Isolation and identification of the antimicrobial substance included in tempeh using Rhizopus stolonifer NBRC 30816 for fermentation

Masahiro Ito; Takashi Ito; Hideyuki Aoki; Koshi Nishioka; Tsugumi Shiokawa; Hiroko Tada; Yuki Takeuchi; Nobuyuki Takeyasu; Tadashi Yamamoto; Shogo Takashiba

doi:10.1016/j.ijfoodmicro.2020.108645

Isolation and identification of the antimicrobial substance included in tempeh using Rhizopus stolonifer NBRC 30816 for fermentation

Int J Food Microbiol. 2020 Jul 16:325:108645. doi: 10.1016/j.ijfoodmicro.2020.108645. Epub 2020 Apr 22.

Authors

Affiliations

¹ Department of Pathophysiology - Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, Okayama 700-8525, Japan. Electronic address: m.ito.0617@s.okayama-u.ac.jp.
² Department of Pathophysiology - Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, Okayama 700-8525, Japan. Electronic address: itakashi@okayama-u.ac.jp.
³ Ikeda Food Research Co., Ltd., 95-7 Minooki-cho, Fukuyama, Hiroshima 721-0956, Japan. Electronic address: h.aoki@ikedatohka.co.jp.
⁴ Ikeda Food Research Co., Ltd., 95-7 Minooki-cho, Fukuyama, Hiroshima 721-0956, Japan. Electronic address: k.nishioka@ikedatohka.co.jp.
⁵ Division of Instrumental Analysis, Department of Instrumental Analysis and Cryogenics, Advanced Science Research Center, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama, Okayama 700-8530, Japan. Electronic address: shioka-t@okayama-u.ac.jp.
⁶ Division of Instrumental Analysis, Department of Instrumental Analysis and Cryogenics, Advanced Science Research Center, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama, Okayama 700-8530, Japan. Electronic address: tadahrk@okayama-u.ac.jp.
⁷ Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama, Okayama 700-8530, Japan. Electronic address: pdja8fvt@s.okayama-u.ac.jp.
⁸ Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama, Okayama 700-8530, Japan. Electronic address: takeyasu@okayama-u.ac.jp.
⁹ Department of Pathophysiology - Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, Okayama 700-8525, Japan. Electronic address: tadashii@md.okayama-u.ac.jp.
¹⁰ Department of Pathophysiology - Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, Okayama 700-8525, Japan. Electronic address: stakashi@okayama-u.ac.jp.

PMID: 32353648
DOI: 10.1016/j.ijfoodmicro.2020.108645

Abstract

In this study, we focus on the antimicrobial properties of tempeh, a soybean fermented food, against oral bacteria. Tempeh showed antimicrobial activity against dental caries pathogenic bacterium Streptococcus mutans at a final concentration of 1 mg/mL. An antimicrobial substance contained in tempeh was present in the 100 kDa or greater fraction generated by ultrafiltration, but it was found not to be proteinaceous by native-PAGE, SDS-PAGE and protein degradation tests. Next, when the fraction was purified with an ODS column, the 80% and 100% methanol eluates showed antimicrobial activity against S. mutans. The 100% methanol eluate was further subjected to a 2nd column purification, and isolation of the target was confirmed by HPLC. When the isolated material was analyzed by ESI-MS, the m/z was 279.234. Further analysis by Raman spectroscopy revealed a peak similar to linoleic acid. This substance also possessed antimicrobial properties equivalent to linoleic acid.

Keywords: Antibacterial; Fermented soybean food; Linoleic acid; Oral infection.

MeSH terms

Anti-Infective Agents / analysis
Anti-Infective Agents / isolation & purification*
Dental Caries / microbiology
Dental Caries / prevention & control
Fermentation
Fermented Foods / analysis
Glycine max / metabolism*
Glycine max / microbiology
Plant Extracts / pharmacology*
Rhizopus / metabolism*
Soy Foods / analysis
Streptococcus mutans / drug effects*

Substances

Anti-Infective Agents
Plant Extracts