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View the document 16 Continuous Production of Soy Sauce in a Bioreactor

16 Continuous Production of Soy Sauce in a Bioreactor

Takashi Hamada, Yaichi Fukushima, and Hiroshi Motai

Soy sauce is a traditional all-purpose seasoning with a salty taste and sharp Havor. In the conventional method of brewing soy sauce (Figure 1), cooked soybeans and roasted wheat are mixed with spores of Aspergillus species and fermented in solid culture for 2 days to produce koji. The koji is then mixed with brine to make moromi, the mash that ferments to produce soy sauce. Over time the soybeans and wheat are hydrolyzed by enzymes such as proteinases, peptidases, and amylases. During the first stage of moromi fermentation, Pediococcus halophilus grows and produces lactic acid, which lowers the pH. Accompanying the decrease in pH, vigorous alcohol fermentation by Zygosaccharomy-ces rouxii occurs. As a result, 2 to 3 percent ethanol and many kinds of aroma components are produced by this yeast. At the same time, phenolic compounds such as 4-ethylguaiacol (4EG) and 4-ethylphenol, which add characteristic aroma to soy sauce, are produced by other types of yeasts such as Candida versatilis and Candida etchellsii.

It takes over 6 months for the entire fermentation and aging of the morommi mash. Therefore, shortening this period is important and new processes for soy sauce brewing are desirable. This paper describes the continuous production of soy sauce in a bioreactor system, which consists of reactors containing immobilized glutaminase and immobilized cells of P. halophilus, Z. rouxii, and C. versatilis.


The processes for soy sauce production using the conventional and bioreactor methods are shown in Figure 1. The bioreactor method differs from the conventional one in the following ways: (a) proteases from continuous submerged culture are used (1), (b) fermentation is carried out in the liquid state, and (c) the fermentation period is considerably shorter. It takes several months for the conventional fermentation but only about 2 days for the bioreactor method.

In the bioreactor method, raw liquid was successively passed through, first, a glutaminase reactor to increase glutamic acid; second, a P. halophilus reactor to carry out lactic acid fermentation; and, third, a Z. rouxii reactor to carry out alcohol fermentation and a C. versatilis reactor to produce phenolic compounds such as 4-ethylguaiacol. Two reactors containing immobilized yeast cells were set in parallel, and the flow rate of the feed solution to the Z. rouxii and C. versatilis reactors was set in a ratio of 10 to 1. Carrier, packed gel volume, and operating conditions such as residence time, temperature, and aeration in each reactor are shown in Table 1.

FIGURE 1 Manufacturing processes for soy sauce by conventional and bioreactor methods.

TABLE 1 Conditions of Fermentation in Each Reactor



Packed Gel







Reactor Carrier





Temperature, C









P. halophilus







Z. rouxli







C. versatilis







AS, Alginate-colloidal silica. Al, Alginpte.


A profile of continuous fermentation by immobilized cells of P. halophilus, Z. round, and C. r~ersatilis is shown in Figure 2. The fermentation continued for over 100 days without any microbial contamination. A consistent increased level of glutamic acid (in the range of 0.3 to 0.4 percent) was found in the effluent from glutaminase reactor, with a residence time of 0.7 hours. Lactic acid was produced by immobilized cells of P. halophilus in quantities of 0.7 to 1.0 percent at a residence time of about 6 hours, and consequently the pH declined to 4.9 to 5.0, similar to that of conventionally brewed soy sauce. Ethanol was produced constantly by immobilized cells of Z. rotlxti in quantities of 2.5 to 2.7 percent at a residence time of about 26 hours. This is the standard ethanol content in soy sauce. About 10 ppm (parts per million) of 4-ethylguaiacol was produced by immobilized cells of C. versatilis at a residence time of about 10 hours, and the final 4ethylguaiacol content after mixing the two fermented liquids from the reactors of Z. rouxii and C. versatilis was about 1 ppm, which is the optimum concentration in conventional soy sauce. The total residence time for lactic acid and alcohol fermentation was about 30 hours in this system. This was considerably shorter than the conventional fermentation period of 3 to 4 months required to produce the same amounts of lactic acid and ethanol.

High numbers of viable cells were present in the gel and liquid in each reactor. The number was 10- to 100-fold higher in moromi mash. The shortening of the fermentation period in the bioreactor method is possibly due to the high density of immobilized cells in the gel and free cells in the liquid.

The main chemical components of the fermented liquid from the bioreactors were examined, including lactic acid, glucose, ethanol, and nitrogenous compounds.

FIGURE 2 Profile of continuous fermentation of soy sauce by a bioreactor system. (), lactic acid; (a), glutamic acid (values indicate the increase in the amount of glutamic acid); (v) pH; (•), ethanol; (D), glucose; 4-KG after passing through the C. versatilis reactor; 4-KG in the final product.


The organic acids and aroma components in the bioreactor soy sauce were examined. The proportions of organic acids except citric acid were not much different between the bioreactor soy sauce and the conventional one, although the former was a little lower in acetic acid and succinic acid. It appears that the high residual content of citric acid in the bioreactor soy sauce arises from the inability of P. halophilus to utilize citric acid. Aroma components present in both the bioreactor and conventional soy sauces were not qualitatively different. However, the former was higher in isoamyl alcohol and acetoin and lower in isobutyl alcohol, ethyl lactate, 4-hydroxy-2(orS)-ethyl-5(or2)-methyl3(2H)-furanone, and 4-hydroxy-5-methyl-3(2H)-furanone.

To evaluate the aesthetic qualities of the bioreactor-produced soy sauce, sensory tests were carried out. For example' the intensity of the alcoholic, fresh, sweet, acid, and sharp odors as well as the special lZiga (baking aroma) and bushoshu (foul fermented aroma) were compared between the bioreactor and conventional soy sauces. The odors are important for the quality of soy sauce. Although the bioreactor soy sauce was a little weaker in aroma and fresh odor than the conventional soy sauce, the quality of the former was generally judged to be similar to that of the latter.

The total time required for the production of soy sauce by the bioreactor system, including enzymatic hydrolysis of the raw materials, fermentation with immobilized whole cells, and the refining process, is only about 2 weeks (2). This is considerably shorter than the 6 months with the conventional method of soy sauce brewing consisting of koji making, fermentation and aging of morons, and refining.

From these results we conclude that the quality of the bioreactor soy sauce was very similar to that of the conventional soy sauce from both chemical and sensory evaluations and that the bioreactor system is practical for the production of soy sauce.


1. Fukushima, Y., H. Itoh, T. Fukase, and 1I. Motai. 1989. Applied Microbiology and Biotechnology 30:604-608.

2. Hamada, T., M. Sugishita, Y. Fukushima, and H. Motai. 1991. Process Biochemistry 26:39-45.