Establishment of High-Throughput Detection Method for Phytic Acid Content in Sesame Seeds and Screening of Low Phytic Acid Germplasms
[Objective]Phytic acid is one of the main anti-nutritional components in sesame.To explore the optimal conditions for efficient extraction of phytic acid from sesame seeds,establish a high-throughput detection method for phytic acid content,and apply it to the detection of phytic acid content variation in sesame population materials and the screening of low phytic acid germplasm resources,so as to promote the basic research of phytic acid in sesame and the breeding of sesame varieties with low phytic acid content.[Method]Using 0.4 mol·L-1 HCl as the extraction solvent,single factor tests such as seed weight,crusher time,crusher frequency and extraction solution volume were carried out to extract phytic acid from sesame seeds,and the content of phytic acid was determined in a high-throughput manner using modified iron precipitation method.On the basis of the single factor test,the response surface experiment of Box-Behnken four factors and three levels was carried out.The quadratic polynomial regression equation model with phytic acid yield as the response value was established,and the response surface plot and contour plot were drawn.The main factors affecting phytic acid yield and the interaction between the factors were analyzed to determine the optimal extraction conditions for the detection of phytic acid content.Using this parameter condition,the phytic acid content of 200 sesame germplasm resources planted in two environments was determined to screen low phytic acid germplasm.[Result]Analysis of variance(ANOVA)showed that the established regression model was highly significant(P＜0.0001),the lack of fit was not significant(P＞0.05),the equation fitted the test well,and this regression equation could be used to optimize the extraction of phytic acid from sesame seeds.The linear term of the four factors had a very significant effect on phytic acid yield,and the order of influence on the yield of phytic acid was seed weight＞crusher time＞crusher frequency＞extract solution volume.The response surface analysis diagram showed that there were some interactions between seed weight and crusher time,seed weight and crusher frequency,crusher time and crusher frequency,crusher time and extraction solution volume,and crusher frequency and extraction solution volume.The optimal phytic acid extraction conditions optimized by single factor test and response surface design were as follows:seed weight of 30 mg,crusher time of 6.5 min,crusher frequency of 50 Hz,extraction solution volume of 435 μL.The predicted phytic acid yield under this condition was 14.03 mg·g-1,and the actual average phytic acid yield was 14.14 mg·g-1,the model prediction was very close to the actual value of the test.Three stable low phytic acid sesame germplasms were screened out from 200 germplasm resources,with an average content of 11.63 mg·g-1.[Conclusion]An efficient phytic acid extraction and detection technology for sesame seeds was established,which could significantly reduce the experimental time and the amount of samples used,and provided a feasible method for the high-throughput detection of phytic acid content in sesame seeds with good reproducibility and high accuracy.
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