605 SKIN MICROBIOME INNOVATIONS
count technique. The activity of the test material inoculated is evaluated at 30 seconds, 1,
5, 10, and 30 minutes after initial inoculation to quantitatively determine the concentration
of viable microorganisms remaining after incubation time. Cell viability is evaluated by
calculating the log reduction between the measured initial microbial population and the
surviving microbial population after each time interval. Results from the time kill test are
evaluated two days later.
RESULTS
The area under the curve (AUC) is calculated by adding the areas under the line between
each consecutive absorbance measurement using the following equation:
Equation 1.
AUC t TKR
i i i i+1
i=0
n-1
=-+
+
∑(t 1
2 1 )(TKR )
The AUC between the values TKR1 and TKR2 at times t1 and t2 are equivalent to the
product of difference in time and the average of the two time kill result values.
Table III and Figure 3 below present the time-kill results for an antimicrobial-free,
yeast-derived extract. This active ingredient was developed through the fermentation
of Saccharomyces cerevisiae in a defined medium under controlled temperature and time
conditions. During fermentation, the Saccharomyces cells were exposed to UV radiation at
specific wavelengths, followed by cell lysis. After an aqueous extraction was performed,
Lactobacillus ferment was introduced to the solution, which was then filtered, resulting
in a yeast-derived active free from antimicrobial agents. This assay was run in triplicate
and the data obtained from this run meets the criteria for a valid assay as the control
performed as anticipated across all the tested microorganisms. Dendritic cells incubated
Table III
Time Kill Results: Percent Reduction in Viable Organisms After Inoculation and Sampling Time Intervals,
Inoculum Concentration CFU/mL
Microorganism Sample Inoculum
concentration
30
seconds
1 minute 5 minutes 10
minutes
30
minutes
CM (Control) 80.5% 81.2% 81.0% 83.3% 85.1%
S. aureus 0.01% active
0.1% active
2.5 × 106 99.9%
99.9%
99.9%
99.9%
99.9%
99.9%
99.9%
99.9%
99.9%
99.9%
1.0% active 99.9% 99.9% 99.9% 99.9% 99.9%
CM (Control) 73.5% 78.1% 81.2% 83.3% 84.3%
P. aeruginosa 0.01% active 4.1 × 106 99.9% 99.9% 99.9% 99.9% 99.9%
0.1% active 99.9% 99.9% 99.9% 99.9% 99.9%
1.0% active 99.9% 99.9% 99.9% 99.9% 99.9%
CM (Control) 38.3% 56.1% 60.2% 75.2% 62.3%
S. epidermidis 0.01% active
0.1% active
1.2 × 106 23.2%
25.3%
38.1%
36.1%
42.1%
35.1%
43.3%
42.4%
43.9%
44.5%
1.0% active 22.3% 43.1% 45.3% 46.2% 43.5%
CFU: Colony Forming Units.

606 JOURNAL OF COSMETIC SCIENCE
with the antimicrobial-free yeast-derived active at 0.01%, 0.1%, and 1.0% in vitro prevented
the growth of detrimental microorganisms assessed within 30 seconds and maintained a
commensal microbiota environment.
As displayed in Table III and Figure 3 above, the signaling molecules from the untreated
dendritic cells are unable to kill 100% of all the microorganisms assessed at any of the time
intervals. Additionally, the untreated dendritic cells at the thirty-minute time interval
killed 85.1%, 84.3%, and 62.3% of S. aureus, P. aeruginosa, and S. epidermidis, respectively.
These results indicate that the signaling molecules from untreated dendritic cells allowed
for growth of both the detrimental and commensal microorganisms evaluated.
Conversely, the signaling molecules from the treated dendritic cells with any of the
concentrations prevents all growth of the detrimental microorganisms, S. aureus and P.
aeruginosa, at every time point. Additionally, the media from dendritic cells treated with
0.01%, 0.1%, and 1.0% of the antimicrobial-free yeast-derived active left the S. epidermidis
alive at the 30-minute time interval by 56.1%, 55.5%, and 56.5%, respectively. By allowing
the commensal microbiota to remain on the skin, it is beneficially repairing the skin’s
microbiome and normalizing the epidermal barrier.
At this moment, there is not a full understanding of how the antimicrobial-free yeast-
derived active is interacting with the commensal bacteria and selectively destroying the
pathogenic bacteria. However, the working hypothesis is the ingredient activates dendritic
cells to release certain signaling molecules that target pathogenic bacteria. There are
currently more experiments being conducted to identify and characterize the types and
amounts of signaling molecules released from dendritic cells.
However, the basis and outcomes of the dermal microbiome-immunology assay are highly
significant, marking a revolutionary step forward in the emerging field of immunocosmetics.
By unraveling the complex crosstalk within the skin microbiome and immunity, it provides
a crucial tool for understanding how to effectively modulate and enhance skin health.
This innovation enables researchers and skincare developers to unlock the mysteries of the
skin microbiome, leading to targeted treatments and personalized cosmetics that leverage
microbiome manipulation for optimal skin wellness.
Figure 3. Time kill results for dendritic cells treated with an antimicrobial-free yeast-derived active
inoculated with the tested microorganism populations across time intervals. Values indicate the percent of
microorganisms killed.