The Effects of Vital Wheat Gluten and Transglutaminase on the Thermomechanical and Dynamic Rheological Properties of Buckwheat Dough

The improvement of the processing properties of gluten-free cereals is a challenging issue for cereal technologists. Vital wheat gluten (VWG), transglutaminase (TGase), or combinations of the two have been successfully used to improve the processing properties of food materials. In this study, a Mixolab system and a rheometer were used to investigate the effects of TGase on the thermomechanical and dynamic rheological properties of buckwheat flour dough (BF dough) and a buckwheat flour dough containing 15% VWG (BF-VWG dough). The results indicated that the addition of 1.0% TGase decreased the water absorption and significantly increased the stability, storage modulus (G′), and complex modulus (|G*|) of the BF dough. The presence of 15% VWG had a synergistic effect, enhancing the effectiveness of the TGase in improving the thermomechanical and dynamic rheological properties of the BF-VWG dough. Quantification of the free amino groups showed that the amino groups were more effectively bonded in the BF-VWG dough than in the BF dough when the TGase level exceeded 1.0%. Confocal laser scanning microscopy (CLSM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed that the TGase promoted the formation of protein complexes. These data indicate that the combination of VWG and TGase can be used to improve the properties of buckwheat flour for buckwheat-based food production.     

Enzymatic properties of transglutaminase produced by a new strain of Bacillus circulans BL32 and its action over food proteins

The aim of this study was to physicochemically characterize transglutaminase (TGase) from Bacillus circulans BL32, a strain recently isolated from the Amazon basin region, for its application in food systems. The effects of pH and temperature on the enzyme activity were determined by Central Composite Rotatable Design (CCRD), with maximal TGase activities obtained for pH between 5.7 and 8.7 and temperatures of 25-45 °C. This microbial TGase showed to be remarkably stable: over 90% of its activity was retained after 120 min of incubation at 50 °C. The Ca²⁺ and Mg²⁺ cations enhanced enzyme activity and its thermal stability when in concentrations of up to 2 and 1 mol L⁻¹, respectively. Casein, isolated soy protein, and hydrolysed animal protein were treated with this TGase. The decrease in the amount of free amino groups, especially for casein, showed the cross-linking of protein catalysed by this enzyme, while the emulsifying properties of these proteins were improved with treatment. These results suggest that this microbial TGase has a good potential to be used in food and other industrial applications.     

Modification of pork-skin jelly by enzymatic cross-linking: melting resistance and quality improvement

Improvements of melting resistance and quality by modification of pork-skin jelly through enzymatic cross-linking were studied, and the mechanism of quality improvement was discussed in this work. Gel strength, springiness and chewiness of modified gel increased significantly (P < 0.05). Transglutaminase (TGase) also improved the viscoelasticity, stability and melting resistance of gel system, as proved by rheological analysis. Sensory evaluation showed that increase in texture need to be moderate and the utilisation of 0.6% TGase was the most appropriate for pork-skin jelly. Significant effects of TGase on inducing protein cross-link and aggregation were confirmed by determining rheology during enzyme treatment and cross-linking extent of pork-skin soup. Correlation analysis showed TGase could improve melting temperature and texture by facilitating cross-linking. Covalent interaction based on ε-(γ-glutamyl)-lysine induced by TGase could play the main role in these improvements. This study suggested that TGase could be applied to design gelatin-based food for tailored quality properties through enzymatic cross-linking.     

Emulsifying properties of the transglutaminase-treated crosslinked product between peanut protein and fish (Decapterus maruadsi) protein hydrolysates

BACKGROUND: Defatted peanut meal, a protein‐rich by‐product from the oil extraction industry, is underutilised owing to its inferior functional properties. In this study, transglutaminase (TGase) crosslinking and proteolysis were used to improve the emulsifying properties of peanut protein isolate (PPI) extracted from the meal. PPI and PPI hydrolysate (PPIH) were conjugated separately with fish (Decapterus maruadsi) protein hydrolysate (DPH), catalysed by TGase to obtain improvements in the emulsifying properties. RESULTS: Analyses by electrophoresis and high‐performance liquid chromatography indicated that polymers were formed in all TGase‐treated samples. In emulsions of PPIH, PPI‐DPH and PPIH‐DPH the volume/surface average particle diameter (d₃₂), creaming and instability phenomenon were decreased and the zeta‐potential was increased after TGase treatment, showing improved emulsifying activity and emulsion stability. In the case of PPI, TGase treatment had no effect on the emulsifying activity, but the emulsion stability of TGase‐treated PPI was improved. CONCLUSION: The study showed that TGase crosslinking and proteolysis could improve the emulsifying properties of PPI, while proteolysis followed by TGase crosslinking proved more efficient. The emulsifying properties of the heterologous protein systems of PPI‐DPH and PPIH‐DPH were also improved by TGase treatment. Copyright © 2010 Society of Chemical Industry     

原生质体融合提高产谷氨酰胺转氨酶菌株产量

目的：研究并建立产谷氨酰胺转氨酶茂原链霉菌原生质体制备技术,通过原生质体融合技术筛选高产菌株。方法：以溶菌酶处理茂原链霉菌获得原生质体,采用原生质体融合技术,通过96 孔板高通量初筛、试管复筛、摇瓶验证选育高产菌株。结果：茂原链霉菌形成的原生质体再生出的菌落直径比较小,而菌丝生成的菌落直径比较大,两种形态的菌落96 孔板发酵后酶活力有显著性差异。不同的茂原链霉菌株制备原生质体,酶解程度、原生质体纯度、再生率有很大的差异,再生率最高可达1 804.25%,最低仅为12.76%。原生质融合后的高产菌株,选取96 孔板初筛酶活力前3%的菌株进行试管发酵,得到高产菌株比例为32.2%,酶活力比亲本高22.4%,经摇瓶验证产酶比对照提高16.28%。结论：利用基因组重排技术,可以初步对茂原链霉菌进行高产菌株选育。     

蛹虫草中谷氨酰胺转氨酶基因的克隆及在毕赤酵母中的表达

谷氨酰胺转氨酶(TGase或TG)是一种可催化蛋白质间形成异肽键,使蛋白质改性的天然酶制剂。本文以蛹虫草基因组为模板,通过PCR扩增得到TGase相似蛋白的基因片段tgM,将其与大肠杆菌-毕赤酵母穿梭表达载体pPIC9K连接,构建重组表达载体pPIC9K-TG,在毕赤酵母中进行表达,实现目的蛋白的胞外分泌表达,结果发酵液中重组TGase的酶活力为100 U/L。本研究为TGase的异源表达及潜在的工业应用提供参考。     

Effect of cross-linking with transglutaminase on the heat stability and some functional characteristics of sodium caseinate

The effect of heating (140 °C, 0–60 min between pH 6.0 and 7.0) on the turbidity, pH 4.6 soluble amino group content and urea PAGE profiles of sodium caseinate (NaCN) and transglutaminase (TGase)-treated NaCN was determined. pH-dependent heat-induced changes in the turbidity and urea PAGE profiles of NaCN were initially attributed to casein aggregation followed by subsequent degradation on extended heating. Cross-linked NaCN samples (incubated with TGase at 20 °C, [E:S] of 1:50 and 1:20 for 185 min) were generally less turbid and had lower pH 4.6 soluble amino group content on heating than unmodified NaCN. The nitrogen solubility of cross-linked NaCN was improved at pH 2.0, 3.0 and 5.0. Some improvements in emulsifying activity index and stability of cross-linked NaCN were observed at pH 5.0 and 10.0. The improved heat stability and nitrogen solubility observed after TGase cross-linking may help extend the range of applications for NaCN.     

Heterologous production of bacteriocins by lactic acid bacteria

Over the last two decades, bacteriocins produced by lactic acid bacteria (LAB) have been the subject of considerable research and industrial interest due to their potential as food biopreservatives. The development of heterologous expression systems for such antimicrobial compounds may offer a number of advantages over native systems, such as facilitating the control of bacteriocin gene expression or achieving higher production levels. In addition, the heterologous production by food-grade LAB offers an attractive method for overcoming some of the adverse situations that may affect the effectiveness of some bacteriocins in food systems. Construction of multibacteriocinogenic strains or acquisition of antimicrobial properties by industrial strains are further objectives that can be achieved through the use of heterologous gene expression systems. The development of new biotechnological tools and recent advances in LAB genetics account for the escalating number of studies dealing with heterologous production of bacteriocins by such hosts. This paper reviews the literature published on the subject and compares the different experimental strategies that have been used up to the present for this purpose.     

Purification and characterisation of transglutaminase from four fish species: Effect of added transglutaminase on the viscoelastic behaviour of fish mince

The transglutaminase (TGase) enzyme from four different fish species, namely bigeye snapper, Indian oil sardine, tilapia and common carp have been isolated and characterised. The specific activity of the enzyme was higher in tilapia followed by oil sardine, common carp and bigeye snapper. The molecular weight of pure TGase was found to be in the range of 73–95 kD for four different fish species. The temperature optima for maximum activity of TGase varied amongst four species studied. The effect of activator (calcium chloride) and inhibitors (ethylenediaminetetraacetic acid (EDTA), ammonium chloride and free lysine–hydrochloride) at different concentrations on the TGase enzyme activity have been evaluated. The addition of isolated TGase to fish mince from Cynoglossus sp. was attempted in order to evaluate the setting and gelling ability. The setting and gelling ability of fish mince in presence of TGase improved considerably as revealed by small strain and large strain test.     

Effects of pectic fat mimetics and transglutaminase on the regularity of protein and fat degradation and flavour compounds in Cheddar cheese during ripening

The effect of simultaneous transglutaminase (TGase) treatment with pectic fat mimetics (PFM) addition on regularity of protein and fat degradation and flavour compound variations in Cheddar cheese during ripening was investigated. In the early stages of fermentation, the cheese with 20% PFM and 15 U/L TGase had a higher amino acid content. In the middle stage of fermentation, cheese with high concentration of TGase hydrolysed more proteins. Cheese supplemented with PFM and TGase showed increased content of long-chain polyunsaturated fatty acids, especially linoleic acid (C18:2). In addition, PFM and TGase compensated the flavour deficiencies of low-fat cheeses and had positive effects on volatile compounds such as alcohols, acids and methyl ketones. Overall, the characteristics and flavours of Cheddar cheese with the addition of PFM and TGase were superior to the control group, which could provide a theoretical basis for the application of PFM and TGase in cheese production.     

植物来源谷氨酰胺转氨酶的研究进展

植物来源谷氨酰胺转氨酶(TGase)是一种在植物中广泛存在的催化酰基转移反应的蛋白酶,已被证实具有不同的理化性质和生理功能。结合最新的研究,本文综述了植物来源TGase在不同组织、器官中的分布,性质及其在植物生长发育过程中起到的作用。同时,为了获得植物来源TGase进行功能特性及其在食品、医学和其它工业中的应用研究,本文介绍了植物来源TGase异源表达的研究进展,并为该方法的改进提出了建设性意见。     

Bioutilisation of agro‐industrial waste for lactic acid production

With the advancement in science and technology, the importance of bioconversion of industrial waste into value‐added products through biotechnological route has been realised. Moreover, to make the process economic and environment responsive, agro‐industrial waste/by‐products can be used as substrates in fermentation processes as an inexpensive raw material. In the field of food biotechnology, organic acids represent the third largest category among the biological products, which can be produced chemically or by fermentation process. Among organic acids, lactic acid holds an important position because of its multivariate applications in food, pharmaceutical, cosmetics, textiles and other industries. Traditionally, lactic acid was produced from pure substrates, but agricultural feedstocks and industrial by‐products have evoked the interest of researchers to use them as raw materials for the biotechnological production of lactic acid. The utilisation of biomass has gained major attention due to environmental hazards, scarcity of fossil fuels and meet the increasing world food and feed demand. Integrated biorefinery approaches have also been adopted for the production of lactic acid along with primary product to minimise the cost. This review provides comprehensive information on the recent advances in biotechnological production of lactic acid utilising agro‐industrial waste/by‐products.     

Use of micellar casein concentrate and milk protein concentrate treated with transglutaminase in imitation cheese products—Unmelted texture

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.     

Transglutaminase treatment of brown rice flour: A chromatographic, electrophoretic and spectroscopic study of protein modifications

Recently, it was shown that transglutaminase (TGase) treatment of brown rice (BR) flour results in textural improvements of gluten-free bread. In this study, changes in the protein profiles of BR flour and protein fractions induced by TGase treatment were investigated to better understand the activity and specificity of the enzyme. Size-exclusion HPLC (SE-HPLC) profiles of flour extracts, under reducing conditions, revealed the presence of macromolecular protein complexes, as well as low molecular weight proteins. After TGase treatments (10 U/g of proteins) a general reduction in peak intensities indicated the polymerisation of BR proteins into larger, insoluble complexes. Microchip capillary electrophoresis and two-dimensional (2D) gel electrophoresis revealed that the α and β glutelin subunits were primary substrates for the polymerisation reaction, whereas albumins and globulins were only slightly affected. SE-HPLC of the protein fractions revealed glutelins’ polymerisation into high molecular weight structures after TGase treatment. Dynamic light scattering measurements showed that new supramolecular aggregates of glutelins co-existed with the macromolecular complexes already present in the untreated fraction. Front-face fluorescence approaches indicated that TGase treatment caused a decrease in protein surface hydrophobicity of BR flour, but not of the glutelin suspensions. It is concluded that the large protein complexes resulting from glutelin polymerisation and the stronger hydrophobic interactions among proteins result in the improved textural properties of TGase-treated BR bread.     

茂原链霉菌谷氨酰胺转氨酶合成与菌体形态分化的关系

目的：研究茂原链霉菌（Streptomyces mobaraensis）合成谷氨酰胺转氨酶（transglutaminase,TGase）与菌体分化之间的关系,以及TGase的生理功能。方法：以S. mobaraensis为出发菌株,通过向培养基中添加乙二胺四乙酸（ethylenediaminetetraacetic acid,EDTA）抑制TGase激活关键蛋白酶的活性从而调控成熟TGase的合成,同时利用激光共聚焦显微镜和扫描电镜观察发酵过程中菌体的活力和形态变化。结果：对照组随着TGase产量的升高,菌体生存活力呈先上升后下降的趋势,并且菌体表现出复杂的形态变化;实验组自加入EDTA后TGase产量不再升高,菌体生存活力也逐渐减弱,菌体形态分化滞后。结论：TGase的合成会影响茂原链霉菌生存活力及其形态分化。     

Effect of using transglutaminase on physical,chemical and sensory properties of set-type yoghurt

This study was conducted in order to evaluate the effects of transglutaminase (TGase) addition on some properties of set-type yoghurts such as titratable acidity, lactic acid, tyrosine, viscosity, gel firmness, syneresis, aroma compounds, sensory analysis, and micro-structural properties. The enzyme was added to yoghurt-milk at different production steps (after homogenization, after pasteurization and together with starter culture addition) and two different incubation times (10 min and 1 h) were used. Five TGase treated yoghurt samples and control sample were analyzed on 1st, 10th, and 20th days of storage. TGase addition did not cause significant changes on chemical properties of yoghurts. However, enzyme addition after pasteurization increased the gel strength and decreased the syneresis. Results of electron microscope showed that enzyme addition led proteins to be distributed more evenly in gel network due to the formation of cross-links between proteins.     

Improving protein secretion of a transglutaminase-secreting Corynebacterium glutamicum recombinant strain on the basis of 13C metabolic flux analysis

Corynebacterium glutamicum is known as a host species for amino acid production. This microorganism was recently noticed as a host that produces secreted proteins. In this study, we performed ¹³C metabolic flux analysis (¹³C-MFA) on a recombinant C. glutamicum strain that secretes a heterologous transglutaminase (TGase) to improve TGase secretion. For the ¹³C-MFA of a TGase-secreting C. glutamicum strain in batch cultivation, a ¹³C-labeling experiment and measurement of mass isotopomer distributions of proteinogenic amino acids were performed, and metabolic fluxes were determined considering the changes in fractional ¹³C-labeling of proteinogenic amino acids with respect to culture time. The TGase yield increased at the stationary phase but decreased toward its end. The results of ¹³C-MFA revealed that the flux from glycolysis to the TCA cycle gradually increased during TGase secretion. We speculate that the NADH/NAD⁺ ratio in the cells increases and that as a result, the specific glucose uptake rate decreases in the stationary phase because of the increased flux of the TCA cycle. Since it is expected that a decrease in the NADH/NAD⁺ ratio would improve the TGase yield, we tried to enhance lactate production in a TGase-secreting C. glutamicum strain to decrease cellular NADH levels by increasing the pH level in the culture. The TGase yield increased in 1.4-fold by increasing the pH from 6.7 to 7.2, indicating that the TGase yield was successfully improved on the basis of the ¹³C-MFA.     

In vitro antioxidant and immunomodulatory activity of transglutaminase-treated sodium caseinate hydrolysates

Sodium caseinate (NaCN) was incubated prior to and after hydrolysis with a microbial transglutaminase (TGase) and hydrolysed with Prolyve 1000. The resultant hydrolysates were tested for their immunomodulatory and antioxidant activity. TGase-treated hydrolysates significantly reduced (p < 0.05) the production of IL-6 at 0.5 and 1 mg mL⁻¹ and the non-TGase treated hydrolysate reduced the production of IL-6 at 1 mg mL⁻¹ in concanavalin (ConA) stimulated Jurkat T cells. None of the samples had an effect on IL-2. The hydrolysates showed higher oxygen radical absorbance capacity assay and ferric reducing antioxidant power activity than unhydrolysed NaCN, but no significant (p > 0.05) differences were found between the TGase-treated and non-TGase-treated samples. In the presence of hydrogen peroxide, the non-TGase-treated sample exhibited the highest DNA protective effect in U937 cells. These findings suggest that NaCN derived hydrolysates with and without treatment with TGase may exert specific antioxidant, genoprotective and anti-inflammatory effects.     

Cross-linking activity of sarcoplasmic fraction from bigeye snapper (Priacanthus tayenus) muscle

Addition of sarcoplasmic fraction from bigeye snapper (Priacanthus tayenus) into natural actomyosin in combination with setting at 40°C resulted in the cross-linking of myosin heavy chain (MHC). Higher amount of sarcoplasmic fraction and extended setting time resulted in a higher cross-linking, indicating the presence of endogenous transglutaminase (TGase) in bigeye snapper muscle. TGase activity was activated by calcium ion and reducing agents (β-mercaptoethanol and dithiotreitol), but was inhibited by N-ethylmaleimide (NEM), NH₄Cl and EDTA. TGase in the sarcoplasmic fraction was not stable when heated at temperature above 40°C, particularly with an increasing heating time. TGase was stable at pH ranging from 5.0 to 7.0, in which more than 70% activity was retained. Therefore, sarcoplasmic fraction possessed a cross-linking activity caused by TGase and its recovery for further uses should be considered.