Formation of Lipid and Protein Oxidation Products during In Vitro Gastrointestinal Digestion of Dry-Cured Loins with Different Contents of Nitrate/Nitrite Added

Abstract

The effect of nitrate/nitrite (0, 37.5, 75, and 150 mg/kg) in the dry-cured loin formulation on the formation of lipid and protein oxidation products during in vitro digestion was evaluated. Dry-cured loins formulated with nitrate/nitrite resulted in significantly less lipid and protein oxidation than uncured loins before and after simulated digestion. Compared to loins added with 0 mg/kg nitrate/nitrite, dry-cured loins with 37.5, 75, and 150 mg/kg contained a significantly lower content of conjugated dienes, malondialdehyde, carbonyls, and non-heme iron, and higher amounts of nitrosylmioglobin and thiols. During in vitro digestion, the content of conjugated dienes, malondialdehyde, and carbonyls increased, while thiol content decreased, indicating the development of lipid and protein oxidative processes. At the end of the intestinal phase, the 75 mg/kg digests had a significantly higher content of conjugated dienes, while no differences were found among the other digests. During the in vitro intestinal phase (180 and 240 min), nitrate/nitrite curing resulted in significantly lower malondialdehyde concentrations in the 37.5, 75, and 150 mg/kg loin digests than in the uncured loin digests. No significant differences were observed at the end of the intestinal digestion phase between the cured loin digests. Digests of dried loins without nitrate/nitrite addition showed higher carbonyl contents than the nitrate/nitrite cured counterparts. The loss of thiols was significantly higher in loin digests without added nitrate/nitrite than in loin digests with different amounts of curing salts. The addition of 37.5 mg/kg nitrate/nitrite in the cured loin formulation prevents the formation of lipid peroxidation products and carbonyls from protein oxidation and thiol loss during digestion.

Keywords: carbonyls; conjugated dienes; dry-cured loin; in vitro digestion; malondialdehyde; nitrate; nitrite; thiols.

Figure 1

Flow diagram of the INFOGEST in vitro digestion method and sampling times in the experiment. Acronyms: SSF: Simulated Salivary Fluid, SGF: Simulated Gastric Fluid, and SIF: Simulated Intestinal Fluid. Enzyme activities are in units per mL of final digestion mixture at each corresponding digestion phase.

Figure 2

Nitrite content (nmol/mL of digest) in digest at the end of the oral (2 min), gastric (120 min), and intestinal phase (240 min). n.s.: not significant; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001. a,b: Values for the same time of sampling bearing different letters are significantly different at p ≤ 0.05, Tukey’s test.

Figure 3

Non-protein nitrogen content (mg Gly equiv/mL of digest) in digest at the end of the oral (2 min), gastric (120 min), and intestinal phase (240 min). *: p < 0.05. a,b: Values for the same time of sampling bearing different letters are significantly different at p ≤ 0.05, Tukey’s test.

Figure 4

Changes in lipid oxidation products in dry-cured loins formulated with different amounts of NO₂Na/NO₃K (0, 37.5, 75, and 150 mg/kg) during in vitro gastrointestinal digestion: (a) conjugated dienes; (b) malondialdehyde. The time 2 min represents the end of the oral phase, time 120 min, the end of the gastric phase; meanwhile, times 150 and 180 min represent intermediate times of the intestinal phase that ends at time 240 min. *: p < 0.05; **: p < 0.01; ***: p < 0.001. a,b,c: Means for the same time of sampling with different letters are significantly different (p ≤ 0.05, Tukey’s test).

Figure 5

Changes in protein oxidation products in dry-cured loins formulated with different amounts of NO₂Na/NO₃K (0, 37.5, 75, and 150 mg/kg) during in vitro gastrointestinal digestion: (a) protein carbonyls; (b) thiols. The time 2 min represents the end of the oral phase, time 120 min, the end of the gastric phase; meanwhile, times 150 and 180 min represent intermediate times of the intestinal phase that ends at time 240 min. *: p < 0.05; **: p < 0.01; ***: p < 0.001. a,b: Means for the same time of sampling with different letters are significantly different (p ≤ 0.05, Tukey’s test).