Search Results (68)

This study investigated the effect of a concentrated kombucha inoculum and transglutaminase (TG) on the rheological and textural properties of fermented milk products and compared their average production costs to commercial yoghurt. Semi-skimmed milk was used, to which microbial TG was added at a level of 0.02% w/w. The kombucha inoculum, prepared from black tea, was concentrated to 55.6% total solids. Four samples were produced: two with TG and two without. The TG-containing samples showed significantly higher textural properties, including firmness and consistency, than the non-TG samples. They also exhibited the largest hysteresis loop area and the highest yield stress, indicating a stronger gel structure. The Herschel–Bulkley model successfully described the flow behaviour of all samples and confirmed their shear-thinning, non-Newtonian nature. Principal Component Analysis (PCA) showed that both TG addition and inoculum concentration significantly influenced the product properties. TG improved the rheological and textural properties and increased the stability during storage. However, the production costs for TG-treated samples were higher than those for non-TG-treated samples and commercial yoghurt. Nevertheless, the higher costs could be justified by the perceived additional nutritional benefits for consumers. Overall, the results show that the combination of concentrated kombucha inoculum with transglutaminase can improve the structural and rheological quality of fermented dairy products, which is potentially of commercial importance. Full article

The effects of different levels of microbial transglutaminase (MTGase) at 0% (control), 0.25%, 0.50% and 1% on the physicochemical properties, volatile compounds and free amino acid composition of pastırma, a Turkish dry-cured meat product, were investigated. The MTGase treatment had no significant effect on the a_w, L* and b* values of pastırma. The thiobarbituric acid reactive substances value decreased as the MTGase level increased. The maximum cutting force was found to be higher in enzyme-treated pastırma groups compared with the control. Enzyme treatment increased the maximum stress–relaxation force, but no statistical difference was observed between the 0.50% and 1% enzyme treatments. No significant differences were observed between groups in the volatile compound profile. However, in the correlation analysis, the control group showed a close correlation with the 0.25% MTGase group. This was also the case for the 0.5% and 1% MTGase groups. In the samples, glutamic acid, arginine, alanine, cystine and valine were determined to be the dominant free amino acids, and glutamic acid showed a close correlation with valine; lysine with arginine; and cystine with serine. MTGase had no significant effect on the total free amino acid content. Full article

The mechanical properties of the extracellular matrix critically influence cell behavior in both physiological and pathophysiological states, including cardiac fibrosis. In vitro models have played a critical role in assessing biological mechanisms. In this study, we characterized mechanically tunable enzymatically crosslinked gelatin-microbial transglutaminase (mTG) hydrogels for modeling cardiovascular diseases. Gelatin hydrogels were fabricated via direct mixing or immersion crosslinking methods. Hydrogel formulations were assessed using the Piuma nanoindenter and Instron systems. This study investigates the effects of fabrication methods, UV ozone (UVO) sterilization, crosslinking methods, and incubation media on hydrogel stiffness. Further, this study examined the response of murine cardiac fibroblasts to hydrogel stiffness. The hydrogels exhibited modulus ranges relevant to both healthy and fibrotic cardiac tissues. UVO exposure led to slight decreases in hydrogel modulus, while the fabrication method had a significant impact on the modulus. Hydrogels incubated in phosphate buffered saline (PBS) were stiffer than those incubated in Medium 199 (M199), which correlated with lower pH in PBS. Fibroblasts cultured on stiffer hydrogels display enhanced smooth muscle actin (SMA) expression, suggesting sensitivity to material stiffness. These findings highlight how fabrication parameters influence the modulus of gelatin-mTG hydrogels for cardiac tissue models. Full article

Microbial transglutaminase (mTG) is most frequently utilized in order to increase the gelling properties of soybean protein isolate (SPI), but there are still some limitations of mTG-based hydrogel fabrication technology. Therefore, we aimed to develop a dual modification technique based on enzyme plus organic acid treatment to fabricate SPI hydrogels with high gel strength and stability. Our results showed that mTG plus glucose-δ-lactone (GDL), lactobionic acid (LBA) or maltobionic acid (MBA) treatment could significantly improve the gel strength, textural properties, and water-holding capacity of SPI hydrogels. Also, the addition of these organic acids remarkably reduced the surface hydrophobicity (H₀) and intrinsic fluorescence as well as increased the storage modulus (G′), loss modulus (G″) values, average particle size, and the absolute value of zeta potential of samples. GDL, LBA, or MBA greatly increased the β-sheet level and decreased the α-helix level in hydrogels, as well as dissociated 11S subunits of SPI into 7S subunits. Notably, covalent interactions, hydrogen bonding, and hydrophobic interactions of three organic acids with SPI, as well as the effects of organic acids on the interactions among the intramolecular and intermolecular forces of SPI molecules, contributed to their promoting effects on the formation of hydrogels. The LF-NMR and SEM analyses confirmed the effects of GDL, LBA, and MBA on converting the free water into immobilized and bound water as well as forming a dense stacked aggregate structure. Therefore, GDL, LBA, and MBA are promising agents to be combined with mTG in the fabrication of SPI hydrogels with high gel strength and stability. Full article

In this study, chitooligosaccharides (COS) were introduced into black soybean protein (BSP) using transglutaminase (TG) as a biocatalyst. The film-processing properties and physiological activities of the enzymatically glycosylated black soybean protein (EGBSP) were studied. The results showed that glycosylation decreased the surface hydrophobicity, absolute value of the zeta potential, its minimum solubility, and film permeability of BSP by 69.86%, 6.04%, 36.68%, and 14.91%, respectively, while increasing the tensile strength and elongation at break of its protein film by 56.57% and 172.68%, respectively. The gel time was shortened, and the acid-induced gel properties of EGBSP were similar to those of BSP. The anticancer effect of EGBSP was evaluated by the tumor inhibition rate, flow cytometry, and morphology observation of an ascites tumor in H₂₂ tumor-bearing mice. The immune organs (spleen, thymus), immune cells (lymphocytes, NK cells), and immune factors (IL-2, IL-12) of H₂₂ tumor-bearing mice were detected to evaluate the immunomodulatory effects of EGBSP. The results showed that medium and high doses of BSP had positive effects on immune enhancement and anti-cancer activity of H₂₂ tumor-bearing mice, while almost all doses of EGBSP showed significant effects. These results indicated that glycosylation significantly improved the anti-cancer effect and immunomodulatory activity of H₂₂ tumor-bearing mice while prolonging their overall survival. In conclusion, the glycosylation method using microbial transglutaminase to catalyze the introduction of chitooligosaccharides into black bean protein can improve the film-processing properties and biological activities of BSP more effectively than the enzyme crosslinking method. Full article

Microbial transglutaminase (mTG) is a bacterial survival factor, which is frequently used as a food additive. This results in the formation of immunogenic epitopes that may cause autoimmunity. Primary biliary cholangitis (PBC) is a cholestatic, autoimmune liver disease characterized by the presence of characteristic autoantibodies. The aim of this work was to determine epitope similarity and cross-reactivity between mTG- and PBC-specific antigens and to investigate whether the microbial enzyme may be associated with the induction of autoimmunity due to epitope similarity and cross-reactivity. Monoclonal and polyclonal antibodies against mTG were applied to nine different PBC-specific antigens using ELISA technique. They reacted significantly with four out of nine antigens. This reaction was most pronounced for gp210 and PML protein. We also performed in vitro studies on the impact of the mTG on the specific antigen–antibody binding using sera of PBC patients. We found four PBC-specific antigens that share homology with mTG sequences. We noticed inhibition of this specific binding by the mTG to the PDC M2, gp210, PML, and KLHL12 protein. Microbial mimics may be the major targets of cross-reactivity with human-specific antigens. Cross-reactivity may indicate a link between mTG and the development of autoimmune diseases. Full article

This study sheds light on the potential of microbial transglutaminase (mTG)-mediated modification to enhance the properties of heat-denatured whey protein-based films. In this study, we investigated the biochemical modification of heat-denatured whey proteins (WPs) using mTG, an enzyme known for the ability of crosslinking reactions. By introducing ε-(γ-glutamyl)-lysine crosslinks via an acyl transfer reaction, mTG enhances the properties of bio-based materials. In this research, heated WPs were demonstrated to effectively serve as mTG substrates. The preparation of crosslinked bio-based material was achieved using a casting method under alkaline conditions (pH 12) in the presence of glycerol (40% w/w), which was added as a plasticizer to the film-forming solution (FFS). A comprehensive characterization of the FFSs and the resulting materials was carried out. The FFSs were quite stable as evidenced by Zeta potential values that were always around 30/40 mV regardless of the presence of the enzyme. The enzymatic modification increased the elongation at break of the materials from 10.4 ± 4.9 MPa to 27.6 ± 8.9 MPa, while decreasing both tensile strength and Young’s modulus, thereby making the resulting material more extensible. On the other hand, the enzyme affected both the CO₂ and O₂ barrier properties, with permeability values for these gases being 0.90 cm³ mm m⁻² day⁻¹ kPa⁻ and 0.26 cm³ mm m⁻² day⁻¹ kPa, respectively, when the films were cast without the enzymatic treatment, but decreasing to 0.14 ± 0.02 cm³ mm m⁻² day⁻¹ kPa (CO₂) and 0.02 ± 0.02 cm³ mm m⁻² day⁻¹ kPa (O₂) in the presence of 24 U/g of mTG. These novel materials, prepared from renewable sources, could potentially be used in the food packaging field to replace/reduce the highly pollutant petroleum-based ones. Full article

With growing environmental and health concerns surrounding meat consumption, meat analogs have emerged as sustainable and health-conscious alternatives. A major challenge in developing these products is replicating the fibrous, elastic texture of meat, where microbial transglutaminase (MTG) has shown significant potential. MTG catalyzes protein cross-linking, enhancing the structural integrity of meat analogs. This study aimed to evaluate the effects of MTG concentrations (0%, 0.5%, and 1%) and incubation times (0, 1.5, and 3 h) on the quality and rheological properties of meat analogs. Analogs were tested for pH, protein content, dry matter, fat retention, and thermal loss. Textural properties, including hardness, cohesiveness, gumminess, springiness, and chewiness, were determined using texture profile analysis, while leakage parameters were evaluated through water and fat content tests. Results revealed that higher MTG concentrations and longer incubation times improved protein content (14.34% to 15.55%), dry matter (29.61% to 32.53%), and reduced total leakage (1.262% to 0.634%). Textural properties, including hardness (57.08 N to 83.14 N), gumminess (19.40 N to 30.00 N), and chewiness (17.60 N × mm to 29.58 N × mm), also significantly improved with increasing MTG levels. Thermal loss ranged from 98.37% to 100.9%, showing enhanced retention at higher MTG concentrations. These results support the role of MTG in creating meat analogs with improved meat-like textures, achieved through enhanced protein cross-linking and moisture retention. Full article

Homogeneous antibody–drug conjugates (ADCs) exhibit significantly improved pharmacological properties compared to their heterogeneous counterparts. Site-specific conjugation of the payload to the IgG required for homogeneity can be achieved using enzymes. One example is microbial transglutaminase (MTGase), which can selectively perform transamidation on the Q295 residue of human Fc when N297 glycans are removed. As a result, two modifications can be introduced per IgG molecule; however, achieving higher drug-to-antibody ratios (DARs) requires the use of branched linkers. While several such linkers have been reported, little information is available on the relationship between linker structure and ADC properties. To address this gap, we synthesized two branched amino triazide linkers, differing by a PEG₄ fragment inserted after the branching point, which were used to prepare two homogeneous trastuzumab-based DAR 6 ADCs (a “short” and a “long” one). This was achieved by a two-step process consisting of enzymatic linker conjugation followed by bioorthogonal coupling with a cleavable linker bearing monomethyl auristatin E (MMAE). Two other trastuzumab–MMAE conjugates were used as controls: a heterogeneous DAR 6 ADC, made using conventional thiol–maleimide chemistry, and a homogeneous DAR 2 ADC. We found that, while the four conjugates had identical affinity for HER2, their cytotoxicity differed significantly: the “long” homogeneous DAR 6 ADC was just as active as its heterogeneous counterpart, but the “short” DAR 6 ADC was an order of magnitude less potent, inferior even to the DAR 2 conjugate. Our findings indicate that the length of the branched linker critically affects the cytotoxic activity of ADCs, possibly due to steric hindrance influencing the rate of linker cleavage by lysosomal enzymes. Full article

In the era of the blue bio-economy, which promotes the sustainable utilization and exploitation of marine resources for economic growth and development, the fisheries and aquaculture industries still face huge sustainability issues. One of the major challenges of these industries is associated with the generation and management of wastes, which pose a serious threat to human health and the environment if not properly treated. In the best-case scenario, fishery and aquaculture waste is processed into low-value commodities such as fishmeal and fish oil. However, this renewable organic biomass contains a number of highly valuable bioproducts, including enzymes, bioactive peptides, as well as functional proteins and polysaccharides. Marine-derived enzymes are known to have unique physical, chemical and catalytic characteristics and are reported to be superior to those from plant and animal origins. Moreover, it has been established that enzymes from marine species possess cold-adapted properties, which makes them interesting from technological, economic and sustainability points of view. Therefore, this review centers around enzymes from fishery and aquaculture waste, with a special focus on proteases, lipases, carbohydrases, chitinases and transglutaminases. Additionally, the use of fishery and aquaculture waste as a substrate for the production of industrially relevant microbial enzymes is discussed. The application of emerging technologies (i.e., artificial intelligence and machine learning) in microbial enzyme production is also presented. Full article

Objective: The goal of this systematic review was to identify the mechanisms associated with the enzymatic degradation of collagen and gelatin biomaterials and the possible associated flaws. Methods: Four databases (PubMed, B-On, Cochrane Library, and ResearchGate) were used for the bibliographic search of articles. The research question was formulated using the PCC method, (P): collagen or gelatin sponges, hydrogels, and scaffolds; concept (C): enzymatic degradation of collagen or gelatin sponges, hydrogels, and scaffolds; and context (C): effect of enzymatic action on degradation time of collagen or gelatin sponges, hydrogels, and scaffolds. The search was contextualized according to PRISMA recommendations. The identification and exclusion of evidence followed the PRISMA criteria, with specific inclusion and exclusion factors being stipulated for the selection of articles. The risk of bias assessment was performed using the QUIN Scale. Results: The initial search was composed of 13,830 articles after removing duplicates; 56 articles followed for the full-text reading; 45 were excluded; then, 11 articles were obtained, constituting the results of this systematic review. All studies evaluated the materials using gravimetric analysis, and collagenases were the proteases used for the degradation solution. The materials tested were as follows: human-like collagen (HLC) hydrogel with microbial transglutaminase (MTGase), gelatin sponges subjected to different types of crosslinking, and collagen scaffolds with different types of crosslinking. The period of analysis varied between 0.25 h and 35 days. It was possible to highlight the lack of uniformity in the protocols used, which varied largely, thus influencing the degradation times. The risk of bias was low in nine studies and medium in two studies. Conclusions: This systematic review identified a gap in the literature, highlighting the absence of in vitro studies using human saliva and a collagenase concentration close to the physiological levels to simulate oral dynamics. However, based on existing literature, the mechanisms associated with collagen enzymatic degradation in collagen and gelatin biomaterials were comprehensively understood, answering the first research question postulated. In response to the second research question, the main shortcomings identified in the laboratory evaluation of mechanisms associated with collagen enzymatic degradation in collagen and gelatin biomaterials included the lack of standardization in degradation test protocols; this limited inter-study comparisons, which increased heterogeneity. Additionally, variations in collagenase concentrations and types influenced collagen degradation rates, and inappropriate evaluation intervals hindered the identification of total degradation time. Full article

Mastitis-causing bacteria can establish persistent infections in the mammary glands of commercially important dairy animals despite the presence of strong specific humoral and cellular immune mechanisms. We investigated the effect of vitamin D₃ in the diet at a set level, but in two different forms (i.e., unencapsulated and encapsulated by complex coacervation with sulfur-saturated bovine lactoferrin-alginate using microbial transglutaminase-catalyzed crosslinking) on the immune response in late-lactating dairy goats. Dairy goats (n = 18) were randomly assigned to three experimental groups (n = 6). Dairy goats were orally administered 0.35 mg of vitamin D₃/day in the unencapsulated form and 0.35 mg of vitamin D₃/day in the encapsulated powder form. Another group received the basal diet. The experimental period lasted 6 weeks. The blood serum concentrations of 25-hydroxyvitamin D₃ [25-(OH)-D₃], lactoferrin, immunoglobulin A (IgA), and interferon-gamma (INF-γ) were measured. There were major differences in these parameters between dietary groups. However, the delivery of vitamin D₃ in the encapsulated powder form to dairy goats resulted in a marked increase in 25-(OH)-D₃ concentration in serum, while the serum level of lactoferrin also increased. Alternatively, the serum levels of IgA and the immunomodulatory cytokine INF-γ were elevated following supplementation with the encapsulated vitamin D₃. The observed effects suggest that the deliverable form of dietary vitamin D₃ results in differences in the immune response in late-lactating dairy goats. Full article

To address the limitations of alginate and gelatin as separate hydrogels, partially oxidized alginate, alginate dialdehyde (ADA), is usually combined with gelatin to prepare ADA-GEL hydrogels. These hydrogels offer tunable properties, controllable degradation, and suitable stiffness for 3D bioprinting and tissue engineering applications. Several processing variables affect the final properties of the hydrogel, including degree of oxidation, gelatin content and type of crosslinking agent. In addition, in 3D-printed structures, pore size and the possible addition of a filler to make a hydrogel composite also affect the final physical and biological properties. This study utilized datasets from 13 research papers, encompassing 33 unique combinations of ADA concentration, gelatin concentration, CaCl₂ and microbial transglutaminase (mTG) concentrations (as crosslinkers), pore size, bioactive glass (BG) filler content, and one identified target property of the hydrogels, stiffness, utilizing the Extreme Boost (XGB) machine learning algorithm to create a predictive model for understanding the combined influence of these parameters on hydrogel stiffness. The stiffness of ADA-GEL hydrogels is notably affected by the ADA to GEL ratio, and higher gelatin content for different ADA gel concentrations weakens the scaffold, likely due to the presence of unbound gelatin. Pore size and the inclusion of a BG particulate filler also have a significant impact on stiffness; smaller pore sizes and higher BG content lead to increased stiffness. The optimization of ADA-GEL composition and the inclusion of BG fillers are key determinants to tailor the stiffness of these 3D printed hydrogels, as found by the analysis of the available data. Full article

This study investigated the effects of high-pressure processing (HPP; 600 MPa/15 min) and microbial transglutaminase-catalyzed (MTG; 30 U·g of protein⁻¹) crosslinking on the concentration of dissolved proteins (SOL), free sulfhydryl groups (SH), surface hydrophobicity (H₀), and viscosity of pea protein isolates (PPI) at different concentrations (1–13%; w/v). The SOL increased by increasing protein concentration (max. 29%). MTG slightly affected SOL. HPP decreased SOL with increasing protein concentration, and the combination MTG + HPP resulted in a lower SOL than HPP alone. The concentration of SH in untreated PPI increased with increasing protein concentration, reaching a maximum of 8.3 μmol·mg prot⁻¹. MTG increased SH at higher protein concentrations. HPP lowered SH, but its concentration increased by increasing protein concentration. HPP + MTG offset the effect of MTG, yielding lower SH. MTG did not affect H₀ at 1% concentration but increased it for concentrations from 3–5%, and there was a decrease with 7–9%. HPP increased H₀ up to 37% for intermediate protein concentrations but did not affect it at higher concentrations. MTG + HPP decreased H₀ at all protein concentrations. The viscosity of the dispersions increased with protein concentration. HPP increased the viscosity of the dispersions for concentrations above 7%, while MTG only caused changes above 9%. Combined MTG + HPP resulted in viscosity increase. The results underscore the opportunity for innovative development of high-protein products with improved properties or textures for industrial application. Full article

Gelatin-based hydrogels with excellent mechanical properties and conductivities are desirable, but their fabrication is challenging. In this work, an innovative approach for the preparation of gelatin-based conductive hydrogels is presented that improves the mechanical and conductive properties of hydrogels by integrating Z–Gln–Gly into gelatin polymers via enzymatic crosslinking. In these hydrogels (Gel–TG–ZQG), dynamic π–π stacking interactions are created by the introduction of carbobenzoxy groups, which can increase the elasticity and toughness of the hydrogel and improve the conductivity sensitivity by forming effective electronic pathways. Moreover, the mechanical properties and conductivity of the obtained hydrogel can be controlled by tuning the molar ratio of Z–Gln–Gly to the primary amino groups in gelatin. The hydrogel with the optimal mechanical properties (Gel–TG–ZQG (0.25)) exhibits a high storage modulus, compressive strength, tensile strength, and elongation at break of 7.8 MPa at 10 °C, 0.15 MPa at 80% strain, 0.343 MPa, and 218.30%, respectively. The obtained Gel–TG–ZQG (0.25) strain sensor exhibits a short response/recovery time (260.37 ms/130.02 ms) and high sensitivity (0.138 kPa⁻¹) in small pressure ranges (0–2.3 kPa). The Gel–TG–ZQG (0.25) hydrogel-based sensors can detect full-range human activities, such as swallowing, fist clenching, knee bending and finger pressing, with high sensitivity and stability, yielding highly reproducible and repeatable sensor responses. Additionally, the Gel–TG–ZQG hydrogels are noncytotoxic. All the results demonstrate that the Gel–TG–ZQG hydrogel has potential as a biosensor for wearable devices and health-monitoring systems. Full article