Screening of Salt-Tolerant Protease Producing Koji Mold and Its Application in Soy Sauce Manufacture

• Main Authors: NAN-WEI SU, 蘇南維
• Other Authors: MIN-HSIUNG LEE
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/03663297658765886247

博士 === 國立臺灣大學 === 農業化學研究所 === 89 === A mold strain isolated by enrichment culture with sodium chloride from the soil in the Taipei area of Taiwan were compared with a commercial strain of Aspergillus oryzae for their proteolytic activities in an 18% NaCl aqueous solution system. This isolated strain subsequently identified and designated as Aspergillus sp. FC-10 produced protease with superior saline tolerance. In aflatoxin tests, this strain did not produce detectable aflatoxin after growing on steamed grain polished rice substrate for 24 days. Two types of extracellular proteases were preliminarily fractionated by means of DEAE-Sepharose CL-6B anionic exchange chromatography. Proteolytic activity of the non-adsorbed protease (P-I) was reduced to 9.4% in the 18% NaCl solution as compared to its original activity determined in the buffer solution. However, the adsorbed protease (P-II) was particularly salt-tolerant and stable, with 50% proteolytic activity retained throughout the six-hour stability test in 18% NaCl solution. P-II was further purified to homogeneity through preparative isoelectric-focusing electrophoresis and gel filtration chromatography, with an overall recovery of 12.7%. This salt-tolerant protease demonstrated an optimum pH range of 7.0-9.0 for activity, with a stable pH range of 5.0-9.0. The optimum process temperature at pH 7.0 was 65℃. The enzyme revealed a molecular mass of 28 kDa and was deduced as a monomer with an isoelectric point of 3.75. The enzyme activity was strongly inhibited by 5 mM of HgCl2 and FeCl3, and significantly inhibited by 5 mM of CuSO4, FeSO4, and MnCl2. The activity of this purified protease was obviously inhibited by Na2•EDTA; however, leupeptin, pepstatin A, PMSF, and E-64 did not affect the activity. Based on the N-terminal amino acid sequence and amino acid composition, this purified protease should possibly be classified as a member of the deuterolysin family. The gene sequence of this purified salt-tolerant protease was still not clear and needed further research. Two kinds of soy sauce fermentation were performed with Aspergillus sp. FC-10, i.e., traditional treatment soy sauce and fast hydrolysis at 45℃ for 48 hours and then aging at room temperature. After the fermentation and aging, the general compositions of Aspergillus sp. FC-10 soy sauces were respectively described as following total nitrogen, degree of hydrolysis (FN/TN), and total nitrogen utilization ratio (TNUR) in order, 1.68％, 54.2％ and 71.6％ in the traditional treatment soy sauce, and 1.73％, 55.1％ and 73.7％ in the fast hydrolysis soy sauce. Based on above data, there were significant differences between both Aspergillus sp. FC-10 soy sauces and the comparison soy sauce. The free amino acid contents of Aspergillus sp. FC-10 soy sauces were 36.6 mg/mL in the traditional treatment soy sauce and 39.7 mg/mL in the fast hydrolysis soy sauce. The major kinds of free amino acid in both Aspergillus sp. FC-10 soy sauces were glutamic acid and aspartic acid, and their relative compositions by weight were respective 11.6% and 11.1% in the traditional treatment soy sauce, and 9.3% and 10.7% in the fast hydrolysis soy sauce. The major flavor compounds in Aspergillus sp. FC-10 soy sauce and traditional fermented soy sauce were considered that there were no significant differences, but the contents of individual flavor compound in two kinds of soy sauces were quite different. According to the results, the total nitrogen could be promoted by Aspergillus sp. FC-10 koji in the earlier mash fermentation. However, this isolated strain might not be suitable to use directly in soy sauce manufacturing. Further studies involving mixed fermentation used the koji from the present factory and Aspergillus sp. FC-10 koji, and/or preparing the enzyme extract form Aspergillus sp. FC-10 to shorten the time of hydrolysis as improving the process of soy sauce manufacturing.