Poscosecha de ciruela variedad angeleno : conservación frigorífica tradicional y en atmósfera modificada

Objetivos: reducir pérdidas durante la conservación frigorífica, emplear atmósfera modificada como método suplementario a la refrigeración, alargar el período de aptitud comercial. Metodología: se trabajó con fruta acondicionada a 0±1 °C y 90±5 % HR, según las siguientes variantes: 1. testigo: 20 kg fruta a granel sin seleccionar en caja plástica; 2. granel + film PVC: 10 kg de fruta a granel en bandejas de madera más cartón corrugado recubierta con film de PVC; 3. celpack: bandejas de madera recubiertas de cartón corrugado con dos celpack de 23 frutos cada uno; 4. celpack + atmósfera modificada: ídem anterior pero cada celpack en bolsa de polietileno de baja densidad de 20 μ. A partir de los 30 días de conservación se extrajo semanalmente, durante 9 semanas, una muestra de 46 frutos, de los cuales 23 fueron analizados al momento de ser extraídos y los 23 restantes luego de 48 horas de comercialización simulada (sc). Para la evaluación estadística se aplicó análisis de la varianza con el programa SAS (Statistical Analysis System) y se determinaron las diferencias entre tratamientos con el test de Duncan. Para sabor, en cambio, se aplicó una prueba de homogeneidad de P2. La evaluación de sabor se realizó mediante degustación con panel de 5 catadores entrenados. Resultados: Los frutos tenían las siguientes características al inicio de conservación: calibre 61.4 mm, peso 117.8 g, firmeza de pulpa 3.1 kgf, sabor agridulce, contenido de sólidos solubles 17.5 °Bx, acidez 0.78 g ác. málico%g, % cubrimiento 83.69 %. Luego de la conservación frigorífica (97días): % de color de cobertura 95 %. La firmeza de la pulpa en el tratamiento celpack + bolsa se diferencia con valores más altos, media de 2.8 kgf , el resto con media 2.6 kgf. En sc la firmeza es inferior y esta disminución es menor en celpack + bolsa. Sólidos solubles, media 17.21 °Bx, en sc valores con media de un 0.3 % más. Acidez titulable: disminución progresiva, de 0.68 a 0.47 g%g al fin de conservación. Sabor: a partir de los 59 días aumentan los frutos insípidos y desagradables excepto en celpack + bolsa. Síntomas de deshidratación: a partir de los 79 días la única variante que no presenta síntomas es celpack + bolsa. Conclusiones: El acondicionamiento en celpack redujo la incidencia de ataque por mohos (fue el único tratamiento sin ataque durante 94 días); tampoco presentó sabores desagradables y su limitación en conservación se debió a la deshidratación evidente a partir de 74 días. La fruta embalada en celpack + bolsa tuvo mayores valores de resistencia a la presión y 100 % de frutos sin deshidratación a los 94 días de conservación; a partir de 80 días es evidente el ataque de mohos y frutos con sabores desagradables. Las variantes granel y granel + film presentan deterioro por deshidratación a partir de 74 días. La conservación no debería superar 80 días. Celpack + bolsa muestra mejores resultados, con mayores valores de resistencia a la presión que los otros tratamientos; con respecto al sabor, mantiene una mayor proporción de sabor dulce.

Physiological features of Schizosaccharomyces pombe of interest in the making of white wines

This work studies the physiology of Schizosaccharomyces pombe strain 938 in the production of white wine with high malic acid levels as the sole fermentative yeast, as well as in mixed and sequential fermentations with Saccharomyces cerevisiae Cru Blanc. The induction of controlled maloalcoholic fermentation through the use of Schizosaccharomyces spp. is now being viewed with much interest. The acetic, malic and pyruvic acid concentrations, relative density and pH of the musts were measured over the entire fermentation period. In all fermentations in which Schizo. pombe 938 was involved, nearly all the malic acid was consumed and moderate acetic concentrations produced. The urea content and alcohol level of these wines were notably lower than in those made with Sacch. cerevisiae Cru Blanc alone. The pyruvic acid concentration was significantly higher in Schizo. pombe fermentations. The sensorial properties of the different final wines varied widely.

Schizosaccharomyces selective differential media

This study discusses the optimisation of a selectiv e and differential medium which would facilitate the isolation of Schizosaccharomyces (a genus with a low incidence compared to other microorganisms) to select individuals from this genus for industrial purposes, especially in light of the recent approval of the use of yeasts from this genus in the wine industry by the International Organisation of Vine and Wine, or to detect the presence of such yeasts, for those many authors who consider them food spoilers. To this end, we studied various selective differential agents based on the main thephysiological characteristics of this species, such as its high resistance to high concentrations of sugar, sulfur dioxide, sorbic acid, benzoic acid, acetic acid or malo ethanolic fermentation. This selective medium is based on the resistance of the genus to the antibiotic actidione and its high resistance to inhibitory agents such as benzoic acid compared to possible microorganisms which can give rise to false positive results. Malic acid was used as a differential fact or due to the ability of this genus to metabolise it to ethanol, which allows detecting of the degradation of this compound. Lastly, the medium was successfully used to isolate strains of Schizosaccharomyces pombe from honey.

Análisis de las diferencias en el crecimiento vegetativo, en los componentes del rendimiento y en la composición de las uvas y vinos del cv. cabernet sauvignon (vitis vinifera L.), producidos por el déficit hídrico durante pre-envero y post-envero

La presente Tesis Doctoral se realizó con el fin de estimar conjuntamente la respuesta agronómica y fisiológica de la vid (Vitis vinifera L.), así como los efectos sobre la evolución de la maduración, composición y la calidad de la uva y del vino, bajo la aplicación de diferentes déficit hídricos en pre-envero y post-envero, dentro de un marco de referencia de cambio climático. La variación climática que prevén los estudios sobre el cambio climático, resulta un factor decisivo en la eficiencia del uso del agua en la vid. En zonas cálidas, las estrategias de cultivo del viñedo frente al cambio climático deben de ir dirigidas a atenuar sus efectos sobre el crecimiento y el desarrollo de la vid, haciéndose imprescindible el estudio pormenorizado del déficit hídrico como factor decisivo en la obtención de las uvas adecuadas, ya que son la clave indispensable para el éxito en la elaboración del vino, y de forma muy especial en los vinos enfocados a un sector de alta calidad. El ensayo se llevó a cabo en un viñedo comercial de Bodegas Licinia, en la Comunidad de Madrid, durante los años 2010 y 2011. La variedad estudiada fue Cabernet sauvignon / 41 B, plantada a un marco de plantación de 3 m x 1 m, con un guiado vertical de la vegetación. El dispositivo experimental fue totalmente al azar, y se establecieron 4 tratamientos experimentales con 4 grados de disponibilidad hídrica, déficit moderado continuo (T0,45-0,6), déficit severo continuo (T0-0,3), déficit severo después de envero (T0,45-0,3) y déficit severo antes de envero (T0-0,6). En cada tratamiento se distribuyeron 3 repeticiones. El año 2010 fue el más lluvioso de los años de ensayo, con 478 mm de precipitaciones anuales, lo que supuso 146 mm más que en el año 2011. Su distribución a lo largo del ciclo fue más homogénea en el año 2010, mientras que en 2011 las precipitaciones contabilizadas en el período de maduración de la uva fueron nulas. La temperatura media subió 0,9ºC en 2011, respecto a 2010 y en cuanto a la integral térmica eficaz, en 2011 se acumularon, desde el 1 de abril hasta el final de ciclo, 217 grados•día más que en 2010. El déficit hídrico en pre-envero, modificó notablemente el crecimiento vegetativo y la producción de cosecha de la parcela de ensayo, no así la fertilidad de las yemas. El tratamiento con mayor disponibilidad hídrica (T0,45-0,6) obtuvo el mayor peso de baya, y los tratamientos con menor déficit hídrico en pre-envero (T0,45-0,6 y T0,45-0,3) registraron los mayores rendimientos de cosecha, mientras que las menores tasas de cuajado correspondieron al tratamiento con un déficit severo continuo (T0-0,3). La parcela de ensayo se caracterizó por un exceso de vigor y un alto crecimiento vegetativo. El pH del mosto se vio afectado por el déficit hídrico, disminuyendo su valor en el tratamiento de déficit hídrico severo antes de envero (T0-0,6). Organolépticamente, no se percibieron diferencias significativas en los vinos elaborados en función del déficit hídrico, y respecto a su composición físico-química, solo existieron diferencias en la concentración de ácido L-Málico, con mayores concentraciones en los tratamientos sin déficit hídrico en pre-envero, T0,45-0,6 y T0,45-0,3. El déficit hídrico modificó notablemente el color del vino, aumentando los valores de las coordenadas CIELAB a* y b*, la luminosidad (L*), croma (C*) y tonalidad (H*), para los tratamientos con un déficit severo en pre-envero (T0-0,3 y T0-0,6) y disminuyendo estas en el tratamiento con mayor disponibilidad hídrica (T0,45-0,6). Del mismo modo, mediante el análisis de color por métodos tradicionales, IPT e IC de los vinos, aumentó en los tratamientos con mayor déficit hídrico en pre-envero (T0-0,3 y T0-0,6), respecto a los tratamientos de mayor disponibilidad (T0,45-0,6 y T0,45-0,3). La concentración de taninos de la baya en vendimia, no se vio afectada por el déficit hídrico, aunque sí estuvo relacionada positivamente con el tamaño de las bayas. Organolépticamente, los hollejos del año 2011 resultaron con menor frescura, acidez, afrutado, sensación herbácea e intensidad tánica, aunque con mayor astringencia respecto a 2010. Las pepitas fueron más astringentes y aromáticas pero menos crujientes, sin llegar a los niveles de madurez del año 2010. El catador relacionó los taninos con la calidad del vino, asociándolos con un mayor cuerpo, acidez, intensidad, equilibrio gustativo, amargor y menor astringencia en la fase gustativa. La concentración de taninos en los vinos se vio favorecida con el déficit hídrico en pre-envero y post-envero. Los tratamientos con mayor déficit hídrico en pre-envero, T0-0,6 y T0-0,3, obtuvieron las menores concentraciones de potasio en mostos y vinos. Las relaciones entre la concentración de potasio, ácido L-Málico y el porcentaje de color rojo puro (dA(%)) resultaron altamente significativas, de modo que las mayores tasas de potasio en el vino se asociaron a los valores más bajos de color rojo y a los mayores de ácido L-Málico. ABSTRACT The present Doctoral Thesis has been done in order to estimate the grapevine (Vitis vinifera L.) agronomic and physiologic performance or response as well as the impact in the grape and wine maturity, composition and quality evolution, with different water deficits. The variation in climate that the global warming studies for seen is a key factor for the grapevine water use efficiency. In warm areas the farming vineyards strategy to face the climatic change, should be focused on diminish the effects on the grapevine growth and development, so that the water deficit detailed analysis becomes decisive to obtain the appropriate grapes, that are the main subject for a successful wine production and especially for top quality wines. The trial was carried out in a commercial vineyard in Chinchón (Madrid), Licinia winery, during the 2010 and 2011 seasons. The grape variety studied was Cabernet Sauvignon grafted onto 41B with a vine spacing 3m x 1m trained as VSP. Experimental design consisted on 4 irrigation treatments with 3 replications totally randomized. Irrigation treatments were: moderate regulated deficit (T0,45-0,6), severe continuous deficit (T0-0,3), severe post-veraison deficit (T0,45-0,3) and severe pre veraison deficit (T0-0,6). The 2010 was rainier year than the 2011; Total annual rain in 2010 was 478 mm, which resulted in 146 mm more than in 2011. The distribution along the vine cycle was more homogeneous in the 2010, whereas precipitations in 2011 along the grape maturity period were nonexistent. The average temperature in 2011 was 0,9ºC higher than that of the 2010 and regarding to the thermal integral, in the 2011 from 1st April to the end of the growing cycle, was 217 degrees•day higher than that in 2010. Water deficit significantly modified the vegetative growth and yield but, it did not modified bud fertility. The treatment with the highest water availability (T0,45-0,6) got the highest berry size, the lowest berry set rates were found in the severe continuous deficit treatment (T0-0,3). The plot studied in this trial was characterized by both excessive vigour and vegetative growth. Water deficit modified the pH must by, reducing it in the severe water deficit during pre-veraison (T0-0,6). There were not differences in wine tasting between the water deficits treatments. Regarding to the physical-chemical composition, it only existed differences in the L-malic acid concentration, resulting higher concentrations in the water deficit pre-veraison treatments: T0,45-0,6 y T0,45-0,3. Water deficit significantly modified wine colour by, increasing the CIELAB coordinates a* and b*, the brightness (L*), croma (C*) and tonality (H*), in the lower water availability pre-veraison treatments (T0-0,3 y T0-0,6), and reducing them in the in the moderate continuous water deficit ones (T0,45-0,6). By means of traditional wine colour parameters analyses, red colour percentage, TPI, they became higher in the lower water availability pre-veraison treatments (T0-0,3 y T0-0,6), than in those with higher availability (T0,45-0,6 y T0,45-0,3). At harvest, berry tannins concentrations was not affected by the water deficit although it did in a positive way, in the berry size. Berry tasting in 2011, resulted in a lower freshness, acidity, fruity, herbaceous flavour and tannic intensity, but with higher astringency respect to the 2010 season. Seeds, in 2011, were more astringent and aromatic as in the 2010, but less crunchy, without getting to the point of maturity. The taster linked the tannins to wine quality, associating them with a higher bodiest wine, acidity, intensity, taste balance, bitterness and with a lower astringency in the tasting stage. Treatments with a higher water deficit up to veraison T0-0,6 y T0-0,3 got less musts and wines potassium concentration. The relation between L-malic acid and the full red color percentage (dA(%)), were highly related, resulting the higher potassium content the lower wine quality.

New Schizosaccharomyces applications in red wines alcoholic fermentation

The Schizosaccharomyces strains consumed less primary amino nitrogen and produced less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ?wine? colour than the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.

1H HR MAS NMR metabolomic and non-destructive 2D NMR relaxometry to assess internal quality in apples.

NMR can be considered a multi-scale multidimensional technology in the sense that it provides both spatial insight at macroscopic (MRI) or microscopic level (relaxometry), together with chemical characterization (HR-MAS). In this study 296 apples (from 4 cultivars) were MRI screened (20 slices per fruit) among which 7 fruits were used for metabolomic study by 1H HR MAS in order to assess various chemical shifts: malic acid, sucrose, glucose, fructose and ethanol. On the first season, tissue samples were taken from the sound and affected apples (near the core, centre and outer part of the mesocarp) belonging to sound and affected locations, while on the second season, tissue samples were focused on the comparison between sound and affected tissue. Beside, MRI and 2D non-destructive relaxometry (on whole fruits, and localized tissue) where performed on 72 and 12 apples respectively in order to compare features at macroscopic (tissue) and microscopic (subcellular) level. HR MAS shows higher content of ?-glucose, ?-glucose, malic acid and aromatic compounds in watercore affected tissues from both seasons, while sound tissue reflects higher sucrose. Microscopic (subcellular) degradation of tissue varies according to disorder development and is in good accordance with macroscopic characterization with MRI.

Combine use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production

Most red wines commercialized in the market use the malolactic fermentationprocess in order to ensure stability from a microbiological point of view. In this secondfermentation, malic acid is converted into L-lactic acid under controlled setups. Howeverthis process is not free from possible collateral effects that on some occasions produceoff-flavors, wine quality loss and human health problems. In warm viticulture regions suchas the south of Spain, the risk of suffering a deviation during the malolactic fermentationprocess increases due to the high must pH. This contributes to produce wines with highvolatile acidity and biogenic amine values. This manuscript develops a new red winemakingmethodology that consists of combining the use of two non-Saccharomyces yeast strains asan alternative to the traditional malolactic fermentation. In this method, malic acid is totallyconsumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilizationobjective, while Lachancea thermotolerans produces lactic acid in order not to reduce andeven increase the acidity of wines produced from low acidity musts. This technique reducesthe risks inherent to the malolactic fermentation process when performed in warm regions.The result is more fruity wines that contain less acetic acid and biogenic amines than thetraditional controls that have undergone the classical malolactic fermentation.

Potenciación de la respuesta productiva en rumiantes mediante el empleo de proteínas protegidas

El principal objetivo de esta tesis fue incrementar la eficiencia proteica en las dietas de rumiantes mediante el uso de proteínas protegidas (harina de girasol y guisante de primavera), así como mejorar la predicción de los aportes de proteína microbiana. Una partida de harinas comerciales de girasol (HG) y de guisante de primavera (GP) fueron tratadas con soluciones 4 N de ácido málico (268,2 g/L) o ácido ortofosfórico (130,6 g/L). Para cada harina, ácido y día de tratamiento, dos fracciones de 12,5 kg fueron pulverizadas sucesivamente en una hormigonera con la solución de ácido correspondiente mediante un pulverizador de campo. Las dos fracciones fueron mezcladas posteriormente y se dejaron reposar durante 1 h a temperatura ambiente. La mezcla fue luego secada en una estufa de aire forzado a 120 ºC durante 1 h. La estufa fue apagada inmediatamente después y el material tratado se mantuvo dentro de ésta hasta la mañana siguiente. El material fue removido durante el proceso de secado cada 30 min durante las primeras 2 h y cada 60 min durante las 5 h posteriores. Este proceso se repitió hasta conseguir las cantidades de harinas tratadas necesarias en los distintos ensayos. En el primer experimento (capitulo 3) se llevaron a cabo estudios de digestión ruminal e intestinal para evaluar los efectos de la aplicación de las soluciones ácidas indicadas y calor a fin de proteger las proteínas de HG y GP contra la degradación ruminal. Estos estudios se realizaron con tres corderos canulados en el rumen y en el duodeno. El estudio de digestión ruminal fue realizado en tres periodos experimentales en los que los corderos fueron alimentados sucesivamente con tres dietas isoproteicas que incluían HG y GP, sin tratar o tratadas con ácidos málico u ortofosfórico. Cada periodo experimental de 21 días incluyó sucesivamente: 10 días de adaptación a las dietas, un estudio del tránsito ruminal de las partículas de HG y GP (días 11 a 14), y la incubación de las muestras de ambos alimentos en bolsas de nailon (días 15–21). Las harinas incubadas en cada periodo experimental correspondieron a las que fueron incluidas en las dietas. Las bacterias ruminales fueron marcadas desde el día 11 hasta el día 21 del periodo experimental mediante infusión intra-ruminal continua con una fuente de 15N. Tras finalizar las incubaciones in situ el día 21 el rumen fue vaciado en cada periodo para aislar las bacterias asociadas a la fase sólida y liquida del rumen. El estudio de digestión intestinal fue realizado veinte días después del final del estudio ruminal a fin de eliminar el enriquecimiento en 15N de la digesta. En este estudio se incubaron muestras compuestas obtenidas mediante la combinación de los diferentes residuos no degradados en el rumen de forma que fuesen representativas de la composición química de la fracción no degradada en el rumen (RU). En esta fase los corderos fueron alimentados con la dieta sin tratar para determinar la digestibilidad de las harinas tanto tratadas como sin tratar mediante la técnica de las bolsas móviles. Además, las proteínas contenidas en las harinas tratadas y sin tratar, así como en las muestras correspondientes a los residuos a 0 h, las muestras compuestas anteriormente indicadas y las muestras no digeridas intestinalmente fueron extraídas y sometidas a electroforesis para determinar el sitio de digestión de las diferentes fracciones proteicas. Las estimaciones de la RU y la digestibilidad intestinal de la materia seca, la materia orgánica (solamente para RU), la proteína bruta (PB) y el almidón (solamente en GP) fueron obtenidos considerando la contaminación microbiana y las tasas de conminución y salida de partículas. Las estimaciones de RU y de la digestibilidad intestinal disminuyeron en todas las fracciones evaluadas de ambos alimentos al corregir por la contaminación microbiana acaecida en el rumen. Todas las estimaciones de RU aumentaron con los tratamientos de protección, incrementándose también la digestibilidad intestinal de la materia seca en la HG. Los bajos valores de la digestibilidad de la proteína de GP tratado y sin tratar sugieren la presencia de algún factor antitripsico no termolábil es esta harina. Los tratamientos de protección incrementaron consistentemente la fracción de materia seca y PB digerida intestinalmente en los dos alimentos, mientras que la fracción de almidón en la muestra de GP solamente aumentó numéricamente (60,5% de media). Sin embargo, los tratamientos también redujeron la fermentación de la materia orgánica, lo cual podría disminuir la síntesis de proteína microbiana. Los estudios de electroforesis muestran la práctica desaparición de la albumina por la degradación ruminal en ambos alimentos, así como que los cambios en otras proteínas de la muestra RU fueron más pronunciados en GP que en HG. La composición de las bacterias asociadas con las fases de digesta ruminal sólida (BAS) y líquida (BAL) fue estudiada para revisar la precisión de un sistema de predicción previo que determinaba la infravaloración del aporte de nutrientes correspondiente a las BAS cuando de usa 15N como marcador y las BAL como referencia microbiana (capitulo 4). Al comparar con BAS, BAL mostraron menores contenidos en materia orgánica, polisacáridos de glucosa y lípidos totales y un mayor contenido en PB, así como un mayor enriquecimiento en 15N. Los datos obtenidos en el estudio actual se ajustan bien a la ecuación previa que predice el enriquecimiento en 15N de las BAS a partir del mismo valor en BAL. Esta nueva ecuación permite establecer que se produce una infravaloración de un 22% en el aporte de PB al animal a partir de las BAS sintetizadas si las BAL son usadas como muestras de referencia. Una segunda relación calculada utilizando los valores medios por dieta expuestos en numerosos trabajos encontrados en la literatura confirma la magnitud de este error. Esta infravaloración asociada al uso de BAL como referencia fue mayor para el aporte de glucosa (43,1%) y todavía mayor para el aporte de lípidos (59,9%), como consecuencia de los menores contenidos de ambas fracciones en BAL frente a SAB. Estos errores deberían ser considerados para obtener mayor precisión en la estimación del aporte de nutrientes microbianos y mejorar la nutrición de los rumiantes. En el experimento 2 se realizó un estudio de producción (capitulo 5) para evaluar los efectos del tratamiento de las harinas HG y GP con soluciones de ácido málico o ácido ortofosfórico sobre el crecimiento, el consumo de concentrado y el rendimiento y engrasamiento de las canales de corderos de engorde. Noventa corderos machos de cruce entrefino procedentes de tres granjas comerciales (peso inicial medio = 14,6, 15,3 y 13,3 kg, respectivamente) fueron asignados aleatoriamente a cinco dietas con diferentes niveles de proteína y diferentes tratamientos con ácidos y engordados hasta un peso medio al sacrificio de 25 kg. Las fuentes de proteína en el pienso control (C; PB=18,0%) fueron harina de soja, HG y GP sin tratar. En tres de los piensos experimentales, las harinas tratadas con ácido ortofosfórico sustituyeron a las de HG y GP sin tratar (Control Ortofosfórico, PC; PB=18,0% sobre materia seca), sustituyéndose, además, la harina de soja parcialmente (Sustitución Media Ortofosfórico, MSP; PB=16,7%) o totalmente (Sustitución Total Ortofosfórico, TSP; PB=15,6%). Finalmente, en uno de los piensos el ácido ortofosfórico fue reemplazo por acido málico para proteger ambas harinas (Sustitución Media Málico, MSM; PB= 16,7%). La paja de trigo (fuente de forraje) y el concentrado fueron ofrecidos ad libitum. Dieciocho corderos fueron distribuidos en seis cubículos con tres animales para cada dieta. Los datos fueron analizados según un análisis factorial considerando el peso inicial como covariable y la granja de procedencia como bloque. Los datos de consumo de concentrado y eficiencia de conversión fueron analizados usando el cubículo como unidad experimental, mientras que los datos sobre ganancia media diaria, rendimiento a la canal, grasa dorsal y grasa pélvico renal fueron analizados usando el cordero como unidad experimental. No se encontró ningún efecto asociado con el nivel de PB sobre ninguna variable estudiada. Esto sugiere que usando proteínas protegidas es posible utilizar concentrados con 15,6% de PB (sobre materia seca) disminuyendo así la cantidad de concentrados de proteína vegetal a incluir en los piensos y la calidad de los concentrados proteicos. Los corderos alimentados con la dieta MSM tuvieron mayores ganancias medias diarias (15,2%; P= 0,042), y mejores rendimiento a la canal en caliente (1,3 unidades porcentuales; P= 0,037) que los corderos alimentados con el concentrado MSP. Esto podría ser explicado por los efectos benéficos ruminales del malato o por el mayor efecto de protección conseguido con el ácido málico. ABSTRACT The main objective of this thesis project was to increase the protein efficiency in ruminant diets by using protected protein (sunflower meal and spring pea), and improving the prediction of microbial protein supply. Commercial sunflower meal (SFM) and spring pea (SP) were treated with 4 N solutions (200 mL/kg) of malic acid (268.2 g/L) or orthophosphoric acid (130.6 g/L). Daily, two fractions of 12.5 kg of one of these meals were successively sprayed with the tested acid solution in a concrete mixer using a sprayer. Both fractions were then mixed and allowed to rest for 1 h at room temperature. The blend was then dried in a forced air oven at 120 ºC for 1 h. Then the oven was turned off and the treated material was left in the oven overnight. During the drying process, the material was stirred every 30 min during the first 2 h and then every 60 min for the subsequent 5 h. This process was repeated until the amounts of treated flour needed for the different trials performed. In the first experiment (chapter 3), ruminal and intestinal digestion trials were conducted to study the effects of the application of these acid solutions and heat to protect proteins of SFM and SP against ruminal degradation using three wethers fitted with rumen and duodenum cannulae. The ruminal digestion study was carried out in three experimental periods in which the wethers were successively fed three isoproteic diets including SFM and SP, untreated or treated with malic or orthophosphoric acids. The experimental periods of 21 days included successively: 10 days of diet adaptation, SFM and SP particle ruminal transit study (days 11–14) and ruminal nylon-bag incubations (days 15–21). The meals incubated in each experimental period were those corresponding to the associated diet. Rumen bacteria were labelled from days 11 to 21 by continuous intra-ruminal infusion of a 15N source and the rumen was emptied at the end of in situ incubations in each period to isolate solid adherent bacteria and liquid associate bacteria. The intestinal digestion trial was conducted twenty days after the end of the ruminal studies to eliminate the 15N enrichment in the digesta. The tested samples were composite samples obtained pooling the different ruminally undegraded residues to be representative of the chemical composition of the ruminally undegraded fraction (RU). Wethers were fed the untreated diet to determine the intestinal digestibility of untreated and treated meals using the mobile nylon bag technique. In addition, protein in untreated and treated meals and their 0 h, composite and intestinally undigested samples were extracted and subjected to electrophoresis to determine the digestion site of the different protein fractions. Estimates of the RU and its intestinal digestibility of dry matter, organic matter (only for RU), crude protein (CP) and starch (only in SP) were obtained considering ruminal microbial contamination and particle comminution and outflow rates. When corrected for the microbial contamination taking place in the rumen, estimates of RU and intestinal digestibility decreased in all tested fractions for both feeds. All RU estimates increased with the protective treatments, whereas intestinal digestibility-dry matter also increased in SFM. Low intestinal digestibility-CP values in untreated and treated samples suggested the presence of non-heat labile antitrypsin factors in SP. Protective treatments of both feeds led to consistent increases in the intestinal digested fraction of dry matter and CP, being only numerically different for SP-starch (60.5% as average). However, treatments also reduced the organic matter fermentation, which may decrease ruminal microbial protein synthesis. Electrophoretic studies showed albumin disappearance in both SFM and SP, whereas changes in other RU proteins were more pronounced in SP than SFM. The chemical composition of bacteria associated with solid (SAB) and liquid (LAB) rumen-digesta phases was studied to examine the accuracy of a previous regression system determining the underevaluation of SAB-nutrient supply using 15N as marker and LAB as microbial reference (chapter 4). Compared with SAB, LAB showed lower contents of organic matter, polysaccharide-glucose and total lipids and the opposite for the CP content and the 15N enrichment. Present data fitted well to the previous relationship predicting the 15N enrichment of SAB from the same value in LAB. This new equation allows establishing an underevaluation in the supply of CP from the synthesized SAB in 22.0% if LAB is used as reference. Another relationship calculated using mean diet values from the literature confirmed the magnitude of this error. This underevaluation was higher for the supply of glucose (43.1%) and still higher for the lipid supply (59.9%) as a consequence of the lower contents of these both fractions in LAB than in SAB. These errors should be considered to obtain more accurate estimates of the microbial nutrient supply and to improve ruminant nutrition. A production study was performed in experiment 2 (chapter 5) to examine the effects of treating SFM and SP meals with orthophosphoric or malic acid solutions on growth performance, concentrate intake, and carcass yield and fatness of growing-fattening lambs. Ninety "Entrefino" cross male lambs from three commercial farms (average initial body weights (BW) = 14.6, 15.3 and 13.3 kg) were randomly assigned to five diets with different acid treatment and protein levels, and fattened to an average slaughter weight of 25 kg. Protein sources in the control concentrate (C; CP=18%) were soybean meal and untreated SFM and SP. In three of the experimental concentrates, orthophosphoric acid-treated meals substituted untreated SFM and SP (Orthophosphoric Control, PC; CP=18% dry matter basis), and soybean meal was partially (Medium Substitution Orthophosphoric, MSP; CP=16.7%) or totally removed (Total Substitution Orthophosphoric, TSP; CP=15.6%). In addition, in one concentrate orthophosphoric acid was replaced by malic acid to protect these meals (Medium Substitution Malic, MSM; CP= 16.7%). Wheat straw (roughage source) and concentrate were offered ad libitum. Eighteen lambs were allocated to six pens of three animals on each diet. Data were analyzed using a factorial analysis with initial body weight BW as covariate and farm of origin as block. Data on concentrate intake and feed conversion efficiency were analyzed using pen as experimental unit, while data on average daily gain, carcass yield, dorsal fat, and kidney-pelvic-fat were analyzed with lamb as experimental unit. No effect associated with the CP level was observed on any parameter. This suggests that with protected proteins it is possible to feed concentrates with 15.6% CP (dry matter basis) reducing the quantity of vegetable protein meals to include in the concentrate as well as the quality of the protein concentrates. Lambs feed MSM had higher average daily gains (15.2%; P= 0.042), and better hot carcass yields (1.3 percentage points; P= 0.037) than lambs feed MSP. This probably can be explained by ruminal malate actions and by greater protection effects obtained with malic acid.

Dynamic analysis of physiological properties of Torulaspora delbrueckii in wine fermentations and its incidence on wine quality

This work examines the physiology of a new commercial strain of Torulaspora delbrueckii in the production of red wine following different combined fermentation strategies. For a detailed comparison, several yeast metabolites and the strains implantation were measured over the entire fermentation period. In all fermentations in which T. delbrueckii was involved, the ethanol concentration was reduced; some malic acid was consumed; more pyruvic acid was released, and fewer amounts of higher alcohols were produced. The sensorial properties of final wines varied widely, emphasising the structure of wine in sequential fermentations with T. delbrueckii. These wines presented the maximum overall impression and were preferred by tasters. Semi-industrial assays were carried out confirming these differences at a higher scale. No important differences were observed in volatile aroma composition between fermentations. However, differences in mouthfeel properties were observed in semi-industrial fermentations, which were correlated with an increase in the mannoprotein content of red wines fermented sequentially with T. delbrueckii.

Efecto del tratamiento de harina de girasol con ácido mólico y calor sobre la producción de metano y fermetnación in vitro

Incubations were carried out with batch cultures of ruminal micro-organisms to study the effects of the treatment of sunflower meal (SFM) with malic acid at 150 ºC for 1 (SFM1) or 3 (SFM3) hours on in vitro fermentation. There were no differences (P>0.05) between SFM and SFM1 in the amount of gas and volatile fatty acids (VFA) produced and the disappearance of organic matter (OMD), but CH4 and NH3-N concentrations were reduced (P<0.05) by 11.3 and 14.5% with the malic treatment at 150 ºC for 1 hour, respectively. In contrast, SFM3 treatment reduced when compared to SFM gas and VFA production and OMD by 27.4, 32.5 and 49.6 (P<0.05), respectively, indicating decreased fermentability of SFM. The results indicate that combining malic acid and heat treatment (150ºC) for 1 h could be an effective means to reduce both protein degradability and CH4 production, but increasing the length of the treatment to 3 h resulted in reductions of SFM degradability and VFA production.

Bio-renewable enantioselective aldol reaction in natural deep eutectic solvents

Among the deep eutectic solvents (DES), natural deep eutectic solvents (NADES) formed by D-glucose and racemic malic acid are suitable media to perform the enantioselective L-proline catalyzed intermolecular aldol reaction, creating simultaneously and selectively a C–C bond and a new stereocenter. The scope of the reaction was found to be broad, with products being obtained with good levels of diastereo- and enantioselectivities. Furthermore, when the reaction was performed at a large scale, the catalyst together with the reaction media can be recovered by simple water extraction and reused at least three times affording similar results. Therefore, the use of NADES as reaction media to carry out a VOC-free selective process has been demonstrated for the first time. The process is clean, cheap, simple and scalable and meets most of the criteria to be considered as a sustainable and bio-renewable process, with the reaction media and catalyst arising directly from Nature.

Desenvolvimento e aplicação de modelo de calibração multivariada para determinação de açúcares e ácidos orgânicos em bebidas comerciais utilizando espectroscopia no infravermelho

The routine analysis for quantization of organic acids and sugars are generally slow methods that involve the use and preparation of several reagents, require trained professional, the availability of special equipment and is expensive. In this context, it has been increasing investment in research whose purpose is the development of substitutive methods to reference, which are faster, cheap and simple, and infrared spectroscopy have been highlighted in this regard. The present study developed multivariate calibration models for the simultaneous and quantitative determination of ascorbic acid, citric, malic and tartaric and sugars sucrose, glucose and fructose, and soluble solids in juices and fruit nectars and classification models for ACP. We used methods of spectroscopy in the near infrared (Near Infrared, NIR) in association with the method regression of partial least squares (PLS). Were used 42 samples between juices and fruit nectars commercially available in local shops. For the construction of the models were performed with reference analysis using high-performance liquid chromatography (HPLC) and refractometry for the analysis of soluble solids. Subsequently, the acquisition of the spectra was done in triplicate, in the spectral range 12500 to 4000 cm-1. The best models were applied to the quantification of analytes in study on natural juices and juice samples produced in the Paraná Southwest Region. The juices used in the application of the models also underwent physical and chemical analysis. Validation of chromatographic methodology has shown satisfactory results, since the external calibration curve obtained R-square value (R2) above 0.98 and coefficient of variation (%CV) for intermediate precision and repeatability below 8.83%. Through the Principal Component Analysis (PCA) was possible to separate samples of juices into two major groups, grape and apple and tangerine and orange, while for nectars groups separated guava and grape, and pineapple and apple. Different validation methods, and pre-processes that were used separately and in combination, were obtained with multivariate calibration models with average forecast square error (RMSEP) and cross validation (RMSECV) errors below 1.33 and 1.53 g.100 mL-1, respectively and R2 above 0.771, except for malic acid. The physicochemical analysis enabled the characterization of drinks, including the pH working range (variation of 2.83 to 5.79) and acidity within the parameters Regulation for each flavor. Regression models have demonstrated the possibility of determining both ascorbic acids, citric, malic and tartaric with successfully, besides sucrose, glucose and fructose by means of only a spectrum, suggesting that the models are economically viable for quality control and product standardization in the fruit juice and nectars processing industry.

Utilização de bagaço de maçã como ingrediente alimentar em iogurte com potencial efeito benéfico para a saúde

O desenvolvimento de alimentos funcionais evoluiu consideravelmente ao longo dos anos e a capacidade tecnológica para produzir um alimento com compostos fisiologicamente ativos tem crescido significativamente. O presente trabalho teve como objetivo criar um novo alimento, utilizando um subproduto proveniente da indústria agroalimentar. O alimento seleccionado foi o iogurte natural, o qual foi enriquecido com um extrato de bagaço de maçã. O bagaço de maçã contém compostos fenólicos e fibra, mostrando atividade antioxidante significativa e por conseguinte apresenta um potencial efeito positivo na saúde. Avaliaram-se características químicas do bagaço de maçã e das farinhas obtidas a partir deste subproduto, designadamente: acidez, teor em açúcares totais e redutores, cinza, matéria gorda, humidade, proteína bruta, fibra dietética insolúvel e solúvel. O extrato aquoso obtido a partir do subproduto foi também caracterizado quanto ao seu conteúdo fenólico e atividade antioxidante. O iogurte produzido com incorporação do extrato de bagaço de maçã foi estudado do ponto de vista de parâmetros químicos tais como acidez, açúcares totais, cinza, humidade, proteína bruta, pH e fibra bruta, conteúdo em fenólicos totais e atividade antioxidante. O subproduto, os extratos e o iogurte foram também avaliados quanto à sua carga microbiológica. Na caracterização química do bagaço foram obtidos os seguintes valores, expressos em base seca: 2,0±0,01% de acidez (expressa em equivalentes de ácido málico); 15,96±1,53% de açúcares totais; 13,35±1,91% de açúcares redutores; 1,88±0,07% de cinza, 2,49±0,5% de matéria gorda, 81,17±1,98% de humidade e 5,01±0,01% de proteína bruta. Quanto ao seu conteúdo em fibra dietética, o bagaço contém na sua composição 65,80% de fibra dietética insolúvel e 4,90% de fibra dietética solúvel. A farinha obtida após secagem a 60 °C do bagaço de maçã apresentou, na base seca, 1,9±0,04% de acidez, 10,57±1,31% de açúcares totais; 8,50±1,00% de açúcares redutores; 2,22±0,04%de cinza, 4,73±0,11% de matéria gorda, 6,34±0,62% de humidade e 5,40±0,26% de proteína. A atividade antioxidante do extrato aquoso, (AQ_6X10) obtido do bagaço de maçã, utilizado para incorporação no iogurte, determinada através do método ABTS foi de 5,00±1,28µmol TE/g amostra e apresentou um teor em compostos fenólicos de 221,42±0,734 mg EAG/ 100g de extrato, na base seca. Ao nível microbiológico o extrato revelou parâmetros aceitáveis, de acordo com a tabela 13 (anexos 3), para utilização como ingrediente alimentar. Os iogurtes produzidos foram analisados quimicamente. O iogurte, com extrato aquoso de bagaço de maçã incorporado, apresentou 89,64±0,00% de humidade, 0,93±0,00% de acidez (expressa em equivalentes de ácido láctico); 6,42±0,26% de açúcares totais; 0,74%±0,00% de cinza, 3,83±0,00% de proteína bruta e 0,2±0,00% de fibra bruta. O seu conteúdo em compostos fenólicos foi de 12,41±1,69mg EAG/ 100g de iogurte, e a sua atividade antioxidante foi 54,84±4,40 µmol TE/g iogurte. Avaliou-se ainda o iogurte no que diz respeito ao crescimento de bactérias lácticas e constatou-se, por comparação com o iogurte de controlo, que estas se desenvolveram normalmente ao longo do processo de fabrico. A análise microbiológica revelou ainda que o iogurte é seguro do ponto de vista alimentar de acordo com a Tabela 15 (anexos 3). A análise sensorial realizada aos iogurtes demonstrou que o iogurte fortificado apresentou uma aceitação muito boa pelo painel de provadores. O presente estudo demonstrou que o bagaço de maçã, um subproduto das indústrias agroalimentares, é seguro do ponto de vista microbiológico, podendo ser utilizado na preparação de ingredientes alimentares para serem incorporados, por exemplo em iogurte, conferindo-lhe características que se destacam pelo maior teor em fibra e atividade antioxidante em relação ao iogurte não enriquecido, e atributos sensoriais apelativos em termos de textura e sabor.

Chromatographic analysis of antioxidant and related compounds in Polyporus squamosus from different origins

The antioxidant potential of mushrooms is mainly attributed to their composition in polysaccharides, phenolic compounds, tocopherols and some organic acids [1]. Phenolic compounds contribute directly to the antioxidative action and play an important role in stabilizing lipid peroxidation [2]; exhibit a wide range of bioactive properties such as anti-allergenic, anti-inflammatory and antimicrobial, which have been in part related to their antioxidant activity [3]. Tocopherols are important fatsoluble antioxidants, acting in the cellular membrane; due to their role as scavenger of free radicals protecting human cells against degenerative malfunctions [4]. Some organic acids are very common in natural matrices; malic acid contributes to a pleasantly sour taste and is often used as a food additive; citric acid is known due to its antibacterial and antioxidant properties and fumaric acid is important because of its antioxidant, anti-inflammatory, antimicrobial and acidifying properties [5]. The purpose of the present study was to analyze antioxidant and related compounds (phenolic compounds, tocopherols and organic acids) of Polyporus squamosus (Huds.) Fr. samples originated from two different origins (Portugal and Serbia). Specimens of P. squamosus were collected in Bragança (Northeast Portugal) and Jabučki rit (Northern Serbia) during April 2015 and 2012, respectively. Phenolic compounds, organic acids and tocopherols were determined by high performance liquid chromatograph (HPLC) coupled to a diode array detector (DAD), in the two first cases, and a fluorescence detector in the last one. With respect to phenolic and related compounds, p-hydroxybenzoic and cinnamic acids were identified in both samples; the first one predominates in the sample from Portugal, while cinnamic acid was more abundant in the sample from Serbia. Tocopherols (α-, β and γ-isoforms) were found in the sample from Serbia, but in the sample from Portugal, γ-tocopherol was not identified. This sample showed the highest total tocopherols content, and revealed the highest level of β-tocopherol; γ- tocopherol predominated in the sample from Serbia. Among organic acids, it was possible to quantify oxalic, malic and fumaric acids in both samples. Malic acid was found in higher amounts in the sample from Serbia. Overall, the present study shows that mushroom samples from different origins have dissimilar results, but are both rich in bioactive compounds, being a valuable source for the development of natural medicines and nutraceuticals.

Postharvest changes in profiles of sugars, organic acids and tocopherols in leafy vegetables monitored by chromatographic techniques

After harvest, plants remain living organisms with the capacity to carry out metabolic processes. Thus, from the moment they are detached from the source of nutrients, they become entirely dependent on their own organic reserves [1]. Postharvest changes cannot be stopped, but they can be slowed within certain limits. Therefore, this study was conducted to evaluate the effects induced by storage in the profiles of sugars, organic acids and tocopherols of two leafy vegetables. Wild samples of watercress (Nasturtium officinale R. Br.) and buckler sorrel (Rumex induratus Boiss. & Reut.), from the Northeastern region of Portugal, were analyzed after harvest (control) and after storage in sterilized packages (using the passive modification mode) at 4ºC for 7 or 12 days, respectively. Analyses were performed by high-performance liquid chromatography (HPLC) using different detectors, i.e., a refraction index detector (RID) for free sugars, a photodiode array detector (PDA) for organic acids, and a fluorescence (FP) detector for tocopherols. The storage time decreased the levels of fructose, glucose and total sugars in both leafy vegetables and increased the total organic acids content. The decrease of these sugars can be related to its use by the plant to produce the required energy. Ascorbic acid was detected in buckler sorrel and decreased with storage; while the amount of malic acid increased in both species. Curiously, all the tocopherol isoforms increased in watercress, while buckler sorrel just present higher values of γ- and δ- tocopherols. In fact, the de novo synthesis of these bioactives compounds can be a plant strategy to fight against the reactive species that are produced during storage. The knowledge of the behavior of these compounds during storage that was achieved with this study [2] may contribute to the development of more effective preservation strategies for leafy vegetables.