PROBACILLUS REVIVE. Probiotic + Optimizes Moisturization + Promotes Homeostasis + Exfoliates. Tomorrow s Vision Today!

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1 PROBACILLUS REVIVE Probiotic + Optimizes Moisturization + Promotes Homeostasis + Exfoliates Tomorrow s Vision Today!

2 Probacillus Revive Technical Information: Product Code: INCI Name: Lactobacillus Bulgaricus Ferment Filtrate INCI Status: Approved Suggested Use Levels: 1.0% % Suggested Applications: Collagen Production, Barrier Function, Cellular Renewal, Optimizes Moisturization, Probiotic, Promotes Homeostasis

3 Health and Wellness Prebiotics Non-digestible food ingredients that beneficially affect the host by selectively stimulating the grown and/or activity of bacteria Optimizes the balance of the bacteria present on the skin to promote the health

4 Prebiotics + Cosmetic Ingredients Manufacturing the Active Ferment derived from Lactobacillus bulgaricus Prebiotics (oligosaccharides) derived from soy and chicory are used to supplement the culture media Optimizes the growth conditions for the organism The media stimulates the synergistically active compounds that are produced by the bacteria Various filtration techniques are used to isolate and extract the synergistically active compounds

5 Probacillus Revive Benefits Enhances cellular renewal Improves epidermal integrity Enhances barrier function Speeds repair Increases collagen synthesis Improves skin tone Reduces the appearance of fine lines and wrinkles Moisturizes Suitable for products designed for marketability coinciding with the probiotic trend

6 Probacillus Revive Increase in Collagen Type I Production Collagen I is a major component of the skin Human fibroblasts cultured ~6,000 cells per dish in vitro in 10.0% Fetal Calf Serum (FCS) medium for 24 hours Cells were removed, placed into amino acid deficient medium and supplements with 1.0%, 2.0% or 4.0% Probacillus Revive and amino acid deficient control Incubated for 48 hours Elisa Assay: Immunolabed with primary and secondary antibodies (murine anti-collagen I monoclonal antibody and murine anti-igg antibody) in peroxidase/ TMB substrate (3, 3, 5, 5 tetramethylbenzidine) visualization system

7 Probacillus Revive Collagen Production Assay Figure 1. Increase in collagen type I production when using different concentrations of test material

8 Probacillus Revive Dansyl Chloride Assay A 5.0% Dansyl Chloride was prepared by dispersing Dansyl Chloride 95.0% (Sigma) in petrolatum ~2.0g of the ointment was applied to three 2cm x 2cm locations on the volar forearm of each panellist The material was allowed to remain in place for 24 hours at which time any excess ointment was removed ~50mL of product was applied to the appropriate test site once per day The sites were examined daily under ultraviolet light (SL-3660 Long Wave Ultra Violet, Black Light Easter Corp) for fluorescence The test was continued until no fluorescence was detected at any site

9 Probacillus Revive Dansyl Chloride Assay Test Materials Unloaded Vehicle 4.0% Glycolic Acid 10.0% Probacillus Revive The values listed reflect the average time for each product Figure 2. Average number of days taken for complete cellular turnover in relation to test material applied

10 Probacillus Revive Hydration Assay Protocol 10 m/f subjects, ages Novameter (9003 DPM) was used to measure skin hydration over the course of 30 days 2mg of each test material was applied twice daily to designated areas on the volar forearms of panelists Figure 3. Percent increase of skin hydration levels for each test material applied

11 Viability (% of control) Probacillus Revive Cellular Viability 150% 140% 130% 120% % Protocol Human dermal fibroblasts were seeded into 96-well tissue culture plates and allowed to grow to confluency in complete DMEM 110% 100% 90% 80% 70% 60% 50% % % 1% 0.10% 0.01% Concentration of Sample Concentrations: 0.10%, 0.01%, 1.0% Ten microliters of viability reagent was added to 90µL of cell culture media in culture wells Probacillus Revive exhibited positive results by increasing cell metabolism Figure 4. Cellular Metabolism of Probacillus Revive-treated fibroblasts expressed in terms of percent of control

12 Antioxidant Concentrations (µmte) Probacillus Revive ORAC Assay Figure 5. Antioxidant capacities µM Trolox 12.5µM Trolox 0.5% Probacillus Revive % Probacillus Revive Protocol Solutions of Probacillus Revive and Trolox (positive control) were prepared in 75mM potassium phosphate buffer Concentrations: 0.1%, 0.5% 25μL of test material & Trolox were combined with 150μL of fluorescein in 75mM potassium phosphate buffer & incubated in the Synergy HT Microplate reader at 37 C for 30 minutes Probacillus Revive began exhibiting antioxidant activity at a 0.1% concentration

13 IL-6 Concentration (pg/ml) Probacillus Revive IL-6 ELISA Protocol Human dermal fibroblasts were seeded into 12-well tissue culture plates and allowed to grow to confluency in complete DMEM 1500 Concentrations: 0.10%, 0.01%, 1.0% % % % Probacillus ReviveProbacillus ReviveProbacillus Revive Positive Control Negative Control Probacillus Revive was not able to decrease IL-6 production compared to our positive control at higher concentrations but at low concentrations has some antiinflammatory activity Figure 6. Probacillus Revive -treated fibroblasts IL-6 concentrations

14 Percent Change (%) Probacillus Revive IL-6 ELISA % Probacillus Revive 0.1% Probacillus Revive 0.01% Probacillus Revive Series Protocol Human dermal fibroblasts were seeded into 12-well tissue culture plates and allowed to grow to confluency in complete DMEM Concentrations: 0.10%, 0.01%, 1.0% Probacillus Revive was not able to decrease IL-6 production compared to our positive control at higher concentrations but at low concentrations has some antiinflammatory activity Figure 7. Percent change in IL-6 production compared to positive control

15 Protein Concentration (µg) Probacillus Revive Sirius Red/Fast Green Collagen Collagen Proteins Non-Collagen Proteins Protocol Human dermal fibroblasts were seeded into 24-well tissue culture plates and allowed to grow to confluency in complete DMEM Concentrations: 0.10%, 0.01%, 1.0% 10 Concentrations of Probacillus Revive were added to the serum-free DMEM and incubated with fibroblasts for 24 hours 1 100µg/mL 50ng/mL 1% 0.1% 0.01% Ascorbic Acid IGF-1 Complete Media Probacillus Revive Probacillus Revive exhibited positive effects on collagen synthesis Figure 8. Collagen and non-collagen protein concentrations

16 % Collagen/Non-Collagen Proteins Probacillus Revive Sirius Red/Fast Green Collagen 20% 15% Protocol Human dermal fibroblasts were seeded into 24-well tissue culture plates and allowed to grow to confluency in complete DMEM 10% Concentrations: 0.10%, 0.01%, 1.0% 5% 3.92% 3.76% 4.99% 3.62% 3.78% 3.79% Concentrations of Probacillus Revive were added to the serum-free DMEM and incubated with fibroblasts for 24 hours 0% 100µg/mL 50ng/mL 1% 0.1% 0.01% Probacillus Revive exhibited positive effects on collagen synthesis Ascorbic Acid IGF-1 Complete Media Probacillus Revive Figure 9. Percent collagen compared to non-collagen proteins

17 Probacillus Revive Technical Information: Product Code: INCI Name: Lactobacillus Bulgaricus Ferment Filtrate INCI Status: Approved Suggested Use Levels: 1.0% % Suggested Applications: Collagen Production, Barrier Function, Cellular Renewal, Optimizes Moisturization, Probiotic, Promotes Homeostasis

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