The Study Of Preparation,Debittering And Antioxidant Activity Of Pea Peptides

Pea protein is hypoallergenic and rich in amino acid composition,making it a high-quality source of vegetable protein for enzymatic preparation of antioxidant active peptides.However,pea proteins still suffer from low enzymatic efficiency and unacceptable bitterness of the hydrolysate.Therefore,in this paper,we prepared pea antioxidant active peptides in high yield by heat treatment combined with a stepwise dual enzymatic method and compared the bitterness intensity,composition and structure of the pea peptides under different debittering treatments.The main findings are as follows the main studies and results are as follows.Firstly,the effects of enzyme type and addition method on the hydrolysis of pea proteins were analyzed,and it was found that stepwise enzymatic digestion by Alcalase and Protemax was better than single enzyme and simultaneous enzymatic digestion.To further improve the efficiency of enzymatic digestion,pea proteins were pretreated using heat treatment and high-speed shear.Secondary structure analysis by Fourier infrared spectroscopy（FTIR）revealed that the pretreatment reduced the orderliness of the protein,unfolded the molecular structure and increased the number of contact sites with the enzyme.At the same time,scanning electron microscopy（SEM）observed an increase in pores and breaks on the protein surface,compromised integrity,increased contact area with the enzyme and much higher hydrolysis efficiency.In particular,the effect of heat treatment was more significant.After heat treatment,the protein recovery of the hydrolysates increased by 5.5%,the proportion of insoluble peptides decreased by 9.6%,the amino acid content increased by 18.4%,and peptides with molecular weights below 1000 Da accounted for 82.8%.The final enzymatic conditions were determined as follows:pea protein was heat-treated at 95°C for 10 min and then enzymatically digested for 2.5 h and 1 h with the addition of alkaline protease（p H 8.5,55°C,3%）and complex protease（p H 7.0,50°C,1%）.Secondly,after comparison with the bitterness intensity of commercially available pea peptides,all were found to have varying degrees of bitterness.To this end,the effect of several food-grade mild reagents（sec-butanol,flavour protease andβ-Cyclodextrin（β-CD））on the removal of bitterness from pea peptides at different concentration gradients was compared.A partial least squares regression（PLSR）model was also developed to explore the correlation between the sensory indices of pea peptides and the free amino acid composition and molecular weight distribution.The results showed that the pea peptides were significantly detoxified under successive treatments with sec-butanol（1:4,w/w）andβ-CD（1.5%,w/v）,with the lowest bitterness score（0.3）,electronic tongue bitterness response value（1.15）,the lowest content of bitter amino acids（1007.57 mg/100 g）and the lowest proportion of peptides with molecular weights of 180-500Da（5.43%）.Therefore,this condition was selected as the final debittering process and structurally characterised.results from FTIR and 1H-NMR spectroscopy indicated that sec-butanol treatment not only removed a portion of the bitter peptide,but also promoted sufficient exposure of the hydrophobic domain,favouring the encapsulation ofβ-CD with the hydrophobic amino acids in the pea peptide to mask the bitterness.Finally,the effect of combined debittering on the antioxidant activity of pea peptides was evaluated.The results showed that the IC50 values of DPPH radical scavenging activity,ABTS radical scavenging activity and Fe²⁺binding activity of pea peptides after de bittering increased from 2.63 mg/m L,0.85 mg/m L and 1.69 mg/m L to 3.63 mg/m L,1.67 mg/m L and 2.95 mg/m L,respectively.an H₂O₂ oxidative damage model was developed to determine H₂O₂ concentration of 600μM,the cell survival rate was 50.80%under this condition,and the protective effect of pea peptides on cell oxidative damage was further investigated.In this model,pea peptides before and after debittering could protect Hep G2 cells from H₂O₂ by reducing reactive oxygen species（ROS）and malondialdehyde（MDA）;promoting the expression of superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-Px）.In particular,the pea peptides after debittering were able to achieve almost equivalent activity to that before debittering at higher concentrations.Therefore,the pea peptides before and after debittering can be used in pharmaceuticals or functional foods with different organoleptic requirements.