Preparation Of Soluble Eggshell Membrane Hydrolysate And Its Anti-oxidaitve Stress And Anti-inflammatory Effects

The gastrointestinal tract is always exposed to extraneous antigens, microorganisms, anddeleterious molecules, which cause the excess production of reactive oxygen species (ROS)and pro-inflammatory cytokines. ROS causes the imbalance of redox status to induceoxidative stress thereby leading to macromolecular damage whilst superfluouspro-inflammatory cytokines initiate immune response related to the intestinal inflammation.Therefore, promising strategies against oxidative stress and inflammation include inhibitingoxidative stress via antioxidant defences, reducing inflammation by anti-inflammatorydefences, or both. The egg production in China accounts more than40%of the globleproduction. As an abundant resource of proteins, eggshell membrane (ESM) is composed ofnumerous proteins. However, the limited utilisation of ESM is due to its insoluble nature andto the presence of countless cross-linked disulphide bonds. So far, the preparation methods ofESM hydrolysates are limited and the researches for bioactivities of ESM hydrolysates arevery few. Hereby, a new method needs to be investigated to prepare bioactive ESMhydrolysates for human health and the mechanisms of their activities need to be studied.Firstly, soluble ESM hydrolysates AL-PS were prepared using digestion by acombination of Alcalase (AL) and Protease S (PS). AL-PS was fractionated throughultrafiltration membranes to produce its fractions AL-PS-I (molecular weight <5kDa),AL-PS-II (5-10kDa), and AL-PS-III (>10kDa). According to chemical antioxidant assays,AL-PS-I was found to exhibit better reducing power (1mg/mL，OD700nm0.308), DPPHradical scavenging activity (IC500.6mg/mL), and OH·radical scavenging activity (IC500.34mg/mL). AL-PS-III had very good Fe2+chelating activity (IC501.8mg/mL). In cellularantioxidant assay, AL-PS-I showed significant radical scavenging activity in Caco-2cells.H2O2-induced Caco-2cell model was established to study the anti-oxidative stressactivity of AL-PS and AL-PS-I. AL-PS and AL-PS-I can promote the glutathione (GSH)concentration, reduce IL-8secretion, and inhibit the oxidation of lipids and proteins. Cellularantioxidant enzyme activities and GSH level was investigated. The results showed that AL-PSand AL-PS-I can up-regulate γ-glutamylcysteine synthetase (γ-GCS) activity and its mRNAexpression, increase GSH synthesis and the ratio of GSH/GSSG. In normal cells, AL-PS andAL-PS-I improved glutathione reductase (GR) and glutathione S transferase (GST) activities.In H2O2-induced cells, they enhanced glutathione peroxidase (GPx), GR, and GST activities.Tumor necrosis factor (TNF)-or lipopolysaccharide (LPS)-induced Caco-2and HT-29cell models were used to study the anti-inflammatory activity of AL-PS and its fractions.AL-PS and AL-PS-I were found to significantly inhibit the IL-8secretion (P <0.05) inTNF--induced Caco-2and HT-29cells and in LPS-induced Caco-2cells. AL-PS-III significantly suppressed IL-8secretion (P <0.05) in LPS-induced HT-29cells and theinhibition rate was up to60%. The results from RT-PCR showed that AL-PS and AL-PS-I candown-regulate the expressions of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, andTNF-, whilst up-regulate anti-inflammatory cytokine IL-10to reduce inflammatory reactionin TNF--induced cells. AL-PS and AL-PS-III inhibited IL-1β, IL-6, IL-8, IL-10, IL-12,TNF-, and TLR4expressions in LPS-induced HT-29cells. AL-PS-III may regulate the TLR4receptor to prevent or reduce the inflammatory reaction. The results from Western blot (WB)showed that AL-PS, AL-PS-I, and AL-PS-III reduced the activation of NF-κB and inhibitedNF-κB signaling pathway.In DSS-induced colitic mice, the anti-inflammatory activity of AL-PS was studied invivo. From the results, administration of AL-PS (200mg/kg body weight, BW) amelioratedthe weight loss and clinical signs and reduced the DSS-induced secretion of TNF-and IL-6.There was a significant positive correlation between protein level and gene expression ofTNF-or IL-6(r=0.98, P <0.05; r=0.98, P <0.05). Especially the inhibition of IL-6secretion was significant (P <0.05). AL-PS can down-regulate the expressions of variousinflammatory cytokines, including pro-inflammatory cytokine IL-6and anti-inflammatorycytokine IL-10(P <0.05). In addition, AL-PS significantly promoted the pro-apoptotic Baxgene (P <0.05) and the ratio of Bax/Bcl-2was in an increased trend.In conclusion, soluble ESM hydrolysates had good chemical antioxidant activity andcellular anti-oxidative stress activity in intestinal cells through improving cellular antioxidantenzyme activitys and the reduction or synthesis of antioxidant (GSH) level thereby inhibitingthe oxidation of cellular macromolecular (lipids and proteins) and restoring the homeostasisof redox status. In addition, soluble ESM hydrolysates had noticeable anti-inflammatoryactivity in vitro and in vivo to suppress the activation of NF-κB to mediate thedown-regulation of pro-inflammatory cytokines and the up-regulation of anti-inflammatorycytokines. They exhibited anti-inflammatory activity to reduce IL-8secretion in stimulatedcells. ESM hydrolysates may mediate the IL-6trans-signaling pathway to activate theapoptosis of T cells and restore the intestinal immune balance in DSS-induced colitic mice.Therefore, soluble ESM hydrolysates presented anti-oxidative stress activity andanti-inflammatory activity to protect intestinal health and prevent inflammatory boweldiseases (IBD). This study provided a novel method to produce high value-added materialsfrom ESM and offered a scientific enlightenment for developing edible bioactive additivesderived from ESM.