CN109837323A - A kind of screening technique of lactic acid bacteria - Google Patents
A kind of screening technique of lactic acid bacteria Download PDFInfo
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Abstract
The present invention relates to microbe to screen technical field, specially a kind of screening technique of lactic acid bacteria.The method of the present invention includes: first to be ground sample, is then expanded culture；Grow it on culture medium with tilt-pour process to separate lactic acid bacteria single colonie later；Single bacterium drop point bacterium is connected on the MRS plate of indicator bacteria, and is covered with MRS soft agar；The bacterial strain for picking out fungistatic effect carries out single colonie purifying and preservation；Finally verified again.The influence of other miscellaneous bacterias early period can be not only reduced with the bacterial strain that this method separation screening comes out, the lactic acid bacteria that can also exclude no fungistatic effect simultaneously inhibits the bacterial strain of pathogen needed for not only can reducing workload while can also more targetedly filtering out.
Description
Technical field
The invention belongs to microbe to screen technical fields, and in particular to a kind of screening technique of lactic acid bacteria.
Background technique
In recent years, with the continuous development of China's economy, very big change is had occurred in the dietary structure of people, is rationally drunk
Food, nutrition equilibrium become the main care factor of people.Aquatic livestock is numerous in variety, is that nutrition content is in admirable proportion
One group food, they not only contain the Abundant protein being easily absorbed by the human body, but also fat content is low, while also having needed by human body
Various vitamin and minerals, firmly get liking for numerous people, thus in recent years China's marine industry development it is very swift and violent.For
High yield is obtained, people gradually develop culture fishery to high density, the intensive direction of height, but this is just faced with water body richness battalion
Feedingization, aquatic environment is complicated, so as to cause being on the rise for disease.Antibiotic has fabulous curative effect to aquatic pathogenic bacteria,
Simultaneously also along with drug tolerance of strain the problems such as highlight increasingly, so being become with the substance substitute antibiotics of green non-pollution
Hot topic.
Lactic acid bacteria is the common name of a kind of bacterium that a large amount of lactic acid are generated using fermentable carbohydrates.Lactic acid bacteria was growing
Cheng Zhonghui metabolism generates substances, these metabolites such as organic acid, bacteriocin, special enzyme and hydrogen peroxide and can kill or inhibit
The growth of pathogen, such method will not make bacterium generate drug resistance, therefore quilt is it is believed that be one kind of alternative antibiotic
Natural free of contamination bacterial strain.
There are many screening operations that researcher is engaged in lactic acid bacteria at present, but current screening technique is mostly first high-volume
Screening, then surveys physicochemical property, primarily determines to carry out 16s sequencing after lactic acid bacteria, determines its strain name, then pressed down
Bacterium experiment, the physicochemical property that large batch of screening just expends workload, and carries out later very much in the process determine not only
It takes time and effort, while being also a kind of waste of cost.
Summary of the invention
In consideration of it, the screening side for having the lactic acid bacteria of inhibiting effect it is necessary to provide a kind of pair of pathogenic bacteria regarding to the issue above
Method, wherein this method can be reduced as non-lactic acid bacteria and workload brought by the lactic acid bacteria without fungistatic effect, increase have suppression
The identified amount of the lactic acid bacteria of bacterium effect.
The present invention is achieved by the following technical solutions:
A kind of screening technique of lactic acid bacteria, comprising steps of
1) sampling originally, after being ground to crushing, is added in MRS meat soup, stationary culture；
2) it after taking step 1) to cultivate resulting bacterium solution dilution, is mixed with the MRS culture medium with indicator, after condensation, culture；
3) preparation instruction bacterium solution；
4) the resulting single colonie of culture in picking step 2), point bacterium is on MRS plate, pouring step 3) made from indicator bacteria
Liquid, after condensation, constant temperature incubation；
5) point takes the bacterial strain for having obvious inhibition zone that culture obtains in step 4), be inoculated in it is another newly have indicator
In MRS plate, culture；
6) single colonie that the culture of picking step 5) obtains lines on MRS plate, after culture；Single bacterium obtained by picking is fallen within again
It is cultivated in MRS meat soup, preservation is stand-by.
Further, the screening technique further includes step 7): verification step 6) in culture after bacterial strain with the presence or absence of suppression
Bacterium effect.
Further, in the step 1), sample first grinds (glass homogenizer) with physiological saline, then by treated
Sample is added in MRS meat soup, and sample accounts for 10% or so of MRS broth volume, can use 15ml centrifuge tube, stationary culture 8-10h.
Further, the indicator that MRS culture medium used has in the step 2) is 1-2% bromocresol purple solution, 30
DEG C or 37 DEG C of constant temperature incubation 16-18h.
Further, the preparation method of indicator bacteria includes: to be incubated overnight three ride of indicator bacteria in the step 3)；
Picking single bacterium is fallen in TSB meat soup, 37 DEG C of 8-10h of 180rpm；8-10h is cultivated extremely in 1-3% inoculum concentration switching TSB meat soup again
Mid-log phase surveys its OD595Value, by bacterium solution OD595Value is diluted to 1.0；1ml is taken to be added to have sterilized and be cooled to 50 DEG C or so
It is mixed in TSB culture medium.
Further, indicator bacteria used in the step 3) must have stronger vigor, i.e., raw after activating repeatedly
The long bacterial strain to the logarithm middle and later periods.
Further, TSB culture medium used is necessary for soft agar medium, agar content 0.7% in the step 3).
Further, step 4) midpoint bacterium stands 1-2h, then pouring step 3 after on MRS plate) indicator bacteria
Liquid, after condensation, inversion is incubated in 30 DEG C or 37 DEG C of constant incubators, cultivates 16-18h.
Further, when the step 5) point takes bacterium colony, can be inserted into culture medium in carry out picking, as indicator bacteria cover with it is flat
Plate please deducts entire flat-plate inverted to come, and point takes single colonie again.
Further, 30 DEG C or 37 DEG C cultures after the step 5) inoculation is good, 16-18h.
Further, 20% glycerol tube of bacterium colony after expanding culture in the step 6) (uses 60% glycerol (to match with water
Set): bacterium solution=2:1 is made) in -80 DEG C of preservations.
Further, it is further verified in the step 7) using filter paper enzyme or Odontothrips loti.
The invention has the advantages that:
The present invention is to increase to bacteriostatic experiment during bacterial strain screening simultaneously on the lactic acid bacterial screening method on basis
It carries out, keeps the specific aim of bacterial strain screening stronger, avoid the waste of manpower and goods and materials, increase the screening accuracy of purpose lactic acid bacteria.
This method is to determine acid-producing bacteria strain by bromocresol purple development process, then by picking single bacterium drop point bacterium in indicator bacteria
On plate, while to guarantee that lactobacter growth upper layer should be covered with MRS culture medium, it is to be screened go out have the bacterial strain for inhibiting indicator bacteria after, then
Single strain needed for being obtained using the methods of conventional point bacterium, three rides, enrichment, is made finally by filter paper enzyme of its supernatant
Bacteriostatic experiment has reached the effect of verifying.
Detailed description of the invention
The screening technique Main Stage state diagram of lactic acid bacteria Fig. 1 of the invention；Wherein: 1. use the method culture sample poured into
Product；2. the bacterium of point flavescence color covers one layer of indicator bacteria on MRS plate；3. choosing the bacterium point bacterium of fungistatic effect in band
On the MRS culture medium of indicator；4. three ride of bacterial strain of picking colour changed into yellow is on MRS culture medium；5. fluid enlargement culture；
6. filter paper enzyme is verified.
Fig. 2 is with V.h as the point bacterium plate of indicator bacteria；Bottom platform is MRS culture medium, and grid is pulled behind plate,
Point has a single colonie in each grid, topples over indicator bacteria after standing 1h, culture is inverted, if occurred around bacterium
Transparent circle, then it represents that the bacterial strain has inhibitory effect to V.h, picks out and carries out next step research.
Fig. 3 is with V.p as the point bacterium plate of indicator bacteria；Bottom platform is MRS culture medium, and grid is pulled behind plate,
Point has a single colonie in each grid, topples over indicator bacteria after standing 1h, culture is inverted, if occurred around bacterium
Transparent circle, then it represents that the bacterial strain has inhibitory effect to V.p, picks out and carries out next step research.
Fig. 4 is with S.a as the point bacterium plate of indicator bacteria.Bottom platform is MRS culture medium, and grid is pulled behind plate,
Point has a single colonie in each grid, topples over indicator bacteria after standing 1h, culture is inverted, if occurred around bacterium
Transparent circle, then it represents that the bacterial strain has inhibitory effect to S.a, picks out and carries out next step research.
Fig. 5 is with A.h as the point bacterium plate of indicator bacteria.Bottom platform is MRS culture medium, and grid is pulled behind plate,
Point has a single colonie in each grid, topples over indicator bacteria after standing 1h, culture is inverted, if occurred around bacterium
Transparent circle, then it represents that the bacterial strain has inhibitory effect to A.h, picks out and carries out next step research.
Fig. 6 is given instruction the confirmatory experiment of bacterium with V.h；The plate is V.h indicator bacteria plate, and filter paper is affixed on plate,
The lactic acid bacteria strains supernatant for selecting 10 μ l again, is incubated overnight, and obvious fungistatic effect such as occurs, that is, indicates that the bacterial strain is that can inhibit
The lactic acid bacteria of V.h.
Fig. 7 is given instruction the confirmatory experiment of bacterium with V.p；The plate is V.p indicator bacteria plate, and filter paper is affixed on plate,
The lactic acid bacteria strains supernatant for selecting 10 μ l again, is incubated overnight, and obvious fungistatic effect such as occurs, that is, indicates that the bacterial strain is that can inhibit
The lactic acid bacteria of V.p.
Fig. 8 is given instruction the confirmatory experiment of bacterium with S.a；The plate is S.a indicator bacteria plate, and filter paper is affixed on plate,
The lactic acid bacteria strains supernatant for selecting 10 μ l again, is incubated overnight, and obvious fungistatic effect such as occurs, that is, indicates that the bacterial strain is that can inhibit
The lactic acid bacteria of S.a.
Fig. 9 is given instruction the confirmatory experiment of bacterium with A.h；The plate is A.h indicator bacteria plate, and filter paper is affixed on plate,
The lactic acid bacteria strains supernatant for selecting 10 μ l again, is incubated overnight, and obvious fungistatic effect such as occurs, that is, indicates that the bacterial strain is that can inhibit
The lactic acid bacteria of A.h.
Specific embodiment
The problem of solved in order to better illustrate the present invention, used technical solution and effect achieved, are now tied
It closes specific embodiment and related data is further described.It should be noted that the content of present invention is including but not limited to following implementation
Example and combinations thereof embodiment.
Particular technique or condition are not specified in the embodiment of the present invention, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available etc.
The conventional products that approach obtains.
1. embodiment used medium and agent prescription:
1) MRS culture medium used in embodiment:
Peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 20.0g, Tween-80 1.0mL, hydrogen citrate
Diammonium 2.0g, anhydrous sodium acetate 5.0g, K2HPO4 2.0g、MnSO4·H2O 0.25g、MgSO4·7H2O 0.58g, agar 15g,
Distilled water 1.0L adjusts pH to 6.5 with NaOH.
2) the MRS culture medium with indicator used in embodiment:
Peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 20.0g, Tween-80 1.0mL, hydrogen citrate
Diammonium 2.0g, anhydrous sodium acetate 5.0g, K2HPO4 2.0g、MnSO4·H2O 0.25g、MgSO4·7H2O 0.58g, agar 7g,
Distilled water 1.0L adds 2% bromocresol purple solution of 1ml.
3) preparation of bromocresol purple used in embodiment
The bromocresol purple solution of 10-20g/L, i.e. the bromocresol purple solution of 1-2% are prepared with 95% ethyl alcohol and bromocresol purple.
4) MRS broth bouillon used in embodiment:
Peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 20.0g, Tween-80 1.0mL, hydrogen citrate
Diammonium 2.0g, anhydrous sodium acetate 5.0g, K2HPO4 2.0g、MnSO4·H2O 0.25g、MgSO4·7H2O 0.58g, distilled water
1.0L。
5) TSB culture medium used in embodiment:
Tryptone 17.0g, phytone 3.0g, NaCl 5.0g, K2HPO42.0g, glucose 2.5g, agar powder
15g, distilled water 1L.
6) TSB broth bouillon used in embodiment:
Tryptone 17.0g, phytone 3.0g, NaCl 5.0g, K2HPO42.0g, glucose 2.5g, distilled water
1L。
7) TSB soft agar medium used in embodiment:
Tryptone 17.0g, phytone 3.0g, NaCl 5.0g, K2HPO42.0g, glucose 2.5g, agar powder
7g, distilled water 1L.
2. screening technique
Experimental method process such as Fig. 1
1) 1g aquatic livestock intestinal samples are taken, 1ml physiological saline is added, is ground to crushing with tissue grinder, will crush
Sample afterwards is added in the 15ml centrifuge tube for being placed with 14ml MRS meat soup, stationary culture 8-10h；Wherein treated sample with
Culture medium should fill up centrifuge tube.
2) 10 times of eight pipes of dilution method dilution of 1ml step 1) bacterium solution physiological saline are taken, take last each 1ml of three pipes solution points
It is not added in culture dish, is toppled over tilt-pour process and have cooled to 55 DEG C of the sterilized MRS culture medium with indicator, mix,
After to be condensed, inversion is incubated in 30 DEG C of constant incubators, 16-18h.
3) indicator bacteria prepare: by indicator bacteria on TSB culture medium (TSB plate) three rides, be incubated overnight；Picking single bacterium
It falls in TSB meat soup, 37 DEG C of 8h of 180rpm；Culture 8h surveys its OD to mid-log phase in 1% inoculum concentration switching TSB meat soup again595
Value, by bacterium solution OD595Value is diluted to 1.0；It takes 1ml to be added to 100ml and sterilized and is cooled to 50 DEG C of TSB soft agar medium (fine jade
Rouge content 0.7%) in mix；
4) with the single colonie on plate in toothpick picking step 2), bacterium is put on MRS plate, stands 1h, pouring step 3)
It is mixed with the TSB culture medium 8ml-10ml of indicator bacteria, after to be condensed, inversion is incubated in 37 DEG C of constant incubators, 16-18h；
5) a new MRS plate with indicator is separately taken, the bacterial strain of obvious inhibition zone is taken with sterilized toothpick point
It is inoculated in the MRS plate with indicator, 30 DEG C of cultures, 16-18h；
6) three ride of picking step 5) single colonie is on MRS plate, and after 37 DEG C are incubated overnight, picking single bacterium falls within MRS
In meat soup, 37 DEG C of culture 16h, -80 DEG C of preservations of 20% glycerol tube of gained bacterium solution；
7) bacterial strain after expanding culture verifies whether that there are fungistatic effects with filter paper enzyme.
Embodiment 1:
There is the bacterial strain of fungistatic effect as indicator bacteria screening with Vibrio harveyi (Vibrio harveyi, V.h), is inclined
16h is cultivated on germy MRS culture medium in having put, experimental result such as Fig. 2 is isolated to be verified with filter paper after single colonie and be tied
Fruit such as Fig. 6.
Embodiment 2:
There is the bacterium of fungistatic effect as indicator bacteria screening with vibrio parahaemolytious (Vibrioparahaemolyticus, V.p)
Strain is poured upon cultivating 16h on germy MRS culture medium in having put, experimental result such as Fig. 3, isolates after single colonie with filter
Scraps of paper verification result such as Fig. 7.
Embodiment 3:
There is fungistatic effect as indicator bacteria screening with staphylococcus aureus (Staphylococcus aureus, S.a)
Bacterial strain is poured upon cultivating 16h on germy MRS culture medium in having put, and experimental result such as Fig. 4 is used after isolating single colonie
Filter paper verification result such as Fig. 8.
Embodiment 4:
There is the bacterium of fungistatic effect as indicator bacteria screening with Aeromonas hydrophila (Aeromonas hydrophila, A.h)
Strain is poured upon cultivating 16h on germy MRS culture medium in having put, experimental result such as Fig. 5, isolates after single colonie with filter
Scraps of paper verification result such as Fig. 9.
The separation that no fungistatic effect bacterial strain can be reduced in initial step with such method, not only reduces work
Amount, while the accuracy rate of screening can also be increased, the further experiment for after provides reliable basis.
In present embodiment only by taking aquatic livestock enteron aisle sample as an example, do not illustrate that screening technique of the present invention is only used for pair
The screening of lactic acid bacteria in aquatic livestock intestinal flora.Screening technique of the invention can also be used in having antibacterial effect in different samples
The original samples in step 1) are replaced in the screening of the lactic acid bacteria of fruit.Other samples include the animal intestinal tracts such as livestock and poultry, vegetable
Dish, fruit, soil equal samples are feasible.
Screening technique of the present invention can also be by selecting different indicator bacterias, and screening has the lactic acid of fungistatic effect to specific strain
Bacterium, however it is not limited to the indicator bacteria that present embodiment is enumerated.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of screening technique of lactic acid bacteria, comprising steps of
1) sampling originally, after crushing, is added in MRS meat soup, stationary culture；
2) it after taking step 1) to cultivate resulting bacterium solution dilution, is mixed with the MRS culture medium with indicator, after condensation, culture；
3) preparation instruction bacterium solution；
4) single colonie in picking step 2), point bacterium is on MRS plate, pouring step 3) it is obtained indicate bacterium solution, after condensation, training
It supports；
5) point takes the bacterial strain for having obvious inhibition zone that culture obtains in step 4), is inoculated in the MRS plate with indicator, trains
It supports；
6) single colonie that the culture of picking step 5) obtains lines on MRS plate, after culture；Single bacterium obtained by picking falls within MRS again
In meat soup cultivate to get.
2. screening technique according to claim 1, which is characterized in that the screening technique further includes step 7): verifying step
The fungistatic effect of bacterial strain after rapid 6) middle culture.
3. screening technique according to claim 2, which is characterized in that use filter paper enzyme or Oxford cup in the step 7)
Method is further verified.
4. screening technique according to claim 1 to 3, which is characterized in that in the step 1), sample is first used
Physiological saline grinding, then by treated, sample is added in MRS meat soup, and sample accounts for the 10% of MRS broth volume, stationary culture
8-10h。
5. screening technique according to claim 1 to 3, which is characterized in that the screening technique step 2) and step
It is rapid 5) in the indicator that has of MRS culture medium used be 1-2% bromocresol purple solution；Condition of culture are as follows: 30 DEG C or 37 DEG C cultures
16-18h。
6. screening technique according to claim 1 to 3, which is characterized in that the system of indicator bacteria in the step 3)
Preparation Method includes: to be incubated overnight three ride of indicator bacteria；Picking single bacterium is fallen in TSB meat soup, 37 DEG C of 8-10h of 180rpm；
The switching of 1-3% inoculum concentration cultivates 8-10h to mid-log phase in TSB meat soup again, surveys its OD595Value, by bacterium solution OD595Value is diluted to
1.0；It takes 1ml to be added in the TSB culture medium for having sterilized and be cooled to 50 DEG C to mix.
7. screening technique according to claim 6, which is characterized in that indicator bacteria used in the step 3) is to live repeatedly
The bacterial strain of logarithm middle and later periods is grown to after change.
8. screening technique according to claim 6, which is characterized in that TSB culture used in the screening technique step 3)
Base is soft agar medium, agar content 0.7%.
9. screening technique according to claim 1 to 3, which is characterized in that step 4) midpoint bacterium is in MRS
On plate after stand 1-2h, then pouring step 3) instruction bacterium solution, after condensation, inversion be incubated in 30 DEG C or 37 DEG C of incubators,
Cultivate 16-18h.
10. screening technique according to claim 1 to 3, which is characterized in that the bacterium colony in step 6) after culture
Using -80 DEG C of preservations of 20% glycerol tube.
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