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Kikukawa et al. Microbiome Res Rep 2023;2:4 https://dx.doi.org/10.20517/mrr.2022.24 Page 7 of 12

Figure 3. Antibacterial activities of C16:1 authentic standards against S. aureus (A) and S. epidermidis (B) strains. MIC values of 6-cis-
C16:1, 7-cis-C16:1, and 9-cis-C16:1 isomers for S. aureus and S. epidermidis were determined. Data are mean ± SD of three independent
assays, represented by the bars and whiskers. MIC: Minimum inhibitory concentration.

than grown in MRS medium [Figure 4A]. Then, we supplemented the MRS medium with each of TOS
medium components or supplemented the TOS medium with each of MRS medium components to
examine how each of the components affects the 7-cis-C16:1 content in the cells [Figure 4A]. The results
showed that neither of the TOS components, (NH ) SO , L-cysteine, galactooligosaccharide, nor K HPO did
4
4 2
2
4
increase 7-cis-C16:1 content of the cells when MRS-based medium was used. However, the addition of the
MRS components, Tween 80, citrate, and glucose to the TOS medium markedly decreased 7-cis-C16:1
content both in the amount and in the ratio in TFA. Especially, the addition of Tween 80, a surfactant,
drastically reduced the content by 10-fold (1.1 mg/L and 5.5% in TFA to 0.2 mg/L and 0.6% in TFA) to a
level similar to that obtained when grown in MRS. The effect of Tween 80 on the 7-cis-C16:1 productivity
was dose-dependent, but its addition to the TOS medium did not affect post-culture cell biomass
[Figure 4B].

Antibacterial ability of TFA prepared from the JCM 7042 cells
Finally, an antibacterial assay was performed using acid-hydrolyzed TFA preparations obtained from the
cells of the JCM 7042 strain [Figure 5]. To understand how the presence of Tween 80 in the medium affects
lipid metabolism and antibacterial activity, the strain was grown either in TOS medium or in TOS medium
supplemented with 5 g/L of the surfactant, and used for TFA preparation.

With respect to the TFA prepared from cells grown in TOS, high antibacterial activity was observed against
S. aureus NBRC 100910 and NBRC 12732 with MICs of 62.5 µg/mL and 31.3 µg/mL, respectively, while the
T
activity was not very high against S. aureus NBRC 13276 and NBRC 14462 with MICs of about 400 µg/mL
for both strains. The MIC values towards S. epidermidis were in a range between 660 and 1000 µg/mL. The
TFA prepared from cells grown in TOS + Tween 80 did not show marked antibacterial activity for S. aureus
T
NBRC 100910 , NBRC 13276, and NBRC 14462 (MICs >1660 µg/mL), while it showed weak activity
towards NBRC 12732 with a MIC of about 830 µg/mL. No activity was observed against S. epidermidis
(MICs of > 2000 µg/mL).

Fatty acid composition analysis revealed that the addition of Tween 80 to the medium not only decreased
the total amount of TFA by 2-fold but also reduced the ratio of C12:0, C14:0, 7-cis-C16:1, and 9-cis-C16:1
and increased C16:0 and 9-cis-C18:1 in TFA [Table 2]. The low abundance of each C16:1, especially 7-cis-
C16:1, could be responsible for the low antibacterial activity of the TFA prepared from the cells grown in
the presence of Tween 80.