CN110106114A - A kind of freeze drying protectant and freeze-drying store method and application for Listeria monocytogenes standard substance - Google Patents
A kind of freeze drying protectant and freeze-drying store method and application for Listeria monocytogenes standard substance Download PDFInfo
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- CN110106114A CN110106114A CN201910388287.2A CN201910388287A CN110106114A CN 110106114 A CN110106114 A CN 110106114A CN 201910388287 A CN201910388287 A CN 201910388287A CN 110106114 A CN110106114 A CN 110106114A
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Abstract
The present invention relates to a kind of freeze drying protectant for Listeria monocytogenes standard substance and freeze-drying store method and applications; freeze drying protectant according to the present invention is the dedicated freeze drying protectant for being directed to Listeria monocytogenes standard substance; cooperated by four kinds of skim milk, polyvinylpyrrolidone, sucrose and tyrosine special components with specific mass percentage; it can be synergistic; Listeria monocytogenes standard substance after making freeze-drying reaches higher viable bacteria rate, and its viable bacteria rate can still maintain higher level after through a long time storage.
Description
Technical field
The invention belongs to microorganism aid technical fields, and in particular to a kind of jelly for Listeria monocytogenes standard substance
Dry protective agent and freeze-drying store method and application.
Background technique
With the development of biotechnology, it is related to the relevant microbial standard substance such as bio-safety, food safety, microorganism
Detection method, effectively analysis measurement means and magnitude tracing technology are still the important topic that we face.Due to micro- life
Analyte detection process is influenced by individual difference, especially sensitive simultaneously for environmental pollution, for same sample when most
It can not recheck, so being badly in need of reinforcing microbial standard substance R&D capability, by using close practical with clear magnitude, matrix
The microbial cells standard substance of sample realizes the control of microorganism detection outcome quality, guarantees the accuracy, just of testing result
Property.
Listeria Monocytogenes abbreviation Listeria monocytogenes are a kind of pathogens of zoonosis.Its energy
The disease for causing Lee Salmonella of people and animals, is mainly shown as septicemia, meningitis and monocytosis after infection.It is widely present in
Singly increasing Lee Salmonella in nature, present in food has danger to the safety of the mankind, which can still grow in 4 DEG C of environment
Breeding is one of the main pathogenic fungi that chilled food threatens human health, therefore, in food hygiene Micro biological Tests, it is necessary to
Paid attention to.
CN108485979A discloses a kind of dedicated bacterium freeze drying protectant of ensilage, is resisted by dopamine, benzoic acid
Bad hematic acid ester, phosphatide, activation chitosan, urea, saltcake and calcium salt composition first mix saltcake and calcium salt in configuration process
Afterwards, it dries in baking oven to constant weight, obtains dry mixed salt, then be passed through nitrogen into batch mixer, under nitrogen protection state, to mixed
It is mixed that dopamine, benzoic acid acid ascorbyl ester, phosphatide, activation chitosan, urea and dry mixed salt, stirring are sequentially added in material machine
It closes uniformly, discharging encapsulates to get the dedicated bacterium freeze drying protectant of ensilage.The dedicated bacterium of gained ensilage of the invention is frozen
Dry protective agent has excellent protecting effect.
CN107365705A discloses freeze drying protectant and its application of a kind of oil-soluble probiotics freeze-dried vaccine powder, the freeze-drying
Protective agent includes the sugar of the unsaturated fatty acid of 5-20%, 5-25% based on mass fraction, and surplus is water, is used for and probiotics
Freeze-drying prepares oil-soluble probiotics freeze-dried vaccine powder after mixing.The freeze drying protectant of oil-soluble probiotics freeze-dried vaccine powder of the invention,
Still have in separate sources oil product and quickly dissolve and make the evenly dispersed ability in different media of cell, and cell is in length
Possess excellent activity, stability and resistance under phase high temperature storage.
CN103468572A discloses a kind of freeze drying protectant of photogen, which is by following parts by weight
Primary raw material be prepared: 42-55 parts of galactolipin, 19-26 parts of trehalose, 98-105 parts of skimmed milk.Above-mentioned freeze drying protectant
It can be used for the preparation of photogen freeze-dried powder, when being used to prepare the freeze-dried powder of photogen and the optimum volume ratio of bacterium solution to be 1:(7-
13) between.The freeze drying protectant can improve every gram in photogen freeze-dried powder of bacteria containing amount, and can guarantee in photogen freeze-dried powder
Bacteria containing amount stability and raising photogen recovery characteristic.
The special of Listeria monocytogenes standard substance is directed to excellent protective value there are no a kind of in the prior art
With freeze drying protectant, therefore, develops and a kind of be directed to the special of Listeria monocytogenes standard substance with excellent protective value
It is necessary with freeze drying protectant.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of for Listeria monocytogenes standard substance
Freeze drying protectant and freeze-drying store method and application.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, and the freeze-drying is protected
Agent is protected based on mass percentage including following component: skim milk 40-60%, polyvinylpyrrolidone 0.2-0.8%, sucrose
2-6%, tyrosine 0.2-0.8%, solvent are sterile distilled water.
Freeze drying protectant according to the present invention is the dedicated freeze drying protectant for being directed to Listeria monocytogenes standard substance,
The mechanism of each ingredient protection bacterium is different in protective agent.The present invention by skim milk, polyvinylpyrrolidone (PVP),
Four kinds of special components of sucrose and tyrosine are cooperated with specific mass percentage, wherein the milk protein in skim milk
(predominantly casein) can protect cells from solution damage and ice crystal damage, play a protective role；Polyvinylpyrrolidone
(PVP) there is the biggish degree of polymerization, have higher glass transition temperature, with the increase of PVP concentration, the viscosity of aqueous solution is therewith
Become larger, the glass transition temperature of sucrose is significantly raised, and the protective effect risen to the protein of the cell in refrigerating process is consequently also
It increased, but when concentration is excessive will increase the moisture of final products, so that protein becomes more unstable in product, therefore need
Want the PVP of certain concentration as protectant adding ingredient；Sucrose is made of a molecule glucose and a molecule fructose
Disaccharide, chemical property are stablized, and are in undefined structure more, to preventing secondary protein structure from changing, during frozen dried and storage
The stretching, extension of hiding phase internal protein and remarkable effect is assembled；Tyrosine can increase sample collapse temperature prevent freeze-drying process in because
The destruction of pharmaceutical grade protein caused by collapsing.These four protective agent ingredients are realized from different level to Listeria monocytogenes standard
The protection of substance, and can be synergistic, the Listeria monocytogenes standard substance after making freeze-drying reaches higher viable bacteria rate, and passes through
Its viable bacteria rate can still maintain higher level after long-time storage.
Preferably, the freeze drying protectant is based on mass percentage including following component: skim milk 50%, polyethylene
Pyrrolidones 0.5%, sucrose 4%, tyrosine 0.5%, solvent are sterile distilled water.
On the other hand, the present invention provides a kind of freeze-drying store method of Listeria monocytogenes standard substance, and the freeze-drying is protected
Deposit method are as follows: pre-freeze after mixing Listeria monocytogenes suspension with the protective agent as described above after sterilizing, then carry out freezing and do
It is dry, obtain freeze-drying prods.
Preferably, the Listeria monocytogenes suspension and protectant volume ratio are 1:(50-200)；
The Listeria monocytogenes suspension and the specific selection of protectant volume ratio within the above range, are compared because being less than this
Example range meeting bacterial density is excessively high, and protection agent content is not enough to protect excessive bacterium, and bacterium protective rate is caused to decline, and is more than this
Proportional region can make bacterial density too low, and protection agent content is too high, and sample can be with the reduction of cell density, the survival rate of thallus
Sharply decline.Protective agent only sufficiently could be merged and be uniformly distributed in above-mentioned volume ratio range with thallus, could enhance bacterium
Tolerance of the body to hyperosmosis and drying condition.
Preferably, the cell density of the Listeria monocytogenes suspension is 1 × 107-1×109CFU/mL。
In the present invention, the sterilizing uses high pressure steam sterilization mode.
Common sterilization method has high pressure steam sterilization, filtration sterilization, radiation sterilization etc., and wherein autoclaving is
A kind of sterilizing methods of all microorganisms including brood cell can be killed；Sterilization by filtration is that cannot have by densification with bacterium
Method of the principle of hole filter material to remove microorganism in gas or liquid；Radiation sterilization is to utilize ultraviolet radioactive and ionising radiation
Kill microorganism.The specific sterilization method that the present invention selects is high pressure steam sterilization, and this sterilization method can make the list after freeze-drying
The viable bacteria rate for increasing Listeria standard substance maintains higher level.
Preferably, the temperature of the high pressure steam sterilization be 115-121 DEG C, such as 115 DEG C, 116 DEG C, 117 DEG C, 118 DEG C,
119 DEG C, 120 DEG C or 121 DEG C etc..
Preferably, two kinds of components of sucrose and skim milk sterilize at 115 DEG C；Polyvinylpyrrolidone and tyrosine exist
It sterilizes at 121 DEG C, this sterilization method can guarantee more preferably sterilization effect.
Preferably, the time of the high pressure steam sterilization be 15-25min, such as 15min, 16min, 17min, 18min,
19min, 20min, 22min, 24min or 25min etc..
Preferably, the temperature of the pre-freeze be -85~-75 DEG C, such as -85 DEG C, -84 DEG C, -83 DEG C, -82 DEG C, -81 DEG C, -
80 DEG C, -79 DEG C, -78 DEG C or -75 DEG C etc., preferably -80 DEG C.
The specific selection of the temperature of the pre-freeze is within the scope of -85~-75 DEG C, because will lead to economic cost lower than this range
It is excessively high, sample can be prevented from quick pre-freeze in a short time higher than this range.
The advantage of the row ultralow temperature pre-freeze advanced before being freeze-dried is: the viable bacteria in sample at low temperature may be used
It slowly to grow, is such as directly put in freeze dryer and is lyophilized according to conventional freeze-drying program, sample temperature, which generally requires, spends 1-2
Drop to zubzero temperature in hour, time mistake that conventional freezing temperature decline spend lower containing protectant sample crystallization point
Long, Listeria monocytogenes can slowly be grown at low temperature, and bacterial number changes during may result in.Therefore, originally
Application allows sample most quick using being in advance quickly put into prepared sample in -85~-75 DEG C of ultra-low temperature surroundings
Degree freezing, reduce because cool down in freeze drier it is relatively slow caused by bacterial number variation in sample.For example, if sample be placed in-
Pre-freeze is carried out at 20 DEG C, then sample needs the longer pre-freeze time, and sample is easy to thaw, and leads to the form of subsequent freeze-drying sample
Irregular, bacterial number changes in sample, influences survival rate.
Preferably, the time of the pre-freeze be 2-10h, such as 2h, 2.5h, 2.8h, 3h, 3.5h, 4h, 5h, 6h, 7h, 8h,
9h or 10h etc., preferably 8h.
Preferably, it is described obtain freeze-drying prods after vacuum gland sealing is carried out to it.
The sealing means of freeze-drying prods generally use the sealing of vacuum gland or the sealing of antivacuum gland, use vacuum pressure herein
Lid sealing is so that the viable bacteria rate of final freeze-drying Listeria monocytogenes standard substance maintains higher level in a long time.
As the preferred technical solution of the present invention, the freeze-drying store method are as follows:
Protective agent is subjected to high pressure steam sterilization 15-25min at 115-121 DEG C, then by cell density is 1 × 107-1
×109The Listeria monocytogenes suspension of CFU/mL is 1:(50-200 with volume ratio with protective agent) it is mixed, mixture is obtained,
Mixture is carried out to pre-freeze 1-4h at -75~-85 DEG C again, is finally freeze-dried, obtains freeze-drying prods, obtained freeze-drying is produced
Product carry out the sealing of vacuum gland；The protective agent includes following component by mass percentage: skim milk 40-60%, poly- second
Alkene pyrrolidone 0.2-0.8%, sucrose 2-6%, tyrosine 0.2-0.8%, solvent are water.
In another aspect, the present invention provides a kind of freeze drying protectant as described above in freeze-drying Listeria monocytogenes standard substance
In application.
Compared with the existing technology, the invention has the following advantages:
Freeze drying protectant according to the present invention is the freeze drying protectant for being directed to Listeria monocytogenes standard substance, is passed through
Four kinds of skim milk, polyvinylpyrrolidone, sucrose and tyrosine special components are cooperated with specific mass percentage,
Can be synergistic, the Listeria monocytogenes standard substance after making freeze-drying reaches higher viable bacteria rate, and after through a long time storage its
Viable bacteria rate can still maintain higher level.Freeze-drying store method simple possible according to the present invention, it is easily operated, make
The viable bacteria rate of Listeria monocytogenes standard substance freeze-drying prods made from this method is higher.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with preferred implementation of the invention
Example to further illustrate the technical scheme of the present invention, but the present invention is not limited in scope of embodiments.
Key instrument involved in following all examples and comparative example includes the following: with reagent
Biohazard Safety Equipment (Labconco company, the U.S.)；Freeze drier (Labconco company, the U.S.)；Constant incubator
(Shanghai Hui Tai instrument manufacturing Co., Ltd)；High-pressure sterilizing pot (Shenan Medical Appliances Factory, Shanghai)；Milli-Q pure water meter (the U.S.
Merck & Co., Inc.)；Electronic balance (Mettler Toled company, the U.S.)；Ultra low temperature freezer (thermo company, the U.S.)；Pai Dashi is equal
Matter device (French Interscience company).
Listeria Monocytogenes (Listeria monocytogenes) reference culture (ATCC strain number
19115)；Listeria spp chromogenic culture medium (Beijing Luqiao Technology Co., Ltd.)；Listeria enrichment broth base (LB1, LB2)
(Beijing Luqiao Technology Co., Ltd.)；Trypticase soy yeast extract agar (TSA-YE) (the limited duty of Beijing overpass technology
Ren company)；Phosphate buffer (Beijing Luqiao Technology Co., Ltd.)；0.85% sterile saline (Beijing overpass skill
Art Co., Ltd)；Digested tankage (Shanghai City metrological testing technology research self-control)；Skim milk (De Yun company)；Polyethylene pyrrole
Pyrrolidone (Chinese medicines group)；Tyrosine (Chinese medicines group)；Sucrose (Chinese medicines group).
Embodiment 1
The present embodiment carries out resurrection, identification and the culture of strain, and concrete operations are as follows:
The standard Listeria Monocytogenes strain of freezen protective is crossed resurrection on nutrient agar, 37 DEG C of cultures
18h-24h.It picks from the plate single Listeria monocytogenes and falls and be inoculated into nutrient broth, 37 DEG C, cultivate 18h-24h, take bacterium
Liquid is inoculated on TSA-YE culture medium, and 37 DEG C to after growing bacterium colony, carry out after Gram's staining the bacterium arrangement in micro- sem observation
In rod-short, size is about (0.4 μm -0.5 μm) × (0.5 μm -2.0 μm), random picking colony VITEK full-automatic biochemical point
Analyzer is identified that the single bacterium colony of picking pure culture punctures SIM dynamic culture base, 30 DEG C of culture 48h, on SIM culture medium
Side is grown in umbrella, and the single bacterium colony of picking pure culture is punctured on blood plate, 37 DEG C of culture 48h, haemolysis circle is narrow, it is clear,
Bright, to sum up experimental result, is finally accredited as Listeria monocytogenes.
Embodiment 2
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant is pressed
Mass percentage meter includes following component: skim milk 50%, polyvinylpyrrolidone 0.5%, sucrose 4%, tyrosine
0.5%, solvent is sterile distilled water.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, is included the following steps:
Protective agent is subjected to high pressure steam sterilization, then by cell density is 1 × 108The Listeria monocytogenes suspension of CFU/mL
It is mixed with protective agent with volume ratio for 1:100, obtains mixture, then mixture is dispensed with sterile pipette to brown west
In woods bottle, every bottle of 1mL carries out pre-freeze 8h at -80 DEG C, is freeze-dried 36h under 0.015mbar vacuum degree, obtains freeze-drying and produces
Product carry out the sealing of vacuum gland to the standard substance being lyophilized.
Embodiment 3
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant is pressed
Mass percentage meter includes following component: skim milk 40%, polyvinylpyrrolidone 0.2%, sucrose 6%, tyrosine
0.8%, solvent is sterile distilled water.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Embodiment 4
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant is pressed
Mass percentage meter includes following component: skim milk 60%, polyvinylpyrrolidone 0.8%, sucrose 2%, tyrosine
0.2%, solvent is sterile distilled water.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Comparative example 1
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant is pressed
Mass percentage meter includes following component: skim milk 25%, polyvinylpyrrolidone 1%, sucrose 1%, tyrosine 1%,
Solvent is sterile distilled water.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Comparative example 2
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant is pressed
Mass percentage meter includes following component: skim milk 70%, polyvinylpyrrolidone 0.1%, sucrose 8%, tyrosine
0.1%, solvent is sterile distilled water.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Comparative example 3
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant with
The difference of embodiment 2 is only that without containing polyvinylpyrrolidone, other are consistent.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Comparative example 4
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant with
The difference of embodiment 2 is only that without containing sucrose, other are consistent.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Comparative example 5
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant with
The difference of embodiment 2 is only that without containing tyrosine, other are consistent.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, operating procedure and embodiment 2 are consistent.
Embodiment 5
The present embodiment provides a kind of freeze drying protectant for Listeria monocytogenes standard substance, the freeze drying protectant is pressed
Mass percentage meter includes following component: skim milk 50%, polyvinylpyrrolidone 0.5%, sucrose 4%, tyrosine
0.5%, solvent is sterile distilled water.
Listeria monocytogenes standard substance is lyophilized using this freeze drying protectant, is included the following steps:
Protective agent is subjected to high pressure steam sterilization, then by cell density is 1 × 108The Listeria monocytogenes suspension of CFU/mL
It is mixed with protective agent with volume ratio for 1:100, obtains mixture, then mixture is dispensed with sterile pipette to brown west
In woods bottle, every bottle of 1mL carries out pre-freeze 8h at -80 DEG C, is freeze-dried 36h under 0.015mbar vacuum degree, obtains freeze-drying and produces
Product carry out antivacuum gland sealing to the standard substance being lyophilized.
Evaluation test:
(1) bacterium freeze-drying survival rate evaluation
8 bottles of samples are randomly selected in the product made from embodiment 2-5 and comparative example 1-5 respectively, to freeze-drying front and back
Viable count be measured, obtain mean viable rate (viable bacteria rate (%)=[viable bacteria before viable count/freeze-drying after freeze-drying
Number] × 100%), the wherein measuring method of viable count are as follows: single to increase Liszt and develop the color colony counting method.
The results are shown in Table 1, and mean viable rate of the viable bacteria after freeze-drying is 30% in embodiment 2, and survival rate is most
It is high；By the data result of comparative example 1 and 2 it is found that working as the quality hundred of skim milk, polyvinylpyrrolidone, sucrose and tyrosine
When point content is unsatisfactory for 40-60%, 0.2-0.8%, 2-6%, 0.2-0.8%, viable bacteria rate can be remarkably decreased；By comparative example 3-5
Data result it is found that when lacking any one in polyvinylpyrrolidone, sucrose and tyrosine in protective agent, viable bacteria rate
Also it can be remarkably decreased.
Table 1
(2) estimation of stability under long-time storage
The the 0th, 3,7,15,30 day after the completion of freeze-drying respectively, in the product made from embodiment 1-7 and comparative example 1-5
It randomly selects 3 bottles of samples, calculates mean viable rate (viable count before viable count/freeze-drying after survival rate (%)=freeze-drying
× 100%), wherein the measuring method of viable count is consistent with the above.
The results are shown in Table 2, and it is 30% or so that the viable bacteria rate of embodiment 2 maintains stable level in 30 days, can protect
Hold prolonged stability.
Table 2
| Group | 0 day | 3 days | 7 days | 15 days | 30 days |
| Embodiment 2 | 30% | 32.3% | 30% | 32.5% | 31.5% |
| Embodiment 3 | 17.5% | 10% | 0% | 0% | 0% |
| Embodiment 4 | 24.3% | 12% | 0% | 0% | 0% |
| Embodiment 5 | 7.8% | 2% | 0% | 0% | 0% |
| Comparative example 1 | 1.2% | 0% | 0% | 0% | 0% |
| Comparative example 2 | 8.8% | 3% | 0% | 0% | 0% |
| Comparative example 3 | 2.9% | 1% | 0% | 0% | 0% |
| Comparative example 4 | 5.0% | 2% | 0% | 0% | 0% |
| Comparative example 5 | 8.1% | 4.2% | 0% | 0% | 0% |
The Applicant declares that the present invention is explained by the above embodiments of the invention is used for Listeria monocytogenes standard substance
Freeze drying protectant and freeze-drying store method and application, but the present invention is not limited to the above embodiments, that is, does not mean that this hair
The bright above-described embodiment that must rely on could be implemented.It should be clear to those skilled in the art, any changes to of the invention
Into addition, selection of concrete mode of equivalence replacement and auxiliary element to each raw material of product of the present invention etc. all fall within the present invention
Protection scope and the open scope within.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
Claims (10)
1. a kind of freeze drying protectant for Listeria monocytogenes standard substance, which is characterized in that the freeze drying protectant presses matter
Measuring percentage composition meter includes following component: skim milk 40-60%, polyvinylpyrrolidone 0.2-0.8%, sucrose 2-6%, junket
Propylhomoserin 0.2-0.8%, solvent are sterile distilled water.
2. freeze drying protectant as described in claim 1, which is characterized in that the freeze drying protectant wraps based on mass percentage
Include following component: skim milk 50%, polyvinylpyrrolidone 0.5%, sucrose 4%, tyrosine 0.5%, solvent are sterile steaming
Distilled water.
3. a kind of freeze-drying store method of Listeria monocytogenes standard substance, which is characterized in that the freeze-drying store method are as follows: will
Pre-freeze after Listeria monocytogenes suspension is mixed with the protective agent as claimed in claim 1 or 2 after sterilizing, then carry out freezing and do
It is dry, obtain freeze-drying prods.
4. as claimed in claim 3 freeze-drying store method, which is characterized in that the Listeria monocytogenes suspension with it is protectant
Volume ratio is 1:(50-200)；
Preferably, the Listeria monocytogenes suspension and protectant volume ratio are 1:100；
Preferably, cell density is 1 × 107-1×109 CFU/mL。
5. freeze-drying store method as described in claim 3 or 4, which is characterized in that the sterilizing uses high pressure steam sterilization side
Formula；
Preferably, the temperature of the high pressure steam sterilization is 115-121 DEG C；
Preferably, the time of the high pressure steam sterilization is 15-25min.
6. the freeze-drying store method as described in any one of claim 3-5, which is characterized in that the temperature of the pre-freeze is -85
~-75 DEG C.
7. the freeze-drying store method as described in any one of claim 3-6, which is characterized in that the temperature of the pre-freeze is -80
℃；
Preferably, the time of the pre-freeze is 2-10h, preferably 8h.
8. the freeze-drying store method as described in any one of claim 3-7, which is characterized in that it is described obtain it is right after freeze-drying prods
It carries out the sealing of vacuum gland.
9. the freeze-drying store method as described in any one of claim 3-8, which is characterized in that the freeze-drying store method are as follows:
Protective agent is subjected to high pressure steam sterilization 15-25min at 115-121 DEG C, then by cell density is 1 × 107-1×
109The Listeria monocytogenes suspension of CFU/mL is 1:(50-200 with volume ratio with protective agent) it is mixed, mixture is obtained, then
Mixture is subjected to pre-freeze 1-4h at -75~-85 DEG C, is finally freeze-dried, obtains freeze-drying prods, to obtained freeze-drying prods
Carry out the sealing of vacuum gland；The protective agent includes following component by mass percentage: skim milk 40-60%, polyethylene
Pyrrolidones 0.2-0.8%, sucrose 2-6%, tyrosine 0.2-0.8%, solvent are water.
10. application of the freeze drying protectant as claimed in claim 1 or 2 in freeze-drying Listeria monocytogenes standard substance.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910388287.2A CN110106114A (en) | 2019-05-10 | 2019-05-10 | A kind of freeze drying protectant and freeze-drying store method and application for Listeria monocytogenes standard substance |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910388287.2A CN110106114A (en) | 2019-05-10 | 2019-05-10 | A kind of freeze drying protectant and freeze-drying store method and application for Listeria monocytogenes standard substance |
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| CN110106114A true CN110106114A (en) | 2019-08-09 |
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| CN111719007A (en) * | 2020-08-06 | 2020-09-29 | 南方医科大学 | Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof |
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| CN111719007A (en) * | 2020-08-06 | 2020-09-29 | 南方医科大学 | Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof |
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