Abstract

In this study, papain-generated casein hydrolysates (CH) with a degree of hydrolysis of 13.7% were subjected to a papain-mediated plastein reaction in the absence or presence of one of the exogenous amino acids—Gly, Pro, and Hyp—to prepare four plastein modifiers, or mixed with one of three amino acids to prepare three mixtures. The assay results confirmed that the reaction reduced free NH2 for the modifiers and caused amino acid incorporation and peptide condensation. When RAW264.7 macrophages were exposed to the CH, modifiers, and mixtures, these samples promoted macrophage growth and phagocytosis in a dose-dependent manner. In addition, the CH shared similar activity in the cells as the mixtures, while the modifiers (especially the PCH-Hyp prepared with Hyp addition) exerted higher potential than CH, the mixtures, and PCH (the modifier prepared without amino acid addition). The plastein reaction thus enhanced CH bioactivity in the cells. When RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS), the inflammatory cells produced more lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) formation, and caused more four inflammatory mediators (NO, PGE2, TNF-α, and IL-6) and two anti-inflammatory mediators (TGF-β1 and IL-10). However, the PCH-Hyp, PCH, and CH at dose levels of 100 μg/mL could combat against the LPS-induced inflammation. Overall, the PCH-Hyp was more active than the CH and PCH in reducing LDH release, ROS formation, and the secretion of these inflammatory mediators, or in increasing the secretion of the anti-inflammatory mediators. The qPCR and Western blot analysis results further confirmed that these samples had anti-inflammatory effects on the stimulated cells by suppressing the LPS-induced activation of the NF-κB signaling pathway, via regulating the mRNA/miRNA expression of iNOS, IL-6, TNF-α, IL-1β, COX-2, TLR4, IL-10, TGF-β1, miR-181a, miR-30d, miR-155, and miR-148, as well as the protein expression of MyD88, p-IKKα, p-IκBα, p-NF-κB p65, and iNOS, involved in this signaling pathway. In addition, the immunofluorescence assay results revealed that these samples could block the LPS-mediated nuclear translocation of the p65 protein and displayed the same function as the NF-κB inhibitor BAY 11-7082. It was concluded that CH could be endowed with higher anti-inflammatory activity to the macrophages by performing a plastein reaction, particularly that in the presence of exogenous Hyp.

Highlights

  • The plastein reaction was discovered by Danilevski and Okuneff in 1902, when the addition of rennet into the protein hydrolysate led to the production of a precipitate [1]

  • It was observed that the plastein reaction of the chicken plasma protein and sea cucumber hydrolysates led to improved inhibition of the angiotensin-converting enzyme (ACE) [8,9], and that of soy protein hydrolysates resulted in higher anti-platelet activity [10]

  • This study aimed to reveal whether the plastein reaction could impact the anti-inflammatory potential of protein hydrolysates efficiently and whether the three exogenous amino acids might cause different anti-inflammatory efficiencies

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Summary

Introduction

The plastein reaction was discovered by Danilevski and Okuneff in 1902, when the addition of rennet into the protein hydrolysate led to the production of a precipitate [1] This reaction meant a process in which a high substrate concentration (20–50%, w/v) of protein hydrolysate or polypeptide was catalyzed by peptide bonding mode in the presence of an appropriate protease to form an insoluble, high molecular weight, proteinlike precipitate, thixotropic colloids, or thixotropic viscous gels [2,3]. The plastein products enriched with exogenous phenylalanine (Phe) or tyrosine (Tyr) could provide a protective activity in the hepatocytes against the H2O2- or ethanol-induced damage [11,12] This reaction was evident to elevate the anti-oxidative and hypolipidemic effects of protein hydrolysates [13,14]. To further reveal the possible application of this unique chemical reaction on protein hydrolysates or peptides, it is necessary to deeply understand and investigate elements of the plastein reaction, such as the critical anti-inflammatory function of protein hydrolysates or peptides

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