The antimicrobial properties of cis-6-hexadecenoic acid (C16:1Δ6), a component of the innate human metabolome, were studied and its application to cosmetic products was investigated in detail. A variety of the resident and transient microbial flora of the skin, oral cavity, and intestine was applied to an investigation of the antimicrobial activity of C16:1Δ6. C16:1Δ6 showed selective antibacterial activity against human microbial pathogens such as Clostridium perfringens (one of the most common causes of food poisoning) and Streptococcus mutans (one of the tooth-decaying bacteria), as well as Staphylococcus aureus. C16:1Δ6 seemed to possess a “species-selective” antibacterial activity against bacteria belonging to the genera Staphylococcus and Clostridium. We performed a preliminary assessment of the application of C16:1Δ6 as an antimicrobial component in a liquid lip gloss by performing preservation efficacy and home-use tests. We noted that the amount of p-hydroxybenzoate was needed in an oily cosmetic, especially liquid lip gloss, could be reduced by replacing p-hydroxybenzoate with C16:1Δ6. We suggest that C16:1Δ6 may find application as a stable antimicrobial substance gentle enough for use in consumer products by its selective antimicrobial characteristics.
Cis-6-hexadecenoic acid (C16:1Δ6) (
human sebum and is ubiquitous in human skin and hair [
This fatty acid is also called sapienic acid because the presence of C16:1Δ6 is restricted to humans among hair-bearing animals [
As reported elsewhere, we have developed methods for the convenient production of C16:1Δ6 by means of industrial processes [
In a study of the relationship between sebum composition and skin disease, we found that there was a significant decrease in free C16:1Δ6 content in the skin of patients with atopic dermatitis (AD) compared with that of healthy controls [
In the present paper, we characterize the antimicrobial properties of C16:1Δ6 against the resident and transient microbial flora of humans. Additionally, we suggest the feasibility of using C16:1Δ6 with p-hydroxybenzoate as an antimicrobial substance in cosmetic products. Though p-hydroxybenzoate is widely used as a chemically synthesized preservative in a variety of cosmetic and food products, the suggested combination with this antimicrobial substance would decrease the use of p-hydroxybenzoate, which may be advantageous from the point of view of safety and ecology. Thus, the use of C16:1Δ6, an innate component of the human metabolome, may find application in the suppression of microorganisms harmful to human health while reducing the need for synthesis and dissemination of synthetic preservatives.
This study used Rhodococcus sp. strain KSM-T64 (FERM P-18182 in NITE), a mutant impaired for esterase production that was derived from the alkane-as- similating Rhodococcus sp. strain KSM-B-3 (FERM BP-1531 in NITE) [
Microorganisms mentioned below were used for the investigation of the antimicrobial efficacy of C16:1Δ6. Representatives of the bacteria typically found on the human skin were as follows: Staphylococcus epidermidis JCM 2414, Staphylococcus saprophyticus JCM 2427, S. aureus ATCC 12600, Micrococcus luteus JCM 1464, Micrococcus varians IFO 15358, Corynebacterium xerosis JCM 1971, and Propionibacterium acnes ATCC 6919. Representatives of the bacteria found in the human oral cavity were as follows: Streptococcus mutans JCM 5705, Streptococcus salivarius JCM 5707, Streptococcus sanguis JCM 5708, Actinomyces viscosus ATCC 15987, and Actinomyces naeslundii ATCC 12104. Representatives of bacteria found in the human intestine were as follows: Clostridium perfringens JCM 1290, Clostridium butyricum JCM 1391, Escherichia coli ATCC 11775, and Enterobacter cloacae JCM 1232. Representatives of spore-forming bacteria broadly detected in the environment were as follows: Bacillus subtilis IFO 13719 and Bacillus cereus JCM 2152. Representatives of bacteria generally found in aqueous environments were as follows: Pseudomonas aeruginosa IFO 12689, Pseudomonas putida ATCC 14164, Pseudomonas cepacia JCM 5964, and Pseudomonas fluorescens JCM 5963. Representatives of the yeasts were as follows: Candida albicans JCM 1542, Saccharomyces cerevisiae JCM 7255, and Cryptococcus laurentii IFO 0609.
In addition, three unidentified environmental strains (isolated during home- use testing of liquid lip gloss) were employed as the test strains for the preservative efficacy testing. These strains consisted of two Gram-positive coccal bacteria (Staphylococcus sp. and Streptococcus sp. designated 030411-1-1 and 030411- 1-2) as well as a Gram-positive rod-shaped bacterium (Bacillus sp. designated 030411-1-3).
SCD and SCDLP agar media purchased from Nihon Pharmaceutical (Tokyo, Japan) were used for bacterial cultivation, except for P. acnes, which used GAM Agar Modified “Nissui” purchased from Nissui Pharmaceutical (Tokyo, Japan). GP and GPLP agar medium purchased from Nihon Pharmaceutical were used for culturing yeasts.
Chemicals for cultivation of Rhodococcus sp. strain KSM-T64 were of analytical grade and obtained from Wako Pure Chemical (Osaka, Japan) unless otherwise noted. Oleic acid (C18:1Δ9) used for the study of antimicrobial activity was obtained as an analytical grade compound from Sigma (St. Louis, MO, USA), and other materials formulated in cosmetic formulations were of cosmetic grade.
Freshly growing (2 - 3 days on SCD agar at 30˚C) microbes were suspended at 106 cfu/mL in 50 mM phosphate buffer (pH 7.0), and suspensions were mixed well. C16:1Δ6 was prepared as a 10% (v/v) solution in ethanol and added to each bacterial suspension at a final concentration of 25 or 50 mg/L. After a 1-h incubation at 30˚C, a portion of the suspension was sampled. After inactivation of antimicrobial activity of C16:1Δ6 in the sample by Lecithin and Polysorbate 80 (LP) diluent “Daigo” (Nihon Pharmaceutical), the sample was diluted in LP diluent and aliquots of the inactivated sample were spread onto an appropriate agar medium supplemented with LP diluent “Daigo”. After incubation at 30˚C for 2 - 3 days (except for P. acnes, which was incubated at 37˚C for 3 - 5 days), the number of colonies on agar plates were counted, used to calculate cell densities, and then compared to that of control without C16:1Δ6. Cultivation of anaerobic microbes was performed using the “Anaero Pack” anaerobic cultivation system (Mitsubishi Gas Chemical, Tokyo, Japan).
Freshly growing (2 - 3 days on SCD agar at 30˚C) microbes were suspended at 107 cfu/mL in saline and used as inocula at 1% (v/v) in individual aliquots of 50 mM phosphate buffer (pH 6.0). C16:1Δ6 and oleic acid (18:1, Δ9) were prepared as a 10% (v/v) solution in ethanol and added to each bacterial suspension at a final concentration of 10, 25, or 50 mg/L. Following inoculation, the sample was incubated at 30˚C and sampled at 1, 4, and 24 hours in order to confirm the antimicrobial efficacy of each reagent in a short period. Specimens from respective time points were diluted by LP diluent, plated, incubated, and counted as above. An acceptable range of deviation of 0.5 log, which was mentioned in ISO11930, was applied to this preservative efficacy test.
Cosmetic products such as a liquid lip gloss typically are supplied in a container with a built-in brush, and used in a manner in which the application brush is expected to back-contaminate the container with skin-derived materials and microorganisms. To inhibit the growth of microorganisms, appropriate counter-plans must be constructed. To evaluate the antimicrobial efficacy of C16:1Δ6 under these application conditions, model liquid lip glosses with different preservative components were assayed using a preservative efficacy test (a general laboratory testing method [
Preservative efficacy test: Model liquid lip gloss was prepared as a combination of 60% heavy fluid isoparaffin, 8% ceresin, 0.5% stearyl glycyrrhetinate, 2% red No. 202, 2% oxidized titanium, 7% mica coated with oxidized titanium, 0.5% hyaluronic acid, and squalene (balance); this is the base control formulation 1. Aliquots with different preservative system were formulated to include either the combination: formulation 3 with ethyl paraben and propyl paraben (preservatives commonly used in personal care and cosmetic formulations) at 0.05% (w/ w) each or formulation 2 with C16:1Δ6 at 0.1% (w/w) (
Bacterial strains consisted of Gram-positive environmental isolates 030411-1- 1, 030411-1-2, or 030411-1-3, which were derived from home-use testing of liquid lip gloss. Freshly growing bacteria (cultivated on SCD agar plates at 30˚C for 2 - 3 days) were suspended at 108 cfu/mL in saline and used as inocula at 1% (v/w) in individual aliquots of liquid lip gloss. Following inoculation, the lip gloss was incubated at 30˚C and sampled at 1, 2, 3, and 5 days in order to confirm the efficacy of preservative system in a short period. Specimens from respective time points were diluted by LP diluent, plated, incubated, and counted as above. An acceptable range of deviation of 0.5 log, which was mentioned in ISO11930, was applied to this preservative efficacy test.
Home-use test: Liquid lip gloss was formulated as above but using (as preservatives) one of the three following preservative systems (
Preservative system for preservative efficacy test | Preservative system for home-use test | |||||
---|---|---|---|---|---|---|
Ingredient (% (w/v)) | 1 | 2 | 3 | 4 | 5 | 6 |
C16:1Δ6 | - | 0.1 | - | 0.5 | - | - |
Oleic acid | - | - | - | - | - | 0.5 |
Ethylparaben | - | - | 0.05 | - | 0.1 | 0.1 |
Propylparaben | - | - | 0.05 | 0.1 | 0.1 | 0.1 |
applied a given formulation twice per day for 2 weeks. The same group then applied (in the same manner) a separate formulation on each of two subsequent two-week intervals. Written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki following an explanation of the experimental protocols. The study and protocols were approved by our institutional ethics committee. After each 2-week interval, the numbers of panel members who reported incompatibility (e.g., increased irritation) were recorded, and malodor of the returned samples was evaluated by sensual inspection. Water evaporation from one lip of each panel member was determined (both before and after application of 0.05 g of each formulation) using a humidity sensor with a quartz crystal (Skicon-200, IBS Co. Ltd, Shizuoka, Japan) at 30˚C with 30% RH. And the efficacy of suppression of water evaporation from the lip was calculated by using below Equation (1).
Significant differences between experimental values for the number of malodorous samples were determined using chi-square test for goodness of fit, and p-values < 0.05 were considered as the level of significance.
Antimicrobial activity of C16:1Δ6 against 25 kinds of microorganisms corresponding to the resident and transient human flora was evaluated in detail at two different concentrations; 25 or 50 mg/L. It was reported that C16:1Δ6 had a synergistic effect with high concentration of ethanol [
The compound exhibited a very unique “species-selective” antimicrobial activity (that is, antimicrobial effectiveness that differed among species in the same genus), as shown by comparison between C. perfringens and C. butyricum and between S. epidermidis and S. aureus; “species-selectivity” for S. epidermidis and S. aureus was already found by Takigawa et al. [
No. | Test strain | Ratio (%)a | |
---|---|---|---|
Skin microbe | 1 | Staphylococcus epidermidis JCM 2414 | 16.7 |
2 | Staphylococcus saprophyticus JCM 2427 | 18.9 | |
3 | Staphylococcus aureus ATCC 12600 | 1.90 | |
4 | Micrococcus luteus JCM 1464 | <0.100 | |
5 | Micrococcus varians IFO 15358 | <0.100 | |
6 | Corynebacterium xerosis JCM 1971 | <0.100 | |
7 | Propionibacterium acnes ATCC 6919 | <0.100 | |
Oral microbe | 8 | Streptococcus mutans JCM 5705 | <0.100 |
9 | Streptococcus salivarius JCM 5707 | <0.100 | |
10 | Streptococcus sanguis JCM 5708 | <0.100 | |
11 | Actinomyces viscosus ATCC 15987 | <0.100 | |
12 | Actinomyces naesulundii ATCC 12104 | <0.100 | |
Intestinal microbe | 13 | Clostridium perfringens JCM 1290 | <0.100 |
14 | Clostridium butyricum JCM 1391 | 42.2 | |
15 | Escherichia coli ATCC 11775 | 16.4 | |
16 | Enterobacter cloacae JCM 1232 | 26.7 | |
Spore-forming microbe | 17 | Bacillus subtilis IFO 13719 | <0.100 |
18 | Bacillus cereus JCM 2152 | <0.100 | |
Environmental microbe | 19 | Pseudomonas aeruginosa IFO 12689 | 32.5 |
20 | Pseudomonas putida ATCC 14164 | 37.3 | |
21 | Pseudomonas cepacia JCM 5964 | 4.10 | |
22 | Pseudomonas fluorescens JCM 5963 | 10.0 | |
Yeast | 23 | Candida albicans JCM 1542 | 2.20 |
24 | Saccharomyces cerevisiae JCM 7255 | <0.100 | |
25 | Cryptococcus laurentii IFO 0609 | <0.100 |
aValue shows ratio of surviving microorganisms. Reaction was performed at 30˚C for 1 h at the final concentration of 50 mg/L (pH 7.0); initial inoculum was 106 cfu/mL.
Species-selective antimicrobial activity is known in the field of antifungal derivatives against Tricophyton species. At low concentration, miconazole nitrate, a popular imidazole antifungal derivative, shows good antimicrobial efficacy against Trichophyton mentagrophytes but not against Trichophyton rubrum [
We also found that C16:1Δ6 had high efficacy against P. acnes, which is known to be a factor in acne, and against C. albicans, which is known to be a main cause of candidiasis. Given that C16:1Δ6 application to the skin of AD patients has been shown to inhibit colonization by S. aureus [
Considering our results in the context of the literature, we infer that C16:1Δ6 maintains normal skin conditions both by promoting water retention and by playing a defensive role via mild antimicrobial efficacy. This proposed mechanism would be similar to that of sphingosine and related ceramide lipid derivatives, which are known to contribute to the normalization of skin; those compounds show antimicrobial efficacy against various microbes on the skin and in the oral cavity [
We compared the antimicrobial activity of C16:1Δ6 and oleic acid, which was frequently used in cosmetic products, against S. aureus and S. epidermidis at 10, 25, and 50 mg/L respectively. As a result of this experiment, C16:1Δ6 showed higher antimicrobial efficacy against S. aureus than oleic acid at 1 and 4 hours with significant differences. On the other hand, both unsaturated fatty acid showed less antimicrobial efficacy against S. epidermidis compared to S. aureus. This “species selective antimicrobial efficacy” was confirmed at any concentrations, so the result at 50 mg/L was shown in
From this result, we found that human innate C16:1Δ6 had significantly higher selective antimicrobial activity than widely-used oleic acid for the first time.
There are some reports for antimicrobial activity of oleic acid. High antimicrobial efficacy against oral microorganisms [
compositions of S. aureus and S. epidermidis. The linear-chain fatty acid content of S. aureus was higher in than that of S. epidermidis. In contrast, branched- chain fatty acids content was lower in S. aureus compared to S. epidermidis, with values for iso-forms and for anteiso-forms (data not shown). Fatty acids are known to be the main components of the cell membrane, and have been shown to change in response to antimicrobial stressors such as antibiotics. We inferred that the high ratio of anteiso-fatty acids in S. epidermidis contributes to decreased susceptibility to C16:1Δ6 compared to that of S. aureus. In addition, it was reported that the decrease of branched-chain membrane fatty acid content increased susceptibility to a variety of stresses by producing more-rigid membrane in S. aureus [
In general, non-aqueous formulations do not contain water and show low water activity (Aw: <0.75); incorporated microorganisms cannot grow in such formulations, and cell counts decrease gradually even in formulations that lack preservatives [
In a preservation efficacy test using any of three Gram-positive bacterial isolates, it was found that the formulation 2 including C16:1Δ6 at the concentration of 0.1% (w/w) showed a rapid decrease in cell number compared to the control formulation 1 without preservatives (
A similar preservative efficacy was observed in a formulation 3 that incorporated ethyl and propyl parabens at concentrations of 0.05% (w/w) each (
confirmed to have an adequate preservative efficacy for these microorganisms (data not shown).
Home-use tests were used to compare the use of three distinct preservative systems: formulation 4 was 0.5% (w/w) C16:1Δ6 + 0.1% (w/w) propyl paraben; formulation 5 was the combination of ethyl and propyl parabens at 0.1% (w/w) each; and formulation 6 was 0.1% (w/w) ethyl and propyl parabens + 0.5% (w/w) oleic acid. None of the C16:1Δ6-containing samples displayed a malodor after two weeks of home use; in contrast, the other two formulations (parabens only and parabens + oleic acid) exhibited malodor in 7 of 20 and 4 of 20 samples respectively. By using chi-square test for goodness of fit, it was found that there was a significant difference with p-value < 0.05 for the generation of malodor in these formulations. In addition, no panel members reported an irritation during two weeks of twice-daily application (
The formulation 4 with C16:1Δ6 showed a higher antimicrobial efficacy in the home-use test compared to the formulation with oleic acid. Based on these results, human sebaceous fatty acid C16:1Δ6 functioned effectively in a real-use situation while providing mild preservative efficacy for users. We also found that the preservative system with C16:1Δ6 showed good suppression of water evaporation from the lip compared to the system using paraben esters only or paraben esters + oleic acid; formulation 4 showed 52%, formulation 5 showed 45%, and formulation 6 showed 38% as an average ratio of suppression of water evaporation from the lip. We envision a bright future for the development of products
Formulation for home-use test | |||
---|---|---|---|
4 | 5 | 6 | |
Number of malodorous samplesa (Ratio of malodorous samples) | 0 of 20 (0%) | 7 of 20 (35%) | 4 of 20 (20%) |
Number of people felt irritation | 0 of 20 | 0 of 20 | 0 of 20 |
aSignificant differences between experimental values for the number of malodorous samples were determined using chi-square test for goodness of fit, with p < 0.05 considered significance. In this experiment, chi-square test for goodness of fit was 6.73 and the p-value was 0.03.
incorporating this unique unsaturated fatty acid as an antimicrobial agent.
Cosmetics for lips, especially liquid lip gloss, are used for long periods; as a result, saliva, components from lips, and microbes on skin and in the oral cavity might be introduced into a formulation. In that situation, contaminants might negatively influence product quality (e.g., create malodor), depending on the product type, package design, miscibility with water, product usage, etc. To repress a malodor because of growth of microorganisms, it is very effective to add preservatives into a formulation. On the other hand, the lips represent very sensitive mucous membranes; incorporation of preservatives could result in increased irritation and/or damage to the lips. We aimed to improve the preservative efficacy of a formulation in oily liquid lip cosmetics by including C16:1Δ6, a compound that already is present on human skin and displays appealing antimicrobial efficacy. There are many Gram-positive microbes that are resident flora on the skin (e.g., Staphylococcus species) and in the oral cavity (e.g., Streptococcus species). The addition of C16:1Δ6 to cosmetics such as lip gloss is expected to be a very useful approach, and we confirmed the actual effectiveness of this fatty acid. GC analyses show that C16:1Δ6 is stable in oily liquid cosmetics (e.g., liquid lip gloss), which are composed of various oily raw materials, and that C16:1Δ6 is stable when formulated as a simple solution that incorporates an anti-oxidizing agent such as dibutylhydroxytoluene (data not shown). Our clarification of the antimicrobial properties of C16:1Δ6 should facilitate the application of this molecule as a component of consumer products.
C16:1Δ6 had species-selective antimicrobial efficacy against not only staphylococcal but also clostridial species. Notably, C16:1Δ6 showed an appealing profile of antimicrobial activity against objectionable microbes that have potential pathogenicity in humans, including those found on skin and in the oral cavity. By performing preservation efficacy and home-use tests, formulations incorporating C16:1Δ6 inhibited growth of spiked Gram-positive bacteria contaminants, and did not exhibit malodor or irritation during two weeks of human use. Based on these results, we suggest that this molecule may find application as a stable antimicrobial substance gentle enough for use in consumer products.
We thank Dr. K. Yamamoto of Osaka Prefecture University (Osaka, Japan) for the investigation of an antimicrobial mechanism by analyzing bacterial fatty acid composition.
The authors declare that they have no conflict of interest.
Araki, H., Hajime, M., Tamura, E., Hagihara, H. and Takigawa, H. (2017) Human Sebaceous Cis-6-Hex- adecenoic Acid: Possible Application of an Innate Antimicrobial Substance to Cosmetic Products for Mucous Membrane. Journal of Cosmetics, Dermatological Sciences and A- pplications, 7, 133-147. https://doi.org/10.4236/jcdsa.2017.72012