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 30°C 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)

	Control 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

	Medium* (g 100/ml, 100 g dry mycelia)	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 30°C for 72 hours
*** T.N. x6.25