PRODUCTION OF NATURAL AROMA COMPOUNDS BY BIOCATALYSIS

Gli esteri svolgono un ruolo rilevante nell'industria alimentare, sono tra i composti piu\u300 importanti e versatili di aromi e fragranze naturali in alimenti, bevande e cosmetici. La loro preparazione da substrati naturali e l'utilizzo di bioprocessi (eg. fermentazione o reazioni enzimatiche) e\u300 attrattivo, perche\u301 il prodotto finale puo\u300 essere etichettato e commercializzato in UE e USA come naturale. Pertanto, nuovi approcci biotecnologici per ottenere aromi sono desiderati, una volta che siano efficienti e sostenibili. Molti esteri con proprieta\u300 aromatiche possono essere ottenuti enzimaticamente usando lipasi che catalizzano reazioni di esterificazione, transesterificazione o interesterificazione. In questa tesi di dottorato abbiamo sviluppato due sistemi per la produzione di esteri con proprieta\u300 aromatiche: 1) Un metodo biocatalitico per la preparazione enzimatica di diversi esteri con proprieta\u300 aromatiche da alcoli primari (isoamilico, n-esilico, geranilico, cinnamilico, 2-feniletilico e benzilico) ed esteri etilici naturalmente disponibili (formiato, acetato, propionato e butirrato). Le biotrasformazioni sono catalizzate da un'aciltransferasi di Mycobacterium smegmatis (MsAcT) e precedute da eccellenti rese (80- 97%) e brevi tempi di reazione (30-120 minuti), anche quando sono state utilizzate concentrazioni di substrato elevate (fino a 0,5 M). Questa strategia enzimatica rappresenta un'alternativa efficace all'applicazione delle lipasi nei solventi organici e un miglioramento significativo rispetto ai metodi gia\u300 noti in termini di uso ridotto di solventi organici, aprendo la strada a una preparazione sostenibile ed efficiente degli agenti aromatizzanti naturali. 2) Un metodo biocatalitico con la lipasi legata al micelio liofilizzato di Aspergillus oryzae che catalizza l'esterificazione diretta di alcoli e acido acetico in solvente organico. Ha mostrato un'elevata stabilita\u300 verso substrati e prodotti. L'acqua prodotta durante l'esterificazione non ha influenzato in modo significativo l'equilibrio della reazione, consentendo conversioni elevate. Queste caratteristiche sono state sfruttate per preparare esteri con proprieta\u300 aromatiche dell'acetato (isoamil e cinnamil acetato) in sistemi in batch e continui. E\u300 stato sviluppato un continuous stirred tank membrane reactor (CST-MR) ovvero un reattore continuo a membrana sotto agitazione per garantire una buona produttivita\u300 e un'elevata stabilita\u300 del biocatalizzatore. Entrambi i sistemi di produzione sono promettenti, rappresentano due diverse alternative e possono essere ulteriormente ottimizzati e scalati per gli interessi del settore.Esters play a significant role in the food industry, they are among the most important and versatile components of natural flavours and fragrances in food, drinks and cosmetics Their preparation starting from natural substrates and using bioprocesses (e.g., fermentation or enzymatic reactions) is appealing, since the final product can be labelled and commercialized in EU and USA as natural. Therefore, new biotechnological approaches for obtaining flavours are highly demanded as long as they are efficient and sustainable. Many flavour/fragrance esters can be enzymatically obtained using lipases that catalyse esterification, transesterification or interesterification reactions. In this PhD thesis we studied two systems for production of flavours esters: 1) A straightforward biocatalytic method for the enzymatic preparation of different flavour esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalysed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and preceded with excellent yields (80-97%) and short reaction times (30-120 minutes), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared to already known methods in terms of reduced use of organic solvents, paving the way to a sustainable and efficient preparation of natural flavouring agents. 2) Mycelium-bound lipase of dry mycelium of Aspergillus oryzae catalysed direct esterification of alcohols and acetic acid in organic solvent, showing high stability towards substrates and products. Water produced during the esterification did not significantly affect the equilibrium of the reaction, allowing for high conversions. These features were exploited for preparing flavour-active acetate esters (e.g., isoamyl and cinnamyl acetate) in batch and continuous systems. A continuous stirred tank membrane reactor (CST-MR) was developed securing good reactor productivity and high biocatalyst stability. Both production systems are promising, represent two different alternatives and can be further optimized and scaled up for the interests of the industry

Quantitative analysis of parent-of-origin effect in reproductive and morphological selection criteria in the Pura Raza Española horse

It is generally assumed that parents make a genetically equal contribution to their offspring, but this assumption might not always hold. This is because the expression of a gene can be blocked by methylation during gametogenesis, and the degree of methylation can depend on the origin of the parental gene (imprinting) or by preferential management associated with genetic merit. The first consequences of this for quantitative genetics is that the mean phenotypes of reciprocal heterozygotes need no longer be the same, as would be expected according to Mendelian heritage. We analysed three mare reproductive traits (reproductive efficiency, age at first foaling and foaling number) and three morphological traits (height at withers, thoracic circumference, and scapula‐ischial length) in the Pura Raza Española (PRE) horse population, which possesses a deep and reliable pedigree, making it a perfect breed for analysing the quantitative effect of parent‐of‐origin. The number of animals analysed ranged from 44,038 to 144,191, all of them with both parents known. The model comparison between a model without parent‐of‐origin effects and three different models with parent‐of‐origin effects revealed that both maternal and paternal gametic effects influence all the analysed traits. The maternal gametic effect had a higher influence on most traits, accounting for between 3% and 11% of the total phenotypic variance, while the paternal gametic effect accounted for a higher proportion of variance in one trait, age at first foaling (4%). As expected, the Pearson's correlations between additive breeding values of models that consider parent‐of‐origin and that do not consider parent‐of‐origin were very high; however, the percentage of coincident animals slightly decreases when comparing animals with the highest estimated breeding values. Ultimately, this work demonstrates that parent‐of‐origin effects exist in horse gene transmission from a quantitative point of view. Additionally, including an estimate of the parent‐of‐origin effect within the PRE horse breeding program could be a great tool for a better parent's selection and that could be of interest for breeders, as this value will determine whether the animals acquire genetic categories and are much more highly valued

Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials

Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials

Genetic Structure Analysis of the Pura Raza Español Horse Population through Partial Inbreeding Coefficient Estimation

The aim of this work was to analyze genetic parameters such as the inbreeding coe cient (F), relatedness coe cient (AR) and partial inbreeding coe cient (Fij) of the whole PRE population, and the ancestors which account for 50% of the total genetic variability of the current population, from genealogical information. The average F of the whole PRE population (328,706 animals) has decreased from 8.45% to 7.51% in the least 20 years. The Fij was estimated for the whole PRE population, resulting in a database of 58,772,533 records containing one record for each Fij that each animal receives from a certain common ancestor (CA). A total of 10,244 CAs contributed to the Fij with an average of 5370 descendants, with each descendant having an average of 170 CAs. Over the generations, the number of CAs has increased, while the proportion of Fij by each one has decreased. In addition, the contributions of the more influential ancestors have changed. The increased census, the limited use of artificial insemination and our increased knowledge about inbreeding depression and the animals’ breeding values allow breeders to select horses more for their functionality and conformation than for their pedigree reputation, which is the cause of all these changes

Orthotopic kidney transplantation survival and complications: systematic review and meta-analysis

Purpose: To determine graft and patient survival and adverse events in patients who undergo orthotopic kidney transplantation. Methods: We performed a systematic review and meta-analysis. We search in Medline, Embase, and Central from inception to nowadays. We included observational studies with patients who undergo orthotopic kidney transplantation. The primary outcomes were overall patient and graft survival. We pooled the information in a frequency meta-analysis with a 95% CI. We analyzed bias with the STROBE statement. Results: Of the 106 papers initially retrieved, four met the inclusion criteria. Vascular and urinary tract complications were reported in 19% and 15%, respectively. The overall patient survival was 92% 95%CI (88% to 95%), I2 = 0%, and the overall graft survival was 88% 95 CI (83% to 91%), I2 = 0%. Conclusion: Our analysis showed a high survival rate in patients and kidney grafts after orthotopic kidney transplantation, with a similar complication rate compared to a heterotopic kidney transplant

Opacity for realistic 3D MHD simulations of cool stellar atmospheres

Context. Realistic 3D time-dependent simulations of stellar near-surface convection employ the opacity binning method for efficient and accurate computation of the radiative energy exchange. The method provides several orders of magnitude of speed-up, but its implementation includes a number of free parameters. Aims. Our aim is to evaluate the accuracy of the opacity binning method as a function of the choice of these free parameters. Methods. The monochromatic opacities computed with the SYNSPEC code are used to construct opacity distribution function (ODF) that is then verified through detailed comparison with the results of the ATLAS code. The opacity binning method is implemented with the SYNSPEC opacities for four representative cool main-sequence stellar spectral types (F3V, G2V, K0V, and M2V). Results. The ODFs from SYNSPEC and ATLAS show consistent results for the opacity and bolometric radiative energy exchange rate Q in case of the F, G, and K -- type stars. Significant differences, coming mainly from the molecular line lists, are found for the M -- type star. It is possible to optimise a small number of bins to reduce the deviation of the results coming from the opacity grouping with respect to the ODF for the F, G, and K -- type stars. In the case of the M -- type star, the inclusion of splitting in wavelength is needed in the grouping to get similar results, with a subsequent increase in computing time. In the limit of a large number of bins, the deviation for all the binning configurations tested saturates and the results do not converge to the ODF solution. Due to this saturation, the Q rate cannot be improved by increasing the number of bins to more than about 20 bins. The more effective strategy is to select the optimal location of fewer bins.Comment: 26 pages, 29 figures. Accepted for publication in section 15 (Numerical methods and codes) of Astronomy and Astrophysic

Survey of Risk Factors and Genetic Characterization of Ewe Neck in a World Population of Pura Raza Español Horses

Ewe Neck is a relatively common morphological defect in Pura Raza Español (PRE) horses and other Baroque type horse breeds, which adversely affects the breeding industry; (1) objectives: to establish the within-breed prevalence, possible associated factors, and heritability of Ewe Neck in PRE horses; (2) methods: the database included evaluations of 35,267 PRE horses. The Ewe Neck defect, 16 morphological traits, and 4 body indices were recorded. A Bayesian genetic animal model included the following systematic effects: sex, age, coat color, geographical area of the stud, and birth stud size were used; (3) results: in this PRE population, a total of 27.12% was affected. All the risk factors studied were significantly associated with the Ewe Neck score. The heritability coefficient for Ewe Neck score ranged from 0.23 to 0.34. Morphological traits (height at chest, length of back, head-neck junction, and bottom neck-body junction) and the indices (head and thoracic index) were those most closely related with the appearance of Ewe Neck; (4) conclusions: Ewe Neck is a relatively frequent defect in PRE horses, associated with risk factors and other morphological traits, with a moderate level of heritability. Breeding to select against this condition may therefore be beneficial in this breed