Obtention of the bacterial ferment

Lactobacillus sp. ferment (Lectroglaze^TM biotech ingredient, Lipotec S.A.U., Lubrizol Life Science, Beauty) was developed from a Lactobacillus strain isolated from fermented beets. The biotechnological process to produce the material involved in the fermentation in stirred tank bioreactors, followed by clarification to remove biomass. Ferment broth contains Oligosaccharides (fructo and galactooligosaccharides) (1-3 KDa), lactic acid, and peptides (0.5-1.4 KDa).

Unless otherwise stated, all statistical analysis in vitro were calculated using an unpaired Student’s t-test, and in vivo, a Student’s t-test or Wilcoxon test after checking the normality distributions by Shapiro-Wilk test.

Inducing SCFAs production, electricity and antioxidant capacity of S. epidermidisin vitro.

SCFAs production was measured in aculture containing S. epidermidis treated with Lactobacillus extract for 17h. Acetic acid, isobutyric acid, isovaleric acid and methylbutyric acid were measured by means of a Shimadzu gas-chromatograph equipped with Flame-ionization detector, comparing each analyte to a known control (Scharlab, Sigma-Aldrich) (32,33).

For measuring the potential of theextract to induce electricity generation in S. epidermidis, the bacteria were incubated in the presence/absence of the extract for 24h. Electricity produced by bacteria was detected by using a chamber equipped with a cathode and an anode. The cathode has wrapped up to a Nafion membrane which served as a proton exchange membrane. Voltage changes were recorded by a digital multimeter (Lutron) for 300 seconds. The highest voltage value was used for quantification.

To evaluate the antioxidant activity, S. epidermidis was treated with Lactobacillus extract for 24h. Culture medium alone and with the bacteria were used as basal control. The antioxidant efficacy was evaluated by FRAP (Ferric reducing antioxidant power) assay.

Inhibition of collagen degradation in a skin biopsy model for stress-induced premature senescence (SIPS) in the presence of S. epidermidis.

The rejuvenating effect of Lactobacillus extract was tested ex vivo on human skin explants, in which the SIPS phenotype was induced in the presence of S. epidermidis. Biopsies were treated on the surface with S. epidermidis (4h) and then H₂O₂ (2mM) was added to the culture medium (2h, systemically). Lactobacillusextract was then applied systemically and topically (24h) daily for 3 days (day 4=collection). Sections were incubated with collagen hybridizing peptide (CHP) Cy3-Conjugate for quantification of denatured collagen by measuring fluorescence intensity.

Clinical evaluation of a cream containing 2% of Lactobacillus extract for the assessment of youthful looking smooth and glowing skin.

A clinical test was carried out on 161 female and male volunteers from Spain and China. The volunteers from Spain were comprised of 95 subjects which were split into a younger group (20-30 y.o.) and an older group (40-60 y.o.). The volunteers from China were comprised of 66 older volunteers only (40-60 y.o). All the volunteers applied either a cream containing 2% of the active ingredient or a placebo cream on the whole face, twice a day for 28 days. After the treatment, pores changes were evaluated.

Specific inclusion criteria:

• Age: between 20 and 60 years old (±1 year).

• Skin type: Phototype II–III.

• Subjects showing pores on cheek area.

• Subjects showing light skin tone (Individual Typology Angle > 35).
  
  

Evaluation of pores

The number of total pores and the pores index (accounting for pore depth, size and density) were evaluated on the cheek area with Antera 3D® system (Miravex). With age, pores change in shape and become enlarged and elongated, which is mainly caused by the reduced skin collagen and elasticity due to aging (34). The length and area of the age-related pores were analyzed using computer vision and were compared between younger and older groups of volunteers. Briefly, from the images obtained, circular pores were subtracted obtaining an image where only the elongated pores could be analyzed. A second filter for pore size was applied to ensure that only visible age-related pores remained. For each image, the mean length and the percentage of area covered by elongated aged pores were measured.

Another clinical study was conducted with 95 male and female participants from Spain, following the same protocol and assessment criteria as described in the preceding study. After 28 days of treatment, changes in skin translucency, luminosity and hydration were evaluated.

Measurement of skin translucency

Skin translucency was evaluated by measuring the diffuse light scattered within the skin through Translucency Meter TLS (Dia-Stron). A skin translucency 3D map was generated by creating a surface plot with a colormap based on the average measurement values obtained on the cheek from all the volunteers.

Skin luminosity assessment

The luminosity of the skin was evaluated on the cheek through the L* parameter in the cieLab color scale using Antera 3D® system (Miravex).

Measurement of skin hydration

Skin moisturization was assessed using a Corneometer® CM 825 (Courage & Khazaka). A 3D skin hydration map was generated by creating a surface plot with a colormap, based on the average values measured at 5 distinct points on one hemi-face of each volunteer.

Inducing SCFAs production, electricity and antioxidant capacity of S. epidermidisin vitro.

The capacity of Lactobacillus extract to induce SCFAs in S. epidermidis was demonstrated in vitro, as SCFAs act as electron donors and are thereby able to promote electricity production (5,6,7).

As shown in Figure 1, treatment with ​​​​​​​Lactobacillus extract induced acetic acid, isobutyric acid, isovaleric acid and methylbutyric acid release in vitro in the presence of S. epidermidis.

Figure 1. % Induction of SCFAs release measured by HPLC (vs. Control: *p < 0.05; **p < 0.01).

As it is hypothesized that SCFAs production contribute to electricity generation by acting as electron donors (1,2), the capacity of the ingredient to induce electrogenic activity in the bacteria was next evaluated. A significant increase in the levels of electricity production of the S. epidermidis culture in the presence of the Lactobacillus extract was observed compared to the control (Figure 2).

Figure 2. A) % Highest point of electricity in the presence of S. epidermidis (vs Control: *** p < 0.001). B) Representative image showing the changes in electricity along time measured by a digital multimeter.

As electricity may play a key role in neutralizing ROS (7, 9), we evaluated if the Lactobacillus extract enhances bacterial antioxidant activity. As shown in Figure 3, the active ingredient exhibited some antioxidant efficacy on its own. However, when combined with S. epidermidis, the induction of the antioxidant capacity was superior compared to the antioxidant efficacy of either the active ingredient or S. epidermidis alone.

Figure 3. % Antioxidant efficacy measured by FRAP analysis (vs. Control: **** p < 0.0001; vs. Control with S. epidermidis: **** p < 0.0001).

Inhibition of collagen degradation in a skin biopsy model for SIPS in the presence of S. epidermidis.

Lactobacillus extract was able to significantly reduce SIPS induced collagen degradation, minimizing the age-induced collagen disruption for a rejuvenating effect (Figure 4).

Figure 4. A) % Denatured collagen levels (vs. Senescence: **** p < 0.0001). B) Representative images of collagen degradation in human skin biopsies measured by fluorescence measurement of CHP binding.

Taken together, these results demonstrate that Lactobacillus extract increases SCFAs production in S. epidermidis, thus inducing the generation of bioelectricity and promoting an antioxidant effect to effectively protect the skin from damage caused by oxidative stress.

Clinical efficacy of a 2% Lactobacillus extract cream for the assessment of youthful looking smooth and glowing skin.

Reducing the appearance of pores and age-related pores.

Pore assessment showed a significant decrease in the number of pores and pores index (which accounts for pore depth, size, and density) for smoother skin (Figure 5) after the application of the cream containing 2% of Lactobacillus extract.

Figure 5. Variation (%) in the: A) Number of pores (*p<0.05 vs. initial time and placebo) B) Pores index (low significance 0.05<p<0.1 vs. initial time on the cheek area using the Antera 3D® system (Miravex). C) Representative images showing minimized appearance of pores in a volunteer from Spainand D) from China.​​​​​​​

We then analyzed pore length and area finding an 80% higher prevalence of age-related pores in the older group compared to the younger group (data not shown). Further, the effect of the active cream treatment on the pore’s phenotype was analyzed, showing a significant reduction in elongation, contributing to a younger looking complexion (Figure 6).

Figure 6. Variation (%) in the A) length of elongated pores (*p<0.05 vs. initial time and placebo), and B) the pore area of elongated pores (*p<0.05 vs. initial time) on the cheek area. C) Representative example of image processing data analysis using Antera 3D® system (Miravex), in which age-related pores were identified by detecting elongated and enlarged pores through computer vision techniques D) Representative images showing the minimized appearance of enlarged pores in a volunteer from Spain.

Increasing skin translucency.

Skin translucency was evaluated, and the results showed a significant increase contributing to a radiant skin complexion and a glow from within effect (Figure 7) after application of the active cream.

Figure 7. A) Variation (%) in skin translucency (****p<0.0001 vs. initial time) on the cheek area B) Representation of the 3D map of skin translucency generated from the average measurement values of all volunteers using the Translucency Meter TLS (Dia-Stron).

A significant increase in skin luminosity contributing to a glowing skin complexion was observed (Figure 8, 9).

Figure 8. Variation (%) in skin luminosity (**p<0.01 vs. initial time and placebo)evaluated on the cheek area through the L* parameter in the cieLab color scale using Antera 3D® system (Miravex).

Figure 9. Representative images showing an increase in skin glow in female volunteer from Spain.

Increase in skin hydration

Hydration is also key to achieving natural glow. When skin is dehydrated, it can appear dull and dry,

losing radiance (35). A skin hydration 3D map was generated based on the measurement values obtained at different regions of the face. A significant increase in hydration was observed on the cheek area (Figure 10) after active cream application.

Figure 10. A) Variation (%) in skin hydration (**p<0.01 vs. initial time and *p<0.05 vs. placebo) on the cheek area B) Representation of the 3D map of skin hydration generated from the average measurement values of all volunteers using the Corneometer® CM 825 (Courage&Khazaka).

The novel Lactobacillus extract emerges as an innovative and comprehensive solution to reduce the visibility of age-related pores and improve dull and dry skin. Inspired by the functionality of electrogenic bacteria, a novel active ingredient has been developed to boost the skin’s natural electricity levels, acting as ROS scavengers and thereby offering a significant antioxidant effect. In vitro and ex vivo studies demonstrated the ability of the Lactobacillus extract to protect skin cells from the damaging effects of cellular senescence, helping also to minimize oxidative stress and collagen degradation under these conditions. On volunteers, a cream containing 2% of Lactobacillus extract helped obtain a glowy skin appearance by improving skin translucency, luminosity and hydration, as well as by decreasing the appearance of all pores and of the more specific age‑related pores.

The development of skincare products targeting skin microbiota represents a paradigm shift in the cosmetic field. This approach moves away from conventional methods focused solely on surface‑level concerns to more holistic strategies addressing the underlying causes of skin aging and damage. By leveraging the natural symbiotic relationship between the skin and its resident bacteria, this innovative solution has the potential to revolutionize skincare, promoting healthier, more resilient skin.

Incorporating the Lactobacillus extract into anti-aging skin cosmetic formulations will allow brands and manufacturers to offer consumers a powerful solution that promotes overall skin and microbiome health, combating the aging process by minimizing age-related changes in pore visibility, glow, and hydration.