In vitro digestion of nitrite and nitrate preserved fermented sausages - New understandings of nitroso-compounds' chemical reactivity in the digestive tract.

In vitro digestions of dry-cured sausages formulated with four different rates of added sodium nitrite and sodium nitrate (NaNO2 / NaNO3, in ppm: 0/0; 80/80; 120/120; 0/200) were performed with a dynamic gastrointestinal digester (DIDGI®). The chemical reactivity of the potentially toxic nitroso-compounds (NOCs), oxidation reactions products and different iron types were evaluated over time. No nitrite nor nitrate dose effect was observed on NOCs' chemical reactivity. Nitrosothiols were scarce, and nitrosylheme was destabilized for every conditions, possibly leading to free iron release in the digestive tract. Total noN-volatile N-nitrosamines concentrations increased in the gastric compartment while residual nitrites and nitrates remained stable. The minimal rate of 80/80 ppm nitrite/nitrate was enough to protect against lipid oxidation in the digestive tract. The present results provide new insights into the digestive chemistry of dry sausages, and into new reasonable arguments to reduce the load of additives in formulations.

Determination of nitroso-compounds in food products.

Nitrite and nitrate are present in many foods. Nitrate can be converted into nitrite in human body. Nitrite can react with secondary amines to form secondary amines and with thiols to form nitrosothiols. Some nitrosamines are cancers suspect. Because of their importance in terms of human health, research on these compounds is still topical and the use of a rapid and reproducible method for determination and quantification of these compounds is necessary. This article presents a method to study the chemical reactivity of nitrite in meat products through the analysis of non-volatile nitrosamines and nitrosothiols based on:•A specific alkaline and heat extraction of nitro-compounds followed by deprotenization by ultrafiltration•NO detection by the Griess reaction•NO released from S-NO and N-NO bonds by UV light followed by a specific cleavage of S-NO bonds with HgCl2This method, validated on cured meat products, could be developed in the same way on all products containing nitrite and nitrate and leading to the formation of nitroso-compounds. The limit of detection for these compounds are of the order of the micromole per liter.

Chemical reactivity of nitrite and ascorbate in a cured and cooked meat model implication in nitrosation, nitrosylation and oxidation.

Nitrite, added to cured meat for its bacteriological and technological properties, is implicated in the formation of nitroso compounds (NOCs), such as nitrosylheme, nitrosamines and nitrosothiols, suspected to have a potential impact on human health. The mechanisms involved in NOC formation are studied in regard with the dose-response relationship of added nitrite and its interaction with ascorbate on NOC formation in a cured and cooked meat model. The impact of a second cooking stage on nitrosation was evaluated. The addition of nitrite in the cured and cooked model promoted heme iron nitrosylation and S-nitrosation but not N-nitrosation. Nitrite reduced lipid oxidation without an additional ascorbate effect. The second cooking sharply increased the nitrosamine content while the presence of ascorbate considerably lowered their levels and protected nitrosothiols from degradation. This study gives new insights on the chemical reactivity of NOCs in a cured meat model.

New Insights into the Chemical Reactivity of Dry-Cured Fermented Sausages: Focus on Nitrosation, Nitrosylation and Oxidation.

Nitrite and nitrate are added to cured meat for their bacteriological, technological and sensorial properties. However, they are suspected to be involved in the formation of nitroso compounds (NOCs), such as potentially mutagenic nitrosamines, nitrosylheme and nitrosothiols. Controlling the sanitary and sensorial qualities of cured meat products by reducing these additives requires elucidating the mechanisms involved in the formation of NOCs. To this end, we studied the dose-response relationship of added sodium nitrite and/or sodium nitrate (0/0, 80/80, 0/200, and 120/120 ppm) on the formation of NOCs in dry cured fermented sausages. The results showed a basal heme iron nitrosylation in the absence of NaNO2/NaNO3 due to starter cultures. This reaction was promoted by the addition of NaNO2/NaNO3 in the other conditions. Reducing the dose to 80/80 ppm still limits lipid oxidation without the formation of non-volatile nitrosamines. Conversely, the addition of NO2/NO3 slightly increases protein oxidation through higher carbonyl content. The use of 80/80 ppm could be a means of reducing these additives in dry-cured fermented meat products.

Design of an In Vitro Model to Screen the Chemical Reactivity Induced by Polyphenols and Vitamins during Digestion: An Application to Processed Meat.

Processed meats' nutritional quality may be enhanced by bioactive vegetable molecules, by preventing the synthesis of nitrosamines from N-nitrosation, and harmful aldehydes from lipid oxidation, through their reformulation. Both reactions occur during digestion. The precise effect of these molecules during processed meats' digestion must be deepened to wisely select the most efficient vegetable compounds. The aim of this study was to design an in vitro experimental method, allowing to foresee polyphenols and vitamins' effects on the chemical reactivity linked to processed meats' digestion. The method measured the modulation of end products formation (specific nitroso-tryptophan and thiobarbituric acid reactive substances (TBARS)), by differential UV-visible spectrophotometry, according to the presence or not of phenolic compounds (chlorogenic acid, rutin, naringin, naringenin) or vitamins (ascorbic acid and trolox). The reactional medium was supported by an oil in water emulsion mimicking the physico-chemical environment of the gastric compartment. The model was optimized to uphold the reactions in a stable and simplified model featuring processed meat composition. Rutin, chlorogenic acid, naringin, and naringenin significantly inhibited lipid oxidation. N-nitrosation was inhibited by the presence of lipids and ascorbate. This methodology paves the way for an accurate selection of molecules within the framework of processed meat products reformulation.

Investigation by Synchrotron Radiation Circular Dichroism of the Secondary Structure Evolution of Pepsin under Oxidative Environment.

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.