The objective of this work is to identify an isolate Td of yielding high nattokinase, and to analyze the molecular characteristics of its nattokinase. The morphologic, biochemical and physiological characterizations, and molecular biological methods were used to identify the taxonomic position of isolate Td ;mass spectrometric determination and analysis of MALDI-TOF, SDS-PAGE and fibrinolytic activity were employed to measure the characteristics of nattokinase, and PCR method was adopted to clone the full length of nattokinase gene. The bacterial strain Td was identified as Bacillus subtilis subsp. subtilis according to the results of morphologic, biochemical and physiological characterizations, as well as 16S rDNA, the sequences of 16S rDNA gyrA, hybrid rate of DNA-DNA. The yield of nattokinase produced by strain Td reached 300 mg/L, accounted for over 40% of the total protein. Fibrinolytic activity was over 230 U/mL. Amino acid sequence of nattokinase produced by Td showed the highest similarity to the subtilisin E. and the full length of the gene was 1143 bp. Conclusively, as a high-yield and high-activity strain for nattokinase, B. subtilis subsp. subtilis Td is a promising candidate for industrial development and utilization.

在河南省汝阳县伏牛山区首次发现了蛹虫草的生长与分布.调查了蛹虫草的生长环境并对蛹虫草菌株进行了分离纯化、形态学观察和分子鉴定.结果表明,野生蜗虫草主要分布在山体的背阴面,毒生于针、阔叶混交林地表土层鳞翅目昆虫的蛹体上,子座呈1个或多个分支,长3～8 cm,颜色呈橘黄至棕黄色,子囊壳丰富.分子鉴定表明,蛹虫草的ITS1-5.8SrDNA-ITS2序列长度为478 bp,GC含量为56.4％.经GenBank数据库同源性检索比对,与蛹虫草有高度同源性,达到100％.分离自野生蛹虫草的菌株ycc-ry-01在大米综合培养基上只能形成原基,不能长出子座,而对照菌株ycc-jzc可以培养出子座;经液体发酵培养后的菌株ycc-ry-01与对照菌株ycc-jzc均表现出纤溶活性,菌丝体提取液的活性大于发酵液的活性.

通过对蛹虫草(Cordyceps militaris)菌丝液体发酵产纤溶酶的实验研究,获得了蛹虫草纤溶酶液体发酵的适宜参数.结果表明,蛹虫草菌丝液体发酵产纤溶酶的适宜碳源为玉米粉和蔗糖,豆饼粉和较高浓度的玉米浆(8％～12％)作为氮源是适宜的,培养基中适量添加MgSO4(0.05％)、KH2PO4(0.1％)和CaCl2(0.05％)、MnSO4(0.05％)等无机盐有利于蛹虫草纤溶酶产生;发酵pH值以4.5～5.5为宜,温度以22～24℃为宜,培养时间以6～7 d为宜.

The nattokinase crudes were prepared and drying with different methods,and the influence of drying temperature and treating times on the nattokinase crudes were tested.It was found that the activity of nattokinase and electrophoretic spectrometry of different strains were different from each other.Both of freeze-drying and static microwave vacuum drying methods were suitable for nattokinase drying.The during time and the temperature(50 ℃ or above)have a serious influence on nattokinase activity of the crudes,and have 80% activity for 24 monthes preservation under the room conditions.

To increase the DH of chickpea protein,and provide the basis for industrialization of the enzymatic production of chickpea producing oligopeptides,the optimal condition for alcalase hydrolysis of chickpea protein was studied. The degree of hydrolysis(DH) was determined according to the pH-state method. Through a single-factor test and an orthogonal designed experiment,the alcalase hydrolysis of chickpea protein was systematically studied. Then,the three proteases of alcalase,papain and bromelain commonly hydrolyzing chickpea protein was carried out to increase the DH of chickpea protein. The results showed that the obtained optimum hydrolysis with alcalase condition was pH 8.5,temperature(T) 55℃,concentration of substrate [S]2%,and ratio of enzyme and substate([E]/[S]) 2%. Under this condition,the degree of hydrolysis(DH) was27.86%. Under each optimum conditions of the three proteases respectively(The obtained optimum hydrolysis condition of alcalase was pH 8.5,temperature(T) 55℃,concentration of substrate [S] 2%,and ratio of enzyme and substate([E]/[S]) 2%. The obtained optimum hydrolysis condition of papain and bromelain was pH 7.2,temperature(T) 55℃,concentration of substrate [S] 2%,and ratio of enzyme and substate([E]/[S]) 2%),the chickpea protein hydrolyzing by the alcalase,papain and bromelain in turn was carried out,and the DH couldreach 34.64%. The DH of chickpea protein hydrolyzing by the three proteases above-mentioned under each optimum condition respectively in turn was much higher than that of the DH of chickpea protein hydrolyzing by the three proteases simultaneously. Furthermore,the yield of oligopeptides was significantly improved.

The nattokinase crudes were treated with γ-ray irradiation at different absorbed doses,and the total ginsenosides,nattokinase activity and the vital microbes were tested.It was found that the inf luences of the γ-ray irradiation on the total ginsenosides and nattokinase activity were slightly,while the lethal effect of γ-ray irradiation on the vital microbes such as Bacillus subtilus Natto,Staphylococcus aureus and coliform bacteria were seriously(decreased 99%).This studies provided some experimental data for irradiation methods applicated on the nattokinase crudes disinfection and preservation.

In this paper,we determined the degree of hydrolysis(DH)of the chickpea protein with three proteolytic enzymes of alcalase,papain and bromelain(domestic)at different times,and analysed the electrophoretic pattern of the hydrolytic products.The result shows that after treated with the proteases for three hours,the chickpea proteins were mostly hydrolysed into oligopeptides,and the DH value(35.42%)of chickpea protein hydrolyzing by the three proteases above-mentioned under each optimum conditions respectively in order was much higher than that of the DH of chickpea protein hydrolyzing by the three proteases simultaneously.

Grifola frondosa is a edible and medicinal fungi.We optimalizated the carbon and nitrogen sources for liquid culture of Grifola frondosa,then used the hot water extraction method,to extract the extracellular polysaccharides(EPS)from the culture media at 60 ℃ for 2 hours.The results showed that the suitable carbon and nitrogen sources were glucose and yeast.The best content of Grifola frondosa EPS was 9 g/L.This study laid a good foundation for further Grifola frondosa EPS study.