Application of bacillus subtilis lipopeptide sodium and preservative composition containing bacillus subtilis lipopeptide sodium

Abstract

The invention relates to application of sodium surfactin and a preservative composition containing the sodium surfactin. The invention relates to the technical field of product corrosion prevention, in particular to application of sodium surfactin to improvement of influence of preservative components on skin microecology and application of sodium surfactin to preparation of a preservative composition friendly to skin microecology.

Description

Technical Field

[0001] The present application relates to the technical field of product preservation, and in particular to the use of sodium subtilisin and a preservative composition containing the same. Background Art

[0002] Topical skin products contain other ingredients such as water and functional ingredients, which are easily infected and deteriorated by microorganisms. For example, in the field of cosmetics, lotions or emulsions without preservatives are prone to breed bacteria, causing them to deteriorate and may cause damage to the skin. Therefore, adding an appropriate amount of preservatives can protect the quality and safety of the product. However, based on the fact that preservatives need to indiscriminately kill microorganisms in the product to ensure their preservative effect, they will inevitably kill beneficial microorganisms on the skin indiscriminately during the use of the product. This may destroy the original micro-ecological system of the skin on the one hand, and on the other hand, it may also destroy the survival and growth of bacteria that are beneficial to the skin, leading to skin problems due to flora disorders, such as barrier damage.

[0003] Therefore, how to improve the adverse effects of preservatives on skin flora and develop a preservative composition that is friendly to the skin microecology remains a technical problem that needs to be solved. Summary of the invention

[0004] In view of the above technical problems, the present application found through in-depth research that it is not necessary to add ingredients that are beneficial to the skin microecology to the commonly used preservatives, such as the β-glucan tested in the present application, to improve the adverse effects of preservatives on the skin flora. At the same time, the present application unexpectedly found that sodium subtilisin can effectively improve the adverse effects of preservative ingredients on the skin flora without affecting the preservative effect of the preservative ingredients themselves, thereby completing the present application.

[0005] This application involves the following:

[0006] 1. Use of sodium subtilisin in improving the effects of preservative ingredients on skin microecology.

[0007] 2. The use according to item 1, comprising making the surviving number of beneficial skin bacteria greater than the surviving number of harmful skin bacteria, preferably, the surviving number of beneficial skin bacteria / the surviving number of harmful skin bacteria ≥20.

[0008] 3. Use of sodium subtilisin in preparing a skin microecological friendly preservative composition.

[0009] 4. The use according to item 3, wherein the antiseptic composition comprises:

[0010] (a) sodium subtilisin, and

[0011] (b) at least one preservative ingredient.

[0012] 5. The use according to item 3 or 4, wherein when the antiseptic composition remains on the skin, the surviving number of beneficial bacteria on the skin is greater than the surviving number of harmful bacteria on the skin. Preferably, the surviving number of beneficial bacteria on the skin / the surviving number of harmful bacteria on the skin is ≥20.

[0013] 6. The use according to item 3 or 4, wherein when the antiseptic composition remains on the skin, the killing ability of harmful bacteria on the skin is higher than the killing ability of beneficial bacteria on the skin. Preferably, the killing ability of harmful bacteria on the skin / the killing ability of beneficial bacteria on the skin is ≥20.

[0014] 7. The use according to item 3 or 4, wherein the preservative composition is used to inhibit the survival and growth of microorganisms in the product.

[0015] 8. The use according to item 3, wherein the mass ratio of component (a) to component (b) is (0.01-20):1.

[0016] 9. A microecologically friendly antiseptic composition, the components of the composition are:

[0017] (a) sodium subtilisin, and

[0018] (b) at least one preservative ingredient.

[0019] 10. The composition according to item 9, wherein the mass ratio of component (a) to component (b) is (0.01-20):1.

[0020] Beneficial effects of this application:

[0021] The present application, by compounding sodium subtilisin and preservative ingredients, can change the killing behavior of the preservative ingredients on microorganisms during the time the product remains on the skin, for example, weaken the killing effect of the preservative ingredients on beneficial microorganisms and improve the adverse effects of the preservative ingredients on the skin flora;

[0022] Furthermore, the present application can achieve indiscriminate killing of microorganisms in the product over a long period of time by compounding sodium subtilisin and preservative ingredients, thereby not affecting the preservative effect of the preservative ingredients on the long-term storage of the product. Specific implementation methods

[0024] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in components' functions as the criterion for distinction.

[0025] As mentioned throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0026] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments. References to "about" a value or parameter herein include (and describe) variations to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0027] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0028] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. This statement is therefore intended to serve as antecedent basis for the use of exclusive terminology such as "only", "only" and the like in connection with the recitation of claim elements, or the use of a limitation of "no".

[0029] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] The present application provides the use of sodium subtilisin in improving the effects of preservative ingredients on skin microecology.

[0031] Subtilis lipopeptide sodium (INCI: SODIUM SURFACTIN) is produced by fermentation of Bacillus subtilis strains and is known as one of the most efficient biosurfactants. It is a lipopeptide biosurfactant consisting of a hydrophilic head composed of a peptide chain ring and an aliphatic hydrocarbon chain. Subtilis lipopeptide sodium in this application can be directly commercially available or homemade.

[0032] In some embodiments, the sodium subtilisin is obtained by self-production, for example, by fermentation with Bacillus subtilis.

[0033] In a specific embodiment, the fermentation medium used in the production of sodium subtilisin includes fatty alcohol or its derivatives. For example, the fatty alcohol includes dodecanol, tridecanol, tetradecanol, pentadecanol, heptadecanol, hexadecanol, cetostearyl alcohol, etc.; the fatty alcohol derivatives include fatty acids and / or fatty aldehydes; the fatty acids include hexadecanoic acid, octadecanoic acid, etc., and the fatty aldehydes include tetradecanoic acid, pentadecanal, etc.

[0034] In cosmetics, the role of preservatives is to protect the product from microbial contamination, extend the shelf life of the product, and ensure the safety of the product. Preservatives include permitted preservatives and unrestricted antimicrobial raw materials. Permitted preservatives are obviously irritating or toxic, so their dosage or scope of use is restricted. Unrestricted antimicrobial raw materials have certain antibacterial effects, and their irritation or toxicity is relatively small. Their dosage is not restricted in the cosmetic safety technical specifications. Therefore, in order to reduce the irritation or toxicity of permitted preservatives, permitted preservatives and unrestricted antimicrobial raw materials are often used together, or even only unrestricted antimicrobial raw materials are used as preservatives.

[0035] Permitted preservatives include: aldehydes (imidazolidinyl urea, bis(hydroxymethyl)imidazolidinyl urea, etc.); esters (parabens); quaternary ammonium salts (quaternium-15, stearyl trimethyl ammonium chloride, etc.); acids and their salts (benzoic acid, sorbic acid, etc.); phenols (benzylchlorophenol, phenylphenol, etc.); alcohol ethers (benzyl alcohol, phenoxyethanol, etc.), oxazolidines (5-bromo-5-nitro-1,3-dioxane, 7-ethylbicyclooxazolidine, etc.); inorganic salts (thiophene, silver chloride deposited on titanium dioxide, etc.); and others (methylisothiazolinone, sodium N-hydroxymethylglycinate, etc.).

[0036] Unrestricted antibacterial raw materials include: alcohols (ethanol, isopropanol, etc.); 1,2-diols (1,2-pentanediol, ethylhexylglycerol, etc.); organic acids (ferulic acid, caprylhydroxamic acid, etc.); medium-chain fatty acid monoglycerides (glyceryl caprylate, sorbitan caprylate, etc.); aromatic phenols and aromatic alcohols (phenylethyl alcohol, thymol, etc.); plant extracts (neem seed oil, tea tree essential oil, etc.); others (parahydroxyacetophenone, etc.).

[0037] The antiseptic ingredients that can be used in the present application are not limited to those listed above, and any substance with an antiseptic effect is included in the scope of the antiseptic ingredients of the present application.

[0038] In some embodiments, the use comprises increasing the number of viable skin-friendly bacteria to a greater number than the number of viable skin-harmful bacteria.

[0039] In some preferred embodiments, the surviving number of the beneficial skin bacteria / the surviving number of the harmful skin bacteria is ≥20, for example, the surviving number of the beneficial skin bacteria / the surviving number of the harmful skin bacteria can be ≥20, ≥30, ≥50, ≥100, ≥200, ≥300, ≥500, ≥600, ≥700, ≥800, ≥900, ≥1000, ≥1500, ≥2000, ≥2500, ≥3000, ≥3500, ≥4000, ≥4500, ≥5000, ≥5500, ≥6000, ≥6500, ≥7000, ≥7500, ≥8000, ≥8500, ≥9000, ≥9500, ≥10000, or even higher.

[0040] In some embodiments, the skin deleterious bacteria include Staphylococcus aureus.

[0041] In some embodiments, the beneficial skin bacteria include Staphylococcus epidermidis.

[0042] Those skilled in the art should understand that the sodium subtilisin peptide of the present application has similar effects in improving the effects of preservative ingredients on the skin microecology for beneficial skin bacteria with similar properties to Staphylococcus epidermidis, or harmful skin bacteria with similar properties to Staphylococcus aureus; for example, non-limiting optional beneficial skin bacteria and harmful skin bacteria may also include Staphylococcus capitis, diphtheroid bacteria, Pseudomonas aeruginosa, Propionibacterium, Candida albicans, non-pathogenic mycobacteria, etc.

[0043] The present application provides a use of sodium subtilisin in preparing a skin microecological friendly preservative composition.

[0044] In some embodiments, the preservative composition comprises:

[0045] (a) sodium subtilisin, and

[0046] (b) at least one preservative ingredient.

[0047] In some embodiments, the antiseptic composition, when left on the skin, allows the viable number of beneficial skin bacteria to be greater than the viable number of harmful skin bacteria.

[0048] In some preferred embodiments, the surviving number of the beneficial skin bacteria / the surviving number of the harmful skin bacteria is ≥20, for example, the surviving number of the beneficial skin bacteria / the surviving number of the harmful skin bacteria can be ≥20, ≥30, ≥50, ≥100, ≥200, ≥300, ≥500, ≥600, ≥700, ≥800, ≥900, ≥1000, ≥1500, ≥2000, ≥2500, ≥3000, ≥3500, ≥4000, ≥4500, ≥5000, ≥5500, ≥6000, ≥6500, ≥7000, ≥7500, ≥8000, ≥8500, ≥9000, ≥9500, ≥10000, or even higher.

[0049] In some embodiments, the antiseptic composition, when left on the skin, has a higher ability to kill bacteria harmful to the skin than to kill bacteria beneficial to the skin.

[0050] In some preferred embodiments, the killing ability of harmful bacteria to the skin / the killing ability of beneficial bacteria to the skin is ≥20, for example, the killing ability of harmful bacteria to the skin / the killing ability of beneficial bacteria to the skin can be ≥20, ≥30, ≥50, ≥100, ≥200, ≥300, ≥500, ≥600, ≥700, ≥800, ≥900, ≥1000, ≥1500, ≥2000, ≥2500, ≥3000, ≥3500, ≥4000, ≥4500, ≥5000, ≥5500, ≥6000, ≥6500, ≥7000, ≥7500, ≥8000, ≥8500, ≥9000, ≥9500, ≥10000, or even higher.

[0051] In the present application, the killing ability refers to the ability to inhibit the survival of microorganisms. For example, the killing ability can be evaluated by measuring the number of surviving microorganisms after mixing and culturing the microorganisms with the antiseptic composition of the present application.

[0052] In some embodiments, the value of "the ability to kill skin-harmful bacteria / the ability to kill skin-beneficial bacteria" is the value of "the surviving number of the skin-beneficial bacteria / the surviving number of the skin-harmful bacteria".

[0053] In some embodiments, the preservative composition is used to inhibit the survival and growth of microorganisms in a product.

[0054] The mass ratio of sodium subtilisin and preservative provided in the present application is set to improve the adverse effects of preservatives on skin flora. When sodium subtilisin needs to play other roles in the product, such as an emulsifier, it can be further added. The product is not limited to the mass ratio range provided in the present application.

[0055] In some embodiments, the mass ratio of the component (a) to the component (b) is any mass ratio range within the range of (0.01-20):1, (0.01-17):1, (0.01-8):1, (0.01-6):1 or (0.01-20):1; for example, in a specific embodiment, the mass ratio of the component (a) to the component (b) is 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0. 1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or any mass ratio in the range of (0.01-20):1.

[0056] In some embodiments, the preservative ingredient is added in an amount allowed to be added in cosmetics. In some embodiments, the preservative ingredient is added in an amount of 0.01-5wt%, 0.05-5wt%, 0.1-5wt%, 0.5-5wt%, 1-5wt%, 2-4wt%, 2.5-3.5wt% or any addition range within the range of 0.01-5wt%. For example, in a specific embodiment, the amount of the preservative component added is 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt% or any addition amount range within the range of 0.01-5wt%.

[0057] The present application also provides a microecologically friendly antiseptic composition, the components of which are:

[0058] (a) sodium subtilisin, and

[0059] (b) at least one preservative ingredient.

[0060] In some embodiments, the mass ratio of component (a) to component (b) is (0.01-20):1.

[0061] The limitations of sodium subtilisin and the preservative component, and the mass ratio are as described above. Example

[0062] The specific embodiments of the present application will be described in more detail below. Although the specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0063] Subtilisin sodium 1 was purchased from Bloomage Biotech Co., Ltd., and subtilisin sodium 2 was purchased from Kaneka Corporation.

[0064] Experimental Example 1 Microbial co-cultivation experiment

[0065] 1. Experimental Principle

[0066] The test sample was quantitatively divided into sterile test tubes, and a certain amount of skin pathogens (Staphylococcus aureus, see Table 1 for details) and representative strains of beneficial bacteria (Staphylococcus epidermidis, see Table 1 for details) were inoculated at the same time. A sterile PBS buffer solution was used as the control group, and the mixture was evenly mixed. After a certain period of time, the sample and the control group were taken out and quantitatively poured into sterile PBS buffer solution, and then mixed evenly by an oscillator, and the sample liquid was drawn into the culture medium for culture and counting. Finally, the two different strains of the sample group and the control group were counted separately to obtain the number ratio CFU (Staphylococcus epidermidis) / CFU (Staphylococcus aureus), which was compared with the control group CFU (Staphylococcus epidermidis) / CFU (Staphylococcus aureus). If the number of Staphylococcus epidermidis (resident bacteria) increased significantly, and the number of Staphylococcus aureus (pathogenic bacteria) decreased significantly, it was preliminarily considered that it was beneficial to the balance of skin microecology.

[0067] Table 1 Bacterial strain information

[0068] name Strain No. Staphylococcus aureus ATCC 6538 Staphylococcus epidermidis ATCC 12228

[0069] 2. Experimental steps

[0070] 1) Sample preparation

[0071] Sample set: Prepare sample solution according to Table 5.

[0072] Among them, embodiments 1-14 are configured as follows:

[0073] Weigh p-hydroxyacetophenone and add it into water, heat it to 70-80°C, stir it evenly, cool it to room temperature, discharge it, and then add different concentrations of lipopeptide sodium 1 into it.

[0074] Example 15 is configured as follows:

[0075] Weigh p-hydroxyacetophenone and hexylene glycol and add them into water, heat to 70-80°C, stir evenly, cool to room temperature, discharge, and then add 1% of subtilisin sodium.

[0076] Example 16 is configured as follows:

[0077] Weigh butanediol, pentanediol and ethylhexylglycerol and add them into water, heat to 70-80°C, stir evenly, cool to room temperature, discharge, and then add subtilisin sodium 1.

[0078] Example 17 is configured as follows:

[0079] Weigh p-hydroxyacetophenone and add it into water, heat it to 70-80°C, stir it evenly, cool it down to room temperature, discharge it, and then add subtilisin sodium 2.

[0080] Examples 18-21, Comparative Examples 3 and 4 were configured as follows:

[0081] Referring to the raw materials and contents in Table 2, add the raw materials of phase A into a beaker, stir quickly and evenly, and heat to 70-80°C. Heat the raw materials of phase B to 70-80°C. Add the raw materials of phase B to phase A and homogenize at 3000-5000RPM for 3-5 minutes. Cool to room temperature and discharge.

[0082] Table 2 Emulsion ingredients

[0083]

[0084] Embodiments 22-23 are configured as follows:

[0085] Referring to the raw materials and contents in Table 3, weigh all the raw materials and add them into a beaker, heat to 70-80°C, stir evenly, cool to room temperature, and discharge.

[0086] Table 3 Ingredients of aqueous solution

[0087]

[0088]

[0089] Comparative Example 1 was configured as follows:

[0090] Weigh p-hydroxyacetophenone and add it into water, heat it to 70-80℃, stir it evenly, cool it down to room temperature, and discharge it.

[0091] Comparative Example 2 is configured as follows:

[0092] Weigh p-hydroxyacetophenone and hexylene glycol and add them into water, heat to 70-80℃, stir evenly, cool to room temperature and discharge.

[0093] Comparative Examples 5 and 6 are configured as follows:

[0094] Weigh p-hydroxyacetophenone and add it into water, heat it to 70-80°C, stir it evenly, cool it to room temperature, discharge it, and then add different concentrations of β-glucan into it.

[0095] Control group: PBS buffer.

[0096] 2) Sample measurement and inoculation

[0097] Accurately measure the liquid sample into a sterile screw-cap test tube in the clean bench, 5 mL for each sample. Use an inoculation loop to pick up half a ring of bacteria from the Staphylococcus aureus plate and Staphylococcus epidermidis plate, add them to 10 mL of sterile saline, and mix them evenly. At this time, the concentration of the bacterial suspension is 10 7 -10 8 Then take 1 mL of the bacterial suspension and dilute it 10 times in 9 mL of sterile saline, mix well, and obtain the inoculated bacterial suspension. At this time, the concentration of the bacterial suspension is 10 6 -10 7 Then, 100 μL of Staphylococcus aureus and Staphylococcus epidermidis bacterial solution were added to the sample.

[0098] 3) Add bacteria content

[0099] The final inoculum size for all samples should be kept as consistent as possible and should be within 10 g of product. 5 CFU to 10 6 Between CFU.

[0100] 4) Sample storage after inoculation

[0101] During the test, the inoculated samples should be kept in an environment of 20-25°C.

[0102] 5) Sample and count the product at different time points

[0103] Mix carefully before each sampling to ensure homogeneity.

[0104] Take 0.5mL to 4.5mL PBS buffer and shake on an oscillator for 30s-1min to mix it thoroughly. This is a 10-fold dilution. Repeat this process to make 10-fold serial dilutions in the form of 1:100, 1:1000, etc. Pipette 1mL of the sample solution into a TSA solid plate. The plate count is 10-1. Use a coating rod to spread it evenly, and so on, until the appropriate gradient is counted.

[0105] Serial dilutions were performed using the spreading rod method.

[0106] 6) Colony culture after sampling and counting

[0107] Microorganisms will be grown on agar plates using the media, time and temperature shown in Table 4 below.

[0108] Table 4

[0109] Staphylococcus aureus 32.5±2.5℃ TSA culture for 3-5 days Staphylococcus epidermidis 32.5±2.5℃ TSA culture for 3-5 days

[0110] 7) Count the number of colonies after culture

[0111] If several dilutions show growth, use the dilution that shows an acceptable count range. Bacteria 30-300 CFU / plate. For specific colony counting principles, refer to the colony counting method in Chapter 5 Microbiological Testing Methods of the "Cosmetic Safety Technical Specifications 2015 Edition".

[0112] 8) Result evaluation

[0113] CFU of the growth control group in the same period of time 表皮葡萄球菌 / CFU 金黄色葡萄球菌 For comparison, if CFU 表皮葡萄球菌 / CFU 金黄色葡萄球菌 If the ratio of CFU U increases significantly, it means that the sample is conducive to the growth of Staphylococcus epidermidis and unfavorable to the growth of Staphylococcus aureus. It is preliminarily believed that the sample is conducive to the skin microecology. On the contrary, if CFU 表皮葡萄球菌 / CFU 金黄色葡萄球菌 If the ratio of is significantly reduced, it means that the sample is not conducive to the growth of Staphylococcus epidermidis but is more conducive to the growth of Staphylococcus aureus. It is preliminarily believed that the sample is not conducive to the skin microecology.

[0114] 3. Experimental results and conclusions

[0115] Table 5 Composition of individual preservative ingredients and sodium subtilisin

[0116]

[0117]

[0118] Summary: Using the co-culture method of Staphylococcus aureus and Staphylococcus epidermidis, at 6 hours, the CFU of Examples 1-23 表皮葡萄球菌 / CFU 金黄色葡萄球菌 The CFU of the control group was much larger than that of the control group. 表皮葡萄球菌 / CFU 金黄色葡萄球菌 , indicating that after adding sodium subtilisin to the preservative ingredients, the killing effect on Staphylococcus aureus is greater than that on Staphylococcus epidermidis, delaying the killing of Staphylococcus epidermidis, which is beneficial to the skin microecology. In contrast, after adding β-glucan to the preservative ingredients in Comparative Examples 5 and 6, the protection against Staphylococcus aureus and Staphylococcus epidermidis is indiscriminate, CFU 表皮葡萄球菌 / CFU 金黄色葡萄球菌 No significant changes.

[0119] Test Example 2 Anticorrosion Challenge Test

[0120] 1. Experimental methods:

[0121] (1) Sample sterility test.

[0122] (2) Transfer 20 g of sample to a sterile container. Vortex the bacterial suspension tube on a vortex shaker for about 15 seconds and inoculate once. The inoculation volume is 1%. Mix the product thoroughly for about 1 minute immediately after inoculation.

[0123] (3) The final inoculum volume is 10 per gram of product. 5 CFU to 10 6 During the CFU test, the inoculated samples were kept at 20-25°C.

[0124] (4) Sampling and recovery of products at different time points

[0125] Sampling recovery and counting were carried out at 7d, 14d and 28d respectively.

[0126] (5) Before each sampling, mix to ensure homogeneity.

[0127] (6) The minimum sample volume is 1 ml of product + 9 ml of neutralizer in a test tube containing glass beads. Shake, vortex, and invert until evenly mixed.

[0128] (7) Plate counts for serial dilutions were performed in sterile saline. After serial dilutions were completed, plates were cast in duplicate.

[0129] (8) Microorganisms will be grown on agar plates and cultured according to the medium, time and temperature shown in Table 6 below.

[0130] Table 6

[0131] Yeast 22.5±2.5℃ SDA or SDATL culture for 3-5 days Mold 22.5±2.5℃ SDA or SDATL culture for 3-7 days bacteria 32.5±2.5℃ TSA or TSATL culture for 3-5 days

[0132] Count colonies after incubation: Use a dilution that shows an acceptable range of counts: 30-300 CFU / plate for bacteria and yeasts, 5-50 CFU / plate for molds.

[0133] (9) Result evaluation

[0134] The log reduction was determined using the calculated log reduction challenge control counts (inoculation concentration per gram of product at the starting point) and the log recovery values ​​at different time points. All data are log reductions.

[0135] (10) Reference standard: ISO11930-2019, see Table 7 for details.

[0136] Table 7

[0137]

[0138]

[0139] 2. Experimental results, see Table 8 for details.

[0140] Table 8 Summary of anticorrosion challenge results

[0141]

[0142] For specific anti-corrosion results, see Table 9-Table 21.

[0143] Specifically, it involves Comparative Example 1, Examples 1-5, Example 11, and Examples 18-23 in Test Example 1.

[0144] Table 9 Anticorrosion Challenge Test Results of Comparative Example 1

[0145]

[0146]

[0147] It can be seen from the data results in Table 9 that the anticorrosion effect of the composition of Comparative Example 1 meets the requirements of ISO11930-2012 Standard A.

[0148] Table 10 Anticorrosion Challenge Test Results of Example 1

[0149]

[0150] It can be seen from the data results in Table 10 that the anti-corrosion effect of the composition of Example 1 meets the requirements of ISO11930-2012 Standard A.

[0151] Table 11 Anticorrosion Challenge Test Results of Example 2

[0152]

[0153] It can be seen from the data results in Table 11 that the anti-corrosion effect of the composition of Example 2 meets the requirements of ISO11930-2012 Standard A.

[0154] Table 12 Anticorrosion Challenge Test Results of Example 3

[0155]

[0156] It can be seen from the data results in Table 12 that the anti-corrosion effect of the composition of Example 3 meets the requirements of ISO11930-2012 Standard A.

[0157] Table 13 Anticorrosion Challenge Test Results of Example 4

[0158]

[0159] It can be seen from the data results in Table 13 that the anticorrosion effect of the composition of Example 4 meets the requirements of ISO11930-2012 Standard A.

[0160] Table 14 Anticorrosion Challenge Test Results of Example 5

[0161]

[0162] It can be seen from the data results in Table 14 that the anti-corrosion effect of the composition of Example 5 meets the requirements of ISO11930-2012 Standard A.

[0163] Table 15 Anticorrosion Challenge Test Results of Example 11

[0164]

[0165] It can be seen from the data results in Table 15 that the anti-corrosion effect of the composition of Example 11 meets the requirements of ISO11930-2012 Standard A.

[0166] Table 16 Anticorrosion Challenge Test Results of Example 18

[0167]

[0168] It can be seen from the data results in Table 16 that the anti-corrosion effect of the composition of Example 18 meets the requirements of ISO11930-2012 Standard A.

[0169] Table 17 Anticorrosion Challenge Test Results of Example 19

[0170]

[0171] It can be seen from the data results in Table 17 that the anti-corrosion effect of the composition of Example 19 meets the requirements of ISO11930-2012 Standard A.

[0172] Table 18 Anticorrosion Challenge Test Results of Example 20

[0173]

[0174] It can be seen from the data results in Table 18 that the anti-corrosion effect of the composition of Example 20 meets the requirements of ISO11930-2012 Standard A.

[0175] Table 19 Anticorrosion Challenge Test Results of Example 21

[0176]

[0177]

[0178] It can be seen from the data results in Table 19 that the anti-corrosion effect of the composition of Example 21 meets the requirements of ISO11930-2012 Standard A.

[0179] Table 20 Anticorrosion Challenge Test Results of Example 22

[0180]

[0181] It can be seen from the data results in Table 20 that the anti-corrosion effect of the composition of Example 22 meets the requirements of ISO11930-2012 Standard A.

[0182] Table 21 Anticorrosion Challenge Test Results of Example 23

[0183]

[0184] It can be seen from the data results in Table 21 that the anti-corrosion effect of the composition of Example 23 meets the requirements of ISO11930-2012 Standard A.

[0185] Examples 1-5, Example 11, and Examples 18-23 can all pass the antiseptic challenge test and can achieve the antiseptic effect required for long-term storage of the product.

[0186] The description of the present disclosure is given for the purpose of illustration and description, and is not intended to be exhaustive or to limit the present disclosure to the disclosed form. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present disclosure, and to enable those of ordinary skill in the art to understand the present disclosure and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. Use of sodium subtilisin in improving the effects of preservative ingredients on skin microecology.

2. The use according to claim 1, comprising making the surviving number of beneficial skin bacteria greater than the surviving number of harmful skin bacteria, preferably, the surviving number of beneficial skin bacteria / the surviving number of harmful skin bacteria ≥ 20.

3. Use of sodium subtilisin in preparing a skin microecological friendly preservative composition.

4. The use according to claim 3, wherein the antiseptic composition comprises: (a) sodium subtilisin, and (b) at least one preservative ingredient.

5. According to the use according to claim 3 or 4, when the antiseptic composition remains on the skin, the surviving number of beneficial bacteria on the skin is greater than the surviving number of harmful bacteria on the skin. Preferably, the surviving number of beneficial bacteria on the skin / the surviving number of harmful bacteria on the skin is ≥20.

6. The use according to claim 3 or 4, wherein when the antiseptic composition remains on the skin, the killing ability of the antiseptic composition on skin-harmful bacteria is higher than the killing ability on skin-beneficial bacteria. Preferably, the killing ability on skin-harmful bacteria / the killing ability on skin-beneficial bacteria is ≥20.

7. The use according to claim 3 or 4, wherein the preservative composition is used to inhibit the survival and growth of microorganisms in the product.

8. The use according to claim 3, wherein the mass ratio of component (a) to component (b) is (0.01-20):

1.

9. A microecologically friendly antiseptic composition, the components of the composition are: (a) sodium subtilisin, and (b) at least one preservative ingredient.

10. The composition according to claim 9, wherein the mass ratio of component (a) to component (b) is (0.01-20):1.

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