Study on the effects of pre-slaughter transport stress on water holding capacity of pork: Insights from oxidation, structure, function, and degradation properties of protein

Abstract

This work systematically investigated the effects of pre-slaughter transport stress on pork water holding capacity (WHC) during aging from the perspectives of oxidation, structure, function, and degradation properties of protein. Pigs were randomly divided into three-hour transport (Transport-induced stress, T group) and three-hour transport followed by three-hour resting (Control, TR group). Results demonstrated that T treatment markedly declined pork WHC. Compared with TR group, T group presented increased oxidation levels. Meanwhile, T treatment exacerbated the shift of protein secondary structure from α-helix to random coil and protein unfolding levels. The decreased solubility, thermal stability, and degraded levels of proteins were also observed in T group. Additionally, muscle contractions of T group were more severe than TR group. This study supported that pre-slaughter transport stress altered physicochemical properties and structures of postmortem muscle proteins, which reduced pork WHC via impairing the interactions between protein and water molecules and changing the muscle fiber structure.

Keywords: Microstructure; Pre-slaughter transport stress; Protein degradation; Protein oxidation; Protein structure; Water holding capacity.

Fig. 1

Effect of pre-slaughter transport stress on the water holding capacity and water distribution of pork during postmortem aging, (A) drip loss, (B) cooking loss, (C) LF-NMR relaxation time spectra, (D) T₂₁ and T₂₂ relaxation times, and (E) P₂₁ and P₂₂. Different letters (A-C) indicate a significant difference among aging time points at the same treatment (p < 0.05); Different letters (a, b) indicate a significant difference between two treatments at the same aging time (p < 0.05). TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 2

Effects of pre-slaughter transport stress on the antioxidant enzyme activities and ROS levels in postmortem muscles, (A) GSH-Px, (B) SOD, (C) CAT, and (D) ROS levels. “*” indicate a significant difference at p < 0.05, “**” indicate a significant difference at p < 0.01. TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 3

Effects of pre-slaughter transport stress on the oxidation levels of pork during postmortem aging, (A) carbonyl content, (B) total SH content, and (C) free SH content. Different letters (A–C) indicate a significant difference among aging time points at the same treatment (p < 0.05); Different letters (a, b) indicate a significant difference between two treatments at the same aging time (p < 0.05). TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 4

Effects of pre-slaughter transport stress on the structure of pork MPs during postmortem aging, (A) CD spectra, (B) secondary structure ratio, (C) the amount of bound BPB, (D) intrinsic fluorescence spectra, and (E) particle size distribution spectra and average particle size. Different letters (A–C) indicate a significant difference among aging time points at the same treatment (p < 0.05); Different letters (a, b) indicate a significant difference between two treatments at the same aging time (p < 0.05). TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 5

Effects of pre-slaughter transport stress on the functional properties of pork protein during postmortem aging, (A) protein solubility, (B) DSC thermogram, and (C) maximum denaturation temperature (T_max). Different letters (A-C) indicate a significant difference among aging time points at the same treatment (p < 0.05); Different letters (a, b) indicate a significant difference between two treatments at the same aging time (p < 0.05). TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 6

Effects of pre-slaughter transport stress on the μ-calpain, caspase-3, and critical cytoskeleton proteins of pork LT muscle at 3 d of postmortem aging, (A) representative immunoblot bands and (B) intensity of target proteins and caspase-3 activity. “*” indicate a significant difference (p < 0.05), “**” indicate a significant difference (p < 0.01), and “ns” mean no significant difference. TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 7

Effects of pre-slaughter transport stress on the microstructure of pork LT muscle during postmortem aging, (A) representative H&E staining images, (B) average muscle fiber gap, and (C) representative SEM images. Different letters (A-C) indicate a significant difference among aging time points at the same treatment (p < 0.05); Different letters (a, b) indicate a significant difference between two treatments at the same aging time (p < 0.05). TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment.

Fig. 8

PCA of the indicators of pork samples with different pre-slaughter handling methods and aging time as well as a schematic diagram of pre-slaughter transport stress impairing the water holding capacity of pork during postmortem aging, (A) loading plot, (B) scores plot, and (C) schematic diagram. TR: three-hour transport followed by three-hour resting treatment; T: three-hour transport treatment; WHC-d: drip loss; WHC-c: cooking loss; T-SH: total sulfhydryl; F-SH: free sulfhydryl; TPS: total protein solubility; SPS: sarcoplasmic protein solubility; BPB: surface hydrophobicity; P-size: particle size.