CN101225430A - Isolation and screening method of antibacterial and anti-tuberculosis plant endophytes - Google Patents

Abstract

The invention discloses a method for isolating and screening antibacterial and anti-tuberculosis plant endophytes. The method includes preparation of antibacterial and anti-tuberculosis plant tissues, surface disinfection and sterility detection, separation and purification of plant endophytes, fermentation of endophytic fungi, endophytic bacteria Fermentation, treatment of fermentation broth, treatment of bacteria, screening of effective antibacterial strains, screening of effective anti-tuberculosis strains and preservation of strains. The invention provides a guarantee for obtaining effective antibacterial and anti-tuberculosis substances from microorganisms, and obtains a batch of effective bacterial strains through the invention.

Description

Isolation and screening method of antibacterial and anti-tuberculosis plant endophytes

technical field

The invention relates to the technical field of plant drug screening, in particular to a method for separating and screening antibacterial and anti-tuberculosis plant endophytes.

Background technique

In recent years, due to the scarcity of plant resources, plant endophytes can produce some special physiologically active substances, so domestic and foreign pharmacists have paid extensive attention to the research of endophytes. In the isolation process of plant endophytes, the disinfection methods used mainly include physical disinfection and chemical disinfection. There are several combinations in the selection of chemical disinfectants: 75% alcohol; 75% alcohol + mercury chloride; 75% alcohol + sodium hypochlorite. Because the disinfection is not thorough, so the selection of physical disinfection and alcohol alone as a disinfectant is less. The combination of the latter two is commonly used. Foreign scholars often use the combination of 75% alcohol + sodium hypochlorite, mainly considering that sodium hypochlorite is non-toxic compared to mercury chloride. However, this combination is usually not as thorough as the combination of 75% alcohol + mercuric chloride, and the treatment of explants takes a long time, which is likely to cause the death of endophytic bacteria. Domestic scholars often use the disinfectant combination of 75% alcohol + mercuric chloride for surface disinfection. However, due to different preferences of researchers, different types of host plants and different types of host tissues, this combination is also different. Therefore, a rapid and effective chemical disinfection combination is crucial for the isolation of endophytes.

The antibacterial and anti-tumor activity of plant endophytes, many scientists have carried out in-depth research, and obtained a series of active monomers, and through the optimized surface disinfection method, the study of plant endophytes such as camphor and angustifolia The antibacterial and anti-tuberculosis activity, through fermentation to obtain secondary metabolites that have an inhibitory effect on human and animal pathogens and Mycobacterium tuberculosis, thereby obtaining antibacterial and anti-tuberculosis active strains, which has not received much attention in the field of endophytic bacteria research.

Contents of the invention

The purpose of the present invention is to propose a method for separating and screening secondary metabolites that have obvious inhibitory effects on human and animal pathogens and mycobacterium tuberculosis from antibacterial and anti-tuberculosis plant tissues by optimized chemical methods, so that Plant endophytic bacteria resources are more effectively utilized, so that antibacterial and anti-tuberculosis strains can be screened from microorganisms, and antibacterial and anti-tuberculosis substances can be extracted from microbial fermentation products.

In order to achieve the above object, the present invention is achieved through the following technical solutions.

1. A method for isolating and screening antibacterial and anti-tuberculosis plant endophytes, characterized in that the steps are as follows:

(1) Preparation of antibacterial and anti-tuberculosis plant tissues: collecting plant roots, stems, leaves, fruits and other tissues;

(2) Surface disinfection and sterility testing: In a sterile workbench, rinse each part of the tissue in step (1) three times with sterile water, use 75% alcohol + 0.1% mercuric chloride for surface disinfection, and use sterile water After washing 5 times, they were planted in water agar medium and incubated at a constant temperature of 27°C. Use sterile water for the last rinse of surface disinfection as a blank control;

(3) Separation and purification of plant endophytes: the endophytes grown in step (2), pick colonies of different shapes, fungi to PDA medium, bacteria to beef extract solid medium, and cultured by streaking until After purification, store it in the corresponding test tube slope for later use;

(4) Fermentation of endophytic fungi: subpackage the PDA liquid medium in conical flasks, pick a little mycelium of endophytic fungi purified in step (3) into culture flasks with an inoculation loop, put them on a shaker at 27°C, and set them at 150r/h min, shaking culture for 6-7 days, the fermentation broth was centrifuged at 4000r/min for 10-15min, and the supernatant was taken;

(5) Fermentation of endophytic bacteria: subpack the beef extract liquid culture medium into conical flasks, pick a small amount of endophytic bacterial colonies purified in step (3) with an inoculation loop, put them in a culture flask, place them on a shaker at 37°C, and set them at 150r /min, shaking culture for 2-3 days, the fermentation broth was centrifuged at 4000r/min for 10-15min, and the supernatant was taken;

(6) Processing of fermented liquid: add 95% ethanol to the supernatant of step (4) (5), make ethanol concentration be 75% after mixing, leave standstill overnight, treat liquid 4000r/min centrifugal 10-15min, The supernatant was taken, distilled under reduced pressure at 40°C, dried in vacuum and stored in a refrigerator at 4°C for later use;

(7) Treatment of thalline: place the thalli of step (4) (5) in a garden bottom flask and use an equal amount of acetone or ethyl acetate water bath to reflux for extraction for 2 hours, extract twice, and centrifuge at 4000r/min for 20min, 60 Distilled under reduced pressure at ℃, concentrated and dried, and stored in a refrigerator at 4 ℃ for later use;

(8) Screening of effective antibacterial strains: Weigh the powder of step (6) (7), add a certain amount of sterile water to make the liquid concentration 10 mg/ml, use the improved K-B disc method, and judge the inhibitory effect by measuring the size of the bacterial circle ;

(9) Screening of effective anti-tuberculosis strains: After mixing 10 mg/ml liquid medicine with a certain modified Roche medium to form a slant, the inoculation concentration is 103 mg/ml of Mycobacterium tuberculosis human, and the inoculation volume is 0.1 ml. Incubate at 37°C and observe the appearance of colonies;

(10) Preservation of strains: inoculate the endophytic strains with high inhibition efficiency screened in step (7) in 25% sterile glycerol, store at -80°C, and set aside.

Step (2) of the present invention disinfects each part of antibacterial and anti-tuberculosis plant tissue time as follows: roots and stems are soaked in 75% alcohol for 1 min, rinsed with sterile water for 3 times, and rinsed with 0.1% mercury liter for 1.5 min; leaves and fruits are soaked in 75% alcohol for 1 min , sterile water 3 times, 0.1% mercury liter rinse 0.5-1min. The water agar medium consists of 1.5 g of agar, 1000 ml of distilled water, and a natural pH.

The PDA culture medium described in steps (3), (4) and (5) of the present invention has components and contents as follows: 200g of potatoes, 20g of glucose, 15g of agar, 1000ml of distilled water, and about pH7.0. The components and contents of the beef extract medium are as follows: 3g of beef extract, 10g of peptone, 5g of sodium chloride, 15g of agar, 1000ml of distilled water, and a pH of about 7.0.

The test bacteria described in (8) and (9) of the present invention are Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (CMCC (B) 63501), Escherichia coli (CMCC ( One or more Several (the above are common pathogenic and epidemic pathogenic bacteria for humans and animals).

The improved KB disc method adopted in step (8) of the present invention is to dilute the activated test bacteria into 1.0×10 ⁶ CFU according to McFarland turbidimetry, and mix it with beef extract solid medium cooled to about 50°C, After solidification, place four sterile paper sheets with a diameter of 5mm on each medium plane, pour 15ul of the prepared medicinal solution into the paper sheets, and place in a 37°C incubator for cultivation.

The improved Roche culture medium described in step (9) of the present invention: monosodium glutamate (more than 95% of sodium glutamate) 7.2g, potassium dihydrogen phosphate 2.4g, magnesium sulfate 0.24g, magnesium citrate 0.6g, glycerol 12mL, distilled water 600mL, Potato starch 30g, whole egg liquid 1000mL, 2% malachite green 20mL; Preparation method: After adding potassium dihydrogen phosphate, magnesium sulfate, magnesium citrate, monosodium glutamate and glycerin to distilled water, heat and dissolve in boiling water, and remove the solution from the boiling water bath Take it out, add potato starch after cooling down slightly, put it in a boiling water bath and continue heating, and keep stirring or shaking to make it into a uniform paste. Take fresh eggs and wash them with soap, soak them in 75% alcohol for 30 minutes, take them out, dry them with sterile gauze and break the egg shells, collect the egg yolks and albumen, put them together in a sterilized beaker, and beat them with a sterilized glass rod. Homogenize the egg liquid, filter it with sterilized gauze, collect 1000ml of egg liquid, add the potato paste cooled to below 60°C, and add malachite green aqueous solution. After thorough mixing, divide into sterilized test tubes, place them on an inclined plane in an oven, and sterilize them intermittently at 85°C for 60 minutes twice (once a day). The drug-containing culture is based on the quantitative addition before sterilization. After sterilization, the drug-free medium is placed in a 37°C incubator for sterility test, and it can be used after it proves sterile growth.

The present invention is based on the difference in surface disinfection time of antibacterial and anti-tuberculosis plants, through chemical methods to carry out disinfection treatment on different tissues of different plants, to determine a set of fast and feasible schemes, and to provide a method for screening endophytic bacteria of antibacterial and anti-tuberculosis plants; The present invention tracked and measured the inhibitory effect of the secondary metabolites of the isolated endophytic strains on the tested human and animal pathogens and Mycobacterium tuberculosis, thereby obtaining effective strains and providing a batch of starting strains for the research and development of antibacterial and anti-tuberculosis drugs; Third, the present invention is simple and easy to operate.

Detailed ways

The method for isolating and screening antibacterial and anti-tuberculosis plant endophytes of the present invention will be further described below in conjunction with examples.

Example 1:

The tissues of various parts of the ten genus Angustifolia collected from the campus of Chongqing University were selected as the host samples of endophytes, and the surface was disinfected according to the following procedures:

(1) In the aseptic operating table, the host sample of the plant endophyte is washed 3 times with sterile water;

(2) Use the disinfection combination of 75% alcohol + 0.1% mercury liter, soak in 75% alcohol for 1-5min (interval 1min), rinse with sterile water 3 times, rinse in 0.1% mercury liter for 0.5-2min (interval 0.5min) min), rinsed with sterile water for 5 times, and collected the sterile water used to rinse the plant tissue for the last time as a control.

The results showed: Roots and stems were soaked in 75% alcohol for 1 min, washed 3 times with sterile water, rinsed with 0.1% mercury liter for 1.5 min, and 5 times with sterile water; leaves and fruits were soaked in 75% alcohol for 1 min, washed 3 times with 0.1% mercury chloride Rinse with mercury chloride for 1.5-1min, 5 times with sterile water. The endophytic bacteria isolated under this condition had no interference from other bacteria.

Example 2:

(1) Through the disinfection scheme of Example 1, the endophytic bacteria grown from the antibacterial and anti-tuberculosis plant tissues were picked and inoculated on the beef extract solid medium according to different colony forms, and kept at a constant temperature of 37°C Cultivation: pick endophytic fungi and inoculate them on the PDA medium by streaking, and culture at a constant temperature of 27°C. After purification, inoculate the corresponding slant and save it for later use;

(2) Carry out liquid fermentation culture of the above-mentioned endophytic bacterial strains: the endophytic fungus medium is PDA liquid medium, the endophytic bacteria is beef extract liquid medium, the medium is subpackaged in the Erlenmeyer flask, picked with an inoculation loop Take a small amount of purified endophytic bacteria in a culture bottle, put the fungus on a shaker at 27°C, 150r/min, and shake it for 6-7 days, put the bacteria on a shaker at 37°C, 150r/min, shake it for 2-3 days, the fermentation broth After centrifugation at 4000r/min for 10-15min, take the supernatant;

(3) Fermentation liquid treatment: add 95% ethanol to the supernatant, make the ethanol concentration 75% after mixing, let stand overnight, treat the liquid 4000r/min centrifugal 10-15min, get the supernatant, decompression distillation, vacuum After drying, the powder was collected and stored in a refrigerator at 4°C for later use;

(4) Treatment of the bacteria: place the bacteria in a garden bottom flask and use an equal amount of acetone or ethyl acetate for reflux extraction in a water bath for 2 hours, extract twice, centrifuge at 4000r/min for 20min, distill under reduced pressure, concentrate and dry, and store in 4 ℃ refrigerator spare;

(5) Determination of antibacterial activity: the powder is prepared into a liquid medicine with a concentration of 10 mg/ml, and the activated test bacteria are mixed with beef extract solid medium cooled to about 50 ° C according to McFarland turbidimetry, and the test bacteria The bacterial concentration reaches 1.0×10 ⁶ CFU. After solidification, place four sterile paper sheets with a diameter of 5mm on each medium plane, pour the prepared liquid medicine into the paper sheets in an amount of 15ul, and place in a 37°C incubator. After culturing for one day, measure the size of the bacterial circle to judge the inhibitory effect;

(6) The results showed that one strain of endophytic fungi was isolated from the stem of Phellodendron amurense (PE-S11), the root of golden buckwheat (FE-R9), and the root of coltsfoot (TE-R7). All have obvious inhibitory effects, and the size of the inhibition zone is as follows:

Indicator bacteria Endophytic fungi inhibition zone (mm) FE-R9 TE-R7 PE-S11 Staphylococcus aureus Bacillus subtilis Streptococcus pneumoniae Escherichia coli Pseudomonas aeruginosa Candida albicans 10.8±0.415.3±0.219.5±1.123.2±1.314.9±0.815.4±0.3 12.7±1.418.3±1.822.5±0.925.3±1.623.5±1.719.7±0.5 21.4±0.916.2±1.424.2±1.615.7±0.530.0±0.718.5±0.3

Example 3:

(1) Through the disinfection scheme of Example 1, the endophytic bacteria grown from the medicinal plant tissue were picked and inoculated on the beef extract solid medium according to different colony forms, and cultured at a constant temperature of 37°C ; Pick the endophytic fungi and inoculate them on the PDA medium by streaking, and cultivate them at a constant temperature of 27°C. After purification, inoculate the corresponding slant and save it for later use;

(2) Carry out liquid fermentation culture of the above-mentioned endophytic bacterial strains: the endophytic fungus medium is PDA liquid medium, the endophytic bacteria is beef extract liquid medium, the medium is subpackaged in the Erlenmeyer flask, picked with an inoculation loop Take a small amount of purified endophytic bacteria in a culture bottle, put the fungus on a shaker at 27°C, 150r/min, and shake it for 6-7 days, put the bacteria on a shaker at 37°C, 150r/min, shake it for 2-3 days, the fermentation broth After centrifugation at 4000r/min for 10-15min, take the supernatant;

(3) Fermentation liquid treatment: add 95% ethanol to the supernatant, make the ethanol concentration 75% after mixing, let stand overnight, treat the liquid 4000r/min centrifugal 10-15min, get the supernatant, decompression distillation, vacuum After drying, the powder was collected and stored in a refrigerator at 4°C for later use;

(4) Treatment of the bacteria: place the bacteria in a garden bottom flask and use an equal amount of acetone or ethyl acetate for reflux extraction in a water bath for 2 hours, extract twice, centrifuge at 4000r/min for 20min, distill under reduced pressure, concentrate and dry, and store in 4 ℃ refrigerator spare;

(5) Preparation of improved Roche medium: 7.2g monosodium glutamate (sodium glutamate 95%), 2.4g potassium dihydrogen phosphate, 0.24g magnesium sulfate, 0.6g magnesium citrate, 12mL glycerol, 600mL distilled water, 30g potato starch , whole egg liquid 1000mL, 2% malachite green 20mL; Preparation method: After adding distilled water to potassium dihydrogen phosphate, magnesium sulfate, magnesium citrate, monosodium glutamate and glycerin, heat in boiling water to dissolve, take the solution out of the boiling water bath, After cooling, add potato starch, put it in a boiling water bath and continue heating, and keep stirring or shaking to make it into a uniform paste. Take fresh eggs and wash them with soap, soak them in 75% alcohol for 30 minutes, take them out, dry them with sterile gauze and break the egg shells, collect the egg yolks and albumen, put them together in a sterilized beaker, and beat them with a sterilized glass rod. Homogenize the egg liquid, filter it with sterilized gauze, collect 1000ml of egg liquid, add the potato paste cooled to below 60°C, and add malachite green aqueous solution. After thorough mixing, divide into sterilized test tubes, place them on an inclined plane in an oven, and sterilize them intermittently at 85°C for 60 minutes twice (once a day). The drug-containing culture is based on the quantitative addition before sterilization. After sterilization, the drug-free medium is placed in a 37°C incubator for sterility test, and it can be used after it proves sterile growth;

(6) Determination of anti-tuberculosis activity: After mixing 10 mg/ml liquid medicine with a certain modified Roche medium to form a slant, inoculate Mycobacterium tuberculosis human at a concentration of 10 ³ mg/ml, and the inoculation volume is 0.1 ml. Incubate at 37°C and observe the appearance of colonies.

(7) The results showed that: one endophytic fungus was obtained from the stem of pokeweed (ES-S23), the leaf of the ten major contributions of narrow leaves (EM-L40), and the stem of Cinnamomum camphora (EC-S44). The metabolites have a certain inhibitory effect on human Mycobacterium tuberculosis, and also have obvious inhibitory effects on four human and animal pathogenic bacteria.

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.