Preservatives are a very important part of the cosmetics industry. With the variety of cosmetic types and dosage forms, and the increasing use of high-tech bio-materials, many cosmetic companies are actively researching preservatives (including their variety and concentration) and constantly establishing new anti-corrosion systems. Among the practical effects of evaluating preservatives, the most widely used and classical research method is the microbial challenge test (m icrob ialchallenge test ing). This test method is based on the possible contamination (primary and secondary pollution) in cosmetic production and consumer use, artificially adding a certain amount of microorganisms (bacteria and mold) to the product, and then analyzing the product at intervals. The effect of preservatives on microorganisms is based on the amount of bacteria added to the bacteria during the test to judge whether the preservative has antiseptic effect or not. This method has high reference value in evaluating the antiseptic effect of preservatives.
First, materials and methods
1. Anti-corrosion system commonly used in cosmetics
The antiseptic system commonly used in 8 kinds of cosmetics was selected and added to the cream in the conventional way, and 2 control creams (without any preservatives) were set at the same time. Antiseptic system 1 is 011% domestic fungus mollusc; system 2 is 011% domestic fungus, 012% methylparaben, 011% propylparaben; system 3 is 0104% domestic CY21; system 5 is domestic CY21 012% methylparaben, 011% propylparaben; system 5 is a traditional methylparaben and propyl ester compatible with uranium; preservative systems 6, 7 and 8 contain the same preservatives, System 6 is at a low concentration, system 7 is at a high concentration, and system 8 is at a high concentration compatible with other preservatives. Shanghai Chuangsai Technology provides methylparaben, methylparaben, Methylparaben, >99%, USP, 99-76-3, commodity number: C84-2041-25G, price 140 yuan.
2. Microbial challenge experiment
1 test microorganism
Bacteria include: Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Bacillus megaterium, Pseudomonas aeruginosa. Molds include: Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium citrinum, Paecilomyces sphaeroides, Rhizoctonia solani, Chaetomium globosa, Trichoderma viride
Before the experiment, each strain was inoculated into a suitable medium and cultured at 37 ° C (bacteria) or 28 ° C (molar). After 2 days of bacterial culture, after 5 days of mold culture, select appropriate amount of colony in sterile physiological saline to prepare a certain concentration of mixed bacteria (1 × 108 ömL) or mixed mold suspension (1 × 107 ömL). Store at 4 ° C for later use. Shanghai Chuangsai Technology provides 7220-81-7, aflatoxin B2 (>98%, BR), Aflatoxin B2, commodity number: C84-0627-1 mg, and the price is 1482 yuan.
2 28-day microbial challenge test for one-time addition
30 g of each test sample was weighed, and mixed bacteria or mixed mold suspension was added, and the final bacterial amount per gram of the test cream was 5 × 106 bacteria and 3 × 105 molds, respectively. Then mix well and place at 28 °C. Sampling and analysis on 0, 7, 14, 21 and 28 days of inoculation: Accurately weigh 3g of sample, add it to a sterile conical flask containing glass beads, add 27mL of sterile physiological saline, and mix thoroughly. The suspension was diluted 1:10; then diluted 10 times with sterile saline. The amount of bacteria contained in the test article was counted according to the flat plate pouring method. The bacterial culture was carried out at 37 ° C for 24 h to 48 h, and the mold culture was carried out at 28 ° C for 3 to 5 days. This experiment was used to judge the effectiveness of the preservative. Shanghai Chuangsai Technology provides 94-13-3, propylparaben, GCS, commodity number: D16-1010228-5ml, the price is 98 yuan.
The evaluation criteria are as follows: When the bacteria are inoculated once per gram of sample (1×106 bacteria and 1×105 mold), the amount of viable bacteria on the 14th day is reduced to no more than 011% of the initial concentration, and then gradually decreases, at 28 The day is 0. The standard is preservative effective (tested), non-compliant with preservatives (not tested). Shanghai Chuangsai Technology provides MUG rapid test E. coli reagent, BR, commodity number: D16-1024759-250g, inquiry.
3 repeated 3 times of microbial challenge test
This method is based on the microbiological challenge test recommended by the International CTFA (International Cosmetics, Fragrance and Detergent Association). Weigh 30g of the test sample and add it once every 2 weeks (ie, the first, third and fifth weeks of the experiment), adding 1×106～1×107 ög samples and 1×105 molds each time. ö g～ 1×106 ög samples. Samples were analyzed for samples containing bacteria at 0, 7, and 14 days after the addition of bacteria (before the addition of bacteria). The method was the same as before. This method can divide the tested samples into three categories:
(1) Excellent antiseptic effect (W), that is, after three additions, on the 7th or 14th day after each addition, the amount of viable bacteria is reduced to no more than 0101% of the initial concentration (ie ≤100 ög Or mL sample, passed the test).
(2) The antiseptic effect is still acceptable (M), that is, after three additions, the amount of viable bacteria is reduced to no more than 011% of the initial concentration on the 7th or 14th day after each addition (ie ≤1000) A sample of ög or mL, passed the test).
(3) Poor antiseptic effect (P), that is, after three additions, on the 7th or 14th day after each addition, the amount of viable bacteria is >1000 ög or mL samples (not tested).
Second, the results
From the results, the evaluation results of the two methods of adding anti-corrosion system are basically the same. The three-time addition can also make a difference in the degree of effective anti-corrosion system: W: Well p reservat ive and anti-corrosion (M :M arginal p reservat ive). In addition, within 1 to 2 weeks after one addition, the amount of bacteria in the sample can be reduced to 011% of the added bacteria, and the antibacterial activity of the bacteria can be passed through three addition experiments, such as preservative systems 5 and 8. If it is greater than 011%, even if there is a tendency to gradually decrease to 0 in the later test period, it may be difficult to pass the three-time addition experiment, such as the preservative system 7 for the anti-corrosion of bacteria.
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