CN107236784A - Staphylococcus aureus standard sample and preparation method thereof in milk powder - Google Patents
Staphylococcus aureus standard sample and preparation method thereof in milk powder Download PDFInfo
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- CN107236784A CN107236784A CN201710512083.6A CN201710512083A CN107236784A CN 107236784 A CN107236784 A CN 107236784A CN 201710512083 A CN201710512083 A CN 201710512083A CN 107236784 A CN107236784 A CN 107236784A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- C12N1/20—Bacteria; Culture media therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
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Abstract
Staphylococcus aureus standard sample and preparation method thereof in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of milk powder.Staphylococcus aureus standard sample includes object bacteria and background flora in milk powder, and the object bacteria is staphylococcus aureus, and the background flora is made up of citrobacter freundii, ETEC, Klebsiella Pneumoniae, Bacillus cereus.Uniformity of the present invention, stability meet standard sample requirement, the stability of standard sample can at utmost be ensured, set about from the condition for meeting special transport, research by the research of special process, stability and uniformity etc. forms the sample preparation technology of a set of perfect standard sample, it is suitable for preparing staphylococcus aureus standard sample in the milk powder for meeting standard sample requirement, it is adaptable to the purposes such as quality control, the method validation in laboratory.
Description
Technical field
Golden yellow grape in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of milk powder
Coccus standard sample and preparation method thereof.
Background technology
Milk powder microbiological Test field is very special.At present, it at home and abroad there is no mechanism and prepare milk powder microbiology mark
Quasi- sample and the precedent of large-scale production, still belong to blank field.Though there are some mechanisms to prepare the standard sample of microbiology class at present,
But the uniformity and stability of standard sample can hardly meet requirement, transport extremely difficult；Or standard sample is only to contain mesh
The pure bacterial strain of bacterium is marked, it is larger with actual sample gap, it is impossible to meet the needs such as routine experimentation room quality control.
Staphylococcus aureus be a kind of common gram-positive bacteria, be distributed widely in nature, can cause people and
The infection of animal.After human infection staphylococcus aureus, you can cause the local pyogenic infection of skin, it can also cause serious
Internal organs infection, or even septicemia, toxic shock etc..The enterotoxin that staphylococcus aureus produces can be infected with food and
Cause food poisoning.If in the staphylococcus aureus sample prepared, object bacteria easily changes (breeding and death etc.), will
Cause the storage life of sample short, the uniformity and stability of sample are poor, influence quality control effect.Therefore, bacterium is considered
Biochemical characteristic, choose the metastable flora of property as object bacteria to ensure sample uniformity and stability it is very heavy
Will.The process specifications of staphylococcus aureus sample preparation are higher, and the uniformity and stability of sample are with freezing
Strain, lyophilized process conditions, the use of freeze drying protectant, the medium of rehydration etc. have close relationship.
Staphylococcus aureus index in milk powder, directly reflects the hygienic quality and potential pathogenic risk of milk powder.If
Staphylococcus aureus number may result in food origin disease more than a threshold quantity.Therefore, prepare uniformity and stability is all
Good staphylococcus aureus standard sample, the random and uncertainty that staphylococcus aureus can be avoided to examine, effectively
Ground ensures the accurate credible of testing result.This ensures milk powder safety, even breaks state to improving Good Laboratory controlled level
Border trade barrier, raising China milk powder international competitiveness all have special important meaning.
The content of the invention
The purpose of the present invention is the viable bacteria number for overcoming object bacteria in staphylococcus aureus standard sample in transport, storage
There is provided staphylococcus aureus standard sample in a kind of milk powder for the problem of can all being changed with total clump count during test etc.
Product, uniformity, stability meet standard sample requirement, and it is a further object to provide the preparation method of the sample, work
Skill is simple, and success rate is high.
The technical scheme that is used to achieve the above object of the present invention is：Staphylococcus aureus standard sample in milk powder,
It is characterized in that：Including object bacteria and background flora, the object bacteria is staphylococcus aureus (Staphylococcus
Aureus), the background flora is by citrobacter freundii (Citrobacterfreundii), ETEC
(E.coil), Klebsiella Pneumoniae (Klebsiellapnenmoniae), Bacillus cereus (Bacillus cereus) group
Into.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein the volume fraction of trehalose is 12%, taken off
The volume fraction of fat milk powder is 15%.
The aimed concn of object bacteria is 10 in the sample2~103CFU/mL, the aimed concn of background flora is 103CFU/
mL。
The preparation method of staphylococcus aureus standard sample in milk powder, it is characterized in that：The choosing of bacterial strain is added including sample
Select, the preparation of freeze drying protectant, sample are lyophilized, four steps of the uniformity of sample and stability test, wherein sample was freezed
Cheng Wei：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, and detailed process is：
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
(2) bacterium is increased
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2~103CFU/mL and the back of the body
The aimed concn 10 of scape flora3CFU/mL prepares bacteria suspension, and the bacteria suspension of object bacteria is prepared respectively and the bacterium of 4 background bacterium is hanged
Liquid, background bacterium presses 1:1 volume ratio is mixed to form background flora suspension, then target bacteria suspension and background flora suspension are pressed into 1：
1 volume ratio is mixed, and is placed in be stirred continuously down on magnetic stirring apparatus and is dispensed into sample bottle, adds bottle stopper, but to leave space, no
Cover reality；
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 50~55h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample is issued to laboratory of participating in the experiment from whole samples
Or carry out uniformity and stability test.
The freeze drying protectant is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Marine alga
Sugar 12%, skimmed milk power 15%.
The uniformity of the sample and stability test are according to GB/T 15000.3-2008《Standard sample work directive/guide (3)
The rule and statistical method of standard sample definite value》Carry out.
The selection of strain of the present invention uses staphylococcus aureus as object bacteria, with citrobacter freundii, large intestine angstrom
Uncommon Salmonella, Klebsiella Pneumoniae, Bacillus cereus at utmost ensure the stability of test sample as background flora, and
The detection of staphylococcus aureus is not influenceed.
The staphylococcus aureus standard sample of the present invention has the advantage that characteristic：Microorganism living, quantity do not occur
Change, biochemical character do not morph, set about from the condition for meeting special transport, by the research of special process, stability and
Research of uniformity etc. forms the technology of preparing of a set of perfect standard sample, is suitable for staphylococcus aureus standard in milk powder
The preparation of sample.
Brief description of the drawings
Fig. 1 is present invention process flow chart.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment is described in further detail to the present invention, but the invention is not limited in tool
Body embodiment.
Embodiment 1
Staphylococcus aureus standard sample in milk powder, including object bacteria and background flora, the object bacteria are golden yellow grape
Coccus (Staphylococcus aureus), the background flora is by citrobacter freundii
(Citrobacterfreundii), ETEC (E.coil), Klebsiella Pneumoniae (Klebsiella
Pnenmoniae), Bacillus cereus (Bacillus cereus) is constituted.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein the volume fraction of trehalose is 12%, taken off
The volume fraction of fat milk powder is 15%.
The aimed concn of object bacteria is 10 in the sample2~103CFU/mL, the aimed concn of background flora is 103CFU/
mL。
Embodiment 2
As shown in figure 1, in a kind of embodiment 1 in milk powder staphylococcus aureus standard sample preparation method, including sample adds
Plus the selection of bacterial strain, the preparation of freeze drying protectant, sample are lyophilized, four steps of the uniformity of sample and stability test, specifically
Step is as follows：
1st, sample adds the selection of bacterial strain
According to object bacteria：Staphylococcus aureus (Staphylococcus aureus), background flora：ETEC
(E.coil), Klebsiella Pneumoniae (Klebsiellapnenmoniae), Bacillus cereus (Bacillus cereus), not
Family name's citric acid bacillus (Citrobacterfreundii) selection standard bacterial strain, all reference cultures are purchased from the machine that government specifies
Structure, and with bacterial strain certificate, it is ensured that the traceability of bacterial strain.
2nd, the preparation of freeze drying protectant
Sample is using trehalose, skimmed milk power and sterilized water as matrix (volume fraction：Trehalose 12%, skimmed milk power 15%) prepare
Freeze drying protectant.
3rd, sample is freezed
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
(2) bacterium is increased
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL
The bacterium solution of corresponding single culture is made in freeze drying protectant；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2~103CFU/mL and the back of the body
The aimed concn 10 of scape flora3CFU/mL prepares bacteria suspension.To ensure in obtained final sample containing above aimed concn
According to object bacteria 10 in bacterial strain, the present embodiment3CFU/mL prepares the bacteria suspension of object bacteria；Background bacterium presses 103CFU/mL prepares 4
The bacteria suspension of background bacterium, by 1:1 volume ratio mixing, forms background flora suspension, then by target bacteria suspension and background flora
Suspension presses 1：1 volume ratio is mixed, and obtains plastc ring, and bacterial strain content is checked using turbidimetry；It is placed on magnetic stirring apparatus not
Take 1.0mL to be dispensed into sample bottle (cillin bottle) under disconnected stirring, add bottle stopper, but to leave space, reality should not be covered；(4) freeze
And packaging
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, and freeze drier parameter is by following setting：
Chilling rate (Cooling Rate) 0.5 DEG C/min
- 1 DEG C of 15min of early stage cold point (Incipient Freezing Point)
- 40 DEG C of cryogenic temperature (Cooling temperature)
- 29 DEG C of eutectic point (Melting Point Eutectic temperature)
20 DEG C of 120min of heating-up temperature (Heating temperature)
Start freeze drier, machine is directly entered the freezing dry process of sequencing, whole freeze-drying process 55h.
After sample freeze-drying terminates, directly carry out jumping a queue in case；Closing machine.The not tight cillin bottle of plug is rejected,
Sample is vacuum state in cillin bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, and the sample of preservation suitably manage and detect, from whole sample
Random selected sample is issued to laboratory or carries out uniformity and stability test in product.
4th, the uniformity of sample and stability test
Uniformity and stability inspection to object bacteria in sample are the main methods of verification sample preparation process validity.Check
Sample homogeneity and stability are according to GB/T 15000.3-2008《Standard sample work directive/guide (3) standard sample definite value it is general
Principle and statistical method》Carry out.Gained sample homogeneity and Detection of Stability method and result are as follows：
12 samples are randomly selected respectively, using SN/T 1895-2007 staphylococcus aureus method of testings, in repeat condition
Test the staphylococcus aureus of 2 × 12 parts of samples.Result data carries out statistical disposition, statistic procedure and knot with method of analysis of variance
Fruit is as follows：
The variance analysis formula of table 1
In upper table,
The sample homogeneity testing result of table 2
The sample homogeneity of table 3 tests the results of analysis of variance
Conclusion：Under 95% fiducial probability, compared with influence of the other factors to test result, the inhomogeneities of sample is to connect
Receive.
Using two kinds of stability test：One kind is the stability test under storage temperature (4 DEG C), another to be
Stability test at high temperature (traffic condition of analog sample), from three temperature spots, respectively 20 DEG C, 36 DEG C and
42℃.Periodic detection sample, 3 samples are tested for different temperature points different holding time, by 2 × 3 parts of sample results
Average (after logarithmic transformed) carries out statistical disposition, F values with method of analysis of variance<FCritical value, then the stability of description standard sample meet
It is required that, while determining to meet the most long holding time of standard sample requirement under condition of different temperatures.Stability test result is shown in
Table 4 and table 5.
The sample short-term stability of table 4 tests the results of analysis of variance
The sample short-term stability of table 5 tests the results of analysis of variance
| SS | The free degree | MS | F values | F critical values | Fiducial probability | Sbb | |
| Between group | 0.114502 | 44 | 0.002602 | 1.41 | 1.64 | 0.95 | 0.019 |
| In group | 0.082971 | 45 | 0.001844 |
Embodiment 3
In milk powder described in the present embodiment each step of the preparation method of staphylococcus aureus standard sample with embodiment 2
In it is identical, different technical parameters are：During bacteria suspension is prepared, the concentration of object bacteria is according to 2 × 10 in target bacteria suspension4CFU/mL
Prepare；Freeze-drying process 50h.
Embodiment 4
In milk powder described in the present embodiment each step of the preparation method of staphylococcus aureus standard sample with embodiment 3
In it is identical, different technical parameters are：During bacteria suspension is prepared, the concentration of object bacteria is according to 4 × 10 in target bacteria suspension3CFU/mL
Prepare；Freeze-drying process 52h.
Claims (6)
1. staphylococcus aureus standard sample in milk powder, it is characterized in that：Including object bacteria and background flora, the object bacteria is
Staphylococcus aureus, the background flora is by citrobacter freundii, ETEC, Klebsiella Pneumoniae, waxy bud
Born of the same parents bacillus constitutes.
2. staphylococcus aureus standard sample in milk powder according to claim 1, it is characterized in that：The sample is with marine alga
Sugar, skimmed milk power and sterilized water are matrix, and the volume fraction of wherein trehalose is that the volume fraction of 12%, skimmed milk power is 15%.
3. staphylococcus aureus standard sample in milk powder according to claim 1 or 2, it is characterized in that：In the sample
The aimed concn of object bacteria is 102~103 CFU/mL, the aimed concn of background flora is 103 CFU/mL。
4. the preparation method of staphylococcus aureus standard sample in milk powder according to claim 1, it is characterized in that：Including
The selection of sample addition bacterial strain, the preparation of freeze drying protectant, lyophilized sample, four steps of the uniformity of sample and stability test
Suddenly, wherein sample freeze-drying process is：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, specific mistake
Cheng Wei：
（1）Bacterial strain recovery passage
Reference culture is added in nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
（2）Increase bacterium
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
（3）Bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2~103CFU/mL and the back of the body
The aimed concn 10 of scape flora3 CFU/mL prepares bacteria suspension, and the bacteria suspension of object bacteria is prepared respectively and the bacterium of 4 background bacterium is hanged
Liquid, background bacterium presses 1:1 volume ratio is mixed to form background flora suspension, then target bacteria suspension and background flora suspension are pressed into 1：
1 volume ratio is mixed, and is placed in be stirred continuously down on magnetic stirring apparatus and is dispensed into sample bottle, adds bottle stopper, but to leave space, no
Cover reality；
（4）Lyophilized and packaging
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 50~55h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
（5）Storage
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample is issued to laboratory or entered from whole samples
Row uniformity and stability test.
5. the preparation method of staphylococcus aureus standard sample in milk powder according to claim 4, it is characterized in that：It is described
Freeze drying protectant is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Trehalose 12%, defatted milk
Powder 15%.
6. the preparation method of staphylococcus aureus standard sample in milk powder according to claim 4, it is characterized in that：It is described
The uniformity of sample and stability test are according to GB/T 15000.3-2008《Standard sample work directive/guide（3）Standard sample is determined
The rule and statistical method of value》Carry out.
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| CN201710512083.6A CN107236784A (en) | 2017-06-28 | 2017-06-28 | Staphylococcus aureus standard sample and preparation method thereof in milk powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710512083.6A CN107236784A (en) | 2017-06-28 | 2017-06-28 | Staphylococcus aureus standard sample and preparation method thereof in milk powder |
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ID=59990967
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Cited By (5)
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| CN108690807A (en) * | 2018-05-16 | 2018-10-23 | 上海市计量测试技术研究院 | A kind of staphylococcus aureus standard substance and its preparation method and application |
| CN108865922A (en) * | 2018-05-02 | 2018-11-23 | 山东出入境检验检疫局检验检疫技术中心 | listeria monocytogenes standard sample and preparation method thereof |
| CN109762771A (en) * | 2019-02-28 | 2019-05-17 | 中国检验检疫科学研究院 | Staphylococcus aureus qualitative criteria sample and preparation method in water soluble cosmetics |
| CN111690712A (en) * | 2020-07-13 | 2020-09-22 | 北京海关技术中心 | Sample for verifying qualitative detection capability of staphylococcus aureus in mask and preparation method thereof |
| CN116218678A (en) * | 2023-04-14 | 2023-06-06 | 中国食品发酵工业研究院有限公司 | Preparation process of microorganism standard sample for smearing sampling and microorganism standard sample |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108865922A (en) * | 2018-05-02 | 2018-11-23 | 山东出入境检验检疫局检验检疫技术中心 | listeria monocytogenes standard sample and preparation method thereof |
| CN108690807A (en) * | 2018-05-16 | 2018-10-23 | 上海市计量测试技术研究院 | A kind of staphylococcus aureus standard substance and its preparation method and application |
| CN109762771A (en) * | 2019-02-28 | 2019-05-17 | 中国检验检疫科学研究院 | Staphylococcus aureus qualitative criteria sample and preparation method in water soluble cosmetics |
| CN111690712A (en) * | 2020-07-13 | 2020-09-22 | 北京海关技术中心 | Sample for verifying qualitative detection capability of staphylococcus aureus in mask and preparation method thereof |
| CN116218678A (en) * | 2023-04-14 | 2023-06-06 | 中国食品发酵工业研究院有限公司 | Preparation process of microorganism standard sample for smearing sampling and microorganism standard sample |
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