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.

Investigation of Escherichia coli O157:H7 Survival and Interaction with Meal Components during Gastrointestinal Digestion.

Escherichia coli O157:H7 is responsible for foodborne poisoning, incriminating contaminated animal food and especially beef meat. This species can survive in the digestive tract, but, up to now, very few studies have considered its survival during the gastrointestinal digestion of meat. The present study aimed to investigate the survival of the pathogenic strain E. coli O157:H7 CM454 during the gastrointestinal digestion of ground beef meat and its interactions with meal components using a semidynamic digestive model. The CM454 strain in meat survived throughout digestion despite acidic pH (pH 2) and the presence of bile salts. The addition of nitrite and ascorbate in the digestion medium led to a decrease in strain survival. During digestion, a release of free iron was observed, which was accentuated in the presence of the CM454 strain. In addition, the strain modified the Fe2+/Fe3+ ratio, in favor of Fe2+ compared to the noninoculated meat sample. In the presence of nitrite, nitroso compounds such as nitrosamines, nitrosothiols, and nitrosylheme were formed. E. coli O157:H7 CM454 had no impact on N-nitrosation but seemed to decrease S-nitrosation and nitrosylation.

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.

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.

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.

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.

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.

Physicochemical and microbiological characteristics of El-Guedid from meat of different animal species.

El-Guedid is an Algerian traditional meat-based product that is prepared from red meats. It belongs to the wide diversity of salted/dried meat products. This study described the physicochemical and microbiological properties of different products from four animal origins and during all the conservation. Results indicated that these products were mainly characterized by a low moisture with an average decrease of water content between 15.6% and 16.3% for all the samples, and a decrease in water activity ranging from 0.66 to 0.68, while the salt content ranged from 8.8 to 19.3%. A decrease in pH values oscillated from (6.3-6.4) to reach (5.2-5.5) at T0 and T365 consecutively, in all the samples. Microbial analyses revealed the absence of pathogenic bacteria such as Listeria and Salmonella but the sporadic contamination by Staphylococcus aureus up to one month of ripening. Lactic acid bacteria and coagulase negative staphylococci were the dominant populations in El-Guedid with Leuconostoc mesenteroides, Lactobacillus sakei, and Staphylococcus saprophyticus as the main species identified. All these populations decreased along the process and reached low levels (2 log CFU/g) at the end of storage (365 days). The drastic drying of El-Guedid led to safe traditional meat product that could promote its production.

Workflow towards the generation of bioactive hydrolysates from porcine products by combining in silico and in vitro approaches.

Food-derived bioactive peptides have generated an increasing interest in the field of health and well-being research. They can act either against the metabolic syndrome, participate in regulating the oxidation balance or act on the immune system. The aim of this study is to develop a workflow to generate bioactive peptides from three porcine offals namely, heart, liver, and lung and one muscle the Longissimus Dorsi, by combining in silico and in vitro approaches. Bioinformatics tools (e.i. BIOPEP and Uniprot) permitted to orientate the choice of enzymes for generating abundant bioactive peptides from the four studied porcine products. With papain and subtilisin, the main bioactivities potentially released were ACE inhibitors, DPP4 inhibitors and antioxidant peptides. An in vitro validation study using papain and subtilisin demonstrated high DPP4 inhibitors and antioxidant bioactivities for the generation of peptides. This work allowed: i) the identification of all proteins that composed porcine heart, liver, lung and LD muscle that could be useful for the scientific community, ii) the development of a workflow to select most abundant proteins in a product while considering abundance factors and iii) the potential of porcine meat and offals to generate DPP4 inhibitors and antioxidant peptides. However, there is still a need in developing new tools in order to face limitations of mass spectrometry for the identification of peptides with less than six amino acids. Such a work may contribute to the development of the circular economy and the innovative creation of value-added products from animal production.

A comprehensive review of bioactive peptides obtained from animal byproducts and their applications.

Livestock generates high quantities of residues, which has become a major socioeconomic issue for the meat industry. This review focuses on the identification of bioactive peptides (BPs) in animal byproducts and meat wastes. Firstly, the main bioactivities that peptides can have will be described and the methods for their evaluation will be discussed. Secondly, the various origins of these BPs will be studied. Then, the techniques and tools for the generation of BPs will be detailed in order to discuss, in the final part, how peptides could be used and assimilated. BPs possess diverse biological activities and can be strategic candidates for substituting synthetic molecules. In silico potentiality studies are a helpful tool to understand and predict BPs released from proteins and their potential activities. However, in vitro validation is often required. Although BP use is compelled by strict regulations in relation to the field of application, they are also limited by their low bioavailability and bioaccessibility. Therefore, it is important to test peptide stability during gastrointestinal digestion. Protective strategies have been discussed since their use could improve the stability and effectiveness of BPs.

Using a dynamic artificial digestive system to investigate heme iron nitrosylation during gastro-intestinal transit.

The International Agency for Research on Cancer recently classified cured meats as carcinogenic for humans and red meats as probably carcinogenic. Mutagens can be formed during meat process or digestion. In a previous study, we used a dynamic artificial digestive system (DIDGI®) to investigate protein oxidation and N-nitrosation during bovine meat digestion. This new paper completes the previous one by focusing on the endogenous heme iron nitrosylation. Low nitrosylation due to nitrate initially present in meat and to ammonia oxidation in the stomach was observed in the digestive tract even in conditions in which no nitrite was added to the model. The endogenous addition of nitrite (1 mM) considerably increased heme iron nitrosylation while a significant decrease was observed with prior meat cooking (30 min at 60 and 90 °C).

To what extent does the nitrosation of meat proteins influence their digestibility?

Nitrosation can occur during meat digestion due to the physicochemical conditions of the stomach (low pH and reducing conditions). The aim of the present study was to elucidate the link between the nitrosation of proteins from beef meat and their digestibility by comparing cooked meat digested with and without the addition of nitrite. To do this, a dynamic in vitro artificial digestive computer-controlled system (DIDGI®) was used to reflect human gastro-intestinal conditions. Peptides and proteins from gastrointestinal digestion were identified by high-resolution LC-MS/MS mass spectrometry. The results showed a dynamic digestion pattern of meat proteins according to their cellular localization. A combined effect of the digestive compartment and the addition of nitrite was established for the first time on peptides profile, linking the nitrosation of proteins and their digestibility.

Iron-catalysed chemistry in the gastrointestinal tract: Mechanisms, kinetics and consequences. A review.

Chemical changes that occur during the storage and processing of food can affect its nutritional content. During digestion, the exposure of food to considerable variations of pH and high oxygen and peroxide concentrations also participates in the deterioration of nutrients, with a negative impact on the nutritional value of the diet and harmful consequences for human health. Iron plays a key role in gastrointestinal chemistry. Haem iron, which exists only in meat, and non-haem iron, present in most foods, are catalysts of most of the reactions implicated in the deterioration of nutrients. Disintegration of food matrix due to mechanical forces and enzymatic hydrolysis favour this endogenous process. This paper provides a review of what is known in the literature concerning the mechanisms and kinetics of endogenous reactions catalysed by iron. The main consequences on nutrient bioavailability are reported and protective strategies against the deleterious effect of iron are discussed.

Stoichio-Kinetic Modeling of Fenton Chemistry in a Meat-Mimetic Aqueous-Phase Medium.

Fenton reaction kinetics, which involved an Fe(II)/Fe(III) oxidative redox cycle, were studied in a liquid medium that mimics meat composition. Muscle antioxidants (enzymes, peptides, and vitamins) were added one by one in the medium to determine their respective effects on the formation of superoxide and hydroxyl radicals. A stoichio-kinetic mathematical model was used to predict the formation of these radicals under different iron and H2O2 concentrations and temperature conditions. The difference between experimental and predicted results was mainly due to iron reactivity, which had to be taken into account in the model, and to uncertainties on some of the rate constant values introduced in the model. This stoichio-kinetic model will be useful to predict oxidation during meat processes, providing it can be completed to take into account the presence of myoglobin in the muscle.

Mechanisms and kinetics of heme iron nitrosylation in an in vitro gastro-intestinal model.

Fresh red meat and cured meat consumption increases the risk of gastro-intestinal cancers and it is strongly suspected that nitrosylheme is implicated by stimulating the endogenous production of mutagenic aldehydes and N-nitroso compounds. To investigate the extent of endogenous heme iron nitrosylation an experimental in vitro model that mimics the physicochemical conditions of the gastro-intestinal tract was used in association with a mathematical model of chemical reaction kinetics. The combined effect of pH (from 7.2 to 3.2) and myoglobin oxidation state was evaluated in the reaction of nitrite with heme iron, and the observed rate constants of the reactions were determined. Nitrosylation was optimal under mildly acidic conditions (pH 6.5-4.7). Up to 20% of myoglobin can be nitrosylated under gastro-intestinal conditions in this pH range. The effect of various antioxidants (from meat or vegetables) on the endogenous nitrosylation process was also tested.

Oxidation and nitrosation of meat proteins under gastro-intestinal conditions: Consequences in terms of nutritional and health values of meat.

The chemical changes (oxidation/nitrosation) of meat proteins during digestion lead to a decrease in their nutritional value. Moreover, oxidized and nitrosated amino acids are suspected to promote various human pathologies. To investigate the mechanisms and the kinetics of these endogenous protein modifications, we used a dynamic artificial digestive system (DIDGI®) that mimics the physicochemical conditions of digestion. The combined effect of meat cooking and endogenous addition of ascorbate and nitrite was evaluated on protein oxidation (by measuring carbonyl groups), protein nitrosation (by measuring nitrosamines), and proteolysis. Considerable carbonylation was observed in the digestive tract, especially under the acidic conditions of the stomach. Nitrosamines, caused by ammonia oxidation, were formed in conditions in which no nitrite was added, although the addition of nitrite in the model significantly increased their levels. Meat cooking and nitrite addition significantly decreased protein digestion. The interactions between all the changes affecting the proteins are discussed.

Mechanisms and kinetics of tryptophan N-nitrosation in a gastro-intestinal model.

The reaction of nitrite with different amino acids containing secondary amino groups was tested under simulated in-vitro conditions of the digestive tract. After treatment, tryptophan was the only amino acid that exhibited specific UV absorbance of nitrosamines at 335nm, supporting the assumption that it is the main source of endogenous nitrosamines. The combined effect of pH (from 2 to 6.5) and nitrite (from 0.1 to 20mM) was analyzed and the mechanisms and kinetic laws of tryptophan N-nitrosation were determined. The model was then completed by the addition of iron and various antioxidants in concentrations reflecting different diets. The results clearly demonstrated that, in the presence of iron, large amounts of N-nitroso-tryptophan can be formed even at neutral pH, as in the intestine. Antioxidants (ascorbic acid, trolox C, ß carotene, chlorogenic acid, phytic acid and butylated-hydroxytoluene) had various impacts on the extent of N-nitrosation, depending on the iron level.

Impact of the Fenton process in meat digestion as assessed using an in vitro gastro-intestinal model.

The production of oxygen free radicals catalysed by non-haem iron was investigated in an in vitro mimetic model of the digestive tract using specific chemical traps. Superoxide radicals (O2(∗-)) and their protonated form (hydroperoxyl radicals, HO2(∗)) were detected by the reduction of nitroblue tetrazolium into formazan, and hydroxyl radicals (OH(∗)) were detected by the hydroxylation of terephthalate. Under gastric conditions, O2(∗-)/HO2(∗) were detected in higher quantity than OH(∗). Increasing the pH from 3.5 to 6.5 poorly affected the kinetics of free radical production. The oxidations generated by these free radicals were estimated on myofibrils prepared from pork rectus femoris muscle. Myofibrillar lipid and protein oxidation increased with time and oxidant concentration, with a negative impact on the digestibility of myofibrillar proteins. Plant food antioxidants considerably decreased free radical production and lipid oxidation but not protein oxidation.

Effects of pH and NaCl levels in a beef marinade on physicochemical states of lipids and proteins and on tissue microstructure.

Meat slices were incubated for 24h at 10°C in marinade solutions so that their pH reached 6.5, 5.4, or 4.3, and their NaCl contents 0.9% or 2%. The histological modifications were assessed by image analysis using two parameters: mean fibre cross section area (FCSA, µm(2)) and the ratio of the extra-cellular space (ECS, % area). The changes in lipids and proteins were evaluated through oxidation, protein surface hydrophobicity, and protein aggregation by granulometry (size and shape of particles). ANOVA showed that FCSA decreased linearly while ECS increased when pH decreased from 6.5 to 4.3 at either low or high NaCl salt content. Decreasing pH significantly increased TBARS and carbonyl groups, while increasing NaCl level had a significant protective effect on lipids. No marinating effect was observed on free thiols. Protein surface hydrophobicity and aggregation increased significantly with decreasing pH, but were unaffected by NaCl. The effect of these physicochemical changes on the nutritional value of meat is discussed.

Quantitative study of the relationships among proteolysis, lipid oxidation, structure and texture throughout the dry-cured ham process.

Temperature, salt and water contents are key processing factors in dry-cured ham production. They affect how proteolysis, lipid oxidation, structure and texture evolve, and thus determine the sensory properties and final quality of dry-cured ham. The aim of this study was to quantify the interrelationships and the time course of (i) proteolysis, (ii) lipid oxidation, (iii) five textural parameters: hardness, fragility, cohesiveness, springiness and adhesiveness and (iv) four structural parameters: fibre numbers, extracellular spaces, cross section area, and connective tissue area, during the dry-cured ham process. Applying multiple polynomial regression enabled us to build phenomenological models relating proteolysis, salt and water contents to certain textural and structural parameters investigated. A linear relationship between lipid oxidation and proteolysis was also established. All of these models and relationships, once combined with salt penetration, water migration and heat transfer models, can be used to dynamically simulate all of these phenomena throughout dry-cured ham manufacturing.