Profiling and Prebiotics Potential of Gracilaria sp. Hydrolysate Obtained from Optimized Citric Acid-Assisted Hydrolysis

Abstract

Gracilaria sp. is widely recognized as a rich source of agar-type polysaccharides. These complex polysaccharides can be hydrolyzed into oligosaccharides and simpler sugar molecules, yielding a biosugar-rich hydrolysis that serves as a potential substrate for fermentation products. Acid-catalyzed hydrolysis (saccharification) using citric acid offers a simpler and more economical than enzymatic hydrolysis. The study aimed to optimize the hydrolysis process of Gracilaria sp. using citric acid and evaluate the prebiotic potential of resulting hydrolysate. A Box–Behnken design under the Response Surface Methodology (RSM) framework was employed using Design Expert 13 software, with three independent variables: substrate amount (1–4 g), acid concentration (0.01 N–1 N), and hydrolysis time (10, 20, and 30 minutes). The response variable measured was the reducing sugar content. The probiotic activity of the hydrolysate was evaluated based on the growth performance of the probiotic candidates L. acidophilus and L. delbrueckii spp. bulgaricus, along with assessments of the prebiotic effect and prebiotic index. The optimized hydrolysis conditions predicted by the quadratic model were obtained at a substrate amount of 3.209 g, a citric acid concentration of 0.117 N, and a hydrolysis time of 30 minutes. Validation experiments under these conditions produced a hydrolysate with a reducing sugar content of 57.14 mg/100 mL. Furthermore, the prebiotic effect scores of the hydrolysate ranged from 3.01 to 1.025, while its prebiotic index values (1.31 and 1.03) were higher than those of inulin. These findings confirm that citric acid hydrolysis of Gracilaria sp. yields a functional biosugar with promising prebiotic potential, supporting further investigation for food and health applications.