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

Identification of the chemical structure
The TFA from Bifidobacterium sp. JCM 7042 were derivatized into 4,4-Dimethyloxazoline (DMOX) and
subsequently analyzed by gas chromatography-mass spectrometry (GC-MS) as described previously [35,36] .
The position of the double bond of C16:1 was analyzed using the plasma-mediated modification method by
[37]
liquid chromatography-mass spectrometry (LC-MS) as previously reported . For LC-MS analysis, fatty
acid samples were analyzed in the form of free fatty acid (FFA).

Preparation of FFA samples extracted from Bifidobacterium sp. JCM 7042
To obtain FFAs for the LC-MS analysis and antibacterial assay, bacterial cells were harvested by
centrifugation (3260 × g) at 4 °C for 20 min, washed twice with 0.85% NaClaq, and directly acid-hydrolyzed.
Briefly, a pellet of Bifidobacterium cells was dissolved in 400 µL of acetonitrile and 50 μL of 5.0 N HCl in a
glass tube. The sample was lysed by vortex and incubated at 100 °C for one hour. After cooling to room
temperature, 800 μL of t-butyl methyl ether, 100 μL of methanol, and 400 μL of H O were added, and the
2
mixture was vortexed for 1 min. After centrifugation at 3260 × g for 5 min, the upper organic phase was
collected. Subsequently, 800 μL of water was added, and the sample was vortexed again for 1 min. After
phase separation at 3260 × g for 5 min, the upper organic phase containing FFAs was collected. The
collected organic phase was evaporated under a stream of nitrogen gas, and the recovered FFAs were
reconstituted in 100 μL of acetone before being subjected to LC-MS analysis.

Antibacterial assay
The antibacterial activity of FFAs was evaluated by determining minimum inhibitory concentration (MIC)
as described in a previous study . S. aureus and S. epidermidis cells collected at the exponential growth
[38]
4
phase were diluted to a concentration of 2.0 × 10 CFU/mL with the NB medium. The cell suspensions were
dispensed into 96-well titer plate, to which fatty acids dissolved in dimethyl sulfoxide or ethanol (10 mg/ml)
were added. The fatty acid concentrations were varied between 1.0 and 2000 μg/mL. The assays were carried
out in biological triplicate. The lowest concentration (highest dilution) required to prevent the growth of
microorganisms was regarded as the MIC.

RESULTS
Screening of Bifidobacterium strains for C16:1 content
As a preliminary screening, approximately one hundred microbial strains collected from soil samples and
laboratory stocks, including Enterobacteriaceae, Saccharomycetaceae, Bifidobacteriaceae, Nocardiaceae, and
some filamentous fungi, were screened for their content of C16:1; however, no characteristic C16:1
production was observed for any of the strains, except for the ones belonging to the genes Bifidobacterium
(data not shown). Therefore, as the first screening, we examined the fatty acid content of 25 Bifidobacterium
strains by GC [Table 1]. MRS medium was used for cultivation. As a result, several strains of B. adolescentis,
B. boum JCM 1211, and Bifidobacterium sp. JCM 7042 strains were found to show high unknown C16:1
content that is not 9-cis-C16:1. Among them, B. boum JCM 1211 had the highest C16:1 ratio (2.7%) in TFA,
while Bifidobacterium sp. JCM 7042 showed the highest amounts of TFA and C16:1 (106.6 and 2.4 mg/L of
broth, respectively). Other species, such as B. bifidum, B. animalis, B. indicum, and B. longum, did not
contain the C16:1 as a fatty acid constituent.

B. adolescentis 12451, B. adolescentis 12-111, B. boum JCM 1211, and Bifidobacterium sp. JCM 7042 were
then used for the second screening, in which TOS broth was used for cultivation [Figure 1]. B. boum JCM
1211 and Bifidobacterium sp. JCM 7042 showed comparable C16:1 content with Bifidobacterium sp. JCM
7042 being the highest. Based on the results obtained from the first and second screenings, we selected
Bifidobacterium sp. JCM 7042 for further study.