INFLUENCE OF TRANSGLUTAMINASE-INDUCED CROSS-LINKING ON IN VITRO DIGESTIBILITY OF SOY PROTEIN ISOLATE

The influence of covalent cross‐linking by microbial transglutaminase (MTGase) on the sequential in vitro pepsin and trypsin digestion process and the digestibility of soy protein isolate (SPI), was investigated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and nitrogen release analyses. Various subunits of β‐conglycinin and acidic subunits of glycinin were cross‐linked by MTGase to form high molecular weight (MW) biopolymers, while basic subunits of glycinin were unaffected. SDS‐PAGE analysis indicated that the cross‐linking mainly affected in vitro pepsin digestion pattern of various subunits of β‐conglycinin, while the trypsin digestion pattern of native SPI was nearly unaffected. Nitrogen release analysis showed that the in vitro pepsin or/and trypsin digestibility of native SPI (at the end of pepsin or trypsin ingestion) was significantly decreased (P ≤ 0.01) by the MTGase treatment (for more than 2 h). The cross‐linking by MTGase also significantly decreased the in vitro digestibility of preheated SPI. These results suggest that the cross‐linking by means of transglutaminase may negatively affect the nutritional properties of food proteins.     

Effect of heat and transglutaminase on solubility of goat milk protein‐based films

The effect of heat, transglutaminase and combination of heat and transglutaminase treatments on the solubility of films prepared from goat milk casein, goat milk whey proteins and whole goat milk proteins was investigated. Goat milk casein films were less soluble when treated with transglutaminase and combination of heat with transglutaminase compare with heat‐treated caseins alone. Heat treatment was more effective at decreasing the solubility of whey protein films. SDS‐PAGE patterns demonstrated that goat milk caseins were better cross‐linked by transglutaminase, whereas whey proteins were better cross‐linked by heat. The extent of cross‐linking was further enhanced when a combination of heat and transglutaminase was used.     

Changes in the structure,digestibility and immunoreactivities of glycinin induced by the cross‐linking of microbial transglutaminase following heat denaturation

This study aimed to evaluate the effects of microbial transglutaminase (MTG)‐mediated modification on the structure, digestibility and immunoreactivities of glycinin. Glycinin was separated from soya bean and cross‐linked with MTG, and the sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed that the molecular weight of cross‐linked glycinin was higher than that of native glycinin. Individual MTG cross‐linking could maintain stable secondary structures and spatial structure. Sequential heat denaturation and MTG cross‐linking could promote the unfolding of protein structures and reduce their hydrophobicity. The digestibility of glycinin was decreased, and its immunoreactivities were increased because of MTG‐induced structural alteration, including primary and spatial structures.     

Addition of crude tiger nut protein extract affects stiffness of enzymatically cross‐linked dairy proteins

The influence of crude tiger nut protein extract on the gel properties of enzymatically cross‐linked dairy proteins was investigated. Enzymatic cross‐linking of dairy proteins in the presence of crude tiger nut proteins caused the formation of larger casein polymers and increased the degree of polymerisation. Gel stiffness of acidified products containing whey proteins was higher when cross‐linking occurred in the presence of crude tiger nut proteins. The results are relevant for improving the textural characteristics of acidified aqueous tiger nut extract (tiger nut milk) enriched in dairy proteins.     

The effect of transglutaminase on the properties of milk gels and processed cheese

The aim of this study was to investigate the influence of transglutaminase (TG) on rennet coagulation properties, and the impact on the properties of processed cheese. The TG was added before, at the same time as, and after the addition of rennet. The parameters moisture, total solids and proteins and physical parameters (syneresis index, wheying‐off and rheological behaviour) were measured. The manufacturing of the milk gels treated with transglutaminase after rennet addition was shown to be an effective means of improving the physical properties (reduced synereseis index and increased consistency index) of processed cheese, possible by the occurrence of enzymatic cross‐linking.     

Modification of the renneting process in Berridge substrate by transglutaminase

The influence of enzymatic cross‐linking on the rennet coagulation of Berridge substrate was investigated in the rennet gel samples prepared by three different methods using a microbial Tgase. Protein cross‐linking was carried out at each of three stages: before the addition of rennet, simultaneously with rennet and after the addition of rennet. The results showed that the cross‐linking of milk proteins before the addition of rennet inhibited the primary enzymatic phase of rennet coagulation. It was observed that enzymatic cross‐linking affected both the primary and the secondary phases of rennet coagulation. The most suitable application method was the addition of transglutaminase at the 5th minute after the addition of rennet.     

Application of transglutaminase to derivatize proteins: 1. Studies on soluble proteins and preliminary results on wool

The use of enzymes in chemical processing is gaining favour due to the reduction of hazardous chemicals and because it is considered to be environmentally safe. The acyl transfer reaction between primary amines and glutamine residues in proteins is catalysed by the enzyme transglutaminase. The efficiency of microbial transglutaminase to attach functional amines and catalyse inter‐ and intra‐molecular crosslinks was investigated using reduced carboxymethylated κ‐casein, gelatin and wool. Model systems used in this research gave evidence of both cross‐linking of the protein and covalent binding of the primary amine o‐phosphorylethanolamine to the protein. These data agree with earlier publications that show transglutaminase catalyses the formation of covalent cross‐links between the γ‐carboxyamide group of glutamine and the ε‐amino group of lysine and also the incorporation of primary amines into proteins. Preliminary analysis of treated wool indicated the covalent bonding of the functional amine to the protein. Our goal is to increase the value of wool by enzymatic addition of functional groups to the wool fibre. Published in 2004 for SCI by John Wiley & Sons, Ltd.     

Comparative study on protein cross-linking and gel enhancing effect of microbial transglutaminase on surimi from different fish

BACKGROUND: Microbial transglutaminase (MTGase) has been used to increase the gel strength of surimi. Nevertheless, its effectiveness varies with fish species. The aim of this study was to elucidate the effect of MTGase at different levels on protein cross‐linking and gel property of surimi from threadfin bream, Indian mackerel and sardine in the presence and absence of endogenous transglutaminase. RESULT: Breaking force of all surimi gels increased as MTGase levels (0–0.6 U g^?1) increased except for threadfin bream surimi gel, where the breaking force decreased at 0.6 U g^?1 (P < 0.05). In the presence of EDTA, the gel strengthening effect was lower, suggesting the combined effect of endogenous transglutaminase with MTGase. With the addition of MTGase, the gel with the highest increase in breaking force showed highest decrease in myosin heavy chain. When cross‐linking activity of MTGase on natural actomyosin (NAM) was determined, the highest decreasing rate in ε‐amino group content with the concomitant increased formation of cross‐linked proteins was found in NAM from threadfin bream. The reactivity of muscle proteins toward MTGase‐induced cross‐linking was in agreement with surimi gel strengthening. CONCLUSION: The composition and properties of muscle proteins of varying fish species more likely determined protein cross‐linking induced by MTGase, thereby affecting their gel properties. Copyright © 2011 Society of Chemical Industry     

DIFFERENT ARRANGEMENT OF ɛ-(γ-GLUTAMYL)LYSINE CROSS-LINKING IN ALASKA POLLOCK (THERAGRA CHALCOGRAMMA) SURIMI PROTEINS BY STREPTOVERTICILLIUM AND ENDOGENOUS TRANSGLUTAMINASES DURING SUWARI PROCESS

The objective of the present study is to compare the protein cross‐linking reaction in Alaska pollock surimi that is catalyzed by a commercially available microbial transglutaminase and by endogenous Alaska pollock transglutaminase. The endogenous transglutaminase was inhibited by EGTA and activated by CaCl₂ The microbial transglutaminase was added to the salted surimi with and without EGTA and CaCl₂. These surimi pastes were incubated at 25C up to 24 h followed by cooking at 90C. The resultant gels were fractionated into soluble and insoluble (aggregate) fractions by SDS‐urea extraction. Compositional analysis revealed that the aggregate consisted predominantly of cross‐linked myosin heavy chain. The distribution of ?‐(γ‐glutamyl)lysine isopeptide in the soluble and aggregate fractions andpeptide mapping analyses of the aggregate fraction demonstrate that the formation of isopeptide cross‐links in Alaska pollock surimi proteins during suwari process differs when catalyzed by the microbial transglutaminase and endogenous transglutaminase.     

Protein Oxidation in Processed Meat: Mechanisms and Potential Implications on Human Health

Processed meats represent a large percentage of muscle foods consumed in the western world. Various processing steps affect the physicochemical properties of the meat, compromise its nutritional components, or produce some compounds that are of health concern. Hence, the impact of oxidation on human health and the aging process and the influence of diet on these harmful reactions are of growing interest. Past decades have seen more focus on lipid oxidation, microbial deterioration, and pathogenicity, as well as production of carcinogenic compounds during meat processing. The oxidation of protein, which is a major component in meat systems, has received less attention. Protein oxidation has been defined as a covalent modification of protein induced either directly by reactive species or indirectly by reaction with secondary by‐products of oxidative stress. Not only are these modifications critical for technological and sensory properties of muscle foods, they may have implications on human health and safety when consumed. Cooking, for example, has been observed to increase free radical generation while it also decreases the antioxidant protection systems in meat, both of which contribute to protein oxidation. Many other meat processing techniques, as well as other emerging technologies, may significantly affect protein oxidation and protein overall quality. This paper explores the current understanding of meat processing techniques and their possible effects on the status of protein oxidation and nutritional value, as well as their possible implications on human health.     

Microbial Transglutaminase: General Characteristics and Performance in Food Processing Technology

The enzyme microbial transglutaminase (MTGase) (EC 2.3.2.13) catalyzes acyl transfer reactions, deamidation, and inter and intramolecular crosslinks between amino acid residues of glutamine and lysine. Its application in foods alters the technological properties of food proteins, such as their emulsifying capacity, gelation, viscosity, water holding capacity, and foam formation and stability. The commercial application of the enzyme in Brazil is limited almost entirely to the restructured meat industry and dairy beverages, but several studies have demonstrated its potential for commercial application in vegetable products such as soy, which is little exploited in the market. The application of this enzyme to vegetable derivatives allows for the development of new products or improvement of the functional properties of traditional foods. The objective of this article is to offer a comprehensive overview of the general characteristics and function of the enzyme microbial transglutaminase, with emphasis on its application in foods.     

Comparison of the effects of adding microbial transglutaminase to milk and ice cream mixture on the properties of ice cream

In this study, the effect of recombinant microbial transglutaminase enzyme on the physicochemical properties of ice cream produced by two different methods was investigated. For this purpose, different enzyme units (2, 4 and 6 U/g milk protein) were applied to both ice cream mixture and milk. While with the first method, a 10% increase in the overrun value of ice cream samples was observed, a 7% increase was obtained by the second method. All ice cream samples exhibited pseudoplastic behaviour. This study indicated that  transglutaminase concentration is an important factor in terms of improving the physicochemical properties of ice cream.     

Gelation of Food Protein Induced by Recombinant Microbial Transglutaminase

ABSTRACT: The recombinant microbial transglutaminase from Streptoverticillium mobaraense var. (rMTGase) was expressed in Escherichia coli. Specific enzyme activity of rMTGase was comparable to native MTGase. However, the gelation of a sodium caseinate solution induced by rMTGase was slower than that induced by native MTGase. In addition, the mechanical property of kamaboko prepared with rMTGase was weaker than that with native MTGase. In SDS‐PAGE analysis, α‐casein monomers decreased more slowly during the incubation with rMTGase than MTGase. These results confirmed the difference of cross‐linking activity between the 2 enzymes. Furthermore, thermal stability of rMTGase was lower compared to native MTGase. These results suggest that the difference of cross‐linking activity and thermal stability between the 2 enzymes cause differences in gelation activity of protein.     

The preparation of an oligochitosan‐glycosylated and cross‐linked caseinate obtained by a microbial transglutaminase and its functional properties

A glycosylated and cross‐linked caseinate (GCC) with glucosamine amount of 4.74 g/kg protein was generated from caseinate and oligochitosan by a microbial transglutaminase. The applied temperature, pH and molar ratio of acyl donor/acceptor were 37 °C, 7.5 and 1:3, respectively; while caseinate concentration, transglutaminase addition and reaction time selected from single‐factor trials were 50 g/L, 10 kU/kg protein and 3 h, respectively. Electrophoretic analysis revealed the cross‐linking and glycosylation of caseinate. Compared with caseinate, GCC showed improved solubility in pH 4–11, higher digestibility in vitro and water binding capacity, about 3‐fold, but lower surface hydrophobicity and oil binding capacity (34%).     

Moisture-induced aggregation of alpha-lactalbumin: effects of temperature, cations, and pH

Abstract: Alpha‐lactalbumin is an important dairy protein ingredient, and has been widely used in high‐protein foods such as infant formula and nutritional bars for its nutritional and functional properties. The purpose of this study was to investigate the moisture‐induced aggregation of alpha‐lactalbumin in premixed protein dough model systems, and to illustrate the effects of temperature, cations, and pH on the progress of protein aggregation. Our results suggested that storage temperature was a critical factor for protein aggregation in model systems, and the formation of protein aggregates became faster with increases in storage temperature. Calcium significantly improved the thermal stability of alpha‐lactalbumin and slowed down the formation of protein aggregates. The increases in pH accelerated the aggregation of alpha‐lactalbumin. Our results also suggested that the formation of intermolecular disulfide bonds together with noncovalent interactions are the main mechanisms resulting in the moisture‐induced aggregation of alpha‐lactalbumin in model systems. Practical Application: Alpha‐lactalbumin is an important dairy protein ingredient, and has been widely used in high‐protein foods such as infant formula and nutritional bars for its nutritional and functional properties. Our results suggested low storage temperature, the presence of calcium and low pH condition can make high‐protein food products containing alpha‐lactalbumin more stable.     

Casein gelation under simultaneous action of transglutaminase and glucono‐δ‐lactone

Casein solutions (5% w/v) were treated with microbial transglutaminase (MTG) and glucono‐δ‐lactone (GDL) under varying conditions in order to obtain gels. Storage modulus (G ′) and gelation time of the gels were measured by oscillation rheometry, while protein cross‐linking was determined by gel permeation chromatography. The addition of only GDL to milk resulted in very weak gels, while MTG on its own was not able to create gel networks. Simultaneous action of both ingredients led to gels, the firmness of which was linearly related to the added amount of MTG, but passed through a maximum with rising GDL concentrations. Using chromatographical analysis, increasing G ′ values were interrelated with the formation of MTG‐induced oligomers. The gelation time was directly proportional to the GDL concentration but not influenced by the addition of MTG within the studied range of concentration.     

Physicochemical Properties of Non‐thermally Cross‐linked Corn Starch with Phosphorus Oxychloride using Ultra High Pressure (UHP)

Corn starch (20%, w/w) was non‐thermally and conventionally cross‐linked with phosphorus oxychloride (POCl₃; 0.01, 0.05, or 0.1%, based on dry weight of starch) at 400 MPa for 5, 15 and 30 min and at 45°C for 2 h, respectively. Swelling power and solubility of both non‐thermally and conventionally cross‐linked corn starches were relatively lower than those of native corn starch. The pressure holding time did not affect the solubility and swelling power of non‐thermally cross‐linked corn starches. X‐ray diffraction patterns and relative crystallinity were not significantly altered by both conventional and non‐thermal cross‐linking. DSC thermal characteristics of both non‐thermally and conventionally cross‐linked corn starches were not significantly changed indicating that the double helical structure of amylopectin was not influenced by both conventional and non‐thermal cross‐linking reactions. Both non‐thermal and conventional cross‐linking greatly affected the Rapid Visco Analyser (RVA) pasting properties, such as increase in pasting temperature and decrease in peak viscosity compared to native starch. This result suggests that in case of cross‐linking using POCl₃, both non‐thermal and conventional methods result in similar physicochemical properties and non‐thermal cross‐linking with POCl₃ can reduce the reaction time from 2 h to 15 min. This work shows the potential and possibility of non‐thermal starch modification and provides the basic and scientific information on the physicochemical properties of non‐thermally cross‐linked corn starches with phosphorus oxychloride using UHP.     

Flowability and wetting behaviour of milk protein ingredients as influenced by powder composition,particle size and microstructure

This work aimed to provide a comprehensive characterisation of several physicochemical properties of different milk protein powders frequently used as ingredients in dairy‐based food formulations, in particular flowability and wettability. The flow and wetting properties were influenced mainly by the powder composition, less than by other physicochemical properties, such as particle size and microstructure. Powders with high fat content had poor flow and wetting properties. The wetting properties were also influenced by the protein content (mainly by the casein content) of the powders and were poor for powders having poor release of ionic calcium.     

Pectins in Processed Fruits and Vegetables: Part II—Structure–Function Relationships

ABSTRACT: Pectin represents a very heterogeneous biopolymer whose functionality remains largely puzzling. The link between the pectin fine structure and functional properties with relevance to plant growth and development, as well as food processing, is continually being explored. This review describes the current knowledge of pectin structure–function relationships. Key mechanisms dictating pectin structure–function relationships are discussed, including the polymer biosynthesis, cross‐linking mechanisms, enzymatic and nonenzymatic conversion reactions, solubility properties, and more. Insight into the polymer structure–function relationships is highlighted by examining traditional and advanced methodologies used in pectin research. The role of pectin in modulating the quality characteristics of plant‐based foods is underlined while pin‐pointing some of the main challenges and perspectives. An integrated approach using the pectin structure–function relationship in the precision engineering of mechanical properties of tissue‐based systems is proposed.     

TRANSGLUTAMINASE: ITS UTILIZATION IN THE FOOD INDUSTRY

Transglutaminase is an enzyme that forms crosslinks between protein molecules. This crosslinkage has unique effects on protein properties, gelation capability, thermal stability, water-holding capacity, etc. A transglutaminase has been isolated from Streptoverticillium sp., and its practical use in the food industry realized. Transglutaminase is now widely used in seafood, surimi products, meat products, noodles/pasta, dairy products, baked goods, and so on. It has great potential to improve the firmness, elasticity, viscosity, heat stability, and water-holding capacity of prepared foods through the mild enzyme reaction. The overall applications of transglutaminase in the food industry are reviewed.