Optimization of the Microbial–Enzymatic Synergistic Treatment of Cottonseed Protein and Evaluation of the Nutritional Value and Antioxidant Activity of Cottonseed Peptides

This study was conducted to optimize the conditions for the synergistic treatment of cottonseed protein with microorganisms and enzymes and to evaluate the nutritional value and antioxidant activity of the resulting cottonseed peptides, with the ultimate goal of improving the nutritional quality of cottonseed protein. In single-factor experiments, laccase, alkaline protease, Saccharomyces cerevisiae, and Lactobacillus acidophilus were individually applied to cottonseed protein, and the optimal ranges for additive dosage, temperature, moisture content, and treatment duration were established using free gossypol, acid-soluble protein, and pH as response indicators. A Box–Behnken response surface design was subsequently adopted to perform an integrated analysis of the three responses and to determine the optimal conditions for the combined microbial–enzymatic treatment. The nutritional value and antioxidant activity of the cottonseed peptides obtained under these conditions were then evaluated. The optimal process parameters were identified as follows: microbial and enzyme dosages each at 1% (w/w), temperature of 37 °C, 37% moisture content, and treatment time of 96 h. Under the optimized conditions, the free gossypol content of the treated cottonseed protein was reduced to 67.30 mg/kg, representing a decrease of 83.69%; the acid-soluble protein content reached 29.72%, an increase of 25.86 percentage points; the reducing sugar content was 19.49 mg/g, an increase of 13.89 mg/g; and the pH dropped by 1.59 units to 4.91. Analysis of the peptide molecular weight distribution revealed that 99.61% of the cottonseed peptides had a molecular weight below 10,000 Da, and 65.45% were below 1000 Da. The peptides also exhibited excellent antioxidant capacity. In conclusion, the microbial–enzymatic synergistic treatment significantly elevated the contents of acid-soluble protein, reducing sugars, and peptides, enhanced antioxidant capacity, and reduced both free gossypol content and pH, thereby effectively improving the nutritional quality of cottonseed protein.