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Application of soy waste as koji substrate for rice miso manufacturing (5, 6)

Milled rice is used for making rice koji, which supplies the necessary enzymes for the fermentation of rice miso. However, during the 48-hour period of koji preparation, approximately 10 per cent of the solids are consumed by the fungi. Soybeans also lose approximately 10 per cent of their solids, though the rate varies widely, depending on soaking and cooking methods. This investigation, there fore, was designed not only to determine the best way to manufacture rice miso, but also to explore the utilization of soybean cooking waste as a substrate for cultivating the fungus, Aspergillus. Soybean waste contains all the nutrients required by Aspergillus and also promotes the fermentation of miso (7). If successful, the results should yield the following advantages:

1) reduction of the COD value of soybean cooking waste by 80 per cent or more;

2) an up-grading of the biomass to food level, and

3) lowering the amount of rice koji needed, thereby eliminating the necessity of using so much expensive rice as an ingredient.

After screening tests employing 28 strains of fungi, including Aspergillus sp., Rhizopus sp., Penicillium sp., and Paecilomyces sp., we selected Aspergillus oryzae FRI-23 for the experiment. It was isolated from commercial tanekoji (fermented brown rice) and proved to be free of mycotoxins.

Soybean cooking waste with a COD of 20,000 ppm gave the highest rate of growth and the best reduction of COD, as shown in Figure 2. Cultivation was conducted at 30C under conditions of 1 vvm at a stirring rate of 400 rpm for 24 hours. At that time, the proteolytic enzyme activity attained a peak. At this stage, except for amylase, most of the proteolytic enzymes, particularly polypeptidases, were found to remain in the cells. As shown in Table 3, except for acid proteinase and amylase, the activity of essential enzyme produced in the cooking waste from 1,000 kg soy beans was higher than that in rice koji made from 700 kg of rice. This fact suggests the possibility of replacing the koji from rice with the mycelium grown in the waste when miso is made from 1,000 kg of soybeans, 700 kg of rice, and 430 kg of salt, or the same ratio of these ingredients.

TABLE 3. Enzyme Activity of Mycelium Made from Soybean Cooking Waste and Rice Koji

Enzyme   Enzyme activity

Mycelium (x 1,000)

Rice koji (x 1,000)
Proteinase (pH 3) 23,200 42,000
  (pH 6) 36,000 35,280
  (pH 7.5) 17,200 15,960
Acid carboxypeptidase   144 84
Leucine aminopeptidase   188 59
Amylase   1.2 1,176

Figure. 2. Flow Diagram of Miso Fermentation with Supplementation of Mycelium Grown in Cooking Waste

The mycelium was forced through a filter cloth and pressed to an 80.5 per cent moisture level. After chopping and grinding, the mycelium was mixed with green miso, prepared by mixing cooked soybeans and salted rice koji with an inoculum that included salt-resistant lactic acid bacteria and yeast. After fermentation, this new type of miso, containing two to five per cent of wet, living mycelium, showed a more advanced fermentation and degree of ripening than did the conventional miso

As illustrated in Table 4, the amounts of amino acids liberated from the protein of the mycelium-containing miso

TABLE 4. Effect of Mycelia on the Liberation of Free Amino Acids and Amides of Miso (mg/100 g)


0 Days*

67 Days 2% Mycelia

67 Days

5% Mycelia

67 Days

Asp-NH 60 104 176 229
Glu-NH2 103 271 360 428
Lysine 73 188 220 248
Histidine 17 36 42 61
Arginine 133 277 244 221
Asparagine 27 130 154 170
Threonine 30 119 132 188
Serine 41 157 186 232
Glutamine 62 249 311 381
Proline 23 101 109 115
Glycine 12 58 73 92
Alanine 34 125 155 188
Cystine 28 78 71 70
Valine 25 124 149 167
Methionine 17 42 63 70
Isoleucine 18 110 132 153
Leucine 40 223 260 289
Tyrosine 23 143 144 174
Phenylalanine 38 197 189 228

* Immediately after mixing rice koji, soy cooking waste, salt, and water for fermentation.

Mycelium enzyme represents the total amount of enzyme in the mycelium grown in the cooking waste from 1,000 kg of soybeans. Rice koji enzyme represents the total amount of enzyme in the rice koji made from 700 kg of rice were greater than those found in conventional miso. The soy waste mycelium also accelerated the growth and fermentation of the micro-organisms added as starters, thus playing a very important role in the formation of the attractive flavours found in ripened miso.

The amino acid patterns of the mycelium were similar to those in biomass grown on acetate. It is of interest that the content of nucleic acids, including RNA and DNA in mycelium, was 4 per cent on a dry weight basis (Table 5).

TABLE 5. Amino Acid, RNA and DNA Composition of Soybean

Cooking Waste and Mycelia of A. oryzae FRI-23


(g 100/ml, 100 g dry mycelia)

Amino acids    
Asparagine 0.071 3.3
Threonine 0.019 1.6
Serine 0.019 1.7
Glutamine 0.151 4.7
Proline 0.032 1.3
Glycine 0.024 1.5
Alanine 0.020 2.0
Cystine - -
Valine 0.018 1.9
Methionine (0.007) (0.6)
Isoleucine 0.014 1.4
Leucine 0.022 2.3
Tyrosine 0.014 1.2
Phenylalanine 0.019 5.5
Lysine 0.039 3.0
Histidine 0.016 1.0
Arginine 0.058 1.9
Tryptophan - -
RNA - 3.5
DNA - 0.5
Crude protein*** 0. 11 40. 0

* Soybean cooking waste (COD 20,000 ppm)
** Shaking culture at 30C for 72 hours
*** T.N. x6.25


The utilization of the wastes from the food industries is beset with many problems, among which economic feasibility is of prime importance, particularly for comparatively smallscale factories. As an example of one solution for coping with these problems, the use of soybean cooking waste as a substrate for koji-mould cultivation was investigated. The biomass obtained contributed not only protein and other nutrients, but also enhanced enzyme activity for the fermentation of miso, thus providing an apparent economic advantage.


1. Food Agency, Ministry of Agriculture, Forestry, and Fisheries, Japan, Standards for Management of Pollution in Miso Factories, 1977.

2. R. Hendrickson and J.W. Kesterson, "Citrus Molasses," University of Florida Agric. Expt. Stat Bulk 677 (1964).

3. K. Arima and T. Uozumi, "A New Method for Estimation of the Mycelium Weight in Koji," Agric. Biol. Chem. 31: 1-19 (1967)

4. T. Mochizuki, "Utilization of Soybean Cooking Waste as a Medium for Yeast Production," Misono Kagaku to Gijutsu 201: 2 (1970).

5. M. Yanagimoto, H. Saito, and H. Ebine, "Cultivation of Aspergillus oryzae in Soybean Cooking Waste," J. Soc. Brew. Japan 70:424 (1975).

6. S. Nikuni, H. Ito, H. Takagi, and H. Ebine, "Utilization of Soybean Cooking Drain," Sbokuhin Kogyo Gakkasishi 26 (in press).

7. S. Hayashida, N. Minamisato, T. Tei, and M. Hongo, "The High Concentration of Alcohol-Producing Factor in Koji," Nogeikagakukaishi 48 : 529 (1974).

Discussion summary

Questions concerning the extent to which SCP production on secondary juice from citrus peel is applied in Japan, and about the economics thereof, cannot be adequately answered until the project is completed in 1980.

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