Detection of allergenic additives in processed meat products.

Allergic responses to food components are an increasing problem all over the world. It is therefore important to protect people who are vulnerable to food allergens against accidental and unintended consumption of products containing allergic ingredients. The meat industry commonly uses various allergic additives in the production of processed products, such as legumes (soy, peas, beans), milk and egg preparations, cereals containing gluten (wheat, rye, barley and oats), and spices (celery and mustard). These meat additives have specific technological properties, which help to create a texture or flavor profile, or affect the nutritional value, although some of them, such as soy, mustard, milk and egg white proteins, can cause severe allergic reactions. The aim of this paper is to discuss the application of various recently established methods of detection of allergenic additives in processed meat products - for instance cold cuts and sausages. The new methods are based mainly on protein, DNA, and isoflavones or phytic acid analysis. The article also characterizes the latest trends in the development of research on methods that would enable quick and reliable identification of targeted allergens in meat products. © 2018 Society of Chemical Industry.

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

BACKGROUND: Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. RESULTS: The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as ß-conglutin. CONCLUSIONS: The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry.

The effect of the packaging system and storage time on myofibrillar protein degradation and oxidation process in relation to beef tenderness.

This study investigated the impact of packaging systems on the degradation and oxidation of beef proteins regarding beef tenderness of longissimus lumborum (LL) and biceps femoris (BF) muscles stored in vacuum skin packaging (VSP), a modified atmosphere with high oxygen concentration (MAP), and combined of these two methods (VSP+MAP). A significant decrease in the Warner-Bratzler shear force (WBSF) in VSP at D14 and D28 for LL was observed compared to BF. A significant effect of packaging system on troponin-T (Tn-T) and desmin degradation was shown (p≤0.001). A high concentration of oxygen in MAP and VSP+MAP affected protein oxidation, which was reflected in myosin oxidative cross-linking. An increase of WBSF values detected in steaks packed in VSP and VSP+MAP systems could be caused by the intensification of protein oxidation. Furthermore, BF was more susceptible to oxidation compared to LL. The VSP+MAP packaging system has resulted in the maintenance of a bright, red color, however has not improved the beef tenderness.

The influence of thermal processing on the fatty acid profile of pork and lamb meat fed diet with increased levels of unsaturated fatty acids.

The research was carried out on 32 crossbred pigs of Polish Large White × Danish Landrace with Duroc and 80 rams, crossbreds of the Prolific-Dairy Koludzka Sheep with the Ile de France, a meat sheep. The fodder for the animals was enriched with the unsaturated fatty acids originated mainly from linseed and rapeseed oils. The fatty acid profile was determined in cooked longissimus lumborum, roasted triceps brachii and raw ripened rump from pigs as well as in grilled lambs' legs and their corresponding raw materials. Roasting caused the most pronounced increase of the saturated fatty acids and decrease in the polyunsaturated fatty acids of heated pork muscles. The smallest changes were observed in grilled lamb legs. The heating processes applied in this study, in most cases, did not cause essential changes in the indices of pro-health properties of fatty acid, therefore meat in the majority fulfil the latest recommendations of EFSA and FAO/WHO according to human health.

Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion.

New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey) and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages). Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed after 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

Species-specific expression of various proteins in meat tissue: proteomic analysis of raw and cooked meat and meat products made from beef, pork and selected poultry species.

The aim was to search for proteins differentiating the six species (cattle, pig, chicken, turkey, duck and goose) and relatively stable during the meat aging and only slightly degraded in ready-made products. The two-dimensional electrophoresis was used for analysis of the protein profiles from raw meat and frankfurters and sausages (15 products). The observed species-specific differences in protein expression in raw meat were retained in processed products after finishing the entire technological process. Regulatory proteins, metabolic enzymes, some myofibrillar and blood plasma proteins were identified, which were characterised by the electrophoretic mobility specific to the given species. Large differences in the primary structure were observed in serum albumin, apolipoprotein B, HSP27, H-FABP, ATP synthase, cytochrome bc-1 subunit 1 and alpha-ETF. Some of these proteins have potential to be used as markers in authentication of meat products.

Myosin light chain isoforms retain their species-specific electrophoretic mobility after processing, which enables differentiation between six species: 2DE analysis of minced meat and meat products made from beef, pork and poultry.

Investigation of protein changes as well as authentication of meat is particularly difficult in processed meat products due to their different composition, complexity and very often inhomogeneity. The aim of this study was to check if the inter-species differences in the expression of myosin light chain (MLC) isoforms observed in raw meat were retained in meat products. MLCs from mixtures of minced meat (16 variants), frankfurters and sausages (15 products) made from cattle, pig, chicken, turkey, duck and goose were analysed by 2DE. Species-specific patterns of MLC isoforms were observed in all the mixtures and processed meat products. Relatively small degradation was observed in the MLCs after processing. Image analysis enabled species identification of the meat in all samples when the content of meat of one species was not lower than 10%. However, it was impossible to differentiate between all the six species under investigation on the basis of individual isoform. It was possible when the combination of all the three isoforms (myosin light chain 1 fast, myosin light chain 2 fast and myosin light chain 3 fast) was analysed. The results evidenced that MLCs have potential to be used as markers in authentication of meat products made from the analysed six species.

Is authentication of regional and traditional food made of meat possible?

Authentication of regional and traditional food made of meat poses a significant challenge. It continues to be a very difficult task which requires employment of quite advanced analytical techniques. These products, despite a similar process of manufacturing, differ in taste and aroma. This happens due to the use of special breeds of animals, the application of appropriate feeding regimes as well as the effect of the place and climate. In order to perform correct identification of geographical origin, a good solution is to determine both stable isotopes as well as trace elements. It is essential to collect detailed meteorological and geochemical data and information about farming practices and to compare them with the obtained results. In a majority of cases, the performed identification is confined to species and the determination of the animal breed is very limited. In the case of individual breeds a comparative analysis of SNPs appears to present the highest potential, especially genes affecting the coat color of animals may serve as markers. Experiments confirm that genes responsible for pigmentation underwent mutations in individual breeds. Authentication on the basis of the manufacturing process appears to be easier to realize than tracing geographical origins.

Differences in two-dimensional gel electrophoresis patterns of skeletal muscle myosin light chain isoforms between Bos taurus, Sus scrofa and selected poultry species.

BACKGROUND: In this study the interspecies differences in two-dimensional electrophoresis patterns of skeletal muscle myosin light chain (MLC) isoforms between Bos taurus (cattle), Sus scrofa (pig), Gallus gallus (chicken), Meleagris gallopavo (turkey), Anas platyrhynchos (duck) and Anser anser (goose) were characterised on the basis of specific properties of MLCs associated with their structure and mobility in gel. RESULTS: Two-dimensional electrophoresis separations revealed species-specific differences in the molecular weight and pI of individual MLC isoforms (MLC1f, MLC2f and MLC3f). In the case of closely related animal species such as goose and duck or turkey and chicken, significant differences occurred in MLC1f. For MLC2f, differences between cattle and turkey and between pig and chicken were around 1 and 0.3 kDa respectively. It appeared from the comparison of amino acid sequences that even MLCs with only 2% difference in sequences have different electrophoretic mobilities. CONCLUSION: Interspecies differences in skeletal MLC isoforms appeared between cattle, pig, chicken, turkey, duck and goose. The slight changes observed in the course of the aging process confirmed that these proteins are relatively little susceptible to proteolytic enzymes during meat aging.

Impact of polymorphism of the regulatory subunit of the µ-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle.

The objective of this study was to estimate the impact of the polymorphism of µ-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), α-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism.

Changes of myofibrillar and centrifugal drip proteins and shear force of psoas major and minor and semitendinosus muscles from calves, heifers and cows during post-mortem ageing.

Changes in myofibrillar protein content and centrifugal drip proteins of psoas major and minor (PM) and semitendinosus (ST) muscles of calves, heifers and cows taken from carcasses on the 1st, 6th and 12th day of post-mortem cold storage were estimated. Washed myofibrils and centrifugal drip from muscles were analysed using SDS-PAGE 10 and 12% polyacrylamide gels. No significant changes were observed in content of contractile proteins, α-actinin and regulatory proteins (except for TN-T). There were no significant differences between muscles from investigated groups and between muscles aged in chilled conditions. The levels of titin T1 during ageing varied slightly. The 30 kDa-fraction appearance was fastest in calf, slower in heifer and slowest in cow muscles. More pronounced differences in the level of protein degradation with regards to muscle type, age of animals and time of storage were found in the centrifugal drip of meat. In the drip, the level of high molecular weight proteins was higher in muscles from young animals and in the muscles stored longer. The opposite was observed in case of 26-28 kDa proteins. Their amount in muscle drip decreased with increased storage time. The rate of proteolysis and release of cytoskeletal proteins during cold storage of muscles were related to change in shear values of roasted meat. The highest rate of protein degradation was observed in PM calf muscle and the lowest rate in ST cow muscle. The fastest tenderization process was registered in calves muscles and the slowest tenderization in cows.

Changes in structure of psoas major and minor and semitendinosus muscles of calves, heifers and cows during post-mortem ageing.

Changes occurring during post-mortem ageing in structure of psoas major and minor (PM) and semitendinosus (ST) muscles of calves, heifers and cows were observed. Samples from muscles taken 3 h after slaughter and after 6 and 12 days of storage at the temperature of 4 °C were analysed using light and transmission electron microscopy. Three hours after slaughter muscle fibres were close to each other and sarcomeres showed normal structure of A- and I-bands, M-lines and Z-disks. The space between myofibrils and sarcolemma and also between single myofibrils in muscle fibres increased during storage. Structure of sarcomeres underwent continuous degradation, length of sarcomeres increased due to enlargement of I-bands accompanied by Z-disk degradation. During ageing structural changes in myofibrils took place faster, were more intensive in muscles of younger animals and were more extensive and faster in PM than in ST muscles.

Thermal properties of titin from porcine and bovine muscles.

The thermal properties of titin isolated from porcine and bovine longissimus muscles were investigated by differential scanning calorimetry in the temperature range from 20 to 100 °C. A single peak with average maximum temperatures of 75.6 and 78.4 °C characterized porcine and bovine titin denaturation, respectively. The peaks were much broader than those from the other major muscle proteins. Titin denaturation enthalpy values (1.6-2.6 J/g) were only about half those of whole meat and also lower than those previously determined for myosin, actin, or collagen. The relatively high titin denaturation temperature suggests that it may be partially responsible for meat toughening when muscle tissue is heated above 60 °C.