76NISR RESEARCH GRANT
2002 Research Grant

Detection and Analyses of “Difficult to Culture” Microorganisms during Food Processing
Yasuo IGARASHI
Department of Biotechnology, Faculty of Agricultural and Life Science, The University of Tokyo
1.

Introduction

In these years, roles of so called “difficult to culture”
microorganisms in natural environment attract much attention. For example, in soil environment, the percentage of
microorganisms capable of forming colonies on solid media
is estimated as about 1% of the total living microorganisms.
How about in nutritionally rich environment such as food orfood wastes?
In recent years, fortunately, we can follow the transition of microflora in natural environment by using modern
molecular ecological methods such as DGGE or FISH. In
this experiment, we try to confirm the existence of “difficultto culture” microorganisms and clarify the roles of themduring food processing and food decomposition by using
molecular ecological methods in addition to traditional microbiological methods such as single colony isolation technique.
2. Isolation and characterization of a “difficult to culture” microorganism in the decomposition process ofkitchen garbage.
During the analytical process of the microflora duringdecomposition of kitchen garbage, we observed the existence of so called “difficult to culture” microorganism. We
could confirm by DGGE analysis that the major microorganism in the community could not form colonies by normal plating method. Surprisingly, the 16S-rDNA sequenceof the DGGE fragment suggests that the existence of “difficult to culture” microorganisms similar to our “difficult toculture” microorganism had been already reported in otherhabitats such as activated sludge or compost. This microorganism was similar to Bacillus species, and named “B Lx”
After enormous efforts, we could finally isolate BLxby the colony formation method, and identified the strain as
Cerasibacillus quisquilarum gen. nov., sp. nov. after precise

taxonomical researches. By quantitative-PCR and FISH
methods, BLx was shown to be the major habitat of the degrading process of kitchen garbage, and occupied approximately 30–50% of the total population at the middle stageof the process.
Unexpectedly, BLx showed no distinct ability to degrade food components except gelatinase activity. Moreover, BLx could not utilize major food components such as
glucose. On the other hand, BLx showed moderate resistingabilities against high temperature (55°C), high pH (10) andhigh salt concentration (5%). These resisting abilities ofBLx are believed to be one of the reasons that BLx can be
the major habitats of the microbial community, because
these abilities are considered to be essential to survive during the process we adopted.
3. Analyses of microbial communities during fermentation processes of traditional fermented foods.
We analyzed the transition of microbial communities
during Kurozu (black vinegar) making process in
Kagoshima Prefecture, because we were highly interested
in that saccharification, alcohol fermentation and acetic
acid fermentation took place sequentially in one big china
jar in this fermentation process.
At present, our achievement on this research is primitive one, and we are just following the experimental resultsby Prof. Koizumi of Tokyo University of Agriculture,
which had been mainly done by traditional isolation
method. But, by using modern molecular ecological methods such as DGGE, we could observe the dynamic transition of microflora during the process. The existence of “difficult to culture” microorganisms was also suggested in thisprocess. We hope to clarify the structure and functions of
the microbial community during Kurozu making process byusing newly developed molecular techniques in addition totraditional microbiological and biochemical methods.