Study On Freshness Evaluation Of Grass Carp(Ctenopharygodon Idella) Fillets And Development Of Freshness Indicator Protein Test Strips

With the improvement of people’s living standard,the conception of food has changed from "eating enough" to "eating well",thus consumers favor fresh fish fillets with better quality.However,the current national standards for fish only provied the determination of "qualified" or not,without the division of freshness evaluation.The conclusions of freshness determination provided in the literature cannot be compared due to different methods,and some freshness testing methods are complicated.The grass carp in our country was taken as the research object.Sensory evaluation,physical indicators and chemical indicators were used to evaluate the freshness of the grass carp fillets,and Hierarchical Cluster Analysis(HCA)was used to divide different freshness intervals.The freshness indicator proteins were selected which were consistent with the freshness interval of comprehensive evaluation and suitable for colloidal gold immunochromatography.Based on the above,a colloidal gold immunochromatographic test strips for detecting the freshness of grass carp fillets was developed to provide a convenient method for evaluation of grass carp freshness.Firstly,the freshness of chilled and iced grass carp fillets was comprehensively evaluated and divided into intervals,and the freshness indicating proteins suitable for indicating freshness and can be used to make colloidal gold immunochromatographic test strips were screened.The freshness of grass carp fillets was determined by sensory and physicochemical indicators to reflect different aspects of freshness,and the freshness intervals were classified by HCA.The grass carp protein for freshness indicator that were consistent with the intervals classified by the comprehensive evaluation method were further screened by protein Western blotting and purified for the colloidal gold immunochromatographic test strips in the next step.The results show that there are two principal components for both chilled and iced grass carp fillets,with total contribution rates of 92.203% and 94.151% respectively,indicating that they can basically reflect the original variable information of chilled and iced grass carp fillets.According to the results of HCA,the three intervals dividing the freshness of chilled grass carp fillets are 0～1day,2～7 days and 8～10 days,and the three intervals dividing the freshness of iced grass carp fillets are 0～3 days,4～10 days and 11～15 days,respectively.The trends of troponin T,desmin and parvalbumin in chilled and iced grass carp fillets were consistent with the intervals of the comprehensive evaluation of freshness,indicating that all these proteins could be used as freshness indicator proteins for grass carp fillets.The parvalbumin was purified by heat extraction combined with DEAE-Sepharose ion exchange column with a purity of more than95%,which can be used for the colloidal gold immunochromatographic test strips in the next step.Then,a colloidal gold immunochromatographic test strips(GICA)method was developed that can be used for rapid detection of freshness of grass carp fillets.A colloidal gold immunochromatographic test strips were prepared,optimized,and tested for freshness of grass carp fillets based on the selected parvalbumin.The results show that the optimum p H was 7 and the optimum concentration was 24 μg/m L when colloidal gold conguates.The purified colloidal gold-m Ab conjugate optimum volume was 7 μL/pad.The spraying concentrations of parvalbumin and anti-rabbit Ig G were 1.5 mg/m L and 1.0 mg/m L,respectively.The NC membrane is preferably CN 140.The strip has excellent performance in specificity,repeatability,stability and sensitivity.Drip loss protein has a better indicator effect than sarcoplasmic protein solution.According to the determination of drip loss protein,the corresponding T/C values of the first-stage,second-stage and third-stage freshness of chilled grass carp fillets were 0.590 ±0.048 ～ 0.693 ± 0.020,0.464 ± 0.091 ～ 0.590 ± 0.048,0.400 ± 0.059 ～ 0.464 ± 0.091 respectively,and the iced grass carp fillets were 0.606 ± 0.014 ～ 0.698 ± 0.010、0.478 ± 0.026～ 0.606 ± 0.013、0.367 ± 0.029 ～ 0.478 ± 0.026 respectively.The agreement between the measured and theoretical values of the freshness of grass carp detected by colloidal gold immunochromatographic test strips ranged from 87.27% to 111.72% using ELISA method as a control.