Streptomycetes as Microbial Cell Factories for the Biotechnological Production of Melanin.

Melanins are complex, polymeric pigments with interesting properties like UV-light absorbance ability, metal ion chelation capacity, antimicrobial action, redox behaviors, and scavenging properties. Based on these characteristics, melanins might be applied in different industrial fields like food packaging, environmental bioremediation, and bioelectronic fields. The actual melanin manufacturing process is not environmentally friendly as it is based on extraction and purification from cuttlefish. Synthetic melanin is available on the market, but it is more expensive than animal-sourced pigment and it requires long chemical procedures. The biotechnological production of microbial melanin, instead, might be a valid alternative. Streptomycetes synthesize melanins as pigments and as extracellular products. In this review, the melanin biotechnological production processes by different Streptomyces strains have been revised according to papers in the literature. The different fermentation strategies to increase melanin production such as the optimization of growth conditions and medium composition or the use of raw sources as growth substrates are here described. Diverse downstream purification processes are also reported as well as all the different analytical methods used to characterize the melanin produced by Streptomyces strains before its application in different fields.

Mechanical, Barrier and Thermal Properties of Amylose-Argan Proteins-Based Bioplastics in the Presence of Transglutaminase.

The bioeconomy aims to discover new sources for producing energy and materials and to valorize byproducts that otherwise would get wasted. In this work, we investigate the possibility of producing novel bioplastics, made up of argan seed proteins (APs), extracted from argan oilcake, and amylose (AM), obtained from barley plants through an RNA interference technique. Argan, Argania spinosa, is a plant widespread in arid regions of Northern Africa, where it plays a fundamental socio-ecological role. Argan seeds are used to obtain a biologically active and edible oil, producing a byproduct, the oilcake, that is rich in proteins, fibers, and fats, and is generally used as animal food. Recently, argan oilcakes have been attracting attention as a waste to be recovered to obtain high-added-value products. Here, APs were chosen to test the performance of blended bioplastics with AM, because they have the potential to improve the properties of the final product. High-AM-starches present attractive features for use as bioplastics, including a higher gel-forming capacity, a higher thermal stability, and reduced swelling compared to normal starch. It has already been demonstrated that pure AM-based films provide more suitable properties than normal starch-based films. Here, we report on the performance of these novel blended bioplastics in terms of their mechanical, barrier, and thermal properties; and the effect of the enzyme microbial transglutaminase (mTGase) as a reticulating agent for AP's components was also studied. These results contribute to the development of novel sustainable bioplastics with improved properties and confirm the possibility of valorizing the byproduct, APs, using them as a new raw material.

Sustainable Exploitation of Posidonia oceanica Sea Balls (Egagropili): A Review.

Posidonia oceanica (L.) Delile is the main seagrass plant in the Mediterranean basin that forms huge underwater meadows. Its leaves, when decomposed, are transported to the coasts, where they create huge banquettes that protect the beaches from sea erosion. Its roots and rhizome fragments, instead, aggregate into fibrous sea balls, called egagropili, that are shaped and accumulated by the waves along the shoreline. Their presence on the beach is generally disliked by tourists, and, thus, local communities commonly treat them as waste to remove and discard. Posidonia oceanica egagropili might represent a vegetable lignocellulose biomass to be valorized as a renewable substrate to produce added value molecules in biotechnological processes, as bio-absorbents in environmental decontamination, to prepare new bioplastics and biocomposites, or as insulating and reinforcement materials for construction and building. In this review, the structural characteristics, and the biological role of Posidonia oceanica egagropili are described, as well as their applications in different fields as reported in scientific papers published in recent years.

Argan (Argania spinosa L.) Seed Oil Cake as a Potential Source of Protein-Based Film Matrix for Pharmaco-Cosmetic Applications.

Various different agri-food biomasses might be turned into renewable sources for producing biodegradable and edible plastics, potentially attractive for food, agricultural and cosmeceutical sectors. In this regard, different seeds utilized for edible and non-edible oil extraction give rise to high amounts of organic by-products, known as seed oil cakes (SOCs), potentially able to become protein-rich resources useful for the manufacturing of biodegradable films. This study reports the potential of SOC derived from Argania spinosa (argan), a well-known plant containing valuable non-refined oil suitable for food or cosmetic use, to be a promising valuable source for production of a protein-based matrix of biomaterials to be used in the pharmaco-cosmetic sector. Thus, glycerol-plasticized films were prepared by casting and drying using different amounts of argan seed protein concentrate, in the presence of increasing glycerol concentrations, and characterized for their morphological, mechanical, barrier, and hydrophilicity properties. In addition, their antioxidant activity and effects on cell viability and wound healing were investigated. The hydrophobic nature of the argan protein-based films, and their satisfying physicochemical and biological properties, suggest a biorefinery approach for the recycling of argan SOC as valuable raw material for manufacturing new products to be used in the cosmeceutical and food industries.

Microbial Transglutaminase as a Tool to Improve the Features of Hydrocolloid-Based Bioplastics.

Several proteins from animal and plant origin act as microbial transglutaminase substrate, a crosslinking enzyme capable of introducing isopeptide bonds into proteins between the aminoacids glutamines and lysines. This feature has been widely exploited to modify the biological properties of many proteins, such as emulsifying, gelling, viscosity, and foaming. Besides, microbial transglutaminase has been used to prepare bioplastics that, because made of renewable molecules, are able to replace the high polluting plastics of petrochemical origin. In fact, most of the time, it has been shown that the microbial enzyme strengthens the matrix of protein-based bioplastics, thus, influencing the technological characteristics of the derived materials. In this review, an overview of the ability of many proteins to behave as good substrates of the enzyme and their ability to give rise to bioplastics with improved properties is presented. Different applications of this enzyme confirm its important role as an additive to recover high value-added protein containing by-products with a double aim (i) to produce environmentally friendly materials and (ii) to find alternative uses of wastes as renewable, cheap, and non-polluting sources. Both principles are in line with the bio-economy paradigm.

Plasticizing Effects of Polyamines in Protein-Based Films.

Zeta potential and nanoparticle size were determined on film forming solutions of native and heat-denatured proteins of bitter vetch as a function of pH and of different concentrations of the polyamines spermidine and spermine, both in the absence and presence of the plasticizer glycerol. Our results showed that both polyamines decreased the negative zeta potential of all samples under pH 8.0 as a consequence of their ionic interaction with proteins. At the same time, they enhanced the dimension of nanoparticles under pH 8.0 as a result of macromolecular aggregations. By using native protein solutions, handleable films were obtained only from samples containing either a minimum of 33 mM glycerol or 4 mM spermidine, or both compounds together at lower glycerol concentrations. However, 2 mM spermidine was sufficient to obtain handleable film by using heat-treated samples without glycerol. Conversely, brittle materials were obtained by spermine alone, thus indicating that only spermidine was able to act as an ionic plasticizer. Lastly, both polyamines, mainly spermine, were found able to act as "glycerol-like" plasticizers at concentrations higher than 5 mM under experimental conditions at which their amino groups are undissociated. Our findings open new perspectives in obtaining protein-based films by using aliphatic polycations as components.

Trehalose-containing hydrocolloid edible films prepared in the presence of transglutaminase.

In this article, edible hydrocolloid films were prepared by using Citrus pectins and the protein phaseolin in the presence of microbial transglutaminase, an enzyme able to catalyze isopeptide bonds between endo-protein-reactive glutamine and lysine residues. For the first time, trehalose, a nonreducing homodisaccharide into which two glucose units are linked together by a α-1,1-glycosidic linkage, was used as a component of hydrocolloid films constituted of both proteins and carbohydrates. Our data have demonstrated that these films act as very effective barriers to gases, especially to CO2 . They also present a high antioxidant capability as measured by the 2,2-diphenyl-1-picrylhydrazyl scavenging assay. In addition, the films were characterized using Atomic Force Microscopy, a powerful tool used to evaluate film surface topography and roughness. The results of our experiments clearly indicate that the trehalose-containing films prepared both in the presence and absence of transglutaminase are composed of nanoparticles with a smooth surface, having similar roughness values (Rα). In conclusion, according to barrier and antioxidant properties and to their structure, it is possible to consider the trehalose-containing films as innovative bioplastics potentially able to protect different kinds of foods.

Biotechnological Production and Characterization of Extracellular Melanin by Streptomyces nashvillensis.

Melanins are pigments employed in food, cosmetic, and textile industries, manufactured by extraction from cuttlefishes. Their biotechnological production by Streptomycetes, instead, has been poorly investigated so far. In this paper, for the first time, the strain Streptomyces nashvillensis DSM 40314 was tested as an extracellular melanin producer by investigating the influence of diverse temperatures (26, 28, and 30 °C) and pH values (6.0 and 7.0) on bacterial growth, melanin production, and on the activity of the secreted tyrosinase, the first enzyme of the pigment biosynthetic pathway. In physiological 96-h shake flask experiments, the optimal growth parameters resulted to be 28 °C and pH 7.0, at which a maximum biomass of 8.4 ± 0.5 gcdw/L, a melanin concentration of 0.74 ± 0.01 g/L (yield on biomass of 0.09 ± 0.01 g/gcdw and productivity of 0.008 ± 0.001 g/L/h), and a final tyrosinase activity of 10.1 ± 0.1 U/mL were reached. The produced pigment was purified from the broth supernatant with a two-step purification process (75.0 ± 2.0% of purity with 65.0 ± 5.0% of recovery) and tested for its chemical, antioxidant, and photoprotective properties. Finally, characterization by UV-visible and FT-IR spectroscopy, elemental analyses, and mono- and bi-dimensional NMR suggested the eumelanin-like nature of the pigment.

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.

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.

Edible Films Made of Dried Olive Leaf Extract and Chitosan: Characterization and Applications.

Nowadays a possible strategy in food preservation consists of the use of active and functional packaging to improve safety and ensure a longer shelf life of food products. Many studies refer to chitosan-based films because of the already-known chitosan (CH) antibacterial and antifungal activity. In this work, we developed CH-based films containing Dried Olive Leaf Extract (DOLE) obtained by Naviglio extractor, with the aim to investigate the polyphenols yield and the antioxidant activity of this extract entrapped in CH-based-edible films. Olive tree cultivation produces a huge amount of byproducts that are usually simply burned. Phenolic compounds are already studied for their beneficial effects on human health. Some studies reported that phenols isolated from olive leaves have been shown to inhibit the growth of different strains of microorganisms. Thus, the antimicrobial effect of DOLE-containing films against bacterial strains (Salmonella enterica subsp. enterica serovar Typhimurium ATCC® 14028, Salmonella enteritidis RIVM 706, and Enterococcus faecalis ATCC® 29212) was tested in vitro. The DOLE component of the films is effective in inhibiting all the bacteria tested in a dose-dependent manner. Thus, it was demonstrated that these edible films can act as active bioplastics when used to wrap hamburgers in substitution for baking paper, which is normally used.

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.

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.

Hydrocolloid-Based Coatings with Nanoparticles and Transglutaminase Crosslinker as Innovative Strategy to Produce Healthier Fried Kobbah.

This study addresses the effect of coating solutions on fried kobbah. Coating solutions were made of pectin (PEC) and grass pea flour (GPF), treated or not with transglutaminase (TGase) and nanoparticles (NPs)-namely mesoporous silica NPs (MSN) or chitosan NPs (CH-NPs). Acrylamide content (ACR), water, oil content and color of uncoated (control) and coated kobbah were investigated. Zeta potential, Z-average and in vitro digestion experiments were carried out. Zeta potential of CH-NPs was stable from pH 2.0 to pH 6.0 around + 35 mV but decreasing at pH > 6.0. However, the Z-average of CH-NPs increased by increasing the pH. All coating solutions were prepared at pH 6.0. ACR of the coated kobbah with TGase-treated GPF in the presence nanoparticles (MSN or CH-NPs) was reduced by 41.0% and 47.5%, respectively. However, the PEC containing CH-NPs showed the higher reduction of the ACR by 78.0%. Water content was higher in kobbah coated by PEC + CH-NPs solutions, while the oil content was lower. The color analysis indicated that kobbah with lower browning index containing lower ACR. Finally, in vitro digestion studies of both coating solutions and coated kobbah, demonstrated that the coating solutions and kobbah made by means of TGase or nanoparticles were efficiently digested.

Gelling behavior of bio-tofu coagulated by microbial transglutaminase combined with lactic acid bacteria.

The aim of this study was to investigate the gelling behavior of proteins in bio-tofu (soymilk-cow milk mixture gel) coagulated by microbial transglutaminase (MTGase) combined with lactic acid bacteria (LAB). It was shown that MTGase (3.0 U/g protein) treatment of soymilk-cow milk mixture (SCMM) could not induce gelation at 43℃ even if the incubation was lasting 4 h. However, the concomitant use of LAB (0.025 UC/L) along with MTGase could induce the formation of denser and finer gel network with smaller pores and higher storage modulus (G') compared to SCMM treated with only LAB. Electrophoresis and mass spectrometry results indicated that LAB improve MTGase-dependent polymerization of proteins. In addition, this study investigates the effect of LAB and MTGase treatment on the rheology behavior of the derived gel products. In general, the use of both bio-coagulants for the manufacture of a mixed protein gel, might open new horizons in the field of novel nutrional and functional foods.

Molecular farming of human tissue transglutaminase in tobacco plants.

In this study we have utilized Nicotiana tabacum with a molecular farming purpose in attempt of producing transgenic plants expressing the human tissue transglutaminase (htTG). Three plant expression constructs were used enabling targeting and accumulation of the recombinant protein into the plant cell cytosol (cyto), the chloroplasts (chl) and the apoplastic space (apo). Analysis of transgenic T(0) plants revealed that recombinant htTG was detectable in all three transgenic lines and the accumulation levels were in a range of 18-75 microg/g of leaf material. In the T(1) generation, the recombinant htTG was still expressed at high level and a significant catalytic activity was detected into the leaf protein extracts. Southern blot analyses revealed that apo and chl plants of T(1) generation possess a high copy number of the recombinant htTG in their genome, while the cyto plants carry a single copy.

Tobacco BY-2 cells as effective bioreactor for the production of puroindolines.

Puroindolines are two small proteins so called for the presence of an hydrophobic tryptophan-rich domain. Associated to wheat starch granules in Triticum aestivum, puroindolines have been shown to be responsible for the softness of the wheat endosperm. Moreover, have been proved to possess bactericide and anti-fungal properties together with the capacity of forming very stable foams. All these features make puroindolines very attractive for medical, pharmaceutical and food industrial applications. The aim of this study was to explore a plant molecular farming approach for producing a recombinant puroindolines. Three specific recombinant constructs, aimed for the expression in the apoplast and chloroplast compartments, were prepared and used for transformation of Nicotiana tabacum cv BY-2 cells. Recombinant PINB targeted to the chloroplast was obtained as 0.35% of BY-2 cell TSP. Antimicrobial activity experiments demonstrated that at MIC concentration recombinant PINB is responsible for about 91% growth inhibition of E. coli.

Effect of Mesoporous Silica Nanoparticles on The Physicochemical Properties of Pectin Packaging Material for Strawberry Wrapping.

Citrus peel pectin was used to prepare films (cast with or without glycerol) containing mesoporous silica nanoparticles. Nanoparticles reduced significantly the particle size, and had no effect on the Zeta potential of pectin solutions. Mechanical characterization demonstrates that pectin+nanoparticles containing films slightly increased tensile strength and significantly decreased the Young's modulus in comparison to films made only of pectin. However, elongation at the break increased in the pectin+nanoparticles films cast in the presence of glycerol, while both Young's modulus and tensile strength were reduced. Moreover, nanoparticles were able to reduce the barrier properties of pectin films prepared with or without glycerol, whereas positively affected the thermal stability of pectin films and the seal strength. The 0.6% pectin films reinforced or not with 3% nanoparticles in the presence of 30% glycerol were used to wrap strawberries in order to extend the fruit's shelf-life, over a period of eighty days, by improving their physicochemical properties.

Extraction and characterization of Foeniculum vulgare pectins and their use for preparing biopolymer films in the presence of phaseolin protein.

Pectins from Foeniculum vulgare were extracted under acidic conditions. The obtained pectins were mainly composed of uronic acid but also contained traces of rhamnose, galactose, and arabinose. Extracted pectins were used as a carbohydrate source to prepare biopolymer films in the absence and in the presence of phaseolin protein. The swelling characteristics of the films were examined as a function of ionic strength, pH, and the applied osmotic stress. The swelling behavior was dominated by a Donnan-type effect, which decreases with increasing ionic strength and counterion valency. In all cases the swelling of films containing phaseolin was reduced, suggesting a network formation between protein and pectins. Mechanical property studies have also estimated the validity of the obtained novel biopolymer films in terms of mechanical resistance.

Synthesis and resistance to in vitro proteolysis of transglutaminase cross-linked phaseolin, the major storage protein from Phaseolus vulgaris.

The ability of phaseolin to act as an acyl donor and acceptor substrate of transglutaminase was studied by using an enzyme isolated from Streptoverticillium mobarense. Phaseolin, a trimeric storage protein from Phaseolus vulgaris L., was shown to possess both glutamine and lysine residues reactive for the enzyme. The extent of transglutaminase-catalyzed cross-linking has been studied in function of both incubation time and enzyme concentration. Native- and SDS-PAGE demonstrated that phaseolin is intra- and intermolecularly cross-linked by transglutaminase and gives rise to different polymers as well as to modified forms of the protein having a similar molecular weight but lower Stokes radius if compared to unmodified phaseolin. Cross-linked phaseolin was found to be more resistant to proteolytic cleavage than the unmodified counterpart, as demonstrated by in vitro trypsin and pepsin digestion experiments. This behavior could suggest novel possible uses of the transglutaminase-modified phaseolin.