Antioxidant activity stability of Lentil protein hydrolysate against heat and pH treatments

Bioactive peptides are protein fragments with 2 to 20 amino acids that have different biological properties depending on the type of amino acids and peptide sequences, including antioxidant, antihypertensive, and antidiabetic. These peptides are inactive in their parent protein sequences but are released during fermentation, enzymatic hydrolysis, or food processing, and exhibit a positive effect on body function and health being. Lentil protein hydrolysate containing antioxidant peptides can be considered as an ingredient of functional foods. One major challenge in using protein hydrolysate in the formulation of functional foods is their stability against the various processes applied to food such as heat and pH treatments. 

In this study, Lentil protein (Lens esculinaris) was hydrolyzed by Alcalase enzyme under controlled conditions (enzyme/substrate ratio of 90 Anson unit (AU)/ kg protein, 55°C, one hour). The intensity of enzymatic hydrolysis was monitored by the OPA method and antioxidant activity was evaluated based on DPPH and ABTS radical scavenging activity. The heat stability of lentil protein hydrolysate was evaluated by heating samples at 37, 50, 75 (for 15 -60 min), and 90°C (for 5 minutes). The pH stability was investigated by exposing the sample at a pH of 2, 5, 7, 9, And 11 for 1 hr and then adjusting on 7. OPA method was also used to evaluate the possible effect of pH and heat treatments on the content of free amino groups. 

The results showed that hydrolysis of Lentil protein by Alcalase under controlled conditions produced antioxidant peptides. Heating at 37, 50, and 75°C for 15 minutes reduced the DPPH radical scavenging activity by 1.25, 4.9, and 10.17% and ABTS radical scavenging activity by 3.8, 6.8, and 9%, respectively. The results of the OPA assay also showed a significant (P<0.05) decrease in the number of free amino groups in protein hydrolysate exposed to heat treatment. With increasing the time of treatment up to 60 minutes, the antioxidant activity decreased more significantly (P<0.05), simultaneously with a decrease in the content of free amino acid groups in the protein hydrolysate sample. So that, after heat treatment at 37, 50, and 75 ° C for 60 minutes, the free amino acid groups reached from 33/66 μM leucin /mg protein to 29.51, 27.59, and 25.68 μM leucin /mg protein  and the most decrease in antioxidant activity was measured for samples exposed to 75°C for 60 minutes. It caused a 27.2%, and 29.2% reduction in DPPH and ABTS radical scavenging activity, respectively. Also, exposure to heat treatment at 90°C for 5 minutes caused a 15% and 13% decrease in DPPH and ABTS radical scavenging activity. The results obtained from consideration the antioxidant  activity of samples exposed to pH treatment (2, 5, 7, 9, and 11 for 1 hour) showed the highest antioxidant activity of peptides at neutral pH and confirmed that acidic and alkaline conditions caused a significant decrease in antioxidant activity (P<0.05). As exposure to pHs 2 and 11 for one hour led to respectively 16.3 and a 29.2% decrease in DPPH radical scavenging activity and 16 and 18.2% decrease in ABTS radical scavenging activity. The results of the OPA assay also confirmed the role of acidic and basic pH on less exposure of free amino acid groups in protein structure.The results showed the potential of using Alcalase enzyme to hydrolyze Lentil protein and produce antioxidant peptides and the Lentil protein hydrolysate with antioxidant activity exhibited relative stability toward different heat and pH treatments. It was concluded that peptides retained 88% and 76% of antioxidant activity at maximum heat (90 ° C for 5 minutes) and pH treatment ( pH=11, for 1 hour). According to the results of the OPA assay, the observed decrease in antioxidant activity  may be due to the changes that happen in protein and peptide structure when are exposed to heat and pH treatments. Altogether, our results showed that Lentil protein hydrolysate can be considered as a potential food ingredient with stable antioxidant activity.