Effects Of High Hydrostatic Pressure Treatment On The Proteolysis Of Whey Protein Isolate And The Antioxidant Activity Of The Hydrolysates

High hydrostatic pressure treatment (HHP) is a novel non-thermal processing technology, and its application in food processing has gained an increasing attention. It has already been confirmed that the increasing of free radicals concentration is closely related to many human diseases, aging, food spoilage and shelf life shortening etc. However, recent studies have found that the hydrolysates of whey protein which is the main byproduct of cheese processing had some extent of antioxidant activity. Therefore, the development of whey protein for the production of antioxidant peptides is of great significance. In this paper, the hydrolysis of commercial bovine whey protein isolate (WPI) by four proteases during or after HHP treatment and the antioxidant activity of the hydrolysates were studied, and then the antioxidant components of the WPI hydrolysates with the maximum antioxidant activity were also analyzed by a series of separation and purification technology. The purpose of this study is providing some kind of theoretical guidance for the using of HHP to develop whey protein antioxidant peptides.Firstly, the effects of different factors on the hydrolysis of WPI by Alcalase under HHP treatment were studied. The optimum process parameters for WPI hydrolysis under HHP treatment were obtained using the orthogonal experiment, which were substrate concentration 5.0% (w/v), enzyme to substrate ratio of 5.0% (w/w), the hydrolysis time of 30min and the pressurization of 300MPa. The priority of these factors was: hydrolysis time > pressure > enzyme to substrate ratio > substrate concentration. Under the optimum hydrolysis conditions, the degree of hydrolysis of WPI was up to 30.33%.Secondly, in order to study the antioxidant activity of WPI hydrolysates by different proteases during or after HHP treatment, the hydrolysis of WPI by Pepsin, Chymotrypsin, AS1.398 or Alcalase during or after the pressurization of 100~ 600MPa was studied separately, and then the antioxidant activity of the hydrolysates was analyzed using chemiluminescence and pyrogallol autoxidation methods. The results showed that HHP treatment had significantly increased the hydrolysis of WPI by the four proteases and the antioxidant activity of the hydrolysates, compared with that obtained under atmospheric pressure. The WPI hydrolysates obtained by Alcalase under the pressurization of 400MPa had the strongest antioxidant activity among all the hydrolysates, and the water-soluble antioxidant capacity and the scavenging activity of superoxide anion radical were 1.90μg ascorbic acid/ mg and 53.68%, respectively. In addition, the study also found that the surface hydrophobicity of WPI hydrolysates and the degree of hydrolysis of WPI showed a good negative correlation, the surface hydrophobicity of WPI hydrolysates significantly decreased with the increasing of the degree of hydrolysis of WPI.Thirdly, the Alcalase hydrolysates with the highest antioxidant activity were isolated by 10kDa and 3kDa ultrafiltration centrifuge tubes. It was showed that the molecular weight <3kDa of WPI peptides (FractionⅢ) exhibited the strongest antioxidant activity, which had the superoxide anion radical scavenging activity of 75.92%. The results of amino acid composition analysis showed that the content of Tyr, Lys and total hydrophobic amino acids of FractionⅢwas significantly higher than other fractions. Further analysis on FractionⅢusing Sephadex G-25 chromatography found that the eluted fraction ranging from 589~1349Da (E2) made the greatest contribution to the antioxidant activity. MALDI-TOF analysis on E2 carried out in the range of m/z 500~3000 found 5 peptides lower than 1000Da (617.14Da, 673.11Da, 744.02Da, 902.75Da and 929.42Da) and one peptide of 1242.08Da in relatively lower abundance. The results of nano-LC/MS/MS analysis confirmed that two of these peptides which identified as Glu-Gln-Leu-Thr-Lys and Glu-Leu-Lys-Asp-Leu-Lys were originated fromα-La, and the others were originated fromβ-Lg, which were identified as Gly-Leu-Asp-Ile-Gln-Lys, Thr-Lys-Ile-Pro-Ala-Val-Phe-Lys, Lys-Ile-Ile-Ala-Glu-Lys-Thr-Lys and Thr-Pro-Glu-Val-Asp-Asp-Glu- Ala-Leu-Glu-Lys.