| Docosahexaenoic acid?DHA?is an omega-3 series of polyunsaturated fatty acids,a product beneficial for human health.With the increase of its applications in food,health care products and pharmaceutical industries,DHA obtained by traditional extraction method is unable to meet the market demands.DHA fermentation by Schizochytrium has become a hotspot.This thesis aimed to study the effects of nitrogen/carbon sources feeding strategies on DHA fermentation,key enzyme activities,intracellular protein changes of Schizochytrium sp.S31 during DHA accumulation.The major results were summarized as follows:?1?DHA production performance with different initial C/N ratios was examined by batch mode,and the optimal initial C/N ratio was determined as 7:1.Under this condition,final DHA concentration reached 2.66 g·L-1.On this basis,DHA production was further improved by optimzing carbon/nitrogen sources addition:1)the carbon source?glucose?was intermittently fed without nitrogen source addition,DHA concentration increased to 8.88g·L-1;2)using the same carbon feeding strategy as above,but intermittently adding nitrogen source?yeast extract?when the initial nitrogen source exhausted during the first 80 h of fermentation.Each yeast extract addition amount was 5 g·L-1,DHA concentration increased to 11.97 g·L-1;3)using the same nitrogen source feeding strategy and continuously feeding glucose to keep its concentration around 30 g·L-1,DHA concentration reached 16.75 g·L-1.?2?Using analysis methods of enzymatic activity and different protein to explore the physiological mechanisms affecting the accumulation of DHA in different substrate feeding strategies.The study investiagted Schizochytrium sp.S31 disruption by two methods:glass bead breakage and ultrasonic disruption to remove lipid,in combination of using the methods of freeze-thaw,ethanol/acetone precipitations.The results indicated that high-quality intracellular protein could be extracted by methods of glass bead breaking and freeze-thaw.?3?Using intracellular protein extracts to analyze the changes of key enzymes6-phosphoglucomkinase?G6PDH?,malic enzyme?ME?,citrate lyase?ACL?and isocitrate dehydrogenase?ICDH?during DHA synthesis.Results showed that G6PDH activity did not change significantly during the entire DHA synthesis phase.The activity of ME and ACL gradually increased during the early period of DHA synthesis and gradually decreased during the mid-to-late period.ACL activity under different substrate feeding strategies had no significant difference,but ME activity and final DHA yield had positive correlation.?4?The MS technology was used to analyze the differential expression of intracellular proteins during the synthesis of DHA in different substrate feeding strategies.The five differentially expressed proteins identified were heat shock protein Hsp 100,Polyunsaturated fatty acid synthase subunits,pyruvate kinase,glutamate synthetase,and ATP synthase.N-source limitation would increase the expression level of Hsp 100 and enhance cells tolerance against severe environments.The polyunsaturated fatty acid synthase subunits directly related to the synthesis of DHA and had positive correlation with DHA production.Pyruvate kinase and its expression were significantly promoted by constant glucose feeding. |
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