Taste Analysis And Cloning Expression Of Umami Octapeptide

Umami,one of the five basic tastes,not only brings pleasure to people,but also has many benefits for health,which is one of the essential taste needs of human beings.As a kind of potential food peptide flavoring agent developed in recent years,umami peptide could endue food with unique umami flavor,and has the characteristics of safety,nutrition and health,showing the huge market potential.However,most of the recent researches about umami peptides focus on the extraction and identification of umami peptides,but lack of structural features and taste mechanism of umami peptides.In addition,although the biological preparation of umami peptide has many advantages over enzymatic extraction and chemical synthesis,such as low cost and high yield,the research on the production method of umami peptide is still in its infancy and concentrated on the hot umami peptides,therefore,the biological preparation of the newly identified umami peptide needs to be further explored.In this paper,an umami octapeptide derived from Oreochromis spp.?OSP?was selected as the object of study to analyze its taste characteristics and explore its production method.Firstly,the interaction between OSP and umami receptor was studied by molecular simulation technique to resolve OSP structural characteristics and analyze its taste mechanism.Then,the multi-copy tandem strategy was employed to express OSP in the engineered bacteria,and its process conditions were optimized.The main research contents were as follows:?1?Structural modeling,semi-flexible molecular docking screening and molecular dynamics simulation were employed to obtain a stable docking complex between OSP and T1R1/T1R3.By interaction analysis of the docking complex,it was found that OSP formed strong interaction with umami receptor,including hydrogen bonds,salt bonds and?-ion bonds,of which the hydrogen bonds accounted for the main role.Further structural analysis of OSP,its N-terminal basic group and the C-terminal acidic group were mainly responsible for interaction with the umami receptor,and hydrophobic groups were presumed to play an important role in the formation of a particular three-dimensional conformation of OSP.?2?According to molecular simulation analysis,the interaction between 8OSP and umami receptors was strong from the energy and structure point of view,indicating that 8OSP was the most promising tandem octapeptide with umami,so 8OSP was selected to further study.Subsequently,by using molecular techniques such as codon optimization and double restriction enzyme digestion,the recombinant E.coli BL21/pMA09srfA-8OSP was constructed for 8OSP expression.Finally,the sensory evaluation was used to verify the taste characteristics of 8OSP expressed by recombinant bacteria.The results showed that 8OSP had slightly higher intensity than the standard solution without any bad taste such as bitterness and sourness,which was consistent with the calculation in tandem design of umami octapeptide.The above results suggested 8OSP showed high potential to be further developed into umami flavoring products?3?The effects of medium composition?carbon source,nitrogen source?and different culture conditions?inoculum size,culture temperature,culture pH?on the production of 8OSP by recombinant E.coli BL21/pMA09srfA-8OSP were investigated.By optimizing the conditions,the optimal culture conditions were obtained:the carbon source was glucose,the nitrogen source was yeast extract+peptone+?NH₄?₂SO₄,the inoculum size was 1%,the culture temperature was 37°C,and the pH was 7.0.Under this condition,the 8OSP concentration produced by shake flask fermentation was 0.152 mg/mL,achieved its efficient cloning expression.Subsequently,batch fermentation of 8OSP was carried out,and the 8OSP concentration finally reached 1.31 mg/mL,which was 8.6 times higher than that of shake flask fermentation.The above results of conditions optimization and batch fermentation provide a reference for industrial production of umami peptides.