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Background: Corneal collagen cross-linking (CXL) is a procedure for making bonds that connect polymer chains to one another. Corneal CXL aims to slow or stop the progression of keratoconus by using photooxidative therapy so as to increase stromal rigidity. Transglutaminase enzymes are currently widely used in the food industry. Recent studies have shown that mRNA, fibronectin, and transglutaminase were found to be more abundant in human corneal keratocytes treated with UVA and riboflavin. Transglutaminase is considered to reduce discomfort caused by UVA irradiation. Methods: A total of 21 white New Zealand rabbits were divided into three groups, namely, transglutaminase-induced CXL group, epithelial-off CXL group, and transepithelial CXL group. The ocular surface was treated with a 1 U/mL microbial transglutaminase solution, and both the epithelial-off and transepithelial groups were exposed to clinical ultraviolet A-riboflavin (UVA/RF). The efficacy of each group was evaluated on the 14 th day after the procedures. The central corneal thickness (CCT) and intraocular pressure (IOP) were evaluated using Corneal Visualization Scheimpflug Technology (Corvis ST). Results: The transglutaminase-induced CXL group exhibited the highest mean CCT (370.14 ± 38.85) in comparison with the UVA/RF epithelial-off group (368.00 ± 25.48) and the UVA/RF transepithelial group (369.86 ± 23.43). The transglutaminase-induced CXL group had the highest IOP mean (8.50 ± 3.02) compared with the UVA/RF epithelial-off (6.50 ± 3.07) and UVA/RF transepithelial groups (7.00 ± 1.90). There were no significant differences in CCT ( p = 0.990) or IOP ( p = 0.563) between the groups. Conclusions: The findings of this study suggest that there are no significant differences between the transglutaminase-induced CXL group and the UVA/RF CXL group. The safety of transglutaminase-induced CXL could be comparable to that of UVA/RF CXL in terms of altering CCT and IOP, which are two factors contributing to corneal rigidity.

Objective(s): Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples. In the present study, the influence of excess of cations in [177Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied and a protocol to negate the enhancement observed was developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [131I]I-Lipiodol was standardized.Methods: GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]I-Lipiodol various modification in the protocols like orbital vortexing at different rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD.Results: It was observed that at MVD, [177Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes.Conclusions: Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement. Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [131I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs.

Abstract Background Streptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli. Results First, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 ℃ and 94.25% increases in melting temperature and half-life at 60 ℃ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun’s lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL. Conclusions The stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

ABSTRACT Objectives: Evaluate the celiac disease (CD) markers, within the scope of its screening, in a pediatric population with diagnosis of type 1 diabetes (T1D) at Hospital de Braga (HB) and determine the prevalence of CD in the sample. Reflect on CD screening algorithm applied in this pediatric population. Subjects and methods: Retrospective observational study with 94 patients diagnosed with T1D at age 10 years or younger, followed up at the HB Outpatient Diabetology Consultation, including those referred from other hospitals. Record of clinical information, IgA anti-transglutaminase and anti-endomysium and HLA DQ2/DQ8 haplotypes. Results: We obtained positive serological test for CD in 4 patients. This test had 100% sensitivity and specificity. The prevalence of CD was 4.3% (n = 4). Positive HLA screening in 84.6% of patients, with both sensitivity and negative predictive value of 100% and specificity of 16.67%. Diagnosis of CD was made on average 3.40 ± 3.32 years after the diagnosis of TD1. All cases of CD registered non-gastrointestinal manifestations, none had gastrointestinal symptoms. Conclusion: This study proved that there is a higher prevalence of CD in pediatric population with TD1, when compared to general population, and clarified the importance of CD screening. Furthermore, it was observed that serological screening for CD antibodies is an excellent screening test and HLA typing, although not the most suitable first line test, can be useful in excluding the possibility of patients with T1D developing CD.

BackgroundParoxysmal gluten-sensitive dyskinesia is a subtype of movement disorder classified as canine paroxysmal dyskinesia (cPD), which until now has only been described in Border Terriers (BT).ObjectivesOur aim was to report cPD with positive gluten serology in dog breeds other than BT.AnimalsThirty-one client-owned dogs with suspected cPD were examined in this study.MethodsThe hospital records of the dogs where the serum was tested for modified gliadin peptide immunoglobulin G (gliadin IgG) and tissue transglutaminase-2 immunoglobulin A (transglutaminase-2 IgA) were studied. A total of 31 dogs were presented to the clinic with cPD. A work-up consistent with Tier 1 or Tier 2 confidence levels for canine epilepsy was undertaken in all dogs. The dogs' diets and episode descriptions or videos in 16/31 cases were additionally studied. A follow-up was held to inquire about the dogs' wellbeing and response to the diet changes.ResultsFourteen of the 31 dogs tested positive for gluten sensitivity with either gliadin IgG or transglutaminase-2 IgA or both ratios elevated. In seven dogs, serology was classified as questionable with gliadin IgG or transglutaminase ratios mildly elevated. Ten dogs tested negative. According to the owners' reports, five of the dogs that tested positive had no more episodes after changing to a strictly gluten-free diet, with one of the dogs relapsing twice after being fed treats containing gluten. Three dogs had a reduction in episode frequency of >50%, and two dogs had shorter and less intense episodes.ConclusionA considerable subset of dog breeds presented for presumed cPD showed laboratory signs of gluten sensitivity and responded to a gluten-free diet.

IntroductionDermatitis herpetiformis (DH) is a rare autoimmune, polymorphous blistering disorder, characterized by severe itch or burning sensation, which represents the cutaneous manifestation of celiac disease (CD). The current estimation of DH versus CD is around 1:8 and the affected individuals have a genetic predisposition. Pathogenetically, IgA autoantibodies against the epidermal transglutaminase, an essential constituent of the epidermis, cause DH and are reported to develop through cross-reaction with the tissue transglutaminase, with IgA auto-antibodies causing CD. Immunofluorescence techniques allow for a rapid diagnostics of the disease using patient sera. Evaluation of IgA endomysial deposition with indirect immunofluorescence on monkey oesophagus is highly specific, but moderately sensitive, with some operator-dependent variability. Recently, indirect immunofluorescence with monkey liver as a substrate has been proposed as an alternative, well-functioning diagnostic approach with higher sensitivity in CD.MethodsThe objective of our study was to evaluate whether monkey oesophagus or liver tissue shows advantage for diagnostics in patients with DH, compared to CD. To that end, sera of 103 patients with DH (n=16), CD (n=67) and 20 controls ere compared by 4 blinded experienced raters.ResultsFor DH, we found a sensitivity of 94.2% for monkey liver (ML) compared to 96.2% in monkey oesophagus (ME), while specificity in ML was superior (91.6% versus 75%) to ME. In CD, ML had a sensitivity of 76.9% (ME 89.1%) and specificity of 98.3% (ME 94.1%).DiscussionOur data show that ML substrate is well suitable for DH diagnostics.

Celiac disease (CD) is a complex multi-organ disease with a high prevalence of extra-intestinal involvement, including neurological and psychiatric manifestations, such as cerebellar ataxia, peripheral neuropathy, epilepsy, headache, cognitive impairment, and depression. However, the mechanisms behind the neurological involvement in CD remain controversial. Recent evidence shows these can be related to gluten-mediated pathogenesis, including antibody cross-reaction, deposition of immune-complex, direct neurotoxicity, and in severe cases, vitamins or nutrients deficiency. Here, we have summarized new evidence related to gut microbiota and the so-called "gut-liver-brain axis" involved in CD-related neurological manifestations. Additionally, there has yet to be an agreement on whether serological or neurophysiological findings can effectively early diagnose and properly monitor CD-associated neurological involvement; notably, most of them can revert to normal with a rigorous gluten-free diet. Moving from a molecular level to a symptom-based approach, clinical, serological, and neurophysiology data might help to disentangle the many-faceted interactions between the gut and brain in CD. Eventually, the identification of multimodal biomarkers might help diagnose, monitor, and improve the quality of life of patients with "neuroCD". [ABSTRACT FROM AUTHOR]

Meat mimics were prepared from pea protein isolate-alginate gel via 3D printing. The texture of 3D-printed meat mimics was modified by incorporating transglutaminase (TGase) or κ-carrageenan (κc) at 0.3, 0.6 or 0.9% (w/w) into printing material prior to 3D printing. Rheological properties of modified printing material were measured; results were used to support 3D printing results. Textural properties of raw and cooked meat mimics were determined and compared with those of selected animal meats, namely, pork tenderloin, chicken breast, salmon meat and Spanish mackerel. Cooking losses of meat mimics were also determined. G′, G″ and tan δ of TGase-modified material were not significantly different from those of the control. In contrast, increasing κc content resulted in increased G′ and G″; tan δ of all κc-modified samples decreased from that of the control. Addition of TGase at 0.9% into printing material increased the hardness of raw meat mimics, while κc at 0.9% increased hardness of cooked meat mimics. Raw meat mimics treated with 0.9% TGase exhibited texture closest to raw salmon. Texture of cooked meat mimics treated with 0.9% κc was closest to that of cooked salmon. TGase-treated meat mimics tended to experience lower cooking losses, while κc-treated meat mimics exhibited an opposite trend. [ABSTRACT FROM AUTHOR]

Until now, Streptoverticillium mobaraense transglutaminase (TG) is the only commercialized TG, but limited information is known about its selection tendency on crosslinking sites at the protein level, restricting its application in the food industry. Here, four recombinant Bacillus TGs were stable in a broad range of pH (5.0–9.0) and temperatures (<50 °C), exhibiting their maximum activity at 50–60 °C and pH 6.0–7.0. Among them, TG of B. cereus (BCETG) demonstrated the maximal specific activity of 177 U/mg. A structural analysis indicated that the Ala147-Ala156 region in the substrate tunnel of BCETG played a vital role in catalytic activity. Furthermore, bovine serum albumin, as well as nearly all protein ingredients in soy protein isolate and whey protein, could be cross-linked by BCETG, and the internal crosslinking paths of three protein substrates were elucidated. This study demonstrated Bacillus TGs are a candidate for protein crosslinking and provided their crosslinking mechanism at the protein level for applications in food processing. [ABSTRACT FROM AUTHOR]

Introduction: Celiac disease can be associated with other diseases, including neurological disorders. In this study, the relationship between celiac disease and refractory epilepsy was evaluated in patients who were referred to Imam Khomeini Hospital in Urmia. Methods: In this cross-sectional study, patients with refractory epilepsy who were referred to the neurology clinic of Imam Khomeini Hospital in Urmia, during the second half of 2019 and cases with controlled epilepsy were studied as a control group. The statistical population of the present study included 50 patients with refractory seizures and 50 patients with controlled seizures. The mean age of patients was 32.96±11.35 years. Five milliliters of blood samples were taken from the patients, and a serum antitTG test was performed using the ELISA kit. Then, in patients with positive anti-tTG, a duodenal biopsy sample was prepared using an endoscopy. Results: This study showed that the mean serum level of anti-tTG in patients with refractory epilepsy was higher than in patients with controlled epilepsy. Anti-tTG test results were positive in five out of 50 patients with refractory epilepsy, and it was positive in two out of 50 patients with controlled epilepsy. There was no significant difference between the two groups in terms of serum levels of anti-tTG (P=0.14). Also, there was no significant relationship between serum levels of anti-tTG, age, and genus (P>0.05). Biopsy results in three patients in the refractory epilepsy group and one patient in the controlled epilepsy group were in favor of a definitive diagnosis of celiac disease. Patients with confirmed celiac disease using endoscopy had higher anti-tTG levels (P=0.006). Conclusion: There was no significant difference between celiac disease in cases with refractory epilepsy and controlled epilepsy. [ABSTRACT FROM AUTHOR]

Dairy foods are essential in the diet, although in some susceptible individuals they may cause allergy to cow's milk proteins. Therefore, alternative methods are sought to reduce their allergenicity. Transglutaminase (TG) is widely used in dairy products mainly to improve texture. Although it has been claimed that TG can be used to modify the digestibility and allergenicity of foods, its impact within a real matrix has been rarely studied. The aim of this work was to assess the allergenic potential of crosslinked skim milk (SM), milk casein fraction (CN), and whey protein (WP). To this purpose, inhibition ELISA with sera from milk allergic patients, in vitro activation tests of mouse mast cells and splenocytes, and simulated gastrointestinal digestion assays were performed. The results showed that cross-linking increased the binding of IgE to WP, but decreased IgE-binding to SM and CN. However, no differences were observed in the ability of cross-linked proteins to induce mast cell degranulation compared to native proteins. The cross-linking of SM and CN reduced Th2 cytokine release from the splenocytes of sensitized mice. All TG-treated samples exhibited more resistance to in vitro digestion than the untreated proteins and the human IgE binding capacity after digestion was higher. In conclusion, TG treatment of milk proteins does not reduce the risk of eliciting allergic symptoms in cow's milk allergic patients. [ABSTRACT FROM AUTHOR]

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Melt and stretch properties in dairy-based imitation mozzarella cheese (IMC) are affected by the amount of intact casein provided by dairy ingredients in the formulation. Rennet casein (RCN) is the preferred ingredient to provide intact casein in a formulation. Ingredients produced using membrane technology, such as milk protein concentrate (MPC) and micellar casein concentrate (MCC), are unable to provide the required functionality. However, the use of transglutaminase (TGase) has potential to modify the physical properties of MPC or MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC retentates on melt and stretch properties when they are used in IMC and to compare them with IMC made using RCN. The MCC and MPC retentates were produced using 3 different lots of pasteurized skim milk and treated with 3 levels of TGase enzyme: no TGase (control), low TGase: 0.3 units/g of protein, and high TGase: 3.0 units/g of protein. Each of the MCC and MPC treatments was heated to 72°C for 10 min to inactivate TGase and then spray dried. Each MCC, MPC, and RCN powder was then used in an IMC formulation that was standardized to 48% moisture, 21% fat, 20% protein, and 1% salt. The IMC were manufactured in a twin-screw cooker by blending, mixing, and heating various ingredients (4.0 kg). Due to extensive crosslinking, the IMC formulation with the highest TGase level (MCC or MPC) did not form an emulsion. The IMC made from MCC treatments had significantly higher stretchability on pizza compared with their respective MPC treatments. The IMC made from TGase-treated MCC and MPC had significantly lower melt area and significantly higher transition temperature (TT) and stretchability compared with their respective controls. Comparison of IMC made using TGase-treated MCC and MPC to the RCN IMC indicated no difference in TT or texture profile analysis-stretchability; however, the Schreiber melt test area was significantly lower. Our results demonstrated that TGase treatment modifies the melt and stretch characteristics of MCC and MPC in IMC applications, and TGase-treated MPC and MCC can be used to replace RCN in IMC formulations. [ABSTRACT FROM AUTHOR]

The amount of intact casein provided by dairy ingredients is a critical parameter in dairy-based imitation mozzarella cheese (IMC) formulation because it has a significant effect on unmelted textural parameters such as hardness. From a functionality perspective, rennet casein (RCN) is the preferred ingredient. Milk protein concentrate (MPC) and micellar casein concentrate (MCC) cannot provide the required functionality due to the higher steric stability of casein micelle. However, the use of transglutaminase (TGase) has the potential to modify the surface properties of MPC and MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC powders on the unmelted textural properties of IMC and compare them with IMC made using commercially available RCN. Additionally, we studied the degree of crosslinking by TGase in MPC and MCC retentates using capillary gel electrophoresis. Three lots of MCC and MPC retentate were produced from pasteurized skim milk via microfiltration and ultrafiltration, respectively, and randomly assigned to 1 of 3 treatments: no TGase (control); low TGase: 0.3 units/g of protein; and high TGase: 3.0 units/g of protein, followed by inactivation of enzyme (72°C for 10 min), and spray drying. Each MCC, MPC, and RCN was then used to formulate IMC that was standardized to 21% fat, 1% salt, 48% moisture, and 20% protein. The IMC were manufactured by blending, mixing, and heating ingredients (4.0 kg) in a twin-screw cooker. The capillary gel electrophoresis analysis showed extensive inter- and intramolecular crosslinking. The IMC formulation using the highest TGase level in MCC or MPC did not form an emulsion because of extensive crosslinking. In MPC with a high level of TGase, whey protein and casein crosslinking were observed. In contrast, crosslinking and hydrolysis of proteins were observed in MCC. The IMC made from MCC powder had significantly higher texture profile analysis hardness compared with the corresponding MPC powder. Further, many-to-one (multiple) comparisons using the Dunnett test showed no significant differences between IMC made using RCN and treatment powders in hardness. Our results demonstrated that TGase treatment causes crosslinking hydrolysis of MCC and MPC at higher TGase levels, and MPC and MCC have the potential to be used as ingredients in IMC applications. [ABSTRACT FROM AUTHOR]

Current therapeutic treatments for the repair and/or replacement of damaged skin following disease or traumatic injury is severely limited. The chicken eggshell membrane (ESM) is a unique material: its innate physical and mechanical characteristics offer optimal barrier properties and, as a naturally derived extract, it demonstrates inherent biocompatibility/biodegradability. To further enhance its therapeutic and clinical potential, the ESM can be modified with the thermo-responsive polymer, poly(N-isopropylacrylAmide) (PNIPAAm) as well as the incorporation of (drug-loaded) silver nanoparticles (AgNP); essentially, by a simple change in temperature, the release and delivery of the NP can be targeted and controlled. In this study, ESM samples were isolated using a decellularization protocol, and the physical and mechanical characteristics were profiled using SEM, FT-IR, DSC and DMA. PNIPAAm was successfully grafted to the ESM via amidation reactions and confirmed using FT-IR, which demonstrated the distinctive peaks associated with Amide A (3275 cm−1), Amide B (2970 cm−1), Amide I (1630 cm−1), Amide II (1535 cm−1), CH2, CH3 groups, and Amide III (1250 cm−1) peaks. Confirmation of the incorporation of AgNP onto the stratified membrane was confirmed visually with SEM, qualitatively using FT-IR and also via changes in absorbance at 380 nm using UV-Vis spectrophotometry during a controlled release study for 72 h. The biocompatibility and cytotoxicity of the novel constructs were assessed using human dermal fibroblast (HDFa) and mouse dermal fibroblast (L929) cells and standard cell culture assays. Metabolic activity assessment (i.e., MTS assay), LDH-release profiles and Live/Dead staining demonstrated good attachment and spreading to the samples, and high cell viability following 3 days of culture. Interestingly, longer-term viability (>5 days), the ESM-PNIPAAm and ESM-PNIPAAm (AgNP) samples showed a greater and sustained cell viability profile. In summary, the modified and enhanced ESM constructs were successfully prepared and characterized in terms of their physical and mechanical profiles. AgNP were successfully loaded into the construct and demonstrated a desirable release profile dependent on temperature modulation. Fibroblasts cultured on the extracted ESM samples and ESM-PNIPAAm demonstrated high biocompatibility in terms of high cell attachment, spreading, viability and proliferation rates. As such, this work summarizes the development of an enhanced ESM-based construct which may be exploited as a clinical/therapeutic wound dressing as well as a possible application as a novel biomaterial scaffold for drug development. [ABSTRACT FROM AUTHOR]

This article aimed to improve the utilization of by-products of tofu and produce high-quality tofu, on the basis of the production process of acid slurry tofu, bean dregs and transglutaminase (TG enzyme) were added to it. The soybean-water ratio, soybean dregs addition, acid slurry addition and transglutaminase addition were chosen as four factors, sensory score was used as evaluation index to optimize the preparation process of high-fiber acid slurry tofu, and the product was compared with the traditional acid slurry tofu, the dietary fiber content, sensory evaluation, texture characteristics, protein structure and microstructure of the two were analyzed. Results showed that, the optimal process results were as follows: Soybean-water ratio 1:4.6, 15% soybean dregs addition, 21% acid slurry addition, 0.3% transglutaminase addition. The order of influence on the sensory score of high-fiber acid slurry tofu was: Soybean dregs addition amount> transglutaminase addition amount> acid slurry addition amount> soybean-water ratio. Under this condition, the dietary fiber content of high-fiber acid slurry tofu increased from 0.26% to 2.15%. The texture characteristics were similar to traditional one, texture was not hardened due to the increasing of dietary fiber, flavor was strong, internal tissue was uniform and flexible. From FTIR spectrum, it could be seen that transglutaminase enhanced the cross-linking between protein and protein, and the protein gel structure was more compact. The microstructure was more dense and orderly.

Abstract Soybean protein isolate (SPI) is a high‐quality plant protein that is primarily used to process various soybean products coagulated by transglutaminase (TGase). In this study, the degree of hydrolysis (DH), sulfhydryl content (SH), surface hydrophobicity (H0 ), secondary structural constitution, and microstructure of TGase‐treated soybean protein (SPI, 7S, and 11S) were determined, as well as the effects of NaCl, urea, and SDS on the properties and intermolecular forces of SPI gel were analyzed. The results show that the H0 and SH content of SPI, 7S, and 11S decreased significantly with TGase treatment time (p

Beef knuckle contains less amount of fat, which tastes dry and hard after being pan fried. In order to increase the richness of the juice of the steak made of that, promoting the formation of a soft and tender taste as well, this experiment studied the effect of injection of walnut oil emulsion under high-pressure and low-temperature on steak quality. In this experiment, beef knuckle steak added with walnut oil emulsion had been successfully prepared through injection under high-pressure and low-temperature followed vacuum tumbling technology, and the influence of emulsions with different transglutaminase (TGase) content (0%, 0.3%, 0.6%, 0.9%, 1.2%) on the emulsion bonding capacity of steak and the influence of emulsion with different walnut oil content (20%, 30%, 40%, 50%) on the physicochemical indexes and quality indexes of steak were studied. The bonding capacity of steak adding emulsion increased with rising TGase contents in emulsions, and stabilized after TGase contents reached 0.9%. Injection walnut oil emulsions into steak increased its moisture contents by 1.44%~2.31%, and decreased pH value by 0.17~0.08, increased then decreased L* value, decreased a* value and b* value. The shear force of steak were reduced by 26.62%~65.33%, the yield were increased by 3.43%~11.43%, the hardness and chewiness of steak were reduced by 15.44%~77.06% and 26.56%~77.52%, respectively. Sensory evaluation experiments showed that the injection of emulsion improved the softness and juiciness of steak products, yet had little effect on the flavor and tastes. Through the comparison between influence of emulsions contain different amount of TGase and walnut oil on the process performances, physicochemical properties and quality indicators of steak, the emulsion with TGase content of 0.9% and walnut oil content of 40% was most suitable for production practice.

Research background. In industrial ayran production, milk is diluted to the desired protein content (2% (m/V)) prior to fermentation by yogurt starter cultures by partial replacement of cheese whey in reconstituted form with potable water. This may be an alternative way of protein recovery from cheese by-products as well as reducing the production costs since less milk is used in ayran production. On the other hand, the balance between milk caseins and whey proteins is disturbed when cheese whey is added to milk for ayran production, which likely leads to a time-dependent phase separation during cold storage. Modification of ayran matrix by enzymatic crosslinking of proteins may be a solution to overcome this potential physical instability of ayran. This topic has not been explored to date, and the present study was designed to investigate the possibilities of utilization of reconstituted whey powder (RWP) and microbial transglutaminase (MTG) in ayran production. Experimental approach. Milk was diluted to obtain 2% protein level using RWP and potable water. The aim of utilization of RWP was to meet 5, 10 or 15% of the protein content of the final product. RWP solutions were obtained by calculating the amounts of whey powder required to meet the specified ratios and mixing it with the water required for dilution. We prepared eight different ayran samples divided in three groups, namely group A: prepared by partially diluting milk with RWP to obtain 5, 10 or 15% of total protein amount in the product, group AMTG: prepared by adding microbial transglutaminase (0.5 U per g of protein) to group A samples, and control group without RWP and with or without the addition of MTG. The gross composition, physical (phase separation and viscosity, chemical (volatile and peptide profiles and SDS-PAGE electrophoresis patterns) and sensory properties of the samples were evaluated throughout 15 days of storage with weekly intervals. Results and conclusions. Since the amounts of whey powder used to obtain RWP were different, dry matter levels of the samples differed. Using RWP in ayran production increased the phase separation slightly. Incorporation of MTG affected the physical properties of the ayran samples positively and prevented phase separation at a satisfactory level. SDS-PAGE electrophoretograms revealed that cross-linking between proteins triggered by MTG formed intense bonds at high molecular mass regions. The remaining parameters were not affected by MTG. Results revealed that the samples with 10% RWP with and without addition of MTG were determined as the formulations with the highest commercialization potential. Novelty and scientific contribution. Utilizing RWP in the production of ayran to reduce the protein content of the final product to the desired level is a new approach. Since complete replacement of RWP with potable water to dilute milk to reach 2% (m/V) protein likely leads to lower sensory scores, we have investigated a possibility of partial replacement of RWP with potable water. A time-dependent phase separation is the major challenge of industrial ayran production. This physical problem was largely eliminated by means of MTG-mediated cross-linking of milk proteins. The proposed novel ayran production method offers dairy industry reduction of production costs and contributes to sustainability in milk production since smaller volume of milk is used to reach desired protein content in the final product.

The effects of transglutaminase (TGase) addition (0.4–1.2 g/100g), ultrasound (120–720 W, 20 min), and their combination on the gel properties and in vitro digestion characteristics of bay scallop adductor muscle were studied. The gel strength of the gel sample with TGase content of 0.8 g/100g (TG-0.8) was 58.2% higher than that of the control sample (CON). The gel sample treated with ultrasound at 480 W (UT-480) had the highest gel strength. The strength of the gel prepared by combination of 0.8 g/100g TGase and 360 W ultrasound (UT-TG) was 82.3% higher than that of CON. The whiteness and water holding capacity of the gel increased regardless of the addition of TGase or ultrasound treatment. SDS-PAGE patterns showed that the myosin heavy chain of the treated samples became thinner, and the changes of actin and tropomyosin were not significant. The scanning electron microscopy results of gel samples prepared by ultrasound combined with TGase showed a denser structure, which was related to the lowest total sulfhydryl content and TCA-soluble peptide content. The results of dynamic rheology show that the UT-TG sample had the highest G′ value, followed by TG-0.8. The in vitro digestion characteristics of the selected gel samples were also discussed. The degree of protein hydrolysis and the content of free amino acids in TG-0.8 samples were the lowest, which improved after ultrasound treatment. Overall, the combination of appropriate ultrasound treatment and TGase addition provides an effective means for improving gel properties and digestibility of scallop surimi product.

The microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTGase) is widely used for industrial applications. However, in the last decades, TGases from other bacteria have been described. We focused our attention on TGase, from Kutzneria albida (KalbTGase), recently characterized as more selective than MTGase and proposed for applications in drug delivery. By comparison of the crystallographic structures, the volume of the catalytic site results smaller in KalbTGase. We compared KalbTGase and MTGase structural flexibility by molecular dynamics (MD) simulations at different conditions. KalbTGase is more rigid than MTGase at 300 K, but the catalytic site has a preserved conformation in both structures. Preliminary studies at higher temperatures suggest that KalbTGase acquires enhanced conformational flexibility far from the active site region. The volume of the catalytic active site pocket of KalbTGase at room temperature is smaller than that of MTGase, and decreases at 335 K, remaining stable after further temperature increase. On the contrary, in MTGase the pocket volume continues to decrease as the temperature increases. Overall, the results of our study suggest that at room temperature the enhanced specificity of KalbTGase could be related to a more closed catalytic pocket and lower flexibility than MTGase. Moreover, by preliminary results at higher temperature, KalbTGase structural flexibility suggests an adaptability to different substrates not recognized at room temperature. Lower adaptability of MTGase at higher temperature with a reduction of the catalytic pocket, instead, suggests a reduction of its activity.

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus , which was found to be upregulated at the protein level in response to an injection of the β-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l -γ-glutamine- p -nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood. • Pacifastacus leniusculus i-type lysozyme (Pl-ilys) is mainly expressed in hemocytes. • Pl-ilys is deficient of muramidase activity. • Pl-ilys is functioning as a clot degrading or destabilising enzyme. [ABSTRACT FROM AUTHOR]

The poor gelation properties of pea protein isolate (PPI) limit their use in food industries. In the present study, PPI was modified by pH-shifting combined with heat treatment, followed by cold-set gelation of PPI gels induced by transglutaminase. Both pH 2–3 and pH 10–12 promoted the unfolding of PPI molecules and the transformation of secondary structures from ordered to disordered. In particular, PPI molecules with pH 12-shifting tended to expand and unfold, which resulted in the exposure of internal active groups and improvement in surface hydrophobicity. Accordingly, after the addition of 50 U/g transglutaminase, PPI gels with pH 12-shifting and heating treatment exhibited homogeneous and dense gel network with high cross-linking degree (88.12 ± 1.80%) and water holding capacity (92.13 ± 2.40%), since pH 12-shifting and heating treatment promoted the conversion of random coils and β-turns to β-folds and α-helices in PPI gels. However, with pH 10 and 11-shifting, the PPI molecules was partially unfolded and the PPI gels had a high non-network protein content, resulting in low-strength gel networks of PPI gels with rough and loose microstructures and a low WHC. These results showed that TGase-crosslinked cold-set PPI gels after pH 12-shifting and heat treatment could promote the formation of homogeneous and dense gel network structure, so that improved the PPI gelation properties, which had great potential to be used as gelling agents or nutraceutical delivery systems for thermal sensitive bioactive compounds in food and non-food fields. [Display omitted] • TGase-crosslinked PPI cold-set gels were prepared by pH shifting and heat treatment. • pH 12-shifting promoted the formation of Gln-Lys covalent and disulfide bonds in PPI gels. • TGase-crosslinked PPI after pH 12-shifting and heat treatment assisted in the formation of a dense gel network. • Highly strengthened network exhibited high gel strength and water holding capacity. • TGase-crosslinked PPI after pH10 and 11-shifting resulted in rough and loose structures. [ABSTRACT FROM AUTHOR]

Pennycress protein isolate (PcPI) has inferior gelation and water holding properties compared to market leader soy protein, preventing its use in food applications such as meat analogues. Therefore, this work aimed to induce polymerization of PcPI by either cold atmospheric plasma (CAP) or transglutaminase (TG) to improve functionality and texturization potential. Micro-compounding was utilized to determine bench-scale texturization potential of unmodified, CAP, and TG modified PcPI hydrated at 50% moisture. CAP treatment following dielectric barrier discharge (DBD) induced polymerization primarily through disulfide linkages. Whereas, TG resulted in a relatively higher extent of polymerization induced by disulfide linkages and other covalent interactions involving mostly cruciferin acidic subunits. Compared to unmodified PcPI, the gel strength doubled and tripled post CAP and TG treatments, respectively. TG treatment caused a significant increase in water holding capacity by 20%. Unmodified PcPI did not form fibrous structures upon micro-compounding, instead it formed a soft mass with low resilience and cohesiveness. CAP modified PcPI (PcPI-CP) had the lowest water holding properties, which resulted in a relatively hard (significantly highest mechanical energy), dense fibrous structures. Due to high gelation strength and water holding capacity, TG modified PcPI (PcPI-TG) resulted in less dense fibrous structures with more air incorporation (significantly higher void %) relative to PcPI control and PcPI-CP. PcPI-TG texturized mass had significantly higher chewiness that is desirable for meat analogues. This research, for the first time, confirmed the potential success of induced polymerization of PcPI, especially with TG, in enhancing gelation and texturization potential. [Display omitted] • Cold plasma (CP) and transglutaminase (TG) induced pennycress protein crosslinking. • Protein crosslinking by CP occurred through intermolecular disulfide linkages. • Protein crosslinking by TG occurred via intra- and inter-molecular covalent bonds. • Protein crosslinking contributed to enhanced gel strength and texturization. • TG modification resulted in superior properties of texturized pennycress protein. [ABSTRACT FROM AUTHOR]

Fat analogs that precisely simulate the properties of animal fat are urgently required to meet the global demand for sustainable and healthy plant-based meat alternatives. In this study, we engineered a novel curdlan and transglutaminase-based heat-induced emulsion gel that mimics the thermal characteristics and elastic texture of porcine adipose tissue. Emulsion gels were formulated using different transglutaminase (TGase) concentrations (0–300 mg/g soy protein isolate) combined with 20% soybean oil, 2% soy protein isolate, and 7% curdlan. Fourier-transform spectroscopy and microstructural observations revealed that protein cross-linking increased to form a dense gel network as the TGase concentration increased. Textural property analysis revealed that optimized protein cross-linking eliminated the undesirable brittleness often associated with plant-based fat analogs and resulted in an enhanced springy texture reminiscent of traditional animal fat. In addition, our fat analogs demonstrated thermal stability by maintaining their elastic properties during cooking and heating. These findings suggest that our emulsion gels could be feasible plant-based alternatives to animal adipose tissue, and our strategy could promote future research on improving other plant-based meat products. [Display omitted] • Novel emulsion gel replicates animal adipose tissue characteristics via high-set gel structure and protein cross-linking. • The high-set gel structure, made up of a triple helix structure of Curdlan micelles, can exhibit strength comparable to that of a collagen network and thermal stability. • Transglutaminase treatment reduces the brittleness and increases the elasticity of curdlan emulsion gel by promoting intermolecular bonds. • The fat analog, proposed in this study, marks a significant stride in advancing quality standards for plant-based meat alternatives. [ABSTRACT FROM AUTHOR]

Exo70, a key exocyst complex component, is crucial for cell motility and extracellular matrix (ECM) remodeling in cancer metastasis. Despite its potential as a drug target, Exo70's post-translational modifications (PTMs) are poorly characterized. Here, we report that Exo70 is transamidated on Gln5 with Lys56 of cystatin A by transglutaminases TGM1 and TGM3, promoting tumor metastasis. This modification enhances Exo70's association with other exocyst subunits, essential for secreting matrix metalloproteinases, forming invadopodia, and delivering integrins to the leading edge. Tumor suppressor liver kinase B1 (LKB1), whose inactivation accelerates metastasis, phosphorylates TGM1 and TGM3 at Thr386 and Thr282, respectively, to inhibit their interaction with Exo70 and the following transamidation. Cantharidin, a US Food and Drug Administration (FDA)-approved drug, inhibits Exo70 transamidation to restrain tumor cell migration and invasion. Together, our findings highlight Exo70 transamidation as a key molecular mechanism and target and propose cantharidin as a therapeutic strategy with direct clinical translational value for metastatic cancers, especially those with LKB1 loss. [Display omitted] • TGM1 and TGM3 mediate transamidation of Exo70 • Exo70 transamidation promotes cancer metastasis • LKB1 induces TGM1/3 phosphorylation to suppress Exo70 transamidation Hou et al. demonstrate that the transglutaminases TGM1 and TGM3 are responsible for the transamidation of the exocyst component Exo70, thereby regulating tumor cell invasion. Exo70 transamidation can be repressed by LKB1 phosphorylation of TGM1/3 or the application of cantharidin, an FDA-approved drug. [ABSTRACT FROM AUTHOR]

The weak gelling and poor digestion properties of myofibrillar protein (MP) under low-salt conditions constrain the development of functional sodium-reduced meat products. This study investigated how modification with γ-polyglutamic acid (γ-PGA) and transglutaminase (TG) cross-linking influences the conformation, gelation, and in vitro digestion of low-salt MPs pretreated with konjac glucomannan (KGM) and ultrasound. The results revealed that γ-PGA addition effectively facilitated the exposure of tryptophan and tyrosine residues in low salt MPs. The better dispersibility and slightly lower rheological properties of protein sols suggested the circumscribed effect of γ-PGA in augmenting protein gelation. TG crosslinking promoted moderate aggregation of MPs through different sites in myosin fragments, augmented the uniform distribution of sodium, and remarkably enhanced the gelling behavior of MPs. Importantly, the simultaneous incorporation of γ-PGA contributed to the refinement and uniformization of the TG cross-linked protein gel network, especially in the protein gel modified by pre-ultrasound treatment in combination with KGM. This result could also be corroborated by the augmented intermolecular interactions and enhanced digestibility observed in γ-PGA-modified TG crosslinked protein gel. Coupled with the identification of potentially bioactive peptides, it was confirmed that myosin light chain, actin, and myomesin account for the major contribution to the γ-PGA-induced changes in MP gels. Therefore, TG cross-linking combined with γ-PGA could be used to regulate the conformation and gelling properties of low-salt MPs, thus influencing the sodium distribution and digestion properties of low-salt meat products. [Display omitted] • The moisture loss defect in the TG cross-linked MP gel was compensated by γ-PGA. • The protein aggregation and uniform distribution of sodium were improved by TG. • The association of γ-PGA facilitated the refinement of TG cross-linked MP gels. • γ-PGA promoted neck TG cross-linking in MPs modified by pre-ultrasound with KGM. • Myosin light chain, actin, and myomesin caused changes in γ-PGA-modified MP gels. [ABSTRACT FROM AUTHOR]

This investigation mainly aimed to synthesize transglutaminase (TGase)-catalyzed glycosylated ovalbumin (TGaseOVA) and elucidate the effect of diverse enzymatic reaction durations (from 1 h to 5 h) on the physicochemical, structural, and functional properties of glycosylated ovalbumin. Following a 4 h TGase reaction, the level of graft glycosylation reached 36.6%. Electrophoretic analysis provided evidence on the presence of high-molecular-weight glycoprotein polymers in the modified product. The glucosamine (GlcN) content measured 16.6 mg/g during high-performance liquid chromatography. Furthermore, compared with the original protein, TGaseOVA exhibited a stable three-dimensional network structure. After GlcN grafting, structural changes were analyzed in terms of intermolecular and intramolecular covalent crosslinking within the protein, and the results confirm that these modifications considerably improved the functional properties of TGaseOVA particles. This study demonstrates prospects for the development of novel protein compositions to broaden applications of food proteins. [Display omitted] • The physicochemical and functional properties of OVA, modified through enzymatic glycosylation, exhibited a strong correlation with both enzyme activity and enzyme action time. • TGase-catalyzed glycosylation improves the structure of OVA and forms a three-dimensional network structure. • TGase-catalyzed glycosylation confers OVA with improved solubility, emulsification, foaming ability, apparent viscosity, rheological properties and thermal stability. [ABSTRACT FROM AUTHOR]

This study prepared emulsion gels by modifying ovalbumin (OVA)-flaxseed oil (FSO) emulsions with transglutaminase (TGase) and investigated their properties, structure and oxidative stability under different enzyme reaction times. Here, we found prolonged reaction times led to the transformation of α-helix and β-turn into β-sheet and random coil. The elasticity, hardness and water retention of the emulsion gels increased significantly, but the water-holding capacity decreased when the reaction time exceeded 4 h. Confocal laser scanning microscope (CLSM) indicated extended enzyme reaction time fostered oil droplet aggregation with proteins. Emulsion gel reduced FSO oxidation, especially after 4 h of the enzyme reaction, the peroxide value (PV) of the emulsion gel was reduced by 29.16% compared to the control. In summary, the enzyme reaction time of 4 h resulted in the formation of a dense gel structure and enhanced oxidative stability. This study provides the potential applications in functional foods and biomedical fields. [Display omitted] • A novel FSO - encapsulated emulsion gel was constructed by inducing OVA with TGase. • Extended enzymatic reaction time improved emulsion gel properties and structure. • TGase - treated OVA aggregated more on the surface of the oil droplets. • Emulsion gel enhanced the oxidative stability of FSO during storage. [ABSTRACT FROM AUTHOR]

Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance. [Display omitted] • An all-biomass adhesive was developed by enzyme activation and enzyme crosslinking. • A double-enzyme co-activation method targeted protein-carbohydrate complexes in DSM. • TGase crosslinker replaced the traditional chemical crosslinkers. • The adhesives achieved excellent bonding strength for wood and metal. • Low-cost raw materials and simple steps achieved cost effectiveness of adhesives. [ABSTRACT FROM AUTHOR]

The study showed that transglutaminase (TG) significantly affected the thermal properties and retrogradation of wheat flour (WF). Confocal laser scanning microscopy showed that the samples treated with TG had an increased number of starch granules embedded in protein fragments compared with the control sample. Thermal properties were altered, and the swelling power of the samples decreased with increasing enzyme concentration. A change in pasting properties, especially a decrease in the final and setback viscosities, was also observed after adding TG. TG delayed the retrogradation process, with inhibitory effects attained at an enzyme concentration of approximately 0.05%. The inhibitory effects of TG on the retrogradation of WF were mainly hypothesized to be caused by alterations in the water absorption of the starch granules from the effect of TG on the surface. [Display omitted] • TG inhibited the retrogradation of wheat starch. • TG had major impacts on thermal properties and reduced swelling power of WF. • Microstructure revealed the change in protein and starch distribution in WF. [ABSTRACT FROM AUTHOR]

Glycation by transglutaminase (TGase)-type could effectively improve the structure and functional properties of proteins. However, the influence on intestinal inflammation or the underlying mechanisms has not been investigated. The goal of this research was to compare the bioactivities between glycated casein generated from the TGase-catalyzed reaction and oligochitosan as well as casein using a mouse model of dextran sulfate sodium (DSS)-induced intestinal inflammation to examine the protective effects and the underlying mechanism of glycated casein on intestinal inflammation. Eight groups of C57BL/6 mice were randomly assigned in this study: Control group: standard diet for 35 days; Model group: standard diet for 28 days and then colitis induction; Pretreated groups: different levels (200, 400, 800 mg/kg BW) of casein or glycated casein for 28 days before colitis induction. The mice were drinking water containing a 3% DSS solution for seven days of mice to cause colitis. The results indicated that glycated casein and casein at 200–800 mg/kg BW all relieved DSS-induced weight loss, reduced disease activity index (DAI) score, alleviated colon length shortening, weakened the destruction of colonic mucosal structure, decreased serum LPS, and MPO, IL-1β, IL-6 and TNF-α levels in serum and colon, as well as regulated the expression of proteins involved in the TLR4/NF-κB signaling pathway in a concentration-dependent manner. Glycated caseinate showed a better protective effect against DSS-induced colitis than casein, highlighting that the TGase-type glycation of proteins as a potential functional food ingredient might be a helpful method for gut health.

Stabilization and reusability of enzyme transglutaminase (TGM) are important goals for the enzymatic process since immobilizing TGM plays an important role in different technologies and industries. TGM can be used in many applications. In the food industry, it plays a role as a protein-modifying enzyme, while, in biotechnology and pharmaceutical applications, it is used in mediated bioconjugation due to its extraordinary crosslinking ability. TGMs (EC 2.3.2.13) are enzymes that catalyze the formation of a covalent bond between a free amino group of protein-bound or peptide-bound lysine, which acts as an acyl acceptor, and the γ-carboxamide group of protein-bound or peptide-bound glutamine, which acts as an acyl donor. This results in the modification of proteins through either intramolecular or intermolecular crosslinking, which improves the use of the respective proteins significantly.

Transglutaminase 2 (TG2) is a multifunctional protein widely distributed in various tissues and involved in many physiological and pathological processes. However, its actual role in biological processes is often controversial as TG2 shows different effects in these processes depending on its localization, cell type, or experimental conditions. We characterized the enzymatic and functional properties of TG2 proteins expressed in Danio rerio (zebrafish) to provide the basis for using this established animal model as a reliable tool to characterize TG2 functions in vivo. We confirmed the existence of three genes orthologous to human TG2 (zTGs2) in the zebrafish genome and their expression and function during embryonic development. We produced and purified the zTGs2s as recombinant proteins and showed that, like the human enzyme, zTGs2 catalyzes a Ca2+ dependent transamidation reaction that can be inhibited with TG2-specific inhibitors. In a cell model of human fibroblasts, we also demonstrated that zTGs2 can mediate RGD-independent cell adhesion in the extracellular environment. Finally, we transfected and selected zTGs2-overexpressing HEK293 cells and demonstrated that intracellular zTGs2 plays a very comparable protective/damaging role in the apoptotic process, as hTG2. Overall, our results suggest that zTGs2 proteins behave very similarly to the human ortholog and pave the way for future in vivo studies of TG2 functions in zebrafish.

Advances in mass spectrometry-based proteomics now permit analysis of complex cellular structures. Application to epidermis and its appendages (nail plate, hair shaft) has revealed a wealth of information about their protein profiles. The results confirm known site-specific differences in levels of certain keratins and add great depth to our knowledge of site specificity of scores of other proteins, thereby connecting anatomy and pathology. An example is the evident overlap in protein profiles of hair shaft and nail plate, helping rationalize their sharing of certain dystrophic syndromes distinct from epidermis. In addition, interindividual differences in protein level are manifest as would be expected. This approach permits characterization of altered profiles as a result of disease, where the magnitude of perturbation can be quantified and monitored during treatment. Proteomic analysis has also clarified the nature of the isopeptide cross-linked residual insoluble material after vigorous extraction with protein denaturants, nearly intractable to analysis without fragmentation. These structures, including the cross-linked envelope of epidermal corneocytes, are comprised of hundreds of protein constituents, evidence for strengthening the terminal structure complementary to disulphide bonding. Along with other developing technologies, proteomic analysis is anticipated to find use in disease risk stratification, detection, diagnosis and prognosis after the discovery phase and clinical validation.

Abstract Background Patients with celiac disease present with not only gastrointestinal symptoms but also extraintestinal manifestations such as anemia, osteopathy, dermatitis herpetiformis, and celiac neuropathy. Despite a fairly wide range of celiac neuropathies, we report a case of the acrodystrophic variant of celiac polyneuropathy, which has not been previously described. Case presentation A 41-year-old Ukrainian male suffered from symmetric, sensorimotor axonal polyneuropathy and encephalopathy associated with celiac disease, which is characterized by severe trophic disorders in the lower extremities (trophic ulcers, hyperkeratosis, and anhidrosis). Acrodystrophic changes in the lower extremities were due to both neurogenic and direct immunoinflammatory damaging effects. Clinical–electrophysiological dissociation was also noted, which was represented by a gross axonal lesion with the preservation of muscle strength. The absence of enteropathic manifestations was accompanied by the pronounced histological changes in the duodenal mucosa by IIIb stage of Marsh. A gluten-free diet in combination with membrane plasma exchange and intravenous pulse methylprednisolone was prescribed to reduce the severity of sensory disorders and regression of encephalopathy within 7 months. Conclusion Celiac disease may be a potential cause of neuropathy and encephalopathy in adult patients. Further immunosuppressive treatment protocols for both intestinal and extraintestinal manifestations of celiac disease are required.

Abstract Background Chronic kidney disease (CKD) has a high global prevalence and large unmet need. Central to developing new CKD therapies are in vivo models in CKD. However, next‐generation antibody, protein, and gene therapies are highly specific, meaning some do not cross‐react with rodent targets. This complicates preclinical development, as established in vivo rodent models cannot be utilized unless tool therapeutics are also developed. Tool compounds can be difficult to develop and, if available, typically have different epitopes, sequences, and/or altered affinity, making it unclear how efficacious the lead therapeutic may be, or what dosing regimen to investigate. To address this, we aimed to develop a nonhuman primate model of CKD. Methods In vivo rodent unilateral ureteral obstruction (UUO) models kidney fibrosis and is commonly used due to its rapidity, consistency, and ease. We describe translation of this model to the cynomolgus monkey, specifically optimizing the model duration to allow adequate time for assessment of novel therapeutics prior to the fibrotic plateau. Results We demonstrated that disease developed more slowly in cynomolgus monkeys than in rodents post‐UUO, with advanced fibrosis developing by 6 weeks. The tubulointerstitial fibrosis in cynomolgus monkeys was more consistent with human obstructive disease than in rodents, having a more aggressive tubular basement expansion and a higher fibroblast infiltration. The fibrosis was also associated with increased transglutaminase activity, consistent with that seen in patients with CKD. Conclusion This cynomolgus monkey UUO model can be used to test potential human‐specific therapeutics in kidney fibrosis.

Autologous plasma proteins can be used to fabricate patient specific cardiovascular implants but need to be cross-linked to increase their mechanical strength and reduce water solubility. Glutaraldehyde is the state-of-the-art solution but its reaction products have been shown to be cytotoxic and pro-inflammatory. In this work, it has been shown, that cross-linking of plasma proteins with biocompatible alternatives to glutaraldehyde is possible. This was achieved by identifying four candidate substances (thrombin, transglutaminase, genipin, EDC) from current literature and investigating their ability to cross-link porcine plasma proteins in vitro. The degree of crosslinking was examined using calorimetric (DSC) and spectroscopic (FTIR, Raman) methods, mapping the influence of cross-linking on the denaturation temperature and primary amino-group content of the proteins. It could be shown that thrombin, genipin and EDC are able to cross-link plasma proteins to a satisfactory degree and thus represent useful alternatives to glutaraldehyde. Transglutaminase, on the other hand, could not sufficiently cross-link the plasma proteins and was therefore ruled out as an alternative.

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This study investigated the effect of ultrasound and enzymatic cross-linking on the freeze-thaw (FT) stability and release properties of whey protein isolate hydrogels. We evaluated the FT stability by the changes in the microstructure, riboflavin retention, syneresis, water holding capacity (WHC), and texture of gels subjected to 3 FT cycles. High-intensity ultrasound (HUS) and transglutaminase (TGase)-mediated cross-linking improved the FT stability of whey protein isolate hydrogels loaded with riboflavin (WPISAR), as demonstrated by a more uniform and denser porous structure, significantly higher riboflavin retention, WHC, and textural properties, and lower syneresis after 3 FT cycles than those of untreated hydrogels. Furthermore, HUS- and TGase-mediated cross-linking decreased protein erosion and swelling ratio of WPISAR in simulated gastrointestinal fluids (SGIF) and reduced the riboflavin release rate in SGIF both with and without the addition of digestive enzymes. After 3 FT cycles, faster riboflavin release occurred due to a more porous structure induced by ice crystal formation compared with their unfrozen counterparts as detected by confocal laser scanning microscopy. High-intensity ultrasound- and TGase-mediated cross-linking alleviated the FT-induced faster riboflavin release rate in SGIF. High-intensity ultrasound- and TGase-treated gel samples showed that both diffusion and network erosion were responsible for riboflavin release regardless of FT. These results suggest that HUS- and TGase-mediated cross-linking improved the FT stability of WPISAR with a high riboflavin retention, and might be a good candidate as a controlled-release vehicle for riboflavin delivery to overcome undesired FT processing. [Display omitted] [ABSTRACT FROM AUTHOR]

The present study aimed to develop a ready-to-eat restructured European sea bass (Dicentrarchus labrax) meat product. The response variables have been addressed as a function of MTGase addition (0, 0.16, 0.32%), pressure weight (0, 2.54, 5.09 gf/cm2), and setting time (4, 14, 24 h) at 4°C to restructured fish meat using response surface methodology (RSM) based design of experiments. The optimizing process was characterized by using two physical (deviation in diameter, pore) and two sensorial (appearance, texture) analyses. In addition to image processing and sensory analysis, microbiological quality, water activity, pH value, and variations after heat application were examined. The use of MTGase and pressure weight combination improved textural properties and sensory results. The RSM study demonstrated that 17.72 hours of setting time, 3.46 gf/cm2 of pressure weight, and 0.30% MTGase were the optimal parameters of restructured fish meat product. Optimum product results were statistically matched with the solutions; pore level: %0.89±0.01, deviation in diameter: %1.94±0.00, appearance: 8.55±0.07, and texture: 8.45±0.05. These results were also supported by microstructure findings and other quality parameters. The restructured fish product that meets with the positive appreciation of the consumer can be improved by adding spices, forming different shapes, and available for commercial production. [ABSTRACT FROM AUTHOR]

There is a high demand for designing healthy-functional dairy gels with a newly structured protein network in the food industry. Non-fat yogurt gels enriched with stable tarragon essential oil-nanoemulsions (TEO-NEs) using crosslinking of microbial transglutaminase (MTGase) were developed. The gas chromatography-mass spectrometry analysis showed that methyl chavicol (85.66%) was the major component in TEO extracted by the hydrodistillation process. The storage-dependent droplet size and physicochemical stability data of samples at room temperature for 30 days revealed that the TEO-NE containing 0.5% tween-80 and 1:2 TEO/sunflower oil had the lowest peroxide value and droplet growth ratio. The response surface methodology-based formulation optimization of free-fat yogurt gels using MTGase (0.15–0.85 U/g) and the best TEO-NE (0.5–3.02%) using the fitted second-order polynomial models proved that the combination of 0.87% TEO-NE and 0.70 U/g MTGase led to the desired pH (4.569) and acidity (88.3% lactic acid), minimum syneresis (27.03 mL/100 g), and maximum viscosity (6.93 Pa s) and firmness (0.207 N) responses. Scanning electron microscopy images visualized that the MTGase-induced crosslinks improved the gel structure to increase the firmness and viscosity with a reduction in the syneresis rate. The optimal yogurt gel as a nutritious diet not only provided the highest organoleptic scores but also maintained its storage-related quality with the lowest mold/yeast growth and free-radical oxidation changes. [ABSTRACT FROM AUTHOR]

Transglutaminases are protein cross‐linking and protein‐modifying enzymes that have attracted considerable interest due to their causal involvement in various diseases and versatility in industrial applications. In particular, microbial transglutaminases (MTG) from Streptomyces bacteria have managed in recent years to evolve from simple food additives to specialized enzymes for the site‐directed modification of therapeutic proteins. The review summarizes relevant studies from the beginning dealing with the occurrence, production, structure, catalysis, and substrate molecules of MTG enzymes. It also addresses biotechnological procedures with MTG from S. mobaraensis (SmMTG) as the most prominent representative in focus. Reassessment of the available data revealed unexpected insights into catalysis of SmMTG and other transglutaminases, suggesting selection of glutamine donor proteins by subsites at the front vestibule and the existence of distinct lysine pockets. Flexibility of the SmMTG‐accessible glutamine donor substrate regions seems to be more important than the glutamine environment. Nevertheless, residues in close vicinity to glutamines also determine interaction with the SmMTG subsites. The apparent lack of subsites for lysine donor proteins suggests self‐assembly of the substrate proteins prior to enzymatic cross‐linking. The study of natural substrate proteins, especially their mutual interaction, is proposed to further illuminate catalysis of SmMTG. To this end, structure and function of the characterized substrate proteins from S. mobaraensis are discussed in conclusion. [ABSTRACT FROM AUTHOR]

The main aim of the research was to develop a new biocompatible and injectable composite with the potential for application as a bone-to-implant bonding material or as a bone substitute. A composite based on hydroxyapatite, gelatin, and two various types of commercially available transglutaminase (TgBDF/TgSNF), as a cross-linking agent, was proposed. To evaluate the impacts of composite content and processing parameters on various properties of the material, the following research was performed: the morphology was examined by SEM microscopy, the chemical structure by FTIR spectroscopy, the degradation behavior was examined in simulated body fluid, the injectability test was performed using an automatic syringe pump, the mechanical properties using a nanoindentation technique, the surface wettability was examined by an optical tensiometer, and the cell viability was assayed by MTT and LDH. In all cases, a composite paste was successfully obtained. Injectability varied between 8 and 15 min. The type of transglutaminase did not significantly affect the surface topography or chemical composition. All samples demonstrated proper nanomechanical properties with Young's modulus and the hardness close to the values of natural bone. BDF demonstrated better hydrophilic properties and structural stability over 7 days in comparison with SNF. In all cases, the transglutaminase did not lead to cell necrosis, but cellular proliferation was significantly inhibited, especially for the BDF agent. [ABSTRACT FROM AUTHOR]

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Summary: Whey protein and cellulose derivatives are abundant and renewable raw materials that provide an environmentally friendly alternative to fossil fuel sources used for food packaging. A novel biodegradable composite film comprising whey protein concentrates (WPC) aqueous solutions (10%, w/v) with different concentrations of Hydroxypropyl methylcellulose (HPMC) (0, 1, 2, 3, 4 and 5 wt% of WPC) was prepared in the present study. The effect of transglutaminase (TG) on the functional properties of the film was investigated. SDS‐PAGE profiles indicated that TG modulated the formation of intermolecular cross‐linking of WPC. FT‐IR results showed that HPMC modified the mechanical properties of WPC. Incorporation of HPMC decreased the transparency and improved the tensile strength and extensibility of the film. TG addition led to a significant enhancement of the mechanical properties of the film. These findings indicated that TG promoted the formation of WPC‐HPMC composite film with improved mechanical properties. [ABSTRACT FROM AUTHOR]

objective: Strained or concentrated yoghurt is a traditional milk product that its popularity and consumption has increased during recent years due to its higher nutritional value, longer shelf life, desirable textural and sensory properties, and today it is widely produced industrially. This product can be produced from many different animal milks such as buffalo, cow, goat, ewe and camel. The milk of various animals differs in chemical composition and therefore this factor can affect the characteristics of the final product. Identical to the other dairy products, strained yoghurt manufactured from buffalo milk has the higher quality than those produced from cow milk due to the higher total solids and fat content; but its utilization has limited because of its higher cost. Transglutaminase enzyme (TG), by creating inter- and intra-molecular cross linkages among proteins is known as an effective strategy to improve the functional properties of dairy products. TG treatment has been considered as a practical technique instead of increasing the total solids of milk (by adding skim milk powder). Therefore, the aim of this study was investigate the effect of TG enzyme treatment on physicochemical and sensory properties of strained yoghurt prepared from cow and buffalo milk mixture and introduce the best product with the lower buffalo milk substitution (with a more reasonable price). Materials and methods: Strained yoghurt samples with different amounts of buffalo milk replacement with cow (0, 25, 50, 75 and 100%, v/v) and using TG enzyme (0 and 0.015%, w/v) were produced and 10 experimental treatments evaluated for physicochemical (pH, acidity (% lactic acid), syneresis (%) and dry matter (%)) and sensory properties (texture, color, odor and flavor) during 21 days of storage (1, 7, 14 and 21 days) under refrigeration conditions. The mean of obtained values were analyzed using a random factorial design and compared by Duncan test at 5% level. Results: The results showed that increasing the substitution amount of buffalo milk with cow milk caused a significant increase in acidity and dry matter while it expressively reduced the pH and syneresis of yoghurt samples. Also, with increasing the TG enzyme concentration, the amount of acidity and syneresis decreased and the pH increased significantly, but this variable had no significant effect on dry matter. By increasing the storage time, the pH decreased significantly and its initial value of 4.11 at the first day of storage reached to 3.98 at the end of 21 days of storage. The initial values of acidity, dry matter and syneresis as 0.98%, 20.69% and 4.45% increased till the end of storage and reached to 1.23%, 22.16% and 10.35%, respectively. In terms of sensory acceptability, increasing the concentration of enzyme caused a significant increase in texture score of the samples, while the enzyme had no significant effect on other sensory scores of the samples. With increasing the mixing rate of buffalo milk, the texture, colour and flavour scores of the samples increased significantly and the aroma score decreased. Furthermore, the sensory properties of the samples decreased significantly during the storage period. Conclusion: According to the results of this study, by replacing 75% of buffalo milk with cow milk and using transglutaminase enzyme treatment (0.015%), it is possible to produce a strained yoghurt with suitable quality characteristics. [ABSTRACT FROM AUTHOR]

In the past few years, researchers have focused on improving the functional properties and qualities of food products. To this end, they have used crosslinking for enhancing the functional properties of proteins in the food products. Enzymatic or non-enzymatic crosslinking can be used to modify food proteins. Protein crosslinking is efficient in generating novel textures and developing product formulations, while also maintaining the desired texture and mouthfeel of food products. Enzymatic treatments using laccases, transglutaminases, peroxidases, and tyrosinases could help in designing meat replacement products, and developing non-dairy yoghurt and cheeses with good consistency. However, these catalytic mechanisms are accompanied by many technical issues that need to be overcome while developing complex food matrices. [ABSTRACT FROM AUTHOR]