PS6-6. Fraction-specific Changes in Protein-bound Carbonyl Concentrations Following Retort Canning of a Commercial Raw Canine Diet.

Abstract

Formation of protein-bound carbonyls (PBC), irreversible markers of protein oxidation (POx), can lead to the loss of protein function and the reduction of protein digestibility. The impact of the retort canning process on POx in canine diets has not been investigated. We hypothesized that PBC concentrations in a raw canine diet would increase after the retort canning process. Thus, the objective of this study was to evaluate the effect of retort canning on POx in a raw canine diet by measuring PBC content in the soluble and insoluble fractions of the retorted and raw diets. A commercial complete and balanced raw diet was retorted in ten separate batches, targeting a minimum lethality value of 10. A sample of the raw diet was reserved prior to each canning. During the retort process, the food matrix separated into insoluble (RetortSolid, n = 10) and soluble (RetortLiquid, n = 10) fractions, which were collected separately for analysis. Insoluble (RawSolid, n = 10) and soluble (RawLiquid, n = 10) fractions of the reserved raw control were collected after centrifugation at 5000xg (4 °C) for 5 minutes. Solid samples were powdered in liquid nitrogen and homogenized in 0.15M KCl. In three replications, eight aliquots of each solid homogenate or vortexed liquid sample were mixed with 10% (w/v) trichloroacetic acid. Protein precipitates were solubilized in 10mM Sodium Phosphate Buffer, pH 7.0, and 5% SDS (wt./vol), then placed in an ultrasonic bath for 30 minutes (55 °C). Four of the aliquots were derivatized with 10mM 2,4-dinitrophenylhydrazine (DNPH) in 3M HCl, and the remaining four blanks were treated with the same volume of 3M HCl, with normal washing procedures. Samples were re-solubilized in 6M guanidine hydrochloride in 20mM NaH2PO4 (pH 6.5) and sonicated, incubated overnight (4 °C), and sonicated a second time. Carbonyl compounds were detected spectrophotometrically at an absorbance of 370nm. Protein concentration was detected using the Bradford assay. Carbonyl concentrations (nmol/ml) were corrected to the average protein content (mg/ml) of their respective blanks to achieve a corrected carbonyl content (nmol/mg protein). Data were analyzed in R v4.5.1 for ANOVA with diet type (raw vs. retort) as the fixed effect and batch and sample date as random effects. The mean corrected carbonyl content was greater in the soluble (liquid) fractions, regardless of diet type. There was a difference in the mean corrected carbonyl concentration (P < 0.001) between RetortSolid (36.8 nmol/mg) and RawSolid (22.6 nmol/mg) and between RetortLiquid (93.3 nmol/mg) and RawLiquid (59.9 nmol/mg). Overall, these data indicate that retort processing increased detectable protein-bound carbonyl concentrations in this canine diet, with the most pronounced response observed in the soluble (liquid) fraction. These findings suggest that fraction-specific analysis may help clarify how processing influences protein oxidation in wet diets.