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.

Deep UV excited muscle cell autofluorescence varies with the fibre type.

The rat skeletal muscle consists of four pure types of muscle cells called type I, type IIA, type IIX and type IIB, and their hybrids in different proportions. They differ in their contraction speeds and metabolic pathways. The intracellular composition is adapted to the fibre function and therefore to fibre types. Given that small differences in composition are likely to alter the optical properties of the cells, we studied the impact of the cell type on the fluorescence response following excitation in the deep UV region. Rat soleus and extensor digitorum longus (EDL) muscle fibres, previously identified based on their cell types by immunohistofluorescence analysis, were analyzed by synchrotron fluorescence microspectroscopy on stain-free serial muscle cross-sections. Muscle fibres excited at 275 nm showed differences in the fluorescence emission intensity among fibre types at 302, 325, 346 and 410 nm. The 410/325 ratio decreased significantly with contractile and metabolic features in EDL muscle, in the order of I > IIA > IIX > IIB fibres (p < 0.01). Compared to type I fibres, the 346/302 ratio of IIA fibres decreased significantly in both EDL and soleus muscles (p < 0.01). This study highlights the usefulness of autofluorescence spectral signals to characterize histological cross-sections of muscle fibres with no staining chemicals.

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.

The Effects of Postmortem Time on Muscle Trout Biochemical Composition and Structure.

Fish industry operators have to process fish that arrive at various postmortem times. Postmortem time constrains processing and impacts product quality, safety, and economic value. The objective identification of biomarkers is desirable to predict the postmortem day of aging and this requires a comprehensive longitudinal characterisation of postmortem aging. We analysed the postmortem aging process in trout over a 15-day window. Quantitative physicochemical measurements (pH, colour, texture, aw, proteolysis, and myofibrillar protein solubility) performed on the same fish over time revealed the levels of protein denaturation, solubility, and pH, among other parameters, change very little when assessed by conventional chemical methods. Histological analyses were performed on thin sections and revealed fibre ruptures after 7 days of storage on ice. Ultrastructures were observed by transmission electronic microscopy (TEM) and revealed that sarcomere disorganisation occurred more often after 7 days of storage. Label-free FTIR micro-spectroscopy combined with a SVM model accurately predicted the postmortem time. Spectra-based PC-DA models also enable the identification of biomarkers corresponding to Day 7 and Day 15 postmortem. This study provides insights on postmortem aging and raises prospects for the rapid assessment of trout's freshness status from label-free imaging.

Effects of high-pressure treatment on the muscle structure of salmon (Salmo salar).

High pressure (HP) is a non-thermal treatment that is generally used to reduce the microbiological contamination of food products, such as Atlantic salmon (Salmo salar). However, HP is known to alter the stability of proteins and can therefore affect the quality of salmon flesh. In this study, the effects of HP treatment for 5 min at 200, 400 and 600 MPa on the structure of Atlantic salmon were investigated. Transversal histological sections revealed a decrease in the fibre size from 200 MPa associated with an expansion of the extracellular spaces. Connective tissue was found to be modified from 400 MPa, resulting in an increase in its surface area. Fourier transform infrared (FT-IR) microspectroscopy revealed a reduction in the α-helix content and an increase in the aggregated ß-sheet structure content with increasing pressure, reflecting a change in the secondary structure of proteins from 200 MPa.

Effect of dry salt versus brine injection plus dry salt on the physicochemical characteristics of smoked salmon after filleting.

Smoked fish fillets are pre-salted as a food conservation and quality preservation measure. Here we investigated biochemical and sensory aspects of smoked salmon fillets. Left-side salmon fillets were dry-salted while the right-side fillets underwent a mixed salting method consisting of an injection of saturated brine followed by surface application of dry salt. After 6 h of salting, all the fillets were smoked. At each step of the process, quality was evaluated using instrumental measurements (pH, color, texture, water content, salt content, aw), and lipid distribution was visualized by MRI. Mixed-salted fillets had a higher salt content than dry-salted fillets and variability in salt distribution was dependent on the salting process. However, these variations had no effect on pH, color or texture, which showed similar values regardless of salting method. Fatty areas had a lower salt content due to slower diffusion of aqueous salt solutions through them. Mixed salting speeds up the salting of the muscle without significantly affecting the quality traits of the salmon fillet.

Structural changes in local Thai beef during sous-vide cooking.

Thai beef (Bos indicus) samples were sous-vide-cooked at temperatures of 60°C, 70°C or 80°C for 2 to 36 hrs and prepared for microstructure characterization by light and electron microscopy. Muscle fibers showed a first phase of lateral shrinkage during the first 6 hrs of cooking at 60-70°C and the first 2 hrs at 80°C followed by a second phase of significant alternations of shrinkage and swelling independently of water transfers. Swelling peaked at 12 hrs. Microstructural changes were more variable for samples cooked at 60-70°C than for samples cooked at 80°C that showed a larger cross-sectional myofibrillar mass area (CSA). Hypercontracted fibers were evidenced at all temperature-time combinations and were associated with adjacent wavy fibers and a characteristic structural evolution in the mitochondria. The role of thermal denaturation of proteins and the ultrastructural analogy of hypercontracted fibers with cold-shortened fibers are discussed.

Shockwave processing of beef brisket in conjunction with sous vide cooking: Effects on protein structural characteristics and muscle microstructure.

We studied the effect of shockwave processing and subsequent sous vide cooking on meat proteins (molecular size and thermal stability) and muscle structures (molecular, micro- and ultrastructure). Beef briskets were subjected to shockwave (11 kJ/pulse) and were sous vide-cooked at 60 °C for 12 h. Shockwave processing alone decreased the enthalpy and thermal denaturation temperature of the connective tissue proteins (second peak in the DSC thermogram, p < 0.05) compared to the control raw samples, while the protein gel electrophoresis profile remained unaffected. It led to disorganisation of the sarcomere structure and also modified the protein secondary structure. More severe muscle fibre coagulation and denaturation were observed in the shockwave-treated cooked meat compared to the cooked control. The results show that shockwave processing, with and without sous vide cooking, promotes structural changes in meat, and thus may have the potential to improve the organoleptic quality of the tough meat cuts.

Impact of Tumbling Process on the Toughness and Structure of Raw Beef Meat Pieces.

Tenderness is a major factor in consumer perception and acceptability of beef meat. Here we used a laboratory tumbling simulator to investigate the effectiveness of the tumbling process in reducing the toughness of raw beef cuts. Twelve Semitendinosus beef muscles from cows were tumbled according to four programs: T1 (2500 consecutive compression cycles (CC), for about 3 h), T2 (6000 CC, about 7.5 h), T3 (9500 CC, about 12 h), and T4 (13,000 CC, about 16 h). The effect of tumbling on the toughness of raw meat was assessed using compression tests (stresses measured at 20% and 80% of deformation ratios) and microscopic observations made at the periphery and centre of meat samples, and compared against non-tumbled controls. Longer tumbling times significantly reduced the stresses measured at 20% and 80% compression rates, which reflected the toughness of muscle fibres and connective tissue, respectively. At the microscopic level, longer tumbling times led to reduced extracellular spaces, increased degradation of muscle structure, and the emergence of amorphous zones. A 12-h tumbling protocol ultimately makes the best compromise between the process time demand and toughness reduction in beef Semitendinosus meat pieces.

Thermal denaturation of proteins in the muscle fibre and connective tissue from bovine muscles composed of type I (masseter) or type II (cutaneous trunci) fibres: DSC and FTIR microspectroscopy study.

The changes in secondary structure of proteins with heating were characterised and compared for bovine masseter (fibre type I) and cutaneous trunci (fibre type II) muscles by Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR) microspectroscopy. Heating led to a decrease in α- helices, and an increase in aggregated strands, random coils and aromatic side chains in the muscle fibres of both muscles. In the intramuscular connective tissue (IMCT) of both muscles, a decrease in α- helix, turn and unordered structures was complemented with an increase in aggregated strands. At temperatures < 60 °C, the greater thermal denaturation of proteins in cutaneous trunci than in masseter (FTIR), supported by a myosin associated peak at 55.8 °C for cutaneous trunci and no peak for masseter (DSC), indicates that myosin in type II fibres is more sensitive to thermal denaturation than myosin in type I fibres and this should be considered in thermal meat processing.

Deciphering PSE-like muscle defect in cooked hams: A signature from the tissue to the molecular scale.

PSE-like technological defect in the meat industry is of great importance due, to the economic loss it can cause. It has been studied from the biochemical perspective but very few studies have focused on tissular characterization. This study proposes innovative approaches that combine mechanistic elucidation and the discovery of potential biomarkers. This study focused on muscle destructuration using imaging and label-free quantitation. Oxidative stress and apoptotic processes were found to be linked to the specific evolution of the PSE-like destructuration zone, namely 'inner', within hams. Four m/z values were found to be related to the specific localization of the PSE-like defect, and a mass shift of 27 Da suggested a possible connection with oxidation. These potential markers of the PSE-like area in ham provide a new perspective to sort raw material based on the possible development of PSE-like areas.

Antioxidant supplementation restores defective leucine stimulation of protein synthesis in skeletal muscle from old rats.

Aging is characterized by a progressive loss of muscle mass that could be partly explained by a defect in the anabolic effect of food intake. We previously reported that this defect resulted from a decrease in the protein synthesis response to leucine in muscles from old rats. Because aging is associated with changes in oxidative status, we hypothesized that reactive oxygen species-induced oxidative damage may be involved in the impairment of the anabolic effect of leucine with age. The present study assessed the effect of antioxidant supplementation on leucine-regulated protein metabolism in muscles from adult and old rats. Four groups of 8- and 20-mo-old male rats were supplemented or not for 7 wk with an antioxidant mixture containing rutin, vitamin E, vitamin A, zinc, and selenium. At the end of supplementation, muscle protein metabolism was examined in vitro using epitrochlearis muscles incubated with increasing leucine concentrations. In old rats, the ability of leucine to stimulate muscle protein synthesis was significantly decreased compared with adults. This defect was reversed when old rats were supplemented with antioxidants. It was not related to increased oxidative damage to 70-kDa ribosomal protein S6 kinase that is involved in amino acid signaling. These effects could be mediated through a reduction in the inflammatory state, which decreased with antioxidant supplementation. Antioxidant supplementation could benefit muscle protein metabolism during aging, but further studies are needed to determine the mechanism involved and to establish if it could be a useful nutritional tool to slow down sarcopenia with longer supplementation.

Histological Study of Ultrastructural Changes in Muscle Exposed to Various Concentrations of NaCl Brine.

Efforts to reduce NaCl content in meat products hinge on gaining a deeper understanding of how NaCl content shapes product changes during the salting process. Bovine semitendinosus muscle samples were incubated for 5 days in 7 different brine baths at NaCl concentrations of 0 (control), 0.15, 0.30, 0.50, 0.75, 1.0, and 1.5 M, respectively. Histological and ultrastructural analyses showed that after incubation, muscle fiber size increased almost 30% in 0.15 and 0.3 M NaCl brine but decreased in 0.50 and 0.75 M NaCl. At above 0.75 M NaCl, brining led to significantly degraded muscle tissue, preventing the morphological characterization by image analysis. Structural modifications were strongest in samples immersed in 1 M NaCl brine. M and Z lines showed dramatic solubilization from 0.5 M NaCl upwards. Extracted proteins were released into subsarcolemmal space at 0.75 M NaCl then into extracellular space at higher NaCl levels. These findings bring insight into the sequence of structural alterations in meat as a function of ionic strength, making it possible to optimize the meat salting process. PRACTICAL APPLICATION: Knowledge on the relationship between NaCl concentration, in situ structural changes and transfers of salt-extracted proteins can be usefully mobilized to reduce sodium content in cured meat products.

In situ characterization of acidic and thermal protein denaturation by infrared microspectroscopy.

Foods meet acid pH during gastric digestion after cooking. An in situ infrared microspectroscopy approach was developed to detect the effects of heat and acid treatments on protein structure separately. Infrared spectra were obtained from meat samples treated with heat and/or acid, and wavenumbers accounting independently for the treatments were extracted by principal component regression. Extreme-acid treatment (pHinitial 2.0) was well predicted (0.5% error) by a simple ratio of as-observed spectral intensities at 1211 and 1396 cm-1, reflecting a perturbation in the vibration of amino acid residues (phenylalanine, tyrosine and aspartic acid) by protein unfolding and protonation. Using the imaging mode of an IR microscope, meat protein acidification was evidenced with high spatial resolution. The heat effect was well discriminated from the acid effect by the ratio of as-observed intensities at 1666 and 1697 cm-1 (0.9% error), indicating content of aggregated ß-sheets relative to α-helix structure.

Myofiber metabolic type determination by mass spectrometry imaging.

Matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a powerful tool that opens new research opportunities in the field of biology. In this work, predictive model was developed to discriminate metabolic myofiber types using the MALDI spectral data. Rat skeletal muscles are constituted of type I and type IIA fiber, which have an oxidative metabolism for glycogen degradation, and type IIX and type IIB fiber which have a glycolytic metabolism, present in different proportions according to the muscle function and physiological state. So far, myofiber type is determined by histological methods that are time consuming. Thanks to the predictive model, we were able to predict not only the metabolic fiber type but also their location, on the same muscle section that was used for MALDI imaging. Copyright © 2017 John Wiley & Sons, Ltd.

Colonisation of Meat by Escherichia coli O157:H7: Investigating Bacterial Tropism with Respect to the Different Types of Skeletal Muscles, Subtypes of Myofibres, and Postmortem Time.

Escherichia coli O157:H7 is an enterohaemorrhagic E. coli (EHEC) responsible for serious diseases, especially pediatric, and of great concern for the meat industry. Meat contamination by EHEC occurs at slaughtering, especially at dehiding stage, where bacteria can be transferred from hides to carcasses. The skeletal muscle tissues comprise four major types of myofibres, which differ in their contraction velocity and metabolism. Myofibres are surrounded by the extracellular matrix (ECM). Adhesion of E. coli O157:H7 to meat was investigated considering well-defined types of skeletal muscle and their constituent myofibres as well as postmortem changes in muscle, using fluorescence microscopy and immunohistochemical analyses. By analysing the adhesion of E. coli O157:H7 to model oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] skeletal muscles, it first appeared that differential adhesion occurred at the surface of these extreme skeletal muscle types. At a cellular level, bacterial adhesion appeared to occur essentially at the ECM. Considering the different constituent myofibres of types I, IIA, IIX and IIB, no significant differences were observed for adhering bacteria. However, bacterial adhesion to the ECM was significantly influenced by postmortem structural modifications of muscle tissues. By providing information on spatial localisation of E. coli O157:H7 on meat, this investigation clearly demonstrated their ability to adhere to skeletal muscle, especially at the ECM, which consequently resulted in their heterogeneous distribution in meat. As discussed, these new findings should help in reassessing and mitigating the risk of contamination of meat, the food chain and ultimately human infection by EHEC.

Sodium Chloride Diffusion during Muscle Salting Evidenced by Energy-Dispersive X-ray Spectroscopy Imaging.

To better understand the relationship between the muscle structure and NaCl transfers in meat, we used energy-dispersive X-ray spectroscopy (EDS) coupled with scanning electron microscopy (SEM) to analyze brined and dry-salted rat muscles. The muscles were freeze-dried to avoid the delocalization of soluble ions that happens in regular dehydration through a graded series of ethanol. Na and Cl maps were superimposed on SEM images to combine the muscle structure and NaCl diffusion. Brining causes rapid diffusion of NaCl through the tissue. Most brine diffuses in a linear front from the muscle surface, but a small proportion enters through the perimysium network. The muscle area penetrated by brine shows heterogeneous patterns of NaCl retention, with some connective tissue islets containing more NaCl than other parts of perimysium. NaCl penetration is considerably slower after dry salting than after brining.

High pressure processing of meat: effects on ultrastructure and protein digestibility.

The effects of high pressure processing (HPP, at 175 and 600 MPa) on the ultrastructure and in vitro protein digestion of bovine longissimus dorsi muscle meat were studied. HPP caused a significant change in the visual appearance and texture of the meat subjected to HPP at 600 MPa so that it appeared similar to cooked meat, unlike the meat subjected to HPP at 175 MPa that showed no significant visible change in the colour and texture compared to the raw meat. The muscles were subjected to digestion under simulated gastric conditions for 1 h and then under simulated small-intestinal conditions for a further 2 h. The digests were analysed using gel electrophoresis (SDS-PAGE) and ninhydrin assay for amino N. The effect of the acid conditions of the stomach alone was also investigated. Reduced SDS-PAGE results showed that pepsin-digested (60 min) HPP meats showed fewer proteins or peptides of high molecular weight than the pepsin-digested untreated meat, suggesting more breakdown of the parent proteins in HPP-treated meats. This effect was more pronounced in the muscles treated at 600 MPa. These results are in accordance with microscopy results, which showed greater changes in the myofibrillar structure after simulated gastric digestion of the sample processed at 600 MPa than at 175 MPa. Transmission electron microscopy also showed the presence of protein aggregates in the former sample, resulting probably from protein denaturation of sarcoplasmic proteins, in the subcellular space and between myofibrils; along with cell contraction (similar to that caused by heating) in the former.

Hyperspectral deep ultraviolet autofluorescence of muscle fibers is affected by postmortem changes.

After slaughter, muscle cells undergo biochemical and physicochemical changes that may affect their autofluorescence characteristics. The autofluorescent response of different rat extensor digitorum longus (EDL) and soleus muscle fiber types was investigated by deep ultraviolet (UV) synchrotron microspectroscopy immediately after animal sacrifice and after 24 h of storage in a moist chamber at 20 °C. The glycogen content decreased from 23 to 18 µmol/g of fresh muscle in 24 h postmortem. Following a 275 nm excitation wavelength, the spectral muscle fiber autofluorescence response showed discrimination depending upon postmortem time (t0 versus t24 h) on both muscles at 346 and 302 nm and, to a lesser extent, at 408 and 325 nm. Taken individually, all fiber types were discriminated but with variable accuracy, with type IIA showing better separation of t0/t24 h than other fiber types. These results suggest the usefulness of the autofluorescent response of muscle cells for rapid meat-aging characterization.

Protein matrix involved in the lipid retention of foie gras during cooking: a multimodal hyperspectral imaging study.

Denaturation of the protein matrix during heat treatment of duck foie gras was studied in relationship to the amount of fat loss during cooking. A low fat loss group was compared with a high fat loss group by histochemistry, FT-IR, and synchrotron UV microspectroscopy combination to characterize their protein matrix at different scales. After cooking, the high fat loss group showed higher densification of its matrix, higher ultraviolet tyrosine autofluorescence, and an infrared shift of the amide I band. These results revealed a higher level of protein denaturation and aggregation during cooking in high fat loss than in low fat loss foie gras. In addition, the fluorescence and infrared responses of the raw tissue revealed differences according to the level of fat losses after cooking. These findings highlight the importance of the supramolecular state of the protein matrix in determining the fat loss of foie gras.