CN103966098B - The treatment process of a kind of freeze-drying preservation Zao Peizhong colony microorganism - Google Patents
The treatment process of a kind of freeze-drying preservation Zao Peizhong colony microorganism Download PDFInfo
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Abstract
The present invention relates to the treatment process of a kind of freeze-drying preservation Zao Peizhong colony microorganism, key step is: in poor unstrained spirits, add cell cultures PBS damping fluid used carry out repeatedly whirlpool concussion, centrifugation grain unstrained spirits and elutriant, more centrifugal all elutriants obtain bacterium mud; Then pre-freeze and vacuum lyophilization is carried out by collecting the bacterium mud obtained.By analyzing microorganism rate of loss and microorganism structure in each sample after former poor unstrained spirits and process, result shows: adopt treatment process of the present invention, the rate of loss of bacterium and fungi total amount is within 2.0%, the rate of loss of single strain is within 2.5%, and the bacterium mud collected has biological activity, microorganism structure is consistent with former poor unstrained spirits.This pretreatment process is used to collect the bacterium mud obtained, be convenient to be loaded lyophilize and later stage vacuum melting-sealed in ampoul tube, extend the preservation time, the method can be widely used in other for being separated, collecting colony microorganism and keep bioactive purposes, for colony's rate of loss estimation of Analysis of Microbial Diversity provides important evidence.
Description
Technical field
The present invention relates to a kind of collection method of living microorganism, particularly a kind for the treatment of process based on Freeze Drying Technique preservation Zao Peizhong colony microorganism.
Background technology
Microorganism is the valuable source of country, and microbial preservation is the necessary technology means of microbiological research and related production application.Vacuum Freezing & Drying Technology has the principal element of low temperature, drying and three culture presevation of isolated air concurrently, and be one of the most safe and reliable method of the long-term preservation microbial strains of generally acknowledging at present, applied widely, preservation term is long, and surviving rate is high.It is research object with single strain that current microorganism preserves main, and very few for the research report of structural colony's microbial preservation.
Grain unstrained spirits is rich in various nutritional condition needed for microbial growth, and it experienced by certain fermentation time and a large amount of microorganism of network, and the heap fermentation of poor unstrained spirits has the effect of secondary koji, and the quality of its fermentation, directly affects local flavor and the quality of white wine.The microorganism structure that the fermented mash of different white wine has it exclusive, the difference of liquor style and liquor odor type comes from the difference of its colony's microbe sort, structure to a great extent.Group preservation is carried out for microorganism wherein, be convenient to the research of microorganism and characteristic colony microorganism producing, industrial application.
This researchist finds in researchs such as Zao Peizhong colony microorganism structure, activity, collections; if directly attached for poor unstrained spirits matrix is carried out preservation; due to the existence of poor unstrained spirits particle; sublimation drying will be extended; be unfavorable for freeze-dried homogeneous available protecting; also be unfavorable for follow-up vacuum melting-sealed preservation, thus reduce the survival rate of microorganism, shorten the preservation time of microorganism.In prior art, the main purpose of elution method transfer sample microbial extracts microbial genome to carry out diversity analysis, part microorganism inactivation in this process, and do not analyze microorganism rate of loss, and the entirety representativeness of microbial information cannot be estimated.Therefore how complete migrating out colony microorganism wherein and do not affect activity will become crucial.This researchist is by different experimental studies, and find to be used by poor unstrained spirits cell cultures damping fluid used to carry out repeatedly vortex oscillation, wash-out, centrifugation grain unstrained spirits and microorganism, finally obtain the active bacteria mud consistent with poor unstrained spirits microorganism structure; Then the bacterium mud of collection is carried out lyophilize, and directly poor unstrained spirits is carried out compared with freeze-drying preservation, not only reducing the cryodesiccated time, also improving the survival rate of microorganism.
Summary of the invention
Therefore, the invention provides the treatment process of a kind of freeze-drying preservation Zao Peizhong colony microorganism, the method carries out preservation based on Vacuum Freezing & Drying Technology to Zao Peizhong colony microorganism, object is by complete being as far as possible shifted out of the microorganism in poor unstrained spirits, and do not damage its activity, to improve its survival rate, be convenient to later stage vacuum melting-sealed preservation and extend the preservation time, make colony's preservation of microorganism in poor unstrained spirits feasible.
For realizing this object, technical scheme is as follows:
A treatment process for freeze-drying preservation Zao Peizhong colony microorganism, step is as follows:
1) wash-out, transfer step:
A, take poor unstrained spirits in sterile centrifugation tube, add the sterile PBS buffer of sterile glass beads and cell cultures, whirlpool concussion 4 ~ 6min, centrifugal 5 ~ 7min in whizzer, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add sterile PBS buffer, whirlpool concussion 3 ~ 4min, centrifugal 5 ~ 7min in whizzer, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 150 ~ 255ml;
D, merge the elutriant of above-mentioned a, b, c, centrifugal 6 ~ 8min in whizzer, collect supernatant liquor and bacterium mud;
2) vacuum-freeze-dry step:
A, the bacterium mud collected in step 1) is added hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:1 ~ 1:2, and mixing, is placed in ampoul tube, pre-freeze 6 ~ 8h under-80 ~ 4 DEG C of temperature condition;
B, by above-mentioned pre-freeze sample vacuum lyophilization 19-23h under-50 ~-40 DEG C of temperature condition, after freeze-drying terminates, detect Microbial survival rate, to obtain final product.
Above-mentioned treatment process, in step 1) a, sterile glass beads add-on is 3 ~ 5, diameter 6mm;
In step 1) a, b, the working parameter of whizzer is: 4 DEG C, 300 ~ 500rpm; In step 1) d, the working parameter of whizzer is: 4 DEG C, 6000 ~ 7000rpm;
In step 1), the compound method of PBS damping fluid is: by 8gNaCl, 0.2gKCl, 3.63gNa
2hPO
412H
2o, 0.24gKH
2pO
4dissolve in 1L distilled water, regulate pH to be 7.4, to obtain final product.
Above-mentioned treatment process, step 2) formula of hybrid protection agent is in a: the skimmed milk of 16 ~ 18%, the glucose of 8 ~ 10%, the glycerine of 2 ~ 3%;
Step 2) pre-freeze program is in a: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 3 ~ 5h;
Step 2) vacuum lyophilization program is in b: the dry parameters of trunk is-50 DEG C, 18 ~ 20h, and rear dry parameters is-40 DEG C, 1 ~ 3h.The dry parameters of preferred trunk is-50 DEG C, 19h, and rear dry parameters is-40 DEG C, 2h.
Particularly, the treatment process of a kind of freeze-drying preservation Zao Peizhong colony microorganism, step is as follows:
1) wash-out, transfer step:
A, take 10 ~ 15g grain unstrained spirits in sterile centrifugation tube, add the sterile glass beads of 3 ~ 5 6mm and the sterile PBS buffer of cell cultures, whirlpool concussion 4 ~ 6min, the centrifugal 5 ~ 7min of 300 ~ 500rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add sterile PBS buffer, whirlpool concussion 3 ~ 4min, the centrifugal 5 ~ 7min of 300 ~ 500rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 150 ~ 255ml;
D, merge the elutriant of above-mentioned a, b, c, with the centrifugal 6 ~ 8min of 6000rpm ~ 7000rpm in 4 DEG C of whizzers, collect supernatant liquor and bacterium mud;
2) vacuum-freeze-dry step:
A, the bacterium mud collected is added hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:1 ~ 1:2, and mixing, is placed in ampoul tube, carries out following pre-freeze program: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 3-5h; The formula of described hybrid protection agent is: the skimmed milk of 16 ~ 18%, the glucose of 8 ~ 10%, 2 ~ 3% glycerine;
B, above-mentioned pre-freeze sample is carried out vacuum lyophilization, time of drying is 19 ~ 23h, and the dry parameters of trunk is-50 DEG C, 18 ~ 20h, and rear dry parameters is-40 DEG C, 1 ~ 3h;
After c, drying terminate, use plate dilution method, detect Microbial survival rate, to obtain final product.
For detecting the feasible of this technological method, analyze poor unstrained spirits microorganism rate of loss in elution process, concrete grammar is as follows:
Microorganism detection in a, former poor unstrained spirits: take 10g grain unstrained spirits, add in the triangular flask containing 90ml sterilized water, shaking table concussion 30min, uses plate dilution method to detect kind and the quantity of contained bacterium, fungi in former poor unstrained spirits.
B, wash-out, transfer step obtain the detection of microorganism in bacterium mud: bacterium mud step 1) collected, adds in the triangular flask containing 100ml sterilized water, use plate dilution method to detect kind and the quantity of bacterium, fungi contained by bacterium mud.
C, wash-out, transfer step remain the detection of microorganism in poor unstrained spirits: by residue complete for step 1) wash-out grain unstrained spirits, add in the triangular flask containing 90ml sterilized water, use plate dilution method to detect kind and the quantity of contained bacterium, fungi in the poor unstrained spirits of residue.
D, wash-out, transfer step obtain the detection of microorganism in supernatant liquor: step 1) is collected the centrifuged supernatant after bacterium mud, use plate dilution method to detect bacterium contained by it, the kind of fungi and quantity.
The microbe population of method of calculation microbe population/former poor unstrained spirits contained by the complete residue grain unstrained spirits of: wash-out of the microorganism rate of loss of poor unstrained spirits in above-mentioned elution process;
In above-mentioned centrifugal process, the method for calculation of the microorganism rate of loss of poor unstrained spirits are: the microbe population of the microbe population/former poor unstrained spirits of centrifuged supernatant after collection bacterium mud;
The two rate of loss sum is total microorganism rate of loss of this pretreatment process.
In above-mentioned each microorganism detection, when using plate dilution method to detect microorganism, each extent of dilution all repeats 3 times; Bacteria culture medium used is broth culture, and its composition is: sodium-chlor 10%, peptone 10%, extractum carnis 5%, agar 20%, pH are 7.0, and microbial culture temperature is 37 DEG C, time 24h; Fungi substratum is YEPD substratum, and its composition is: glucose 10%, peptone 10%, yeast extract paste 5%, agar 20%, pH nature, culture temperature is 28 DEG C, time 48h.
Poor unstrained spirits uses cell cultures damping fluid used to carry out repeatedly vortex oscillation, wash-out by the present invention, centrifugation grain unstrained spirits and microorganism, result shows: the rate of loss of bacterium and fungi total amount is within 2.0%, the rate of loss of single strain is within 2.5%, and the bacterium mud collected has biological activity, microorganism structure is consistent with former poor unstrained spirits.The bacterium mud of collection is carried out lyophilize, and directly poor unstrained spirits is carried out compared with freeze-drying preservation, not only reducing the cryodesiccated time, also improving the survival rate of microorganism.Therefore, adopt treatment process of the present invention, both maintained Zao Peizhong colony microbic activity, and rate of loss is low, Microbial survival rate is high, be also convenient to later stage vacuum melting-sealed preservation and extend the preservation time.This treatment process also can be widely used in other for being separated, collecting colony microorganism and keep bioactive purposes, for colony's rate of loss analysis of Analysis of Microbial Diversity provides important evidence.
Concrete embodiment
Embodiment 1:
A treatment process for freeze-drying preservation Zao Peizhong colony microorganism, step is as follows:
1) wash-out, transfer step:
A, take 10g grain unstrained spirits in sterile centrifugation tube, add the sterile PBS buffer of the sterile glass beads of 3 6mm, 50ml cell cultures, whirlpool concussion 4min, with the centrifugal 5min of 500rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add 40ml sterile PBS buffer, whirlpool concussion 3min, with the centrifugal 5min of 500rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, and the 1st time sterile PBS buffer consumption is 40ml, the 2nd 20ml, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 150ml;
D, merge the elutriant of above-mentioned a, b, c, amount to 150ml, with the centrifugal 7min of 6500rpm in 4 DEG C of whizzers, collect supernatant liquor and bacterium mud;
Described PBS buffer method is: by 8gNaCl, 0.2gKCl, 3.63gNa
2hPO
4.12H
2o, 0.24gKH
2pO
4dissolve in 1L distilled water, regulate PH to be 7.4;
2) vacuum-freeze-dry step:
A, the bacterium mud collected is added hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:1, and mixing, is placed in ampoul tube, carries out following pre-freeze program: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 3h; The formula of described hybrid protection agent is: the skimmed milk of 16%, the glucose of 8%, 2% glycerine;
B, above-mentioned pre-freeze sample is carried out vacuum lyophilization, time of drying is 21h, and the dry parameters of trunk is-50 DEG C, 19h, and rear dry parameters is-40 DEG C, 2h;
After c, drying terminate, use plate dilution method, detect Microbial survival rate, to obtain final product.
In addition, take 10g grain unstrained spirits 2 parts, a utilization plate dilution method, detect kind and the quantity of contained bacterium, fungi in former poor unstrained spirits; Other 1 part with above-mentioned step 1) elution process, by collect centrifugal after the complete poor unstrained spirits of supernatant liquor, bacterium mud and wash-out, use plate dilution method, detect the microorganism structure of residue poor unstrained spirits, collection bacterium mud and supernatant liquor.Take 10g grain unstrained spirits, according to step 2 with method) method carry out freeze-drying, obtain dry poor unstrained spirits, use plate dilution method, detect microorganism structure.
After the analytical results of grain unstrained spirits microorganism rate of loss in wash-out transfer process and lyophilize, the comparing result of Microbial survival rate is as follows:
The poor unstrained spirits of table 1 microorganism rate of loss in wash-out transfer process is analyzed
After table 2 lyophilize, the Microbial survival rate of bacterium mud and poor unstrained spirits contrasts
Embodiment 2:
A treatment process for freeze-drying preservation Zao Peizhong colony microorganism, step is as follows:
1) wash-out, transfer step:
A, take 12g grain unstrained spirits in sterile centrifugation tube, add the sterile PBS buffer of the sterile glass beads of 4 6mm, 60ml cell cultures, whirlpool concussion 5min, with the centrifugal 6min of 400rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add 50ml sterile PBS buffer, whirlpool concussion 3.5min, with the centrifugal 6min of 400rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, and the 1st time sterile PBS buffer consumption is 50ml, the 2nd 40ml, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 200ml;
D, merge the elutriant of above-mentioned a, b, c, amount to 200ml, with the centrifugal 6min of 7000rpm in 4 DEG C of whizzers, collect supernatant liquor and bacterium mud;
Described PBS buffer method is: by 8gNaCl, 0.2gKCl, 3.63gNa
2hPO
4.12H
2o, 0.24gKH
2pO
4dissolve in 1L distilled water, regulate PH to be 7.4;
2) vacuum-freeze-dry step:
A, the bacterium mud collected is added hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:1.5, and mixing, is placed in ampoul tube, carries out following pre-freeze program: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 4h; The formula of described hybrid protection agent is: the skimmed milk of 17%, the glucose of 9%, 2.5% glycerine;
B, above-mentioned pre-freeze sample is carried out vacuum lyophilization, time of drying is 19h, and the dry parameters of trunk is-50 DEG C, 18h, and rear dry parameters is-40 DEG C, 1h;
After c, drying terminate, use plate dilution method, detect Microbial survival rate, to obtain final product.
In addition, take 10g grain unstrained spirits, use plate dilution method, detect kind and the quantity of contained bacterium, fungi in former poor unstrained spirits; Take 12g grain unstrained spirits, with above-mentioned step 1) elution process, by collect centrifugal after the complete poor unstrained spirits of supernatant liquor, bacterium mud and wash-out, use plate dilution method, detect the microorganism structure of residue poor unstrained spirits, collection bacterium mud and supernatant liquor.Take 12g grain unstrained spirits, according to step 2 with method) method carry out freeze-drying, obtain dry poor unstrained spirits, use plate dilution method, detect microorganism structure.
After the analytical results of grain unstrained spirits microorganism rate of loss in wash-out transfer process and lyophilize, the comparing result of Microbial survival rate is as follows:
The poor unstrained spirits of table 3 microorganism rate of loss in wash-out transfer process is analyzed
After table 4 lyophilize, the Microbial survival rate of bacterium mud and poor unstrained spirits contrasts
Embodiment 3
A treatment process for freeze-drying preservation Zao Peizhong colony microorganism, step is as follows:
1) wash-out, transfer step:
A, take 15g grain unstrained spirits in sterile centrifugation tube, add the sterile PBS buffer of the sterile glass beads of 5 6mm, 75ml cell cultures, whirlpool concussion 6min, with the centrifugal 7min of 300rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add 75ml sterile PBS buffer, whirlpool concussion 4min, with the centrifugal 7min of 300rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, and the 1st time sterile PBS buffer consumption is 60ml, the 2nd 45ml, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 255ml;
D, merge the elutriant of above-mentioned a, b, c, amount to 255ml, with the centrifugal 8min of 6000rpm in 4 DEG C of whizzers, collect supernatant liquor and bacterium mud;
Described PBS buffer method is: by 8gNaCl, 0.2gKCl, 3.63gNa
2hPO
4.12H
2o, 0.24gKH
2pO
4dissolve in 1L distilled water, regulate PH to be 7.4;
2) vacuum-freeze-dry step:
A, the bacterium mud collected is added hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:2, and mixing, is placed in ampoul tube, carries out following pre-freeze program: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 5h; The formula of described hybrid protection agent is: the skimmed milk of 18%, the glucose of 10%, 3% glycerine;
B, above-mentioned pre-freeze sample is carried out vacuum lyophilization, time of drying is 23h, and the dry parameters of trunk is-50 DEG C, 20h, and rear dry parameters is-40 DEG C, 3h;
After c, drying terminate, use plate dilution method, detect Microbial survival rate, to obtain final product.
In addition, take 10g grain unstrained spirits, use plate dilution method, detect kind and the quantity of contained bacterium, fungi in former poor unstrained spirits; Take 15g grain unstrained spirits, with above-mentioned step 1) elution process, by collect centrifugal after the complete poor unstrained spirits of supernatant liquor, bacterium mud and wash-out, use plate dilution method, detect the microorganism structure of residue poor unstrained spirits, collection bacterium mud and supernatant liquor.Take 15g grain unstrained spirits, according to step 2 with method) method carry out freeze-drying, obtain dry poor unstrained spirits, use plate dilution method, detect microorganism structure.
After the analytical results of grain unstrained spirits microorganism rate of loss in wash-out transfer process and lyophilize, the comparing result of Microbial survival rate is as follows:
The poor unstrained spirits of table 5 microorganism rate of loss in wash-out transfer process is analyzed
After table 6 lyophilize, the Microbial survival rate of bacterium mud and poor unstrained spirits contrasts
Claims (4)
1. a treatment process for freeze-drying preservation Zao Peizhong colony microorganism, step is as follows:
1) wash-out, transfer step:
A, take poor unstrained spirits in sterile centrifugation tube, add the sterile PBS buffer of sterile glass beads and cell cultures, whirlpool concussion 4 ~ 6min, centrifugal 5 ~ 7min in whizzer, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add sterile PBS buffer, whirlpool concussion 3 ~ 4min, centrifugal 5 ~ 7min in whizzer, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 150 ~ 255ml;
D, merge the elutriant of above-mentioned a, b, c, centrifugal 6 ~ 8min in whizzer, collect supernatant liquor and bacterium mud;
2) vacuum-freeze-dry step:
A, by step 1) in the bacterium mud collected add hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:1 ~ 1:2, and mixing, is placed in ampoul tube, pre-freeze 6 ~ 8h under-80 ~ 4 DEG C of temperature condition respectively;
B, by pre-freeze sample vacuum lyophilization 19 ~ 23h under-50 ~-40 DEG C of temperature condition, after freeze-drying terminates, detect Microbial survival rate, to obtain final product;
Step 1) in the compound method of PBS damping fluid be: 8gNaCl, 0.2gKCl, 3.63gNa2HPO412H2O, 0.24gKH2PO4 are dissolved in 1L distilled water, regulate pH to be 7.4, to obtain final product;
Step 1) working parameter of whizzer is in a, b: 4 DEG C, 300 ~ 500rpm; Step 1) working parameter of whizzer is in d: 4 DEG C, 6000 ~ 7000rpm;
Step 2) formula of hybrid protection agent is in a: the skimmed milk of 16 ~ 18%, the glucose of 8 ~ 10%, the glycerine of 2 ~ 3%.
2. treatment process as claimed in claim 1, is characterized in that: step 1) sterile glass beads add-on is 3 ~ 5 in a, diameter 6mm.
3. treatment process as claimed in claim 1, is characterized in that: step 2) pre-freeze program is in a: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 3 ~ 5h;
Step 2) vacuum lyophilization program is in b: the dry parameters of trunk is-50 DEG C, 18 ~ 20h, and rear dry parameters is-40 DEG C, 1 ~ 3h.
4. the treatment process as described in as arbitrary in claim 1-3, is characterized in that: step is as follows:
1) wash-out, transfer step:
A, take 10 ~ 15g grain unstrained spirits in sterile centrifugation tube, add the sterile glass beads of 3 ~ 5 6mm and the sterile PBS buffer of cell cultures, whirlpool concussion 4 ~ 6min, the centrifugal 5 ~ 7min of 300 ~ 500rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
B, by the residue of wash-out in step a grain unstrained spirits add sterile PBS buffer, whirlpool concussion 3 ~ 4min, the centrifugal 5 ~ 7min of 300 ~ 500rpm in 4 DEG C of whizzers, collect elutriant, for subsequent use;
C, above-mentioned steps b repeat 2 times, amount to concussion wash-out grain unstrained spirits 4 times, amount to and add sterile PBS buffer 150 ~ 255ml;
D, merge the elutriant of above-mentioned a, b, c, with the centrifugal 6 ~ 8min of 6000rpm ~ 7000rpm in 4 DEG C of whizzers, collect supernatant liquor and bacterium mud;
2) vacuum-freeze-dry step:
A, the bacterium mud collected is added hybrid protection agent, the mass volume ratio of bacterium mud and hybrid protection agent is 1:1 ~ 1:2, and mixing, is placed in ampoul tube, carries out following pre-freeze program: at 4 DEG C of refrigerations 1h ,-20 DEG C of freezing 2h ,-80 DEG C of freezing 3 ~ 5h; The formula of described hybrid protection agent is: the skimmed milk of 16 ~ 18%, the glucose of 8 ~ 10%, 2 ~ 3% glycerine;
B, pre-freeze sample is carried out vacuum lyophilization, time of drying is 19 ~ 23h, and the dry parameters of trunk is-50 DEG C, 18 ~ 20h, and rear dry parameters is-40 DEG C, 1 ~ 3h;
After c, drying terminate, use plate dilution method, detect Microbial survival rate, to obtain final product.
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| 窑池微生态资源库的建立、管理及菌种保藏;张丽莺;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20070315(第3期);B024-23 * |
| 窑泥糟醅发酵过程微生物多态性特征;张肖克等;《酿酒科技》;20061231(第1期);65-68 * |
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