52 PRESERVATIVES
antibodies and are tagged a second time with another IL-6-specific antibody labelled with horseradish peroxidase (HRP). The addition of the chromagen solution, containing 3,3’,5,5’-tetramethylbenzidine, provides the colorimetric reaction with HRP that is quantitated through optical density (OD) readings on a microplate spectrometer. The standard curve provides a reference from the OD readings for the amount of IL-6 in each sample. Human dermal fibroblasts are seeded into 12-well tissue culture plates and allowed to grow to confluence in complete DMEM. 0.1% and 0.01% concentrations of Lactobacillius Ferment were added to complete DMEM containing 1μg/mL Lipopolysaccharide (LPS) and incubated with fibroblasts for 72 hours. Complete media containing 1μg/mL LPS was used to create an inflammatory environment and dexamethasone (DEX) in the presence of LPS was used as a positive control to quell inflammation. Standards were prepared in concentrations ranging from 2476 pg/mL to 0 pg/mL. 50 μL of Solution B was added to wells for standards and assay controls and 50 μL of Solution A was added to experiment wells. 100μL of standards, controls, and samples were added to appropriate wells. After a one hour incubation at room temperature and washing, 50μL Solution A and 100 μL anti- IL-6 conjugate was added to all wells. Following a one hour incubation and washing, 100 μL chromagen solution was added for the colorimetric reaction. One- hundred μL stop solution was added to stop the reaction after 15 minutes. The optical density was read at 450 nm on the Synergy HT Microplate Reader. A standard curve was created by reducing the data and generating a linear curve fit. The IL-6 concentration of Lactobacillus Ferment treated-fibroblasts was determined by extrapolation from the standard curve and expressed in pg/mL. The results of the ELISA demonstrate that the Lactobacillus Ferment can decrease IL-6 levels in in vitro cultured human dermal fibroblasts. This treatment is equal to or more effective than the comparative positive control of dexamethasone (DEX) (Fig 3). The in vivo moisturisation assay provides
information about the skin’s hydration by measuring the conducting properties of the upper skin layers when subjected to an alternating voltage. The presence of moisture in the skin improves conductance therefore resulting in higher readings than dry skin. Six volunteers M/F between the ages of 23 and 45 who were known to be free of any skin pathologies participated in this study. A DermaLab Corneometer was used to measure the moisture levels on the
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Figure 4: Skin moisturisation and regression, 2% Lactobacillus Ferment in base lotion vs the controls of the base lotion placebo and untreated sites.
subject’s volar forearms. Following initial measurements, all subjects were asked to apply 2 mg of each test material. Measurements were taken immediately after application and then weekly for four weeks. The test material consisted of 2% Lactobacillus Ferment in a base lotion. For added perspective, measurements of an untreated test site and a site treated with the base lotion were recorded. After the initial four week experiment the subjects stopped applying the different test products, however, moisture levels were still recorded at the different test sites at 24 hours, one week and two weeks. By following such a protocol it can be seen that the Lactobacillus Ferment provides an increase in skin hydration compared to both the positive and negative controls. Additionally, moisture retention in the skin remained higher where the Lactobacillus Ferment was used, despite the product no longer being applied (Fig 4). As with all preservative and
antimicrobial systems aspects of safety and sustainability are of utmost importance. The Lactobacillus Ferment is both mild and gentle on the skin, supported by a complete dossier of toxicological data, including but not limited to: Repeat insult patch test, skin sensitisation, mutagenicity, phototoxicity and dermal and ocular irritation. Derived through a sustainable biotechnology from a natural source of lactic acid bacteria this active is also readily biodegradable.
Conclusion Through Lactobacillus fermentation, it is possible to create a multifunctional active which can provide skin benefits of moisturisation and a reduced inflammatory environment while providing broad- spectrum antimicrobial protection of a finished formulation. The natural defence strategy of a commensal probiotic bacteria can reflect similar defence strategies of the commensal microflora that exists on the skin and in the gut. The activity of these postbiotic peptides is consequently more sympathetic to the skin environment reducing the potential of sensitisation. This activity is demonstrated through extensive challenge testing and in vivo/in vitro objectified analysis on the skin and skin cells. By utilising a multifunctional active with
antimicrobial efficacy, cosmetic chemists are able to approach formulating in a more holistic manner, choosing materials with skin and scalp benefits, whilst also providing antimicrobial properties to protect a formulation. Additives, such as the Lactobacillus Ferment, capitalise on the current trends of ‘Probiotic Technology’ and ‘Postbiotic Activity’, helping to support the skin and the skin microflora. Working with non-viable postbiotics secretions vs viable probiotic cells, support these claims without compromising the integrity and safety of a formulation. Lactobacillus Ferment captures a sustainable fermentation technology with a controlled postbiotic approach, which is gentle on both the environment and the skin.
PC February 2020
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