Loredana Mariniello

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.

Thermal stabilization of trypsin by enzymic modification with β-cyclodextrin derivatives

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several β‐cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono‐6‐ethylenediamino‐6‐deoxy‐β‐cyclodextrin, mono‐6‐propylenediamino‐6‐deoxy‐β‐cyclodextrin, mono‐6‐butylenediamino‐6‐deoxy‐β‐cyclodextrin and mono‐6‐hexylenediamino‐6‐deoxy‐β‐cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4–21% after conjugation. The Km values for cyclodextrin–trypsin complexes represented about 58–87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5–10 °C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 °C for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 °C was markedly increased for the oligosaccharide–trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.

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.

Cereal dietary proteins with sites for cross-linking by transglutaminase

Abstract The ability of several cereal proteins to act as substrates for transglutaminase purified from guinea pig liver was investigated. Among the various dietary proteins tested, wheat glutelins and gliadins, as well as purified A-gliadin, were found to be the most effective acyl donor substrates for transglutaminase. In particular, these proteins seemed to be able to produce not only γ(glutamyl) spermidine adducts but also polymeric complexes, probably through intermolecular e(γ-glutamyl)lysine crosslinks. In the case of A-gliadin, the single lysyl residue occurring in the amino acid sequence (Lys-186) is supposed to act as acyl acceptor site. The peptic-tryptic fragments of gliadins and prolamines of different origin behaved as transglutaminase substrates similarly to native gliadin, mostly in giving rise to large Mr polymers. These results are consistent with the hypothesis that animal transglutaminase may be involved in the metabolism of cereal dietary proteins, or of their peptide fragments, when present either in the intestinal lumen or in the mucosal cells.

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.

Characterization of Citrus pectin edible films containing transglutaminase-modified phaseolin

The growing social and economic consequences of pollution derived from plastics are focusing attention on the need to produce novel bioprocesses for enhancing food shelf-life. As a consequence, in recent years the use of edible films for food packaging is generating a huge scientific interest. In this work we report the production of an edible hydrocolloid film made by using Citrus pectin and the protein phaseolin crosslinked by microbial transglutaminase, an enzyme able to covalently modify proteins by formation of isopeptide bonds between glutamine and lysine residues. The films were characterized and their morphology was evaluated by both atomic force microscopy and scanning electron microscopy. Mechanical properties and barrier properties to CO2, O2 and water vapor have demonstrated that these films possess technological features comparable to those possessed by commercial plastics. It is worth noting that these characteristics are maintained even following storage of the films at 4°C or -20°C, suggesting that our bioplastics can be tailored to protect food at low temperature. Moreover, gastric and duodenal digestion studies conducted under the same conditions found in the human digestion system have demonstrated that transglutaminase-containing films are regularly digested encouraging an application of the proposed materials as food coatings.

Solubility and Permeability Properties of Edible Pectin-Soy Flour Films Obtained in the Absence or Presence of Transglutaminase

The permeability characteristics and water-solubility of edible pectin–soy flour films obtained in the absence or presence of the enzyme transglutaminase were investigated and compared with those possessed by both the commonly used high density polyethylene film and the biodegradable Mater-Bi® film. The soy protein transglutaminase-catalyzed crosslinking was found to determine a marked decrease in the solubility of the pectin–soy flour films both at different pH and in different denaturing conditions with respect to the films obtained in the absence of the enzyme, even though their solubility remains higher than that of the commercially available polyethylene and Mater-Bi® films. Transglutaminase treatment was also shown to significantly increase pectin–soy flour film barrier properties to oxygen, carbon dioxide and water vapour. In particular, the films obtained in the presence of the enzyme exhibited a permeability to oxygen and carbon dioxide even lower than that possessed by polyethylene films. Our results suggest a possible use of the transglutaminase-polymerized pectin–soy flour films as wrapping of food products requiring a packaging allowing low gas exchange with the environment. Furthermore, the application of these films as coatings to conventional oral dosage forms could provide a viable means of delivering drugs to the colon.

Transglutaminase-mediated amine incorporation into substance P protects the peptide against proteolysis in vitro☆

The in vitro metabolism of transglutaminase-synthesized substance P analogs has been characterized comparing their stability to that of the parent peptide. The major metabolites have been purified and their structures elucidated by mass spectrometry. Our results demonstrated that gln5 spermidine and spermine analogs of substance P possess an enhanced resistance to the action of proteases. Moreover spermine, a large size hydrophilic compound, specifically prevented the hydrolysis at Phe7-Phe8 bond.