There are several contaminating microorganisms in koji, such as Micrococcus, Streptococcus, Lactobacillus, and Bacillus. However, these contaminants are not resistant to the high concentration of salt. Ishigami and Ishikawa (37) isolated some characteristic yellow-green Aspergillus strains, which produced prominently echinulate conidia and smooth-walled codiophores, from koji used in the production of soy sauce. They reported the Aspergillus strains to be a new species, Aspergillus sojae.
The second step in making fermented soy sauce is brine fermentation. It utilizes the lactic bacterium, Pediococcus halophilus and the yeasts Zygosaccharomyces rouxii and Candida species. Aspergillus sojae cannot grow in a pH over 6.0 in the presence of 18% salt (w/v). The specially selected Pediococcus halophilus is cultured and added to the moromi mash. It is necessary to keep the fermenting mixture at 15 ° C for the first month, allowing the pH of the mash to decrease slowly from 6.5 to 5.0. The cultures of Zygosaccharomyces rouxii and Candida species are added as a starter (38). Candida species such as C. versatilis, C. etchellsii, and the like grow at an early stage, but growth is much slower than that of Zygosaccharomyces rouxii, because Zygosaccharomyces rouxii is more anaerobic than the Candida species. The temperature of the moromi is allowed to rise to nearly 28 °C until vigorous alcoholic fermentation starts.
The lactic acid bacterium Tetragenococcus halophila is the dominant species in Indonesian soy mash. Tetragenococcus halophila growing in continuous and retention cultures under defined glucose-limited conditions showed a switch from homolactic (only lactate produced) to mixed-acid fermentation (two formate, one acetate and one ethanol formed per glucose) at low growth rates (39). Chinese- and Japanese-type fermented soy sauces are made of different plant materials. The lactic acid bacterium Tetragenococcus halophila is present and grows in both types. In the Chinese type, almost all isolates utilized L-arabinose, whereas in the Japanese type only 40% of the isolates did. Also, the population in the Japanese type was more heterogeneous regarding substrate utilization. Random amplified polymorphic DNA analysis revealed that the heterogeneous population at the Japanese-type industrial manufacturer was derived from only three strains at maximum (40).
Soy sauce was fermented in a column-type reactor with immobilized whole cells of Pediococcus halophilus, Saccharomyces rouxii, and Torulopsis versatilis entrapped in Ca alginate gels. Lactic acid and alcohol fermentation in a feed solution proceeded faster for an enzymatic hydrolyzate of koji and defatted soybean meal than did a complex fermentation of soy sauce mash (moromi) in slurry. The refined fermentation products obtained with the immobilized microbial cells were similar to conventional soy sauce in organic acid and aroma composition and possessed an acceptable taste and flavor (41). The optimum conditions for continuous alcohol fermentation of soy sauce with immobilized Zygosac-charomyces rouxii cells were investigated using an airlift reactor. The optimum pH and temperature of the fermentation were 4.5-5.5 and 25-27.5°C, respectively. The products fermented by supplying air (0.02 ppm) had higher content of aroma components than that by supplying only nitrogen gas, and the aroma of the former products was similar to that of conventional soy sauce (42).
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