3. Results
3.1. Chemical parameters during the fermentation process
The vinegar fermentation process in pot 1 was characterized
by the quantification of the fermentation products (Fig. 2). The
glucose concentration did not exceed 1mM throughout the
entire fermentation process. The ethanol concentration increased
just after the initiation of fermentation and reached 1200mM on
day 7, when the fungal mycelium was still observed on the
floating koji. The ethanol concentration decreased thereafter,
and the acetate concentration also started to increase. The acetic
acid production accelerated from day 15 to 20, and the final
concentration on day 90 was approximately 900mM(∼5.3% w/
v). This fermentation profile was also observed in the other pots,
i.e., in pot 2 and pot 3. These observations indicated that the
simultaneous process of saccharification and alcohol fermentation
was followed by the production of acetic acid. Entani and
Masai (1985b) and Koizumi et al. (1989) reported obtaining
similar results in their studies of pot vinegar fermentation in the
1980s. The lactate concentration increased within a few days
after the initiation of fermentation, and the concentration of
lactate was maintained at 100–150mM during the remainder of
the process. The production of lactic acid correlated with the
decrease in pH (b4) at the initial stage of fermentation. The
floating koji gradually sank into the fermentation broth and was
replaced by a thin pellicle approximately 6 weeks after the
initiation of fermentation. The pellicle covered the entire liquid
surface within two weeks and remained there during the
remainder of the fermentation process.
3.2. DGGE profile of fungal community
Fig. 3A1, A2, and A3 shows the DGGE profiles of the fungal
communities in pots 1, 2, and 3, respectively. PCR products
could not be obtained using the samples from days 20–90 from
any of the pots. DNA sequences of two representative bands,
bands a and b, were determined (Table 1), whereas the other
bands indicated by an asterisk were not determined due to the
failure to reproducibly amplify the excised bands. Band a was
derived from Saccharomyces sp. including S. cerevisiae. An
identical DNA sequence related to band b was found among
various species of moulds, e.g., Aspergillus sp. and Eurotium
sp. (Table 1). However, among these species, A. oryzae was the
most probable, because the koji used here is a culture of
A. oryzae. The same succession was observed in the three
Fig. 2. Chemical parameters of the fermentation process in pot 1. Open triangle,
acetate; open circle, lactate; closed circle, glucose; closed triangle, ethanol;
asterisk, pH.