CN109680034A - A kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique - Google Patents
A kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique Download PDFInfo
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- CN109680034A CN109680034A CN201910141356.XA CN201910141356A CN109680034A CN 109680034 A CN109680034 A CN 109680034A CN 201910141356 A CN201910141356 A CN 201910141356A CN 109680034 A CN109680034 A CN 109680034A
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Abstract
The invention discloses a kind of efficient demethylchlortetra cylinum bacterial strain cultivation and screening techniques, including configuration culture mother liquor, strain inoculation, breeding strain, fluorescent marker, screening, and six steps such as purification are concentrated.One aspect of the present invention is easy to operate, it cultivates and screening efficiency is high, and it is low in cost, environmental pollution is small, on the other hand the stability and yield of demethylchlortetra cylinum bacterial strain product quality can effectively be improved, while to greatly improve demethylchlortetra cylinum bacterium production efficiency and quality, good economic benefit and social benefit are separately obtained.
Description
Technical field
The invention belongs to chemical technology fields, and in particular to a kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique.
Background technique
Demethylchlortetra cylinum is mainly used for the production of minocycline and tigecycline.Its antibiotic property is stronger, and antimicrobial spectrum is mould with gold
Element is similar, acts on, than aureomycin more stable, in current biological field of medicaments important raw produce stronger than tetracycline, terramycin,
But it currently, due to foreign countries' technology blockage long-term to China, is gone in carrying out demethylchlortetra cylinum production process so as to cause China
The work of first aureomycin production relatively lags behind, and lacks high efficient and reliable, and low-cost demethylchlortetra cylinum product processes.
And in demethylchlortetra cylinum production link, demethylchlortetra cylinum bacterial strain is cultivated and screening is to ensure that demethylchlortetra cylinum
The important foundation of production efficiency quality, and currently adopted when carrying out to the cultivation of demethylchlortetra cylinum bacterial strain and screening operation
Method and technique are often to use for reference similar or similar breeding strain and screening technique development, although can expire to a certain degree
Sufficient demethylchlortetra cylinum bacterial strain is cultivated and the needs of screening, but working efficiency is low, and the cost is relatively high, is also easy to produce a large amount of pollution
Property waste, and bacterial strain cultivate quality stability and screening precision it is relatively poor, seriously affected the production of first aureomycin product
Amount and quality.
Therefore it is directed to this status, needs to develop the completely new demethylchlortetra cylinum bacterial strain cultivation of one kind and screening technique, with full
The needs actually used enough.
Summary of the invention
It is of the existing technology to solve the invention discloses a kind of efficient demethylchlortetra cylinum bacterial strain cultivation and screening technique
The problems such as production efficiency is low and poor product quality.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
A kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique, includes the following steps:
The first step, configuration culture mother liquor, first according to the needs of use, configuration demethylchlortetra cylinum strain culturing is female in culture kettle
Demethylchlortetra cylinum strain culturing mother liquor is at the uniform velocity warming up to 25 DEG C-35 DEG C and kept the temperature, then by liquid then in 10-30 minutes
Ultrasonic emulsification operation in 30-80 minutes is carried out to demethylchlortetra cylinum strain culturing mother liquor, and after completing ultrasonic emulsification operation
10-30 minutes are stood to the heat preservation of demethylchlortetra cylinum strain culturing mother liquor, prepared culture mother liquor is then divided into three parts,
And be carried in three mutually independent reaction kettles respectively, while adding biomarker in wherein a part culture mother liquor,
Then ultrasonic wave mixes 3-20 minutes, and biomarker liquid is prepared；
Second step, strain inoculation to culture dish and are crossed at carrier progress irradiation inactivated first under the conditions of gnotobasis is protected from light
Reason, then by bacterial classification inoculation apparatus by demethylchlortetra cylinum strain be coated on several culture dish upper surfaces, by each training after allowing
It supports ware staggeredly to be fixed along the vertical direction by carrier, and distribution is parallel to the horizontal plane in each culture dish upper surface
Complete strain inoculation；
Third step, breeding strain will complete the culture dish after being inoculated with and carrier are immersed in its separated in the first step together
In middle a part culture mother liquor, and make culture dish and carrier upper surface lower than at least 5 millimeters of mother liquor liquid level of culture, then 25
It cultivates 1-2 days, is can be obtained demethylchlortetra cylinum bacterial strain under DEG C-35 DEG C isoperibols；And in cultivating process, 3-5 hours/
Secondary frequency carries out ultrasonic wave stirring to culture mother liquor, and ultrasonic wave stirring frequency is 0.2-0.5MHz, each supersonic oscillations
Time is 5-20 minutes；
4th step, fluorescent marker are female by the culture for being used to impregnate culture dish and carrier in culture kettle after completing third step operation
Liquid emptying, and select one of them in each culture dish for target culture dish, it then will be in the culture mother liquor of first step configuration
Another part culture mother liquor is sprayed into each culture dish outside target culture dish, by biomarker liquid spray to target culture dish
In, and cultivate mother liquor and under the conditions of biomarker liquid spray parameter is consistent, carried out with 3-5 times/10 hours frequencies uniform
It is sprayed in each culture dish and target culture dish outside target culture dish, and is persistently cultivated under 25 DEG C -35 DEG C isoperibols
2-5 days, and in cultivating process, the culture mother liquor liquid level of culture dish upper surface be higher by culture dish upper surface by demethylchlortetra cylinum
1-10 millimeters of strain upper surface, and after each completion culture mother liquor spray, 100-160r/min vibration is carried out to each culture dish
Angle is not more than 30 ° between culture face upper surface and horizontal plane when continuing at the uniform velocity to vibrate 10-20 minutes under the conditions of swinging, and vibrating；
Demethylchlortetra cylinum bacterial strain after cultivating in the 4th step in target culture dish is carried out biomarker by the 5th step, screening
Object Concentration Testing, and demethylchlortetra cylinum bacterial strain in target culture dish is divided according to biomarker concentration distributed areas,
Then division distributed architecture is carried out according to demethylchlortetra cylinum bacterial strain in target culture dish, successively to bacterial strain in remaining each culture dish into
The division of row same distribution structure, so that different strains quality, which can be completed, divides screening operation；
6th step, concentration purification, according to the division screening of the 5th step as a result, in other culture dishes in addition to target culture dish
It carries out categorised collection with the demethylchlortetra cylinum bacterial strain in region to converge, then by the demethylchlortetra cylinum in non-compliant region
Bacterial strain is removed and harmless treatment, carries out respectively to the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain in each region dense
Contracting purification, and the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain after purifying concentration in each region be consistent after carry out two
It is secondary converge finished product can be obtained.
Further, in the first step, the following mass fraction substance of culture mother liquor is constituted: glucose 1% -3.5%, egg
White peptone 0.5% -2.3%, EDTA0.1% -0.5%, Ca (ClO)20.5%—1.3%、MgSO41.1%—2.5%、(NH4)2Fe
(SO4)2·6H2O0.8% -4.3%, surplus are physiological saline.
Further, in the first step, when to culture mother liquor distribution, for spraying the culture mother liquor amount of operation part
It is 20%-the 30% of culture mother liquor total amount；The part culture mother liquor amount for being used to prepare biomarker liquid is culture mother liquor total amount
10%—30%；Surplus is for impregnating with culture mother liquor.
Further, the biomarker in the first step is any one in fluorescent marker and isotopic label
It plants or two kinds shares.
Further, concentration of the biomarker in the 4th step in culture mother liquor is 3% -10%.
Further, in described second, carrier is the frame structure of axis and horizontal plane distribution, the culture
Ware includes that cross section is coaxially distributed and in web plate plate knot in the matrix of " Qian " font groove-like structure and in matrix with matrix
The pallet of structure, described matrix lower end surface and carrier are hinged by turntable mechanism, the pallet lower end surface rain base bottom
Between spacing be 0-5 millimeters, upper surface is lower than at least 3 millimeters of culture dish, and between the two neighboring culture dish being distributed from top to bottom
Spacing is 5-20 millimeters.
Further, in the third step, when carrying out ultrasonic wave stirring, spacing is not less than 10 between oscillation source and carrier
Centimetre, concussion direction and carrier axis are in 15 ° -90 ° angles.
Further, in the 6th step, the demethylchlortetra cylinum bacterial strain in non-compliant region carries out innoxious place
When reason, used simultaneously using any one or a few in irradiation inactivated, biofermentation and high temperature incineration.
The utility model has the advantages that
Compared with prior art, the present invention has the advantage that
One aspect of the present invention is easy to operate, cultivates and screening efficiency is high and low in cost, environmental pollution is small, on the other hand may be used
The effective stability and yield for improving demethylchlortetra cylinum bacterial strain product quality, to greatly improve the production of demethylchlortetra cylinum bacterium
While efficiency and quality, good economic benefit and social benefit are separately obtained.
Detailed description of the invention
Fig. 1 is process flow chart of the invention.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
Embodiment 1
As shown in Figure 1, a kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique, include the following steps:
The first step, configuration culture mother liquor, first according to the needs of use, configuration demethylchlortetra cylinum strain culturing is female in culture kettle
Demethylchlortetra cylinum strain culturing mother liquor is at the uniform velocity warming up to 25 DEG C and kept the temperature by liquid then in 10 minutes, then to going first gold mould
Plain strain culturing mother liquor carries out ultrasonic emulsification operation in 30 minutes, and to demethylchlortetra cylinum bacterium after completing ultrasonic emulsification operation
Strain culture mother liquor heat preservation stands 10 minutes, prepared culture mother liquor is then divided into three parts, and be carried on three phases respectively
In mutual independent reaction kettle, while biomarker is added in wherein a part culture mother liquor, then ultrasonic wave mixes 3 points
Biomarker liquid is prepared in clock；
Second step, strain inoculation to culture dish and are crossed at carrier progress irradiation inactivated first under the conditions of gnotobasis is protected from light
Reason, then by bacterial classification inoculation apparatus by demethylchlortetra cylinum strain be coated on several culture dish upper surfaces, by each training after allowing
It supports ware staggeredly to be fixed along the vertical direction by carrier, and distribution is parallel to the horizontal plane in each culture dish upper surface
Complete strain inoculation；
Third step, breeding strain will complete the culture dish after being inoculated with and carrier are immersed in its separated in the first step together
In middle a part culture mother liquor, and make culture dish and carrier upper surface lower than 5 millimeters of mother liquor liquid level of culture, then in 25 DEG C of perseverances
It cultivates 2 days, is can be obtained demethylchlortetra cylinum bacterial strain under warm environment；And in cultivating process, the frequency of 3 hours/time is female to culture
Liquid carries out ultrasonic wave stirring, and ultrasonic wave stirring frequency is 0.2MHz, and each supersonic oscillations time is 20 minutes；
4th step, fluorescent marker are female by the culture for being used to impregnate culture dish and carrier in culture kettle after completing third step operation
Liquid emptying, and select one of them in each culture dish for target culture dish, it then will be in the culture mother liquor of first step configuration
Another part culture mother liquor is sprayed into each culture dish outside target culture dish, by biomarker liquid spray to target culture dish
In, and cultivate mother liquor and biomarker liquid spray parameter be consistent under the conditions of, uniformly sprayed with 5 times/10 hours frequencies
Leaching is persistently cultivated 5 days into each culture dish and target culture dish outside target culture dish under 25 DEG C of isoperibols, and is cultivated
In the process, the culture mother liquor liquid level of culture dish upper surface be higher by culture dish upper surface by the milli of demethylchlortetra cylinum strain upper surface 1
Rice, and after each completion culture mother liquor spray, each culture dish is carried out to continue at the uniform velocity oscillation 10 under 100r/min oscillating condition
Minute, and angle is 30 ° between culture face upper surface and horizontal plane when oscillation；
Demethylchlortetra cylinum bacterial strain after cultivating in the 4th step in target culture dish is carried out biomarker by the 5th step, screening
Object Concentration Testing, and demethylchlortetra cylinum bacterial strain in target culture dish is divided according to biomarker concentration distributed areas,
Then division distributed architecture is carried out according to demethylchlortetra cylinum bacterial strain in target culture dish, successively to bacterial strain in remaining each culture dish into
The division of row same distribution structure, so that different strains quality, which can be completed, divides screening operation；
6th step, concentration purification, according to the division screening of the 5th step as a result, in other culture dishes in addition to target culture dish
It carries out categorised collection with the demethylchlortetra cylinum bacterial strain in region to converge, then by the demethylchlortetra cylinum in non-compliant region
Bacterial strain is removed and harmless treatment, carries out respectively to the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain in each region dense
Contracting purification, and the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain after purifying concentration in each region be consistent after carry out two
It is secondary converge finished product can be obtained.
In the present embodiment, in the first step, the following mass fraction substance of culture mother liquor is constituted: glucose 1% -3.5%,
Peptone 0.5%, EDTA0.1%, Ca (ClO)20.5%、MgSO41.1%、(NH4)2Fe(SO4)2·6H2O0.8%, surplus are physiology
Salt water.
In the present embodiment, in the first step, when to culture mother liquor distribution, for spraying the culture mother liquor of operation part
Amount is the 20% of culture mother liquor total amount；The part culture mother liquor amount for being used to prepare biomarker liquid is cultivate mother liquor total amount 10%；
Surplus is for impregnating with culture mother liquor.
In the present embodiment, biomarker in the first step is fluorescent marker, and the biology in the 4th step
Concentration of the marker in culture mother liquor is 3%.
In the present embodiment, in described second, carrier is the frame structure of axis and horizontal plane distribution, the training
Feeding ware includes that cross section is coaxially distributed and in web plate plate in the matrix of " Qian " font groove-like structure and in matrix with matrix
The pallet of structure, described matrix lower end surface and carrier are hinged by turntable mechanism, pallet lower end surface rain matrix bottom
Spacing is 0 between portion, and upper surface is lower than 3 millimeters of culture dish, and spacing is 5 millis between the two neighboring culture dish being distributed from top to bottom
Rice.
In the present embodiment, in the third step, when carrying out ultrasonic wave stirring, spacing is 10 lis between oscillation source and carrier
Rice, concussion direction and carrier axis are in 15 ° of angles.
In the present embodiment, in the 6th step, the demethylchlortetra cylinum bacterial strain in non-compliant region carries out innoxious
When processing, used simultaneously using any one or a few in irradiation inactivated, biofermentation and high temperature incineration.
Embodiment 2
As shown in Figure 1, a kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique, include the following steps:
The first step, configuration culture mother liquor, first according to the needs of use, configuration demethylchlortetra cylinum strain culturing is female in culture kettle
Demethylchlortetra cylinum strain culturing mother liquor is at the uniform velocity warming up to 35 DEG C and kept the temperature by liquid then in 30 minutes, then to going first gold mould
Plain strain culturing mother liquor carries out ultrasonic emulsification operation in 80 minutes, and to demethylchlortetra cylinum bacterium after completing ultrasonic emulsification operation
Strain culture mother liquor heat preservation stands 30 minutes, prepared culture mother liquor is then divided into three parts, and be carried on three phases respectively
In mutual independent reaction kettle, while biomarker is added in wherein a part culture mother liquor, then ultrasonic wave mixes 20 points
Biomarker liquid is prepared in clock；
Second step, strain inoculation to culture dish and are crossed at carrier progress irradiation inactivated first under the conditions of gnotobasis is protected from light
Reason, then by bacterial classification inoculation apparatus by demethylchlortetra cylinum strain be coated on several culture dish upper surfaces, by each training after allowing
It supports ware staggeredly to be fixed along the vertical direction by carrier, and distribution is parallel to the horizontal plane in each culture dish upper surface
Complete strain inoculation；
Third step, breeding strain will complete the culture dish after being inoculated with and carrier are immersed in its separated in the first step together
In middle a part culture mother liquor, and make culture dish and carrier upper surface lower than 8 millimeters of mother liquor liquid level of culture, then in 35 DEG C of perseverances
It cultivates 1 day, is can be obtained demethylchlortetra cylinum bacterial strain under warm environment；And in cultivating process, the frequency of 5 hours/time is female to culture
Liquid carries out ultrasonic wave stirring, and ultrasonic wave stirring frequency is 0.5MHz, and each supersonic oscillations time is 5 minutes；
4th step, fluorescent marker are female by the culture for being used to impregnate culture dish and carrier in culture kettle after completing third step operation
Liquid emptying, and select one of them in each culture dish for target culture dish, it then will be in the culture mother liquor of first step configuration
Another part culture mother liquor is sprayed into each culture dish outside target culture dish, by biomarker liquid spray to target culture dish
In, and cultivate mother liquor and biomarker liquid spray parameter be consistent under the conditions of, uniformly sprayed with 3 times/10 hours frequencies
Leaching is persistently cultivated 2 days into each culture dish and target culture dish outside target culture dish under 35 DEG C of isoperibols, and is cultivated
In the process, the culture mother liquor liquid level of culture dish upper surface be higher by culture dish upper surface by the milli of demethylchlortetra cylinum strain upper surface 10
Rice, and after each completion culture mother liquor spray, each culture dish is carried out to continue at the uniform velocity oscillation 20 under 160r/min oscillating condition
Minute, and angle is 5 ° between culture face upper surface and horizontal plane when oscillation；
Demethylchlortetra cylinum bacterial strain after cultivating in the 4th step in target culture dish is carried out biomarker by the 5th step, screening
Object Concentration Testing, and demethylchlortetra cylinum bacterial strain in target culture dish is divided according to biomarker concentration distributed areas,
Then division distributed architecture is carried out according to demethylchlortetra cylinum bacterial strain in target culture dish, successively to bacterial strain in remaining each culture dish into
The division of row same distribution structure, so that different strains quality, which can be completed, divides screening operation；
6th step, concentration purification, according to the division screening of the 5th step as a result, in other culture dishes in addition to target culture dish
It carries out categorised collection with the demethylchlortetra cylinum bacterial strain in region to converge, then by the demethylchlortetra cylinum in non-compliant region
Bacterial strain is removed and harmless treatment, carries out respectively to the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain in each region dense
Contracting purification, and the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain after purifying concentration in each region be consistent after carry out two
It is secondary converge finished product can be obtained.
In the present embodiment, in the first step, the following mass fraction substance of culture mother liquor is constituted: glucose 3.5%, albumen
Peptone 2.3%, EDTA0.5%, Ca (ClO)21.3%、MgSO42.5%、(NH4)2Fe(SO4)2·6H2O4.3%, surplus are physiological saline.
In the present embodiment, in the first step, when to culture mother liquor distribution, for spraying the culture mother liquor of operation part
Amount is the 30% of culture mother liquor total amount；The part culture mother liquor amount for being used to prepare biomarker liquid is cultivate mother liquor total amount 30%；
Surplus is for impregnating with culture mother liquor.
In the present embodiment, the biomarker in the first step is isotopic label, the biology in the 4th step
Concentration of the marker in culture mother liquor is 10%.
In the present embodiment, in described second, carrier is the frame structure of axis and horizontal plane distribution, the training
Feeding ware includes that cross section is coaxially distributed and in web plate plate in the matrix of " Qian " font groove-like structure and in matrix with matrix
The pallet of structure, described matrix lower end surface and carrier are hinged by turntable mechanism, pallet lower end surface rain matrix bottom
Spacing is 5 millimeters between portion, and upper surface is lower than 5 millimeters of culture dish, and spacing between the two neighboring culture dish being distributed from top to bottom
It is 20 millimeters.
In the present embodiment, in the third step, when carrying out ultrasonic wave stirring, spacing is 50 lis between oscillation source and carrier
Rice, concussion direction and carrier axis are in 90 ° of angles.
In the present embodiment, in the 6th step, the demethylchlortetra cylinum bacterial strain in non-compliant region carries out innoxious
When processing, used simultaneously using any one or a few in irradiation inactivated, biofermentation and high temperature incineration.
Embodiment 3
As shown in Figure 1, a kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique, include the following steps:
The first step, configuration culture mother liquor, first according to the needs of use, configuration demethylchlortetra cylinum strain culturing is female in culture kettle
Demethylchlortetra cylinum strain culturing mother liquor is at the uniform velocity warming up to 28 DEG C and kept the temperature by liquid then in 20 minutes, then to going first gold mould
Plain strain culturing mother liquor carries out ultrasonic emulsification operation in 45 minutes, and to demethylchlortetra cylinum bacterium after completing ultrasonic emulsification operation
Strain culture mother liquor heat preservation stands 21 minutes, prepared culture mother liquor is then divided into three parts, and be carried on three phases respectively
In mutual independent reaction kettle, while biomarker is added in wherein a part culture mother liquor, then ultrasonic wave mixes 18 points
Biomarker liquid is prepared in clock；
Second step, strain inoculation to culture dish and are crossed at carrier progress irradiation inactivated first under the conditions of gnotobasis is protected from light
Reason, then by bacterial classification inoculation apparatus by demethylchlortetra cylinum strain be coated on several culture dish upper surfaces, by each training after allowing
It supports ware staggeredly to be fixed along the vertical direction by carrier, and distribution is parallel to the horizontal plane in each culture dish upper surface
Complete strain inoculation；
Third step, breeding strain will complete the culture dish after being inoculated with and carrier are immersed in its separated in the first step together
In middle a part culture mother liquor, and make culture dish and carrier upper surface lower than at least 5 millimeters of mother liquor liquid level of culture, then 28
It cultivates 1.5 days, is can be obtained demethylchlortetra cylinum bacterial strain under DEG C isoperibol；And in cultivating process, the frequency pair of 4 hours/time
It cultivates mother liquor and carries out ultrasonic wave stirring, and ultrasonic wave stirring frequency is 0.4MHz, each supersonic oscillations time is 15 minutes；
4th step, fluorescent marker are female by the culture for being used to impregnate culture dish and carrier in culture kettle after completing third step operation
Liquid emptying, and select one of them in each culture dish for target culture dish, it then will be in the culture mother liquor of first step configuration
Another part culture mother liquor is sprayed into each culture dish outside target culture dish, by biomarker liquid spray to target culture dish
In, and cultivate mother liquor and biomarker liquid spray parameter be consistent under the conditions of, uniformly sprayed with 4 times/10 hours frequencies
Leaching is persistently cultivated 4 days into each culture dish and target culture dish outside target culture dish under 28 DEG C of isoperibols, and is cultivated
In the process, the culture mother liquor liquid level of culture dish upper surface be higher by culture dish upper surface by the milli of demethylchlortetra cylinum strain upper surface 5
Rice, and after each completion culture mother liquor spray, each culture dish is carried out to continue at the uniform velocity oscillation 15 under 120r/min oscillating condition
Minute, and angle is 15 ° between culture face upper surface and horizontal plane when oscillation；
Demethylchlortetra cylinum bacterial strain after cultivating in the 4th step in target culture dish is carried out biomarker by the 5th step, screening
Object Concentration Testing, and demethylchlortetra cylinum bacterial strain in target culture dish is divided according to biomarker concentration distributed areas,
Then division distributed architecture is carried out according to demethylchlortetra cylinum bacterial strain in target culture dish, successively to bacterial strain in remaining each culture dish into
The division of row same distribution structure, so that different strains quality, which can be completed, divides screening operation；
6th step, concentration purification, according to the division screening of the 5th step as a result, in other culture dishes in addition to target culture dish
It carries out categorised collection with the demethylchlortetra cylinum bacterial strain in region to converge, then by the demethylchlortetra cylinum in non-compliant region
Bacterial strain is removed and harmless treatment, carries out respectively to the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain in each region dense
Contracting purification, and the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain after purifying concentration in each region be consistent after carry out two
It is secondary converge finished product can be obtained.
In the present embodiment, in the first step, the following mass fraction substance of culture mother liquor is constituted: glucose 2.1%, albumen
Peptone 1.3%, EDTA0.3%, Ca (ClO)20.8%、MgSO41.5%、(NH4)2Fe(SO4)2·6H2O3.3%, surplus are physiological saline.
In the present embodiment, in the first step, when to culture mother liquor distribution, for spraying the culture mother liquor of operation part
Amount is the 25% of culture mother liquor total amount；The part culture mother liquor amount for being used to prepare biomarker liquid is cultivate mother liquor total amount 25%；
Surplus is for impregnating with culture mother liquor.
In the present embodiment, the biomarker in the first step is fluorescent marker and isotopic label with 1:1 ratio
Example is used in mixed way, and concentration of the biomarker in the 4th step in culture mother liquor is 4%.
In the present embodiment, in described second, carrier is the frame structure of axis and horizontal plane distribution, the training
Feeding ware includes that cross section is coaxially distributed and in web plate plate in the matrix of " Qian " font groove-like structure and in matrix with matrix
The pallet of structure, described matrix lower end surface and carrier are hinged by turntable mechanism, pallet lower end surface rain matrix bottom
Spacing is 2.5 millimeters between portion, and upper surface is 5 millimeters lower than culture dish, and between the two neighboring culture dish being distributed from top to bottom
Spacing is 8 millimeters.
In the present embodiment, in the third step, when carrying out ultrasonic wave stirring, spacing is 14 lis between oscillation source and carrier
Rice, concussion direction and carrier axis are in 45 ° of angles.
In the present embodiment, in the 6th step, the demethylchlortetra cylinum bacterial strain in non-compliant region carries out innoxious
When processing, used simultaneously using any one or a few in irradiation inactivated, biofermentation and high temperature incineration.
Embodiment 4
As shown in Figure 1, a kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique, include the following steps:
The first step, configuration culture mother liquor, first according to the needs of use, configuration demethylchlortetra cylinum strain culturing is female in culture kettle
Demethylchlortetra cylinum strain culturing mother liquor is at the uniform velocity warming up to 30 DEG C and kept the temperature by liquid then in 11 minutes, then to going first gold mould
Plain strain culturing mother liquor carries out ultrasonic emulsification operation in 50 minutes, and to demethylchlortetra cylinum bacterium after completing ultrasonic emulsification operation
Strain culture mother liquor heat preservation stands 19 minutes, prepared culture mother liquor is then divided into three parts, and be carried on three phases respectively
In mutual independent reaction kettle, while biomarker is added in wherein a part culture mother liquor, then ultrasonic wave mixes 16 points
Biomarker liquid is prepared in clock；
Second step, strain inoculation to culture dish and are crossed at carrier progress irradiation inactivated first under the conditions of gnotobasis is protected from light
Reason, then by bacterial classification inoculation apparatus by demethylchlortetra cylinum strain be coated on several culture dish upper surfaces, by each training after allowing
It supports ware staggeredly to be fixed along the vertical direction by carrier, and distribution is parallel to the horizontal plane in each culture dish upper surface
Complete strain inoculation；
Third step, breeding strain will complete the culture dish after being inoculated with and carrier are immersed in its separated in the first step together
In middle a part culture mother liquor, and make culture dish and carrier upper surface lower than 11 millimeters of mother liquor liquid level of culture, then at 30 DEG C
It cultivates 1 day, is can be obtained demethylchlortetra cylinum bacterial strain under isoperibol；And in cultivating process, the frequency of 4 hours/time is to culture
Mother liquor carries out ultrasonic wave stirring, and ultrasonic wave stirring frequency is 0.3MHz, and each supersonic oscillations time is 16 minutes；
4th step, fluorescent marker are female by the culture for being used to impregnate culture dish and carrier in culture kettle after completing third step operation
Liquid emptying, and select one of them in each culture dish for target culture dish, it then will be in the culture mother liquor of first step configuration
Another part culture mother liquor is sprayed into each culture dish outside target culture dish, by biomarker liquid spray to target culture dish
In, and cultivate mother liquor and biomarker liquid spray parameter be consistent under the conditions of, uniformly sprayed with 4 times/10 hours frequencies
Leaching persistently cultivates 3.5 into each culture dish and target culture dish outside target culture dish, and under 25 DEG C -35 DEG C isoperibols
It, and in cultivating process, the culture mother liquor liquid level of culture dish upper surface be higher by culture dish upper surface by demethylchlortetra cylinum strain
6 millimeters of upper surface, and after each completion culture mother liquor spray, each culture dish continue under 120r/min oscillating condition even
Speed oscillation 15 minutes, and angle is 20 ° between culture face upper surface and horizontal plane when oscillation；
Demethylchlortetra cylinum bacterial strain after cultivating in the 4th step in target culture dish is carried out biomarker by the 5th step, screening
Object Concentration Testing, and demethylchlortetra cylinum bacterial strain in target culture dish is divided according to biomarker concentration distributed areas,
Then division distributed architecture is carried out according to demethylchlortetra cylinum bacterial strain in target culture dish, successively to bacterial strain in remaining each culture dish into
The division of row same distribution structure, so that different strains quality, which can be completed, divides screening operation；
6th step, concentration purification, according to the division screening of the 5th step as a result, in other culture dishes in addition to target culture dish
It carries out categorised collection with the demethylchlortetra cylinum bacterial strain in region to converge, then by the demethylchlortetra cylinum in non-compliant region
Bacterial strain is removed and harmless treatment, carries out respectively to the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain in each region dense
Contracting purification, and the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain after purifying concentration in each region be consistent after carry out two
It is secondary converge finished product can be obtained.
In the present embodiment, in the first step, the following mass fraction substance of culture mother liquor is constituted: glucose 2.8%, albumen
Peptone 1.9%, EDTA0.4%, Ca (ClO)20.7%、MgSO42%、(NH4)2Fe(SO4)2·6H2O3.8%, surplus are physiological saline.
In the present embodiment, in the first step, when to culture mother liquor distribution, for spraying the culture mother liquor of operation part
Amount is the 22% of culture mother liquor total amount；The part culture mother liquor amount for being used to prepare biomarker liquid is cultivate mother liquor total amount 28%；
Surplus is for impregnating with culture mother liquor.
In the present embodiment, the biomarker in the first step is fluorescent marker and isotopic label with 1:2.5
Ratio mixes, and concentration of the biomarker in the 4th step in culture mother liquor is 6.5%.
In the present embodiment, in described second, carrier is the frame structure of axis and horizontal plane distribution, the training
Feeding ware includes that cross section is coaxially distributed and in web plate plate in the matrix of " Qian " font groove-like structure and in matrix with matrix
The pallet of structure, described matrix lower end surface and carrier are hinged by turntable mechanism, pallet lower end surface rain matrix bottom
Between portion spacing be 3.5 millimeters, upper surface be lower than 6 millimeters of culture dish, and between the two neighboring culture dish being distributed from top to bottom between
Away from being 14 millimeters.
In the present embodiment, in the third step, when carrying out ultrasonic wave stirring, spacing is 20 lis between oscillation source and carrier
Rice, concussion direction and carrier axis are in 60 ° of angles.
In the present embodiment, in the 6th step, the demethylchlortetra cylinum bacterial strain in non-compliant region carries out innoxious
When processing, used simultaneously using any one or a few in irradiation inactivated, biofermentation and high temperature incineration.
The utility model has the advantages that
Compared with prior art, the present invention has the advantage that
One aspect of the present invention is easy to operate, cultivates and screening efficiency is high and low in cost, environmental pollution is small, on the other hand may be used
The effective stability and yield for improving demethylchlortetra cylinum bacterial strain product quality, to greatly improve the production of demethylchlortetra cylinum bacterium
While efficiency and quality, good economic benefit and social benefit are separately obtained.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (8)
1. a kind of efficient demethylchlortetra cylinum bacterial strain is cultivated and screening technique, which is characterized in that the efficient demethylchlortetra cylinum bacterium
Strain is cultivated and screening technique includes the following steps:
The first step, configuration culture mother liquor, first according to the needs of use, configuration demethylchlortetra cylinum strain culturing is female in culture kettle
Demethylchlortetra cylinum strain culturing mother liquor is at the uniform velocity warming up to 25 DEG C-35 DEG C and kept the temperature, then by liquid then in 10-30 minutes
Ultrasonic emulsification operation in 30-80 minutes is carried out to demethylchlortetra cylinum strain culturing mother liquor, and after completing ultrasonic emulsification operation
10-30 minutes are stood to the heat preservation of demethylchlortetra cylinum strain culturing mother liquor, prepared culture mother liquor is then divided into three parts,
And be carried in three mutually independent reaction kettles respectively, while adding biomarker in wherein a part culture mother liquor,
Then ultrasonic wave mixes 3-20 minutes, and biomarker liquid is prepared；
Second step, strain inoculation to culture dish and are crossed at carrier progress irradiation inactivated first under the conditions of gnotobasis is protected from light
Reason, then by bacterial classification inoculation apparatus by demethylchlortetra cylinum strain be coated on several culture dish upper surfaces, by each training after allowing
It supports ware staggeredly to be fixed along the vertical direction by carrier, and distribution is parallel to the horizontal plane in each culture dish upper surface
Complete strain inoculation；
Third step, breeding strain will complete the culture dish after being inoculated with and carrier are immersed in its separated in the first step together
In middle a part culture mother liquor, and make culture dish and carrier upper surface lower than at least 5 millimeters of mother liquor liquid level of culture, then 25
It cultivates 1-2 days, is can be obtained demethylchlortetra cylinum bacterial strain under DEG C-35 DEG C isoperibols；And in cultivating process, 3-5 hours/
Secondary frequency carries out ultrasonic wave stirring to culture mother liquor, and ultrasonic wave stirring frequency is 0.2-0.5MHz, each supersonic oscillations
Time is 5-20 minutes；
4th step, fluorescent marker are female by the culture for being used to impregnate culture dish and carrier in culture kettle after completing third step operation
Liquid emptying, and select one of them in each culture dish for target culture dish, it then will be in the culture mother liquor of first step configuration
Another part culture mother liquor is sprayed into each culture dish outside target culture dish, by biomarker liquid spray to target culture dish
In, and cultivate mother liquor and under the conditions of biomarker liquid spray parameter is consistent, carried out with 3-5 times/10 hours frequencies uniform
It is sprayed in each culture dish and target culture dish outside target culture dish, and is persistently cultivated under 25 DEG C -35 DEG C isoperibols
2-5 days, and in cultivating process, the culture mother liquor liquid level of culture dish upper surface be higher by culture dish upper surface by demethylchlortetra cylinum
1-10 millimeters of strain upper surface, and after each completion culture mother liquor spray, 100-160r/min vibration is carried out to each culture dish
Angle is not more than 30 ° between culture face upper surface and horizontal plane when continuing at the uniform velocity to vibrate 10-20 minutes under the conditions of swinging, and vibrating；
Demethylchlortetra cylinum bacterial strain after cultivating in the 4th step in target culture dish is carried out biomarker by the 5th step, screening
Object Concentration Testing, and demethylchlortetra cylinum bacterial strain in target culture dish is divided according to biomarker concentration distributed areas,
Then division distributed architecture is carried out according to demethylchlortetra cylinum bacterial strain in target culture dish, successively to bacterial strain in remaining each culture dish into
The division of row same distribution structure, so that different strains quality, which can be completed, divides screening operation；
6th step, concentration purification, according to the division screening of the 5th step as a result, in other culture dishes in addition to target culture dish
It carries out categorised collection with the demethylchlortetra cylinum bacterial strain in region to converge, then by the demethylchlortetra cylinum in non-compliant region
Bacterial strain is removed and harmless treatment, carries out respectively to the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain in each region dense
Contracting purification, and the concentration machine developmental condition of the demethylchlortetra cylinum bacterial strain after purifying concentration in each region be consistent after carry out two
It is secondary converge finished product can be obtained.
2. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that first
In step, the following mass fraction substance of the culture mother liquor is constituted: glucose 1% -3.5%, peptone 0.5% -2.3%,
EDTA0.1%—0.5%、Ca(ClO)20.5%—1.3%、MgSO41.1%—2.5%、(NH4)2Fe(SO4)2·6H2O0.8%—
4.3%, surplus is physiological saline.
3. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that described
In the first step, when to culture mother liquor distribution, the culture mother liquor amount for spraying operation part is cultivate mother liquor total amount 20%-
30%；The part culture mother liquor amount for being used to prepare biomarker liquid is cultivate mother liquor total amount 10% -30%；Surplus is for impregnating
With culture mother liquor.
4. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that described
Biomarker in the first step is that any one or two kinds in fluorescent marker and isotopic label share.
5. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that described
Concentration of the biomarker in culture mother liquor in 4th step is 3% -10%.
6. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that described
In second, carrier is the frame structure of axis and horizontal plane distribution, and the culture dish includes cross section in " Qian " word
The matrix of type groove-like structure and the pallet for being coaxially distributed with matrix in matrix and be in web plate plate structure, described matrix lower end
Face and carrier are hinged by turntable mechanism, and spacing is 0-5 millimeters between the pallet lower end surface rain base bottom, upper end
Face is lower than at least 3 millimeters of culture dish, and spacing is 5-20 millimeters between the two neighboring culture dish being distributed from top to bottom.
7. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that described
In third step, when carrying out ultrasonic wave stirring, spacing is not less than 10 centimetres between oscillation source and carrier, shakes direction and carrier
Axis is in 15 ° -90 ° angles.
8. a kind of efficient demethylchlortetra cylinum bacterial strain according to claim 1 is cultivated and screening technique, which is characterized in that described
In 6th step, when the demethylchlortetra cylinum bacterial strain in non-compliant region carries out harmless treatment, using irradiation inactivated, biology
Any one or a few in fermentation and high temperature incineration uses simultaneously.
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