Research On Umami Taste Evaluation Via Mouse Taste Receptor Cell-based Biosensor

Umami,one of the five basic taste quality,is typically produced from L-monosodium glutamate(MSG),which serves as an important indicator for flavor evaluation.Umami intensity also reflects the content of nitrogen in foods.Therefore,it is of great importance to evaluate umami taste.The existing methods to test umami including sensory evaluation,HPLC and electronic tongue have limitations when compared with human taste system.Biosensor utilizing taste sensing components such as taste receptor,taste cells,taste tissues is an emerging technology to detect taste substances,which truly mirrors the taste mechanism within animals and human beings.However,the research of biosensor mainly focuses on bitterness or sweetness and rarely pays attention to umami taste.Additionally,how to obtain sufficient natural materials to support the extensive research of biosensor becomes a major problem.In this paper,mouse taste stem cells were successfully isolated and cultured in vitro.Organoids were formed by differentiation and proliferation of single stem cells,which comprised matured taste receptor cells(TRCs).Utilizing the matured TRCs as recognition element,we optimized and characterized the construction process of TRCs-based biosensor.Finally,the prepared biosensor was embedded into three-electrodes system to detect five common umami tastants.1)The papillae tissues were isolated,digested and mechanically separated into single cells.The single cells were suspended in 3%Matrigel-containing medium and grew bigger as taste organoids.After 12?15 days,taste organoids were digested with 0.25%trypsin-EDTA.Single cells continued to proliferate and differentiate rapidly,and grew into large numbers of taste organoids.The results of immunofluorescence and Western Blot showed that T1R1/T1R3,m Glu R1,m Glu R4 and?-gustducin were apparently expressed in the organoids,which play a vital role in umami transduction.Calcium imaging revealed concentration-response effect of MSG,indicating that the matured TRCs responded specifically to umami stimuli,such as MSG.2)In order to mount mouse TRCs onto the surface of glassy carbon electrode(GCE),gold nanoparticles(Au NPs)were deposited by electrochemical reduction method.Then poly-L-lysine was modified self-assembly at 37°C to increase the biocompatibility and adhesion.It can be seen from SEM images that Au NPs prepared by electrochemical method were more compact.Electrochemical characterization told that the signal amplification was more obvious than other electrodes.Au NPs treated GCE was scanned by cyclic voltammetry(CV)at different scanning rates.The redox peak current values showed a good linear relationship(R~2=0.99954and 0.99852)with the square root of the scanning rates,which implied the treatment was available and effective for further study.3)Using change rates of response current as the readout signal,five classic umami tastants were detected.The interaction laws were similar to the kinetic features of enzyme-substrates.A linear relationship was perceived between the change in current and concentration of tastants within the micromolar range.Calculation of EC50 values of five ligands to TRC showed that the concentration required for each substance to activate taste cells to produce 50%maximum physiological effect was MSG>MSG+IMP>MSG+GMP>BET>WSA,octapeptide>heptapeptide>hexapeptide.