120NISR RESEARCH GRANT
2003 Young Investigator Research Grant

Microbial Ecological Studies on Traditional FoodFermentation
Shin HARUTA
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo
Research aims
Many kinds of fermented foods have been manufactured
by traditional procedure all over the world. In many cases,
appropriate microorganisms grow selectively during the fermentation process without strict microbiological regulation.
However, the microbial composition and their association
are difficult to clarify by cultivation-isolation methods. Inaddition to the culture-based methods, in this study, molecular microbial ecological approaches were applied to obtainfurther insight into a traditional brewing technology.
Methods
Pot vinegar has been manufactured in Kagoshima prefecture since 1805. After steamed rice, water and rice-koji areadded into a ceramic pot, additional rice-koji is spread onto
the liquid surface (floating koji). The pot is placed outdoors
for around 3 months. The fermentation broth was collectedfrom three fermentation pots several times during the fermentation period. Total DNA was extracted from the harvested cells and used for PCR-denaturing gradient gel electrophoresis (DGGE). The procedures of DNA extraction
and PCR-DGGE were described previously1).

characterized by the bands which were related to Lactobacillus fermentum (bands 1–4), Lactococcus lactis (band6), or Pediococcus acidilactici (band 8). And then, thebands related to Acetobacter pasteurianus (band 9) andLactobacillus acetotolerans (band 7) were detected in theacetate fermentation process. In contrast, Lactobacillus
casei and Lactobacillus plantarum were isolated from thesaccharification/alcohol fermentation process. The previous
studies also reported the isolation of Lactobacillus casei
subsp. casei and Lactobacillus plantarum with high frequency3,4). However, these lactic acid bacteria were not detected in the major bands of the PCR-DGGE profile. Furthermore, the low similarity of band 1, 2, or 3 to the closest
relative may indicate that these bacteria are novel Lactobacillus species.

Results
Chemical analyses of the fermentation broth indicatedthat saccharification and alcohol production simultaneously
proceeded for around 20 days after starting the fermentation. And then, acetic acid concentration increased gradually with the consumption of ethanol and reached 5% at theend of fermentation (day 90). Koizumi et al. reported thesimilar results in 19892). Lactic acid concentration increased in a few days after starting the fermentation andwas kept at 100–150 mM during the process. The production of lactic acid correlated with the decrease in pH below
4 at the initial stage.
PCR-DGGE profile indicated the succession of the bacterial community around day 15–30 when acetate concentration started to increase (Fig. 1). The similar banding profile was observed in three fermentation pots. Although thebanding profiles of the starting materials (day 0) were different from one another, the three pots showed the nearlysame bands on day 1. This indicates the rapid growth of favorable bacteria in a day to achieve the stable fermentation.
The saccharificaion/alcohol fermentation process wasFig. 1. PCR-DGGE profile during the manufacturing process
The arrow represents the linear gradient of polyacrylamide concentration(PA 6–12%) and DNA denaturants (UF 25–50%). The bands indicated by
the circled number were excised to determine the DNA sequences. The
closest relative in DNA databases to each band is as follows. 1, Lactobacillus fermentum (97%); 2, L. fermentum (97%); 3, L. fermentum (97%);4, L. fermentum (98%); 5, Aspergillus oryzae (98%); 6, Lactococcus lactis(100%); 7, Lactobacillus acetotolerans (100%); 8, Pediococcus acidilactici (100%); 9, Acetobacter pasteurianus (100%).

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Conclusion

References

PCR-DGGE analysis represented the succession of bacterial community during the pot vinegar fermentation
process. The change in the fermentation pattern was clearly
characterized by the change in the banding profile. The
same succession was observed in the three pots examined.
The bacteria detected as major bands were different fromthe isolated microorganisms.

1)
2)
3)
4)

Haruta, S. et al., Appl. Microbiol. Biotechnol., 60: 224–231 (2002).
Koizumi, Y. et al., Nippon Shokuhin Kogyo Gakkaishi, 36: 237–244(1989).
Entani, E. and Masai, H., J. Brew. Soc. Japan, 80: 200–205 (1985).
Koizumi, Y. et al., Nippon Shokuhin Kagaku Kogaku Kaishi, 43:347–356 (1996).