570 JOURNAL OF COSMETIC SCIENCE
was set at 32°C. The pH (6.5) was the minimal acidity that all bacteria could tolerate
(data not shown). The incubation time was adjusted to obtain the correct proportions of
bacteria. The evolution of the five species in the consortium was monitored for 24 hours,
with sampling at 2, 4, 8, and 24 hours (Figure 2).
After 2 hours of incubation, C xerosis and S epidermidis concentrations were inferior to the
detection limit (102 CFU/mL). However, they rapidly grew (4 × 107 and 2.1 × 108 CFU/
mL respectively after 8 hours) so that their proportions reached 4% for C xerosis and 21%
for S epidermidis. The proportion of S mitis also increased steadily from 2.0% to 30.2%
at 24 hours. The concentration of C acnes increased from 2 to 8 hours (from 4.1 × 107
and 5.2 × 108 CFU/mL respectively), but its proportion decreased during that incubation
time due to the strong increase of the other species. More specifically, after 24 hours, the
proportion of C acnes was only 8.9%.
In general, after an incubation of 8 hours, the proportion of the five species was close to
what is generally observed on the skin of the cheek: C acnes 50%, S epidermidis 20%, S mitis
20%, and M luteus and C xerosis 10% each. Since 8 hours is also the daytime duration,
and thus a relevant contact time with cosmetic products on the skin, it was used to test
cosmetic ingredients.
Next, the optimal atmosphere for the growth of all species was selected by incubating the
consortium in aerobiosis or in anaerobiosis. The proportion of bacteria was calculated based
on their concentrations and results are presented in Figure 3.
With oxygen, the proportion of C acnes was 6% compared to 41% without oxygen. On the
contrary, S epidermidis and S mitis represent 50% and 33% of the population respectively
in the presence of oxygen. Anaerobiosis offers better condition for C acnes while preserving
the growth of the other strains. This condition was chosen to grow the model consortium
in subsequent assays.
Figure 2. Proportions evolution of the five species in the consortium according to time (total bacteria
concentration 1.0 × 109 CFU/mL at 8 h, 2.5 × 109 CFU/mL at 24 h). Species proportion (%)in the consortium
is the ratio between a species concentration (in CFU/mL) and the concentration of total bacteria (in CFU/mL)
multiplied by 100.

571 COSMETIC INGREDIENTS THAT RESPECT SKIN MICROBIOTA
Finally, the adequate protocol to grow the five species consortium was at a pH of 6.5, a
temperature of 32°C, under anaerobic atmosphere for 8 hours. The reproducibility of this
method was assessed by proceeding to 13 independent assays. Bacteria concentrations in
the consortium were measured and their proportions were calculated (Figure 4A and B).
In terms of population ratios (%,Figure 4A), the most important variation was observed
on C acnes (CI 95% =31.6–42.8%). The least concentrated bacteria (C xerosis and M luteus)
Figure 3. Proportions of bacteria in the consortium containing C acnes (dark blue), S epidermidis (light blue),
S mitis (purple), M luteus (red) and C xerosis (orange) with or without oxygen. Species proportion (%)in the
consortium is the ratio between a species concentration (in CFU/mL) and the concentration of total bacteria
(in CFU/mL) multiplied by 100.
Figure 4. Reproducibility of the consortium in 13 independent assays. (A). Mean species proportions in the
consortium (%,n =13). The whiskers are the confidence intervals (CI 95%). (B). Mean concentrations (log
CFU/mL) of the five bacterial species ±SD (n =13). Marks represent individual values. The coefficients of
variations are indicated above the data. C acnes (dark blue), S epidermidis (light blue), S mitis (purple), M luteus
(red), and C xerosis (orange).