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Kikukawa et al. Microbiome Res Rep 2023;2:4 https://dx.doi.org/10.20517/mrr.2022.24 Page 3 of 12

Recent studies have revealed that C16 monounsaturated fatty acids (hexadecenoic acid, C16:1) such as 6-cis-
C16:1 (sapienic acid) and 9-cis-C16:1 (palmitoleic acid) possess a selective antibacterial activity against
S. aureus [28-31] , which has made C16:1 an attractive target of pharmaceutical therapy. Sapienic acid, which is
present in human sebum, is considered to control the skin microbiota as a natural antimicrobial agent.
Indeed, the level of sapienic acid was lower in atopic dermatitis patients than in healthy individuals and was
inversely correlated to the abundance of S. aureus [32,33] . This compound is, however, not easily obtained in
nature because it is scarcely found in natural oils. Palmitoleic acid is a known component of several natural
oil such as macadamia nut oil and sea buckthorn fruit oil, but the amount is too low to meet industrial use,
either.

Given this, in this study, we first screened bifidobacterial strains for their ability to produce C16:1. Then, the
antibacterial activity towards Staphylococcus strains was examined by using total fatty acids (TFA) extracted
from the selected strain. The results revealed the possibility of the strain for practical use to develop skin
care cosmetics and functional foods.

METHODS
Strains and culture conditions
Bacterial strains were purchased from the Biological Resource Center, National Institute of Technology and
Evaluation (NBRC) (Tokyo, Japan), and the Japan Collection of Microorganisms in Riken BioResource
Research Center (Ibaraki, Japan). Bifidobacterial type-strain cultures (JCM 1190, 1192, 1194, 1195, 1200,
1207, 1211, 1222, 1255, 1275, 1302, 6291, 7042, 8222, 10602, 12451, and 31944, and NCC 2705), B.
adolescentis labo-stock cultures (12451, 3-117, 12-114, 12-111, 4-2, 4-16, 4-58, and 9-124), and staphylococci
T
T
(S. aureus NBRC 100910 , 12732, 13276, and 14462, and S. epidermidis NBRC 100911 and 12993, and
ATCC 49134) were used in this study.

Bifidobacterium strains were cultured in 12 mL of MRS medium for two days at 37 °C and used for
screening for C16:1 content. TOS medium consisting of 10 g/L galactooligosaccharide, 10 g/L Tryptone, 1.0
g/L Yeast extract, 3.0 g/L K HPO , 4.8 g/L KH PO , 3.0 g/L (NH ) SO , and 0.2 g/L MgSO ·7H O
2
4
4
2
4
2
4 2
4
supplemented with 0.5 g/L L-cysteine was also used for cultivation. NB medium was used for the cultivation
of staphylococci and for dilution of the staphylococci cultures in an antibacterial assay which contained
0.5% extra bonito, 1.0% hi-polypeptone, and 0.5% NaCl (pH: 6.0).
Lactobacilli MRS broth, Tryptone, Yeast extract, and galactooligosaccharide were purchased from Difco,
Becton, Dickinson and Co. (Sparks, MD, USA). Other reagents and solvents were purchased from
FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
Fatty acid analysis
Fatty acids extracted in the form of methyl ester from bifidobacteria were prepared and analyzed using a
[34]
previously reported method with minor modifications . TFA is represented by the extracted fatty acid
methyl ester. Following cultivation, bacterial cells were harvested by centrifugation (3260 × g) at 4 °C for 20
min, washed twice with 0.85% NaClaq, and dried at 100 °C for 2.5 h. After measuring the dried cell weight,
the dried cells were directly transmethylated with 10% methanolic HCl at 55 °C for 2.5 h. Tricosanoic acid
(C23:0) was used as an internal standard. The resultant fatty acid methyl esters were extracted with n-
hexane before being concentrated and then analyzed by gas chromatography (GC) equipped with a TC-70
capillary column (GL Sciences Inc., Tokyo, Japan) and a flame ionization detector. The initial column
temperature of 160 °C was raised to 230 °C at 2 °C/min and maintained for 10 min. The injector and
detector were operated at 250 °C.