CN115369039A - Freeze-drying preservation method for quantitative working quality control strains - Google Patents
Freeze-drying preservation method for quantitative working quality control strains Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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Abstract
The invention provides a freeze-drying preservation method of quantitative working quality control strains, which comprises the following steps: s1, culturing microorganisms to prepare a bacterial suspension; s2, carrying out viable bacteria counting treatment on the bacterial suspension; s3, freeze-drying the bacterial suspension; and S4, dissolving the freeze-dried substance for use. The invention adopts a one-time freeze-drying mode for operation, simplifies the operation procedure, shortens the exposure time of the freeze-dried substance at normal temperature, reduces the loss of microorganisms in the whole process, and prevents the activity of the microorganisms from being reduced due to overlong subpackaging time after freeze-drying is finished. The related freeze-drying protective solution can be used as a solid agent, has larger pores and provides better support for freeze-dried substances; providing a better micro-ecological environment for microorganisms; and after freeze-drying is finished, the plugging can be directly carried out in a freeze-drying machine without additional split charging, so that the water content of the freeze-dried microbial substance is kept at an extremely low level.
Description
Technical Field
The invention relates to the field of microbial preservation, in particular to a freeze-drying preservation method of a quantitative working quality control strain.
Background
In the food and pharmaceutical industries, many inspection projects (such as sensitivity tests, growth promotion tests, sterility tests, culture medium suitability tests, bacteria control tests, bacterial challenges, disinfection efficacy verification, neutralizer verification, positive controls, teaching training and the like) use specific microorganism suspensions with certain content ranges. For example, the content of the added microorganism is required to be less than 100cfu when the suitability of the culture medium is checked, and the requirement of the disinfection effectiveness verification carrier method is that the content of the microorganism on the carrier is 5 × 105cfu-5 × 106cfu. However, the bacterial content in the bacterial suspension prepared by manual dilution cannot be stable, for example, the content of the bacterial suspension prepared by the method is required to be less than 100cfu/0.1ml, and the difference between the two dilutions prepared in two consecutive times is large, for example, 10cfu/0.1ml is needed once, and 90cfu/0.1ml is needed once, which causes a large deviation between the test results. Even the content of the prepared bacterial suspension may exceed the specified range, so that the detection data is not credible, the detection result needs to be subjected to waste treatment, deviation evaluation is needed, and detection is carried out again, so that time and labor are wasted, and the use of raw materials or the delivery of finished products is delayed.
The bacterial suspension prepared by the product has accurate bacterial content range, can reduce the error of each test and greatly increase the success rate of the test.
Similar products on the market are all particles with certain shapes and are filled in a common penicillin bottle, and the products in the mode must be lyophilized by adopting a mold and then subpackaged. This process can make the freeze-drying thing moisture absorption certainly, makes the freeze-drying thing water content increase, influences the activity of microorganism in the freeze-drying thing, and transient high temperature environment can make the freeze-drying thing liquefaction or atrophy promptly, if want to avoid the negative effect that brings when partial shipment, needs special customization closing device to guarantee aseptic nature and the aridity of partial shipment environment, perhaps needs to reform transform the freeze dryer, greatly increased the cost input of producing this type of product.
The freeze-dried quality control working strain needs to be dissolved by a small amount of dissolving liquid in the using process, if the dissolved quality control working strain contains too many nutrient substances, the stability of the number of bacteria and the accuracy of a test can be influenced, and the existing freeze-drying maintaining method is too complex to operate when in use, long-time freeze-dried substances are exposed at normal temperature to cause the loss of microorganisms, so that the subpackaging time is too long, and the activity of the microorganisms is easily reduced.
Disclosure of Invention
In order to make up for the defects, the invention provides a freeze-drying preservation method of a quantitative working quality control strain, aiming at improving the quality of a freeze-drying liquid, reducing pollution, loss and moisture, realizing the protection of a bacterial suspension by a freeze-drying protection liquid and the like through simple steps.
The invention is realized by the following steps:
a freeze-drying preservation method of quantitative working quality control strains comprises the following steps:
s1, culturing microorganisms to prepare a bacterial suspension: culturing single microorganism under appropriate conditions, eluting thallus Porphyrae with sterile eluate, and mixing; diluting the mixture into bacterial suspension with required concentration by using sterile diluent;
s2, carrying out viable bacteria counting treatment on the bacterial suspension: dyeing the microorganism in the bacterial suspension by using a methylene blue dye, dripping the dyed bacterial suspension in a counting plate for viable bacteria counting, and determining the concentration of the microorganism in the suspension;
s3, freeze-drying the bacterial suspension: after the number of the viable bacteria in the S2 is calculated, diluting the original bacteria suspension to a proper concentration, mixing the diluted bacteria suspension with a freeze-drying protective solution 1:1, subpackaging the mixture into a special penicillin bottle, and freeze-drying the mixture to finally prepare a freeze-dried substance with an accurate microorganism concentration range;
s4, dissolving the freeze-dried substance for use: when in use, sodium chloride peptone solution with the pH value of 7.0 is adopted to dissolve the freeze-dried substance, and bacterial suspension with precise microorganism concentration range is prepared after dissolution and then is directly used for various tests.
In one embodiment of the present invention, the preparation steps of the sterile eluent in S1 are as follows:
adding distilled water or deionized water into the eluate dry powder, stirring, heating, boiling to dissolve completely, packaging into triangular flask, and sterilizing the prepared solution at 100-130 deg.C under high pressure for 15min; then the sterilized solution is cooled to 50 ℃ under the aseptic environment at normal temperature, and the prepared aseptic eluent is stored for one day at room temperature or for several days in a refrigerator and is ensured under the environment of being protected from light and 4 ℃.
In one embodiment of the present invention, the sterile diluent in S1 is prepared by the following steps:
s101, preparing distilled water in a distillation mode, and storing the distilled water in a sealed mode for later use;
s102, preparation of 0.85% physiological saline: weighing 8.5g of sodium chloride, dissolving the sodium chloride in 1000ml of distilled water, and uniformly stirring the sodium chloride by using a glass stirring rod to quickly and uniformly dissolve the sodium chloride;
s103, sterilization of physiological saline: sterilizing the prepared normal saline at 121 ℃ for 15min, then placing the normal saline in an aseptic cooling area for natural cooling, and ensuring that the pressure of a sterilization pot is reduced to 0 when the normal saline is taken out;
and S104, refrigerating and storing the physiological saline.
In an embodiment of the present invention, in the viable bacteria counting process in S2, a special staining method is adopted to stain viable bacteria in a bacterial suspension, and counting is performed under a high power microscope, specifically including the following steps:
s201, bacterial suspension dilution: diluting the bacterial suspension to a certain concentration, and preferably, the total bacterial count on the cell counting plate is about 500-1000 cfu;
s202, viable bacteria dyeing: taking 0.9ml of methylene blue dye solution, taking 0.1ml of diluted bacterium solution in S201, uniformly mixing and dyeing in a reagent tube for 10-15 minutes;
s203, counting the plate by a counting plate: covering a cover glass on a counting chamber, shaking the dyed bacterial suspension, sucking a small amount of bacterial suspension by using a pipettor, injecting the bacterial suspension into a counting plate, filling the counting area with the bacterial suspension, standing for a moment, and starting counting after the thalli are naturally settled and stabilized;
s204, counting: placing a blood counting chamber on a microscope objective table, observing and counting by using a microscope, randomly taking 3-5 middle lattices, counting the number of the undyed viable bacteria, repeating for 3 times, and calculating the average number of the viable bacteria in each middle lattice;
s205, conversion: and converting the obtained viable bacteria content in each middle lattice into the viable bacteria content in each milliliter of the dropped bacterial suspension according to the volume size of each middle lattice marked in the description of the counting plate.
In an embodiment of the present invention, the scaling method in S205 is as follows:
calculating the bacteria content in the bacterial suspension according to the bacteria content of a final required product, wherein if the bacteria content in a penicillin bottle is 0.1ml, the requirement of the bacteria content is 100-1000cfu, and a 500cfu value is selected as a target value, the bacteria content in the prepared freeze-drying liquid is required to be about 500cfu/0.1ml, namely 5000cfu/ml, and when the freeze-drying liquid is prepared, the bacteria content of the bacterial suspension and the freeze-drying protective liquid need to be 1:1, mixing, so that the bacterium content in the bacterium suspension is required to be about 10000cfu/ml; and the calculation can be carried out according to the idea when preparing the freeze-dried products with other bacteria contents.
In an embodiment of the present invention, when the bacterial suspension in S3 is subjected to lyophilization treatment, a lyophilization protection solution is added to the bacterial suspension, and a ratio of the lyophilization protection solution to the bacterial suspension is 1:1, fully and uniformly mixing, namely after the freeze-drying protective solution is added, mixing and stirring by adopting a glass stirring rod, and improving the uniform mixing of the freeze-drying protective solution and the bacterial suspension.
In an embodiment of the present invention, the ratio of the lyophilized protection solution is as follows:
1-5% of Arabic gum, 0-3% of bovine serum, 0-5% of lactic acid, 1-2% of glycerol, 2-5% of glycine, 3-8% of arginine, 1-5% of polyethylene glycol, 1-3% of D-sodium erythorbate and 1-10% of skim milk.
In an embodiment of the present invention, the formulation of the lyoprotectant is further selected as follows:
2% of Arabic gum, 1% of bovine serum, 2% of lactic acid, 1% of glycerol, 2% of glycine, 5% of arginine, 1% of polyethylene glycol, 2% of D-sodium erythorbate and 5% of skim milk;
the preparation process of the freeze-drying protective solution is as follows, taking 100ml of freeze-drying protective solution as an example;
s301, measuring 100ml of purified water in a beaker, and heating to boil;
s302, sequentially adding 2g of Arabic gum, 1g of bovine serum, 2g of lactic acid, 1g of glycerol, 2g of glycine, 5g of arginine, 1g of polyethylene glycol and 2g of D-sodium erythorbate into a beaker, and adding the next reagent after the former reagent is dissolved;
s303, adjusting the pH value to 7.2 +/-0.5 by using 1M sodium hydroxide solution, finally adding 5g of skim milk, and fully stirring and uniformly mixing;
s304, filling the mixture into a 100ml infusion bottle or a wide-mouth bottle, carrying out damp-heat sterilization at 116 ℃ for 20 minutes, and placing the mixture in a low-temperature and dark environment at 2-8 ℃ for later use after the sterilization is finished.
In an embodiment of the present invention, the lyophilization process of the lyophilization solution in S3 is as follows:
mixing the prepared bacterial suspension with the prepared freeze-drying protective solution 1:1, mixing to prepare a freeze-dried solution, subpackaging the freeze-dried solution into special penicillin bottles in an amount of 0.05ml-0.5ml per bottle, performing semi-tamponade packaging by using a rubber plug, placing the penicillin bottles subpackaged with the freeze-dried solution into a freeze dryer, pre-freezing for 2 hours at-40 ℃, drying, raising the temperature to 25 ℃, taking the solution for 18-24 hours, and judging that the pressure rise speed in the freeze dryer is less than 2pa/10min after the drying is finished; and (4) after the freeze-drying is finished, performing a tamponade operation on the product, and storing at the temperature of minus 20 ℃.
The invention has the beneficial effects that: the invention provides a freeze-drying preservation method of quantitative working quality control strains, which is operated by adopting a one-time freeze-drying mode, simplifies the operation procedure, shortens the exposure time of a freeze-dried substance at normal temperature, and can be preserved at low temperature after the freeze-dried substance is taken out of a freeze dryer, thereby greatly reducing the loss of microorganisms in the whole process and preventing the activity of the microorganisms from being reduced due to overlong subpackaging time after the freeze-drying is finished. In the process, the split charging is not needed, the risk of contaminating the mixed bacteria is reduced, and the purity of the product is further improved;
the related freeze-drying protective solution consists of several common substances in the market, and the more targeted substances are used: the Arabic gum can be used as a solid preparation, can form larger pores compared with gelatin, and provides better support for a freeze-dried substance; the bovine serum contains various trace elements and can provide a better micro-ecological environment for microorganisms; glycerin, which can prevent the early formation of ice crystals in the early stage of freeze-drying; lactic acid, glycine, arginine and polyethylene glycol, and can prevent the drastic change of pH in the freeze-drying process after mixing; d-sodium erythorbate as antioxidant for prolonging activity of microorganism surface substance; the skim milk can wrap microorganisms, so that active ingredients such as protease on the surfaces of the microorganisms are prevented from being damaged due to severe environmental changes during freeze drying.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic flow chart of steps provided by an embodiment of the present invention;
FIG. 2 is a schematic flow chart illustrating the steps for preparing a sterile diluent according to an embodiment of the present invention;
FIG. 3 is a schematic flow chart of a viable bacteria counting step according to an embodiment of the present invention;
FIG. 4 is a schematic flow chart of the steps for preparing the lyophilized protection solution according to the embodiment of the present invention;
FIG. 5 is a schematic diagram showing the relationship between the storage time at-20 ℃ and the bacteria content of various microbial lyophilized products provided by the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive efforts based on the embodiments of the present invention, are within the scope of protection of the present invention.
Examples
Referring to fig. 1-4, the present invention provides a technical solution: a freeze-drying preservation method for quantitative working quality control strains comprises the following steps:
s1, culturing microorganisms to prepare a bacterial suspension: culturing single microorganism under appropriate conditions, eluting thallus Porphyrae with sterile eluate, and mixing; diluting the mixture into bacterial suspension with required concentration by using sterile diluent;
s2, carrying out viable bacteria counting treatment on the bacterial suspension: dyeing the microorganism in the bacterial suspension by using a methylene blue dye, dripping the dyed bacterial suspension in a counting plate for viable bacteria counting, and determining the concentration of the microorganism in the suspension;
s3, freeze-drying the bacterial suspension: after the number of the viable bacteria in the S2 is calculated, diluting the original bacteria suspension to a proper concentration, mixing the diluted bacteria suspension with a freeze-drying protective solution 1:1, subpackaging the mixture into a special penicillin bottle, and freeze-drying the mixture to finally prepare a freeze-dried substance with an accurate microorganism concentration range;
s4, dissolving the freeze-dried substance for use: when in use, sodium chloride peptone solution with the pH value of 7.0 is adopted to dissolve the freeze-dried substance, and bacterial suspension with precise microorganism concentration range is prepared after dissolution and then is directly used for various tests.
In one embodiment of the present invention, the preparation steps of the sterile eluent in S1 are as follows:
adding distilled water or deionized water into the eluate dry powder, stirring, heating, boiling to dissolve completely, packaging into triangular flask, and sterilizing the prepared solution at 100-130 deg.C under high pressure for 15min; then cooling the sterilized solution to 50 ℃ at normal temperature in a sterile environment, storing the prepared sterile eluent at room temperature for one day or in a refrigerator for several days, and ensuring the sterilized solution to be protected from light and at the temperature of 4 ℃; in order to realize elution treatment of the lawn, effectively realize preparation treatment of the eluent, and prepare the eluent under an aseptic environment, thereby ensuring the sterility of the eluent.
In one embodiment of the present invention, the sterile diluent in S1 is prepared by the following steps:
s101, preparing distilled water in a distillation mode, and storing the distilled water in a sealed mode for later use;
s102, preparation of 0.85% physiological saline: weighing 8.5g of sodium chloride, dissolving the sodium chloride in 1000ml of distilled water, and uniformly stirring the sodium chloride by using a glass stirring rod to quickly and uniformly dissolve the sodium chloride;
s103, sterilization of physiological saline: sterilizing the prepared normal saline at 121 ℃ for 15min, then placing the normal saline in an aseptic cooling area for natural cooling, and ensuring that the pressure of a sterilization pot is reduced to 0 when the normal saline is taken out;
s104, refrigerating and storing the physiological saline;
in order to prepare the microorganism into the bacterial suspension, the diluent is effectively diluted, in order to prevent pollution, the diluent is prepared in an aseptic environment, and after preparation, high-temperature sterilization is carried out, so that the sterility is improved, and the pollution is prevented.
In an embodiment of the present invention, in the viable bacteria counting process in S2, a special staining method is adopted to stain viable bacteria in a bacterial suspension, and counting is performed under a high power microscope, specifically including the following steps:
s201, bacterial suspension dilution: diluting the bacterial suspension to a certain concentration, and preferably, the total bacterial count on the cell counting plate is about 500-1000 cfu;
s202, viable bacteria dyeing: taking 0.9ml of methylene blue dye solution, taking 0.1ml of diluted bacterium solution in S201, uniformly mixing and dyeing in a reagent tube for 10-15 minutes;
s203, counting the counting plate: covering a cover glass on a counting chamber, shaking the dyed bacterial suspension, sucking a small amount of bacterial suspension by using a pipettor, injecting the bacterial suspension into a counting plate, filling the counting area with the bacterial suspension, standing for a moment, and starting counting after the thalli are naturally settled and stabilized;
s204, counting: placing a blood counting chamber on a microscope objective table, observing and counting by using a microscope, randomly taking 3-5 middle lattices, counting the number of the unstained viable bacteria, repeating for 3 times, and calculating the average number of the viable bacteria in each middle lattice;
s205, conversion: converting the obtained viable bacteria content in each middle lattice into the content of viable bacteria in each milliliter of bacterial suspension dropwise added according to the volume size of each middle lattice marked in the description of the counting plate;
in order to realize accurate counting treatment of viable bacteria and effective calculation and conversion treatment of the viable bacteria content in the bacterial suspension, the accurate content of the bacterial suspension can be obtained.
In an embodiment of the present invention, the scaling method in S205 is as follows:
calculating the bacteria content in the bacterial suspension according to the bacteria content of a final required product, wherein if the bacteria content in a penicillin bottle is 0.1ml, the requirement of the bacteria content is 100-1000cfu, and a 500cfu value is selected as a target value, the bacteria content in the prepared freeze-drying liquid is required to be about 500cfu/0.1ml, namely 5000cfu/ml, and when the freeze-drying liquid is prepared, the bacteria content of the bacterial suspension and the freeze-drying protective liquid need to be 1:1, mixing, so that the bacterium content in the bacterium suspension is required to be about 10000cfu/ml; and the freeze-dried substance with other bacteria content can be calculated according to the thought; in order to accurately convert the content of the bacterial suspension, the examples were used to perform accurate calculation.
In an embodiment of the present invention, when the bacterial suspension in S3 is subjected to lyophilization treatment, a lyophilization protection solution is added to the bacterial suspension, and a ratio of the lyophilization protection solution to the bacterial suspension is 1:1, fully and uniformly mixing, namely after the freeze-drying protective solution is added, mixing and stirring by adopting a glass stirring rod, and improving the uniform mixing of the freeze-drying protective solution and the bacterial suspension; in order to realize the protection and freeze-drying treatment of the bacterial suspension, the protection and freeze-drying treatment is carried out by adding a freeze-drying protection solution.
In an embodiment of the present invention, the ratio of the lyoprotectant is as follows:
1-5% of Arabic gum, 0-3% of bovine serum, 0-5% of lactic acid, 1-2% of glycerol, 2-5% of glycine, 3-8% of arginine, 1-5% of polyethylene glycol, 1-3% of D-sodium erythorbate and 1-10% of skim milk; the selection and configuration setting of materials are performed on the freeze-drying protection liquid.
In an embodiment of the present invention, the formulation of the lyoprotectant is further selected as follows:
2% of Arabic gum, 1% of bovine serum, 2% of lactic acid, 1% of glycerol, 2% of glycine, 5% of arginine, 1% of polyethylene glycol, 2% of D-sodium erythorbate and 5% of skim milk;
the preparation process of the freeze-drying protective solution is as follows, taking 100ml of freeze-drying protective solution as an example;
s301, measuring 100ml of purified water in a beaker, and heating to boil;
s302, sequentially adding 2g of Arabic gum, 1g of bovine serum, 2g of lactic acid, 1g of glycerol, 2g of glycine, 5g of arginine, 1g of polyethylene glycol and 2g of D-sodium erythorbate into a beaker, and adding the next reagent after the former reagent is dissolved;
s303, adjusting the pH value to 7.2 +/-0.5 by using 1M sodium hydroxide solution, finally adding 5g of skim milk, and fully stirring and uniformly mixing;
s304, filling the mixture into a 100ml infusion bottle or a wide-mouth bottle, carrying out damp-heat sterilization at 116 ℃ for 20 minutes, and placing the mixture in a low-temperature and light-proof environment at 2-8 ℃ for later use after the sterilization is finished;
in order to realize accurate proportioning treatment on the freeze-drying protective solution and realize configuration treatment on the freeze-drying protective solution.
In an embodiment of the present invention, the lyophilization process of the lyophilization liquid in S3 is as follows:
mixing the prepared bacterial suspension with the prepared freeze-drying protective solution 1:1, mixing to prepare a freeze-dried solution, subpackaging the freeze-dried solution into special penicillin bottles in an amount of 0.05ml-0.5ml per bottle, performing semi-tamponade packaging by using a rubber plug, placing the penicillin bottles subpackaged with the freeze-dried solution into a freeze dryer, pre-freezing for 2 hours at-40 ℃, drying, raising the temperature to 25 ℃, taking the solution for 18-24 hours, and judging that the pressure rise speed in the freeze dryer is less than 2pa/10min after the drying is finished; after the freeze-drying is finished, performing a tamponade operation on the product, and storing at-20 ℃; in order to realize the freeze-drying treatment of the freeze-drying liquid, the freeze-drying liquid can be directly plugged in a freeze-drying machine after being freeze-dried, the split charging is not needed, the water content of the freeze-dried microbial substance is kept at an extremely low level, and the product quality is ensured.
Specifically, the freeze-drying preservation method of the quantitative working quality control strain comprises the following steps:
step one, adding distilled water or deionized water into eluent dry powder, stirring, heating, boiling until the eluent is completely dissolved, subpackaging triangular flasks, and sterilizing the prepared solution at 100-130 ℃ for 15min under high pressure; then cooling the sterilized solution to 50 ℃ at normal temperature in a sterile environment, storing the prepared sterile eluent at room temperature for one day or in a refrigerator for several days, and ensuring the sterilized solution to be protected from light and at the temperature of 4 ℃; in order to realize elution treatment of the lawn, effectively realize preparation treatment of the eluent and preparation under an aseptic environment, and ensure the sterility of the eluent;
the second step is that: preparing distilled water by means of distillation, and hermetically storing the distilled water for standby; preparation of 0.85% physiological saline: weighing 8.5g of sodium chloride, dissolving the sodium chloride in 1000ml of distilled water, and uniformly stirring the sodium chloride by using a glass stirring rod to quickly and uniformly dissolve the sodium chloride; sterilization of physiological saline: sterilizing the prepared normal saline at 121 ℃ for 15min, then placing the normal saline in an aseptic cooling area for natural cooling, and ensuring that the pressure of a sterilization pot is reduced to 0 when the normal saline is taken out; the normal saline is refrigerated for storage;
step three, culturing the microorganisms to prepare a bacterial suspension: culturing single microorganism under suitable conditions, eluting thallus Porphyrae with sterile eluate, and mixing; diluting the mixture into bacterial suspension with required concentration by using sterile diluent;
fourthly, viable bacteria counting treatment is carried out on the bacterial suspension: dyeing the microorganism in the bacterial suspension by using a methylene blue dye, dripping the dyed bacterial suspension in a counting plate for viable bacteria counting, and determining the concentration of the microorganism in the suspension;
fifthly, diluting the bacterial suspension: diluting the bacterial suspension to a certain concentration, and preferably, the total bacterial count on the cell counting plate is about 500-1000 cfu; taking 0.9ml of methylene blue dye solution, taking 0.1ml of diluted bacterium solution in S201, uniformly mixing and dyeing in a reagent tube for 10-15 minutes; the counting plate carries out counting treatment: covering a cover glass on a counting chamber, shaking the dyed bacterial suspension, sucking a small amount of bacterial suspension by using a pipettor, injecting the bacterial suspension into a counting plate, filling the counting area with the bacterial suspension, standing for a moment, and starting counting after the thalli are naturally settled and stabilized; placing a blood counting chamber on a microscope objective table, observing and counting by using a microscope, randomly taking 3-5 middle lattices, counting the number of the undyed viable bacteria, repeating for 3 times, and calculating the average number of the viable bacteria in each middle lattice; converting the obtained viable bacteria content in each middle lattice into the content of viable bacteria in each milliliter of bacterial suspension dropwise added according to the volume size of each middle lattice marked in the description of the counting plate;
and sixthly, freeze-drying the bacterial suspension: after the number of the viable bacteria in the S2 is calculated, diluting the original bacteria suspension to a proper concentration, mixing the diluted bacteria suspension with a freeze-drying protective solution 1:1, subpackaging the mixture into a special penicillin bottle, and freeze-drying the mixture to finally prepare a freeze-dried substance with an accurate microorganism concentration range;
step seven, preparing freeze-drying protective solution: weighing 100ml of purified water in a beaker, and heating to boil; sequentially adding 2g of Arabic gum, 1g of bovine serum, 2g of lactic acid, 1g of glycerol, 2g of glycine, 5g of arginine, 1g of polyethylene glycol and 2g of D-sodium erythorbate into a beaker, and adding the next reagent after the former reagent is dissolved; adjusting pH to 7.2 + -0.5 with 1M sodium hydroxide solution, adding skimmed milk 5g, stirring thoroughly; placing into 100ml infusion bottle or wide-mouth bottle, sterilizing at 116 deg.C for 20 min, and placing in low temperature and dark environment at 2-8 deg.C;
eighthly, preparing freeze-drying liquid, subpackaging the freeze-drying liquid into special penicillin bottles in an amount of 0.05ml to 0.5ml per bottle, performing semi-tamponade packaging by using a rubber plug, placing the penicillin bottles subpackaged with the freeze-drying liquid into a freeze dryer, pre-freezing for 2 hours at the temperature of minus 40 ℃, drying, increasing the temperature to 25 ℃, taking the penicillin bottles for 18 to 24 hours, and judging that the pressure increase speed in the freeze dryer is less than 2pa/10min after the drying is finished; after the freeze-drying is finished, performing a tamponade operation on the product, and storing at-20 ℃; in order to realize freeze-drying treatment of the freeze-dried liquid, and directly perform tamponade in a freeze-drying machine after freeze-drying is finished, additional split charging is not needed, the water content of a microorganism freeze-dried substance is kept at an extremely low level, and the product quality is ensured;
and ninth step, dissolving the freeze-dried substance for use: when in use, sodium chloride peptone solution with the pH value of 7.0 is adopted to dissolve the freeze-dried substance, and bacterial suspension containing precise microorganism concentration range is prepared after dissolution and then is directly used for various tests;
tenth step, counting the bacteria content of the freeze-dried product, taking the bacteria content of about 500cfu in the freeze-dried product as an example: taking out the freeze-dried product, dissolving the freeze-dried product with 1ml of sterile water, mixing uniformly, taking 0.1ml of bacterial suspension, carrying out plate counting, culturing under appropriate conditions, forming colonies by a single microorganism, and counting the number of the colonies, wherein if the average number of the colonies on the plate is 45 colonies, the freeze-dried product in the bottle contains 45cfu 10=450cfu.
According to the above-mentioned idea, after 1ml of sterile water is dissolved in the freeze-dried material with other concentration, the freeze-dried material can be undergone the process of ten times of gradient dilution so as to make the colony number on the final plate be 30-300, then the colony content in the freeze-dried material can be obtained by multiplying dilution times.
According to the method of the above invention, a plurality of microbial lyophilized products were prepared with a bacterial content of about 500cfu per vial of lyophilized product and stored at-20 ℃, and FIG. 5 shows the results after storage.
The present invention includes, but is not limited to, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, candida albicans, aspergillus niger, clostridium sporogenes, salmonella paratyphi B, bacillus subtilis var niger (Bacillus atrophaeus), etc., including most other bacterial propagules, bacterial spores, fungal mycelia, and fungal spores.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A freeze-drying preservation method for quantitative working quality control strains is characterized by comprising the following steps:
s1, culturing microorganisms to prepare a bacterial suspension: culturing single microorganism under suitable conditions, eluting thallus Porphyrae with sterile eluate, and mixing; diluting the mixture into bacterial suspension with required concentration by using sterile diluent;
s2, carrying out viable bacteria counting treatment on the bacterial suspension: dyeing the microorganism in the bacterial suspension by using a methylene blue dye, dripping the dyed bacterial suspension in a counting plate for viable bacteria counting, and determining the concentration of the microorganism in the suspension;
s3, freeze-drying the bacterial suspension: after the number of the viable bacteria in the S2 is calculated, diluting the original bacteria suspension to a proper concentration, mixing the diluted bacteria suspension with a freeze-drying protective solution 1:1, subpackaging the mixture into a special penicillin bottle, and freeze-drying the mixture to finally prepare a freeze-dried substance with an accurate microorganism concentration range;
s4, dissolving the freeze-dried substance for use: when in use, sodium chloride peptone solution with the pH value of 7.0 is adopted to dissolve the freeze-dried substance, and bacterial suspension with precise microorganism concentration range is prepared after dissolution and then is directly used for various tests.
2. The freeze-drying preservation method of quantitative working quality control strains according to claim 1, wherein the preparation steps of the sterile eluent in the S1 are as follows:
adding distilled water or deionized water into the dry powder of the eluate, stirring, heating, boiling to dissolve completely, packaging into triangular flask, and sterilizing the prepared solution at 100-130 deg.C under high pressure for 15min; then the sterilized solution is cooled to 50 ℃ under the aseptic environment at normal temperature, and the prepared aseptic eluent is stored for one day at room temperature or for several days in a refrigerator and is ensured under the environment of being protected from light and 4 ℃.
3. The method for freeze-drying preservation of quantitative working quality control strains according to claim 1, wherein the preparation of the sterile diluent in S1 comprises the following steps:
s101, preparing distilled water in a distillation mode, and storing the distilled water in a sealed mode for later use;
s102, preparation of 0.85% physiological saline: weighing 8.5g of sodium chloride, dissolving the sodium chloride in 1000ml of distilled water, and uniformly stirring the sodium chloride by using a glass stirring rod to quickly and uniformly dissolve the sodium chloride;
s103, sterilization of physiological saline: sterilizing the prepared normal saline at 121 ℃ for 15min, then placing the normal saline in an aseptic cooling area for natural cooling, and ensuring that the pressure of a sterilization pot is reduced to 0 when the normal saline is taken out;
and S104, refrigerating and storing the physiological saline.
4. The freeze-drying preservation method for quantitative working quality control strains according to claim 1, wherein the viable bacteria counting treatment in the S2 is to dye viable bacteria in a bacterial suspension by a special dyeing method and count the viable bacteria under a high power microscope, and the specific steps are as follows:
s201, bacterial suspension dilution: diluting the bacterial suspension to a certain concentration, and preferably, the total bacterial count on the cell counting plate is about 500-1000 cfu;
s202, viable bacteria dyeing: taking 0.9ml of methylene blue dye solution, taking 0.1ml of diluted bacterium solution in S201, uniformly mixing and dyeing in a reagent tube for 10-15 minutes;
s203, counting the plate by a counting plate: covering a cover glass on a counting chamber, shaking the dyed bacterial suspension, sucking a small amount of bacterial suspension by using a pipettor, injecting the bacterial suspension into a counting plate, filling the counting area with the bacterial suspension, standing for a moment, and starting counting after the thalli are naturally settled and stabilized;
s204, counting: placing a blood counting chamber on a microscope objective table, observing and counting by using a microscope, randomly taking 3-5 middle lattices, counting the number of the undyed viable bacteria, repeating for 3 times, and calculating the average number of the viable bacteria in each middle lattice;
s205, conversion: and converting the obtained viable bacteria content in each middle lattice into the viable bacteria content in each milliliter of the dropped bacterial suspension according to the volume size of each middle lattice marked in the description of the counting plate.
5. The method for freeze-drying preservation of quantitative working quality control strains according to claim 4, wherein the conversion method in S205 is as follows:
calculating the bacteria content in the bacterial suspension according to the bacteria content of a final required product, wherein if the bacteria content in a penicillin bottle is 0.1ml, the requirement of the bacteria content is 100-1000cfu, and a 500cfu value is selected as a target value, the bacteria content in the prepared freeze-drying liquid is required to be about 500cfu/0.1ml, namely 5000cfu/ml, and when the freeze-drying liquid is prepared, the bacteria content of the bacterial suspension and the freeze-drying protective liquid need to be 1:1, mixing, so that the bacterium content in the bacterium suspension is required to be about 10000cfu/ml; and the freeze-dried substance with other bacteria content can be calculated according to the thought.
6. The freeze-drying preservation method for quantitative working quality control strains according to claim 5, wherein when the bacterial suspension in S3 is subjected to freeze-drying treatment, a freeze-drying protective solution is added to the bacterial suspension, and the ratio of the freeze-drying protective solution to the bacterial suspension is 1:1, fully and uniformly mixing, namely after the freeze-drying protective solution is added, mixing and stirring by adopting a glass stirring rod, and improving the uniform mixing of the freeze-drying protective solution and the bacterial suspension.
7. The freeze-drying preservation method of quantitative working quality control strains according to claim 6, wherein the proportion of the freeze-drying protective solution is as follows:
1-5% of Arabic gum, 0-3% of bovine serum, 0-5% of lactic acid, 1-2% of glycerol, 2-5% of glycine, 3-8% of arginine, 1-5% of polyethylene glycol, 1-3% of D-sodium erythorbate and 1-10% of skim milk.
8. The freeze-drying preservation method for quantitative working quality control strains according to claim 7, wherein the proportion of the freeze-drying protective solution is further selected as follows:
2% of Arabic gum, 1% of bovine serum, 2% of lactic acid, 1% of glycerol, 2% of glycine, 5% of arginine, 1% of polyethylene glycol, 2% of D-sodium erythorbate and 5% of skim milk;
the preparation process of the freeze-drying protective solution is as follows, taking 100ml of freeze-drying protective solution as an example;
s301, measuring 100ml of purified water in a beaker, and heating to boil;
s302, sequentially adding 2g of Arabic gum, 1g of bovine serum, 2g of lactic acid, 1g of glycerol, 2g of glycine, 5g of arginine, 1g of polyethylene glycol and 2g of D-sodium erythorbate into a beaker, and adding the next reagent after the former reagent is dissolved;
s303, adjusting the pH value to 7.2 +/-0.5 by using a 1M sodium hydroxide solution, finally adding 5g of skimmed milk, and fully stirring and uniformly mixing;
s304, filling the mixture into a 100ml infusion bottle or a wide-mouth bottle, carrying out damp-heat sterilization at 116 ℃ for 20 minutes, and placing the mixture in a low-temperature and dark environment at 2-8 ℃ for later use after the sterilization is finished.
9. The method for freeze-drying preservation of quantitative working quality control strains according to claim 1, wherein the freeze-drying process of the freeze-drying liquid in the step S3 is as follows:
mixing the prepared bacterial suspension with the prepared freeze-drying protective solution 1:1, mixing to prepare a freeze-drying liquid, subpackaging the freeze-drying liquid into special penicillin bottles in an amount of 0.05ml-0.5ml per bottle, performing semi-stoppering by using a rubber plug, pre-freezing the penicillin bottles subpackaged with the freeze-drying liquid in a freeze dryer for 2 hours at-40 ℃, drying the penicillin bottles at the temperature of 25 ℃, wherein the total use time is 18-24 hours, and the judgment mode of drying is that the pressure rise speed in the freeze dryer is less than 2pa/10min; and (4) after the freeze-drying is finished, performing a tamponade operation on the product, and storing at the temperature of minus 20 ℃.
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| CN106635802A (en) * | 2016-11-22 | 2017-05-10 | 浙江泰林生物技术股份有限公司 | Solid-state freeze-dried product of quantitative strain and preparation method and using method thereof |
| WO2022029303A1 (en) * | 2020-08-06 | 2022-02-10 | 4D Pharma Plc | Lyophilisation process |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106635802A (en) * | 2016-11-22 | 2017-05-10 | 浙江泰林生物技术股份有限公司 | Solid-state freeze-dried product of quantitative strain and preparation method and using method thereof |
| WO2022029303A1 (en) * | 2020-08-06 | 2022-02-10 | 4D Pharma Plc | Lyophilisation process |
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| Title |
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| 陈騊声 等主编: "《微生物工程》", 化学工业出版社, pages: 75 - 79 * |
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