CN108776222B - Method for detecting antibacterial activity of princess lingmycin and application - Google Patents
Method for detecting antibacterial activity of princess lingmycin and application Download PDFInfo
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- CN108776222B CN108776222B CN201810612023.6A CN201810612023A CN108776222B CN 108776222 B CN108776222 B CN 108776222B CN 201810612023 A CN201810612023 A CN 201810612023A CN 108776222 B CN108776222 B CN 108776222B
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Abstract
The invention provides a method for detecting the bacteriostatic activity of gonggingmycin and application thereof, relating to the technical field of methods for detecting the bacteriostatic activity, wherein the method comprises the following steps: the method provided by the invention has the advantages of good intuition, high sensitivity, good stability, simplicity, convenience, short time consumption, wide application range, safety, reliability and lower cost, and provides a new way for the antibacterial activity detection of the Briggrin.
Description
Technical Field
The invention relates to the technical field of bacteriostatic activity detection methods, in particular to a method for detecting bacteriostatic activity of gonggingling mycin and application thereof.
Background
"769" is a streptomyces agriculturally used strain separated and screened from the soil of princess ridge in 1967 by the specialist of agricultural academy of Jilin province, and "769" is a metabolite of "769" and a natural biosynthetic mixed preparation.
At present, an important index for evaluating the quality of the princess lingmycin is the antibacterial activity, and the measuring method comprises a physicochemical method and a microbiological method. The physicochemical method is simple and rapid, but has high requirement on the purity of the Bridgkin mycin, otherwise, the method causes great deviation, and the measuring instrument is expensive and the detection cost is high. The microbiological method comprises a dilution method, a turbidimetric method, a tube butterfly method, a respiratory measure method and a dehydrogenation method, and the methods have high sensitivity, safety and reliability and are widely applied, but the microbiological detection method has multiple operation steps, long test process, more influencing factors and difficult grasp of detection personnel.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting the bacteriostatic activity of the Briggrin, so as to solve the technical problems that the existing physical and chemical detection method has high requirements, and the microbiological method is not easy to operate.
The invention provides a method for detecting the bacteriostatic activity of Briggrin, which comprises the following steps:
(a) providing a princess lingmycin water extract and a test bacterium suspension;
(b) mixing the water extract of the Briggrin in different volume ratios with the bacterial suspension of the test bacteria, respectively investigating the growth conditions of the test bacteria to obtain the critical bacteriostatic volume ratio of the water extract of the Briggrin to the bacterial suspension of the test bacteria, and determining the bacteriostatic activity of the water extract of the Briggrin to the test bacteria according to the critical bacteriostatic volume ratio of the water extract of the Briggrin to the bacterial suspension of the test bacteria.
Further, in the step (b), the water extract of the Briggrin mycin and the bacterial suspension of the test bacteria with different volume ratios are mixed, cultured for 2-6 days, and then the growth conditions of the test bacteria are respectively investigated.
Further, the test bacteria are fungi;
preferably, the test bacterium is selected from one of northern leaf blight bacterium, alternaria alternata or beauveria bassiana.
Further, in the step (b), the OD value of the bacterial suspension of the test bacteria is 0.3-0.35.
Further, the water extract of the princess lingmycin is prepared according to the following steps:
(m) inoculating the farm antibiotic 769 strain into a culture medium, and completely culturing to obtain a mixture of the Brilliant mycin and the culture medium;
(n) drying the mixture of the Briggrin and the culture medium to obtain a solid Briggrin, crushing the solid Briggrin, and leaching with water to obtain a Briggrin leaching solution;
(s) centrifuging the Brigguin lingmycin leaching liquor, and taking supernate to obtain the Brigguin lingmycin water extracting liquor.
Further, in step (m), the culture time is 5 to 7 days, preferably 6 days.
Further, in the step (n), the leaching time is 24-48h, preferably 36 h.
Further, in the step (n), the volume ratio of the crushed material of the solid of the Briggrin to the water is 1:2-10, preferably 1: 2.
Further, in step (m), the culture medium is corn grit;
preferably, the corn dregs are soaked, drained and sterilized;
preferably, the corn dregs are soaked by water, and the soaking time of the corn dregs is 1.5-8h, preferably 3 h;
preferably, the sterilization treatment time is 15-25min, preferably 20 min.
The second purpose of the invention is to provide the application of the princess lingmycin and the bacteriostatic activity detection method in antibiotic bacteriostatic activity detection.
The method for detecting the antibacterial activity of the Briggrin has the following beneficial effects that:
(1) the antibacterial activity of the water extract of the gentamycin from the princess ridge on the test bacteria is obtained by observing the growth condition of the test bacteria after the water extract of the gentamycin and the suspension of the test bacteria in different volume ratios are mixed, and the antibacterial activity is more visual, more sensitive and more stable;
(2) no toxic and harmful reagent is introduced in the operation process, so that the method is safer and more reliable, and the detection cost is saved;
(3) the method has the advantages of few operation steps, short consumed time, few influence factors and easy grasp by detection personnel;
(4) the application range is wide, and the kit can be used for detecting the antibacterial activity of other antibiotics.
Drawings
FIG. 1 is a graph showing the growth of the strains of example 1 after mixed culture of suspensions of Mycosphaerella zeae and aqueous extract of Briggrin at different volume ratios for 4 days;
FIG. 2 is a graph showing the growth of the bacterial strains of example 2 after culturing the mixture of 160. mu.L of the bacterial suspension of northern leaf blight and different volumes of aqueous extract of Briggrin for 4 days;
FIG. 3 is a graph showing the growth of the bacterial strains of example 3 after 6 days of mixed culture of 200. mu.L of aqueous extract of Briggrin with different volumes of suspensions of Exserohilum turcicum;
FIG. 4 is a diagram showing the growth of the mixed culture of the bacterial suspensions of Alternaria alternata in comparative example 1 in water extract of Briggrin mycin;
FIG. 5 is a diagram showing the growth of the mixed culture of different volumes of the bacterial suspensions of Cortinarius maydis in comparative example 2 in the aqueous extract of Briggrin mycin;
FIG. 6 is a graph showing the growth of the bacterial strains of example 4 after culturing 160. mu.L of a bacterial suspension of Mycosphaerella zeae in combination with 10 aqueous extracts of Briggrin at different concentrations for 6 days;
FIG. 7 is a graph showing the growth of the cultured strains in example 5 after mixed culture of beauveria bassiana suspensions and an aqueous extract of Briggrin.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
At present, an important index for evaluating the quality of the princess lingmycin is the antibacterial activity, and the measuring method comprises a physicochemical method and a microbiological method. The physicochemical method is simple and rapid, but has high requirement on the purity of the Bridgkin mycin, otherwise, the method causes great deviation, and the measuring instrument is expensive and the detection cost is high. The microbiological method comprises a dilution method, a turbidimetry method, a tube butterfly method, a respiratory measurement method and a dehydrogenation method, and the methods are high in sensitivity, safe, reliable and widely applied, but the microbiological detection methods have the defects of multiple operation steps, long test process, more influence factors and difficulty in being mastered by detection personnel, and errors caused by bacteria quantity difference of a test bacterium are not eliminated in the methods.
According to one aspect of the invention, the invention provides a method for detecting the bacteriostatic activity of gonggingmycin, which comprises the following steps:
(a) providing a princess lingmycin water extract and a test bacterium suspension;
(b) mixing the water extract of the Briggrin in different volume ratios with the bacterial suspension of the test bacteria, respectively investigating the growth conditions of the test bacteria to obtain the critical bacteriostatic volume ratio of the water extract of the Briggrin to the bacterial suspension of the test bacteria, and determining the bacteriostatic activity of the water extract of the Briggrin to the test bacteria according to the critical bacteriostatic volume ratio of the water extract of the Briggrin to the bacterial suspension of the test bacteria.
In the invention, in the step (b), the total growth amount of the test bacteria is reduced along with the increase of the volume ratio of the water extract of the Briggrin to the suspension of the test bacteria, and when the volume ratio of the water extract of the Briggrin to the suspension of the test bacteria is increased to a certain degree and no target fungi are observed to grow, the volume ratio of the water extract of the Briggrin to the suspension of the test bacteria is the critical bacteriostasis volume ratio.
In the invention, the critical bacteriostatic volume of the water extract of the Bridgman mycin is used for judging the level of the bacteriostatic activity of the water extract of the Bridgman mycin, and the smaller the critical bacteriostatic volume is, the better the bacteriostatic activity of the water extract of the Bridgman mycin on test bacteria is.
The method for detecting the antibacterial activity of the Briggrin has the following beneficial effects that:
(1) the critical bacteriostatic volume of the water extract of the Briggrin mycin to the test bacteria is obtained by detecting the growth condition of the test bacteria after the water extract of the Briggrin mycin and the suspension of the test bacteria in different volume ratios are mixed, so that the bacteriostatic activity of the water extract of the Briggrin mycin to the test bacteria is obtained, and the method is more visual, sensitive and stable;
(2) no toxic and harmful reagent is introduced in the operation process, so that the method is safer and more reliable, and the detection cost is saved;
(3) the operation steps are few, the consumed time is short, the influence of the bacteria quantity of the tested bacteria is small, and the operation is easier to master by detection personnel;
(4) the application range is wide, and the kit can be used for detecting the antibacterial activity of other antibiotics.
In a preferred embodiment of the present invention, in step (b), the aqueous extract of Briggrin and the bacterial suspension of the test bacteria are mixed at different volume ratios, cultured for 2-6 days, and then the growth of the test bacteria is detected respectively.
In a typical but non-limiting embodiment of the invention, in step (b), the cultivation time is, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7 days.
In a preferred embodiment of the invention, the test bacterium is a fungus.
In the present invention, fungi include plant fungi, other plant pathogenic fungi or non-pathogenic fungi.
In a further preferred embodiment of the invention, the test bacterium is selected from one of the group consisting of alternaria turciosa, alternaria tabaci and beauveria bassiana, and particularly when the test bacterium is alternaria turcicola or alternaria tabaci, the determination method is more accurate, more stable and more intuitive.
In a preferred embodiment of the present invention, in step (b), the OD of the test bacterial suspension is 0.3-0.35.
In typical, but non-limiting embodiments of the invention, the test bacterial suspension has an OD of, for example, 0.3, 0.31, 0.32, 0.33, 0.34, or 0.35.
In a preferred embodiment of the present invention, the preparation method of the test bacterial suspension comprises the following steps:
adding the test bacteria into a Tween 80 solution with a ratio of 1:1000, and preparing a bacterial suspension with an OD value of 0.3-0.35 for later use.
In a preferred embodiment of the present invention, the test bacteria are cultured using PDA medium.
PDA culture medium is the short name for potato glucose agar culture medium.
In a preferred embodiment of the present invention, the aqueous extract of Briggrin is prepared by the following steps:
(m) inoculating the farm antibiotic 769 strain into a culture medium, and completely culturing to obtain a mixture of the Brilliant mycin and the culture medium;
(n) drying the mixture of the Briggrin and the culture medium to obtain a solid Briggrin, crushing the solid Briggrin, and leaching with water to obtain a Briggrin leaching solution;
(s) centrifuging the Brigguin lingmycin leaching liquor, and taking supernate to obtain the Brigguin lingmycin water extracting liquor.
In the invention, the agricultural resistance "769" is obtained by separating and screening from the soil of princess ridge in 1967 by an expert of agricultural academy of Jilin province, is identified by a Raney secondary researcher of the institute of microbiology of Chinese academy of sciences, is named as a new variety of streptomyces hygroscopicus princess ridge, is also called agricultural resistance "769" in production, and the produced antibiotic is called princess ridge mycin.
In a preferred embodiment of the present invention, in step (m), the culture time is 5 to 7 days, preferably 6 days.
In a typical but non-limiting embodiment of the invention, the agro-resistant "769" strain is inoculated into a culture medium for a period of time such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7 days.
In a preferred embodiment of the present invention, in the step (n), the time for extracting the pulverized solid of the Briggrin with water is 24 to 48 hours.
In the preferred embodiment of the present invention, the time for extraction is set to 24-48h, so that the tomb-mycin in the solid is completely extracted.
In typical but non-limiting embodiments of the invention, the time of leaching is, for example, 24, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45 or 48 h.
In a preferred embodiment of the present invention, in the step (n), the volume ratio of the crushed extract of the princess lingmycin solid to the water is 1: 2-10.
By limiting the volume ratio of the crushed material to water to 1:2-10, the water can completely submerge the crushed material, so that the princess lingmycin can be completely extracted.
In typical but non-limiting embodiments of the invention, the volume of the pulverizate and water is, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1: 10.
In a preferred embodiment of the present invention, in step (m), the culture substrate is corn grit.
The corn residue is selected as the culture medium, so that the culture medium is cheap and easy to obtain, and the cost can be reduced.
In a preferred embodiment of the invention, the corn grits are subjected to soaking, draining and sterilization.
The corn grit is soaked, drained and sterilized to remove impurities and mixed bacteria in the corn grit so as to avoid influencing the growth of the agricultural antibiotic 769 strain.
In a preferred embodiment of the invention, the corn residue is soaked in clear water for 1.5-8 h.
The impurities in the corn residue are removed by soaking the corn residue for 1.5-8h and then draining.
In typical, but non-limiting embodiments of the invention, the corn grit steeping time is, for example, 1, 2, 3, 4, 5, 6, 7, or 8 hours.
In a preferred embodiment of the invention, the time of the sterilization treatment is 15 to 25 min.
The sterilization time is set to be 15-25min, so that the mixed bacteria in the corn dregs are killed.
In typical but non-limiting embodiments of the invention, the sterilization time is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 min.
According to another aspect of the invention, the method for detecting the bacteriostatic activity of the Briggrin mycin is applied to the detection of the bacteriostatic activity of antibiotics.
The method for detecting the bacteriostatic activity of the Briggrin mycin provided by the invention has wide application range and can also be used for detecting the bacteriostatic activity of other antibiotics.
The technical solution provided by the present invention is further described below with reference to examples and comparative examples.
Example 1
The embodiment provides a method for detecting the bacteriostatic activity of princess ridge mycin, which comprises the following steps:
(1) providing a suspension of test bacteria
Taking the northern leaf blight as a test bacterium, adding the northern leaf blight into a mixture of 1:1000 Tween 80, mixing, and measuring OD value with ultraviolet spectrophotometer, wherein the OD value is 0.30-0.35 to obtain test bacterial suspension.
(2) Providing a water extract of princess lingmycin;
activating the strain "769" of the pesticide resistance, inoculating the strain into a corn residue substrate which is soaked, drained and sterilized, culturing for 6 days, then sequentially drying and crushing, adding clear water according to the volume ratio of the crushed material to water of 1:2 for leaching for 36 hours to obtain a tomaymycin leaching liquor, centrifuging the tomaymycin leaching liquor, and taking supernatant to obtain the tomaymycin water leaching liquor.
(3) Determining critical bacteriostasis volume of water extract of princess lingmycin
Taking a PDA culture medium as a culture medium, and adding 160 mu L of the corn northern leaf blight bacterium suspension into 40mL of the culture medium; the growth of northern leaf blight on plates was observed after 4 days by mixing aqueous solutions of Briggrin mycin in volumes of 0. mu.L, 80. mu.L, 100. mu.L, 120. mu.L, 140. mu.L, 160. mu.L, 180. mu.L, 200. mu.L, 220. mu.L, 240. mu.L, 260. mu.L and 280. mu.L, respectively, plating, and culturing at 28 ℃ as shown in Table 1 and FIG. 1.
TABLE 1
Note: + represents that the growth speed of the strain is high and the total amount of the thalli is large;
+ represents that the strain grows faster and the total amount of the bacteria is more;
+ + + represents a slightly slow growth of the strain and a slightly less total amount of the bacteria;
the + and + represents that the strain grows slowly and the total amount of the thalli is small;
and ++++ represents sterile seed growth.
FIG. 1 is a strain growth diagram of different volume ratios of bacterial suspension to water extract, wherein from left to right, from top to bottom, the strain growth diagrams of groups 1-12 are shown, and it can be seen from FIG. 1 and Table 1 that the volume ratio of bacterial suspension to water extract in group 10 is 160 μ L: when 240 mu L, the total amount of the strains is less, and the volume ratio of the bacterial suspension to the water extract from the 11 th group is 160 mu L: at 260. mu.L, no growth of the strain was observed, and as the volume of the aqueous extract increased, the volume ratio of the bacterial suspension to the aqueous extract in group 12 was 160. mu.L: 280 mu L, no strain growth is observed, that is, the volume ratio of the bacterial suspension and the water extract in the 11 th group is determined to be the critical bacteriostatic volume ratio of the water extract of the Briggrin in this embodiment, that is, the critical bacteriostatic volume of the water extract of the Briggrin in this embodiment.
Example 2
The embodiment provides a method for detecting the bacteriostatic activity of princess ridge mycin, which comprises the following steps: the difference between this example and example 1 is that the aqueous extract of gentamicin in the example and the aqueous extract of gentamicin in the example were in different batches. In the determination of the critical inhibitory concentration of the aqueous extract of Brilliant Linnamycin in this example, 160. mu.L of the suspension of Megaeumannomyces maydis was mixed with the aqueous extract of Brilliant Linnamycin in the volume of 0. mu.L, 160. mu.L, 240. mu.L, 320. mu.L, 400. mu.L, 480. mu.L, 560. mu.L, 640. mu.L, 720. mu.L, 800. mu.L, 880. mu.L and 960. mu.L, respectively, and the mixture was plated and cultured at 28 ℃ for 4 days, and then the growth of Megaeumannomyces maydis on the plates was observed, and the results are shown.
TABLE 2
Note: the labeling in Table is the same as in Table 1 and will not be described further.
FIG. 2 is a strain growth diagram of different volume ratios of bacterial suspension to water extract, wherein the strain growth diagrams of groups 1-12 are from left to right and from top to bottom, and as can be seen from FIG. 2 and Table 2, the volume ratio of bacterial suspension to water extract in groups 9-11 is 160 μ L: 720-880. mu.L, the total amount of the strain is small, and the volume ratio of the suspension to the water extract from the 12 th group is 160. mu.L: 960 μ L, the growth of the strain cannot be observed, that is, the volume ratio of the bacterial suspension and the water extract of the 12 th group is determined to be the critical bacteriostatic volume ratio of the batch of the water extract of the Brilliant mycin in this embodiment, that is, the critical bacteriostatic volume of the batch of the water extract of the Brilliant mycin in this embodiment.
Example 3
The embodiment provides a method for detecting the bacteriostatic activity of princess ridge mycin, which comprises the following steps: providing a princess ridge mycin water extract and a bacterial suspension of the corn northern leaf blight, wherein the preparation methods of the princess ridge mycin water extract and the corn northern leaf blight bacterial suspension are the same as that of the embodiment 1, and are not repeated; in this example, to illustrate the effect of bacterial load on critical inhibitory volume, the critical inhibitory volume ratio of the batch of the princess ridge mycin to the bacterial suspension of northern leaf blight fungus is 200 μ L to 160 μ L. In this example, when critical inhibitory volume measurement of a princess ridge mycin water extract on a northern leaf blight bacterial suspension is performed, 200 μ L of the princess ridge mycin water extract is mixed with 160 μ L, 240 μ L, 320 μ L, 400 μ L, 480 μ L, 560 μ L, 640 μ L, 720 μ L and 800 μ L of a corn northern leaf blight bacterial suspension, and the mixture is spread on a plate and cultured at 28 ℃, and after 6 days, growth conditions of the northern leaf blight bacteria on the plate are observed, as shown in fig. 3, the bacterial quantities of the northern leaf blight bacteria in the first row are sequentially increased from left to right in fig. 3, but the growth conditions of the northern leaf blight bacteria are not inhibited by the princess ridge mycin, and the growth conditions of the northern leaf blight bacteria are inhibited by the princess ridge mycin, and it is obvious from the figure that the increase of the average bacterial quantity of the northern leaf blight bacteria does not bring about the critical inhibitory volume. As can be seen from fig. 3, when the detection method provided by the present invention is used to determine the critical bacteriostatic volume of the aqueous extract of gentamicin, the bacterial load of the test bacteria does not significantly affect the bacteriostatic activity of the aqueous extract of gentamicin, and the bacteriostatic effect does not significantly change within 5 times of the bacterial load of the test bacteria.
Comparative example 1
The preparation method of the princess ridge mycin water extract is the same as the example 1, and the preparation method of the tobacco red spot disease bacterium suspension is the same as the preparation method of the corn big spot disease bacterium suspension, so that the details are not repeated; the method specifically comprises the following steps: taking Alternaria alternata as a test bacterium, accurately weighing 40mLPDA culture medium, respectively adding 160 muL, 320 muL and 480 muL of Alternaria alternata bacterial suspension, uniformly mixing and paving, taking a 7mm filter paper disc, placing the disc in a Brilliant lingmycin water extract with antagonistic action, and soaking for 10 min. The filter paper disc was removed by tweezers with sterile forceps and excess water extract was drained off the wall of the bottle. Pasting 4 pieces of filter paper pieces on a flat plate along a diagonal line 2cm away from the edge of the flat plate, culturing for 6 days at 28 ℃ by taking the flat plate without the filter paper pieces as a contrast, comparing the size and the definition of a bacteriostatic circle, and repeating the test for three times at each concentration under the set three concentrations. FIG. 4 shows the determination of the bacteriostatic activity of the aqueous extract of Briggrin Linesmycin against Alternaria alternata based on the diameter of the zone of inhibition. The first to third rows were three replicates from left to right at the same bacterial source concentration, and the fourth row was an experimental control without the addition of a filter paper with an aqueous extract of Brilliant Linesmycin.
As can be seen from fig. 4, the biological activity of the princess's ridge mycin on the alternaria alternate is measured by the bacteriostatic circle method, the size of the bacteriostatic circle has a direct relation with the bacterial quantity contained in the culture medium, the size of the bacteriostatic circle with 1-3 times of bacterial quantity has a significant difference according to the bacterial quantity, the larger the bacterial quantity is, the smaller the diameter of the bacteriostatic circle is, the more the embodied bacteriostatic activity brings errors according to the bacterial quantity of the test bacteria, and the bacteriostatic activity of the princess's ridge mycin on the alternaria alternate cannot be accurately embodied.
Comparative example 2
The comparative example provides a method for detecting the bacteriostatic activity of Briggrin on northern leaf blight of corn by adopting a bacteriostatic circle method, and the difference between the comparative example and the comparative example 1 is that northern leaf blight of corn is taken as a test bacterium, other steps are the same as those in the comparative example 1, and are not repeated, and the result is shown in FIG. 5.
As can be seen from fig. 5, the biological activity of the princess ridge mycin on the corn northern leaf blight is measured by the bacteriostatic circle method, the size of the bacteriostatic circle has a direct relation with the bacterial quantity contained in the culture medium, and the larger the bacterial quantity is, the smaller the diameter of the bacteriostatic circle is, the more the bacterial quantity is, the different the bacterial quantity is, the antibacterial activity of the princess ridge mycin on the corn northern leaf blight cannot be accurately reflected.
Example 4
The embodiment provides a method for detecting antibacterial activity of Briggrin, which comprises the following steps: (1) the preparation method is the same as that of example 1 and is not repeated herein, wherein the test bacterium suspension is prepared by taking the northern leaf blight of corn as a test bacterium;
(2) providing different concentrations of water extract of Briggrin;
activating a farm antibiotic '769' strain, inoculating the strain into a corn residue substrate which is soaked, drained and sterilized, completely culturing for 6 days, taking out the strain from an incubator, drying and crushing the strain in sequence, adding clear water according to the volume ratio of crushed substances to water of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, leaching the crushed substances for 36 hours to obtain 10 princess lincomycin leaching solutions with different concentrations, centrifuging the 10 princess lincomycin leaching solutions with different concentrations, and taking supernatant to obtain 10 princess lincomycin water leaching solutions with different concentrations;
(3) determining critical inhibitory concentration of different concentrations of water extract of Briggrin
160 μ L of the bacterial suspension of the northern leaf blight of maize was mixed with the aqueous extract of the 10 different concentrations of the Briggrin, and the mixture was spread on a plate, cultured at 28 ℃ and observed for 6 days, and the growth of the northern leaf blight of maize on the plate was observed, and the results are shown in FIG. 6.
As can be seen from fig. 6, the water extracts of the princess lincomycin with the same volume and different concentrations have different growth amounts of the northern leaf blight, i.e., the water extracts of the princess lincomycin with different concentrations have different critical bacteriostatic volume ratios, so that the level of the bacteriostatic activity of the water extracts of the princess lincomycin can be accurately determined.
Example 5
The embodiment provides a method for detecting antibacterial activity of Briggrin, which comprises the following steps:
(1) providing a water extract of Briggrin, the preparation method is the same as that of example 1, and the details are not repeated herein;
(2) providing beauveria bassiana suspension, and preparing the beauveria bassiana suspension by the same method as the preparation of the beauveria bassiana suspension in example 1, which is not repeated herein, wherein the OD value of the beauveria bassiana suspension stock solution is 0.32.
(3) The critical bacteriostasis volume of the water extract of the Briggrin mycin is respectively determined by adopting different culture mediums and beauveria bassiana suspensions with different concentrations, and an improved Gaoshi No. 1 culture medium (5 g of peptone, 5g of sodium chloride, 10g of glucose, 10g of soluble starch, 20g of agar, and adding water to 1000mL) and a PDA culture medium are respectively adopted, the dosage is 40mL, and the beauveria bassiana suspension stock solution is sequentially diluted by 10 times, 100 times, 1000 times and 10000 times. Wherein, beauveria bassiana suspension and the princess lingmycin aqueous extract with different concentrations under different culture medium conditions are both 100 μ L, the steps are not repeated as in example 1, and the growth condition of the strain is shown in FIG. 7.
In FIG. 7, the first row and the second row both use modified Gauss No. 1 culture medium as culture medium, and the first row is a graph showing the growth of the strains mixed with the aqueous extract of Briggrin mycin; the second row is a strain growth condition diagram of the beauveria bassiana suspension with different dilution concentrations without adding the water extract of the Briggrin mycin; the third row and the fourth row both use a PDA culture medium as a culture medium, and the third row is a strain growth condition diagram after being mixed with the water extract of the Briggrin mycin; the fourth row is a strain growth condition chart of the beauveria bassiana suspension with different dilution concentrations without adding the water extract of the Briggrin mycin; as can be seen from FIG. 7, the water extract of the Brigguin cinquefoil has an inhibiting effect on the beauveria bassiana, and the complete inhibiting effect of the Brigguin cinquefoil on the beauveria bassiana can be visually observed by using 100 times of the diluent of the beauveria bassiana suspension.
The 769 medium in FIG. 7 is a commercially available modified Gao's No. I medium.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (11)
1. A method for detecting antibacterial activity of Briggrin is characterized by comprising the following steps:
(a) providing a princess lingmycin water extract and a test bacterium suspension;
(b) mixing the water extract of the Briggrin in different volume ratios with the bacterial suspension of the test bacteria, respectively investigating the growth conditions of the test bacteria to obtain the critical bacteriostatic volume ratio of the water extract of the Briggrin to the bacterial suspension of the test bacteria, and determining the bacteriostatic activity of the water extract of the Briggrin to the test bacteria according to the critical bacteriostatic volume ratio of the water extract of the Briggrin to the bacterial suspension of the test bacteria;
in the step (b), mixing the water extract of the Briggrin mycin and the bacterial suspension of the test bacteria in different volume ratios, culturing for 2-6 days, and then respectively investigating the growth conditions of the test bacteria in different volume ratios;
the princess lingmycin water extract is prepared by the following steps:
(m) inoculating the farm antibiotic 769 strain into a culture medium, and completely culturing to obtain a mixture of the Brilliant mycin and the culture medium;
(n) drying and crushing the mixture of the princess lingmycin and the culture medium, and leaching with clear water to obtain a princess lingmycin leaching liquor;
(s) centrifuging the Brigguin lingmycin leaching liquor, and taking supernate to obtain the Brigguin lingmycin water extracting liquor;
in step (m), the culture medium is corn grit;
the test bacteria are fungi;
in the step (b), the OD value of the test bacterium suspension is 0.3-0.35;
in the step (m), the cultivation time is 5 to 7 days;
in the step (n), the leaching time is 12-48 h;
the volume ratio of the crushed material obtained by crushing the mixture of the Briggrin and the culture medium in the step (n) to water is 1: 2-10.
2. The method for detecting the bacteriostatic activity of the Briggrin mycin according to claim 1,
the test bacteria is selected from one of corn northern leaf blight bacteria, tobacco red star disease bacteria or beauveria bassiana.
3. The method for detecting inhibitory activity of gentamicin according to claim 1, wherein in step (m), the cultivation time is 6 days.
4. The method for detecting the bacteriostatic activity of the Briggrin mycin according to claim 1, wherein in the step (n), the leaching time is 36 h.
5. The method for detecting the bacteriostatic activity of the princess ridge-mycin according to claim 1, wherein the volume ratio of the crushed material to the water after the mixture of the princess ridge-mycin and the culture substrate is crushed in step (n) is 1: 2.
6. The method for detecting the bacteriostatic activity of the Briggrin mycin according to claim 1,
the corn dregs are soaked, drained and sterilized.
7. The method for detecting the bacteriostatic activity of the princess ridge mycin according to claim 6, characterized in that the corn grits are soaked in water for 1.5-8 h.
8. The method for detecting the bacteriostatic activity of the princess ridge mycin according to claim 7, characterized in that the soaking time of the corn grit is 3 hours.
9. The method for detecting the bacteriostatic activity of the Briggrin mycin according to claim 6, wherein the time of the sterilization treatment is 15-25 min.
10. The method for detecting the bacteriostatic activity of the Briggrin mycin according to claim 9, wherein the time of the sterilization treatment is 20 min.
11. The use of the method for detecting the bacteriostatic activity of the princess ridge mycin according to any one of claims 1-10 in the detection of the bacteriostatic activity of antibiotics.
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