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PRESERVATIVES 51


screening results warranted further testing to confirm the product’s ability to support cosmetic product preservation. An antimicrobial efficacy test was completed following the PCPC Section 20 Method 3 (Determination of Preservation Adequacy of Water- Miscible Personal Care Products). Fifty grams of a generic cream formula containing 4% Lactobacillus Ferment with a pH 5 was weighed into five individual containers. Each container was inoculated with one of the five test organisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis, and Candida albicans). The inoculum concentration for each organism was standardised using the 0.5 McFarland turbidity standard and further diluted to yield approximately 106 to 108 microorganisms/ml. The amount of each inoculum added to each sample was no more than 1% of the product weight, as to not alter the product composition. The inoculated samples were evaluated at 0, 7, 14, 21, and 28 days after the initial inoculation to determine quantitatively the number of viable microorganisms remaining. On the 28th day of testing the samples were re-inoculated and evaluated at 7, 14, 21, and 28 days after the second exposure to determine the number of viable microorganisms. The results of this double challenge test demonstrate the effectiveness of the antimicrobial system. As shown in Figure 2, the bacteria, yeast and mould were reduced to 0 (>99.9%, 3 log) within the first 24 hours and remained at this level throughout the remaining cycle. These results indicate that the Lactobacillus Ferment has an efficient activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis, and Candida albicans within a generic formulation at pH 5. Further evaluations of the antimicrobial


active were also conducted under different pH environments and at concentration levels of 2% and 4% in order to assess activity under varying conditions of use. Following this it was determined that the Lactobacillus Ferment provided effective activity even when exposed to pH 3 – 7.


Multifunctional efficacy As shown the Lactobacillus Ferment can be used in a formulation to provide an antimicrobial activity. However, this is not its only role. This multifunctional material can also help to deliver moisturising and conditioning properties while limiting the potential of an inflammatory environment within skin cells. Both in vivo and in vitro studies help to support these claims. Interleukin-6 (IL-6) is a multifunctional cytokine, which influences many different cellular and physiological processes.


February 2020 300 250 200 150 100 50 0.96 0 0.15%


Lactobacillus Fernent


0.01%


Lactobacillus Fernent


1 ug/ml LPS + DEX 1 ug/ml LPS Complete Media 0.14 41.68 4.73 278.29


Figure 2: Antimicrobial efficacy test, summary results of 4% Lactobacillus Ferment, pH 5, PCPC Section 20, method 3, initial inoculation.


 Untreated Control  Base Lotion Control  2% Lactobacillus Ferment in Base Lotion 300


200


100


0 T=0 T=24 hours T=1 week T=2 weeks T=3 weeks T=4 weeks


Figure 3: IL-6 concentration change in human dermal fibroblasts following 0.1 and 0.01% Lactobacillus Ferment treatment vs DEX as the positive control and LPS as the negative control


Normal levels of cytokines and other signalling molecules are essential for the homeostasis of inflammatory processes. When an injury occurs IL-6 expression is increased helping to promote the pro- inflammatory response. This leads to adaptive and innate immune responses, antibody production and inflammation. Under normal conditions the increase in IL- 6 expression is restored to normal levels once the trauma is resolved. Under certain conditions elevated levels of IL-6 can lead to disruption and consequent chronic inflammation or disease progression, highlighting its importance in ageing and age-related diseases. IL-6 signals through the nuclear factor- kappa B (NF-κB) pathway that results in the transcription of inflammatory mediators,


including matrix metalloproteinase-1 (MMP- 1). MMPs are responsible for breaking down the extracellular matrix and collagen in the skin leading to wrinkles, fine lines, and loss of skin elasticity. Reducing the level of IL-6 and other inflammatory mediators is believed to slow down degradation of the skin matrix and, possibly, stimulate its replenishment.


The Interleukin-6 ELISA was conducted to assess the changes in IL-6 levels in in vitro cultured human dermal fibroblasts following treatment with the Lactobacillus Ferment. The IL-6 ELISA utilises a colorimetric reaction employing antibodies with antigen specificity to human IL-6. Monoclonal antibodies specific for IL-6 epitopes are coated on a microtiter plate. In positive samples, IL-6 will bind to these


PERSONAL CARE EUROPE


IL-6 concentration (pg/mL)


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