(Editor's note: Please note the present article originally appeared incorrectly referencing "meadowfoam" as the extract source, when in fact it is "meadowsweet." C&T regrets the error). ... A meadowsweet extract is described here to control Staphylococcus hominis and its enzymatic activity that causes axillary malodor. Novel meta sequencing and culturomics techniques were used to verify its effects and those of a test deodorant containing it.
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(Editor's note: Please note the present article originally appeared incorrectly referencing "meadowfoam" as the extract source, when in fact it is "meadowsweet." C&T regrets the error). ... A meadowsweet extract is described here to control Staphylococcus hominis and its enzymatic activity that causes axillary malodor. Novel meta sequencing and culturomics techniques were used to verify its effects and those of a test deodorant containing it.
From a physiological point of view, perspiration is a natural process that regulates body temperature via the secretion of sweat.1 It is produced by sweat glands, epidermal structures that can exist as two types: eccrine and apocrine. Eccrine glands are found over the entire body at birth and produce sweat composed primarily of water and ions.2 Apocrine glands, on the other hand, are associated with hair follicles and develop at the time of puberty in certain specific zones such as the underarms, mammary areolas or genital regions.
In addition to water and ions, the sweat released by these glands also contains organic compounds such as proteins, sugars and lipids. Underarm apocrine glands are therefore an important source of nutrients for the cutaneous microbiota, promoting bacterial proliferation.
Sweat is odorless when it is secreted because its constituent molecules themselves are odorless. Axillary malodor is caused by the enzymatic degradation of sweat molecules by the cutaneous microbiota.11-13
Among the broad diversity of bacteria composing the cutaneous microbiota, some species stand out for their ability to transform odorless secretions into volatile odor products.3, 4 This is the case of Staphylococcus hominis. This odor-generating property results from its ability to metabolize sweat compounds thanks to specific enzymatic activities such as those of C-S lyase.
The enzyme C-S lyase reduces Cys-Gly-3M3SH into cysteine, glycine and 3M3SH, the latter being a particularly malodorous volatile compound belonging to the family of thiols. The thiol nature of 3M3SH gives it a significantly lower olfactory threshold than other volatile compounds, making it the principal agent responsible for the intensity of perspiration odors.3, 10
Three main strategies have been used for decades to prevent odors arising from perspiration:
- preventing the secretion of sweat,
- masking odors or
- eliminating bacteria of the axillary microbiota.5
Nevertheless, these strategies are currently contested.6 The antiperspirant strategy is not physiological since it prevents sweat from being released onto the surface of the skin. It also often requires the use of aluminum salts that are controversial ingredients.7
The fragrance compounds used to hide perspiration odors can be a source of irritation for the underarm skin due to their alcohol content. Finally, the antiseptic strategy uses broad-spectrum antimicrobial agents and does not respect the balance of the axillary cutaneous microbiota. In addition, antiseptic products used can irritate skin, be allergenic or are suspected of being endocrine disruptors.5, 6 These different issues explain the need to develop new anti-odor strategies that are risk-free, natural and that specifically target the biological origin of unpleasant axillary odors.
In this context, the current authors sought to develop an active ingredient targeting the mechanisms involved in odor generationa. To achieve such, a novel test model was developed to determine whether the production of malodorous molecules was the result of a quantitative modification of the microbiota (dysbiosis) and/or a qualitative modification (modified enzymatic activity of the microbiota). These studies are described here.
Materials and Methods: Microbiota and Odor Analysis
Test model: First, 24 Caucasian volunteers underwent an indoor calibrated sport session to stimulate the generation of perspiration odor of distinctive intensity. The olfactory evaluation of t-shirt samples was conducted by a panel of experts qualified for sensorial measurement and trained to note the presence and intensity of odors of a sample on a scale from 0 to 4. This evaluation was conducted on the same day as the physical activity. This analysis established a non-odorous group and a malodorous group (see Figure 1, below).
Microbiota analysis, culturomics: The microbiota of the axillary zone of each volunteer was sampled for a quantitative and qualitative analysis. The relative abundance of bacterial species responsible for odors was studied. Among the species quantifiable under the conditions of this study, the results obtained by 16S rRNA gene sequencing showed an increase of the S. hominis relative abundance in the malodorous group, while the relative abundance of Cutibacterium avidum was not significantly modified (see Figure 2a, below).
Cutaneous microbiota samples were then collected by swabbing the 24 volunteers after 1 hr of indoor cycling. The samples were processed using a culturomics-derived methodology, wherein selective nutrient agar — a custom-made culture medium adapted to isolate some S. hominis clones and promote the growth of this strain — served as the growth medium. After a 48 hr incubation at 37°C, the plates were analyzed and more than 66 colonies with an S. hominis-like phenotype were picked up.
Each isolate was then identified by Internal transcribed spacer-PCR and 28 isolates were confirmed as S. hominis strains. Crude extracts were obtained from these isolates and the activity of the C-S lyase enzyme, involved in unpleasant odors (thiols) generation, was studied.
Interestingly, results highlighted significantly higher C-S lyase activity in S. hominis crude extracts from malodorous volunteers (see Figure 2b, below), compared with S. hominis crude extracts sampled from non-odorous subjects. These results identified the importance of the microbiota as well as the intrinsic activity of its C-S lyase in odor generation.
Natural Active to Target S. Hominis and C-S Lyase Activity
With the aim of developing a natural active to target S. hominis and its C-S lyase activity, 150 candidates were screened for their potential to inhibit the enzymatic activity responsible for thiols generation. Metabolomic analysis revealed meadowsweet extract holds a unique polyphenol profile with the capacity to inhibit thiol production thanks to its families of procyanidins and tellimagrandins. A test deodorant containing the extract was therefore developed for additional in vivo testing.
Meadowsweet Deodorant Study and Meta Sequencing
To assess the efficacy of meadowsweet for deodorant applications, 12 non-smoking Caucasian volunteers (mean age: 52 ± 11 years) who had slight to very intense perspiration odor intensity after a sport session were enrolled. Subjects gave a written informed consent for their participation in this clinical study.
Test protocol: All subjects were instructed to stop using antiperspirants three weeks before the study and to use the provided test deodorant (see Formula 1, below) during the two first weeks of the study. Volunteers also used a normalizing shower gel and shampoo provided during the three weeks preceding the study. In addition, wearing perfume, using body lotion, eating spicy or strong-smelling foods (garlic, onions, asparagus, etc.) and drinking alcohol were prohibited three days before the physical activity test session.
Volunteers were instructed to apply the deodorant containing the meadowsweet extract at 1% in the morning after washing. At each test appointment, the volunteers wore a provided cotton t-shirt professionally laundered, machine washed (90°C) for one hour without any additional detergent. After the cycling session, subjects waited one hour in a temperate room under standardized conditions, still wearing the t-shirts to allow odors to form. Cutaneous microbiota samples were taken from the underarms with sterile swabs.
Microbiota sampling: Bacterial genomic DNA was extracted from the swabs, and samples of cutaneous microbiota were analyzed by 16S rRNA gene sequencing. After sequencing, sequences were demultiplexed and used for downstream data analysis. Bioinformatic analyses consist in cleaning sequences, clustering them in operational taxonomy units (OTUs) and assignment of OTUs.
Results demonstrated that the application of the natural active deodorant before the sport session significant decreased the S. hominis relative abundance without impacting the C. avidum relative abundance (see Figure 3a, below). Moreover, the diversity of axillary bacterial communities was not significantly modified by the natural active deodorant, thereby preserving the balance in this skin zone (see Figure 3b, below).
Culturomics study and C-S lyase activity: Isolates and crude extracts of S. hominis from malodorous volunteers were also obtained as previously detailed. The crude extracts (containing the S. hominis C-S lyase), a substrate of this enzyme (benzylcysteine) and a molecule required for the fluorescence reaction (monobromobimane) were mixed and incubated for 30 min. For this test, fluorescence intensity is correlated with thiol release and thereby related to C-S lyase activity.
Crude extracts of S. hominis were pre-incubated with the meadowsweet active at 0.25%, 0.50%, 0.75% or 1.00% for 15 min. The specific activity of C-S lyase was calculated by dividing the fluorescence increase (DF) after 30 min of reaction by the quantity of proteins (mg) in the crude extract and was expressed as DF s-1 mg-1.
The effect of the active ingredient on enzymatic activity was determined by comparing the specific activities of the bacterial extract in the presence and absence of the active ingredient. The results showed that compared with D0, the active significantly reduced enzymatic activity in a dose-dependent manner; at 1%, it reduced the activity of the enzyme by 52% (see Figure 4, below). The ingredient therefore demonstrated the capacity to regulate the activity of S. hominis that generates perspiration odors.
Discussion and Conclusion
By reducing the relative abundance of S. hominis and limiting its C-S lyase activity, the meadowsweet-based active ingredient and test deodorant containing it significantly reduced the quantity of malodorous volatile thiol compounds. Indeed, as of the first application in the morning before the physical activity session, the perspiration odors released after a physical activity session and measured by a sniff-test were significantly reduced by 58.0% (p < 0.01).
After daily use for 7 days and without having applied the deodorant in the morning before the physical activity session, a reduction in the intensity of perspiration odors after a physical activity was also observed with a sniff test (-39.8%, p < 0.01). Interestingly, the volunteers in this study also positively rated the deodorant at 7.6/10 for its daily efficacy on body odors and 7.5/10 for its capacity to limit perspiration odors after physical activity (data not shown).
The described meadowsweet extract is therefore proposed as a natural active to specifically target the mechanisms behind axillary odor production. In addition, it demonstrated no negative impact on the overall skin microbiome balance.
a Deolya (INCI: Spiraea Ulmaria (Meadowsweet) Extract is a product of Silab.
References
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