Dependence of microbial transglutaminase on meat type in myofibrillar proteins cross-linking

The objectives of this study were to determine the factors that cause differences in the improvements of gel strength and ε(γ-glutamyl)lysine (G-L) content in chicken and beef (Japanese black cattle) myofibrillar proteins after adding microbial transglutaminase (MTG). As the amount of MTG added increased, the breaking strength increased progressively (p < 0.01) in chicken and beef samples, with the exception of chicken samples treated at 40 °C. The values of elasticity in the chicken samples were lower than those of the beef samples (p < 0.01). Surprisingly, the elasticity level, ε(γ-glutamyl)lysine contents and myosin heavy chain (MHC) band sizes of chicken and beef at all levels of MTG were significantly different (p < 0.01). The results of this study suggest that MTG activity was affected by MTG inhibitors; that MTG develops the texture of myofibrils differently in different species. However, the activity is limited and inconstant among meat proteins, as suggested by the data collected from the chicken samples. As a result, when the transferable amino acid residues are depleted (cross-linked) by MTG activity, the function of MTG will be insignificant. The correlation between MTG and different sources of meat protein is quite unstable but it is strong, which was observed when chicken and beef responded differently to MTG because their chemical and physiological properties were different. The remarkable rate of formation of cross-linked proteins and the discrepancy between the expected and observed amount of dipeptide raises the possibility that there are enzymes capable of reversing the reaction induced by transglutaminase in chicken and beef myofibrils. In summary, our results suggest that access of MTG to chicken and beef myofibrils is different because it depends on physiological (muscles and their fibre types), biological (substrates) and biochemical (inhibitors and amino acids) variables.     

Effect of microbial transglutaminase on the natural actomyosin cross-linking in chicken and beef

The objective of this research was to investigate the difference between chicken and beef in the interaction of actomyosin (myosin B) with microbial transglutaminase (MTG). The gel strength of myosin B was improved in both species and was significantly greater in beef than in chicken (P < 0.01). The degree of protein viscosity and the ε(γ-glutamyl)lysine (G–L) content were significantly higher in beef than in chicken (P < 0.01). Myosin heavy chain (MHC) bands visualized by SDS–PAGE revealed that the same proteins in various meat species vary in their size and structure. Scanning electron microscope images (SEMI) revealed that myosin B in both species was polymerized, and formed multi-projection structures of G–L; surprisingly, more of these structures were found in beef than in chicken. It is possible that the proteins in chicken are folded into a strand shape that tightly encases a considerable number of glutamine and lysine residues, whereas MTG substrate cannot couple glutamine and lysine. This suggests that the reactivity of MTG is dependent on the residual amino acids present on the surface of myosin B in meat. Some protein components (peptides with long reiterated methylene groups attached) joined by disulfide bonds (cysteine) in chicken samples were inhibitory and reduced MTG activity. SEMI also suggested that all MTG-dependent mega-structures of protein molecules generated in chicken and beef may vary greatly in size, configuration and complexity after treatment with MTG. We concluded that the optimal cross-links in myosin B induced by MTG are heterogeneous in chicken and beef.     

Gelation properties of myofibrillar/pea protein mixtures induced by transglutaminase crosslinking

Gelation properties of mixtures of myofibrillar protein isolate (MPI)/pea protein isolate (PPI) were studied using a dynamic oscillatory rheometer and a texture analyzer to evaluate PPI as a possible meat product additive. The inclusion of microbial transglutaminase (MTG) increased the gel strength of MPI/PPI mixture (3% + 1%) more than it did for MPI (3%), but less than a 3% MPI, 1% soy protein isolate combination. The direct evidence of interaction between muscle and pea proteins in the form of new sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) bands was not found; however, the improvement in gel strength or gel peak force for the MPI/PPI mixture (3% + 1%) with inclusion of MTG suggested that some ? (γ-glutamyl) lysine (G-L) crosslinking occurred between muscle and pea proteins. It likely that pea protein acted as a non-gelling component and interspersed throughout the primary MPI gel network and the addition of MTG promoted partial crosslinking of MPI. Consequently, MTG is useful in improving gelation properties of heat-induced MPI/PPI gel.     

Gelation properties of chicken myofibrillar protein induced by transglutaminase crosslinking

Gelation properties of chicken myofibrillar protein isolate (MPI) and the effect of microbial transglutaminase (MTG) were studied using a dynamic oscillatory rheometer and a texture analyzer. Final heating temperature had a great impact on gel stiffness and the maximum gel stiffness was obtained at 95 °C. pH and ionic strength also influenced gel stiffness and the maximum gel stiffness was achieved at pH 6, 0.9 M NaCl; however, less stiff gels were formed in 0.6 and 1.2 M NaCl. In the MPI concentration range of ∼0.5-5%, a positive correlation was observed between gel stiffness or gel peak force and MPI concentration. When MTG was included at levels of ∼0 to 12-15 U, positive linear relations were found between gel stiffness or peak force and MTG levels. However, negative correlations for these parameters were observed at higher MTG concentrations. When MTG level was greater than 15 U, gel stiffness or peak force tended to decrease. The improvement in gel strength or gel peak force for the MPI with inclusion of MTG suggested that some ε (γ-glutamyl) lysine (G-L) crosslinking occurred among myofibrillar molecules. Thus, MTG is useful in improving gelation properties of heat-induced MPI gel and provides new opportunities to expand the utilization of low value meat in muscle foods.     

Impact of transglutaminase on the textural, physicochemical, and structural properties of chicken skeletal, smooth, and cardiac muscles

This study examined the effects of microbial transglutaminase (MTG; 3.1 mg/ml) on chicken skeletal, smooth, and cardiac muscles; the meat containing the different muscle types was shaped into sausages and treated at 40 °C and/or 78 °C for 30 min. Although the three muscle types were obtained from the same bird, the effects of MTG addition were not uniform. All the muscle types showed a significant increase in the breaking strength (P < 0.01), but skeletal muscle exhibited the maximum increase. All samples showed a decrease in the fluorescence intensity and a significant reduction in the concentration of proteins that were extracted in a high ionic strength solution (P < 0.05). Scanning electron microscopy images and histological studies revealed that different muscle types had different physical structures and frameworks after MTG treatment, which is a reflection of the differences in the reaction specificity of MTG with different muscle proteins. Histological studies revealed that the reactions of MTG with meat proteins are both exogenous and endogenous. Cooking loss data suggested that MTG did not have any negative effect on water retention during cooking. MTG appears to be a functional and contributive substance since the results suggest that MTG can function on all muscle types that are mechanically processed for different industrial applications. MTG aggregates muscle proteins in different ways that improve their organoleptic properties such as texture, appearance, and water retention.     

Impact of transglutaminase on the textural,physicochemical, and structural properties of chicken skeletal,smooth, and cardiac muscles

This study examined the effects of microbial transglutaminase (MTG; 3.1 mg/ml) on chicken skeletal, smooth, and cardiac muscles; the meat containing the different muscle types was shaped into sausages and treated at 40 °C and/or 78 °C for 30 min. Although the three muscle types were obtained from the same bird, the effects of MTG addition were not uniform. All the muscle types showed a significant increase in the breaking strength (P < 0.01), but skeletal muscle exhibited the maximum increase. All samples showed a decrease in the fluorescence intensity and a significant reduction in the concentration of proteins that were extracted in a high ionic strength solution (P < 0.05). Scanning electron microscopy images and histological studies revealed that different muscle types had different physical structures and frameworks after MTG treatment, which is a reflection of the differences in the reaction specificity of MTG with different muscle proteins. Histological studies revealed that the reactions of MTG with meat proteins are both exogenous and endogenous. Cooking loss data suggested that MTG did not have any negative effect on water retention during cooking. MTG appears to be a functional and contributive substance since the results suggest that MTG can function on all muscle types that are mechanically processed for different industrial applications. MTG aggregates muscle proteins in different ways that improve their organoleptic properties such as texture, appearance, and water retention.     

Extruded meat alternatives made from Maillard-reacted beef bone hydrolysate and plant proteins. Part II – application in sausages

This study investigated the physicochemical properties of sausages made from meat alternatives made from Maillard-reacted beef bone hydrolysate and plant proteins (soy protein and wheat gluten) at different moisture contents (MC; S49%MC, S52%MC, S56%MC and S60%MC). S49%MC had the highest hardness and chewiness, observed with chunks of long fibres under SEM. The hardness and chewiness of sausages decreased as MC increased. S60%MC exhibited soft and mushy texture, and no fibre structure was observed. Sausages made from meat alternatives had higher protein oxidation as compared with reference sausage made from chicken breast (SCB), which could be due to longer storage period, as meat alternatives were extruded, frozen and stored before making into sausages. Sensory results showed that SCB obtained the highest scores for all attributes except for appearance, among all sausages. Overall results showed that further improvements can be made when using extruded meat alternatives to make sausages.     

高压和食盐对鸡肉肠品质特性的影响

在不同压强条件（0.1、200、400 MPa）下对不同食盐添加量（质量分数0%、1%、2%）的生鸡肉肠加热（60 ℃）处理30 min,测定处理后鸡肉肠的持水力、水分分布、质构特性和微观结构,分析高压和食盐对鸡肉肠热诱导凝胶特性的影响。结果发现：常压下,减少食盐添加量会降低鸡肉肠的保水性和质构特性,但高压处理能降低食盐对鸡肉肠的影响。相比常压组,200 MPa高压处理能显著提高产品的保水性和质构特性（P＜0.05）,而400 MPa的高压处理则显著降低产品的保水性和质构特性（P＜0.05）。高压能促进肉糜中的肌纤维分解,使不易流动水的比例升高,自由水比例下降;200 MPa下能形成致密的凝胶网络,而400 MPa则阻碍凝胶网络的形成。高压结合加热处理可用于提高低盐鸡肉肠的品质特性。     

Thermally Induced Changes in Protein Extractability of Postrigor Turkey Breast Meat Measured by Two Methods

Extractability of sarcoplasmic and myofibrillar proteins was reduced in postrigor turkey breast meat held at 40 °C for as short as 30 min. These reductions in protein extractability were successfully detected, using either a filtration-based methodology or one based on centrifugation. However, the coefficient of variation for the filtration method was as much as 4 times greater than for centrifugation. Additionally, the filtration method overestimated sarcoplasmic protein extractability due to the inability to exclude myofibrillar proteins. Centrifugation results indicated the extractability of proteins in 0.55 M KCl was reduced to 52% of controls for samples held at 40 °C for 120 min. Additionally, holding postrigor turkey breast muscle at 40 °C resulted in increased myosin degradation.     

Prevalence and antimicrobial susceptibility of Salmonella isolated from a variety of raw meat sausages in Gaborone (Botswana) retail stores

The objective of the study was to provide baseline data on the prevalence and antimicrobial susceptibility of Salmonella in different types of raw meat sausages directly accessible to the consumers in Gaborone, Botswana. A total of 300 raw sausages comprising 79 beef, 78 pork, 72 chicken, and 71 mutton samples were concurrently analyzed for the presence of Salmonella using a conventional culture method and a validated PCR method. The PCR assay results were in full concordance with those of the conventional culture method for the detection of Salmonella. Sixty-five (21.7%) of 300 samples were positive for Salmonella by both the conventional culture method and PCR assay. Even though more chicken samples contained Salmonella than did any other sausage type, the difference in the presence of Salmonella among the four sausages types was not significant. Eleven serotypes were identified, and Salmonella enterica subsp. salamae II was most prevalent in all the sausage types. Beef sausages generally had higher mesophilic bacterial counts than did the other three sausage types. However, higher microbial counts were not reflective of the presence of salmonellae. Susceptibility of the Salmonella enterica serotypes to 20 antimicrobial agents was determined, and Salmonella Muenchen was resistant to the widest array of agents and was mostly isolated from chicken sausages. Regardless of the meat of origin, all 65 Salmonella isolates were resistant to at least four antimicrobial agents: amikacin, gentamicin, cefuroxime, and tombramycin. This resistance profile group was the most common in all four sausage types, comprising 90% of all Salmonella isolates from beef, 71% from pork, 63% from mutton, and 35% from chicken. These results suggest that raw sausages pose a risk of transmitting multidrug-resistant Salmonella isolates to consumers.     

Use of high pressure to reduce cook loss and improve texture of low-salt beef sausage batters

Methods were investigated to reduce the salt content of beef-containing smallgoods as high-salt intake has been identified as a public health risk for most individuals. Raw meat batters were manufactured from retail beef mince (4–7% fat) using various NaCl concentrations (0–2%), and were packed into casings and subjected to high pressure processing (up to 400 MPa for 2 min at 10 °C). Following pressure treatment, samples were cooked to an internal temperature of 72 °C and cooled. Cooked products were assessed for cooking loss, colour and physical consistency by texture profile analysis. Flavour and overall acceptability were assessed by sensory panels. High pressure processing (HPP) was found to produce a dramatic improvement in the moisture retention of the cooked products. Control (unpressurised) sausages containing 2% NaCl had a similar cook loss (9.3%) to pressure-treated sausages containing just 1% NaCl, whereas unpressurised samples with 1% NaCl had a cook loss of 24.9%. The hardness and gumminess of pressure-treated samples was higher compared to untreated samples, at all salt concentrations. The greatest differences in texture with pressure treatment were seen in the 1% NaCl samples. Pressure treatment generally caused no changes in the colour of either the raw or cooked product; however there was a slight increase in “whiteness” with pressure treatment. Sensory panels reported a greater acceptability in both appearance and texture of pressure-treated sausages of lower salt content compared with non-pressure-treated samples. Examination of extracted proteins using SDS-PAGE and of muscle proteins by thermal analysis indicated that pressure contributed to enhanced binding through protein solubilisation and gelation through partial protein unfolding. The application of high pressure to beef sausages with low-salt content resulted in reduced cooking losses and improved texture.

Industrial relevance

Enhanced meat binding through extraction of salt-soluble proteins is an essential step in the formulation of meat products such as sausages and emulsion-type products. The ability to reduce salt and achieve high binding and water retention through use of HPP is important in being able to produce healthier foods.     

Effects of sodium tripolyphosphate on functional properties of low‐salt single‐step high‐pressure processed chicken breast sausage

Influences of sodium tripolyphosphate (STPP) contents (0.1, 0.2, 0.3 and 0.4%) on water holding capacity (WHC) and texture properties of low‐salt (1.2% NaCl) single‐step high‐pressure processed chicken breast sausages (LSSS‐HPP sausages) were evaluated. Results showed that WHC was improved (4–5%) by the addition of STPP. However, the STPP contents customarily used for cooked sausages (0.3–0.5%) were excessive for LSSS‐HPP sausages, causing a soft and tacky texture. Sausages containing 0.1% of STPP had the best taste according to the sensory evaluations. Chemical interactions plus Raman spectroscopic analysis revealed that STPP addition partly changed native structures of myofibrillar proteins. Furthermore, higher STPP contents in the meat batter prevented those proteins from high pressure denaturing and aggregating in the subsequent single‐step HPP procedure. Increased hydrogen bonds and decreased hydrophobic interactions explained the better WHC and softer texture. Therefore, 0.1% of STPP is the optimal content in the processing of new‐type LSSS‐HPP sausages.     

Proteolysis,physicochemical characteristics and free fatty acid composition of dry sausages made with deer (Cervus elaphus) or wild boar (Sus scrofa) meat: A preliminary study

In order to contribute to typifying delicatessen made with game meat, the proteolysis, physicochemical characteristic and free fatty acid composition were determined in 10 commercial dry sausages, chorizos and saucissons, made with deer or wild boar meat. The a_w and pH values were similar for all the samples; however, the results for dry matter, protein nitrogen, fat, ash, sodium chloride, phosphorus, and sodium nitrite content showed great variation among the samples tested. The myofibrillar protein content was higher than the sarcoplasmic protein content in all samples analysed. The electrophoretic profiles of sarcoplasmic and myofibrillar proteins were different among samples. Principal components analysis, run on the relative density of myofibrillar and sarcoplasmic proteins, separated the chorizo and saucisson samples. Chorizo samples were a homogeneous group in the analysis of myofibrillar proteins, which indicated similar proteolysis effects for all samples. The majority acids were oleic, palmitic, linoleic and stearic in all samples. Chorizos differed from saucissons in the greater quantity (P < 0.05) of polyunsaturated fatty acids.     

Biochemical and technological characteristics of hot chicken meat

In the hot breast and leg muscles of broiler chicken the level of ATP, the 'R' value, the lactic acid content, the pH value, the length of sarcomers, the water and fat retention capacity, the fat emulsion stability, thermal drip, and the extractability of protein fraction were investigated. It was found that in the breast muscles the onset of rigor mortis commenced within 30–60 min, and in the leg muscles as early as 15–30 min after killing of the birds. The deepest rigor mortis occurred between the first and fourth hour, and then gradually declined, sooner in the leg than in the breast muscles. The addition of sodium chloride (2.0–2.5%) to the minced pre-rigor meat not later than 40 min after slaughter, or better, an injection of NaCl brine into intact muscles 15 min after slaughter of birds, preserved their good technological properties.
The tenderness and the thermal drip of hot salted and chilled salted muscles showed no significant differences, but water retention and fat emulsifying capacity were better in the hot salted meat samples. The hot salted and cooked muscles were preferred by the sensory panel to corresponding samples of chilled muscles.
From the hot salted chicken meat more sarcoplasmic and myofibrillar proteins were extracted than from meat salted after chilling. However, after frozen storage the extractability of myofibrillar proteins were higher in the salted chilled meat.     

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.     

Quantification of beef,pork, chicken and turkey proportions in sausages: use of matrix-adapted standards and comparison of single versus multiplex PCR in an interlaboratory trial

Quantitative PCR methods for the determination of beef, pork, chicken and turkey proportions in sausage were tested in an interlaboratory trial. Twelve different laboratories analysed six meat products each made of different compositions of beef, pork, chicken and turkey. Two kinds of calibrators were used: sausages of known proportions of meat and DNA from muscle tissue. Results generated using calibration sausages were more accurate than those resulting from the use of muscle tissue DNA. Regardless of the method used (either multiplex or single PCR), when using calibration sausages, it was always possible to quantify the proportions of meats in the unknown samples (in the range of 0.5–80%) with high precision and accuracy.     

Evaluation of plasma and plasma-alginate fibres for use in sausages

Fresh beef sausages were made in which 0,20 or 40% of the meat protein was replaced by protein from bovine blood plasma in order to determine the effect this had on stability, shrinkage, texture, juiciness, flavour, colour and general acceptability. Both the plasma proteins as isolated, and after spinning into fibres, were examined.

Sausages in which 20 or 40% of the meat protein had been replaced by plasma-alginate fibres were preferred less than those made at the same levels of replacement using plasma protein as isolated, taste panel members disliking their flavour and colour.

On the other hand, sausages incorporating plasma-alginate fibres were drier than those containing corresponding replacement levels of plasma protein as isolated, and their texture was preferred.     

风干肠加工和贮藏过程中蛋白质的降解规律

以总氮、非蛋白氮、氨基酸态氮、挥发性盐基氮、游离氨基酸以及不同种类蛋白质（肌浆蛋白、肌原纤维蛋白、碱溶蛋白和碱不溶蛋白）为指标,分析研究风干肠加工和贮藏过程中蛋白质的降解规律。结果表明：风干肠加工和贮藏过程中总氮含量没有显著变化;非蛋白氮含量持续升高,从原料肉的469.4 mg/100 g增加到成品肠的786.4 mg/100 g,贮藏240 d后增加至1 256.1 mg/100 g;多肽氮和氨基酸态氮含量在加工和贮藏过程中均持续增加,挥发性盐基氮含量在风干过程中没有显著变化,在贮藏180 d后迅速增加;大部分游离氨基酸含量经过加工和贮藏均有所提高,只有半胱氨酸含量明显降低;在加工过程中,肌浆蛋白和肌原纤维蛋白含量均因降解而减少,基质蛋白含量明显增加;在贮藏过程中,肌浆蛋白含量持续降低,肌原纤维蛋白含量在贮藏前180 d无明显变化,贮藏至240 d明显减少,基质蛋白含量没有显著变化。     

Analysis of texture and dynamics of water binding in enzymatically modified myofibrillar preparation obtained from washed mechanically recovered poultry meat

Experiments were conducted on a myofibrillar preparation, obtained from washed mechanically recovered poultry meat. An enzymatic preparation containing microbial transglutaminase (MTG) was added to samples of the myofibril isolate. The binding of water contained in the protein preparation with added MTG was assessed using low field nuclear magnetic resonance (NMR). Moreover, texture was analyzed in myofibril samples with the addition of transglutaminase pre-incubated for 0.5, 1.5, 3, 4.5 and 6 h. All measurements were taken at 7 ± 0.2 °C. Samples with added transglutaminase exhibited improved mechanical failure strength and better water binding capacity. The most dynamic increase of texture parameter values was observed in the interval from 1.5 to 3 h pre-incubation of the preparation with the added enzyme. Based on NMR (T ₁) testing it was established that the highest amount of water was bound by protein in the period from approximately 1 to 1.5 h pre-incubation. After that time free water content in the sample was again found to increase. This means that water was displaced from the system as a result of protein–protein interactions dominating over protein–water interactions. The above suggests that the enzymatic modification of the protein preparation contributed to the intensification of cross-linking between proteins in the preparation.     

Organic Acids as Tenderizers of Collagen in Restructured Beef

Acetic, citric and lactic acid were incorporated into restructured beef steaks to determine their effect on collagen. These steaks were analyzed for collagen solubility, total collagen content, shear force behavior, sarcoplasmic and myofibrillar protein extractability, thermal stability and color. These results were compared to those obtained from control samples containing high collagen and low collagen. Results indicate that these acids increased collagen solubility, total collagen content and shear force values when compared with the control samples. No differences (P > 0.05) were found in sarcoplasmic protein extractability although myofibrillar protein extractability declined. Acid treatment decreased the thermal stability of collagen. The Hunter Color values of the uncooked and cooked acid-treated steaks differed (P 0.05) from the controls.