In Vitro Activities Of Hydrolysates From Abalone Viscera Using Subcritical Water-assissted Enzymatic Hydrolysis

Abalone viscera was extracted by subcritical water-assisted enzymatic hydrolysis.The physicochemical properties and in vitro antioxidant activities were determined.Then,the in vitro immune activity were determined by using RAW264.7 cells.Finally,the transwell model was established by using Caco-2 cells and the in vitro antioxidant activities after transportation were determined.First of all,Antioxidant hydrolysates were prepared from abalone viscera using subcritical water?AVS?-assisted by enzymatic hydrolysis with papain?AVSE-P?,bromelain?AVSE-B?,neutral protease?AVSE-N?,and flavourzyme?AVSE-F?.The protein and carbohydrate contents reached 38.33%and 24.36%,respectively.When AVS was digested by any of the proteases,the protein content increased,but carbohydrate content decreased.The main amino acids of AVSEs included alanine,glycine,and aspartic acid.The IC₅₀0 values of ferric reducing antioxidant power,and 2,2-diphenyl-1-pcrylhydrazyl?DPPH?^·,2,2'-Azino-bis?3-ethylbenzothiazoline-6-sulfonicacid??ABTS?^·+,N,N-Dimethyl-p-phenylenediaminedihydrochloride?DMPD?^·+,and hydroxyl radical?^·OH?scavenging abilities of AVS were 2.93,1.48,1.61,3.72,and 5.51 mg/mL,respectively,which decreased after enzymatic hydrolysis by any of the proteases.The DMPD^·+and^·OH scavenging abilities of AVSE-P and AVSE-B were higher than those of others,whereas the opposite was observed in lipid peroxidation inhibition efficiency,DPPH^·,and ABTS^·+scavenging abilities.Hence,antioxidant activities of AVS could be enhanced by enzymatic hydrolysis,but the influence depends on the type of protease.Secondly,the immune activity of AVS and AVSEs were determined.The abalone viscera subcritical water extract promoted the proliferation and phagocytosis of RAW264.7 cells,but had no significant effect on the phagocytosis of LPS-induced RAW264.7 cells.After enzymatic hydrolysis,except AVSE-B,all AVSEs enhanced the proliferation activity and phagocytosis activity of RAW264.7 cells as well as the phagocytosis activity of LPS-induced RAW264.7 cells.AVSE-N can promote the proliferation of mouse macrophages about 37%.In addition,the AVS inhibited the secretory activity of TNF-?in RAW264.7 cells and LPS-induced RAW264.7 cells,but promoted the secretory activity of IL-10.After enzymatic hydrolysis,AVSE-P,AVSE-B and AVSE-F all had a promoting effect on TNF-?secretory activity and IL-10 secretory activity,and the promoting effect was enhanced.While,AVSE-N still inhibited TNF-?secretory activity and the inhibition was enhanced.In addition,both AVS and AVSEs can inhibit the secretion of NO by inhibiting the activity of iNOS.Therefore,the hydrolysate prepared by enzymatic hydrolysis of abalone viscera with subcritical water has good in vitro immune activity and can play a certain anti-inflammatory effect.Finally,in vitro transportation and absorption experiments were carried out using Caco-2 single cell membrane model.Caco-2 cells were seeded in Transwell Plates and incubated for 21 days,cell connected tightly and the monolayer film was formed.And TEER value was reached 501.25?·cm²,alkaline phosphatase levels of AP side reached 2.67 King Unit/100 mL,the ratio of alkaline phosphatase content of AP side and BL side reached 5:2.The cell membrane transwell model was successfully established.The in vitro antioxidant activities of AVS and AVSEs were determined after transportation and absorption experiments.The total antioxidant capacity of AVSE-N and AVSE-F was higher than that of avse-p and avse-b.The content of reduced glutathione and superoxide dismutase was the highest in AVSE-N,but the content of malondialdehyde was the lowest.Therefore,AVSE-N still showed the best antioxidant activity after transportation and absorption.The results show that the proposed approach can be used for treating abalone viscera,and the obtained antioxidant hydrolysates could be used in nutraceutical and pharmaceutical industries.