Study On Fermentation Process Control And Extraction Technology Of L-tryptophan

As an essential amino acid for human and animals,L-tryptophan is widely used in medicine,food and feed fields,and the market demand is constantly expanding.Therefore,it is of great practical significance to carry out research on its production technology.Guided by the theory of metabolic engineering and taking L-tryptophan producing bacteria E.coli TRTH as the main research object,the key technologies in the control and extraction of L-tryptophan fermentation process were studied in depth.The main research contents are as follows:The process of L-tryptophan mixed-bacteria fermentation was studied.An L-tryptophan mixed-bacteria fermentation system mainly composed of E.coli TRTH and C.glutamicum TQ2223 as a supplement was constructed,and C.glutamicum was used to metabolize the by-products produced by E.coli such as acetic acid and lactic acid.The toxicity of inhibitory by-products to E.coli was weakened,and the growth and acid-producing activity of bacteria were enhanced.The optimum inoculation interval for mixed fermentation was determined by single factor and orthogonal experiments to be 14 h,the inoculation amount of Corynebacterium glutamate TQ2223 was 7.5%,the culture temperature was 36 C,and the pH was 7.0.A small test of 30 L fermenter was carried out under the optimum conditions.Finally,compared with the common fermentation,the biomass of mixed fermentation increased by 8.2%,L-tryptophan production increased by 7.5%,sugar-acid conversion increased by 7.4%,acetic acid accumulation decreased by 77.0%,and lactic acid accumulation decreased by 82.9%.The key influencing factors of L-tryptophan fermentation process were optimized.On the basis of the original fermentation process,the fermentation inoculation amount and the substrate KH2PO4 addition amount were successively optimized,and the focus was on investigating the effects of substrate KH2PO4 addition amount on cell growth,acid production,phosphate consumption,sugar acid conversion rate and by-product accumulation.The test results showed that under the condition of inoculation amount of 20%and substrate KH2PO4 addition amount of 10 g/L,the fermentation efficiency was greatly improved.Finally,the highest cell density reached 65.39 g/L,and the yield of L-tryptophan was 59.55 g/L,which were 45.99%and 31.17%higher than before optimization.The semi-continuous fermentation of L-tryptophan was studied.On the basis of fed-batch fermentation,a single-stage semi-continuous fermentation process of L-tryptophan was constructed,and part of the fermentation broth was released regularly,and then fresh medium was added to continue the culture,for a total of five cycles.On the basis of single-stage and semi-continuous fermentation,two-stage and semi-continuous fermentation tests were carried out,and the discharged fermentation broth was introduced into the secondary tank for culture,so that the fungi and nutrients were fully utilized and the maximum fermentation income was achieved.Finally,the single-stage and two-stage semi-continuous fermentation harvested 524.5 g and 1006.5 g of L-tryptophan,which were 95.6%and 275.3%higher than the fed-batch fermentation(268.2 g),and the sugar-acid conversion rates reached 18.7%and 18.5%,which were 6.86%and 5.71%higher than the fed-batch fermentation(17.5%).The new extraction process of L-tryptophan was studied.All fermentation components including L-tryptophan,bacterial cells and fermentation broth were processed through pretreatment,enzymolysis,reduced pressure concentration,spray drying and other steps,and finally the finished product of tryptophan protein peptide feed additive was obtained.The optimum conditions of ultrasound-assisted trypsin enzymatic hydrolysis were studied,and the optimum ultrasonic amplitude of 30%,continuous ultrasound time of 5 min,and ultrasound interval of 5 min were determined.The results showed that the reaction time was shortened by 60%,the single-step proteolysis rate was increased by 25.52%,the total proteolysis rate was increased by 17.79%and the soluble protein was further increased by ultrasound-assisted enzymatic hydrolysis compared with common enzymatic hydrolysis.Compound protease was used for the second step of enzymolysis to increase the content of free amino acids in the fermentation broth,and the total amount increased from 52.29 g/L to 66.26 g/L.The key conditions of spray drying were explored,and the optimal inlet air temperature of 160?,hot air flow of 34 m3/h and compressed air flow of 480?500 L/h were determined.Under the optimized conditions,the total yield of L-tryptophan can reach 86.85%,and the final product of protein peptide feed additive with L-tryptophan content of 49.96%,water content of 3.49%and crude protein content of 36.75%is obtained.