Detection of Frozen-Thawed Duck Fatty Liver by MALDI-TOF Mass Spectrometry: A Chemometrics Study.

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.

Redox Potential and Antioxidant Capacity of Bovine Bone Collagen Peptides towards Stable Free Radicals, and Bovine Meat Lipids and Proteins. Effect of Animal Age, Bone Anatomy and Proteases-A Step Forward towards Collagen-Rich Tissue Valorisation.

Collagen antioxidant peptides are being widely studied. However, no research has paid attention to biological parameters such as the age and anatomy of collagen-rich tissues, which can determine a change in tissue structure and composition, and then in bioactivity. Moreover, only few research works have studied and assessed peptides antioxidant activity on the food matrix. This work aimed to investigate the effect of bovine's bone age and anatomy, and of six different enzymes, on the antioxidant activity of collagen peptides. Collagen was extracted from young and old bovine femur and tibia; six different enzymes were used for peptides' release. The redox potential, the quenching of stable free radicals, and the antioxidant capacity on bovine meat lipids and proteins was evaluated, under heating from ambient temperature to 80 °C. Age and anatomy showed a significant effect; the influence of anatomy becomes most important with age. Each enzyme's effectiveness toward age and anatomy was not the same. The greatest amount of peptides was released from young bones' collagen hydrolysed with papain. The antioxidant activity was higher at higher temperatures, except for meat proteins. Assessing the effect of age and anatomy of collagen-rich tissues can promote a better application of collagen bioactive peptides.

Investigation of secondary structure evolution of micellar casein powder upon aging by FTIR and SRCD: consequences on solubility.

BACKGROUND: Synchrotron radiation circular dichroism (SRCD) and Fourier transform infrared (FTIR) spectroscopy were used to examine the conformation evolution of micellar casein (MC) powder during storage and to determine whether the spectral changes could be related to their solubility evolution. RESULTS: A loss in intensity of SRCD spectra as a function of storage time has been observed. Quantification of secondary structures revealed losses of α-helix content during storage. Moreover, a redshift of the amide I band in the FTIR spectrum was demonstrated during the storage and was interpreted as a rearrangement of the secondary structure of the protein, which is in line with the SRCD results. The qualitative results obtained by FTIR clearly support the quantitative evolution of the secondary structure obtained by the analysis of SRCD spectra. Principal component analysis (PCA) of FTIR spectra permits a good separation of samples according to the storage time. PCA shows that the evolution of secondary structures and solubility loss are closely linked. CONCLUSION: With the quantitative data provided by SRCD spectra, it was established that, whatever the storage conditions, a unique curve exists between loss of α-helix content and loss in solubility, showing that loss of α-helix content is a marker of solubility loss for the MC powders studied. © 2017 Society of Chemical Industry.

Compared with Raw Bovine Meat, Boiling but Not Grilling, Barbecuing, or Roasting Decreases Protein Digestibility without Any Major Consequences for Intestinal Mucosa in Rats, although the Daily Ingestion of Bovine Meat Induces Histologic Modifications in the Colon.

BACKGROUND: Cooking may impair meat protein digestibility. When undigested proteins are fermented by the colon microbiota, they can generate compounds that potentially are harmful to the mucosa. OBJECTIVES: This study addressed the effects of typical cooking processes and the amount of bovine meat intake on the quantity of undigested proteins entering the colon, as well as their effects on the intestinal mucosa. METHODS: Male Wistar rats (n = 88) aged 8 wk were fed 11 different diets containing protein as 20% of energy. In 10 diets, bovine meat proteins represented 5% [low-meat diet (LMD)] or 15% [high-meat diet (HMD)] of energy, with the rest as total milk proteins. Meat was raw or cooked according to 4 processes (boiled, barbecued, grilled, or roasted). A meat-free diet contained only milk proteins. After 3 wk, rats ingested a (15)N-labeled meat meal and were killed 6 h later after receiving a (13)C-valine injection. Meat protein digestibility was determined from (15)N enrichments in intestinal contents. Cecal short- and branched-chain fatty acids and hydrogen sulfide were measured. Intestinal tissues were used for the assessment of protein synthesis rates, inflammation, and histopathology. RESULTS: Meat protein digestibility was lower in rats fed boiled meat (94.5% ± 0.281%) than in the other 4 groups (97.5% ± 0.0581%, P < 0.001). Cecal and colonic bacterial metabolites, inflammation indicators, and protein synthesis rates were not affected by cooking processes. The meat protein amount had a significant effect on cecal protein synthesis rates (LMD > HMD) and on myeloperoxidase activity in the proximal colon (HMD > LMD), but not on other outcomes. The ingestion of bovine meat, whatever the cooking process and the intake amount, resulted in discrete histologic modifications of the colon (epithelium abrasion, excessive mucus secretion, and inflammation). CONCLUSIONS: Boiling bovine meat at a high temperature (100°C) for a long time (3 h) moderately lowered protein digestibility compared with raw meat and other cooking processes, but did not affect cecal bacterial metabolites related to protein fermentation. The daily ingestion of raw or cooked bovine meat had no marked effect on intestinal tissues, despite some slight histologic modifications on distal colon.

High True Ileal Digestibility but Not Postprandial Utilization of Nitrogen from Bovine Meat Protein in Humans Is Moderately Decreased by High-Temperature, Long-Duration Cooking.

BACKGROUND: Meat protein digestibility can be impaired because of indigestible protein aggregates that form during cooking. When the aggregates are subsequently fermented by the microbiota, they can generate potentially harmful compounds for the colonic mucosa. OBJECTIVE: This study evaluated the quantity of bovine meat protein escaping digestion in the human small intestine and the metabolic fate of exogenous nitrogen, depending on cooking processes. METHODS: Sixteen volunteers (5 women and 11 men; aged 28 ± 8 y) were equipped with a double lumen intestinal tube positioned at the ileal level. They received a test meal exclusively composed of 120 g of intrinsically (15)N-labeled bovine meat, cooked either at 55°C for 5 min (n = 8) or at 90°C for 30 min (n = 8). Ileal effluents and blood and urine samples were collected over an 8-h period after the meal ingestion, and (15)N enrichments were measured to assess the digestibility of meat proteins and the transfer of dietary nitrogen into the metabolic pools. RESULTS: Proteins tended to be less digestible for the meat cooked at 90°C for 30 min than at 55°C for 5 min (90.1% ± 2.1% vs. 94.1% ± 0.7% of ingested N; P = 0.08). However, the particle number and size in ileal digesta did not differ between groups. The appearance of variable amounts of intact fibers was observed by microscopy. The kinetics of (15)N appearance in plasma proteins, amino acids, and urea were similar between groups. The amount of exogenous nitrogen lost through deamination did not differ between groups (21.2% ± 0.8% of ingested N). CONCLUSIONS: Cooking bovine meat at a high temperature for a long time can moderately decrease protein digestibility compared with cooking at a lower temperature for a short time and does not affect postprandial exogenous protein metabolism in young adults. The study was registered at www.clinicaltrials.gov as NCT01685307.

Utilizing the influence of protein enrichment of meal components as a strategy to possibly prevent undernutrition in the elderly: an in vitro approach.

Nutritional strategies are required to limit the prevalence of denutrition in the elderly. With this in mind, fortified meals can provide more protein, but their digestibility must be ensured. Using a dynamic in vitro digester, DIDGI®, programmed with the digestion conditions of the elderly, we evaluated the supplementation of each component of a meal and assessed protein digestibility, amino acid profile, micro-nutrients and vitamins bioaccessibility for a full course meal. Higher protein digestibility was evidenced for the fortified meal, with higher release of essential amino acids. Moreover the large increase of leucine released was comparable to the range advocated for the elderly to favour protein anabolism. This in vitro study underlines the interest of using dish formulations to meet the nutritional needs of seniors, which is why this work will be completed by a clinical study in nursing home.

Fava bean (Vicia faba L.) protein concentrate added to beef burgers improves the bioaccessibility of some free essential amino acids after in vitro oral and gastrointestinal digestion.

The influence of partial replacement of animal protein by plant-based ingredients on the protein digestibility of beef burgers was investigated. Beef burgers were supplemented with fava bean protein concentrate (FB) or a mixture of FB and flaxseed flour (FBFS), both processed by extrusion, at different levels: 0 (control), 10, 15, and 20 % (w/w). A pilot sensory analysis was conducted to select the percentage of flour inclusion for further assays: control, 10 % FB, and 10 % FBFS. Protein digestibility, amino acid profile, and protein secondary structure of these burgers after in vitro oral and gastrointestinal digestion were studied. In vitro boluses were prepared with the AM2 masticator, simulating normal mastication, and static in vitro digestion of boluses was performed according to the INFOGEST method. Inclusion of 10 % FB in beef burgers did not alter their flavour or tenderness compared to the control, whereas tenderness and juiciness scored slightly higher for the 10 % FBFS burgers compared to 15 % and 20 % FBFS ones. Poor lipid oxidative stability during storage was observed with 10 % FBFS burgers. Total protein content was significantly higher (p < 0.05) in 10 % FB burgers than in control burgers after in vitro oral digestion. Additionally, 10 % FB burgers presented higher amounts of free essential amino acids like isoleucine, leucine, phenylalanine, and valine at the end of digestion, as well as methionine, tyrosine, and histidine. Partial substitution of meat protein by 10 % FB improves the nutritional profile of beef burgers, without altering their sensory qualities.

Oral and gastrointestinal nutrient bioaccessibility of gluten-free bread is slightly affected by deficient mastication in the elderly.

The main goal of this work was to investigate the impact of impaired mastication on nutrient bioaccessibility of gluten-free bread in the elderly. In vitro boluses were produced with the AM2 masticator by using two types of programming: normal mastication (NM) and deficient mastication (DM). Static in vitro gastrointestinal digestion was performed with the digestive physiology conditions of the elderly. Subsequently, the granulometric properties of the in vitro boluses produced, their starch and protein digestibility, and lipid peroxidation after in vitro oral and gastrointestinal digestion were evaluated. DM boluses presented higher proportions of large particles, resulting in insufficiently fragmented boluses. A delay in oral starch digestion was observed in DM boluses, probably due to the presence of larger particles that limited the bolus-saliva exchanges. Furthermore, DM boluses exhibited a lower degree of protein hydrolysis at the end of gastric digestion, whereas no differences were observed for protein hydrolysis, sugar release, and lipid peroxidation at the end of digestion (intestinal phase). The results of this study show that impaired mastication somewhat delays the nutrient bioaccessibility of the gluten-free bread tested. Such understanding of the effect of oral decline on the nutrient bioaccessibility of foods is crucial when designing food commodities with enhanced functionalities for the elderly.

Effects of sodium nitrite reduction, removal or replacement on cured and cooked meat for microbiological growth, food safety, colon ecosystem, and colorectal carcinogenesis in Fischer 344 rats.

Epidemiological and experimental evidence indicated that processed meat consumption is associated with colorectal cancer risks. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Compared to the reference level (120 mg/kg of ham), sodium nitrite removal and reduction (90 mg/kg) similarly decreased preneoplastic lesions in F344 rats, but only reduction had an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level and an effective lipid peroxidation control. Among the three nitrite salt alternatives tested, none of them led to a significant gain when compared to the reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions in rats despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions in rats but the concomitant presence of nitrosyl iron in feces. Except the vegetable stock, other alternatives were less efficient than sodium nitrite in reducing L. monocytogenes growth.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

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.

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.

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.

Development of a Statistical Workflow for Screening Protein Extracts Based on their Nutritional Composition and Digestibility: Application to Elderly.

The objective of the study is to develop a workflow to screen protein extracts and identify their nutritional potential as high quality nutritional culinary aids for recipes for the elderly. Twenty-seven protein extracts of animal, vegetable, and dairy origin were characterized. We studied their fate by monitoring static in vitro digestion, mimicking the physiological digestion conditions of the elderly. At the end of the gastric and intestinal phase, global measurements of digestibility and antioxidant bioactivities were performed. The statistical analysis workflow developed allowed: (i) synthesizing the compositional and nutritional information of each protein extract by creating latent variables, and (ii) comparing them. The links between variables and similarities between protein extracts were visualized using a heat map. A hierarchical cluster analysis allowed reducing the 48 quantitative variables into 15 qualitative latent variables (clusters). The application of the k-means method on each cluster enable to classify the protein extracts by level. This defined level was used as categorical value. Multiple correspondence analysis revealed groups of protein extracts with varied patterns. This workflow allowed the comparison/hierarchization between protein extracts and the creation of a tool to select the most interesting ones on the basis of their nutritional quality.

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.

Time course of peptide fingerprints in semimembranosus and biceps femoris muscles during Bayonne ham processing.

The aim of this work was to define reliable markers of muscle and processing time in dry-cured ham using a rapid, precise semi quantitative method for the protein fraction soluble in low ionic strength buffer. For this purpose protein labchip Agilent was used to separate proteins and peptides and accurately determine their molecular weights and concentrations electrophoretically. In this way the protein fingerprinting of dry-cured ham at different process times was characterised, together with targets and products of proteolysis. In addition, the comparison of all the electrophoregrams indicated muscle and dry-curing process markers.