Studies On Submerged Fermentation Of Selenium-riched Aspergillus Oryzae

With the development of biomedical technology, the trace element Selenium is receiving more and more attention, for it is one of the essential nutrients for human bodies. As natural foods contain low level of Selenium, only eating them to absorb Selenium cannot meet human bodies'demands for it. However, it is found that there is higher level of Selenium in inorganic substances like Sodium Selenite, which is so poisonous that it cannot be added to foods directly. Therefore, it is significant to produce a kind of edible food which is rich in Selenium. It is reported in scientific journals that Aspergillus Oryzae is a better carrier of Selenium which can convert the inorganic chemical, Sodium Selenite into organic Selenium by fermentation. With this method, Aspergillus Oryzae can be employed in food production to work as a medium to convert inorganic Selenium into organic Selenium that can be absorbed by human bodies. The aim of the thesis is to explore an easy way for those suffering the lack of selenium.When VIS and UV Spectrophotometry is applied to measure the content of Selenium, Selenium tends to be crystallized. Besides, the samples'heating time and digestion time are not given an account in journals, so they are not exact. Due to the problems mentioned above, some experiments in this thesis were conducted to test such factors as complexion temperatures, complexion time, digestion time, heating time, light period and intensity, and the concentration of O~Phenylene diamine. After many attempts, the optimal conditions for the measurement of the Selenium content were determined: complexion temperature is 25℃, complexion time is 1.5 hours, digestion time is 24 hours, heating time is 10 minutes, the best operating environment is in the dark room and the concentration of O~Phenylene diamine is 1%. Then, a repetitive experiment and recovery test were performed. The results of the two experiments show that RSD are 0.499% and 2.19% respectively, which prove that these conditions are accurate and reliable.The thesis first introduces the studies on the special quality of Aspergillus Oryzae, Selenium tolerance. Sodium Selenite is a kind of rank poison. When it is used as food additive, its amount has to be kept in a certain limit. The concentration of Sodium Selenite in the culture medium has a direct effect on the growth of Aspergillus Oryzae and its ability to absorb Selenium. Through the studies, the highest concentration of Sodium Selenite was determined as 100mg/L.After measuring the content of Selenium, response surface methodology was employed to optimize the liquid medium used in fermentation of Selenium~riched Aspergillus Oryzae strains. Saccharose and yeast cream were selected as the optimal carbon and nitrogen source from the tested carbon and nitrogen source. Following that, full factorial design was used to test the effects of 6 related factors, including Saccharose, yeast cream, calcium chloride, magnesium sulphate, potassiumd hydrogenphosphate, and Vitamin B1, among which yeast cream, calcium chloride, and Vitamin B1 were proven to play the biggest role. Then, the experiment of Steepest Ascent was carried out to approach the optimal response region of the above three factors. Finally, the Central Composite Designs and response surface analysis were used to determine the optimal concentrations of three main influential factors: yeast cream is 28.4g/L, calcium chloride is 0.25g/L, and Vitamin B1 is 0.015g/L.On the basis of the optimization of the culture medium, single factor experiments and response surface methodology were performed again to optimize other cultivation conditions of Aspergillus Oryzae, including the amount of broth in flask, inoculation quantity, fermentation temperature, rotational speed, the initial value of PH, among which three factors, which play a more important role were picked out and optimized by the Central Composite Designs. The optimal values of the five factors were finally got: the amount of broth in flask is 10 %, inoculation quantity is 80mL/250mL, fermentation temperature is 37℃, the initial value of PH is 5, and rotational speed is 180r/min. Based on these experiments, the amount of broth in flask, inoculation quantity and rotational speed were determined to be the most influential factors. And then in three levels, the Central Composite Designs of three factors were conducted to determine their values. The optimal values of the three factors are as follows: the amount of broth in flask is 91mL/250mL, inoculation quantity is 10.5% and rotational speed is 181r/min. Then verification experiments were performed under optimal fermentation condition. After three parallel experiments, the average content of Selenium contained in the cells of Aspergillus Oryzae measured 2.512. Although it was 0.67% less than the theoretical value 2.529, it remained within the error range of the model, which showed that the model could predict the situation of industrial fermentation fairly well, and that the results of the experiments could be applied to practice. On the basis of the optimization of the shake culture mentioned above, fermentation was conducted under the optimal culture conditions in batches to investigate dry weight of Selenium~riched Aspergillus Oryzae, the concentration of left Saccharose and the content of Selenium contained in the cells of Aspergillus Oryzae. The content of selenium reached its maximum: 2.896mg/g after 32 hours'cultivation, which rose 15% from the previous shake~flask culture. Meanwhile, the maximum of dry weight of Aspergillus Oryzae was 9.8g/L. And then, expotional Fed~batch was made in accordance with parameters got from the batch fermentation. The addition of carbon source was beneficial for increasing the content of selenium contained in the cells of Aspergillus Oryzae. At the thirtieth hour of fermentation, the content of selenium reached its maximum: 3.3mg/L, which rose 14% as compared with that in the batch fermentation. Meanwhile, the maximum of dry weight of Aspergillus Oryzae was 12.6g/L, which rose 28% from the batch fermentation. These studies showed that the circle of selenium~enriched Aspergillus Oryzae fermentation with expotional Fed~batch costed less time and the content of selenium and dry weight could reach their maximum at the thirtieth hour.