Postbiotics anti-ageing care: COSSMA

Photo: Mikala Skovgaard/Shutterstock.com

Recently, the use of living probiotics-- tyndallized bacteria and lysates-- and prebiotics has been blooming. However, the production of stable, reproducible and safe formulations containing most of these ingredients remains a topic of discussion. Big effort is currently devoted in elucidating the interactions between beneficial microbes and skin.

figure 1: Echogenicity change after 6 weeks. Photo: www.kalichem.it

figure 2: Skin framework digital image treated with postbiotics (white bands are indicator of redensified areas). Photo: www.kalichem.it

The skin is an ecosystem where about a thousand microbe species live in symbiosis with each other and with the host. The balanced pattern of commensal and symbiotic microorganisms forms the skin microbiota, essential for skin health^1,2. Many of these microbial communities are harmless and some even provide functions not evolved by the human genome. Symbiotic microbes support skin barrier function and modulate immune response protecting the skin against pathogens, allergens etc. Since the skin is a finely organized ecosystem, a perfect balance between host and microbe communities exists to preserve homeostasis and wellness.

Disruption of this delicate balance can induce perturbations in skin barrier function, onset of dermatological disorders associated with photoageing, acne, eczema, psoriasis, dermatitis etc³. Moreover, significant change in the composition and distribution of skin microbe communities occurs during ageing, thus affecting balance between host and microbiota⁴. Solutions aimed at preserving balance of the skin’s ecosystem have become one of cosmetic industry priorities.

figure 3: Wrinkle depth reduction and surface regularization after 6 week treatment. Photo: www.kalichem.it

An elegant way to provide the skin with microbial actives

Studies show that the culture medium of these bacteria, like Lactobacilli, promotes antipathogenic and wound healing effects in the host⁵. Molecules produced by bacteria and released in the medium can exert these effects. It has been postulated that bacteria interact with one another and the skin cells, using a complex network of molecules, the “postbiotics”, which represent a new class of bacteria derived factors, helpful in keeping the skin microbiota balance, reinforcing skin defences, promoting barrier function and other beneficial effects⁶. Due to their reproducible composition, organoleptic features, long shelf life, safety, handling and efficacy, using postbiotics is an elegant and efficient approach to provide the skin with microbial actives. Through a patented biofermentation process (PB Tech by Postbiotica srl) Kalibiome postbiotics for cosmetic applications were designed and produced starting from L. Paracasei, species known for its beneficial skin effects^7,8. Kalibiome postbiotics are naturally produced and released by living microbes and extracted by a technique which avoids bacterial fragments or the presence of toxins.

By modulating biofermentation conditions, it is possible to obtain different molecular patterns for postbiotics. This means that their production is customisable to obtain different target molecules.

figure 4: Postbiotics vs competitor. Photo: www.kalichem.it

figure 5: Kalibiome repairing effect on wounded fibroblasts. Photo: www.kalichem.it

Materials and methods

In vivo tests were performed using Kalibiome in emulsion at 0.2% vs placebo twice daily, on twenty women aged between 40 and 70, to assess skin echogenicity (measurement of the ratio between upper-lower dermis ultrasonography, digital images of skin density by Ultrasound Dermascan C), wrinkle depth (Ra and Rz ratio, image obtained by high performance CCD camera COHU), elasticity (Cutometer MPA580 Khazaka), TEWL, hydration (Corneometer CM 825), skin redness (measurement of a* from CIE L*a*b*, after 24h SLS treatment).

In vitro tests on hyaluronic acid and pro-collagen I expression were carried out by Elisa, comparing postbiotics vs benchmarks. A scratch test was performed using EGF as control with analytic and photographic assessment of wounded fibroblast layer repair. The skin barrier was analysed by immunofluorescence microscopy.

The pathogen biofilm formation was assessed analysing the postbiotics interference with S. aureus biofilm onset vs control. The postbiotic solution was added in microchannels by syringe pump and the biofilm formation was observed through glass surface and recorded by microscope and live imaging incubator.

The antioxidant activity was tested on cells after ROS induction with the genotoxic mitomycin C. Cells were treated with GSH (glutathione) and postbiotics to compare the antioxidant force. Superoxide levels were measured as an oxidation index of a red fluorogenic reagent by mitochondrial superoxide of living cells.

Anti-inflammatory effect was evaluated by ELISA of proinflammatory cytokine expression (IL6, IL8, CCL MCP1) inducing inflammation on keratinocytes by poly I:C.

Atopic dermatitis soothing effect on TSLP itching isoform expression in keratinocytes was evaluated by RT-PCR in presence of poly I:C. The postbiotics were subject to skin irritation potential, occlusive patch test, ocular irritation, and phototoxicity tests. Sensitization or irritation potential were not found in any of those (data not shown).

In vivo-evaluation of photoaging

The postbiotics gave significant results in redensifying the dermis framework in skin echogenicity tests. Echogenicity is the ratio between the upper and lower dermis as tested by high frequency ultrasonography, used to estimate the protection against photoageing9. The postbiotics treatment at 0.2% showed an increase of +15.7% compared to the placebo (Fig.1) and a significantly visible dermis redensifying action, seen in the digital imaging with larger white bands highlighting collagen expression increase (Fig. 2).

In vivo: anti wrinkle tests

The postbiotics at 0.2% were tested in crow’s feet area to investigate effectiveness on wrinkle depth reduction. The treatment results show -13,1% wrinkle depth decrease compared to the placebo, along with surface regularisation as shown in Fig. 3.

figure 8: immunofluorescence microscopy images (ZO-1 green, keratinocytes blue). Photo: www.kalichem.it

figure 9: S.Aureus biofilm evolution, control vs postbiotics. Photo: www.kalichem.it

Effects on hyaluronic acid, procollagen I and fibroblast

The postbiotics antiageing action is ascribable to the dualistic effect observed on skin targets like the epidermal keratinocytes hyaluronic acid involved in skin hydration, cells turnover, wound repair, and the procollagen I involved in restoring the dermis framework (Fig.4). In both cases, the postbiotics outperformed the benchmark.

They show the same efficiency as EGF (Fig.5) in promoting fibroblasts migration after wounding, with results visible after 24 h.

In vitro: immune system modulation

The postbiotics proved modulatory activity on pro-inflammatory cytokines (Fig.6) IL-6 and CCL-2 MCP-1 through a dose dependent expression reduction¹⁰. The same pattern was observed with the proinflammatory chemokine IL-8, whose expression was reduced vs control. The above mechanisms explain the decrease in skin redness and photoageing effects detected in vivo. The immunomodulation activity pattern¹¹ confirms their suitability in sensitive skin products.

Further tests have proven the reduction of long TSLP (thymic stromal lymphoietin) isoform, a cytokine typically occurring in atopic dermatitis and mouth inflammations¹², and increased expression of IL-1010,¹³. The short TSLP boasts ecosystem balancing activity on skin microbiota¹⁴. The postbiotics TSLP regulation (Fig.7) leads to long TSLP isoform decrease and homeostatic short isoform increase.

figure 10: Postbiotics vs Glutathione (GSH) superoxide reduction. Photo: www.kalichem.it

In vitro: skin barrier restoring

The postbiotics increase the expression of the tight junction zonula occludens-1 (ZO-1), a key component involved in keratinocytes adhesion^15,16,17analysed by immunofluorescence microscopy. The keratinocytes were treated with SLS and then with postbiotics vs control. Results show the postbiotics’ superior effects on keratinocytes tightening and sealing, promoting decreased permeability to pathogens and pollutants (Fig. 8).

In vitro: interference with biofilm formation

The postbiotics inhibit the formation of S.Aureus biofilm, (Fig. 9) involved in inflammation diseases such as atopic dermatitis. Their biosurfactants promote physical disruption of the pathogen biofilm, confirming the results of recent studies^18,19 and their suitability for applications where bacterial biofilm presence is matter of concern.

In vitro: antioxidant action

Additional tests analysed the mithocondrial ROS (O2· superoxide) in living cells (Fig. 10). The postbiotics reduced the superoxide radical found in the cells subject to oxidation more efficiently than GSH (glutathione).

The decrease in oxidative stress contributes to the immune system modulation, as it downregulates the activation of pathways involved in the induction of lipid peroxidation, membrane disruption, and protein denaturation that lead to nucleic acid damage, mithocondrial damage and oxidative explosion. These inflammation cascades are triggered at different physiological levels, as recent studies show20 and their negative modulation seems a promising strategy for soothing mucosal targets.

In vivo: redness, elasticity enhancement, skin hydration, TEWL

The postbiotics were tested at 0.2% on different parameters vs a placebo formulation, showing in each case significant benefits (Tab.1). The decrease in skin redness may be linked to an immune system response to pollutants and cytokine modulation control, as previously shown. The elasticity increase is likely related to the proven dermis redensifying action. The restoration of the skin barrer function and the hydration effect reported are linkable to the activity on tight junctions and hyaluronic acid production.

Kalibiome, an innovation in the field of microbiota care

Kalibiome postbiotics are biotech actives developed by a patented fermentation technique which creates, with standardized reproducibility, bacterial active metabolites with proven safety. The patented fermentation process allows the postbiotics to be customised for personal care use. The combined immune system modulation and the anti-ageing features position Kalibiome postbiotics as one of the deepest innovations in the field of microbiota care.

Currently, Kalibiome is recommended for applications in anti-ageing and soothing the skin. Kalibiome postbiotics increase skin compactness, redesify the dermis, reduce wrinkle depth, inrease elasticity, likely ascribable to the stimulatory action on dermal-epidermal framework components (ZO-1 tight junction, hyaluronic acid and procollagen I). As for the soothing effect, the postbiotics displayed an anti-inflammatory action through cytokine release modulation, itching reduction in atopic dermatitis and sensitive skin through TSLP modulation, inhibitory activity on pathogen bacteria biofilm formation and antioxidant effect on stressed cells.

These mechanisms open a new frontier for soothing and antipollution treatments for skin care, medical device applications (oral care, gynecological etc.), and skin disorders support treatments. Further studies are in progress with the goal of extending the applications of this biotech solution.

References

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