Rapid Quantitative Detection Method Of Umami Substances And Quality Analysis During Sufu Fermentation

As an important part of traditional fermented foods in China,sufu was deeply loved by consumers because of its unique flavor,delicious taste and high nutritional value.Aspartic acid（Asp）,glutamic acid（Glu）,5’-inosine acid（5’-IMP）,5’-guanylate（5’-GMP）were important amino acids and nucleotides that had an important effect on the taste quality of sufu.The detection methods of common umami substances had the disadvantages of complex sample preparation,harsh instrument detection conditions,high price,and long detection time.Therefore,it was particularly important to find ways to quickly quantify the umami substances of fermented foods.Consumers’acceptance of sufu depended on external and internal factors,such as price and brand reputation,and internal factors such as nutrition,taste,appearance,etc.,were related to the physical and chemical characteristics of sufu.The physical and chemical properties of food were often affected by external factors,such as production processes,production and storage environmental conditions.Fermentation was one of the important links in the production process of sufu,accompanied by a variety of complex physical and chemical reactions.In order to better analyze and control the quality of sufu,it was necessary to systematically and comprehensively understand the changes of components and their interaction relationships during sufu fermentation.Therefore,based on the electrochemical mechanism and the structural analysis of umami substances,an electrochemical sensor device capable of rapid quantitative detection of umami amino acids and nucleotides was prepared and applied to the detection of umami substances in sufu samples.At the same time,the characteristics and correlation of physicochemical quality changes of sufu during fermentation were studied,in order to provide theoretical basis and support for the analysis and improvement of the quality of sufu.The main research contents and results of this paper were as follows:（1）Application research of N,N-Diphenylthiourea（DPTU）membrane modification sensor for the detection of umami amino acids of sufu during fermentation.A total of 16 free amino acids（FAAs）were detected by using amino acid analyzer during sufu fermentation,of which umami amino acids accounted for40.51%of the total FAAs.Based on the Lewis base-like structure of DPTU and the absorption of electron clouds or corresponding atomic groups,the mathematical relationship between the open circuit potential value and the sum of the concentrations of Glu and Asp was explored by using the DPTU membrane modification sensor,and it was applied to the detection of umami amino acids during sufu fermentation.The experimental results showed that the sensor can quantitatively detect the sum of umami Glu and Asp concentrations in sufu.（2）Research and application of poly methionine（P Met）membrane modification sensor for 5’-IMP detection.A P Met membrane-modified sensor for 5’-IMP detection was prepared by electrochemical polymerization and specific adsorption between amino acid compounds.Through physical and electrochemical characterization,the experimental results showed that the P Met membrane modification sensor can achieve specific adsorption of 5’-IMP.The differential pulsed voltammetry（DPV）and cyclic voltammetry（CV）were used to optimize the conditions for the detection of5’-IMP,and the results showed that the oxidation peak current value of the P Met membrane modification sensor in response to 5’-IMP showed a good linear relationship with its concentration,specifically:in the range of 5’-IMP concentration of 1～15μmol/L,the linear equation was I₁=0.1036（±0.0049）C₁+2.8035（±0.0235）,with a correlation coefficient of 0.9926;In the 5’-IMP concentration range of 15～500μmol/L,the linear equation was I₂=0.0023（±0.0001）C₂+4.3135（±0.0267）,with a correlation coefficient of 0.9938.The lower limit of detection was0.3367μmol/L（S/N=3）.The experimental results showed that the P Met membrane modification sensor had good performance in terms of anti-interference,reproducibility and stability,and can be used in the specific detection of 5’-IMP in sufu samples.（3）Research and application of molecularly imprinted sensors for 5’-GMP detection.With 5’-GMP as the template molecule and o-phenylenediamine（OPD）as the monomer,molecularly imprinted sensors（Ag NPs/SMWCNTs/MIP/GCE）for5’-GMP detection were constructed on the glassy carbon electrode（GCE）modified by carboxylated multi-walled carbon nanotubes（SMWCNTs）by electrochemical polymerization of OPD and deposition of silver nanometers（Ag NPs）.Through physical and electrochemical characterization,the experimental results showed that the molecularly imprinted sensor can achieve specific detection of 5’-GMP.The results showed that the oxidation peak current value of the molecularly imprinted sensor in response to 5’-GMP had a good linear relationship with the logarithm of its concentration,specifically:the linear equation was I₃=0.5557（±0.0197）lg C₃+8.3996（±0.1995）,and the detection range was 0.05～50.00 mmol/L.The correlation coefficient was 0.9938 and the lower detection limit was 0.009 mmol/L（S/N=3）.The experimental results showed that the molecularly imprinted sensor had good performance in terms of interference resistance,reproducibility and stability,and can be used in the specific detection of 5’-GMP in sufu samples.In addition,the sensor combines molecular imprinting with an electrochemical sensor,which not only realized the specific detection of 5’-GMP by the sensor,but also used a fast and simple electrochemical polymeric monomer method to increase the binding site of the template molecule,thereby indirectly improving the detection efficiency of the sensor.（4）Change characteristics of physicochemical composition of sufu during fermentation and its correlation analysis.A number of physicochemical indexes of sufu such as nutritional indicators（crude protein,crude fat,reducing sugars,amino acid nitrogen,total acid,salt and moisture）,umami indicators（Glu,Asp,5’-IMP and5’-GMP）,texture indicators（hardness,viscosity,elasticity,cohesion,adhesion and chewiness）,color indicators（L*,a*and b*）and other indicators（conductivity,turbidity and p H）were dynamically determined and analyzed.The variation law and reasons of the composition of sufu in the fermentation process were revealed,and the nutritional components of sufu in different fermentation stages in the fermentation process were studied on the correlation between umami,texture,color,electrochemistry and other characteristics.Specifically,Glu showed a significant positive correlation with total acid（P<0.05）,Asp showed a significant positive correlation with total acid and reduced sugar（P<0.01）and negative correlation with crude protein and fat（P<0.01）,5’-IMP（P<0.01）,and 5’-GMP with salt（P<0.01）.Hardness,viscosity,elasticity,cohesion,adhesion,and chewiness were significantly positively correlated with crude protein and crude fat（P<0.01）,with amino acid nitrogen and total acid（P<0.01）,and with reduced sugar.L*showed a significant positive correlation with crude protein and crude fat（P<0.01）and a significant negative correlation with amino acid nitrogen and total acid（P<0.01）;a*showed a significant negative correlation with water（P<0.05）;b*showed a significant negative correlation with amino acid nitrogen and total acid（P<0.01）and a negative correlation with crude protein and crude fat（P<0.01）.Turbidity was significantly negatively correlated with water（P<0.05）;conductivity was positively correlated with salt,amino acid nitrogen,total acid,and negatively associated with crude protein,crude fat and water;p H value was positively correlated with salt.