Abstract: [Objective] Establishment of a ninhydrin method for determining free α-amino nitrogen content in hydrolyzed whey protein powder,with identification and optimization of key parameters to achieve higher measured values. [Method] This study optimized the detection conditions for free α-amino nitrogen content through response surface methodology,examining the interactions of four single factors（buffer volume,buffer pH value,ninhydrin volume,and boiling time）. A predictive model was developed and analyzed,followed by validation via a constructed Support Vector Machine（SVM）model,to determine the optimal parameters. [Result] The optimal detection parameters were determined as follows：buffer volume was 2.0 mL,buffer pH value was 6.0,ninhydrin volume was 2.0 mL and the boiling time was 20 min. Analysis of replicated samples under these conditions yielded a mean actual content of 47.48% with a coefficient of variation（CV）of 0.35%. The experimental result showed close agreement with the model-predicted value of 47.718%,demonstrating the model's robustness. Furthermore, the optimized method achieved a 0.35% increase in the determined content compared to the pre-optimized procedure. [Conclusion] The parameters selected by applying the Response Surface Methodology（RSM）yielded experimental results that were in good agreement with the model predictions,providing a theoretical reference for the detection of free α-amino substances content in hydrolyzed whey protein powder.

Key words: hydrolyzed whey protein powder, ninhydrin assay, α-amino nitrogen, response surface method, Support Vector Machine（SVM）, optimization condition