First living probiotic anti-aging active ingredient with effective in vitro and vivo demonstration against placebo
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Presented by: Sabrina Leoty-okombi
The importance of the microbiome for human health is no longer denied. Today, the use of probiotics is not limited to pharmaceutical or nutraceutical applications. Cosmetics is also concerned. Skin microbiota dynamically participates in skin health and beauty. As skin aging is one of the major concerns in cosmetics, we previously designed a clinical study to understand skin microbiome differences between aged and young cohorts. Significantly (p<0.05) higher populations of Lactobacilli were observed in the nonaged cohort and the absence of Lactobacillus crispatus was particularly observed in the wrinkle hollows of the old cohort. These results paved the way for the development of new cosmetic probiotics for anti-aging applications. However, the two major obstacles to the topical use of probiotic are the challenge in ensuring their survival during the manufacturing, storage and application, and in demonstrating their clinical efficacy.
The aim of our study was firstly to demonstrate Lactobacillus crispatus efficacy in vitro, then to determine the best formula that will allow the maintenance of an efficient delivery of the living Lactobacillus crispatus probiotic and significant antiaging clinical results.
The probiotic strain L. crispatus was isolated from healthy skin and identified after full genome sequencing. The strain was fermented and freeze dried after centrifugation to reach a concentration of around 109 colony forming units/g (cfu/g). Its antiaging properties and more particularly its ability to stimulate the synthesis of collagen type I and V which decrease with age was evaluated on human fibroblast culture by DELFIA method.
In order to develop a formula for the skin delivery of the probiotic, several formulating ingredients (emollients, emulsifiers and preservatives) were screened for their impact over a short period of time (1 h to 24h) on the viability of the L. crispatus strain. The ingredients that showed a short-term neutrality toward the viability of the strain were then evaluated over a longer period (up to168 days, at different temperatures). An optimized formulation was finally designed using the most favorable ingredients previously evaluated and it was checked for strain stability at different temperatures. This formulation was used to deliver the probiotic strain for a clinical evaluation on 29 Caucasian women (45-65 years old). The anti-aging efficacy was measured after 3 and 8 weeks thanks to dermis density evaluation by ultrasound imaging coupled to image analysis, and wrinkles analysis by VISIA CR imaging.
The L. crispatus probiotic showed a significant stimulation of collagen I and V content (+133% and +55% respectively) in fibroblast culture versus untreated (p<0.05). Interestingly, we also showed that live bacteria were more effective than the thermally inactivated biomass.
In formulation, the probiotic strain had different viabilities and stabilities according to the nature of the formulating ingredients. It was viable and stable in some natural oil-based emollients for several weeks, it was however only stable for several hours in the presence of some emulsifiers and mild preservatives. These results allowed us to develop an application routine for the clinical test in which an oily serum of concentrated probiotic booster was diluted just before application in a specific neutral emulsion, to deliver a 0.05% dose of probiotic equivalent to 5 105 cfu/g on the skin.
After 2 months of twice-daily application, we measured a significant increase compared to baseline of the density of the sub-epidermal zone by 11% and of the dermis by 6%. The improvement of dermal density was also significant compared to the placebo (+5%). The same tendency was obtained for forehead wrinkles thickness improvement. These results corroborate the in vitro performance previously obtained on collagen stimulation.
The compatibility study of the probiotic strain with the different formula allowed us to develop a galenic which kept the probiotic strain viable for at least 6 months (stability evaluation still ongoing). Moreover, we highlighted the capacity of the Lactobacillus crispatus probiotic strain to densify the dermis of the volunteers thus providing antiaging benefits. These results make this ingredient the first living probiotic anti-aging ingredient with effective in vitro and vivo demonstration against placebo.
The aim of our study was firstly to demonstrate Lactobacillus crispatus efficacy in vitro, then to determine the best formula that will allow the maintenance of an efficient delivery of the living Lactobacillus crispatus probiotic and significant antiaging clinical results.
The probiotic strain L. crispatus was isolated from healthy skin and identified after full genome sequencing. The strain was fermented and freeze dried after centrifugation to reach a concentration of around 109 colony forming units/g (cfu/g). Its antiaging properties and more particularly its ability to stimulate the synthesis of collagen type I and V which decrease with age was evaluated on human fibroblast culture by DELFIA method.
In order to develop a formula for the skin delivery of the probiotic, several formulating ingredients (emollients, emulsifiers and preservatives) were screened for their impact over a short period of time (1 h to 24h) on the viability of the L. crispatus strain. The ingredients that showed a short-term neutrality toward the viability of the strain were then evaluated over a longer period (up to168 days, at different temperatures). An optimized formulation was finally designed using the most favorable ingredients previously evaluated and it was checked for strain stability at different temperatures. This formulation was used to deliver the probiotic strain for a clinical evaluation on 29 Caucasian women (45-65 years old). The anti-aging efficacy was measured after 3 and 8 weeks thanks to dermis density evaluation by ultrasound imaging coupled to image analysis, and wrinkles analysis by VISIA CR imaging.
The L. crispatus probiotic showed a significant stimulation of collagen I and V content (+133% and +55% respectively) in fibroblast culture versus untreated (p<0.05). Interestingly, we also showed that live bacteria were more effective than the thermally inactivated biomass.
In formulation, the probiotic strain had different viabilities and stabilities according to the nature of the formulating ingredients. It was viable and stable in some natural oil-based emollients for several weeks, it was however only stable for several hours in the presence of some emulsifiers and mild preservatives. These results allowed us to develop an application routine for the clinical test in which an oily serum of concentrated probiotic booster was diluted just before application in a specific neutral emulsion, to deliver a 0.05% dose of probiotic equivalent to 5 105 cfu/g on the skin.
After 2 months of twice-daily application, we measured a significant increase compared to baseline of the density of the sub-epidermal zone by 11% and of the dermis by 6%. The improvement of dermal density was also significant compared to the placebo (+5%). The same tendency was obtained for forehead wrinkles thickness improvement. These results corroborate the in vitro performance previously obtained on collagen stimulation.
The compatibility study of the probiotic strain with the different formula allowed us to develop a galenic which kept the probiotic strain viable for at least 6 months (stability evaluation still ongoing). Moreover, we highlighted the capacity of the Lactobacillus crispatus probiotic strain to densify the dermis of the volunteers thus providing antiaging benefits. These results make this ingredient the first living probiotic anti-aging ingredient with effective in vitro and vivo demonstration against placebo.