Evaluating the Ability of the Endothelial Nitric Oxide Synthase Enzyme to Generate Nitric Oxide to Enhance Nitrsohemochromagen Values: Effects of Substrate, Conditions, and Cofactors

Abstract

Meat curing is a traditional preservation method that has been used for decades. While the exact origins of meat curing remain unclear, salt impurities with potassium nitrate play a crucial role in the desired curing attributes. This discovery laid the foundation for modern conventional curing, where sodium nitrite is the main cure ingredient. Sodium nitrite can generate nitric oxide (NO), an intermediate molecule involved in various functions in meat. Sodium nitrite is responsible for the unique pink color, texture, and flavor in addition to enhanced safety of the meat products. However, there has been controversy around using sodium nitrite for curing because of the formation of nitrosamines which could be a potential health risk. Since endothelial nitric oxide synthase enzyme (eNOS) generates NO using the substrate L-arginine and is present in meat, it is believed that NOS can be used as an alternative to the conventional cure to cure meat products. Therefore, the research objectives were (1) to evaluate the impact of amino acid type (L-arginine and L-citrulline), amino acid concentration, temperature, and time on the enzyme activity of the endothelial nitric oxide synthase (eNOS) system and its ability to generate NO as an alternative curing method in post-rigor chicken meat (2) evaluate the impact of the addition of calcium chloride, zinc gluconate, and sodium lactate on eNOS activity to generate nitric oxide and consequently, NO-heme, residual nitrate, and residual nitrite in post-rigor ground chicken and beef meat, and (3) to use response surface methodology to determine the concentrations of calcium chloride, zinc chloride, and sodium lactate that optimize the NO generation and consequently the NO-heme and residual nitrite levels in post-rigor ground chicken and beef meat. NO-heme and other instrument color analyses were conducted according to the meat and muscle biology guide (King et al., 2023). Residual nitrate and nitrite were measured content (ppm) using an Eno-30 analyzer (Amuza Inc, San Diego, CA, USA) equipped with a reverse phase column. The first study's results indicated that the eNOS can generate nitric oxides to form NO- heme and residual nitrite to cure ground chicken meat products. Although the amino acid treatments were inferior to sodium nitrite treatments, it is evident that the enzyme system could be used to cure poultry meat products. This laid the foundation for the second objective aiming to enhance NOS's ability to generate nitric oxide (NO) by using the enzyme’s cofactors in both ground chicken and beef meat. For ground chicken samples, NO-heme values were not significantly different for calcium chloride or zinc gluconate treatments. However, samples treated with low levels of zinc gluconate at 50 and 100 ppm exhibited a slight increase in NO-heme values compared to the negative control. Additionally, the 1.2% sodium lactate treated samples showed a significantly elevated NO-heme value of 11.98 ppm. In ground beef samples, NO-heme values did not differ significantly across treatments. However, higher calcium chloride and sodium lactate concentrations were associated with increased NO-heme levels relative to the negative control. Lower concentrations of zinc gluconate were similarly associated with increased NO-heme levels. Residual nitrate and nitrite levels showed no significant differences between ground chicken and beef, ranging from 4.98 to 6.32 for nitrate and 1.86 to 2.78 for nitrite in chicken and beef, respectively. Narrowing down the ranges of the cofactors in this phase helped construct a central composite design (CCD) to optimize NO-heme and residual nitrite in both meat types. The CCD was utilized to achieve uniform precision throughout the experiment region. While the models were neither significant (P > 0.05) nor sufficient to fully explain the factors’ effects on eNOS, the three factors studied demonstrated some level of influence of the enzyme.