria Green WPB 20mL; Preparation method: behind potassium primary phosphate, sal epsom, magnesium citrate, monosodium glutamate and glycerine adding distil water, put heating for dissolving in the boiling water, solution is taken out from boiling water bath, cold slightly back adds yam starch, put and continue heating in the boiling water bath, and constantly stir or shake, make it into even pasty state.Getting new fresh hen egg soaps in clean rearmounted 75% alcohol and soaks 30min, the taking-up back is dried with sterile gauze and is broken up eggshell, yolk and albumen are collected, be collected in the lump in the sterilization beaker, break into even egg liquid with the sterilization glass rod, filter, collect ovum liquid 1000ml with sterile gauze, add the potato that is cooled to below 60 ℃ and stick with paste, and add malachite green solution.Behind the thorough mixing, be sub-packed in the sterilization test tube, in baking oven, put into the inclined-plane, 85 ℃ of twice of 60min tyndallizations (once a day).Pastille is cultivated based on quantitatively adding before the sterilization, after the sterilization not the pastille substratum put and carry out sterility test in 37 ℃ of incubators, prove that asepsis growth can use;
(6) tuberculosis determination of activity: after 10mg/ml soup and certain modified Russell medium be mixed into the inclined-plane, inoculum density was 10
3The human-like mycobacterium tuberculosis of mg/ml, inoculum size are 0.1ml.37 ℃ of cultivations are observed bacterium colony and situation occurred.
(7) result shows: respectively assign to a strain endogenetic fungus from the stem (EC-S44) of the leaf (EM-L40) of the stem (ES-S23) of Phytolacca acinosa, narrow leaf Mahonia fortunei, fragrant camphor tree, its secondary metabolite has certain restraining effect to human-like mycobacterium tuberculosis, and, four kinds of people and animals pathogenic bacterias are also had the obvious suppression effect.
Claims (4)
1. the separating screening method of an antibiotic antituberculotic plant endophyte is characterized in that step is as follows:
(1) preparation of antibiotic antituberculotic plant tissue: tissues such as the root of herborization, stem, leaf, fruit;
(2) surface sterilization and aseptic detection: in aseptic worktable, with aseptic water washing 3 times of step (1) each several part tissue, adopt 75% alcohol+0.1% mercuric chloride combination carrying out surface sterilization, behind the aseptic water washing 5 times, plant in water agar, 27 ℃ of following constant temperature culture, the last flushing of surface sterilization uses sterilized water as blank;
(3) separation and purification of endophyte of plant: with the endophyte that step (2) grows, the bacterium colony of picking different shape, fungi is to the PDA substratum, and bacterium is to the beef extract solid medium, and is streak culture after pure, is stored in the corresponding test tube slant standby;
(4) endogenetic fungus fermentation: the PDA liquid nutrient medium is divided in the triangular flask, and the endogenetic fungus mycelia of using transfering loop a little step of picking (3) purifying is put 27 ℃ of shaking tables in culturing bottle, 150r/min, shaking culture 6-7 days, fermented liquid was got supernatant liquor through the centrifugal 10-15min of 4000r/min;
(5) endogenetic bacteria fermentation: the beef extract liquid nutrient medium is divided in the triangular flask, and the endogenetic bacteria bacterium colony of using transfering loop a little step of picking (3) purifying is put 37 ℃ of shaking tables in culturing bottle, 150r/min, shaking culture 2-3 days, fermented liquid was got supernatant liquor through the centrifugal 10-15min of 4000r/min;
(6) processing of fermented liquid: the ethanol of adding 95% in the supernatant liquor of step (4), (5), making alcohol concn after the mixing is 75%, standing over night, the centrifugal 10-15min of treatment solution 4000r/min, get supernatant liquor, 40 ℃ of underpressure distillation, it is standby to be stored in 4 ℃ of refrigerators after the vacuum-drying;
(7) processing of thalline: the thalline of step (4), (5) is placed acetone or the ethyl acetate water-bath refluxing extraction 2h of round-bottomed flask with equivalent, lixiviate twice, the centrifugal 20min of 4000r/min, 60 ℃ of underpressure distillation, it is standby to be stored in 4 ℃ of refrigerators behind the concentrate drying;
(8) effective antibiotic bacterial strain screening: take by weighing step (6), (7) powder, add certain sterilized water, making liquor strength is 10mg/ml, adopts and improves the K-B paper disk method, by measuring bacterium circle size, judges and suppresses effect;
(9) effectively the tuberculosis bacterial strain screens: after 10mg/ml soup and certain modified Russell medium were mixed into the inclined-plane, inoculum density was the human-like mycobacterium tuberculosis of 103mg/ml, and inoculum size is 0.1ml, and 37 ℃ of cultivations are observed bacterium colony and situation occurred;
(10) preservation of bacterial classification: the height that step (7) is filtered out suppresses efficient endophyte inoculation in 25% aseptic glycerine, and-80 ℃ of preservations are standby.
2. the separating screening method of a kind of antibiotic antituberculotic plant endophyte according to claim 1, it is characterized in that step (2) is described organizes the each several part disinfecting time as follows to antibiotic antituberculotic plant: root, stem are 75% alcohol-pickled 1min, aseptic water washing 3 times, 0.1% mercuric chloride rinsing 1.5min; Leaf, fruit are 75% alcohol-pickled 1min, sterilized water 3 times, and 0.1% mercuric chloride rinsing 0.5-1min, the water agar component is agar 1.5g, distilled water 1000ml, PH nature.
3. the separating screening method of a kind of antibiotic antituberculotic plant endophyte according to claim 1 is characterized in that the described employing of step (8) improves the K-B paper disk method, is that the examination bacterium that supplies that activation is good is diluted to 1.0 * 10 according to the Maxwell than turbid
6CFU mixes with the beef extract solid medium that is cooled to about 50 ℃, and after waiting to solidify, the aseptic scraps of paper that four diameters are 5mm are put on each substratum plane, and the soup for preparing is squeezed into the scraps of paper by the amount of 15ul, puts 37 ℃ of incubators and cultivates.
4. the separating screening method of a kind of antibiotic antituberculotic plant endophyte according to claim 1, it is characterized in that consisting of of step (9) described modified Russell medium: monosodium glutamate (Sodium Glutamate is more than 95%) 7.2g, potassium primary phosphate 2.4g, sal epsom 0.24g, magnesium citrate 0.6g, glycerine 12mL, distilled water 600mL, yam starch 30g, whole egg liquid 1000mL, 2% Victoria Green WPB 20mL.
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