Microbiological and biochemical survey on the transition of fermentative processes in Fukuyama pot vinegar brewing

Traditional brewing of Fukuyama pot vinegar is a process that has been continued in Fukuyama, Kagoshima, Japan, for almost 200 years. The entire process proceeds from raw materials, including steamed rice, rice koji (steamed rice grown with a fungus, Aspergillus oryzae) and water, to produce vinegar in roughly capped large pots laid in the open air. No special fermentative manipulation is required, except for scattering dried rice koji (called furi-koji) on the surface of the mash to form a cap-like mat on the surface at the start of brewing. As the biochemical mechanism of the natural transition of the fermentative processes during brewing has not been fully explained, we conducted a microbiological and biochemical study on the transition. First, a distinct biochemical change was observed in the brewing of spring preparation; that is, a sharp decline in pH from 6.5 to 3.5 within the first 5 days of brewing was observed due to lactic acid fermentation. Alcoholic fermentation also proceeded with a sharp increase to 4.5% ethanol within the first 5 days under the acidic conditions, suggesting that saccharification and both fermentations proceed in parallel. Acidic conditions and ethanol accumulation restricted the growth of most microorganisms in the mash, and in turn provided a favorable growth condition for acetic acid bacteria which are acid resistant and ethanol-philic. Acetic acid was detected from day 16 and gradually increased in concentration, reaching a maximum of 7% at day 70 that was maintained thereafter. Empirically furi-koji naturally sinks into the mash after around day 40 by an unknown mechanism, allowing acetic acid bacteria to easily form pellicles on the mash surface and promoting efficient acetic acid fermentation. Dominant microbial species involved in the three fermentations were identified by denaturing gradient gel electrophoresis analysis using PCR-amplified defined-regions of small rDNA from microorganisms in the brewing mash or colony direct PCR applied to isolated microorganisms from the mash.