PHILIPPE MOUSSOU, FLORENCE HENRY, AURELIE COURTOIS
BASF Beauty Care Solutions

Enlarged facial pores are a major cosmetic concern, as cited by around 31% of European consumers (1). These enlarged pores are manifested as widened openings of the pilosebaceous follicles, which may appear either as empty funnels or as openings clogged with keratinised plugs known as comedones. A number of intrinsic and extrinsic factors contribute to the development of enlarged pilosebaceous pores, including gender, genetic predisposition, hygiene practices, environmental conditions, ageing, chronic exposure to UV radiation, the presence of comedogenic substances, acne, and seborrhoea (2,3). There is a positive correlation between enlarged pore size and increased sebum production as well as ageing (4, 5). In a multi-ethnic study of 2,585 women, Flament et al. reported that the pore size and density varied greatly with ethnicity, plateaued in most cases with age, and globally corresponded to the subjects’ self-perception (6).

Mushroom extracts have been used for decades in cosmetics to tighten pores due to their rich content of bioactive compounds, including polysaccharides, triterpenoids, and antioxidants. These compounds provide the skin several benefits, including astringent properties, which help to tighten and tone the skin, reduce the appearance of pores, and help to hydrate and maintain the skin’s moisture balance. The recent rise in the use of mushroom extracts in cosmetics is in line with wider holistic wellness and sustainability trends as consumers prioritise ethical sourcing and environmentally friendly formulations. At the same time, the harvesting of wild mushrooms for cosmetic use has raised environmental concerns, driving the search for more responsible sourcing methods, such as cultivation or fermentation. The aim of this paper is to illustrate the improved mode of action of a well-established ingredient based on Fomes officinalis, whose sourcing has been switched for ecological reasons from wild collection to a biotechnological process involving solid-state fermentation.

For 20 years, Butylene Glycol (and) Fomes Officinalis (Mushroom) Extract (and) PEG-40 Hydrogenated Castor Oil, an extract of the fungus Laricifomes officinalis (F. officinalis), also known as agaric, is a proven commercially available ingredient for pore refinement. The mushroom is a wood-inhabiting parasitic fungus that forms distinctive conks and is widespread in the northern hemisphere, mainly in subalpine, montane hypoarctic and boreal old-growth forests on large and old coniferous trees such as larch. However, the population of F. officinalis has decreased significantly, with a decline of about 70-75% of the population. This decline has been attributed to habitat degradation and mass collection (7). In March 2019, the International Union for Conservation of Nature (IUCN) listed the species as endangered and included it in the Red List of Threatened Species (8).

In response to ecological concerns, BASF launched a project in 2018 to transition away from wild collection towards the sourcing of F. officinalis via a more sustainable cultivation method based on solid-state fermentation. The solid-state fermentation technique involves the growth of microorganisms on solid, insoluble materials, such as millet grains, under controlled conditions, in the absence or near absence of free water ((9, 10). The organic nutrients and a certain amount of water are combined in bags to form a solid substrate for mushroom mycelium cultivation. After sterilization through autoclaving at 121°C, the solid substrate is inoculated with the mushroom mycelium and the bags are incubated under controlled conditions of temperature, humidity, CO2 and light until the nutrients are fully consumed and the conversion into mushroom mycelium is completed. This biotechnological production process takes about four months. After solid-state fermentation, the certified organic mushroom mycelium is dried at a temperature below 40°C to protect the heat-sensitive ingredients and ground into a fine powder before being extracted using a combination of water and butylene glycol as a solvent and 0.3% PEG-40 hydrogenated castor oil as a solubiliser.

Fungi are optimal microorganisms for solid-state fermentation, as this biotechnological culture process closely resembles their natural habitat. Fungi typically grow in natural settings on solid substrates such as soil, wood or bark. By bringing cultivated fungi into close contact with the insoluble substrate, the highest nutrient concentration can be achieved for fermentation (9). The principal advantages of this biotechnological culture process compared to harvesting from wild populations are that it has no impact on species population and that it provides controlled, reproductible conditions of production. In comparison to liquid or submerged fermentation, the advantage of solid-state fermentation is its low environmental footprint with low or no water consumption and lower energy requirements (10).

The resulting new generation of this pore minimiser has a naturalness content of 99.7% (according to ISO 16128). Its properties were studied in vivo for pore tightening, astringent sensation, and excess oil removal and ex vivo for its moisturizing properties.

Efficacy on pore tightening
A double-blind, randomised clinical study was carried out on 25 female volunteers, aged between 23 and 63 years, with visible pores and oily skin. The efficacy of the new product (derived from the solid-state fermentation process) at 3% was compared to the previous version (from mushroom harvested in the wild) at 3%. Both were dissolved in water, with a standardised single application of the products by hemi-face and check points before application (baseline) and immediately (5-10 minutes) after application. Volunteers were provided with a circular cotton pad soaked in 5 ml of either the new or previous version of the product at 3% and instructed to swipe the pad over the entire right or left side of their face, as randomly assigned. VISIA imaging and image analysis was used to calculate the pore count and total pore area. The Trichoscience scope with 60x magnification was used to take illustrative macro-images of the pores both at baseline and after using the new product. The volunteers completed self-administered questionnaires to evaluate how they perceived the appearance of their facial pores. The results were then expressed as the mean percentage of change versus the baseline (before treatment), with a negative value indicating a reduction in the pores’ appearance.

Astringent sensation
Astringents are defined as substances that induce the contraction or tightening of soft tissue. They typically elicit sensations of pulling, puckering, or tautness in the skin. The objective of the clinical trial was to assess the immediate perceivable astringent effect of the new version of the active ingredient in comparison to a placebo. The investigation employed a double-blind, placebo-controlled, split-face design along with randomisation for the standardised single application on hemi-facial regions. The trial enlisted 31 female volunteers aged between 23 and 65 years who were provided with a circular cotton pad saturated with either the new product at 3% in water or a placebo solution (water). The immediate perceptions of astringency were evaluated by means of a consumer self-perception questionnaire. Participants rated the intensity of the astringent sensation on a 4-point scale, with response options including: (0) Not perceptible, (1) Barely perceptible, (2) Moderately perceptible, and (3) Dramatically perceptible. 

Removal of excess oil
To assess the impact on skin oiliness and shininess, the same comparative study design was used as the one for assessing the pore-tightening effect on 25 female volunteers aged between 23 and 63 years. The evaluation of skin oiliness and shininess included measuring sebum with the Sebumeter SM 815 and VISIA illustrative imaging five to ten minutes after application (also referred to as immediate measure). A specialised tape was applied to the forehead for 30 seconds to absorb sebum, then affixed to the measuring head of the Sebumeter. Subsequently, the transparency of the tape was quantified using the Sebumeter, with higher transparency indicating an elevated sebum level. To evaluate their perception of the test products’ performance and the resulting skin benefits, the volunteers completed self-administered questionnaires immediately after application. Responses were recorded on a scale ranging from 1 (strongly disagree) to 6 (strongly agree). Mean percentage changes versus the baseline were calculated for each item, with negative values indicating a reduction in the sebum level.

Maintenance of skin hydration
To evaluate the effect on skin hydration, an ex vivo study was conducted on the human stratum corneum. Human skin explants obtained from plastic surgery were used. Epidermal sheets were prepared by peeling the dermis of the explant after briefly heating it. Stratum corneum isolation was achieved through enzymatic digestion. The epidermal sheets were equilibrated under controlled humidity conditions prior to treatment with hydrogels containing the new version of the product (3% or 5%), the previous version (3% or 5%), or a placebo control. Each hydrogel was applied at a rate of 1 mg/cm² three times at 30-minute intervals before measurements were taken. Dielectric conductivity measurements were conducted using the Tagami test, wherein the water content in the stratum corneum was assessed via the dielectric conductivity method using a Skicon-200 hygrometer. Measurements were taken at various intervals over 24 hours. The experiments involved triplicate measurements for each sample and time interval, utilising samples from 10 stratum corneum specimens sourced from three donors. The moisturising effect was quantified by calculating the area under the dielectric conductivity curve over 24 hours. The results, expressed as a percentage ± SEM of the average area under the curve for each product tested against the placebo control, underwent statistical evaluation using Student’s t-test following validation of normality and equality of variances.

Demonstration of minimising effect on visible pores 
The in vivo study revealed that the new generation of the pore minimiser reduced the appearance of visible pores, exhibiting an efficacy that was twice as pronounced as its predecessor. Following its application, the novel product exhibited a reduction (p < 0.01) of 5.4% in the total pore area compared to the baseline, a performance which was significantly (p < 0.05) superior by a factor of 2.8 compared to the previous generation (Fig. 1A). The results for the pore count parameter were also consistent, showing a significant decrease (p < 0.01) of 4.9%, which was also significantly 2.3 times superior to the previous generation. The reduction in pore size is demonstrated visually in macrographs (Fig. 1B)

These results were confirmed by the self-questionnaire, with a significant proportion of volunteers (76%) reporting a reduction in the appearance of their pores following the use of the new product. Compared to the 60% success rate observed with the previous version, the difference between the two product generations is not statistically significant.

Demonstration of increased astringent sensation
The new product demonstrated a statistically significant increase (p < 0.05) in the astringent sensation on the skin immediately after application compared to the placebo, as indicated by the data presented in Figure 2. The average rating attributed by volunteers was 1.5, suggesting a moderate perceived sensation characterised by a discernible but tolerable effect.

Removal of excess oil for less shiny skin 
A notable reduction in sebum levels was observed immediately after application, with a statistically significant decrease (p < 0.001) of 43% compared to the baseline measurements. This reduction in sebum level mirrored the efficacy demonstrated by the preceding generation of the product, which exhibited a substantial decrease of 39% (p < 0.001) relative to the baseline as shown in figure 3A. The tangible reduction in skin shine resulting from the mitigation of excess oil production was visually evident in comparative photographs of volunteers as shown in figure 3B.

The reduction in sebum levels observed in vivo was corroborated by the results of the self-assessment questionnaire. Following the application of both the new and previous version of the product, a majority of volunteers perceived a reduction in excessive skin shine (88% and 72%, respectively) and reported that their skin felt less oily (84% for both formulations).

Maintenance of skin hydration
In the ex vivo setting, the new product exhibited an elevation in the dielectric conductimetry of the human stratum corneum, suggesting a potential role in maintaining skin hydration levels. When applied at concentrations of 3% and 5% in hydrogel formulations, both versions of the product demonstrated a significant increase in the dielectric conductivity of the human stratum corneum compared to the placebo hydrogel. There was a notable hydration effect observed over a 24-hour period, with increases of +40% (SEM ± 6%, p < 0.01) and +100% (SEM ± 22%, p < 0.001) for the new generation at concentrations of 3% and 5%, respectively. These hydration outcomes were comparable to those achieved with the previous version, which showed increases of +47% (± 16%, p < 0.05) and +102% (± 14%, p < 0.001) at concentrations of 3% and 5%, respectively. These findings suggest that the new generation of the active ingredient shows promise in terms of contributing to the preservation of skin moisturisation levels without inducing skin dryness.

Overall, the findings of this study provide evidence that the new generation of the active ingredient based on F. officinalis is even more effective in improving skin texture, reducing pore size, and minimizing excess oil production. Sustainable sourcing through solid-state fermentation underscores its potential as a promising solution for environmentally conscious skincare consumers. Further research and long-term studies may offer additional insights into the sustained efficacy and benefits of the active ingredient in skincare formulations.