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Microscopic Morphology of A. oryzae 

Microscopic Morphology of A. oryzae 

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Soy sauce has been made for centuries by traditional methods, and consumed as the source of protein and vitamins. In this research work, the two fermenting microbes Aspergillus oryzae and Aspergillus flavus were mainly employed to produce soya sauce. Aspergillus oryzae was isolated from moldy soybeans. Also, Aspergillus flavus strain was received f...

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... powder (0.2 gram) was dissolved in 100 ml acetone. 5 ml of sample was put into the separation funnel. 5ml of n-butanol was added into the funnel and shaken vigorously. The layers were separated by placing in standing position for 15 minutes. Then 6-8 the upper layer was taken to use for TLC method. The isolation of A. oryzae was done by the agar plate method in Czapek’s Dox agar, selective agar. The rate of growth was very rapid. The microscopic morphology and cultural characteristics of A. oryzae and A. flavus was shown in Figure 1, 2, 3 and 4. The species was identified by comparing 5 with the reference (Raper and Fennel, 1965). The colony morphology of A. oryzae was examined by plating on four different media and the results were shown in Table 1. Red blood color was obtained by the filtrates of A. oryzae and that of Aspergillus flavus . The flavors were not changed in Kojic acid test. On account of these character the specimen was identified tentatively as Aspergillus oryzae . Four types of soy sauces were produced in this experiment. The yield of the finished products was 90% of the salt brine. The pH and viscosity of soy sauces were measured and the results were described in Table 2. The protein%, fat%, reducing sugar% and alcohol% of soy sauces were measured in this experiment and the results were shown in Table 3. The salt content of soy sauces was described in Table 4. Some colony of Bacillus species were observed in before pasteurized sample. But, no microorganism was observed in after pasteurization sample. By the comparison of R f values of individual standard amino acids and sample hydrolyzate amino acids, the results were shown in Table 5. To obtain the pure culture of Aspergillus oryzae was one of the main research works. Czapek’s Dox medium is a selective medium and were continually employed in subculture preparation until pure culture was obtained. Morphological characteristics such as growth rate, colony size, color and conidiophore formation etc. were enough to confirm the correct strain of Aspergillus oryzae . Moreover, according to Table 1, A. oryzae grow rapidly in Czapek’s Dox agar and Sabouraud dextrose agar. But, potato dextrose agar and malt extract agar gave slower growth rate. This may be due to other contaminating microbes which inhibit the growth of A. oryzae . In the comparison of colony size, the smallest colonial size was found in malt agar. In the study of biochemical test with Kojic acid test, only A. oryzae changed into red blood color. A. flavus remained without any changes. From this point, we can distinguish the species identification of A. oryzae and A. flavus fungal strain. According to the observed data in Table 2, the surest we say was that the smaller the pH value, the higher the viscosity in soy sauce products. But, all pH values existed in the specified range of soy sauce products (within 4.6-5.2). In viscosity measurements, S ory (1.5) and S fla (1.5) had the same value in each product. In the study of protein contents, Table 3 indicated that S ory (3) had better protein content than that of others. Moreover, its fat % and reducing sugar % were reliable for commercial production. In the study of salt content described in Table 4, S ory (1.5) and S fla (1.5) had the same content of 140 ppm respectively. As a part of research work, the presence of amino acids in soy sauce products was detected by Thin Layer Chromatographic technique. According to Table 5, only three amino acids (Valine, Threonine and Tryptophan) were present in A. flavus product. Aspergillus oryzae product contributes four amino acids (Valine, Lysine, Histidine and Tryptophan). However, commercial “Wai Weng” product comprises only the two amino acids (Valine and Histidine). So, A.oryzae product had more suitable for commercial production as a human diet. Soy sauce produced by A. oryzae contains more amino acids than soy sauce produced by A. flavus because A.oryzae produced peptidase and proteinase than A. By comparing the brine ageing time (1.5 months and 3 months), better results were obtained from 3 months. So, brine ageing time was also important for quality improvement of soy sauce. The two fermented fungal strains A. oryzae and A. flavus were mainly employed throughout this study. Not only morphological, microscopical and biochemical test but also culture test on four different media were studied. Analysis of soy sauce product was performed by determining pH, viscosity, and protein %, fat %, reducing sugar %, alcohol %, salt content and finally amino acid composition. Produced soy sauces S ory (1.5) and S fla (1.5) had better viscosity activities (1.6 cp) and S ory (3) had more protein content (3.88) than that of the other types. In A. flavus fermented product, only three amino acids (Valine, Threonine and Tryptophan) were found. A. oryzae fermented soy sauce had four amino acids composition (Valine, Lysine, Histidine and Tryptophan). But, in the analysis of commercial product (Wai Weng), only two amino acids (Valine and Histidine) were found. Analysis of amino acid composition was performed by one dimensional Thin Layer Chromatographic method (TLC). In the overview study of this research work, S ory (3) had better condition than that of the other types. The author would like to express her deepest gratitude to Dr. Mya Mya Oo, Rector, Yangon Technological University and Dr. Khin Maung Saing for their kind help in various ways and invaluable suggestions. The author would like to express special thanks to Ma Yadanar Win Myint (Medical Biotechnology) for help in giving the strain of Aspergillus flavus. 1. Ebine,H. “Advances in microbiology for miso fermentation”. Nippon Jozo Kyokai Zasshi. Japan, 1985. 2. Yokosuka, T. and Sasaki, M. Ferm-ented Protein Foods in the Orient: Shoyu and miso in Japan. “Microbiology of Fer-mented Foods”. Brian, J.B., Wood. Eds. Blackie Academic and Professional, London, 1998. 3. Thom, C., and Raper, K.B. “A Manual of the Aspergilli”. The Willams and Wilkins Company. U.S.A, 1945. 4. Thom, C., and Church, M.B. “The Aspergilli”. Copyright. The Willams and Wilkins Company. U.S.A, 1926. 5. Raper, K.B., and Fennell, D.I. “The Genus Aspergillus ”: Robert. E. Krieger Publishing Company: Hunlignton. New York, 1965. 6. Bui, T. T. “A Study of Vietnamese soy sauce fermentation”. M.Sc. Thesis. University of Western Sydney. Australia, 2003. 7. Kyaw Myo Win. “Isolation and identification of Aspergillus flavus and A. oryzae from local sources”. M.Sc.Thesis. Department of Biotechnology. YTU, Myanmar, 2003. 8. Okazaki, H.; Akiba, M. and Kimura, S. “Investigation on the antimutagenic substance in miso”. Presented in the Annual meeting of the Agricultural Chemistry Society of Japan, Tokyo, ...