Identification of an Antimicrobial Substance from Soybean Fermented "Tempeh"

Objectives: In recent years, increase of elderly people who need long-term care has become a problem not only in Japan but also many countries in the world. Necessity to manage oral infections in a simple manner is a new emerging social issue. Therefore, in order to control oral infections in a simple and long-term manner, an approach using phytochemicals, which regulate attachment and proliferation of resident oral bacteria, would be an economical and effective method by everyday meals. Thus, we identified the ingredients from the foodstuffs which have been shown to be antibacterial against oral bacteria.
Methods: The powders of several foodstuffs including a soybean-fermented food "Tempeh", which have been reported to possess antibacterial properties, were used for this study. A crude extract was prepared by mixing with PBS. Purification from the crude extract was performed by ultrafiltration and ODS-column purification. Streptcoccus mutans ATCC25175 was used as a target bacterium for antibacterial evaluation. The activity was evaluated by turbidity and ATP amount of the bacterial culture solution. Identification of the substance was carried out using HPLC, ESI-MS, and Raman spectroscopy. One-way ANOVA with Tukey’s test was employed for statistical analysis.
Results: Tempeh showed the strongest antimicrobial activity against S. mutans at a final concentration of 1 mg/mL or more compared to other foodstuffs. The crude extract was purified by the methods mentioned above, and antimicrobial property was found in the 100% methanol eluate. Isolation of the target ingredient was confirmed by HPLC. When this ingredient was analyzed by ESI-MS, m/z was 279.234. Further analysis using Raman spectroscopy revealed a peak similar to linoleic acid. It was confirmed that the isolated ingredient had antimicrobial property against S. mutans equivalent to a standard linoleic acid.
Conclusions: Tempeh contains linoleic acid as antimicrobial ingredient and could be a food to reduce oral infections.