Nitrite is a common additive in cured meat formulation that provides microbiological safety, lipid oxidation management and typical organoleptic properties. However, nitrite is also associated with the formation of N-nitrosamines, some of them being associated with colon carcinogenesis. Nitrite addition is thus pointed at by public health agencies and strategies should be established in order to control the formation of N-nitroso-compounds during meat processing and digestion. This work aimed at developing an easy-to-handle test to probe the antinitrosating capacity of plant extracts rich in phenolic compounds. Molecular mechanisms underlying the antinitrosating capacity of phenolic compounds from various classes were further investigated.
Cooked ham was modeled by a 5% sunflower oil-in-water emulsion containing free iron, heme iron, nitrite and N-acetyltryptophan (AcTrp) in levels as justified in Sirvins et al. (2024). The N-nitrosation of AcTrp was evaluated at pH 5 to simulate meat product formulation and the early stage of gastric digestion as well as at pH 2.5 to take into account the final stage of gastric digestion. Commercially available plant extracts were standardized on their contents in reducing compounds (Folin-Ciocalteu method) before performing the evaluation of their antinitrosating capacity. The fate of phenolic compounds and AcTrp as well as the formation of N-nitroso-N-acetyltryptophan (NO-AcTrp) and other reaction products were followed by UHPLC-DAD-MS.
In the absence of phenolic compounds, the rate of AcTrp nitrosation was five times higher at pH 2.5 compared to pH 5 suggesting that the decrease in pH occurring during gastric digestion can favor secondary amine N-nitrosation. Extracts from green tea, a fruit mix, a mix of grape seeds and olive or olive alone, evaluated at the level of 1 mM GAE/g extract, were shown to be among the most antinitrosating extracts at both pH. Their main phenolic contributors were identified and then assessed for their antinitrosating capacity using the same test. They were found to rank, at both pH, as follows: (-)-epigallocatechin gallate = (-)-epicatechin > hydroxytyrosol = rutin = chlorogenic acid. By contrast, ascorbic acid and ascorbic acid-rich extracts from broccoli and acerola were only active at pH 5. In a next step, kinetics for the reaction products between nitrite and these phenolic compounds were acquired highlighting several structure-dependent reaction pathways. These mainly involve nitrite-induced oxidation of the phenolic compound followed by C-nitration, dimerization or coupling with (NO)-AcTrp as well as C-nitrosation in the particular case of flavanols (scheme 1). Most of all, the studied phenolic compounds could scavenge between 2 to 7 nitrite ions per molecule, thus reducing residual nitrite available for the N-nitrosation of AcTrp.
Altogether, these results clearly support a beneficial role for polyphenol-rich plant extracts in the strategy of reduction of nitrate/nitrite in cured meat products but also for their digestion, potentially lessening the burden of carcinogenic N-nitrosamines.