CN109880880A - Moulds and yeasts count standard sample and preparation method in water soluble cosmetics - Google Patents
Moulds and yeasts count standard sample and preparation method in water soluble cosmetics Download PDFInfo
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Abstract
The invention belongs to the field of quality control of Cosmetics microorganism context of detection, in particular to moulds and yeasts count standard sample and preparation method thereof in a kind of water soluble cosmetics.Moulds and yeasts count standard sample includes target flora and background flora in water soluble cosmetics, and the target flora is made of saccharomyces cerevisiae, aspergillus niger, and the background flora is made of Bacillus cereus, escherichia coli, staphylococcus aureus.The composition of flora and content are consistent with actual sample in sample of the present invention；Standard sample is consistent with actual sample matrix composition；Uniformity, stability comply with standard sample requirement, can utmostly guarantee the stability of standard sample；Preparation method, the simple process of sample, success rate are high.
Description
Technical field
The invention belongs to the field of quality control of Cosmetics microorganism context of detection, in particular to a kind of water-soluble makeup
Moulds and yeasts count standard sample and preparation method thereof in product.
Background technique
Cosmetics are the personal care products in many people's daily lifes, and the effect and safety of itself are directly related to
The health safety of user.Microorganism directly reflects the degree that cosmetics are contaminated by bacterial, and production in cosmetics
Raw material used in unit, tool equipment, process flow, operator sanitary condition, be to cosmetics carry out hygienical evaluation
Compositive index.Water soluble cosmetics microbiological Test field is very special.Currently, at home and abroad there is no mechanism preparation is water-soluble
The precedent of Cosmetics microorganism standard sample and large-scale production, still belongs to blank field.It is only standard that the country is existing at present
Bacterial strain or the standard sample suitable for field of food for only containing pure bacterium, it is inconsistent with actual sample, it is unable to satisfy daily reality
Test the needs such as room quality control.
After moulds and yeasts count refers to that cosmetics sample is cultivated under certain condition, institute is dirty in 1g or 1mL cosmetics
The yeast and mold total plate count of the work of dye.If target flora easily changes in the yeast and mold sample of preparation
(breeding and death etc.), the storage life that will lead to sample is short, and the uniformity and stability of sample are poor；Background bacterium and object bacteria contain
The height of amount is also the significant consideration for preparing the standard sample, and content of microorganisms is lower in usual actual sample, therefore institute
Background bacterium and target bacterial content should be consistent with actual sample in the standard sample of preparation.Therefore, comprehensively consider mould and ferment
The biochemical characteristic of female bacterium chooses the metastable reference culture of property as target flora to the uniformity and stabilization for guaranteeing sample
Property is particularly significant, and the uniformity of sample and stability and the strain of freeze-drying, the process conditions of freeze-drying, freeze drying protectant make
There is close relationship with, medium of rehydration etc..
Yeast and mold index in water soluble cosmetics directly reflects cosmetics by yeast and mold pollution level
And its general hygienic status.If yeast and mold is more than a threshold quantity in fruit cosmetic, the generation of injury skin risk is had.
Therefore, preparation uniformity and all good yeast and mold sample of stability can be examined random to avoid yeast and mold
Property and uncertainty, are truly reflected the ability of testing laboratory.This is ensured water-soluble to Good Laboratory controlled level is improved
The quality of cosmetics has special important meaning.
Summary of the invention
The object of the present invention is to provide a kind of and consistent standard samples of actual sample, for matter in cosmetics detection process
Purposes, uniformity, the stability such as amount control, staff training, method validation, culture medium or kit performance evaluation comply with standard sample
The requirement of product, it is a further object to provide the preparation method of the sample, simple process, success rate is high.
Present invention technical solution used for the above purpose is: moulds and yeasts count in water soluble cosmetics
Standard sample, it is characterized in that: including target flora and background flora, the target flora is by saccharomyces cerevisiae (Saccharomyces
Cerevisiae), aspergillus niger (Aspergillus niger) forms, and the background flora is by Bacillus cereus (Bacillus
Cereus), escherichia coli (E.coil), staphylococcus aureus (Staphylococcus aureus) composition.
The sample matrices are toner, and makeup water volume fraction is 0.5%；Protective agent is with trehalose and sterile water group
At wherein trehalose volume fraction is 10%.
The aimed concn of target flora is 10 in the sample2~103CFU/mL, the aimed concn of background flora are 103
CFU/mL。
The preparation method of yeast and mold qualitative criteria sample in water soluble cosmetics, it is characterized in that: including that freeze-drying is protected
Preparation, sample freeze-drying, the uniformity of sample and four stability test, sample definite value steps of agent are protected, wherein sample was lyophilized
Journey are as follows: bacterial strain recovery passage-increasing bacterium-bacteria suspension preparation-freeze-drying-packaging-storage, detailed process are as follows:
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, so that it is recovered and is grown at 36 ± 1 DEG C, and to multiple
The bacterial strain of Soviet Union is identified；
(2) increase bacterium
Target bacteria group culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to
The bacterium solution of corresponding single culture is made in the freeze drying protectant of 10mL；
(3) bacteria suspension is prepared
The bacterium solution that a upper process obtains is mixed with freeze drying protectant, according to target the aimed concn 10 of flora2~103CFU/
The aimed concn 10 of mL and background flora3CFU/mL prepares bacteria suspension, prepares the bacteria suspension and 3 backgrounds of 2 object bacterias respectively
The bacteria suspension of bacterium is mixed to form target flora suspension and background flora suspension by 1:1 volume ratio respectively, then by target flora suspension
It is mixed with background flora suspension by 1:1 volume ratio, obtains plastc ring, be placed on magnetic stirring apparatus and be stirred continuously, 1:100 ratio
Example addition toner, is dispensed into sample bottle after mixing is sufficiently stirred, in addition bottle stopper, but reality is not covered there are gap；
(4) it is lyophilized and packs
Sample bottle equipped with plastc ring is uncapped and is put into freeze dryer, 50~55h is freeze-dried, when sample freezing is dry
It after dry, directly carries out jumping a queue in case, closing machine, sample is vacuum state in sample bottle；
(5) it stores
Sample is placed under the conditions of -15~-20 DEG C and is kept in dark place, random selected sample is issued to reality from whole samples
It tests room or carries out uniformity and stability test.
The sample matrices are toner, and makeup water volume fraction is 0.5%；Protective agent is with trehalose and sterile water group
At wherein trehalose volume fraction is 10%.
" standard sample works directive/guide (3) according to GB/T 15000.3-2008 for the uniformity of the sample and stability test
The rule and statistical method of standard sample definite value " it carries out.
Strain of the present invention is selected using saccharomyces cerevisiae, aspergillus niger as moulds and yeasts count in water soluble cosmetics
The target flora of sample, using Bacillus cereus, escherichia coli, staphylococcus aureus as background flora.In environment
Aspergillus niger is relatively common in common mould, and Blastocystis saccharomyces cerevisiae is relatively conventional.It selects and the consistent makeup of actual sample
Water is matrix, consistent with actual sample；Selecting the trehalose with preferable frozen-dried protective effect is protective agent, and is selected big
Common bacteria is simulated in actual sample between different bacterium as background flora in the environment such as intestines Escherichia and staphylococcus aureus
Competition or disturbance state；The additive amount of target flora and background flora simulates the content of microorganism in actual sample, sufficiently rises
Quality control action is arrived.
Moulds and yeasts count standard sample has the advantage that characteristic in water soluble cosmetics of the invention: sample
Matrix is consistent with actual sample, and the composition of flora and content are consistent with actual sample in sample；Target bacterial content and actual sample
Unanimously；Microorganism living, quantity does not change, biochemical character does not morph, and sets about from the condition for meeting special transport, leads to
It crosses the research of special process, research of stability and uniformity etc. and forms a set of perfect standard sample technology of preparing, be suitable for
The preparation of moulds and yeasts count standard sample in water soluble cosmetics.
Detailed description of the invention
Fig. 1 is process flow chart of the invention.
Specific embodiment
With reference to the accompanying drawing and specific embodiment present invention is further described in detail, but the invention is not limited to tools
Body embodiment.
Embodiment 1
Moulds and yeasts count standard sample in water soluble cosmetics, including target flora and background flora, the mesh
Mark flora is made of saccharomyces cerevisiae (Saccharomyces cerevisiae), aspergillus niger (Aspergillus niger), described
Background flora is by Bacillus cereus (Bacillus cereus), escherichia coli (E.coil), staphylococcus aureus
(Staphylococcus aureus) composition.
The sample matrices are toner, and makeup water volume fraction is 0.5%；Protective agent is with trehalose and sterile water group
At wherein trehalose volume fraction is 10%.
The aimed concn of target flora is 10 in the sample2~103The aimed concn of CFU/mL, background flora is
103CFU/mL。
The sample is for quality control, staff training examination, method validation, culture medium or examination in cosmetics detection process
Agent examination and quality control etc..Such as the culture medium that testing laboratory purchases, needs to verify its validity, can not be verified from appearance,
It then can use the standard sample, provided according to detection method, carry out culture and corresponding detection using culture medium to be verified,
As a result testing result unanimously then illustrates that culture medium can be used by checking and accepting compared with the consistency of the characteristic value of standard sample
In the detection of the project；Otherwise it then needs further to confirm.
Embodiment 2
As shown in Figure 1, in a kind of embodiment 1 in water soluble cosmetics moulds and yeasts count standard sample preparation side
Method, preparation, sample freeze-drying, the uniformity of sample and stability test, standard sample definite value four including sample freeze drying protectant
A step, the specific steps are as follows:
1, the preparation of sample addition bacterial strain
According to target flora: saccharomyces cerevisiae (Saccharomyces cerevisiae), aspergillus niger (Aspergillus
Niger), background flora: Bacillus cereus (Bacillus cereus), escherichia coli (E.coil), golden yellow grape
Coccus (Staphylococcus aureus) selection criteria bacterial strain, all reference cultures are purchased from the qualified bacterium of regular tool
Kind collection, and have bacterial strain certificate, it ensure that the traceability of bacterial strain.
2, the preparation of freeze drying protectant
Sample matrices are toner, and makeup water volume fraction is 0.5%；Protective agent is formed with trehalose and sterile water,
Middle trehalose volume fraction is 10%.
3, sample is lyophilized
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, so that it is recovered and is grown at 36 ± 1 DEG C, and to multiple
The bacterial strain of Soviet Union is identified；
(2) increase bacterium
Target bacteria group culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to
The bacterium solution of corresponding single culture is made in the freeze drying protectant of 10mL；
(3) bacteria suspension is prepared
The bacterium solution that a upper process obtains is mixed with freeze drying protectant, according to target the aimed concn 10 of flora2~103CFU/
The aimed concn 10 of mL and background flora3CFU/mL prepares bacteria suspension.To guarantee to contain the above target in obtained final sample
The bacterial strain of concentration, according to object bacteria 10 in the present embodiment3CFU/mL prepares the bacteria suspension of 4 object bacterias respectively, by 1:1 volume ratio
Mixing forms target flora suspension；Background bacterium presses 104CFU/mL prepares the bacteria suspension of 3 background bacterium, mixes by 1:1 volume ratio,
Form background flora suspension；Background flora suspension is mixed with target flora suspension by 1:1 volume ratio, is obtained plastc ring, is pressed
Toner is added according to 1:100 volume ratio, bacterial strain content is checked using turbidimetry；It is placed on magnetic stirring apparatus to be stirred continuously down and take
1.0mL is dispensed into sample bottle (cillin bottle), in addition bottle stopper, but reality is not covered there are gap；
(4) it is lyophilized and packs
Sample bottle equipped with plastc ring is uncapped and is put into freeze dryer, 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 temperature (Heating temperature)
Start freeze drier, machine is directly entered the freezing dry process of sequencing, entire freeze-drying process 50h.
After sample freeze-drying, 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) it stores
Sample is placed under the conditions of -15~-20 DEG C and is kept in dark place, and the sample of preservation is suitably managed and examined
It surveys, random selected sample is issued to laboratory or carries out uniformity and stability test from whole samples.
4, the uniformity and stability test of sample
Uniformity and stability inspection to object bacteria in sample are the main methods of verification sample preparation process validity.
Checking sample homogeneity and stability, " standard sample works directive/guide (3) standard sample definite value according to GB/T 15000.3-2008
Rule and statistical method " it carries out.Gained sample homogeneity and Detection of Stability method and result are as follows:
12 samples are randomly selected respectively, using cosmetics safety technical specification (2015 editions) test method, are repeating item
Part tests the yeast and mold 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:
1 variance analysis formula of table
In upper table,
2 sample homogeneity testing result of table
3 sample homogeneity of table tests the results of analysis of variance
Conclusion: under 95% fiducial probability, compared with other factors are to the influence of test result, the inhomogeneities of sample is
It is acceptable.
Using two kinds of stability test: one is the stability test under storage temperature (4 DEG C), another kind is
Stability test at high temperature (traffic condition of analog sample), selects three temperature spots, respectively 20 DEG C, 36 DEG C and
45℃.Periodic detection sample tests 3 samples for different temperature points different holding time, by 2 × 3 parts of sample results
Mean value (after logarithmic transformed) carries out statistical disposition, F value < F with method of analysis of varianceCritical value, then the stability of description standard sample meets
It is required that while determining the longest holding time for complying with standard sample requirement under condition of different temperatures.Stability test result is shown in
Table 4 and table 5.
4 sample short-term stability testing result of table
5 sample short-term stability of table tests the results of analysis of variance
5, standard sample definite value
According to the GB/T 15000.3-2008 " rule and statistics of standard sample work directive/guide (3) standard sample definite value
Method " relevant regulations progress, in such a way that definite value is combined in the laboratory Duo Jia, level of organization sample assignment certificate of entrustment and operation refer to
Book is led, each definite value laboratory sends 3 samples, it is desirable that laboratory uses " cosmetics safety technical specification 2015 editions " to carry out gold
The qualitative detection of staphylococcus aureus, each sample feed back 2 retests as a result, counting to the result of laboratory feedback
Macro or mass analysis determines the characteristic value of standard sample.
Embodiment 3
The preparation method of moulds and yeasts count standard sample is each in water soluble cosmetics described in the present embodiment
Step is in the same manner as in Example 2, different technical parameters are as follows: during bacteria suspension is prepared, each object bacteria in target flora suspension
Concentration is according to 5 × 103CFU/mL is prepared, and the concentration of each background bacterium is according to 10 in background flora suspension3CFU/mL is prepared；It was lyophilized
Journey 52h.
Embodiment 4
The preparation method of moulds and yeasts count standard sample is each in water soluble cosmetics described in the present embodiment
Step is in the same manner as in Example 3, different technical parameters are as follows: during bacteria suspension is prepared, each object bacteria in target flora suspension
Concentration is according to 4.5 × 103CFU/mL is prepared；Freeze-drying process 55h.
Claims (6)
1. moulds and yeasts count standard sample in water soluble cosmetics, it is characterized in that: including target flora and background flora,
The target flora is made of saccharomyces cerevisiae, aspergillus niger, and the background flora is by Bacillus cereus, escherichia coli, gold
Staphylococcus aureus composition.
2. moulds and yeasts count standard sample in water soluble cosmetics according to claim 1, it is characterized in that: described
Sample matrices are toner, and makeup water volume fraction is 0.5%；Protective agent is formed with trehalose and sterile water, wherein trehalose body
Fraction is 10%.
3. moulds and yeasts count standard sample in water soluble cosmetics according to claim 1 or 2, it is characterized in that:
The aimed concn of target flora is 10 in the sample2~103CFU/mL, the aimed concn of background flora are 103 CFU/mL。
4. the preparation method of moulds and yeasts count standard sample in water soluble cosmetics according to claim 1,
It is characterized in: preparation, sample freeze-drying, the uniformity of sample and stability test, sample definite value four steps including freeze drying protectant
Suddenly, wherein sample freeze-drying process are as follows: bacterial strain recovery passage-increasing bacterium-bacteria suspension preparation-freeze-drying-packaging-storage, specific mistake
Journey are as follows:
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, so that 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 the jelly of 10mL
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 obtains is mixed with freeze drying protectant, according to target the aimed concn 10 of flora2~103CFU/mL and
The aimed concn 10 of background flora3 CFU/mL prepares bacteria suspension, prepare respectively 2 object bacterias bacteria suspension and 3 background bacterium
Bacteria suspension is mixed to form target flora suspension and background flora suspension by 1:1 volume ratio respectively, then by target flora suspension and carries on the back
Scape flora suspension is mixed by 1:1 volume ratio, is obtained plastc ring, is placed on magnetic stirring apparatus and is stirred continuously, 1:100 ratio adds
Add toner, is dispensed into sample bottle after mixing is sufficiently stirred, in addition bottle stopper, but reality is not covered there are gap；
(4) it is lyophilized and packs
Sample bottle equipped with plastc ring is uncapped and is put into freeze dryer, 50 ~ 55h is freeze-dried, 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) it stores
Sample is placed under the conditions of -15 ~ -20 DEG C and is kept in dark place, random selected sample is issued to laboratory from whole samples
Or carry out uniformity and stability test.
5. the preparation method of moulds and yeasts count standard sample in water soluble cosmetics according to claim 4,
Be characterized in: the sample matrices are toner, and makeup water volume fraction is 0.5%；Protective agent is formed with trehalose and sterile water,
Wherein trehalose volume fraction is 10%.
6. the preparation method of moulds and yeasts count standard sample in water soluble cosmetics according to claim 4,
Be characterized in: 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 " it carries out.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
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- 2019-02-28 CN CN201910149258.0A patent/CN109880880A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
Non-Patent Citations (1)
| Title |
|---|
| 税丕容: "微生物检测在化妆品质量检验中的应用", 《化工管理》 * |
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