Producción biotecnológica de proteasas aspárticas vegetales para la elaboración de nuevos productos lácteos

Todos los cuajos y coagulantes de la leche utilizados para la producción de quesos a nivel industrial son de origen animal o microbiano y pertenecen al grupo de las proteasas aspárticas. Los únicos coagulantes de la leche de origen vegetal utilizados actualmente son los obtenidos a partir de las flores del cardo y su uso es a nivel artesanal. Los quesos producidos con estas proteasas tienen unas características organolépticas diferentes a los producidos con coagulantes de otros orígenes y son muy apreciados por los consumidores. Sin embargo, presentan una serie de problemas como son su elevado precio debido al coste de producción y dificultad en la estandarización de la calidad, que impiden que el producto se comercialice a nivel industrial. Nuestro equipo de investigación está buscando fuentes alternativas de enzimas coagulantes de leche procedentes de especies vegetales para su aplicación en la industria láctea. Antiguamente las especies vegetales objeto de esta tesis eran utilizadas para la producción de quesos; así el nombre científico de la especie Galium verum hace referencia a su capacidad coagulante (en griego galaktos significa leche) y entre los nombres vulgares por los que es conocida figura el de cuajaleches; la otra especie objeto de estudio, Ficus carica (higuera) aparece en la definición de coagulante vegetal, a pesar de lo cual no existe ninguna proteasa aspártica descrita de esta especie. Los objetivos perseguidos con el desarrollo de este trabajo fueron: i) la identificación de genes que codifican para proteasas aspárticas de las especies vegetales G. verum y F. carica y la caracterización de las enzimas que codifican. La elección de las especies vegetales objeto de estudio se realizó en base a un segundo objetivo, ii) la producción en sistemas heterólogos de estas enzimas para ser aplicadas en la elaboración de quesos. El desarrollo de esta tesis doctoral ha contribuido a incrementar el conocimiento sobre las proteasas aspárticas vegetales: hemos identificado y caracterizado in silico la secuencia codificante de dos proteasas aspárticas de G. verum y una de F. carica, así como dos secuencias parciales de esta última especie. Por otra parte, mediante técnicas de ingeniería genética hemos obtenido una levadura (Pichia pastoris) que tiene integrado en su genoma un gen que codifica para una proteasa aspártica de G. verum. La enzima recombinante se obtiene desde el medio de cultivo en su forma activa y el sobrenadante del cultivo de la cepa productora puede ser utilizado directamente como coagulante para la elaboración de quesos. Los quesos de vaca elaborados con este coagulante tienen unas características de textura y sensoriales diferentes a las que presentan los elaborados con la quimosina bovina, enzima tradicionalmente utilizada para la coagulación de la leche

Genetic stabilization of Saccharomyces cerevisiae oenological strains by using benomyl

Wild-type oenological strains of Saccharomyces cerevisiae are usually aneuploid and heterozygotes; thus, when they are used as starters in must fermentation the resulting wine characteristics may vary from year to year. Treatment of a wild-type S. cerevisiae oenological strain with benomyl (methyl-l-butylcarbamoyl-2-benzimidazole carbamate), an antifungal agent shown to cause chromosome loss in yeasts, resulted in a stable starter strain in which the parental oenological traits were unchanged. The oenological S. cerevisiae strain was treated with benomyl in two different ways (A and B), and sporulation ability and spore viability were subsequently assayed. Treatment A resulted in both the highest numbers of tetrads and a reduction in DNA cell content, while treatment B increased spore viability. Fermentation assays were carried out with spore clones obtained from treatment A, and the concentrations of glycerol, lactic acid, acetic acid, and ethanol resulting from the treated strains were found to be similar to those of the parental strain. Benomyl treatment thus achieved stable, highly sporulating oenological S. cerevisiae strains of low ploidy, but preserved the desirable oenological properties of the parental strain

Characterisation of general proteolytic, milk clotting and antifungal activity of Ficus carica latex during fruit ripening

The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening. RESULTSComparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg(-1)). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring. CONCLUSIONFicin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time. (c) 2015 Society of Chemical IndustryThis is peer-reviewed version of the following article: Raskovic, B.; Lazic, J.; Polovic, N. Characterisation of General Proteolytic, Milk Clotting and Antifungal Activity of Ficus Carica Latex during Fruit Ripening. Journal of the Science of Food and Agriculture 2016, 96 (2), 576–582. [https://doi.org/10.1002/jsfa.7126]Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3398

Microencapsulated Chitosan-Based Nanocapsules: A New Platform for Pulmonary Gene Delivery

In this work, we propose chitosan (CS)-based nanocapsules (NCs) for pulmonary gene delivery. Hyaluronic acid (HA) was incorporated in the NCs composition (HA/CS NCs) aiming to promote gene transfection in the lung epithelium. NCs were loaded with a model plasmid (pCMV-βGal) to easily evaluate their transfection capacity. The plasmid encapsulation efficiencies were of approx. 90%. To facilitate their administration to the lungs, the plasmid-loaded NCs were microencapsulated in mannitol (Ma) microspheres (MS) using a simple spray-drying technique, obtaining dry powders of adequate properties. In vivo, the MS reached the deep lung, where the plasmid-loaded CS-based NCs were released and transfected the alveolar cells more homogeneously than the control formulation of plasmid directly microencapsulated in Ma MS. The HA-containing formulation achieved the highest transfection efficiency, in a more extended area and more homogeneously distributed than the rest of tested formulations. The new micro-nanostructured platform proposed in this work represents an efficient strategy for the delivery of genetic material to the lung, with great potential for the treatment of genetic lung diseases

Cloning and functional expression of zeta-carotene desaturase, a novel carotenoid biosynthesis gene from Ficus carica

Carotene desaturation, an essential step in the carotenoid biosynthesis pathway, is catalyzed by two enzymes, phytoene desaturase (PDS) and ζ-carotene desaturase (zeta carotene desaturase, ZDS). Here we describe cloning and E. Coli expression of zds-Fc, a novel Ficus carica ζ-carotene desaturase catalyzing dehydrogenation of ζ-carotene into neurosporene and finally lycopene. The ζ-carotene desaturase (ZDS) gene was amplified from the fig tree by rapid amplification of cDNA ends (RACE) and spanned a 1746 bp open reading frame (ORF), encoding a protein of 582 amino acid residues with a predicted molecular weight of 64kD. The N-terminal region of this polypeptide contained a putative transit sequence for plastid targeting. By phylogenetic and sequence analyses, zds-Fc showed high homology with previously described ζ-carotene desaturases from higher plant species (Al-Babili et al. 1998; Cong et al. 2009; Matthews et al. 2003; Yan et al. 2011). Additionally, sequence analysis revealed a high degree of conservation among plant ZDSs. The deduced ZDS protein, designated FcZDS, also contains an N-terminus dinucleotide-binding, followed by a conserved region identified in other carotene desaturase sequences. These data, taken together, confirm our cloned zds-Fc as an integral part of the ZDS family of protei

Cloning and functional expression of zeta-carotene desaturase, a novel carotenoid biosynthesis gene from Ficus carica

Carotene desaturation, an essential step in the carotenoid biosynthesis pathway, is catalyzed by two enzymes, phytoene desaturase (PDS) and ζ-carotene desaturase (zeta carotene desaturase, ZDS). Here we describe cloning and E. Coli expression of zds-Fc, a novel Ficus carica ζ-carotene desaturase catalyzing dehydrogenation of ζ-carotene into neurosporene and finally lycopene. The ζ-carotene desaturase (ZDS) gene was amplified from the fig tree by rapid amplification of cDNA ends (RACE) and spanned a 1746 bp open reading frame (ORF), encoding a protein of 582 amino acid residues with a predicted molecular weight of 64kD. The N-terminal region of this polypeptide contained a putative transit sequence for plastid targeting. By phylogenetic and sequence analyses, zds-Fc showed high homology with previously described ζ-carotene desaturases from higher plant species (Al-Babili et al. 1998; Cong et al. 2009; Matthews et al. 2003; Yan et al. 2011). Additionally, sequence analysis revealed a high degree of conservation among plant ZDSs. The deduced ZDS protein, designated FcZDS, also contains an N-terminus dinucleotide-binding, followed by a conserved region identified in other carotene desaturase sequences. These data, taken together, confirm our cloned zds-Fc as an integral part of the ZDS family of proteins

Bacillus safensis subsp. osmophilus subsp. nov., isolated from condensed milk, and description of Bacillus safensis subsp. safensis subsp. nov.

7 páginas, 3 figuras, 3 tablasA bacterial strain, designated BC09T, was isolated from a contaminated sample of condensed milk. Phylogenetic analyses based on 16S rRNA gene sequences placed strain BC09T into the genus Bacillus with its closest relatives being Bacillus safensis and Bacillus australimaris with 100 and 99.9 % similarity, respectively. Analysis of the gyrB gene confirmed the closeness of strain BC09T with respect to the species B. safensis since it presented 97.8 and 95.2 % similarity values, respectively, to the type strains of B. safensis and B. australimaris. DNA–DNA hybridization confirmed these results showing averages of 67 and 56 %, respectively, between strain BC09T and the type strains of B. safensis and B. australimaris. Average nucleotide identity blast values obtained for BC09T compared to the closest relative type strains were 95.7 and 67.6 %, respectively, and predicted DNA–DNA hybridization values were 93.1 and 51.9 %, respectively. However, strain BC09T differs from the type strains of its closest relatives in several phenotypic characteristics. MK-7 was the only menaquinone detected and iso-C15:0 and anteiso-C15:0 were the major fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids, two unidentifed glycolipids, three unidentified lipids and one unidentifed phosphoglycolipid. Meso-diaminopimelic acid was detected in the peptidoglycan. The G+C content was 40.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain BC09T represents a new subspecies of B. safensis, for which the name Bacillus safensis subsp. osmophilus subsp. nov. is proposed. The type strain is BC09T (=LMG 30124T, =CECT 9344T).This work has received financial support from the Xunta de Galicia and the European Union (European Social Fund – ESF), by the Spanish Ministry of Economy and Competitivity Project AGL 2.013–48.244 R and by the European Regional Development Fund (ERDF) (2007–2013).Peer reviewe