Angela Sorrentino

Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase.

Whole soy flour and apple pectin were used as raw materials for producing hydrocolloid edible films. The best ratio between the two components (2:1 mg cm(-2), pectin-soy flour) was determined in order to obtain films which could be perfectly handled for their consistence. Films were also prepared in the presence of transglutaminase, an enzyme able to produce isopeptide bonds among the soy polypeptide chains. The latter films showed a smoother surface and higher homogeneity, as demonstrated by microstructural analyses, whereas studies on the mechanical properties indicated that transglutaminase increased their strength and reduced their flexibility. Our results suggest a possible use of the transglutaminase polymerized pectin-soy protein films as edible food or drug coatings.

Incorporation of whey proteins into cheese curd by using transglutaminase

A Ca2+‐independent microbial TGase (transglutaminase) isolated from Streptoverticillium mobaraense was used to obtain whey protein containing novel dairy products. We evaluated the difference both in the curd formation time as well as in the hardness and deformability of the cheese obtained from cows milk in the presence or absence of the enzyme. The results of our experiments showed that the milk coagulation time was dependent on the step in cheese manufacture at which TGase was added. We analysed the deformability and the hardness of the dairy products obtained either by adding both TGase and the milk‐clotting enzyme to the milk sample at the same time or by adding TGase after treating the milk sample for 30 min with the clotting enzyme and cutting the obtained coagulum. TGase treatment conferred a strongly decreased protein content to derived whey. Moreover, when further amounts of whey were added to the milk during the manufacturing process in the presence of TGase, whey‐protein‐enriched dairy products could also be obtained. Our findings may lead to new biotechnologies for the re‐utilization of by‐products from dairy plants and contribute to reduction of environmental pollution from whey‐protein disposal.

Transglutaminase Crosslinked Pectin- and Chitosan-based Edible Films: A Review

The production of biodegradable and edible films with desired mechanical characteristics and gas barrier properties represents one of the most advanced challenges in the field of food wrapping and coating. New edible films can serve not only to provide food with physical protection but also to reduce loss of their moisture, to restrict absorption of oxygen, to lessen migration of lipids, to improve their mechanical handling features, and as materials, to apply in direct contact with internal food to realize a multilayer food packaging. Polymers derived from natural products, like carbohydrates and proteins, offer the greatest opportunities as component of edible films since their biodegradability and environmental compatibility are assured and they can also supplement the nutritional value of specific foods. However, excessive water solubility and poor water vapor barrier properties, and often poor mechanical resistance, have their application limited until the present time. Numerous studies have been carried out to improve their properties by preparing composite and multi-component films or by physically and chemically crosslinking their natural components. In the present review we summarize the main results obtained by crosslinking with the enzyme transglutaminase different proteins contained in multi-component pectin- and chitosan-based edible films, having the aim to create environmentally-friendly “bioplastics” with mechanical and permeability properties similar to the ones exhibited by plastics of petrochemical origin.

Identification of Prunus armeniaca cultivars by RAPD and SCAR markers

Nineteen cultivars of apricot (Prunus armeniaca) were distinguished using random amplified polymorphic DNA (RAPD) markers. One decamer out of 44 used was useful to differentiate cultivars of the Campania Region from those of Northern Italy, North America and Greece. A sequence characterized amplified region (SCAR) marker was obtained. The results provide a protocol to fingerprint DNA of apricots as an efficient way to quality control and fraud prevention.

Transglutaminase-induced chemical and rheological properties of cheese.

Ca2+-independent transglutaminase isolated from Streptoverticillium mobaraense was used to obtain a novel cheese we called “crosslinked cheese.” We characterized both the cheese obtained by adding transglutaminase and milk clotting enzyme at the same time, and the one produced by adding transglutaminase, following cutting of the coagulum. In both cases, water content and cheese yields were higher, whereas protein content significantly increased from 33.8% to 39.5% only in the second type of crosslinked cheese. Isoelectric focusing of the protein extracted from the prepared cheese samples on a thin layer of polyacrylamide gel indicated that mainly β-caseins were involved in the transglutaminase-catalyzed crosslinks occurring in the curd. Few αs1-casein isoforms were entrapped in the curd matrix only when transglutaminase was added at the same time as the coagulant enzyme. Soluble nitrogen determination and HPLC peptide analysis indicated a markedly reduced proteolysis of crosslinked cheese during ripening, probably responsible for the observed enhancement of their hardness in comparison with the control cheese, obtained in the absence of transglutaminase.

Effect of transglutaminase on the mechanical and barrier properties of whey protein/pectin films prepared at complexation pH.

The behavior of pectin and thermally denatured whey proteins at both different protein/polysaccharide ratios and different pH values was investigated. Our findings suggest the formation at pH 5.1 (complexation pH) of transglutaminase-catalyzed cross-links among soluble ionic whey protein/pectin complexes, which could be responsible for the observed increase of both tensile strength (2-fold) and elongation to break (10-fold) of films obtained in the presence of enzyme. Conversely, a significant reduction of elasticity, probably due to the formation of covalent bonds among single whey protein molecules, was observed when the films were prepared in the presence of the enzyme at pH 6.0. In addition, the presence of the enzyme at complexation pH significantly reduced film permeability. Atomic force and scanning electron microscopy revealed significant changes in the microstructure of the films prepared in the presence of TGase as well as in the morphology of their surface.

Transglutaminase-mediated modification of ovomucoid: effects on its trypsin inhibitory activity and antigenic properties

Hen egg can cause food hypersensitivity in infants and young children, and ovomucoid is the most allergenic factor among proteins contained in egg white. Since proteinase treatment, a well-recognized strategy in reducing food allergenicity, is ineffective when applied to ovomucoid because of its ability to act as trypsin inhibitor, we investigated the possibility of reducing the ovomucoid antiprotease activity and antigenic properties by covalently modifying its structure. The present paper reports data showing the ability of the Gln115 residue of ovomucoid to act as an acyl donor substrate for the enzyme transglutaminase and, as a consequence, to give rise to a covalent monodansylcadaverine conjugate of the protein in the presence of both enzyme and the diamine dansylated derivative. Moreover, we demonstrated that the obtained structural modification of ovomucoid significantly reduced the capability of the protein to inhibit trypsin activity, also having impact on its anti-ovomucoid serum-binding properties.

Swelling, Mechanical, and Barrier Properties of Albedo-Based Films Prepared in the Presence of Phaseolin Cross-Linked or Not by Transglutaminase

Edible films were obtained from Citrus paradisi grapefruit albedo homogenates and bean protein phaseolin modified or not by the enzyme transglutaminase. Swelling capability, barrier performance to water vapor, oxygen and carbon dioxide, and mechanical properties of such films were investigated. The addition of the protein, mostly in the presence of transglutaminase, provide films less swellable at pH values above 5 compared to films made by albedo homogenates only, whereas the action of the enzyme clearly improves mechanical properties producing more stretchable and elastic films. Moreover, transglutaminase-mediated cross-linking of phaseolin gives rise to films less permeable to carbon dioxide and able to offer a high barrier to water vapor. These findings suggest that albedo-phaseolin film prepared in the presence of transglutaminase can be a promising candidate to be used as food edible wrap.

Higher susceptibility to amyloid fibril formation of the recombinant ovine prion protein modified by transglutaminase

Prion proteins are known as the main agents of transmissible spongiform encephalopathies affecting humans as well as animals. A recombinant ovine prion protein was found to be in vitro able to act as an effective substrate for a microbial isoform of transglutaminase, an enzyme catalyzing the formation of isopeptide bonds inside the proteins. We proved that transglutaminase modifies the structure of the prion protein by leading to the formation of three intra-molecular crosslinks and that the crosslinked protein form is more competent in amyloid formation compared to the unmodified one. In addition, the crosslinked prion protein was shown also to be more resistant to proteinase K digestion. Our findings suggest a possible use of transglutaminase in stabilizing the prion protein three-dimensional structure in order to investigate the molecular basis of the conversion of the protein into its pathological form.

Putrescine-polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films.

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.