Transglutaminase Concentration Research Articles

Determination of microbial transglutaminase in meat and meat products

Transglutaminase is an enzyme that can be used to cross-link pieces of meat, fish or meat products. The resulting product gives the optical impression of an intact chunk of meat. The usage of transglutaminase as a food additive is permitted in some countries. However, its utilisation has to be declared to ensure transparency for consumers. This paper describes two orthogonal analytical methods suited for the detection of technological relevant transglutaminase concentrations (around 25 mg pure enzyme in 1 kg of product) in meat and meat products. The mass spectrometry-based approach relies on a previous digestion with Achromobacter lyticus protease and LC-MS/MS separation and detection. Sufficient selectivity was obtained by monitoring four different peptides. The orthogonal (complementary and independent), ELISA-based approach relies on two commercially available bacterial transglutaminase-specific antibodies, combined to a sandwich ELISA. The two methods were tested by analysing some 60 samples obtained from the market.

Synthesis and release studies of microalgal oil-containing microcapsules prepared by complex coacervation

Microalgal oil was encapsulated in gelatin-gum Arabic complex coacervated matrices using transglutaminase (TG) as cross-linking agent. The effects of various cross-linking parameters including hardening time, temperature, pH and TG concentration on the oil release rate of the complex coacervation microcapsules (CCMs) were investigated, with sodium dodecyl sulfate (SDS) as release medium. The optimum parameters were as follows: hardening for 6h at 15°C and pH 6.0 with TG concentration of 15U/g gelatin. The microcapsules obtained under optimum conditions had the lowest oil release rate. By analyzing the oil release curves, it was found that the oil release rate did not exactly fit the modified first-order kinetic model but exhibited early time and late time approximations for unsteady state diffusion. A greater initial release of oil was clearly observed whatever cross-linking parameters were, while the release profile became constant indicating some sustained release after the first hour.

Immobilization ofErwiniasp. D12 Cells in Alginate-Gelatin Matrix and Conversion of Sucrose into Isomaltulose Using Response Surface Methodology

Isomaltulose is a noncariogenic reducing disaccharide and also a structural isomer of sucrose and is used by the food industry as a sucrose replacement. It is obtained through enzymatic conversion of microbial sucrose isomerase. An Erwinia sp. D12 strain is capable of converting sucrose into isomaltulose. The experimental design technique was used to study the influence of immobilization parameters on converting sucrose into isomaltulose in a batch process using shaken Erlenmeyer flasks. We assessed the effect of gelatin and transglutaminase addition on increasing the reticulation of granules of Erwinia sp. D12 cells immobilized in alginate. Independent parameters, sodium alginate concentration, cell mass concentration, CaCl2 concentration, gelatin concentration, and transglutaminase concentration had all a significant effect (P < 0.05) on isomaltulose production. Erwinia sp. D12 cells immobilized in 3.0% (w/v) sodium alginate, 47.0% (w/v) cell mass, 0.3 molL−1 CaCl2, 1.7% (w/v) gelatin and 0.15% (w/v) transglutaminase presented sucrose conversion into isomaltulose, of around 50–60% in seven consecutive batches.

Rheological properties and baking performance of rye dough as affected by transglutaminase

Since protein aggregation and formation of a continuous protein matrix in rye dough is very limited, an enzyme-induced protein aggregation method to improve the baking properties was investigated. The effects of microbial transglutaminase (TG) on the properties of rye dough were studied by rheological tests, confocal laser scanning microscopy (CSLM), standard-scale baking tests and crumb texture profile analysis. Addition of TG in the range of 0–4000 Ukg −1 rye flour modified the rheological properties of rye flour dough, resulting in a progressive increase of the complex shear modulus (| G∗|) and in a decrease of the loss factor (tan δ) due to protein cross-linking or aggregation. CLSM image analysis illustrated a TG-induced increase of the size of rye protein complexes. Standard baking tests showed positive effects on loaf volume and crumb texture of rye bread with TG applied up to 500 Ukg −1 rye flour. Higher levels of TG (500 U ≤ TG ≤ 4000 U) had detrimental effects on loaf volume. Increasing TG concentration resulted in an increase of crumb springiness and hardness. In conclusion, the results of this work demonstrated that TG can be used to improve the bread making performance of rye dough by creating a continuous protein network.

Optimization of microbial transglutaminase activity in ice cream using response surface methodology

In order to determine the maximum activity range of transglutaminase (TG) in ice cream, the central composite design was used and the TG concentration along with the reaction time and temperature were evaluated. The rheological behavior of the ice cream mix that provided the best response and the determination of the protein cross-linking by electrophoresis (SDS-PAGE) were evaluated. It was observed that the TG increased the consistency index and favored the pseudoplastic behavior of the ice cream samples. The maximum response (0.69 Pa s n) was obtained using a TG concentration of 4 U g −1 protein at 56.8 °C with a reaction time of 90 min. The protein cross-linking was confirmed by electrophoresis, which showed that κ-casein was more susceptible to attack from TG than whey proteins.

Rheological and baking characteristics of batter and bread prepared from pregelatinised cassava starch and sorghum and modified using microbial transglutaminase

The effect of different concentrations (0, 0.5, 1 and 1.5 U/g) of microbial transglutaminase (MTG) on the creep-recovery properties of gluten-free batter prepared from pregelatinised cassava starch, sorghum and egg white was investigated. The test conducted in the rheometer had an instant loading of 80 Pa for 60 s and recovery of 0 Pa for 140 s. Increasing MTG concentration decreased the batters’ resistance to deformation and compliances but increased zero shear viscosity and elastic recovery. Changes in batter rheological properties were insignificant ( P > 0.05) at MTG concentrations beyond 0.5 U/g. Crumb properties of gluten-free bread baked from the batter revealed that increasing MTG concentration increased ( P < 0.05) crumb firmness and chewiness, whereas increasing incubation time decreased ( P < 0.05) crumb cohesiveness, chewiness and resilience. There were no significant interaction effects ( P > 0.05) between enzyme concentration and incubation time.

Increase of fibrin gel elasticity by enzymes: A kinetic approach

Two enzymes, thrombin and transglutaminase, both participate in the formation of fibrin networks and contribute to the mechanical strength of biogels. A theoretical model built from the available kinetic data showed that a competition may take place between the two enzymes for their common substrate, fibrinogen. To evidence this phenomenon experimentally, the concentrations of the reactants were varied and the rheological properties of the resulting fibrin gels explored. The elasticity of the gels was not a singular function of the transglutaminase concentration, the optimum being also related to fibrinogen and thrombin concentrations. Thrombin concentration influenced the kinetics of gelation, but not the evolution of the mechanical properties of the gel. An indirect relationship between gel elasticity and thrombin concentration was observed upon covalent binding. The liquid phase inside the gel contained a high amount of soluble proteins when a high transglutaminase concentration was used. The impact of this competition between the two enzymes, demonstrated here for the first time, is evaluated for biomaterial elaboration.

Characterization of fish gelatin from surimi processing wastes: Thermal analysis and effect of transglutaminase on gel properties

Fish gelatin extraction from wastes of fish Herring species ( Tenualosa ilisha) was carried out by a series of pretreatment with 0.2 M Ca(OH) 2 followed by 0.1 M citric acid and final water extraction at 50 °C for 3 h. The resulting fish gelatin preparation was evaluated for its dynamic viscoelastic properties, gelling and melting temperatures and gel strength. The gelling and melting temperatures of gelatin samples (at 6.67%, w/v) were obtained from differential scanning calorimetry and rheological studies. The melting temperature of extracted fish gelatin (EFG) obtained ranged from 16.2 to 16.7 °C compared to that of commercial fish gelatin gel (CFG), from 23.7 to 25.6 °C and halal bovine gelatin (HBG), from 26.5 to 28.7 °C. On the other hand, gelling temperatures of EFG, CFG and HBG ranged from 5.1 to 5.2 °C, 11.9 to 17.46 °C, and 12.6 to 19.33 °C, respectively. EFG gave gels with a considerably lower G′ values than CFG and HBG. The bloom strength of EFG gels at 6.67% (w/v) was 69.03 g which was much lower than HBG (336.2 g) and CFG (435.9 g). Enzyme transglutaminase was added in the amounts of 0.5, 1.0, 3.0 and 5.0 mg/g gelatin to modify the gel properties of the extracted fish gelatin. The modified EFG gels obtained had higher gel strengths of 101.1 g and 90.56 g with added transglutaminase of 1.0 and 3.0 mg/g, respectively. However with addition of 5.0 mg/g enzyme the gel strength increased only up to 75.06 g. SDS-PAGE of extracted gelatin gel showed protein band intensities for α1-chains and 53 kDa but in gels added with higher concentration of transglutaminase, these protein band intensities seemed to disappear.

Optimization of cross-linking parameters during production of transglutaminase-hardened spherical multinuclear microcapsules by complex coacervation

Gelatin-gum arabic spherical multinuclear microcapsules (SMMs) encapsulating peppermint oil were prepared by complex coacervation. Transglutaminase (TG) was used to harden the SMMs by complex coacervation instead of traditional reagents such as formaldehyde or glutaraldehyde. The effect of various cross-linking parameters on the hardening effectiveness of SMMs containing peppermint oil was investigated. The optimum parameters were as follows: hardening for 6h at 15 degrees C and pH 6.0 with a TG concentration of 15 U/g gelatin. Compared with formaldehyde, TG exhibits similar microcapsule hardening effectiveness.

Improvement of canola protein gelation properties through enzymatic modification with transglutaminase

Enzymatic modification with transglutaminase (TG) was used to enhance the gelation of canola protein isolate (CPI) and thus improve its potential as a food ingredient. The effects of CPI concentration, TG concentration, treatment temperature and treatment time on CPI gelation properties were evaluated. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. The protein concentration, amount of TG, and treatment temperature were found to significantly impact gel strength. It was found that gelation can be improved by increasing the amounts of protein and TG and by keeping the treatment temperature close to 40 °C. SDS-PAGE showed that cross-linking of subunits occurred through TG treatment thus helping to explain the increase in gel strength observed during texture analysis. Micrographs further corroborated the trends noted during rheological studies.

Influence of transglutaminase protein cross-linking on the rennet coagulation of casein

The influence of protein cross-linking on the rennet coagulation of casein was investigated using a microbial transglutaminase (TG) preparation containing glutathione (GSH). As unheated milk is normally less reactive towards TG, GSH was applied in order to enable non-inhibited cross-linking. Thus, interactions between TG cross-linking and rennet coagulation and their impact on the renneting properties were studied independently from a pre-heat treatment of milk beyond pasteurisation. Protein cross-linking was carried out either prior to or simultaneously with the addition of rennet. Coagulation times t C and gel firmness assessed as the storage modulus G 60 min ′ could be related either to TG pre-incubation times or to TG concentrations. The results show that enzymatic cross-linking affects both the primary and the secondary stage of rennet coagulation. The impact of protein cross-linking on the primary enzymatic phase can be mainly related to increased coagulation times due to inhibition of the caseinomacropeptide release. Consequently, a higher extent of protein cross-linking leads to reduced gel firmness. The simultaneous reaction of TG and rennet, however, results in markedly increased yield after centrifugation due to the enhanced serum binding of the gel network stabilised by additional covalent bonds. In conclusion, the cross-linking has an intense impact on the entire renneting process leading to rennet gels with modified functional properties.

Physico-chemical properties of probiotic yoghurt produced with transglutaminase

The effect of different concentration of transglutaminase - TG (0.02%, 0.06% and 0.12%) on physico-chemical properties of probiotic yoghurt was investigated. Two series of yoghurt were manufactured on a laboratory scale from pasteurised skim milk (0.1 % w/w fat). Series I was produced with TGase activation during a period of 2 h at 40?C, while series II was produced without enzyme activation. Then, the adequate quantities of TGase and probiotic starter culture ABT-4 (Chr. Hansen A/S Denmark) were added. Chemical composition, physico-chemical properties (water holding capacity and whey separation) and sensory characteristics of yoghurt samples were determined after production and during 5 days of storage. Addition of TGase to milk (direct or after activation) for probiotic yoghurt manufacture improved its overall characteristics. Activation of TG in yoghurt production increases water holding capacity as well as decreases syneresis during the storage.

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β- d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.

High-throughput scintillation proximity assay for transglutaminase activity measurement

Members of the transglutaminase enzyme family are involved in a broad range of biological phenomena, including haemostasis, apoptosis, semen coagulation, skin formation, and wound healing. A new and rapid method for measurement of transglutaminase activity is described in this article. The enzyme links tritium-labeled putrescine to biotinylated oligoglutamine, and the tritiated peptide is bound to a streptavidin-coated microtiter plate permanently covered by a thin layer of scintillant. Only the radioisotope incorporated into the peptide substrate is close enough to the scintillant molecules for photons to be produced. The signal generation depends on the transglutaminase activity, and it can be detected by appropriate light-measuring instrumentation without separation steps. The assay is sensitive, specific, linear at concentrations of tissue transglutaminase between 0.05 and 1.6 mU/ml, and suitable for high-throughput measurements.

Use of Low Concentrations of Human IgA Anti-Tissue Transglutaminase to Rule Out Selective IgA Deficiency in Patients with Suspected Celiac Disease,

Selective IgA deficiency (IgAD) is the most common well-defined primary immunodeficiency disorder in humans (1)(2). Patients with IgAD frequently share the haplotype HLA-DQ2, which is also associated with celiac disease (CD) (3), and therefore have a 10- to 20-fold increased risk of CD (4). High concentrations of anti-tissue transglutaminase (h-tTG) IgA antibody are used to diagnose CD (5)(6), but antibodies are not increased in IgAD (7)(8). This has led to the use of assays for total IgA when testing for CD and/or testing for IgG-class antibodies against h-tTG (9). The aim of our study was to assess whether a second-generation IgA anti-h-tTG assay can detect IgAD, as the concentrations of IgA antibodies would be expected to be very low. This could eliminate the expense for additional tests in many individuals. We studied 4 groups of patients. The disease group included 28 patients with IgAD [18 females (median age, 38 years; range, 8–79 years) and 10 males (median age, 24 years; range, 5–75 years)] diagnosed between June 2001 and May 2003. All had total IgA concentrations 0.05 g/L but below the lower limit of the reference interval (0.70 g/L; median IgA, 0.39 g/L; range, 0.07–0.69 g/L). The final diagnoses in the adult diseased controls were multiple myeloma (30 patients), chronic lymphoid leukemia (4), anemia (4), chronic kidney failure (4), Waldestrom disease (3), acute pulmonary edema (2), scleroderma (1), and acute pericarditis (1). The 14 pediatric patients of this group presented with complaints in relation to …

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.

EFFECT OF COLD‐SET BINDERS: ALGINATES AND MICROBIAL TRANSGLUTAMINASE ON THE PHYSICAL PROPERTIES OF RESTRUCTURED SCALLOPS

ABSTRACT Restructured scallops (Argopecten gibbys) were prepared with two cold‐set binders: 1% concentrations of alginate and microbial transglutaminase (MTG), with setting times of 2, 6, 9, 12, 18 and 24 h at 5C. Apparent modulus of elasticity, 5% secant modulus, 10% secant modulus and 20% secant modulus were used to evaluate the effect of setting times on the binding strength of restructured scallops. The binding strength as measured by the 5, 10 and 20% secant modulus was not significantly different (P &gt; 0.05) in MTGase‐restructured scallops, but significantly different in alginate‐restructured scallops at 5 and 10% strain. Apparent modulus of elasticity showed significant differences in the binding strengths (P &lt; 0.05) of restructured scallops prepared from both binders. The alginate gel restructured scallops achieved a binding strength of (158.7 kPa) in a 2‐h setting, while MTG‐restructured scallops reached a binding strength of (336.0 kPa) at a 24 h setting.

Anti-tissue transglutaminase antibodies in inflammatory bowel disease: new evidence

Anti-tissue transglutaminase, previously held to be identical to anti-endomysial antibodies in celiac sprue, has been reported in inflammatory bowel disease patients. To investigate these data further, we evaluated serum and intestinal anti-tissue transglutaminase in inflammatory bowel disease patients, with respect to the Crohn's disease activity index and the integrated disease activity index. Study population comprised: 49 patients with Crohn's disease and 29 patients with ulcerative colitis; 45 patients with celiac sprue and 85 autoimmune patients as disease controls; and 58 volunteers as healthy controls. Immunoglobulin A (IgA) anti-recombinant human tissue transglutaminase and anti-endomysial antibody detection in sera and fecal supernatants were performed. Adsorption of positive sera with recombinant human tissue transglutaminase were also performed. Marked increased anti-tissue transglutaminase concentrations were found in celiac sprue, while low-positive values were also found in Crohn's disease and ulcerative colitis. Anti-endomysial antibodies were detectable only in celiac sprue. Antigen adsorption resulted in a significant reduction of the anti-tissue transglutaminase either in celiac sprue or inflammatory bowel disease sera. A significant correlation between anti-tissue transglutaminase and Crohn's disease activity index or integrated disease activity index scores was found. Anti-tissue transglutaminase was also detectable in fecal supernatants from inflammatory bowel disease patients. Data highlight that both circulating and intestinal anti-tissue transglutaminases are detectable in inflammatory bowel disease, and that they are related to disease activity. These features underline that, in addition to anti-tissue transglutaminase, an anti-endomysial antibody test is necessary in the diagnostic work-up of celiac sprue, especially in patients with known inflammatory bowel disease.

Functionality of rice flour modified with a microbial transglutaminase

Abstract Rice flour is one of the most valuable cereal flours from a nutritional viewpoint. However, its use is limited to unfermented baked products since rice proteins are unable to hold gas produced during fermentation. Protein functionality can be modified by cross-linking. Rice flour was treated with different concentrations of a microbial transglutaminase (TG). The extent of the modification was evaluated by quantifying amino and thiol groups. The addition of TG improved the dynamic rheological properties of rice flour doughs, resulting in a progressive increase of the viscous (G″) and elastic (G′) moduli with increase in TG concentration. The improvement in rice protein functionality became evident in breadmaking, since it was possible to obtain rice bread with an increased specific volume and softer crumb at 1% TG level in the presence of 2% hydroxypropylmethylcellulose. Analyses showed that rice proteins are polymerised through the TG reaction, providing a protein network necessary for holding the gas produced in fermentation.

Studies on Effects of Microbial Transglutaminase on Gluten Proteins of Wheat. II. Rheological Properties

ABSTRACTThe effects of microbial transglutaminase (TG) on the dough properties and the baking performance of wheat flour with strong gluten and high baking quality were studied by farinograph measurements, creep‐recovery tests with dough, extension tests with gluten, and baking tests. A very low concentration of TG caused a significant decrease in the maximum strain of the dough. This decrease was closely related to the TG concentration. Extension tests with gluten showed that increasing concentrations of TG led to a drastic decrease of the extensibility and an increase of the resistance to extension. When concentrations of TG were too high, a complete loss of dough and gluten structure was obtained. On the other hand, with low concentrations of TG, no difference could be detected between the control bread and the bread containing TG in the baking test. With higher concentrations of TG, dough handling became extremely difficult, and the loaf volume decreased substantially due to a loss of structure in the dough. Therefore, the use of TG to improve the dough properties and the baking performance is recommended for flours with weak gluten and poor baking performance.