Yaichi Fukushima

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

SummaryA chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

SummaryThe effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h−1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)−1·h−1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as α-amylase, indicating that the protease production was selectively stimulated by the oils.

Continuous production of soy sauce by a bioreactor system

Abstract Continuous fermentation of soy sauce with immobilized glutaminase and immobilized cells of Pediococcus halophilus, Zygosaccharomyces rouxii , and Candida versatilis was investigated. Glutamic acid production and both lactic acid and alcohol fermentation continued constantly for over 100 days without any problems. The total time required for the production of soy sauce by the bioreactor system was much less than of the conventional method (about 1/10). The soy sauce made by this system was similar to a conventional one in regard to chemical components and the quality of flavour.

Effect of liquid mycelial culture used as a spawn on sawdust cultivation of shiitake (Lentinula edodes)

Mycelium of shiitake (Lentinula edodes) was cultured continuously in liquid medium. The liquid culture was carried out for the production of liquid spawn in the cultivation of this mushroom on synthetic sawdust substrate, and its performance was compared with that of the solid spawn. The initial colonization in culture bags was faster with the solid spawn than with the liquid spawn, but after this stage CO2 production was higher with the liquid spawn than with the solid spawn. For harvesting sufficient amount of good quality mushrooms, 120 d of incubation in bags was needed with the solid spawn, but this was reduced to 90 d for the sawdust blocks using liquid spawn of less than 50 d old. If continuous culture of the liquid spawn was prolonged over 50 d, immature fruit-bodies or their initials formed during the period of bag incubation. The solid subcultures of the liquid spawns retained the fruiting characteristics acquired in the liquid culture. Liquid culture could be a useful tool for breeding of mushrooms.

Continuous Conversion of Glutamine to Glutamate by Immobilized Glutaminase-Producing Yeast

Abstract Yeast cells with a salt-tolerant and thermostable glutaminase were immobilized in silica gel (S gel) and/or alginate-silica complex gel (AS gel). The inhibition rate of the conversion rate of immobilized cells by NaCl were lower than that of free cells. The glutaminases of immobilized cells and free cells were not inactivated by heat treatment at 60°C for 1 h. The half-lives of glutaminase in AS gel were 310 d at 40°C, 40 d at 45°C, and 14 d at 50°C at a constant space velocity (SV) of 0.64. The half-life of the glutaminase activity in cells immobilized in AS gel was longer than that in S gel. By passing a filtrate of wheat gluten hydrolyzed by proteolytic enzymes through the column containing the cells immobilized in AS gel at SV of 0.20, 10 mg/ml of glutamate was continuously produced.

Improved production of viable cells of the salt-tolerant yeast Zygosaccharomyces rouxii by continuous culture

SummaryA continuous culture system of the salt-tolerant yeast Zygosaccharomyces rouxii (soy yeast) was investigated in order to obtain high production efficiency of viable cells. The optimum pH and C/N ratio of the feed medium for cell production were about 5.0 and 16–20, respectively. About a fivefold increase in viable cell number and cell productivity (viable cell number per litre per hour) were obtained in glucose-limited culture at a dilution rate (D) of 0.06 h−1 as compared with batch culture. However, the fermentative activity of the cells from glucose-limited culture was significantly lower than those from batch and dissolved-oxygen (DO)-limited cultures, and the former cells showed lower specific activity of glycolytic enzymes. On the other hand, at the boundary conditions between glucose and DO limitation almost the same cell productivity and higher fermentative activity of the cell were obtained as compared with glucose-limited conditions. The cultivation continued for about 60 days without any problems even if the D was altered. It was found that the continuous cultivation method was suitable for industrial production of viable cells of soy yeasts.

Application of a Bioreactor System to Soy Sauce Production

Continuous fermentation of soy sauce with immobilized glutaminase and immobilized cells of Pediococcus halophilus, Zygosaccharomyces rouxii and Candida versatilis was investigated. Glutamic acid production and both lactic acid and alcohol fermentation continued for over 100 days without any problems. The total time required for the production of soy sauce by the bioreactor system was much less than that of the conventional method (about 1/10). The soy sauce made by this system was similar to a conventional one in regard to chemical components and the quality of flavor.

Efficient Production of Mycelium of Lentinus edodes by a Continuous Culture and the Effect of Lignin on Growth

Abstract In order to obtain a high yield of Lentinus edodes mycelium efficiently used as a liquid spawn for Shiitake mushroom production, a continuous culture system was investigated. The productivity of the mycelium (mg of dry weight of mycelium ml−1h−1) was 14-fold higher at a dilution rate of 0.03 h−1 than that in a batch culture. In the continuous culture, the pulpy form of the mycelium could be easily obtained either with a low dilution rate less than 0.01 h−1 or high agitation conditions of more than 400 rpm. The change in morphology to pulpy form resulted in a increase in the dry weight of the mycelium (DW). The maximum DW was 20 mg/ml at a dilution rate of 0.01 h−1. Lignin, which has been reported to accelerate the growth of L. edodes in a submerged culture, had no effect on mycelium production in the continuous culture, but promoted both the specific glucose consumption rate and maintenance energy. In addition, it was observed that lignin improved the resistance of the mycelium against shear stress. Adding 0.1% acetic acid to the feed medium and maintaining a low pH (about 4.0) in the culture allowed operation for over 70 d without any microbial contamination.

Manufacturing of Solid Culture Media in film Bags for Shiitake Cultivation

A continuous system was developed for manufacturing solid culture media packed in film bags for shiitake mushroom (Lentinus edodes).(1) The medium materials, sawdust mixed with bran, are steam sterilized and cooled, then spawned under agitation. Solid culture media thus produced continuously are packed in two-ply perforated film bags by a packaging machine. The semi-commercial plant has a manufacturing capacity of 430 bags per hour, which contain 1.8–1.9kg spawned sawdust media and have cylindrical shape. The bags are incubated for about 2 weeks until mycelial coat builds up, then delivered to the shiitake growers.