Antiseptic method of cosmetic

Abstract

To provide a method for producing a composition to be applied to skin which has improved antibacterial property, and the composition.SOLUTION: A method for producing a composition to be applied to skin includes: combining a raw material, water and a first agent of a first amount which is effective for suppressing proliferation of microorganisms to obtain a first mixture; and mixing a second agent of a second amount which is effective for suppressing proliferation of microorganisms with the first mixture to obtain a second mixture, where a temperature of the second agent is maintained approximately at 33°C or lower. Further, also provided is a composition to be applied to skin. The composition to be applied to skin contains the first agent of the first amount which is effective for suppressing proliferation of microorganisms and the second agent of the second amount which is effective for suppressing proliferation of microorganisms. The first agent may include a fermentation product, and the second agent may include a probiotic.SELECTED DRAWING: Figure 7

Description

Technical Field

[0001] Related Application Information This patent application claims the priority of U.S. Provisional Patent Application No. 62 / 851,477, filed on May 22, 2019, and U.S. Provisional Patent Application No. 62 / 879,785, filed on July 29, 2019, the entire contents of which are incorporated herein by reference. This patent application also claims the priority of U.S. Non-Provisional Patent Application No. 16 / 821,402, filed on March 17, 2020, the entire contents of which are incorporated herein by reference.

[0002] The subject matter of the present disclosure relates to a method for manufacturing a composition for application to the skin having improved antimicrobial properties, a composition for application to the skin having improved antimicrobial properties and stability, and a method for treating acute skin infections using the composition for application to the skin. The composition for application to the skin may be a cosmetic composition.

Background Art

[0003] Cosmetic compositions containing water and organic / inorganic compounds require anti-corrosion and antibacterial agents against microbial contamination in order to guarantee safety to consumers and enhance preservability. It has been found that the anti-corrosion measures used in the cosmetic industry are either toxic to consumers or ineffective in controlling microorganisms. The most powerful preservatives are highly toxic and are restricted or prohibited by current regulations, but there is still a need for cosmetic products that are not contaminated. As a result, cosmetic manufacturers are seeking new anti-corrosion measures to present safer products in terms of microbiology and toxicology.

[0004] The anti-corrosion method of the present disclosure can be utilized in any composition for application to the skin, such as a cosmetic composition containing water.

[0005] As far as the inventor's knowledge extends, the anti-corrosion method according to the present invention has not been used in the cosmetics industry so far. The anti-corrosion method according to the present invention, as a complete system, enables the compatibility with natural emulsifiers and natural humectants to control the availability of water in the construction of emulsions. The method of the present disclosure does not require complex formulations due to the inclusion of synthetic or chemical and essentially toxic components.

[0006] By adopting the anti-corrosion method of the present disclosure, it is possible to solve the stability of the product as an antimicrobial component, and at the same time, support the skin health state through the improvement of the binding property of epithelial cell tight junctions. In addition, the number of consumers who need natural cosmetics is currently increasing and is expected to increase rapidly in the future. The inventor's discovery may enable the cosmetics industry to meet the needs of an expanding customer base.

Summary of the Invention

[0007] One aspect of the present disclosure is a method for manufacturing a cosmetic composition, which includes combining raw materials, water, and a first amount of a first agent effective for suppressing the growth of microorganisms to obtain a first mixture, and mixing a second amount of a second agent effective for suppressing the growth of microorganisms with the first mixture to obtain a second mixture, and the temperature of the second agent is maintained at about 33°C or lower. The first agent may include a fermented product, cultured sugar, antibacterial protein, antibacterial peptide, or a combination thereof. The second agent may include probiotics such as lactic acid bacteria, preferably Bifidobacterium lactis. The composition may further contain at least one essential oil and / or at least one related terpene.

[0008] Another aspect of the present disclosure relates to a composition for applying to the skin, comprising a first amount of a first agent effective to inhibit the growth of microorganisms and a second amount of a second agent effective to inhibit the growth of microorganisms. The first agent may include a fermentate. The second agent may include probiotics. The composition for applying to the skin may further contain at least one essential oil and / or at least one related terpene. The related terpene may be an isolated terpene.

[0009] Another aspect of the present disclosure relates to a method for treating acute skin infections, comprising administering to a subject in need of treatment for acute skin infections an effective amount of a cosmetic composition according to the present disclosure.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0011] Exemplary and non-limiting embodiments and examples of the methods and compositions of the present disclosure are described in detail below. Unless otherwise indicated, percentages (%) are determined individually using mass (described in gm) for solid materials and volume (described in ml) for liquid materials.

[0012] One aspect of the present disclosure is a method for manufacturing a composition for applying to the skin, comprising combining raw materials, water, and a first amount of a first agent effective to inhibit the growth of microorganisms to obtain a first mixture, and mixing a second amount of a second agent effective to inhibit the growth of microorganisms with the first mixture to obtain a second mixture, wherein the temperature of the second agent is maintained at about 33°C or lower.

[0013] The first agent may include an antifungal agent. The first agent may include a fermented product, a cultured sugar, an antibacterial protein, an antibacterial peptide, or a combination thereof. The fermented product may be a fermentation product of a composition containing lactic acid bacteria. The fermented product may be MicroGARD® 200 (Dupont Nutrition & Health, Delaware DE). Examples of the first agent include cultured sugars (non-limiting examples include lactose, dextrose, fructose, and maltodextrin), proteins (non-limiting examples include casein, whey, soy, and pea, antibacterial peptides), and combinations thereof.

[0014] The second agent may contain probiotics. The probiotics may contain lactic acid bacteria. Some examples of lactic acid bacteria include Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei. The probiotics may include bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lactococcus, bacteria of the genus Streptococcus, bacteria of the genus Pediococcus, and combinations thereof. Preferably, the probiotics include Bifidobacterium lactis HN019. The probiotics can improve the antioxidant system and the ability to reduce radical generation, thereby also supporting another aspect of stability in the formulation. The probiotics can also improve sensory aspects such as odor / aroma and can improve the feel and emulsification of the composition.

[0015] The first effective amount of the first agent for suppressing the growth of microorganisms is not particularly limited. The first effective amount of the first agent for suppressing the growth of microorganisms can be an amount effective for suppressing the growth of mold or yeast. The first effective amount of the first agent for suppressing the growth of microorganisms, as the mass (described in gm) relative to the total volume (described in ml) of the composition for application to the skin, can be about 0.05% or more, about 0.25% or more, about 0.5% or more, about 0.75% or more, about 1% or more, or about 2.75% or more, and can be about 5% or less, about 4.5% or less, about 4% or less, or about 3% or less. The first effective amount of the first agent for suppressing the growth of microorganisms, as the mass relative to the total amount of the composition for application to the skin, can be, for example, about 0.25% to about 5%, or about 0.75% to about 4%, or about 1% to about 3%.

[0016] A second amount of a second agent effective to inhibit the growth of the microorganism may be added to the first mixture to obtain a second mixture, or the first mixture may be added to a second amount of a second agent effective to inhibit the growth of the microorganism to obtain a second mixture, or the second agent and the first mixture may be added simultaneously to obtain a second mixture.

[0017] The second amount of the second agent effective to inhibit the growth of the microorganism is not particularly limited. The second amount effective to inhibit the growth of the microorganism may be about 0.10% or more, about 0.25% or more, about 0.37% or more, or about 0.50% or more, and about 5% or less, about 4% or less, about 2% or less, or about 1% or less, as a mass relative to the total amount of the composition for application to the skin. The second amount effective to inhibit the growth of the microorganism may be, for example, about 0.10% to about 5%, about 0.20% to about 4%, or about 0.25% to about 1%, as a mass (described in gm) relative to the volume (described in ml) of the entire composition for application to the skin.

[0018] After the second agent, for example, the probiotic, is added, the temperature of the second agent may be maintained at about 33°C or lower, about 32°C or lower, about 31°C or lower, about 30°C or lower, or about 29°C or lower. For example, the temperature of the second mixture and the temperature at which each of one or more subsequent steps is performed may be maintained at about 33°C or lower, about 32°C or lower, about 31°C or lower, about 30°C or lower, or about 29°C or lower. For example, the method for producing a composition for application to the skin may include emulsifying the probiotic-containing composition, and the temperature at the end of emulsification of the probiotic-containing composition may be controlled to about 33°C or lower, about 32°C or lower, about 31°C or lower, about 30°C or lower, or about 29°C or lower.

[0019] The method for manufacturing a composition for skin application may further include mixing at least one essential oil and / or at least one related terpene with a second mixture. The related terpene may be an isolated terpene. For example, at least one essential oil and / or at least one terpene may be added to the second mixture. Alternatively, the second mixture may be added to at least one essential oil and / or at least one terpene. Alternatively, the second mixture and at least one essential oil and / or at least one terpene may be added simultaneously, for example, into a container. Examples of at least one essential oil include essential oils from the Burseraceae family such as frankincense (Boswellia sacra, B. carterii, B. frereana, B. serrata, and B. papyrifera), palo santo (Bursera graveolens), myrrh (Commiphora Myrrha or Commiphora Molmol), and copal (Protium copal); essential oils such as tulsi or holy basil (Ocimum tenuifloru), turmeric (Curcuma longa), vetiver (Vetiveria zizanioides), hemp (Cannabis salvia) including seed oil; and essential oils derived from the genus Citrus (Rutaceae) (examples include orange (Citrus Sinensis), Citrus uranium, mandarin orange (Citrus reticulata), lemon (Citrus lemon), lime (Citrus aurantifolia), grapefruit (Citrus x paradise), bergamot (Citrus bergamia), yuzu (Citrus junos), and kumquat (Citrus japonica)), and combinations thereof.For example, a combination of the following essential oils, namely vetiver, palo santo, holy basil, frankincense, and lime, may be used. In certain embodiments, at least one of the above essential oils contains terpenes such as limonene, α-pinene, caryophyllene, myrcene, sabinene, eugenol, β-elemene, α-bulsene, γ-terpinene, terpinolene, and menthofuran. The inclusion of at least one of the above essential oils, or a combination of one or more essential oils, and / or related terpenes may play a symbiotic role in a composition for application to the skin.

[0020] The amount of at least one essential oil and / or at least one related terpene may be, as a volume relative to the volume of the entire composition for application to the skin, about 0.2% or more, about 0.5% or more, about 1% or more, about 2% or more, or about 2.6% or more, and may be about 10% or less, about 5% or less, about 4% or less, or about 3% or less. The amount of at least one essential oil and / or at least one related terpene may be, as a volume relative to the volume of the entire composition for application to the skin, for example, about 0.2% to about 10%, about 0.5% to about 4%, or about 1% to about 3%. For example, if the composition for application to the skin is an emulsion, the amount of at least one essential oil and / or at least one terpene may be about 2.57% as a volume relative to the volume of the entire composition. If the composition for application to the skin is a serum, the amount of at least one essential oil and / or at least one terpene may be about 2.6% as a volume relative to the volume of the entire composition. If the composition for application to the skin is a peeling agent, the amount of at least one essential oil and / or at least one terpene may be about 1.14% as a volume relative to the volume of the entire composition. When two or more essential oils and / or terpenes are used, the amount of at least one essential oil and / or at least one terpene described in the present disclosure means the total amount of the two or more essential oils and / or terpenes.

[0021] In one embodiment, a method for manufacturing a composition for application to the skin according to the present disclosure is (1) Combine the fermented product and the water component to obtain a first composition; (2) Combine the oil component and the wax component, and heat to about 63°C to about 74°C to obtain a second composition; (3) Add the glycerite extract to the first composition and stir to obtain a third composition; (4) Add probiotics to the third composition to obtain a fourth composition; (5) Add the second composition cooled to about 60°C to the fourth composition and emulsify at a temperature of about 33°C or lower; (6) Cool the product of step (5) to 18°C or lower; (7) It may include adding essential oil to the product of step (6).

[0022] The composition for applying to the skin may be a cream. An example of the manufacturing method of the composition for applying to the skin is (1) Combine the fermented product (about 0.75% to about 4%) with the water component (about 50%) and stir; (2) Combine the oil component (about 40%) with the wax component (about 3%) and heat to about 63 - 74°C; (3) Add the glycerite extract (about 2%) to the product of step (1) and stir; (4) Add probiotics (about 0.25% to about 1%) to the product of step (3); (5) Add the oil / wax composition obtained in step (2) and cooled to 60°C to the water / fermented product / glycerite / probiotics composition obtained in step (4), and stir / blend at 33°C or lower to emulsify; (6) Cool the mixture to a temperature of 18°C or lower and higher than 0°C; (7) It may include adding essential oil (about 2%) to the product of step (6) and stirring until incorporated. The final product may be stored at about 10°C to about 18°C, avoiding light.

[0023] In another embodiment, a method for manufacturing a composition for applying to the skin according to the present disclosure is (1) stirring the ferment in a liquid composition containing water and glycerin to obtain a first composition; (2) stirring the probiotics in the first composition to obtain a second composition; (3) adding at least one essential oil to the second composition. It may include. In this embodiment, the method may be carried out at a temperature of about 21° C. or lower.

[0024] The composition for applying to the skin described above may be a liquid composition, for example, a serum. An example of the method for manufacturing the liquid composition may be carried out at a temperature of 21° C. or lower, and (1) combining liquid components, such as glycerite extract (about 28% water and 66% glycerin); (2) stirring the ferment (about 0.75% to about 4%) in the composition obtained in step (1); (3) stirring the probiotics (about 0.25% to about 1%) in the composition obtained in step (2); (4) adding at least one essential oil (about 2.50%). It may include. The resulting final product may be stored at about 10° C. to about 18° C. avoiding light.

[0025] In another embodiment, a method for manufacturing a composition for applying to the skin according to the present disclosure is (1) combining dry ingredients, probiotics and ferment to obtain a first composition; (2) adding the first composition to liquid components containing water and glycerin and stirring to obtain a second composition; (3) combining a dry plant component or a clay component with the second composition and stirring to obtain a third composition; (4) adding a humectant to the third composition and stirring to obtain a fourth composition; (5) adding essential oil to the fourth composition and mixing to obtain a uniform consistency. It may include.

[0026] Another example of the method for producing the above liquid composition may be carried out at a temperature of 21 °C or lower, and (1) combining liquid components, such as glycerite extract (about 11% water, about 26% glycerin), and (2) combining a dry milk-based component (about 1.35%) with probiotics (about 0.25% to about 1%) and fermentate (about 0.75 to about 4%), adding to the composition obtained in step (1), and stirring; and (3) combining a dry plant component or clay component (about 14%), adding to the composition obtained in step (2), and stirring; and (4) adding a humectant, specifically honey (about 45%), but not limited thereto, to the composition obtained in step (3), and stirring; and (5) adding at least one essential oil (about 1.25%), stirring, and mixing to a uniform consistency. The resulting final product may be stored at about 10 °C to about 20 °C, protected from light.

[0027] Another aspect of the present disclosure relates to a composition for applying to the skin, containing a first amount of a first agent effective to inhibit the growth of microorganisms and a second amount of a second agent effective to inhibit the growth of microorganisms.

[0028] The first agent may include a fermentate, a cultured sugar, an antibacterial protein, an antibacterial peptide, or a combination thereof. The fermentate may be a fermentation product of a composition containing lactic acid bacteria. The fermentate may be MicroGARD® 200 (Dupont Nutrition & Health, Delaware, DE). Examples of the first agent include cultured sugars (non-limiting examples include lactose, dextrose, fructose, and maltodextrin), proteins (non-limiting examples include casein, whey, soy, and pea, antibacterial peptides), and combinations thereof.

[0029] The second agent may contain probiotics. The probiotics may contain lactic acid bacteria. Examples of lactic acid bacteria include Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei. The probiotics may include bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lactococcus, bacteria of the genus Streptococcus, bacteria of the genus Pediococcus, and combinations thereof. Preferably, the probiotics contain Bifidobacterium lactis HN019. The probiotics can improve the antioxidant system and the ability to reduce radical generation, thereby also supporting another aspect, namely stability in the formulation. The probiotics can also improve sensory aspects such as odor / aroma and can improve the feel and emulsification of the composition.

[0030] The first effective amount of the first agent for suppressing the growth of microorganisms is not particularly limited. The first effective amount of the first agent for suppressing the growth of microorganisms is not particularly limited. The first effective amount for suppressing the growth of microorganisms can be an amount effective for suppressing the growth of mold or yeast. The first effective amount of the first agent for suppressing the growth of microorganisms, as the mass (described in gm) relative to the total volume (described in ml) of the composition for application to the skin, can be about 0.05% or more, about 0.25% or more, about 0.5% or more, about 0.75% or more, about 1% or more, or about 2.75% or more, and can be about 5% or less, about 4.5% or less, about 4% or less, or about 3% or less. The first effective amount of the first agent for suppressing the growth of microorganisms, as the mass relative to the total amount of the composition for application to the skin, can be, for example, about 0.25% to about 5%, or about 0.75% to about 4%, or about 1% to about 3%.

[0031] The second amount effective for suppressing the growth of microorganisms of the second agent is not particularly limited. The second amount effective for suppressing the growth of microorganisms is, as a mass relative to the total amount of the composition for application to the skin, about 0.10% or more, about 0.25% or more, about 0.37% or more, or about 0.50% or more, and may be about 5% or less, about 4% or less, about 2% or less, or about 1% or less. The second amount effective for suppressing the growth of microorganisms is, as a mass (described in gm) relative to the total volume (described in ml) of the composition for application to the skin, for example, about 0.10% to about 5%, about 0.20% to about 4%, or about 0.25% to about 1%.

[0032] Preferably, the first agent is a fermented product, and the first amount effective for suppressing the growth of microorganisms is, as a mass relative to the total amount of the composition for application to the skin, about 0.75% to about 4%, or about 1% to about 3%.

[0033] Preferably, the second agent is a probiotic, and the second amount effective for suppressing the growth of microorganisms is, as a mass relative to the total amount of the composition for application to the skin, about 0.25% to about 1%.

[0034] The composition for skin application may further contain at least one essential oil and / or at least one terpene related to preventing the growth of microorganisms. The at least one terpene may be at least one isolated terpene. Examples of at least one essential oil include essential oils from the Burseraceae family such as frankincense (Boswellia sacra, Boswellia carterii, Boswellia frereana, Boswellia serrata, and Boswellia papyrifera), palo santo (Bursera graveolens), myrrh (Commiphora Myrrha or Commiphora Molmol), and copal (Protium copal); essential oils such as tulsi or holy basil (Ocimum tenuifloru), turmeric (Curcuma longa), vetiver (Vetiveria zizanioides), hemp (Cannabis salvia) including seed oil; and essential oils derived from the genus Citrus (Rutaceae) (examples include orange (Citrus Sinensis), Citrus uranium, mandarin orange (Citrus reticulata), lemon (Citrus lemon), lime (Citrus aurantifolia), grapefruit (Citrus x paradise), bergamot (Citrus bergamia), yuzu (Citrus junos), kumquat (Citrus japonica)), and combinations thereof. For example, a combination of the following essential oils, namely vetiver, palo santo, holy basil, frankincense, and lime, may be used. In certain embodiments, the at least one essential oil described above contains terpenes such as limonene, α-pinene, caryophyllene, myrcene, sabinene, eugenol, β-elemene, α-bulnesene, γ-terpinene, terpinolene, and menthofuran.The inclusion of at least one of the above essential oils, or a combination of one or more essential oils, and / or related terpenes may play a symbiotic role in a composition for application to the skin.

[0035] The amount of at least one essential oil and / or at least one related terpene may be, as a volume relative to the volume of the entire composition for application to the skin, about 0.2% or more, about 0.5% or more, about 1% or more, about 2% or more, or about 2.6% or more, and may be about 10% or less, about 5% or less, about 4% or less, or about 3% or less. The amount of at least one essential oil and / or at least one related terpene may be, as a volume relative to the volume of the entire composition for application to the skin, for example, about 0.2% to about 10%, about 0.5% to about 4%, or about 1% to about 3%. For example, when the composition for application to the skin is an emulsion, the amount of at least one essential oil and / or at least one terpene may be about 2.57% as a volume relative to the volume of the entire composition. When the composition for application to the skin is a serum, the amount of at least one essential oil and / or at least one terpene may be about 2.6% as a volume relative to the volume of the entire composition. When the composition for application to the skin is a peeling agent, the amount of at least one essential oil and / or at least one terpene may be about 1.14% as a volume relative to the volume of the entire composition. When two or more essential oils and / or terpenes are used, the amount of at least one essential oil and / or at least one terpene described in the present disclosure means the total amount of the two or more essential oils and / or terpenes.

[0036] Another aspect of the present disclosure relates to a method of treating an acute skin infection, comprising administering to a subject in need of treatment for acute skin infection a composition for application to the skin according to the present disclosure in an effective amount. The effective amount of the composition may be an amount sufficient to alleviate, reduce, or improve the symptoms of the acute skin infection. The method of treating the acute skin infection may involve administration of the composition over a long period of time. For example, the composition for application to the skin according to the present disclosure may be administered to the subject for at least 8 hours, at least 12 hours, at least 1 day, at least 1 week, or at least 2 weeks.

Example

[0037] Example 1 The anti-corrosion test was initiated from the use of salts that control unwanted microbial activity by creating an environment that does not promote the growth of microorganisms. In this test, three types of treatments were evaluated: a salt-free control, a low salt concentration (0.05%), and a high salt concentration (5%). Additionally, HOLDBAC® LC (Dupont Nutrition & Health, Delaware DE) was added at a concentration of 0.093% to determine whether this antibacterial agent would produce the desired results in an environment with salt. The initial results are shown in Table 1. The results after one month are shown in Table 2.

[0038] Table 1. Salt test: Initial results JPEG2025111641000002.jpg59153The salt used - organic sea salt that supports clean labeling.

[0039] These results establish clean production. Bacteria, yeasts, and molds appear to have been introduced with the addition of salt.

[0040] Table 2. Salt test: Results after one month JPEG2025111641000003.jpg54153

[0041] Results and Discussion Salt is commonly used as a natural preservative and was proposed as a good and obvious starting point. The results of this experiment showed that clean production could be achieved and that salt did not play a role in controlling yeast, mold, or TPC. In fact, salt seems to have played a harmful role in introducing undesirable bacteria and Y / M during production. In conclusion, salt does not function to control microbial contamination, and the choice was made not to investigate this treatment agent further as a potential solution.

[0042] It was also determined that in these tests, it was not possible to determine whether HOLDBAC® LC was fulfilling its role as an antibacterial agent due to the complex factors introduced by salt. More tests using HOLDBAC® LC are needed to determine its efficacy.

[0043] It was determined that a valid shelf-life test is needed to determine whether this formulation aids in the growth of these potential contaminating bacteria.

[0044] Example 2: Shelf-Life Test 1 Two samples of the emulsion formulation were prepared as follows. (1) A water component (about 50%) was prepared. (2) The oil component (about 40%) was combined with the wax component (about 3%) and heated to about 63 - 74°C. (3) The glycerite extract (about 2%) was added to the water component (1) and stirred. (4) Probiotics were added to the mixture obtained in (3) (the probiotics, Holdbac LC which is Lactobaccillus rhamnosis, was added together with HN019. LC was added at 0.09% and HN019 was added at 0.17%). (5) The oil / wax obtained in (2) and cooled to 60°C was added to the mixture obtained in (4), and stirred and emulsified at 33°C or lower. (6) Essential oil (about 2%) was added, stirred, and then cooled. (7) Cool the mixture to below about 18 °C (without freezing). The samples were stored in the dark at about 10 °C - 18 °C.

[0045] Two types of cosmetic cream samples, 1110001 - MML Emulsion N92117 and 1110002 - MML Emulsion E100517, were sent to Biogen Laboratory Developments, LLC for a preservation efficacy test using Staphylococcus aureus and Candida parapsilosis. These two microorganisms were proposed by this laboratory as they would provide a sufficient bioburden to evaluate the MML formulations. The test started with the emulsion where microbial contamination problems are most likely to occur.

[0046] In Preservation Efficacy Test 1, two types of emulsion samples were tested. For the initial microbial load of these products without inoculation of microorganisms, it was found that bacteria, yeasts, and molds were not present as shown in Table 3 below.

[0047] Table 3. Preservation Efficacy Test 1. Viable count results of products without inoculation of microorganisms JPEG2025111641000004.jpg43153

[0048] Each product was inoculated with Staphylococcus aureus (SA) or Candida parapsilosis (CP) cultured for 24 hours, respectively. The cultured bacteria for 24 hours were tested to determine the total bioburden supplied. The results are as follows: Staphylococcus aureus = 2.6x10 8 cfu / ml Candida parapsilosis = 1.5x10 6 cfu / ml

[0049] For each sample, two 50-gm aliquots were inoculated with either Staphylococcus aureus (SA) or Candida parapsilosis (CP) by adding 1.0 ml of a 24-hour culture to 50 g of the aliquot. Each aliquot inoculated with SA had a final bioburden of 2.6x10 6 cfu / g. Each aliquot inoculated with Cp had a final bioburden of 1.5x10 4 cfu / g.

[0050] Table 4. Preservation efficacy test 1: Microbial counts of MML emulsion JPEG2025111641000005.jpg70153

[0051] Results and discussion Emulsion N92117 contained Bifidobacterium lactis HN019 (a lactic acid bacterium commonly known as a probiotic) at a concentration of 0.17% in addition to HOLDBAC® LC (a probiotic found to be effective against yeasts and molds in the food industry) at a concentration of 0.09% of the formulation. The temperature of emulsification was 99°F (37°C).

[0052] The results are shown in Table 4. The number of Staphylococcus aureus decreased to less than half within 24 hours and by more than 1 log in 2 days in this formulation. This combination had no effect on reducing the number of yeasts; in fact, the number of yeasts increased by more than 2.5 logs during this 2-day study. This indicates that HOLDBAC® LC is not effective in controlling yeasts in these cosmetic formulations.

[0053] Emulsion E100517 contained Bifidobacterium lactis HN019 at a concentration of 0.14% in addition to HOLDBAC® LC at a concentration of 0.09% of the formulation. The emulsification temperature was also 99°F (37°C). This emulsion also contained a high concentration (0.59%) of orange essential oil. This combination had no effect on the yeast count, but a more significant improvement in reducing the Staph. aureus count was observed compared to N92117 (Table 4). This was a result that reconfirmed that HOLDBAC® LC was not effective in controlling yeast in these cosmetic formulations. Also, it was confirmed that adding essential oil could improve the performance of controlling Staph. aureus and could contribute to the control of yeast. More research is needed to understand the role of essential oil.

[0054] The above data show that within the first 2 days of storing both formulations in the ambient environment, the Staph. aureus gradually decreased and decreased by about 1 log. However, in emulsion N92117, the Candida count increased by nearly 1 log within the first 24 hours and further increased by nearly 1 log within 48 hours. Emulsion E100517 maintained a stable state on the second day. The overall recovery rate of these microorganisms in the E100517 emulsion was low, indicating higher resilience than the formulation of N92117. Continue observing these microbial levels and conclude the growth trends of microorganisms in these samples.

[0055] The above data suggest that Staph aureus can ultimately die in both formulations, but Candida can continue to increase in both formulations.

[0056] Example 3: Storage Potency Test 2 This study was designed to test the effect of BioVida (a multi-hurtle antibacterial agent such as mustard seeds and green tea). This antibacterial agent was selected for its clean label properties.

[0057] Emulsion samples were prepared as follows. (1) The ferment (Bio Vida, 0.89%) was combined with the water component (about 50%). (2) The oil component (about 40%) was combined with the wax component (about 3%) and heated to about 63 - 74 °C. (3) The glycerite extract (about 2%) was added to the water component (1) and stirred. (4) Probiotics (HN019, 0.42%) were added to the mixture obtained in (3). (5) The oil / wax obtained in (2) and cooled to 60 °C was added to the mixture obtained in (4), and stirred and emulsified at 33 °C or lower. (6) Essential oil (about 2%) was added, stirred, and then cooled. (7) The mixture was cooled to about 18 °C or lower (not frozen). The samples were stored at about 10 - 18 °C, protected from light.

[0058] This study was conducted on a cosmetic cream sample, 0426001 - MML ONPA 41018. First, a test was conducted on this cream to examine the background microbial count (Table 5). Table 5: Storage efficacy test 2. Background microbial count JPEG2025111641000006.jpg48153

[0059] In this sample, two 50-g aliquots were inoculated by adding 0.5 ml of a 24-hour culture of either Staphylococcus aureus (SA) or Candida parapsilosis (CP) to each 50-g aliquot. The 24-hour cultures were tested to determine the amount of bacteria added to the sample. Staphylococcus aureus: 1.1x10 8 CFU / ml Candida parapsilosis: 1.4x10 7 CFU / ml Each 50-g aliquot inoculated with SA received approximately 1,100,000 CFU / g. Each 50-g aliquot inoculated with CP received approximately 140,000 CFU / g.

[0060] Results: The results are shown in Figure 1 and Table 6 below. Table 6. Microbial Counts in Preservation Potency Test 2 JPEG2025111641000007.jpg114153

[0061] In this study, no decrease in Staphylococcus aureus was observed as seen in the previous experiment. The temperature of the emulsion was 95°F (35°C). Bifidobacterium lactis HN019 was added at a very early stage of the process. No terpene (limonene content of lemon essential oil is 90%) was used in the above formulation. The possible cause could be the addition time of Bifidobacterium lactis HN019. Bifidobacterium lactis HN019 was introduced earlier than in the previous test which showed good results, so the order of addition might have an impact. Additionally, the lack of terpene might also be involved. BioVida does not seem to have a favorable effect on either Staphylococcus aureus or Candida parapsilosis.

[0062] Example 4: Storage Potency Test 3 In this experiment, the effects of fermentates (MG100 (organic milk), MG200 (organic), MG210 (non - organic)), as well as antifungal agents (natamycin at various concentrations), and their effects on Staphylococcus aureus and Candida parapsilosis were compared. Since the process can be involved in the results, the evaluation of specific process conditions (order and temperature) and their possible effects on the microbial results will be included in all future studies. Study: MML - 1: MEN70918 (7 / 9AM) - MG100 MML - 2: EEN71018 (7 / 10AM) - MG200 MML - 3: EVEN71118 (7 / 11AM) - MG210 MML - 4: Morning Emulsion 81418 (8 / 14 / 18AM) - Natamycin (0.025%) MML-5: Evening Emulsion 81818 (8 / 14 / 18 AM) - Natamycin (0.0125%)

[0063] Except for the components, amounts and / or conditions specifically shown in this example, five creams were prepared by the same steps as in Example 3. All the treatment agents contain Bifidobacterium lactis HN019 at a concentration of 0.41 - 0.47%. For each cream, a test was first conducted to determine the background microbial count. The products were tested for APC, yeast and mold (Table 7). Table 7: Background Microbial Counts in Example 4 JPEG2025111641000008.jpg79153

[0064] Each product was inoculated with Staphylococcus aureus (SA) or Candida parapsilosis (CP) cultured for 24 hours, respectively. The cultured bacteria for 24 hours were tested to determine the total bioburden supplied. [[ID=1q]]Staphylococcus aureus = 2.8x10 8 cfu / ml Candida parapsilosis = 3.7x10 6 cfu / ml

[0065] For each sample, two 100 gm vial samples were each inoculated with either Staphylococcus aureus (SA) or Candida parapsilosis (CP) by adding 1.0 ml of the cultured bacteria for 24 hours to the 100 g vial sample. Each 100 gm vial sample inoculated with SA had a final bioburden of 2.8x10 6 cfu / g. Each 100 gm vial sample inoculated with CP had a final bioburden of 37,000 cfu / g.

[0066] Results and Discussion The results of Example 4 are shown in Tables 10 to 12 and Figures 2 and 3. Table 8: Number of microorganisms in MML-1 JPEG2025111641000009.jpg94153 Table 9: Number of microorganisms in MML-2 JPEG2025111641000010.jpg87153 Table 10: Number of microorganisms in MML-3 JPEG2025111641000011.jpg97153 Table 11: Number of microorganisms in MML-4 JPEG2025111641000012.jpg95153 Table 12: Number of microorganisms in MML-5 JPEG2025111641000013.jpg96153

[0067] The order of adding MEN70918MG100 - Bifidobacterium lactis HN019 was correct, but the temperature at the end of the treatment (after emulsification) was 107°F (41.6°C). This treatment agent showed an increase in Staphylococcus aureus (SA) and Candida parapsilosis (CP) at the beginning of 4 weeks and 6 weeks respectively. Temperature may have contributed to the poor result. Since the effect of MG100 was lacking, this component shall be removed from further tests.

[0068] EEN71018 - The product had terpenes (derived from orange essential oil and ylang-ylang). Although not recorded, a hypothesis is put forward that the end temperature on this treatment agent was also high. The number of Staphylococcus aureus was high, which may be consistent with other tests when the temperature rose during emulsification. This treatment agent had a significant effect in reducing yeast. Further research is warranted to evaluate MG200.

[0069] The final temperature after emulsification seems to be related to the effect on Staph aureus (SA) results. Bifidobacterium lactis HN109 was very effective when using temperatures below 101°F (38.3°C).

[0070] The essential oil, Vetiveria zizanioides, shows potential effects as seen in EVEN71118. For 71118, the emulsification temperature was 101°F (38.3°C), the order of addition was correct, and MG210 was used.

[0071] Due to the good performance of MG200 and its clean label (certified organic), this antibacterial agent will be used in future research instead of MG210.

[0072] There may be a relationship between MG200 and the essential oil. In particular, Vetiveria zizanioides may bring about a supporting or symbiotic relationship.

[0073] Natamycin showed good control of Candida parapsilosis (CP), but had negative effects on the product quality such as adverse effects on the aroma and consistency. Also, natamycin is not considered a clean label. For these reasons, this component will be removed in future experiments.

[0074] Example 5: Storage Potency Test 4 In this study, three types of treatment agents were designed to isolate the effects of plants (hemp) and essential oils. To assist in the control of Staph aureus, Bifidobacterium lactis HN019 was added to all treatment agents. This was the first study to evaluate the addition of terpenes after cooling to 65°F (18.3°C). There was a concern that components with cytotoxicity could be induced at high temperatures (those components are temperature-sensitive).

[0075] Samples were prepared by the same procedure as in Example 3, except for the components, amounts and / or conditions specifically shown in this example. Treatment agent: Morning Blend 112918 (Vetiveria zizanioides, essential oil (terpene), hemp, MG200, 88F) Evening Blend 121018 (Vetiveria zizanioides, essential oil (terpene) added after cooling, hemp, MG200, 2.5% NCFM, 88F) Evening E112618 (Vetiveria zizanioides, terpene added after cooling, hemp, MG200, 109F) 112918 - inhibited Staphylococcus aureus by the 4th day and inhibited yeast to 150 by the 5th day.

[0076] 121018 - inhibited Staphylococcus aureus by the 4th day, increased, and <10 by the 7th day. Yeast was 20 by the 7th day. 112618 - Staphylococcus aureus was poorly controlled at 1,500,000 cfu / g by the 7th day, and yeast was 10 cfu / g by the 7th day. The temperature seems to have had a negative impact on Bifidobacterium lactis HN019. It is continuously confirmed that temperature can be the key to solving Staphylococcus aureus results.

[0077] Conclusion: Yeast and Staphylococcus aureus were controlled in two of the three treatment agents. The temperature after emulsification remains an important factor. For the yeast results, the addition of MG200 and essential oil after cooling seems to contribute to positive results.

[0078] Example 6: Storage Potency Test 5 In this study, the role of essential oils in the emulsion formulation was evaluated using a standardized protocol (sequence, temperature, MG200, and Bifidobacterium lactis HN019). Also, in this study, the role of low concentrations of natamycin was evaluated to determine its effect on the yeast count.

[0079] An emulsion formulation was prepared by the same process as in Example 3, except for the components, amounts, and / or conditions specifically shown in this example. First, the ferment was added to water.

[0080] To determine whether the method of preservation, which currently shows positive results in emulsion formulations, can be transferred to other cosmetic formulations, three additional formulations were added to the test, namely a serum (containing water) and two peeling agents (containing components that can contribute to microbial contamination). Both formulations were manufactured via a cold process.

[0081] The serum was prepared as follows by manufacturing at a cold temperature of 21°C or lower. (1) The liquid components, specifically glycerite extract (about 28% water and about 66% glycerin), were combined. (2) Ferment MG200 (about 0.09%) was stirred in the liquid obtained in (1). (3) In the liquid obtained in (2), 0.49% probiotics Bifidobacterium lactis (B. lactis) HN019 and 1.68% Lactobacillus acidophilus NCFM (for skin health) were stirred. (4) Essential oil (about 2.50%) was added. The serum was stored at about 10°C to 18°C, protected from light.

[0082] The peeling agent was prepared by the following process carried out at a temperature of 21°C or lower. (1) A liquid component, for example glycerite extract (about 11% water, about 26% glycerin) was combined. (2) A dry milk-based component (about 1.35%) was combined with probiotics (about 0.25% - about 1%) and fermentates (about 0.75 - about 4%), added to the composition obtained in step (1), and stirred. (3) A dry plant component or clay component (about 14%) was combined, added to the composition obtained in step (2), and stirred. (4) To the composition obtained in step (3), a humectant, specifically honey (about 45%) but not limited to these, was added and stirred. (5) At least one essential oil (about 1.25%) was added, stirred, and mixed to achieve a uniform consistency. The resulting final product was stored at about 10°C - about 20°C, protected from light.

[0083] Four cream blends, two exfoliants, and one serum were provided, and tests were conducted on them against Staphylococcus aureus and Candida parapsilosis.

[0084] For each cream, first, a test was conducted to examine the background microbial level (Table 13). Table 13: Background Microbial Levels in Example 7 JPEG2025111641000014.jpg97153

[0085] For each sample, two sub-samples were each inoculated at 1% by adding 0.4 ml of 24-hour cultured bacteria of either Staphylococcus aureus (SA) or Candida parapsilosis (CP) to 40 g of each sub-sample.

[0086] The 24-hour cultured bacteria were tested to determine how much bacteria was added to the sample. Staphylococcus aureus: 2.1x10 8 CFU / ml Candida parapsilosis: 5.8x10 4 CFU / ml Approximately 2,100,000 CFU / g were added to each 100 g retail sample inoculated with SA. Approximately 580 CFU / g were added to each 100 g retail sample inoculated with CP.

[0087] Results: Table 14: Results of total viable count for each blend of 0510001 - MML: JPEG2025111641000015.jpg Decreased to background level by day 1081532, yeast died on day 0. Table 15: Results of total viable count for each blend of 0510002 - MML: JPEG2025111641000016.jpg Decreased to background level by day 10515321, yeast died on day 6. Table 16: Results of total viable count for each blend of 0510003 - MML: JPEG2025111641000017.jpg Decreased to background level by day 1051536, yeast died on day 1. Table 17: Results of total viable count for each blend of 0510004 - MML: JPEG2025111641000018.jpg Decreased to background level by day 991532, yeast died on day 4. Table 18: Results of total viable count for each blend of 0510005 - MML: JPEG2025111641000019.jpg Decreased to background level by day 1021532, yeast died on day 5. Table 19: Results of total viable count for each blend of 0510006 - MML: JPEG2025111641000020.jpg It decreased to the background level by the 7th day, and the yeast died on the 4th day. Table 20: Results of the total viable count for each blend of 0510007-MML: JPEG2025111641000021.jpg It decreased to the background level by the 21st day, and the yeast died on the 4th day.

[0088] Serum: The serum contains 0.49% Bifidobacterium lactis HN019, 2.6% essential oil (terpene), 1.68% Lactobacillus acidophilus NCFM (for skin health), and 1.09% MG200. This data shows a significant decrease in Staphylococcus aureus on the 1st day and the absence of yeast (see Figure 4). This formulation and process function to reduce the load level.

[0089] Release agent: No water is added to this formulation, but due to other ingredients containing clay and honey, microbial contamination may be likely to occur. The test results are shown in Figure 5.

[0090] Treatment agent 030118 - Although this formulation does not contain Bifidobacterium lactis HN019, it was able to reduce Staphylococcus aureus to background levels by day 21, and yeast was no longer present by day 6. However, with the addition of 0.37% Bifidobacterium lactis HN019 and 2.1% MG200, Staphylococcus aureus reached background levels by day 6, and yeast was no longer present by day 1. This indicates that the addition of Bifidobacterium lactis HN019 and MG200 significantly contributed to the reduction of these microbial contaminations.

[0091] In addition, the inclusion of probiotics and MG200 in this formulation resulted in unexpected quality characteristics. As shown in Figure 6, the addition of these components brought about a permanent and desirable emulsification in the final product. Without these components, the formulation would separate immediately, causing problems when putting out and using this product. This is an important quality improvement.

[0092] Morning emulsion and evening emulsion: This experiment was designed to compare morning and evening formulations with and without essential oils (Formulations 1 and 3 did not contain essential oils). Additionally, the effect of natamycin on the yeast results was evaluated (Morning 2 and Evening 4).

[0093] All emulsions contained 0.4 - 0.6% Bifidobacterium lactis HN019 and 2.83% MG200. Only Evening 1 and Evening 2 also contained 0.0056% natamycin. The morning emulsion did not contain natamycin.

[0094] Results and Discussion The results are shown in Figs. 7 to 13. Fig. 7 shows the test results of Staph aureus of the emulsion of Example 6. Fig. 8 shows the test results of Staph aureus of the evening emulsion of Example 6. Fig. 9 shows the test results of yeast of the evening emulsion of Example 6. Fig. 10 shows the test results of Staph aureus of the morning emulsion of Example 6. Fig. 11 shows the test results of yeast of the morning emulsion of Example 6. Fig. 12 shows the comparison of the test results of Staph aureus between the morning emulsion and the evening emulsion of Example 6, and the effect of natamycin. Fig. 13 shows the comparison of the test results of yeast between the morning emulsion and the evening emulsion of Example 6, and the effect of essential oil.

[0095] This study has found that essential oils (including terpenes) appear to be effective in preserving emulsions. Essential oils are part of a potential hurdle-type technology developed to control microbial growth in cosmetic products. Other studies have shown that essential oils have antibacterial effects. Looking at these results, the plant components in the formulations may not have had their contributions suppressed at this point. More research may be needed to determine the role of the plant components contributing to this preservation method.

[0096] Effect of emulsion temperature: Morning 3 and Morning 4 had an emulsion temperature of 86°F (30°C), and the essential oil was added at 65°F (18.3°C). Evening 1 and Evening 2 had an emulsion temperature of 83°F (28.3°C), and the essential oil was added at 65°F (18.3°C). This result emphasizes the possible effects caused by the temperature difference.

[0097] The evening emulsion contains natamycin. Natamycin has no effect on the number of microorganisms. In fact, the number of yeasts in the formulation containing natamycin remains higher over 14 days than in the formulation without natamycin. In addition, natamycin contributes to having a negative impact on the aroma (unpleasant aroma) and consistency of the emulsion.

Claims

1. A method for manufacturing a composition for applying to the skin, comprising: combining a raw material, water, and a first amount of a first agent effective to inhibit the growth of microorganisms to obtain a first mixture; mixing a second amount of a second agent effective to inhibit the growth of microorganisms with the first mixture to obtain a second mixture; wherein the first agent contains a fermented product; the second agent contains probiotics; and the temperature of the second agent is maintained at about 33°C or lower. A method for manufacturing a composition for applying to the skin, characterized by the above.

2. A composition for applying to the skin, comprising: a first amount of a first agent effective to inhibit the growth of microorganisms and a second amount of a second agent effective to inhibit the growth of microorganisms; wherein the first agent contains a fermented product; and the second agent contains probiotics. A composition for applying to the skin, characterized by the above.

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