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.

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.

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.

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.

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.

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.

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.

Cooking temperature is a key determinant of in vitro meat protein digestion rate: investigation of underlying mechanisms.

The present study aimed to evaluate the digestion rate and nutritional quality of pig muscle proteins in relation to different meat processes (aging, mincing, and cooking). Under our experimental conditions, aging and mincing had little impact on protein digestion. Heat treatments had different temperature-dependent effects on the meat protein digestion rate and degradation potential. At 70 °C, the proteins underwent denaturation that enhanced the speed of pepsin digestion by increasing enzyme accessibility to protein cleavage sites. Above 100 °C, oxidation-related protein aggregation slowed pepsin digestion but improved meat protein overall digestibility. The digestion parameters defined here open new insights on the dynamics governing the in vitro digestion of meat protein. However, the effect of cooking temperature on protein digestion observed in vitro needs to be confirmed in vivo.

Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility.

Effect of pasture- or concentrate-diet on myofibrillar protein oxidation and in vitro digestibility was measured in lamb meat (M. longissimus dorsi) during a refrigerated storage of 7days under gas permeable film. Protein oxidation was measured by the carbonyl content determined chemically using 2,4-dinitrophenylhydrazine (DNPH) and specific targets of oxidation were identified by immunoblotting. Carbonyl content significantly increased during storage and diet affected protein oxidation where animals fed concentrate showed higher carbonyl group levels than animals fed pasture. To evaluate effect of diet and storage time on protein digestibility, myofibrillar proteins were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and α-chymotrypsin) in conditions of pH and temperature which mimic digestive process. The myofibrillar protein digestibility was not influenced by the diet. Storage time had no significant effect on myofibrillar protein susceptibility to pepsin while an important increase in digestibility by trypsin and α-chymotrypsin was detected during storage.

Effect of oxidation on in vitro digestibility of skeletal muscle myofibrillar proteins.

The objective of this study was to investigate the effect of chemical oxidation on myofibrillar protein digestibility. Myofibrils were prepared from pig M. longissimus dorsi and oxidized by a hydroxyl radical generating system. Oxidative modifications of proteins were assessed by the carbonyl content, surface hydrophobicity, electrophoresis, and immunoblotting. Oxidized or nonoxidized myofibrillar proteins were then exposed to proteases of the digestive tract (pepsin, trypsin, and alpha-chymotrypsin). Results showed a direct and quantitative relationship between protein damages by hydroxyl radical and loss of protein digestibility.

Purification of the skeletal muscle protein Endopin 1B and characterization of the genes encoding Endopin 1A and 1B isoforms.

In the present work, a new endopin-like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi-Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino-acids with a molecular mass of 43808Da. The mEndopin 1B gene comprised four coding exons and an additional 5' untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k(ass) approximately 10(6)-10(7) M(-1) s(-1)) suggested that these serpins might play a major role in inflammatory processes.

Revisiting the conversion of muscle into meat and the underlying mechanisms.

The conversion of muscle into meat is a complex process in which all mechanisms responsible for the development of meat qualities are very likely interdependent. Colour and flavour are thus both dependent on oxidative mechanisms. Oxidation and proteolysis are probably two processes involved in the development of meat tenderness. This paper reviewed the consequences of programmed cell death or apoptosis on muscle cells structure and biochemistry and on meat qualities as well. We therefore look at different new hypothesis susceptible to highlight the meat science field and provide new supports for a more dynamic meat research. One of them which would have appeared evident for our purpose since a decade, deals with the fact that, after animal bleeding, muscle cells have no other alternative to only enter the programmed cell death procedure or apoptosis. If we introduce an early phase corresponding to apoptosis, taking place before the rigor onset and overlapping it, we will see that the known consequences of that process bring forward possible answers to still unexplained observations. After an overview of the actual state-of-the-art in meat science, we will introduce the programmed cell death and its underlying mechanisms. We then described the strong analogies between the known consequences of apoptosis and the postmortem changes affecting a set of different muscle characteristics.

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril.

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.

Muscle endopin 1, a muscle intracellular serpin which strongly inhibits elastase: purification, characterization, cellular localization and tissue distribution.

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 microg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41x10(7) M(-1).s(-1)) and trypsin (kass=3.92x10(6) M(-1).s(-1)), whereas plasmin (kass=1.78x10(3) M(-1).s(-1)) and chymotrypsin (kass=1.0x10(2) M(-1).s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.