CN105603042A - Pyocyanine culture medium and method for detecting Pseudomonas aeruginosa in cosmetics - Google Patents

Abstract

The invention discloses a pyocyanin culture medium and a method for detecting pseudomonas aeruginosa in cosmetics. The pyocyanin culture medium of the present invention contains lecithin 0.8～1.2g per liter, Tween-80? 6.0～8.0g, peptone 18.0～20.0g, magnesium chloride 1.4～1.6g, potassium sulfate 8.0～16.0g, trichloro Raw (2,4,4′-trichloro-2′-hydroxydiphenyl ether) 0.05～0.1g, gelatin 25.0～30.0g and agar 13.0～15.0g, the balance is water, pH6.9～7.1. In the present invention, after the cosmetic sample is enriched with SCDLP enrichment liquid, the pyocyanin medium is used to directly conduct a growth test at 42°C to screen and detect Pseudomonas aeruginosa, and observe the screening and training results, such as colonies appearing after screening and training It is further identified in combination with the gelatin liquefaction test. The invention simplifies the operation steps of the Pseudomonas aeruginosa detection method, improves the detection efficiency, and can be used for the detection of Pseudomonas aeruginosa in daily cosmetics.

Description

A kind of pyocyanin culture medium and method for detecting Pseudomonas aeruginosa in cosmetics

technical field

The invention belongs to the field of cosmetic microbial safety detection, and in particular relates to a pyocyanin culture medium and a method for detecting Pseudomonas aeruginosa in cosmetics.

Background technique

Pseudomonas aeruginosa (Pseudomonas aeruginosa) is widely distributed in water, air, soil and the surface of objects in nature, and can also be found in the body surface and intestinal tract of normal humans and animals. Gram-negative bacilli that are resistant to a variety of antibiotics can infect the human body through contact with damaged skin and mucous membranes. They are important opportunistic pathogens that can cause pneumonia and sepsis in patients in severe cases. In recent years, cosmetics have increasingly become a necessity in people's daily life. The nutrients in cosmetics (such as protein, oil, vitamins, etc.) are conducive to the growth and metabolism of Pseudomonas aeruginosa, and their metabolites can decompose some components of cosmetics , causing product corruption and deterioration, and the produced pyocyanin can irritate the user's skin, and even cause allergies or poisoning, seriously endangering the health of consumers. Therefore, "Hygienic Standards for Cosmetics" (2007), ISO22717 and EU76/78/768/EEC stipulate that Pseudomonas aeruginosa should not be detected. At present, when my country conducts hygienic evaluation of cosmetics, it adopts the method stipulated in the "Hygienic Standards for Cosmetics" (2007). It is officially released and will be implemented on December 1, 2016. However, the detection method for Pseudomonas aeruginosa has not been improved or simplified. A simple, fast and effective detection method is very important.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of tedious steps and long detection time in the detection method of Pseudomonas aeruginosa in the prior art, and provide a simple, fast and efficient method for detecting Pseudomonas aeruginosa in cosmetics Pyocyanin media and methods.

The first object of the present invention is to provide a pyocyanin culture medium for detecting Pseudomonas aeruginosa in cosmetics.

The pyocyanin culture medium of the present invention contains lecithin 0.8～1.2g, Tween-806.0～8.0g, peptone 18.0～20.0g, magnesium chloride 1.4～1.6g, potassium sulfate 8.0～16.0g, triclosan ( 2,4,4'-trichloro-2'-hydroxydiphenyl ether) 0.05-0.1g, gelatin 25.0-30.0g, agar 13.0-15.0g, the balance is water, pH 6.9-7.1.

The preparation method of the described pyocyanin culture medium: according to the component content of the described pyocyanin culture medium, lecithin and Tween-80 are heated and dissolved in distilled water first, then add peptone, magnesium chloride, potassium sulfate , triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether), gelatin and agar, mix well, adjust the pH value to 6.9-7.1; autoclave at 121°C for 20min.

The second object of the present invention is to provide a method for detecting Pseudomonas aeruginosa in cosmetics.

The method for detecting Pseudomonas aeruginosa in cosmetics of the present invention is characterized in that the cosmetic samples are taken for enrichment culture to obtain the enrichment culture solution, and then the enrichment culture solution is inserted into the pyocyanin culture medium for cultivation, If no colonies grow on the pyocyanin medium, the cosmetic sample does not contain Pseudomonas aeruginosa; if colonies grow on the pyocyanin medium, add a saturated aqueous solution of ammonium sulfate dropwise to the colonies Carry out gelatin liquefaction test on gelatin liquefaction test, if the gelatin liquefaction test is positive, the cosmetic sample contains Pseudomonas aeruginosa; the described pyocyanin culture medium contains lecithin 0.8～1.2g, Tween-806.0～8.0g, peptone per liter 18.0～20.0g, magnesium chloride 1.4～1.6g, potassium sulfate 8.0～16.0g, triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) 0.05～0.1g, gelatin 25.0～30.0g And agar 13.0 ~ 15.0g, the balance is water, pH6.9 ~ 7.1.

Preferably, adding the enrichment culture solution into the pyocyanin medium for culture is to culture at 42±1°C for 24h-48h.

Preferably, the cosmetic sample is taken for enrichment culture to obtain the enrichment culture solution. When the cosmetic sample is hydrophilic, it is mixed according to the ratio of 1g cosmetic sample: 9mL SCDLP enrichment solution to prepare the cosmetic sample preparation. test solution; when the cosmetic sample is hydrophobic, according to the ratio of 1g cosmetic sample: 8mL SCDLP enrichment solution: 1mL Tween-80, mix well to prepare the cosmetic sample test solution; then prepare the cosmetic sample test solution Place in a constant temperature shaker at 36°C±1°C at 250rpm for enrichment and culture for 18h.

Preferably, the method is also provided with a blank control solution and a positive control solution equal in volume to the cosmetic sample preparation solution, the blank control solution is a SCDLP enrichment solution, and the positive control solution is 100-1000 cfu/ Prepared by mixing the ratio of Pseudomonas aeruginosa solution: 9mL SCDLP enrichment solution.

The Pseudomonas aeruginosa is the standard strain ATCC9027 of Pseudomonas aeruginosa.

The described method of inserting the enrichment culture solution into the pyocyanin medium for cultivation is to take 100 μL of the enrichment culture solution of the cosmetic sample after the enrichment culture and add it to the pyocyanin medium in the petri dish, and spread it evenly , to be cultured after solidification.

In the present invention, after the cosmetic sample is enriched with SCDLP enrichment liquid, the pyocyanin medium is used to directly carry out the growth test at 42°C, to screen and detect Pseudomonas aeruginosa, and to observe the culture results. Combined with gelatin liquefaction test identification.

According to the detection method of the present invention, after being cultivated through the pyocyanin medium, if the pyocyanin medium of the enrichment culture solution of the blank control or the cosmetic sample is added, the bacterium colony of Pseudomonas aeruginosa cannot be cultivated, On the pyocyanin substratum of the enrichment culture solution of the positive control, a colony of Pseudomonas aeruginosa is cultivated, which indicates that there is no Pseudomonas aeruginosa in the cosmetic sample; if the enrichment culture solution of the blank control is added The colony of Pseudomonas aeruginosa could not be cultivated on the pyocyanin medium, but the colony of Pseudomonas aeruginosa could be cultured on the pyocyanin medium of the enrichment medium with cosmetic samples or positive control Colonies, and the colonies are small, green or yellow-green, moist and opaque, then the gelatin liquefaction test is further carried out on the colonies on the pyocyanin medium added with the enrichment culture medium of cosmetic samples for detection, if ammonium sulfate saturated aqueous solution After being added dropwise to the above-mentioned colony for 10 minutes, a clear band appears around the colony, which is positive, indicating that Pseudomonas aeruginosa does exist in the cosmetic sample.

Compared with the prior art, the present invention has the following beneficial effects:

(1) Use pyocyanin medium to carry out strain screening, wherein magnesium chloride and potassium sulfate are pyocyanin chromogenic agents in the pyocyanin medium formula; triclosan (2,4,4'-trichloro- 2'-hydroxydiphenyl ether) is an efficient broad-spectrum antibacterial agent, which has killing and inhibiting effects on Gram-positive bacteria, negative bacteria, yeast and viruses, and its minimum inhibitory concentration on Pseudomonas aeruginosa is far from Higher than other strains; lecithin and Tween-80 are neutralizers, which can further neutralize the preservatives in the sample to be tested and improve detection sensitivity; adding gelatin to the medium can prepare for gelatin liquefaction test, thereby improving detection efficiency .

(2) Put the inoculated pyocyanin culture medium into a biochemical incubator at 42±1°C for cultivation, and directly conduct the growth experiment at 42°C, which simplifies the operation steps and reduces the analysis cost. This method can be used for aeruginosa in daily cosmetics Detection of Pseudomonas.

(3) The blank control solution and positive control solution are added in the sample preparation, which can not only check whether the equipment and culture medium used are completely sterilized and whether the aseptic operation procedures are followed during the inspection process, so as to help find problems in the operation, Moreover, the positive result can be used as a reference to improve the screening efficiency of bacterial strains and ensure the accuracy of the determination results.

detailed description

The following examples are to further illustrate the present invention, rather than limit the present invention.

The SCDLP enrichment solution used in the following examples contains per liter: casein peptone 17g, soybean peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, lecithin 1g, Tween-807g, and the balance is water, adjust the pH to 7.2. The preparation method is that the lecithin is heated and dissolved with distilled water, and then mixed with other ingredients, the pH value is adjusted, and the lecithin is sterilized under high pressure at 121 DEG C for 20 minutes.

Example 1: Detection method of Pseudomonas aeruginosa in toner

Preparation of pyocyanin medium: firstly dissolve lecithin 1.2g and Tween-806.0g with distilled water, then add peptone 20.0g, magnesium chloride 1.4g, potassium sulfate 10.0g, triclosan (2,4,4 '-trichloro-2'-hydroxydiphenyl ether) 0.05g, gelatin 30.0g and agar 13.0g, add water to 1L, stir and mix, adjust the pH value to 6.9, and autoclave at 121°C for 20min.

Detection method:

(1) Bacterial enrichment culture: Aseptically weigh 10g of toner and add it into a glass bottle containing 90mL of SCDLP bacterium enrichment solution, and mix well to prepare a cosmetic sample preparation solution; add 100mL of SCDLP bacterium enrichment solution into another glass bottle, Prepare a blank control solution; add 10 mL of 100 cfu/mL Pseudomonas aeruginosa standard strain ATCC9027 bacteria solution into another glass bottle containing 90 mL of SCDLP enrichment solution, and mix well to prepare a positive control solution. The prepared cosmetic sample preparation solution, blank control solution and positive control solution were respectively placed in a constant temperature shaker at 36°C±1°C at 250rpm to enrich the bacteria for 18h.

(2) Cultivation of pyocyanin medium: take 100 μL each of the enrichment culture solution of the cosmetic sample obtained after enrichment culture, blank control or positive control, and add it to the pyocyanin medium in the plate, spread evenly, After solidification, culture in a constant temperature biochemical incubator at 42±1°C for 48 hours, and count the colonies.

After culturing, Pseudomonas aeruginosa colonies could not be cultivated on the pyocyanin culture medium of the enrichment medium of the blank control, while the pyocyanin culture of the enrichment medium of the positive control Yellow-green colonies were cultured on the base respectively, the colonies were small, moist and opaque. It shows that there are suspicious colonies of Pseudomonas aeruginosa on the pyocyanin medium of the enrichment culture medium added with cosmetic samples. Further verification with gelatin liquefaction test is needed.

(3) Gelatin liquefaction test: the ammonium sulfate saturated aqueous solution was added dropwise to the colonies on the pyocyanin medium of the enrichment culture solution added with cosmetic samples. After 10 minutes, a clear band appeared around the colonies, that is, the gelatin liquefaction test showed Positive. It shows that Pseudomonas aeruginosa does exist in cosmetic samples.

Embodiment 2: the detection method of Pseudomonas aeruginosa in facial cleanser

Preparation of pyocyanin medium: firstly dissolve 1.0g lecithin and Tween-808.0g with distilled water, then add 18.0g peptone, 1.6g magnesium chloride, 8.0g potassium sulfate, triclosan (2,4,4 '-trichloro-2'-hydroxydiphenyl ether) 0.1g, gelatin 28.0g and agar 15.0g, add water to 1L, stir and mix, adjust the pH value to 7.0, and autoclave at 121°C for 20min.

Detection method:

(1) Bacterial enrichment culture: Aseptically weigh 10g of facial cleanser and add it to a glass bottle containing 90mL of SCDLP enrichment solution, and mix well to prepare a test solution for cosmetic samples; add 100mL of SCDLP bacteria enrichment solution into another glass bottle 10mL400cfu/mL Pseudomonas aeruginosa standard strain ATCC9027 bacteria solution was added to another glass bottle containing 90mL SCDLP enrichment solution, and mixed well to prepare a positive control solution. The prepared cosmetic sample preparation solution, blank control solution and positive control solution were respectively placed in a constant temperature shaker at 36°C±1°C at 250rpm to enrich the bacteria for 18h.

(2) Cultivation of pyocyanin culture medium: take 100 μL each of the cosmetic sample obtained after enrichment culture, blank control or positive control enrichment culture solution, add them to the pyocyanin medium in the plate, and spread evenly , after solidification, culture in a constant temperature biochemical incubator at 42±1°C for 35 hours, and count the colonies.

After culturing, Pseudomonas aeruginosa colonies could not be cultivated on the pyocyanin culture medium of the enrichment medium of the blank control, while the pyocyanin culture of the enrichment medium of the positive control Green colonies were cultivated on the base respectively, the colonies were small, moist and opaque. It shows that there are suspicious colonies of Pseudomonas aeruginosa on the pyocyanin medium of the enrichment culture medium added with cosmetic samples. Further verification with gelatin liquefaction test is needed.

(3) Gelatin liquefaction test, ammonium sulfate saturated aqueous solution is added dropwise to the bacterium colony on the pyocyanin culture medium of the enrichment culture solution of cosmetic sample, after 10 minutes, a clear band appears around the bacterium colony, namely gelatin liquefaction test shows Positive. It shows that Pseudomonas aeruginosa does exist in cosmetic samples.

Embodiment 3: the detection method of Pseudomonas aeruginosa in liquid foundation

Preparation of pyocyanin medium: first lecithin 0.8g and Tween-807.0g were heated and dissolved with distilled water, then respectively added peptone 18.0g, magnesium chloride 1.4g, potassium sulfate 12.0g, triclosan (2,4,4 '-trichloro-2'-hydroxydiphenyl ether) 0.1g, gelatin 25.0g and agar 13.0g, add water to 1L, stir and mix, adjust the pH value to 7.1, and autoclave at 121°C for 20min.

Detection method:

(1) Bacterial enrichment culture: Aseptically weigh 10g of foundation liquid and add it to a glass bottle containing 80mL of SCDLP bacterial enrichment solution, add 10mL of Tween-80, and mix well to prepare a cosmetic sample test solution; 100mL of SCDLP bacterial enrichment solution into another glass bottle to prepare a blank control solution; add 10mL of 700cfu/mL Pseudomonas aeruginosa standard strain ATCC9027 bacteria solution into another glass bottle containing 90mL of SCDLP enrichment solution, and mix well to obtain a positive control solution. The prepared cosmetic sample preparation solution, blank control solution and positive control solution were respectively placed in a constant temperature shaker at 36°C±1°C at 250rpm to enrich the bacteria for 18h.

(2) Cultivation of pyocyanin medium: take 100 μL each of the enrichment culture solution of the cosmetic sample obtained after enrichment culture, blank control or positive control, and add it to the pyocyanin medium in the plate, spread evenly, After solidification, culture in a constant temperature biochemical incubator at 42±1°C for 30 hours, and count the colonies.

After culturing, only the pyocyanin culture medium with the positive control enrichment medium could produce Pseudomonas aeruginosa colonies, while the pyocyanin culture with the cosmetic sample or the blank control enrichment medium No colonies were cultivated on the base, indicating that Pseudomonas aeruginosa did not exist in the cosmetic samples.

Embodiment 4: the detection method of Pseudomonas aeruginosa in facial mask cream

Preparation of pyocyanin medium: firstly dissolve 1.0g lecithin and Tween-807.0g with distilled water, then add 20.0g peptone, 1.4g magnesium chloride, 16.0g potassium sulfate, triclosan (2,4,4 '-trichloro-2'-hydroxydiphenyl ether) 0.05g, gelatin 30.0g and agar 14.0g, add water to 1L, stir and mix, adjust the pH value to 7.1, and autoclave at 121°C for 20min.

Detection method:

(1) Bacterial enrichment culture: Aseptically weigh 10g of facial mask cream and add it to a glass bottle containing 80mL of SCDLP bacterial enrichment solution, add 10mL of Tween-80, and mix well to prepare a cosmetic sample test solution; 100mL of SCDLP bacterial enrichment solution into another glass bottle to prepare a blank control solution; add 10mL of 1000cfu/mL Pseudomonas aeruginosa standard strain ATCC9027 bacteria solution into another glass bottle containing 90mL of SCDLP enrichment solution, and mix well to obtain Positive control solution. The prepared cosmetic sample preparation solution, blank control solution and positive control solution were respectively placed in a constant temperature shaker at 36°C±1°C at 250rpm to enrich the bacteria for 18h.

(2) Cultivation of pyocyanin medium: take 100 μL each of the cosmetic sample obtained after the enrichment culture, blank control or positive control solution, and add them to the pyocyanin medium in the plate, spread evenly, and wait for solidification, Place in a constant temperature biochemical incubator at 42±1°C for 24 hours and count the colonies.

After culturing, Pseudomonas aeruginosa colonies could not be cultivated on the pyocyanin culture medium of the enrichment medium of the blank control, while the pyocyanin culture of the enrichment medium of the positive control Yellow-green colonies were cultured on the base respectively, the colonies were small, moist and opaque. It shows that there are suspicious colonies of Pseudomonas aeruginosa on the pyocyanin medium of the enrichment culture medium added with cosmetic samples. Further verification with gelatin liquefaction test is needed.

(3) Gelatin liquefaction test, ammonium sulfate saturated aqueous solution is added dropwise to the bacterium colony on the pyocyanin culture medium of the enrichment culture solution of cosmetic sample, after 10 minutes, a clear band appears around the bacterium colony, namely gelatin liquefaction test shows Positive. It shows that Pseudomonas aeruginosa does exist in cosmetic samples.

Comparative Example 1: Comparison of the inventive method used in Examples 1-4 and the standard method of "Hygienic Standards for Cosmetics" (2007)

In order to verify the correctness and reliability of the detection results utilizing the method of the present invention, the inventors respectively get an additional copy of the cosmetic samples taken in Examples 1-4 under the same conditions and use the "Hygienic Standards for Cosmetics" (2007) standard method to detect them. The tests were carried out, and the specific test results are summarized in Table 1. As can be seen from Table 1, the inventive method only needs to use 2 kinds of mediums, and the time-consuming detection experiment is only half or shorter than that of the standard method, and the detection result is completely consistent with that of the standard method; the inventive method has the advantages of simple operation, The advantages of cost saving, fast and high accuracy.

Table 1 The inventive method compares with standard method detection result

Claims (8)

1. a pyo culture medium, is characterized in that, every liter contain lecithin 0.8～1.2g, Tween-80 6.0～8.0g, peptone 18.0～20.0g, magnesium chloride 1.4～1.6g, potassium sulfate 8.0～16.0g, 2,4, the chloro-2'-hydroxyl two of 4'-tri-Phenylate 0.05～0.1g, gelatin 25.0～30.0g and agar 13.0～15.0g, surplus is water, pH6.9～7.1.

2. a preparation method for pyo culture medium claimed in claim 1, is characterized in that, by described pyoThe constituent content of culture medium, first by lecithin and Tween-80 distilled water heating for dissolving, then adds peptone, magnesium chloride, sulfuric acidPotassium, 2,4, the chloro-2'-dihydroxy diphenyl ether of 4'-tri-, gelatin and agar, mix adjust pH 6.9～7.1.

3. for detection of a method for pseudomonas aeruginosa in cosmetics, it is characterized in that, get cosmetic sample and increase bacteriumCultivation obtains enrichment culture medium, then enrichment culture medium is accessed in pyo culture medium and is cultivated, if pyo is cultivatedOn base, do not grow bacterium colony, in cosmetic sample, do not contain pseudomonas aeruginosa; If grow bacterium colony on pyo culture medium,Ammonium sulfate saturated aqueous solution is dripped and on described bacterium colony, carry out gelatin liquefaction test, gelatin liquefaction test is positive and changesIn adornment product sample, contain pseudomonas aeruginosa; Every liter of described pyo culture medium contain lecithin 0.8～1.2g, tween-806.0～8.0g, peptone 18.0～20.0g, magnesium chloride 1.4～1.6g, potassium sulfate 8.0～16.0g, 2,4,4'-tri-is chloro-2'-dihydroxy diphenyl ether 0.05～0.1g, gelatin 25.0～30.0g and agar 13.0～15.0g, surplus is water, pH6.9～7.1.

4. method according to claim 3, is characterized in that, described pyo culture medium that enrichment culture medium is accessedMiddle cultivation is to be placed in 42 ± 1 DEG C to cultivate 24h～48h.

5. method according to claim 3, is characterized in that, the described cosmetic sample of getting increases bacterium and cultivates and increasedBacteria culture fluid, when described cosmetic sample is while being hydrophilic, in 1g cosmetic sample: the ratio of 9mLSCDLP enrichment liquidMix, prepare the standby inspection of cosmetic sample solution; When described cosmetic sample is while being hydrophobic, by 1g cosmetics sampleProduct: 8mLSCDLP enrichment liquid: the ratio of 1mL Tween-80 mixes, prepare the standby inspection of cosmetic sample solution; Then will make upThe standby inspection of product sample solution is placed in 36 DEG C ± 1 DEG C constant-temperature table 250rpm and increases bacterium cultivation 18h.

6. method according to claim 5, is characterized in that, is also provided with the standby inspection of cosmetic sample solution isopyknicBlank liquid and positive control solution, described blank liquid is SCDLP enrichment liquid, described positive control solution be by1mL100～1000cfu/mL pseudomonas aeruginosa liquid: the ratio of 9mLSCDLP enrichment liquid mixes preparation.

7. method according to claim 6, is characterized in that, described pseudomonas aeruginosa is pseudomonas aeruginosa standardStrains A TCC9027.

8. method according to claim 5, is characterized in that, described pyo culture medium that enrichment culture medium is accessedMiddle cultivation is to get 100 μ L to be added to the pyo cultivation in culture dish through increasing the enrichment culture medium of the cosmetic sample after bacterium is cultivatedOn base, smoothen, after solidifying, cultivate again.