CN106480164A - A kind of method of high flux screening lactein - Google Patents

A kind of method of high flux screening lactein Download PDF

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CN106480164A
CN106480164A CN201610940806.8A CN201610940806A CN106480164A CN 106480164 A CN106480164 A CN 106480164A CN 201610940806 A CN201610940806 A CN 201610940806A CN 106480164 A CN106480164 A CN 106480164A
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culture
cultivated
lactic acid
activated spawn
lactobacillus
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刘陈立
蒙海林
刘明珠
刘复荣
崔金明
何敬愉
张炳照
杨金芳
邓登
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Shenzhen Alpha Feed Agricultural And Pastoral Co ltd
Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Alpha Feed Agricultural And Pastoral Co ltd
Shenzhen Institute of Advanced Technology of CAS
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Abstract

The present invention relates to technical field of bioengineering, and in particular to microbial technology field, more particularly to a kind of method of high flux screening lactein, methods described comprise the steps：(1) pathogen is cultivated；(2) lactic acid bacteria is cultivated；(3) the streptococcus acidi lactici fermented solution supernatant that on porous plate, add LB culture medium, step (1) to cultivate cause of disease fermented liquid and step (2) are cultivated, co-cultures 8 25h, with ELIASA Timing measurement OD on porous plate600Absorbance；Wherein, the porous plate be 24 orifice plates, in 48 orifice plates, 96 orifice plates or 384 orifice plates any one or at least two combination.The present invention is screened on porous plate, can quickly filter out the antibacterial materials such as the lactein that has notable bacteriostatic activity for specific objective pathogen, can drastically increase screening efficiency while select to hundreds of plant of bacterium.

Description

A kind of method of high flux screening lactein
Technical field
The present invention relates to technical field of bioengineering, and in particular to microbial technology field, more particularly to a kind of high flux The method of screening lactobacillus element.
Background technology
Lactein is that a class produced in metabolic process by probiotic lactic acid bacteria has many of relatively strong biological activity Peptide or protein.Lactein due to having efficient bacteriostatic activity and nontoxic to person poultry safety to pathogen, aquaculture, It is used widely in the various fields such as food antiseptic.
In cultivation field, current generally existing abuse of antibiotics, the phenomenon of disinfectant, do not only result in pathogenic bacteria resistance to drugs Increase, and environmental pollution is easily caused, affect health.With the continuous enhancing of drug Resistance of Pathogenic Microorganism from Surface, traditional antibiosis The drawbacks of element is used day by day appears, and the potential safety hazard that antibiotic residue brings causes the great attention of people.And with lactic acid bacteria The plain antibacterial peptide for representative is difficult to make bacterium produce drug resistance because of its unique structure and antibacterial mechanisms；And in animal body may be used Medicament residue, non-immunogenicity and cytotoxicity is avoided, does not damage normal body cell.With respect to other protein-based biologies Preparation, lactein are strong to the tolerance of temperature, soda acid, metal ion, protease.Lactein product be expected to be used alone or Compounded with viable lactic acid bacteria, mix for feed and feed, the normal microecological balance of livestock and poultry animal enteron aisle is maintained, improved its immunity Power, promotes growth, thus increasingly becomes the popular additive of feedstuff industry.
In field of food preservation, lactein also possesses huge market development potential.For example, streptococcus lactis peptide (Nisin) it is a kind of to confirm as safe efficient, reliable food through Food and Agricultural Organization of the United Nations and the World Health Organization and prevent Rotten agent, and be used widely in the whole world.The natural Lactococcus lactis for originating from food-grade of Nisin, to gram-positive bacteria be in Broad-spectrum antibacterial action, such as bacillus, clostridium botulinum, staphylococcus, Listeria, heat-resisting spoilage organisms, bar bacterium, micrococcus, Bright string coccus, mycobacterium etc.；Quickly amino acid is become by proteolytic enzyme digest in alimentary canal after edible, therefore will not be in vivo Residual, will not also change normal intestinal flora.Compared with other antibacterial peptides, the advantage of lactein is can to kill pathogen While, lactic acid bacteria itself is not killed, is thus advantageous to maintain the normal microecological balance of animal and bird intestines.
As lactein has wide application market and potentiality to be exploited, the work of separation screening is carried out for which in recent years Make also more and more.Lactein is naturally occurring in various lactobacillus, thus therefrom can be screened isolated.In prior art Traditional screening technique is screened one by one just for single or several strains of lactic acid bacteria, is resisted by flat board/Odontothrips loti Bacterium screening active ingredients.The feature of the method is that batch is less, with strong points, but is difficult to while screening to up to a hundred plants of bacterium, efficiency Lowly.
Content of the invention
For the deficiencies in the prior art, the invention provides a kind of method of high flux screening lactein, methods described The antibacterial materials such as the lactein that there is notable bacteriostatic activity for specific objective indicator bacteria can be quickly filtered out, is greatly enhanced Screening efficiency.
For reaching this goal of the invention, the present invention is employed the following technical solutions：
In a first aspect, the invention provides a kind of high flux screening lactein is (also known as lactobacillus peptide, lactic acid bacteria antibacterial Peptide) method, methods described comprises the steps：
(1) pathogen is cultivated；
(2) lactic acid bacteria is cultivated；
(3) breast that on porous plate, add LB culture medium, step (1) to cultivate cause of disease fermented liquid and step (2) are cultivated Acid bacteria fermentation liquid supernatant, co-cultures 8-25h, with the absorbance of ELIASA Timing measurement OD600 on porous plate；
Wherein, the porous plate be 24 orifice plates, in 48 orifice plates, 96 orifice plates or 384 orifice plates any one or at least two Combination.
In the present invention, lactein that the lactic acid bacteria produces can be secreted into extracellular, by centrifugation by lactobacillus-fermented The supernatant of liquid is used for detecting that the effect of the lactein that both only can have been tested in supernatant can to exclude lactic acid bacteria thalline itself again To the impact for detecting.
The present invention, step (3) detect when, due to being carried out with porous plate, be easy to do parallel test, can typically do 3 parallel Test, while can also do multiple control groups, the control group for adopting be with 200 μ L sterile LB medium ,+198 μ L of 2 μ L cause of disease bacterium solution Sterile LB medium ,+180 μ L sterile LB medium of 20 μ L streptococcus acidi lactici fermented solution supernatant are control.
Used as optimal technical scheme, the supernatant of the streptococcus acidi lactici fermented solution is obtained by streptococcus acidi lactici fermented solution is carried out centrifugation , the centrifugal rotational speed be 8000-12000g, can be for example 8000g, 8100g, 8200g, 8300g, 8500g, 8600g, 8800g, 10000g, 10100g, 10500g, 10600g, 10800g, 11000g, 11300g, 11500g, 11800g or 12000g, Preferably 10000g；The centrifugation time be 1-15min, can be for example 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min, preferably 3-10min, further preferably For 5min.
In the present invention, the incubation time can be for example 8h, 9h, 10h, 11h, 12h, 13h, 15h, 16h, 18h, 20h, 21h, 23h or 25h.
Preferably, the pathogen described in step (1) is shrimp pathogenic bacteria vibrio parahaemolytious (Vibrioparahaemolyticus), Vibrio harveyi (Vibrio harveyi), Vibrio vulnificus (Vibrio Vulnificus), Aeromonas hydrophila (Aeromonas hydrophila), Vibrio anguillarum (Vibrio anguillarum) or molten In algae vibrios (Vibrio alginolyticus) any one or at least two combination.
Preferably, the lactic acid bacteria is separating obtained lactic acid bacteria from animal alimentary canal and genital tract.
Preferably, the lactic acid bacteria is selected from, but not limited to,：Lactobacillus gasseri, lactobacillus fermenti, curling Bacillus acidi lactici, starch Lactobacillus, lactoenterococcus, Hai Shi enterococcus, enterococcus faecalis, Yue Shi lactobacillus, lactobacillus paracasei, Lactobacillus pentosus, plant Lactobacillus, lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Taiwan galactococcus, Lactococcus lactis, Leuconostoc, Pediococcus acidilactici, Pediococcus pentosaceus, Streptococcus alactolyticus, Streptococcus iniae, Paris streptococcus, streptococcus salivarius, vestibular chain Coccus, Vagococcus, cibarium Wei Si Salmonella, confusus, He Lun Wei Si Salmonella, Young citric acid bacillus, mangrove intestines bar Any one in bacterium, rothia dentocariosa, stick-slip Roche bacterium, shigella flexneri, MRSE or the affiliated subspecies of lactic acid bacteria Or at least two combination.
The subspecies can be the subsp. cremoris of Lactococcus lactis, lactic acid subspecies, Huo Shi subspecies；The bigcatkin willow of Lactobacillus salivarius Plain subspecies, saliva subspecies.
Preferably, activated spawn and Amplification Culture are included the step of culture pathogen described in step (1).
Preferably, the inoculum concentration of the activated spawn is 1-10%, can be for example 1%, 2%, 3%, 4%, 5%, 6%th, 7%, 8%, 9% or 10%, preferably 5%.
Preferably, the Amplification Culture is the expansion by the strain transfer of activation in the LB culture medium of fresh sterilizing In culture, the inoculum concentration of activated spawn is 0.5-5%, can be for example 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%th, 3%, 4% or 5%, preferably 1%.
Preferably, the temperature of the activated spawn and Amplification Culture is 30-40 DEG C, can be for example 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 35-38 DEG C, more preferably 37 DEG C.
Preferably, the rotating speed of the activated spawn and Amplification Culture be 200-300rpm, can be for example 200rpm, 210rpm, 220rpm, 230rpm, 240rpm, 250rpm, 260rpm, 270rpm, 280rpm, 290rpm or 300rpm, preferably 230-280rpm, more preferably 250rpm.
Preferably, the time of the activated spawn and Amplification Culture be 8-25h, can be for example 8h, 9h, 10h, 11h, 12h, 13h, 15h, 16h, 18h, 20h, 21h, 23h or 25h, preferably 10-18h, more preferably 12h.
Preferably, the culture lactic acid bacteria is cultivated using porous plate.
In the present invention, being cultivated using porous plate just can be while cultivates various lactobacillus, can also when test While various lactobacillus are carried out while testing.
Preferably, activated spawn, Amplification Culture and switching culture are included the step of culture lactic acid bacteria described in step (2).
Preferably, the inoculum concentration of the activated spawn is 5-15%, can be for example 5%, 6%, 7%, 8%, 9%, 10%th, 11%, 12%, 13%, 14% or 15%, preferably 10%.
Preferably, the Amplification Culture is the expansion by the strain transfer of activation in the MRS culture medium of fresh sterilizing In culture, the inoculum concentration of activated spawn is 1-10%, for example, can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% Or 10%, preferably 5%.
Preferably, the temperature of the activated spawn and Amplification Culture is 25-33 DEG C, can be for example 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C, 31 DEG C, 32 DEG C, preferably 28-31 DEG C, more preferably 30 DEG C.
Preferably, the time of the activated spawn and Amplification Culture be 8-25h, can be for example 8h, 9h, 10h, 11h, 12h, 13h, 15h, 16h, 18h, 20h, 21h, 23h or 25h, preferably 10-18h, more preferably 12h.
As optimal technical scheme, the switching culture be will be enlarged by cultivate bacterium solution be transferred to fresh sterilizing MRS training In foster base, 25-33 DEG C of culture 3-10h, adds cause of disease bacterium solution, co-cultures 8-25h at 25-33 DEG C.
In the present invention, add cause of disease bacterium solution that the generation of lactein can be induced, make to be difficult to detect bacteriostatic activity originally Part bacterial strain can produce significant bacteriostatic activity after stimulation, increased the possibility for screening more lacteins.
Preferably, described is 28-31 DEG C of training by condition of culture of the culture strain transfer in the MRS culture medium of fresh sterilizing Foster 5-8h, preferably 30 DEG C culture 6h.
Preferably, the condition for co-culturing after the addition cause of disease bacterium solution is 28-31 DEG C of culture 10-18h, preferably 30 DEG C trainings Foster 12h.
Preferably, in the switching culture, the inoculum concentration of Amplification Culture bacterium solution is 1-10%, can be for example 1%, 2%, 3%th, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably 5%.
Preferably, in the switching culture, the inoculum concentration of cause of disease bacterium solution is 0.5-5%, can be for example 0.5%, 0.6%, 0.7%th, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5%, preferably 1%.
Preferably, the volume ratio of the LB culture medium, cause of disease bacterium solution and streptococcus acidi lactici fermented solution supernatant described in step (3) is (60- 100):1:(3-15) can be, for example 60:1:3、61:1:4、62:1:5、65:1:6、68:1:7、70:1:7、73:1:5、75: 1:7、76:1:7、78:1:7、80:1:8、82:1:5、83:1:4、85:1:8、86:1:9、87:1:10、88:1:11、89:1:12、 90:1:6、92:1:3、95:1:4、96:1:5、97:1:6、98:1:11、100:1:12, preferably (70-90):1:(6-12), enter One step is preferably 89:1:10.
Preferably, the time of the co-cultivation is 10-18h, preferably 12h.
Preferably, the temperature of the co-cultivation is 30-40 DEG C, can be for example 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 35-38 DEG C, more preferably 37 DEG C.
Preferably, the rotating speed of the co-cultivation be 200-300rpm, can be for example 200rpm, 210rpm, 220rpm, 230rpm, 240rpm, 250rpm, 260rpm, 270rpm, 280rpm, 290rpm or 300rpm, preferably 230-280rpm, enter One step is preferably 250rpm.
Preferably, the interval time of ELIASA measurement is 1-5h, for example, can be 1h, 2h, 3h, 4h or 5h, preferably For 1-3h, more preferably 2h.
A kind of method of high flux screening lactein, comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：Pathogen is inoculated in the LB culture medium of fresh sterilizing by the inoculum concentration of 1-10%, is placed in 30- 40 DEG C, under the conditions of 200-300rpm, cultivate 8-25h；
2. Amplification Culture：The bacterial classification of activation is inoculated in the LB culture medium of fresh sterilizing by the inoculum concentration of 0.5-5%, puts In 30-40 DEG C, 8-25h under the conditions of 200-300rpm, is cultivated；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Inoculum concentration by 5-15% is placed in the MRS culture medium of lactobacillus inoculum to fresh sterilizing Quiescent culture 8-25h under the conditions of 25-33 DEG C；
2. Amplification Culture：The MRS that activated spawn is inoculated into fresh sterilizing is supported in base by the inoculum concentration of 1-10%, be placed in Quiescent culture 8-25h under the conditions of 25-33 DEG C；
3. switching culture：The bacterial classification that culture be will be enlarged by by the inoculum concentration of 1-10% is inoculated into the MRS culture medium of fresh sterilizing Middle 25-33 DEG C of quiescent culture 3-10h, then the cause of disease bacterium solution of culture is accessed with the inoculum concentration of 0.5-5%, in 25-33 DEG C of standing altogether Culture 8-25h；
(3) lactic acid that the cause of disease fermented liquid that cultivates LB culture medium, step (1) on 96 orifice plates and step (2) are cultivated Fermented liquid supernatant is (60-100) by volume:1:(3-15), 30-40 DEG C on 96 orifice plates, under conditions of 200-300rpm 8-25h is co-cultured, and the absorbance of OD600 is measured with ELIASA per 1-5h.
Compared with prior art, the invention has the advantages that：
(1) present invention is screened on porous plate, can quickly be filtered out and be had notable the suppression for specific objective pathogen The antibacterial materials such as the lactein of bacterium activity, can select to hundreds of plant of bacterium simultaneously, drastically increase screening efficiency；
(2) present invention can induce the generation of lactein by adding cause of disease bacterium solution, make to be difficult to originally to detect antibacterial The part bacterial strain of activity can produce significant bacteriostatic activity after stimulation, increased the possibility for screening more lacteins.
Description of the drawings
Fig. 1 is the design sketch of the antibacterial screening of the present invention；
The curve map that Fig. 2 is screened for the embodiment of the present invention.
Specific embodiment
Technical scheme is further illustrated below by specific embodiment.Those skilled in the art should be bright , the embodiment is only to aid in understanding the present invention, is not construed as the concrete restriction to the present invention.
Experimental technique used in following embodiments if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, all commercially obtain.
Main agents are purchased from Shanghai Sheng Gong bioengineering Co., Ltd；
Lactic acid bacteria culturers storehouse：Applicant's early stage is screened from the aquatic products animal intestinal of the mouth of the Zhujiang River and obtains more than 800 strain of lactic acid bacteria, just Step establishes the aquaculture lactic acid bacteria culturers resources bank of Pearl River Delta.
Pathogen is shrimp pathogenic bacteria vibrio parahaemolytious Vibrio parahaemolyticus ATCC 17802.
Culture medium：
(1) LB culture medium
Tryptone (Tryptone) 10g, yeast extract (Yeast extract) 5g, NaCl 10g, shake container Until solute dissolving, adjusts pH to 7.0 with 5mol/L NaOH, deionized water is settled to 1L, sterilizes under 121 DEG C, 0.1Mpa 20min.It is 2% to add agar powder content when preparing solid medium.
(2) MRS culture medium
Peptone 10.0g, beef leaching thing 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, lemon Lemon acid diamine 2.0g, Tween-80 1.0ml, dipotassium hydrogen phosphate 0.4g, magnesium sulfate 0.58g, manganese sulfate 0.29g, calcium carbonate 20.0g, agar 15.0g, distill water dissolves and constant volume are adjusted to 6.2-6.6 to 1000ml, pH, heat, boil 2min after stirring, 121 DEG C, sterilize under 0.1Mpa 20min.
Embodiment 1
A kind of method of high flux screening lactein, comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：1.9mL sterile LB medium is added toward in 10mL test tube, and accesses the pathogen of 0.1mL defrosting Liquid, puts 37 DEG C, cultivates 12h under the conditions of 250rpm；
2. Amplification Culture：Take 0.25mL bacterium solution access 25mL sterile LB medium 250mL triangular flask in, put 37 DEG C, 12h is cultivated under the conditions of 250rpm；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Load 540 μ L sterilizing MRS culture medium toward in 96 orifice plates per hole, then be respectively connected to 60 μ L defrosting Lactobacillus suspension, quiescent culture 12h at 30 DEG C；
2. Amplification Culture：Taken out per 30 μ L bacterium solution of hole with multichannel pipettor, access and be pre-loaded with 570 μ L of every hole sterilizing MRS In the new orifice plate of culture medium, quiescent culture 12h at 30 DEG C；
3. switching culture：30 μ L bacterium solution are taken per hole, access the new orifice plate for being pre-loaded with 570 μ L of every hole sterilizing MRS culture medium In, quiescent culture 6h at 30 DEG C, 6 μ L cause of disease bacterium solution are added, continues quiescent culture 12h at 30 DEG C；
(3) detect：
1. cultured 96 orifice plate is put in supercentrifuge, is centrifuged 5 minutes under 10000g；
2. new 96 orifice plate is taken, is separately added into 178 μ L sterile LB medium, 2 μ L cause of disease bacterium solution and 20 μ L streptococcus acidi lactici fermented solutions Supernatant, with 200 μ L sterile LB medium ,+198 μ L sterile LB medium of 2 μ L cause of disease bacterium solution, 20 μ L streptococcus acidi lactici fermented solution supernatants+ 180 μ L sterile LB medium are control, arrange 3 repetition orifice plates.
3. 37 DEG C are put, is cultivated 12 hours under the conditions of 250rpm, with not having 2h detection in ELIASA measurement 0-12 hour per hole OD600Numerical value.
As depicted in figs. 1 and 2, wherein Fig. 1 is the integral experiment effect of orifice plate to fungistatic effect, and Fig. 2 is to choose 3 from Fig. 1 Experimental port (D7, E5, E9) and the pathogen OD of control wells (H12)600Versus time curve.Can from the curve of Fig. 2 Go out, compared with control wells H12 (being not added with streptococcus acidi lactici fermented solution supernatant), the streptococcus acidi lactici fermented solution supernatant added by 3 experimental ports is equal There is significant fungistatic effect, its 24 hours bacteriostatic activities are E9>E5>D7.
Embodiment 2
A kind of method of high flux screening lactein, comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：1.9mL sterile LB medium is added toward in 10mL test tube, and accesses the pathogen of 50 μ L defrosting Liquid, puts 30 DEG C, cultivates 25h under the conditions of 300rpm；
2. Amplification Culture：Take 1.25mL bacterium solution access 25mL sterile LB medium 250mL triangular flask in, put 30 DEG C, 25h is cultivated under the conditions of 300rpm；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Load 1080 μ L sterilizing MRS culture medium toward in 48 orifice plates per hole, then be respectively connected to 120 μ L defrosting Lactobacillus suspension, quiescent culture 25h at 25 DEG C；
2. Amplification Culture：Taken out per 60 μ L bacterium solution of hole with multichannel pipettor, access and be pre-loaded with 1140 μ L of every hole sterilizing MRS In the new orifice plate of culture medium, quiescent culture 25h at 25 DEG C；
3. switching culture：60 μ L bacterium solution are taken per hole, access the new orifice plate for being pre-loaded with 1140 μ L of every hole sterilizing MRS culture medium In, quiescent culture 3h at 25 DEG C, 6 μ L cause of disease bacterium solution are added, continues quiescent culture 25h at 25 DEG C；
(3) detect：
1. cultured 48 orifice plate is put in supercentrifuge, is centrifuged 5 minutes under 10000g；
2. new 48 orifice plate is taken, is separately added into 356 μ L sterile LB medium, 4 μ L cause of disease bacterium solution and 40 μ L streptococcus acidi lactici fermented solutions Supernatant, with 400 μ L sterile LB medium ,+396 μ L sterile LB medium of 4 μ L cause of disease bacterium solution, 40 μ L streptococcus acidi lactici fermented solution supernatants+ 360 μ L sterile LB medium are control, arrange 3 repetition orifice plates.
3. 30 DEG C are put, is cultivated 25 hours under the conditions of 300rpm, with not having 2h detection in ELIASA measurement 0-25 hour per hole OD600Numerical value.
Test result is similar to the result of embodiment 1, i.e., streptococcus acidi lactici fermented solution supernatant all has significant fungistatic effect.
Embodiment 3
A kind of method of high flux screening lactein, comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：1.8mL sterile LB medium is added toward in 10mL test tube, and accesses the pathogen of 0.2mL defrosting Liquid, puts 40 DEG C, cultivates 8h under the conditions of 200rpm；
2. Amplification Culture：Take 0.125mL bacterium solution access 25mL sterile LB medium 250mL triangular flask in, put 40 DEG C, 8h is cultivated under the conditions of 200rpm；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Load 570 μ L sterilizing MRS culture medium toward in 96 orifice plates per hole, then be respectively connected to 30 μ L defrosting Lactobacillus suspension, quiescent culture 8h at 33 DEG C；
2. Amplification Culture：Taken out per 60 μ L bacterium solution of hole with multichannel pipettor, access and be pre-loaded with 540 μ L of every hole sterilizing MRS In the new orifice plate of culture medium, quiescent culture 8h at 33 DEG C；
3. switching culture：60 μ L bacterium solution are taken per hole, access the new orifice plate for being pre-loaded with 510 μ L of every hole sterilizing MRS culture medium In, quiescent culture 6h at 33 DEG C, 30 μ L cause of disease bacterium solution are added, continues quiescent culture 8h at 33 DEG C；
(3) detect：
1. cultured 96 orifice plate is put in supercentrifuge, is centrifuged 5 minutes under 10000g；
2. new 96 orifice plate is taken, is separately added into 178 μ L sterile LB medium, 2 μ L cause of disease bacterium solution and 20 μ L streptococcus acidi lactici fermented solutions Supernatant, with 200 μ L sterile LB medium ,+178 μ L sterile LB medium of 2 μ L cause of disease bacterium solution, 20 μ L streptococcus acidi lactici fermented solution supernatants+ 180 μ L sterile LB medium are control, arrange 3 repetition orifice plates；
3. 40 DEG C are put, is cultivated 8 hours under the conditions of 200rpm, with not having 2h detection in ELIASA measurement 0-8 hour per hole OD600Numerical value.
Test result is similar to the result of embodiment 1, i.e., streptococcus acidi lactici fermented solution supernatant all has significant fungistatic effect.
Embodiment 4
A kind of method of high flux screening lactein, comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：1.8mL sterile LB medium is added toward in 10mL test tube, and accesses the pathogen of 0.2mL defrosting Liquid, puts 40 DEG C, cultivates 8h under the conditions of 200rpm；
2. Amplification Culture：Take 0.125mL bacterium solution access 25mL sterile LB medium 250mL triangular flask in, put 40 DEG C, 8h is cultivated under the conditions of 200rpm；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Load 510 μ L sterilizing MRS culture medium toward in 96 orifice plates per hole, then be respectively connected to 90 μ L defrosting Lactobacillus suspension, quiescent culture 12h at 28 DEG C；
2. Amplification Culture：Taken out per 6 μ L bacterium solution of hole with multichannel pipettor, access and be pre-loaded with 594 μ L of every hole sterilizing MRS training In the new orifice plate of foster base, quiescent culture 12h at 28 DEG C；
3. switching culture：6 μ L bacterium solution are taken per hole, access the new orifice plate for being pre-loaded with 574 μ L of every hole sterilizing MRS culture medium In, quiescent culture 8h at 28 DEG C, 20 μ L cause of disease bacterium solution are added, continues quiescent culture 12h at 28 DEG C；
(3) detect：
1. cultured 96 orifice plate is put in supercentrifuge, is centrifuged 5 minutes under 10000g；
2. new 96 orifice plate is taken, is separately added into 178 μ L sterile LB medium, 2 μ L cause of disease bacterium solution and 20 μ L streptococcus acidi lactici fermented solutions Supernatant, with 200 μ L sterile LB medium ,+178 μ L sterile LB medium of 2 μ L cause of disease bacterium solution, 20 μ L streptococcus acidi lactici fermented solution supernatants+ 180 μ L sterile LB medium are control, arrange 3 repetition orifice plates；
3. 40 DEG C are put, is cultivated 12 hours under the conditions of 200rpm, with not having 2h detection in ELIASA measurement 0-12 hour per hole OD600Numerical value.
Test result is similar to the result of embodiment 1, i.e., streptococcus acidi lactici fermented solution supernatant all has significant fungistatic effect.
Embodiment 5
A kind of method of high flux screening lactein, comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：1.9mL sterile LB medium is added toward in 10mL test tube, and accesses the pathogen of 0.1mL defrosting Liquid, puts 37 DEG C, cultivates 12h under the conditions of 250rpm；
2. Amplification Culture：Take 0.25mL bacterium solution access 25mL sterile LB medium 250mL triangular flask in, put 37 DEG C, 12h is cultivated under the conditions of 250rpm；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Load 540 μ L sterilizing MRS culture medium toward in 96 orifice plates per hole, then be respectively connected to 60 μ L defrosting Lactobacillus suspension, quiescent culture 12h at 30 DEG C；
2. Amplification Culture：Taken out per 30 μ L bacterium solution of hole with multichannel pipettor, access and be pre-loaded with 570 μ L of every hole sterilizing MRS In the new orifice plate of culture medium, quiescent culture 12h at 30 DEG C；
3. switching culture：30 μ L bacterium solution are taken per hole, access the new orifice plate for being pre-loaded with 570 μ L of every hole sterilizing MRS culture medium In, quiescent culture 6h at 30 DEG C, 6 μ L cause of disease bacterium solution are added, continues quiescent culture 12h at 30 DEG C；
(3) detect：
1. cultured 96 orifice plate is put in supercentrifuge, is centrifuged 5 minutes under 10000g；
2. new 96 orifice plate is taken, is separately added into 188 μ L sterile LB medium, 2 μ L cause of disease bacterium solution and 10 μ L streptococcus acidi lactici fermented solutions Supernatant, with 200 μ L sterile LB medium ,+198 μ L sterile LB medium of 2 μ L cause of disease bacterium solution, 10 μ L streptococcus acidi lactici fermented solution supernatants+ 190 μ L sterile LB medium are control, arrange 3 repetition orifice plates.
3. 37 DEG C are put, is cultivated 12 hours under the conditions of 250rpm, with not having 2h detection in ELIASA measurement 0-12 hour per hole OD600Numerical value.
Test result is similar to the result of embodiment 1, i.e., streptococcus acidi lactici fermented solution supernatant all has significant fungistatic effect.
Comparative example 1
The comparative example the difference is that only in embodiment 1, is not connect during switching culture in the culture lactic acid bacteria Enter cause of disease bacterium solution, in addition, remaining reagent and reagent dosage and cultural method are all same as Example 1.
As a result show, when cause of disease bacterium solution is not accessed, part streptococcus acidi lactici fermented solution supernatant fungistatic effect is not obvious.Thus Illustrate, the present invention adds a small amount of pathogen energy stimulating lactic acid bacteria to produce corresponding antibacterial material (breast in lactic acid bacteria incubation Sour rhzomorph), thus the lactein being not readily available by conventional method can be screened.
Comparative example 2
The comparative example the difference is that only in embodiment 1, accesses disease during switching culture in the culture lactic acid bacteria The inoculum concentration of original bacteria liquid has exceeded 5%, i.e. inoculum concentration for 8%, in addition, remaining reagent and reagent dosage and cultural method All same as Example 1.
As a result show, when cause of disease bacterium solution access amount is excessive, the growth of lactic acid bacteria is extremely slow, i.e. growth receives suppression System.
Applicant states that the present invention illustrates the process of the present invention by above-described embodiment, but the present invention not office It is limited to above-mentioned processing step, that is, does not mean that the present invention has to rely on above-mentioned processing step and could implement.Art Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement to raw material selected by the present invention and auxiliary element Interpolation, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosure.

Claims (10)

1. a kind of method of high flux screening lactein, it is characterised in that methods described comprises the steps：
(1) pathogen is cultivated；
(2) lactic acid bacteria is cultivated；
(3) lactic acid bacteria that on porous plate, add LB culture medium, step (1) to cultivate cause of disease fermented liquid and step (2) are cultivated Fermented liquid supernatant, co-cultures 8-25h, with ELIASA Timing measurement OD on porous plate600Absorbance；
Wherein, the porous plate be 24 orifice plates, in 48 orifice plates, 96 orifice plates or 384 orifice plates any one or at least two group Close.
2. method according to claim 1, it is characterised in that the pathogen described in step (1) is shrimp pathogenic bacteria pair haemolysis Any one in vibrios, Vibrio harveyi, Vibrio vulnificus, Aeromonas hydrophila, Vibrio anguillarum or vibrio alginolyticus or at least two Combination.
Preferably, the lactic acid bacteria is separating obtained lactic acid bacteria from animal alimentary canal and genital tract；
Preferably, the lactic acid bacteria be lactobacillus gasseri, lactobacillus fermenti, curling Bacillus acidi lactici, starch lactobacillus, lactic acid intestines ball Bacterium, Hai Shi enterococcus, enterococcus faecalis, Yue Shi lactobacillus, lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus plantarum, Luo Yishi breast Bacillus, Lactobacillus rhamnosus, Lactobacillus salivarius, Taiwan galactococcus, Lactococcus lactis, Leuconostoc, Pediococcus acidilactici, pentose Piece coccus, Streptococcus alactolyticus, Streptococcus iniae, Paris streptococcus, streptococcus salivarius, vestibular streptococcus, Vagococcus, Cibarium Wei Si Salmonella, confusus, He Lun Wei Si Salmonella, Young citric acid bacillus, mangrove enterobacteria, rothia dentocariosa, viscous In sliding Roche bacterium, shigella flexneri or MRSE any one or at least two combination.
3. method according to claim 1 and 2, it is characterised in that include the step of culture pathogen described in step (1) Activated spawn and Amplification Culture；
Preferably, the inoculum concentration of the activated spawn is 1-10%, preferably 5%；
Preferably, the Amplification Culture is the Amplification Culture by the strain transfer of activation in the LB culture medium of fresh sterilizing The inoculum concentration of middle activated spawn is 0.5-5%, preferably 1%.
4. method according to claim 3, it is characterised in that the culture lactic acid bacteria is cultivated using porous plate；
Preferably, the temperature of the activated spawn and Amplification Culture is 30-40 DEG C, preferably 35-38 DEG C, more preferably 37 ℃；
Preferably, the rotating speed of the activated spawn and Amplification Culture is 200-300rpm, preferably 230-280rpm, excellent further Elect 250rpm as；
Preferably, the time of the activated spawn and Amplification Culture is 8-25h, preferably 10-18h, more preferably 12h.
5. the method according to any one of claim 1-4, it is characterised in that the culture lactic acid bacteria described in step (2) Step includes activated spawn, Amplification Culture and switching culture；
Preferably, the inoculum concentration of the activated spawn is 5-15%, preferably 10%；
Preferably, the Amplification Culture is the Amplification Culture by the strain transfer of activation in the MRS culture medium of fresh sterilizing The inoculum concentration of middle activated spawn is 1-10%, preferably 5%.
6. method according to claim 5, it is characterised in that the temperature of the activated spawn and Amplification Culture is 25-33 DEG C, preferably 28-31 DEG C, more preferably 30 DEG C；
Preferably, the time of the activated spawn and Amplification Culture is 8-25h, preferably 10-18h, more preferably 12h.
7. the method according to claim 5 or 6, it is characterised in that the switching culture is that the bacterium solution that will be enlarged by cultivating turns 25-33 DEG C of culture 3-10h in the MRS culture medium of fresh sterilizing is connected to, and cause of disease bacterium solution is added, 8- is co-cultured at 25-33 DEG C 25h；
Preferably, described is 28-31 DEG C of culture 5- by condition of culture of the culture strain transfer in the MRS culture medium of fresh sterilizing 8h, preferably 30 DEG C culture 6h；
Preferably, the condition for co-culturing after the addition cause of disease bacterium solution is 28-31 DEG C of culture 10-18h, preferably 30 DEG C cultures 12h.
8. method according to claim 7, it is characterised in that the inoculum concentration of Amplification Culture bacterium solution is in the switching culture 1-10%, preferably 5%；
Preferably, in the switching culture, the inoculum concentration of cause of disease bacterium solution is 0.5-5%, preferably 1%.
9. the method according to any one of claim 5-8, it is characterised in that the LB culture medium described in step (3), cause of disease The volume ratio of bacterium solution and streptococcus acidi lactici fermented solution supernatant is (60-100):1:(3-15), preferably (70-90):1:(6-12), enter one Step is preferably 89:1:10；
Preferably, the time of the co-cultivation is 10-18h, preferably 12h；
Preferably, the temperature of the co-cultivation is 30-40 DEG C, preferably 35-38 DEG C, more preferably 37 DEG C；
Preferably, the rotating speed of the co-cultivation is 200-300rpm, preferably 230-280rpm, more preferably 250rpm；
Preferably, the interval time of the ELIASA measurement is 1-5h, preferably 1-3h, more preferably 2h.
10. the method according to claim 1-9, it is characterised in that methods described comprises the steps：
(1) pathogen is cultivated：
1. activated spawn：Pathogen is inoculated in the LB culture medium of fresh sterilizing by the inoculum concentration of 1-10%, is placed in 30-40 DEG C, 8-25h is cultivated under the conditions of 200-300rpm；
2. Amplification Culture：The bacterial classification of activation is inoculated in the LB culture medium of fresh sterilizing by the inoculum concentration of 0.5-5%, is placed in 30-40 DEG C, under the conditions of 200-300rpm, cultivate 8-25h；
(2) lactic acid bacteria is cultivated：
1. activated spawn：Inoculum concentration by 5-15% is placed in 25-33 by the MRS culture medium of lactobacillus inoculum to fresh sterilizing Quiescent culture 8-25h under the conditions of DEG C；
2. Amplification Culture：The MRS that activated spawn is inoculated into fresh sterilizing is supported in base by the inoculum concentration of 1-10%, be placed in 25-33 Quiescent culture 8-25h under the conditions of DEG C；
3. switching culture：The bacterial classification that will be enlarged by cultivating by the inoculum concentration of 1-10% is inoculated into 25- in the MRS culture medium of fresh sterilizing 33 DEG C of quiescent culture 3-10h, then the cause of disease bacterium solution of culture is accessed with the inoculum concentration of 0.5-5%, 8- is co-cultured in 25-33 DEG C of standing 25h；
(3) lactic acid bacteria that the cause of disease fermented liquid that cultivates LB culture medium, step (1) on 96 orifice plates and step (2) are cultivated sends out Zymotic fluid supernatant is (60-100) by volume:1:(3-15), 30-40 DEG C on 96 orifice plates, trained under conditions of 200-300rpm altogether Foster 8-25h, measures the absorbance of OD600 with ELIASA per 1-5h.
CN201610940806.8A 2016-10-25 2016-10-25 A kind of method of high flux screening lactein Pending CN106480164A (en)

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CN111187729A (en) * 2019-11-14 2020-05-22 华东师范大学 Screening method of propionibacterium and lactobacillus combined bacteria with synergistic mildew inhibition activity
CN111285925A (en) * 2019-12-24 2020-06-16 顾容铖 Separation and purification method of lactobacillus paracasei ZFM54 bacteriocin

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CN107505276A (en) * 2017-08-01 2017-12-22 上海美迪西生物医药股份有限公司 A kind of high throughput assay method of citron acid content in solution
CN110885875A (en) * 2019-11-14 2020-03-17 华东师范大学 Method for high-throughput screening of mould-inhibiting lactic acid bacteria
CN111187729A (en) * 2019-11-14 2020-05-22 华东师范大学 Screening method of propionibacterium and lactobacillus combined bacteria with synergistic mildew inhibition activity
CN111285925A (en) * 2019-12-24 2020-06-16 顾容铖 Separation and purification method of lactobacillus paracasei ZFM54 bacteriocin
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