CN108776222A - Gongzhuling mycin bacteriostatic activity detection method and application - Google Patents
Gongzhuling mycin bacteriostatic activity detection method and application Download PDFInfo
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- CN108776222A CN108776222A CN201810612023.6A CN201810612023A CN108776222A CN 108776222 A CN108776222 A CN 108776222A CN 201810612023 A CN201810612023 A CN 201810612023A CN 108776222 A CN108776222 A CN 108776222A
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Abstract
The present invention provides a kind of Gongzhuling mycin bacteriostatic activity detection method and applications, are related to bacteriostatic activity detection method technical field, described method includes following steps：There is provided Gongzhuling mycin Aqueous extracts and for trying bacterium bacteria suspension, then by different volumes than Gongzhuling mycin Aqueous extracts and for examination bacterium bacteria suspension mix, obtain Gongzhuling mycin Aqueous extracts and the critical antibacterial volume ratio for trying bacterium bacteria suspension, according to the critical antibacterial volume ratio, determine Gongzhuling mycin Aqueous extracts to the bacteriostatic activity for trying bacterium, it alleviates physical chemistry detection method in existing bacteriostatic activity detection method and requires height, microbial method technical problem not easy to operate, method intuitive provided by the invention is good, high sensitivity, stability is good, it is simple and convenient, it takes short, it is applied widely, it is safer, it is more reliable, expense is lower, a kind of new way is provided for the detection of Gongzhuling mycin bacteriostatic activity.
Description
Technical field
The present invention relates to bacteriostatic activity detection method technical fields, are examined more particularly, to a kind of Gongzhuling mycin bacteriostatic activity
Survey method and application.
Background technology
Agriculture resists " 769 " to be by academy of agricultural sciences of Jilin Province expert one plant of agriculture that separation screening obtains from the soil of Gongzhuling in 1967
With streptomycete bacterial strain, Gongzhuling mycin is the metabolite that agriculture resists " 769 ", is a kind of mix preparation of natural biology synthesis.
Currently, an important indicator of evaluation Gongzhuling mycin quality is bacteriostatic activity, assay method has physical chemistry
Method and microbial method.Although physical-chemical process it is easy, quickly, it is very high to Gongzhuling mycin purity requirement, it is no it will cause
Very large deviation, and determining instrument is expensive, testing cost is high.Microbial method includes dilution method, turbidimetry, Guan Diefa, breathing degree
Amount method and dehydriding, these method high sensitivities are securely and reliably widely applied, but these microbiological methods are grasped
It is more to make step, experiment process is long, and influence factor is more, is not easy to testing staff's grasp.
In view of this, special propose the present invention.
Invention content
One of the objects of the present invention is to provide a kind of Gongzhuling mycin bacteriostatic activity detection methods, to alleviate existing object
The technical problems such as the requirement of Physicochemical detection method is high, and microbial method is not easy to operate.
Mycin bacteriostatic activity detection method in Gongzhuling provided by the invention, includes the following steps：
(a) Gongzhuling mycin Aqueous extracts are provided and for trying bacterium bacteria suspension；
(b) by different volumes than Gongzhuling mycin Aqueous extracts and mix for examination bacterium bacteria suspension, investigated respectively for examination bacterium
Growing state obtains Gongzhuling mycin Aqueous extracts and the critical antibacterial volume ratio for trying bacterium bacteria suspension, according to Gongzhuling mycin water
Extract and the critical antibacterial volume ratio for trying bacterium bacteria suspension determine Gongzhuling mycin Aqueous extracts to the bacteriostatic activity for trying bacterium.
Further, in step (b), by different volumes than Gongzhuling mycin Aqueous extracts and for examination bacterium bacteria suspension mix
It closes, after cultivating 2-6 days, then investigates the growing state for trying bacterium respectively.
Further, described for trying bacterium as fungi；
Preferably, described one kind for examination bacterium in Exserohilum turcicum, tobacco brown spot pathogen or beauveria bassiana.
Further, in step (b), the OD values for trying bacterium bacteria suspension are 0.3-0.35.
Further, the Gongzhuling mycin Aqueous extracts are prepared in accordance with the following steps：
(m) resist " 769 " strain to be inoculated into culture substrate agriculture, cultivate completely, obtain Gongzhuling mycin and culture substrate
Mixture；
(n) mixture of Gongzhuling mycin and culture substrate is dried, Gongzhuling mycin solids is obtained, by princess
Ridge mycin solids crushes, and with flooding, obtains Gongzhuling mycin leaching liquor；
(s) Gongzhuling mycin leaching liquor is centrifuged, takes supernatant to get to Gongzhuling mycin Aqueous extracts.
Further, in step (m), incubation time is 5-7 days, preferably 6 days.
Further, in step (n), extraction time 24-48h, preferably 36h.
Further, in step (n), the volume ratio of crushed material and water after the crushing of Gongzhuling mycin solids is 1:2-
10, preferably 1:2.
Further, in step (m), the culture substrate is cut for corn；
Preferably, corn quarrel is by impregnating, draining and sterilization treatment；
Preferably, corn quarrel is impregnated with water, corn is cut soaking time as 1.5-8h, preferably 3h；
Preferably, the sterilization treatment time is 15-25min, preferably 20min.
The second object of the present invention is to provide Gongzhuling mycin provided by the invention and bacteriostatic activity detection method anti-
Application in raw element bacteriostatic activity detection.
Mycin bacteriostatic activity detection method in Gongzhuling provided by the invention has the advantages that：
(1) by observe different volumes than Gongzhuling mycin Aqueous extracts and for examination bacterium bacteria suspension mixing after for examination bacterium growth
Situation, obtain Gongzhuling mycin Aqueous extracts to for try bacterium bacteriostatic activity, it is more intuitively, sensitiveer, it is more stable；
(2) it is introduced without poisonous and harmful reagent in operating process, it is safer, it is more reliable, also more save testing cost；
(3) operating procedure is few, and time-consuming short, influence factor is few, it is easier to which testing staff grasps；
(4) applied widely, it can be used in the bacteriostatic activity for detecting other antibiotic.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Currently, an important indicator of evaluation Gongzhuling mycin quality is bacteriostatic activity, assay method has physical chemistry
Method and microbial method.Although physical-chemical process it is easy, quickly, it is very high to Gongzhuling mycin purity requirement, it is no it will cause
Very large deviation, and determining instrument is expensive, testing cost is high.Microbial method includes dilution method, turbidimetry, Guan Diefa, breathing degree
Amount method and dehydriding, these method high sensitivities are securely and reliably widely applied, but these microbiological methods are grasped
It is more to make step, experiment process is long, and influence factor is more, is not easy to testing staff's grasp, and these methods do not exclude by for examination
The error that bacterium bacterium amount difference is brought.
According to an aspect of the present invention, the present invention provides a kind of Gongzhuling mycin bacteriostatic activity detection methods, including
Following steps：
(a) Gongzhuling mycin Aqueous extracts are provided and for trying bacterium bacteria suspension；
(b) by different volumes than Gongzhuling mycin Aqueous extracts and mix for examination bacterium bacteria suspension, investigated respectively for examination bacterium
Growing state obtains Gongzhuling mycin Aqueous extracts and the critical antibacterial volume ratio for trying bacterium bacteria suspension, according to Gongzhuling mycin water
Extract and the critical antibacterial volume ratio for trying bacterium bacteria suspension determine Gongzhuling mycin Aqueous extracts to the bacteriostatic activity for trying bacterium.
In the present invention, in step (b), with Gongzhuling mycin Aqueous extracts and the increasing for trying bacterium bacteria suspension volume ratio
Adding, the growth total amount for trying bacterium is reduced, when Gongzhuling mycin Aqueous extracts and for trying the increase of bacterium bacteria suspension volume ratio to a certain extent,
When observing the growth of no targeted fungal, Gongzhuling mycin Aqueous extracts and be critical antibacterial body for examination bacterium bacteria suspension volume ratio at this time
Product ratio.
In the present invention, Gongzhuling mycin Aqueous extracts suppression is judged by the critical antibacterial volume of Gongzhuling mycin Aqueous extracts
The active height of bacterium, the critical antibacterial volume is smaller, then illustrates that Gongzhuling mycin Aqueous extracts are better to the bacteriostatic activity for trying bacterium.
Mycin bacteriostatic activity detection method in Gongzhuling provided by the invention has the advantages that：
(1) by detect different volumes than Gongzhuling mycin Aqueous extracts and for examination bacterium bacteria suspension mixing after for examination bacterium growth
Situation obtains Gongzhuling mycin Aqueous extracts to the critical antibacterial volume for trying bacterium, to obtain Gongzhuling mycin Aqueous extracts pair
It is more intuitively, sensitiveer for trying the bacteriostatic activity of bacterium, it is more stable；
(2) it is introduced without poisonous and harmful reagent in operating process, it is safer, it is more reliable, also more save testing cost；
(3) operating procedure is few, time-consuming short, small by being influenced for examination bacterium bacterium amount, it is easier to which that testing staff grasps；
(4) applied widely, it can be used in the bacteriostatic activity for detecting other antibiotic.
In the preferred embodiment of the present invention, in step (b), by different volumes than Gongzhuling mycin water carry
Liquid and for examination bacterium bacteria suspension mixing, culture 2-6 days after, then detect respectively for examination bacterium growing state.
In the typical but non-limiting embodiment of the present invention, in step (b), time of culture as 2,2.5,
3,3.5,4,4.5,5,5.5,6,6.5 or 7 days.
In the preferred embodiment of the present invention, it is fungi for examination bacterium.
In the present invention, fungi includes plant epiphyte, other phytopathogens or non-disease fungus.
In present invention further optimization embodiment, for examination bacterium select Exserohilum turcicum, tobacco brown spot pathogen or
One kind in beauveria bassiana, especially when being corn pinta bacterium or tobacco brown spot pathogen for examination bacterium, the assay method is more accurate
Really, more stable, more intuitively.
In the preferred embodiment of the present invention, in step (b), the OD values for trying bacterium bacteria suspension are 0.3-
0.35。
In the typical but non-limiting embodiment of the present invention, for try the OD values of bacterium bacteria suspension as 0.3,0.31,
0.32,0.33,0.34 or 0.35.
In the preferred embodiment of the present invention, include the following steps for trying the preparation method of bacterium bacteria suspension：
It will be added to 1 for examination bacterium:In 1000 Tween 80 solution, it is spare to be configured to the bacteria suspension that OD values are 0.3-0.35.
In the preferred embodiment of the present invention, it is cultivated using PDA culture medium for examination bacterium.
PDA culture medium is abbreviation of the people to potato dextrose agar.
In the preferred embodiment of the present invention, mycin Aqueous extracts in Gongzhuling are prepared in accordance with the following steps：
(m) resist " 769 " strain to be inoculated into culture substrate agriculture, cultivate completely, obtain Gongzhuling mycin and culture substrate
Mixture；
(n) mixture of Gongzhuling mycin and culture substrate is dried, Gongzhuling mycin solids is obtained, by princess
Ridge mycin solids crushes, and with flooding, obtains Gongzhuling mycin leaching liquor；
(s) Gongzhuling mycin leaching liquor is centrifuged, takes supernatant to get to Gongzhuling mycin Aqueous extracts.
In the present invention, agriculture resists " 769 " separation screening from the soil of Gongzhuling obtained by academy of agricultural sciences of Jilin Province expert 1967
Arrive, it identified through Microbe Inst., Chinese Academy of Sciences Ruan Jisheng researcher, and name for S. ahygroscopicus Gongzhuling it is new
Mutation, also referred to as agriculture resists " 769 " in production, and generated antibiotic is referred to as Gongzhuling mycin.
In the preferred embodiment of the present invention, in step (m), incubation time is 5-7 days, preferably 6 days.
In the typical but non-limiting embodiment of the present invention, agriculture resists " 769 " strain to be inoculated into culture substrate, trains
The foster time is as being 2,2.5,3,3.5,4,4.5,5,5.5,6,6.5 or 7 days.
In the preferred embodiment of the present invention, in step (n), the Gongzhuling mycin solids water after crushing
The time of extraction is 24-48h.
In the preferred embodiment in the present invention, by the way that extraction time is set as 24-48h, so that solids
In Gongzhuling mycin be leached out completely.
In the typical but non-limiting embodiment of the present invention, time of extraction as 24,26,28,30,32,34,
35,36,38,40,42,44,45 or 48h.
In the preferred embodiment of the present invention, in step (n), the smashing after the crushing of Gongzhuling mycin solids
The volume ratio of object and water is 1:2-10.
By the way that the volume ratio of crushed material and water is limited to 1:2-10, so that water can be totally submerged crushed material, so that
Gongzhuling mycin is obtained to be leached out completely.
In the typical but non-limiting embodiment of the present invention, the volume of crushed material and water is such as 1:2,1:3,1:
4,1:5,1:6,1:7,1:8,1:9 or 1:10.
In the preferred embodiment of the present invention, in step (m), culture substrate is cut for corn.
It is cheap and easy to get by selecting corn quarrel to be used as culture substrate, it can more reduce cost.
In the preferred embodiment of the present invention, corn quarrel is by impregnating, draining and sterilization treatment.
By the way that corn quarrel is impregnated, is drained and sterilization treatment, the impurity during corn is cut and miscellaneous bacteria removal, to keep away
Exempt to influence the growth that agriculture resists " 769 " strain.
In the preferred embodiment of the present invention, corn quarrel is impregnated with clear water, the time that corn quarrel is impregnated
For 1.5-8h.
It is drained again by the way that corn is cut after immersion 1.5-8h, the impurity removal during corn is cut.
In the typical but non-limiting embodiment of the present invention, corn is cut soaking time as being 1,2,3,4,5,6,7
Or 8h.
In the preferred embodiment of the present invention, the time of sterilization treatment is 15-25min.
By the way that sterilization time is set as 15-25min, so that the miscellaneous bacteria in corn quarrel is killed.
In the typical but non-limiting embodiment of the present invention, sterilization time as 15,16,17,18,19,20,
21,22,23,24 or 25min.
According to another aspect of the present invention, mycin bacteriostatic activity detection method in Gongzhuling provided by the invention is used for antibiosis
Application in plain bacteriostatic activity detection.
The detection method of mycin bacteriostatic activity in Gongzhuling provided by the invention is applied widely, can be used for other antibiosis
The bacteriostatic activity detection of element.
Technical solution provided by the invention is further described with reference to embodiment and comparative example.
Embodiment 1
A kind of Gongzhuling mycin bacteriostatic activity detection method is present embodiments provided to include the following steps：
(1) bacteria suspension for trying bacterium is provided
It is for trying bacterium, Exserohilum turcicum being added to 1 with Exserohilum turcicum：In 1000 Tween 80s, mixing, use is ultraviolet
Spectrophotometric determination OD values are obtained when between OD values=0.30-0.35 for trying bacteria suspension.
(2) Gongzhuling mycin Aqueous extracts are provided；
Agriculture is resisted into " 769 " actication of culture, and is inoculated into and has been subjected in immersion, the corn for draining and sterilizing quarrel matrix, culture 6
It, is then dried and crushes successively, and is 1 according to the volume ratio of crushed material and water:2, which are added clear water, extracts 36h, obtains public affairs
Main ridge mycin leaching liquor centrifuges Gongzhuling mycin leaching liquor, takes supernatant to get to Gongzhuling mycin Aqueous extracts.
(3) the critical antibacterial volume of Gongzhuling mycin Aqueous extracts is measured
Using PDA culture medium as culture substrate, 160 μ L Exserohilum turcicum bacteria suspensions are added in 40mL culture substrates；Point
It is 0 μ L, 80 μ L, 100 μ L, 120 μ L, 140 μ L, 160 μ L, 180 μ L, 200 μ L, 220 μ L, 240 μ L, 260 μ L and 280 not take volume
The growing state of Exserohilum turcicum on tablet is observed in the Gongzhuling mycin Aqueous extracts mixing of μ L, bed board, 28 DEG C of cultures after 4 days,
As a result as shown in table 1 and Fig. 1.
Table 1
Note：+ represent that growth speed is fast, and thalline total amount is more；
++ represent that growth is very fast, and thalline total amount is more；
+++ represent that growth is slightly slow, and thalline total amount is slightly few；
++++represent growth is slow, and thalline total amount is few；
+++ ++ it represents without growth.
Fig. 1 is the growth figure of different bacteria suspensions and Aqueous extracts volume ratio, wherein from left to right, is followed successively by under upper
The growth figure of 1-12 groups, from Fig. 1 and table 1 as can be seen that the volume ratio of bacteria suspension and Aqueous extracts is 160 μ L in the 10th group：
When 240 μ L, strain total amount is few, and to the volume ratio of the 11st group of bacteria suspension and Aqueous extracts be 160 μ L：When 260 μ L, i.e., it cannot observe
To growth, and it is 160 μ L to the volume ratio of the 12nd group of bacteria suspension and Aqueous extracts with the increase of Aqueous extracts volume：280μ
Growth is also not observed in L, you can determines that the 11st group of bacteria suspension and the volume ratio of Aqueous extracts are public in the present embodiment
The main critical antibacterial volume ratio of ridge mycin Aqueous extracts, i.e., the critical antibacterial volume of Gongzhuling mycin Aqueous extracts in the present embodiment.
Embodiment 2
A kind of Gongzhuling mycin bacteriostatic activity detection method is present embodiments provided to include the following steps：The present embodiment and reality
Apply example 1 the difference is that, the Gongzhuling mycin Aqueous extracts in Gongzhuling mycin Aqueous extracts and embodiment are different batches.?
When measuring the critical Mlc of Gongzhuling mycin Aqueous extracts in the present embodiment, 160 μ L Exserohilum turcicum bacteria suspensions are taken respectively
It is 0 μ L, 160 μ L, 240 μ L, 320 μ L, 400 μ L, 480 μ L, 560 μ L, 640 μ L, 720 μ L, 800 μ L, 880 μ L and 960 μ with volume
The growing state of Exserohilum turcicum on tablet is observed in the Gongzhuling mycin Aqueous extracts mixing of L, bed board, 28 DEG C of cultures after 4 days,
As a result as shown in table 2 and Fig. 2.
Table 2
Note：Mark action is identical as table 1 in table, and details are not described herein.
Fig. 2 is the growth figure of different bacteria suspensions and Aqueous extracts volume ratio, wherein from left to right, is followed successively by under upper
The growth figure of 1-12 groups, from Fig. 2 and table 2 as can be seen that the volume ratio of bacteria suspension and Aqueous extracts is 160 μ in 9-11 groups
L：When 720-880 μ L, strain total amount is few, and to the volume ratio of the 12nd group of bacteria suspension and Aqueous extracts be 160 μ L：When 960 μ L, i.e., not
It is observed that growth, you can determine that the 12nd group of bacteria suspension and the volume ratio of Aqueous extracts are the public affairs of batch in the present embodiment
The main critical antibacterial volume ratio of ridge mycin Aqueous extracts, i.e., the critical antibacterial body of the Gongzhuling mycin Aqueous extracts of batch in the present embodiment
Product.
Embodiment 3
A kind of Gongzhuling mycin bacteriostatic activity detection method is present embodiments provided to include the following steps：It is mould to provide Gongzhuling
The preparation method of the bacteria suspension of plain Aqueous extracts and Exserohilum turcicum, Gongzhuling mycin Aqueous extracts and Exserohilum turcicum bacteria suspension
Same as Example 1, details are not described herein；The present embodiment is the influence to critical antibacterial volume, this batch in order to illustrate bacterium amount
Gongzhuling mycin is 200 μ L to the critical antibacterial volume ratio of Exserohilum turcicum bacteria suspension:160μL.In the present embodiment, it carries out
When Gongzhuling mycin Aqueous extracts are to the critical antibacterial stereometry of Exserohilum Turcicum bacteria suspension, 200 Gongzhuling μ L mycin Aqueous extracts are taken
It is 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 Exserohilum turcicum bacterium with volume
Suspension mixes, bed board, and the growing state of Exserohilum turcicum on tablet is observed in 28 DEG C of cultures after 6 days, the results are shown in Figure 3, figure
In 3 first row from left to right be for examination bacterium bacterium amount successively increase, but without be added Gongzhuling mycin inhibit when for examination bacterium growth
Situation, second row are to be added for the upgrowth situation for trying bacterium after Gongzhuling mycin inhibits, from the graph, it is apparent that the big spot of corn
The increase that germ is measured does not bring apparent influence to antibacterial critical size.From figure 3, it can be seen that using provided by the invention
When detection method measures Gongzhuling mycin Aqueous extracts critical antibacterial volume, for try bacterium bacterium amount number will not be to Gongzhuling mycin
Water body liquid bacteriostatic activity significantly affects, and 5 times do not have significant changes for fungistatic effect in examination bacterium bacterium amount.
Comparative example 1
This comparative example provides a kind of bacteriostatic activity measuring Gongzhuling mycin to tobacco brown spot pathogen using inhibition zone method
Detection method, Gongzhuling mycin Aqueous extracts preparation method is same as Example 1, the preparation side of tobacco brown spot pathogen bacteria suspension
Method is identical as the preparation method of Exserohilum turcicum bacteria suspension, and details are not described herein；Specifically comprise the following steps：With the red star of tobacco
Germ is for trying bacterium, accurately weighing 40mLPDA culture mediums, be separately added into the red star of tobacco that volume is 160 μ L, 320 μ L and 480 μ L
Germ bacteria suspension, mixing bed board take 7mm filter paper dicks, are placed in the Gongzhuling mycin Aqueous extracts with antagonism, impregnate
10min.With aseptic nipper tweezer filter paper dick, extra Aqueous extracts are drained in bottle wall.By 4 filter papers away from plate edge 2cm
Pairs of linea angulata is attached on tablet, and the tablet not put filter paper is control, and 28 DEG C are cultivated 6 days, compares inhibition zone size and clear
Degree is tested under three concentration of setting, and each concentration is set to be repeated three times.Fig. 4 show the size judgement with antibacterial circle diameter
Bacteriostatic activity of the Gongzhuling mycin Aqueous extracts to tobacco brown spot pathogen.First to third row be same bacterium source concentration from left to right
Under three repetitions, the 4th row is that the experiment contrast with Gongzhuling mycin Aqueous extracts filter paper is not added.
From fig. 4, it can be seen that inhibition zone method measures bioactivity of the Gongzhuling mycin to tobacco brown spot pathogen, inhibition zone is big
It is small with culture medium contained in bacterium amount have a direct relation, the inhibition zone size of 1-3 times of bacterium amount because bacterium amount number have it is significant poor
Different, bacterium amount is bigger, and antibacterial circle diameter is smaller, the bacteriostatic activity embodied because for examination bacterium bacterium amount number bring error, Wu Fazhun
Really embody bacteriostatic activity of the Gongzhuling mycin to tobacco brown spot pathogen.
Comparative example 2
This comparative example provides a kind of bacteriostatic activity measuring Gongzhuling mycin to Exserohilum turcicum using inhibition zone method
Detection method, this comparative example and comparative example 1 the difference is that, be for trying bacterium, other steps with Exserohilum turcicum
Identical as comparative example 1, details are not described herein, and the results are shown in Figure 5.
From fig. 5, it can be seen that inhibition zone method measures bioactivity of the Gongzhuling mycin to Exserohilum turcicum, inhibition zone is big
Small to have direct relation with bacterium amount contained in culture medium, for the inhibition zone size of 1-3 times of bacterium amount because of the difference of bacterium amount, bacterium amount is bigger,
Antibacterial circle diameter is smaller, can not accurately embody bacteriostatic activity of the Gongzhuling mycin to Exserohilum turcicum.
Embodiment 4
A kind of Gongzhuling mycin bacteriostatic activity detection method is present embodiments provided, is included the following steps：(1) big with corn
Pinta bacterium is for trying bacterium, preparing for trying bacterium bacteria suspension, the preparation method is the same as that of Example 1, and details are not described herein；
(2) the Gongzhuling mycin Aqueous extracts of various concentration are provided；
It is inoculated into and is had been subjected in immersion, the corn for draining and sterilizing quarrel matrix after resisting " 769 " strain activated agriculture, completely
Culture, takes out from incubator after 6 days, is then dried and crushes successively, and distinguishes according to the volume ratio of crushed material and water
It is 1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9,1:10 are added clear water extraction 36h to get to 10 kinds of various concentrations
Gongzhuling mycin leaching liquor, by the Gongzhuling mycin leaching liquor centrifugation of 10 kinds of various concentrations, take supernatant to get to 10 kinds not
With the Gongzhuling mycin Aqueous extracts of concentration；
(3) the critical Mlc of the Gongzhuling mycin Aqueous extracts of various concentration is measured
Take the Gongzhuling mycin Aqueous extracts of 160 μ L Exserohilum turcicums bacteria suspensions and above-mentioned 10 kinds of various concentrations mixed respectively
It closes, bed board, 28 DEG C of cultures observe the growing state of Exserohilum turcicum on tablet, the results are shown in Figure 6 after 6 days.
From fig. 6, it can be seen that same volume dosage, but the Gongzhuling mycin Aqueous extracts that concentration is different, the leaf blight of corn
The increment of bacterium is different, i.e., the Gongzhuling mycin Aqueous extracts of various concentration have different critical antibacterial volume ratios, thus can be accurate
The really height of the bacteriostatic activity of judgement Gongzhuling mycin Aqueous extracts.
Embodiment 5
A kind of Gongzhuling mycin bacteriostatic activity detection method is present embodiments provided, is included the following steps：
(1) Gongzhuling mycin Aqueous extracts are provided, the preparation method is the same as that of Example 1, and details are not described herein；
(2) beauveria bassiana bacteria suspension is provided, during the preparation method is the same as that of Example 1 prepared by Exserohilum turcicum bacteria suspension,
This is repeated no more, value=0.32 beauveria bassiana bacteria suspension stoste OD.
(3) the beauveria bassiana bacteria suspension of different culture media and various concentration is used to measure Gongzhuling mycin Aqueous extracts respectively
Critical antibacterial volume, No. 1 culture medium of Gao Shi of improvement is respectively adopted, and (peptone 5g, sodium chloride 5g, glucose 10g are soluble
Starch 10g, agar 20g, adds water to 1000mL) and PDA culture medium, dosage 40mL, beauveria bassiana bacteria suspension stoste it is dilute successively
Release 10 times, 100 times, 1000 times, 10000 times.Wherein, under the conditions of different culture media the beauveria bassiana bacteria suspension of various concentration and
Gongzhuling mycin Aqueous extracts are 100 μ L, and details are not described herein with embodiment 1 for step, and growth situation is as shown in Figure 7.
In Fig. 7, using No. 1 culture medium of the Gao Shi of improvement as culture substrate, first row is and princess for first row and second row
The mixed growth situation map of ridge mycin Aqueous extracts；Second row is to be not added with Gongzhuling mycin Aqueous extracts only to add different dilutions dense
The growth situation map of the beauveria bassiana bacteria suspension of degree；Third is arranged and the 4th row is using PDA culture medium as culture substrate, the
Three rows be and the mixed growth situation map of Gongzhuling mycin Aqueous extracts；4th row is to be not added with Gongzhuling mycin Aqueous extracts only
Add the growth situation map of the beauveria bassiana bacteria suspension of different diluted concentrations；From figure 7 it can be seen that Gongzhuling mycin water carries
Liquid is inhibited to beauveria bassiana, and 100 times of dilutions of beauveria bassiana bacteria suspension can intuitively observe public affairs
Main ridge mycin acts on the complete inhibition of beauveria bassiana.
769 culture mediums are the Gause I culture medium of commercially available improvement in above-mentioned Fig. 7.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, it will be understood by those of ordinary skill in the art that：Its according to
So can with technical scheme described in the above embodiments is modified, either to which part or all technical features into
Row equivalent replacement；And these modifications or replacements, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of Gongzhuling mycin bacteriostatic activity detection method, which is characterized in that include the following steps：
(a) Gongzhuling mycin Aqueous extracts are provided and for trying bacterium bacteria suspension；
(b) by different volumes than Gongzhuling mycin Aqueous extracts and for examination bacterium bacteria suspension mix, investigate respectively for try bacterium growth
Situation obtains Gongzhuling mycin Aqueous extracts and the critical antibacterial volume ratio for trying bacterium bacteria suspension, according to Gongzhuling mycin Aqueous extracts
With the critical antibacterial volume ratio for trying bacterium bacteria suspension, determine Gongzhuling mycin Aqueous extracts to the bacteriostatic activity for trying bacterium.
2. mycin bacteriostatic activity detection method in Gongzhuling according to claim 1, which is characterized in that, will in step (b)
Different volumes than Gongzhuling mycin Aqueous extracts and for examination bacterium bacteria suspension mixing, after culture 2-6 days, then investigate different volumes respectively
Than the growing state for trying bacterium.
3. mycin bacteriostatic activity detection method in Gongzhuling according to claim 1, which is characterized in that it is described for examination bacterium be true
Bacterium；
Described one kind for examination bacterium in Exserohilum turcicum, tobacco brown spot pathogen or beauveria bassiana.
4. according to claim 1-3 any one of them Gongzhuling mycin bacteriostatic activity detection method, which is characterized in that in step
(b) in, the OD values for trying bacterium bacteria suspension are 0.3-0.35.
5. mycin bacteriostatic activity detection method in Gongzhuling according to claim 1, which is characterized in that the Gongzhuling mycin
Aqueous extracts are prepared in accordance with the following steps：
(m) resist " 769 " strain to be inoculated into culture substrate agriculture, cultivate completely, obtain the mixed of Gongzhuling mycin and culture substrate
Close object；
(n) mixture of Gongzhuling mycin and culture substrate is dried, crushes, is extracted with clear water, obtain Gongzhuling mycin
Leaching liquor；
(s) Gongzhuling mycin leaching liquor is centrifuged, takes supernatant to get to Gongzhuling mycin Aqueous extracts.
6. mycin bacteriostatic activity detection method in Gongzhuling according to claim 5, which is characterized in that in step (m), training
It is 5-7 days, preferably 6 days to support the time.
7. mycin bacteriostatic activity detection method in Gongzhuling according to claim 5, which is characterized in that in step (n), leaching
It is 12-48h, preferably 36h to carry the time.
8. mycin bacteriostatic activity detection method in Gongzhuling according to claim 5, which is characterized in that in step (n) princess
The volume ratio of crushed material and water after the crushing of the mixture of ridge mycin and culture substrate is 1:2-10, preferably 1:2.
9. the Gongzhuling mycin bacteriostatic activity detection method according to any one of claim 5-8, which is characterized in that in step
Suddenly in (m), the culture substrate is cut for corn；
Preferably, corn quarrel is by impregnating, draining and sterilization treatment；
Preferably, corn quarrel is impregnated with water, corn is cut soaking time as 1.5-8h, preferably 3h；
Preferably, the time of the sterilization treatment is 15-25min, preferably 20min.
10. according to claim 1-9 any one of them Gongzhuling mycin bacteriostatic activity detection method in antibiotic bacteriostatic activity
The application of detection.
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| CN110973147A (en) * | 2019-12-10 | 2020-04-10 | 吉林省农业科学院 | Bactericidal composition for preventing and treating peanut leaf spot disease, application thereof and bactericide |
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