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Use of High Pressure and Ultrasound As a Corrective Measure of The TESIS DE DOCTORAMIENTO Use of High Pressure and Ultrasound as a corrective measure of the pastiness in Dry-Cured Ham Cristina Pérez Santaescolástica 2020 Doctorado internacional Doctorado industrial Cristina Pérez Santaescolástica TESIS of DOCTORAMIENTO: High Use DE Pressure and Ultrasound as a corrective measure of Dry-Cured pastiness in the Ham 2020 Internacional Doctoral School Cristina Pérez Santaescolástica DOCTORAL DISSERTATION “Use of High Pressure and Ultrasound as a corrective measure of the pastiness in Dry-Cured Ham” Supervised by the PhD: José Manuel Lorenzo Rodríguez, Laura Purriños Pérez and Francisco Javier Carballo García Year: 2020 “International mention” “Industrial doctorate” Internacional Doctoral School JOSÉ MANUEL LORENZO RODRÍGUEZ, Researcher and Head of the Department of New Products Development and Food Packaging of the Centro Tecnolóxico da Carne, LAURA PURRIÑOS PÉREZ, Researcher and Head of the Department of Cromatography of the Centro Tecnolóxico da Carne and FRANCISCO JAVIER CARBALLO GARCÍA, Professor of the Area of Food Technology of the University of Vigo, DECLARES that the present work, entitled “Use of High Pressure and Ultrasound as a corrective measure of the pastiness in Dry-Cured Ham”, submitted by Ms CRISTINA PÉREZ SANTAESCOLÁSTICA to obtain the title of Doctor, was carried out under their supervision in the PhD programme “Ciencia y Tecnología Agroalimentaria” and it accomplishes the requirements to obtain the degree of Doctor by the University of Vigo. Ourense, 5th of September 2020 The supervisors José Manuel Lorenzo Rodríguez. PhD Laura Purriños Pérez, PhD Francisco Javier Carballo García To my mother, whom I miss so much and without whom I would not have achieved anything. Her love, understanding and confidence in me have made me what I am. Now, even when she is no longer by my side, I still listen to her words of encouragement that keep me going no matter how difficult. AKNOWLEDGMENTS First of all, I would like to thank my directors, José Manuel Lorenzo Rodriguez, PhD, Laura Purriños Pérez, PhD, and Francisco Javier Carballo García, PhD for all the help received, for the support obtained, for their patience and above all for sharing with me their knowledge and experience. I would also like to thank the Centro Tecnológico de la Carne and its manager, for allowing me to develop my thesis in their facilities and the INIA for granting me the scholarship that made it possible. Secondly, I would like to thank everyone with whom I have had the pleasure of working and who have been part of this long journey, supporting, advising and motivating me. In particular, to my laboratory colleagues for always being available to clear up any doubt and to Manuel Juarez, PhD and Ilse Fraeye, PhD, for inviting me to collaborate in their facilities and who together with their teams made me feel at home despite the distance. Finally, my close friends and my family without whom I would not have made it this far. My sister Elisa and my brother-in-law Jesus supporting me and giving me strength since I was born as if they were my parents. Jesus, nephew and at the same time brother who motivated me to be better every day so that I could be his reference and to guide him whenever he needed on his journey. ABSTRACT This doctoral thesis tried to contribute to solving one of the quality defects whose high incidence leads to great economic losses in the ham industry. In spite of an exhaustive control in the process, 10% of the finished hams present pasty textures, causing technological problems and quality reductions. For this, 200 hams were cured, in which the development of pasty textures was facilitated, being able to carry out the tests of this research work. The objectives were, in a first instance to study the relationship between the proteolysis index and the appearance of texture defects, identifying possible biomarkers associated with proteolytic activity, and also an instrumental determination of its organoleptic consequences. Secondly, from an analytical point of view, to study the effect of two selected non-invasive techniques applied as texture defects corrective measures (power ultrasound and high pressures) on protein structures and organoleptic attributes. At first, adhesiveness and nitrogen fractions were determined to calculate the proteolysis index, being able to classify hams in three intervals: low (<32), medium (32-36) and high (> 36) proteolysis index. Subsequently, free amino acid profile, volatile compound profile and a proteomic study were determined. From the results, it was shown that a high proteolysis index was related to a more adhesiveness as much as an increment in bitter taste and important aromatic losses. The use of power ultrasound could reduce the adhesiveness but it was observed that the taste of samples was turned sweeter, bitterer and more rancid, as well as the typical fatty odour of dry-cured ham could be decreased causing quality reductions. On the other hand, the treatments with high pressures in combination with high temperatures caused intense modifications in the profile of free amino acids and volatile compounds, especially at temperatures of 35 °C. These modifications gave rise to hams apparently with a greater sweet, acid and rancid taste, being the rancid aroma enhanced due to the increased content of aldehyde compounds (especially the hexanal content). However, it was observed that such impact can be minimized when the use of high pressures is combined with temperatures in the range of 0–20 °C. Regarding the proteomic analysis, the main evidences that can be highlighted were the identification of MYH1 and MYH4 protein fragments as suitable proteolysis biomarkers as well as non-fragmented sarcoplasmic proteins namely FABP4 / H, PRDX6, SOD, CBR1 and ACY1 as independent candidate biomarkers of ultrasound treatment. Finally, it was observed that the application of high pressures could promote actin fragmentation. RESUMEN Esta tesis doctoral trató de contribuir a la mejora de la calidad del jamón crudo-curado, intentando solventar uno de los defectos de calidad cuya alta incidencia da lugar a grandes pérdidas económicas. A pesar de un exahustivo control en el proceso de elaboración, un 10% de los jamones curados presentan texturas pastosas, constituyendo un problema tanto para el consumidor, que demanda un producto de calidad, como para el comerciante, que se encuentra con una dificultad en el proceso de loncheado. Para ello se curaron 200 jamones en los que, a partir de variaciones en la temperatura y la humedad del proceso, se facilitó el desarrollo de texturas pastosas pudiendo así llevarse a cabo los estudios que forman parte de esta tesis y cuyos objetivos resumidos fueron los siguientes: 1. Estudio de la relación existente entre el índice de proteólisis y la aparición de defectos de textura, identificando posibles biomarcadores asociados a la actividad proteolítica. 2. Determinación instrumental de las consecuencias organolépticas derivadas de una alta actividad proteolítica. 3. Estudio del efecto a nivel estructural de la aplicación de temperaturas moderadas, asistidas con ultrasonidos de potencia, como medida correctora de la adhesividad. 4. Determinación instrumental de los cambios organolépticos provocados en el producto derivados del uso de temperaturas moderadas asistidas con ultrasonidos de potencia. 5. Estudio de las alteraciones proteicas derivadas del uso de altas presiones como medida correctora de adhesividad. 6. Evaluación instrumental del efecto de altas presiones en la calidad organoléptica del producto, identificando la temperatura óptima para su aplicación. Una vez finalizada la curación de los jamones, éstos fueron deshuesados y loncheados. Para el estudio previo de las muestras se utilizaron cuatro lonchas de las que se extrajo el músculo biceps femoris por ser el más susceptible a sufrir reacciones proteolíticas. Dicho músculo se trituró consiguiendo una mejor homogeneización de la muestra y se envasó al vacío, conservándolo a temperatura de congelación hasta el momento de su análisis. En estas muestras, se determinaron, además de la adhesividad, las fracciones nitrogenadas, a partir de cuyo valor se calculó el índice de proteólisis, consiguiendo clasificar los jamones en tres intervalos: baja (<32), media (32-36) y alta (>36) proteólisis. A continuación, se estudió el perfil de aminoácidos libres y el perfil de compuestos volátiles, a fín de obtener una aproximación de las diferencias sápidas y aromáticas existentes entre los grupos. Paralelamente, dos muestras aleatorias de bajo índice y dos de alto índice de proteólisis fueron sometidas a un estudio proteómico a fín de determinar biomarcadores diferenciales de la actividad proteolítica. Como era de esperar, los valores de nitrógeno no proteico fueron mayores cuanto mayor proteólisis presentaban las muestras. Además, se observó que un índice de proteólisis por encima de 36 % aumentaba de forma significativa (P<0,01) la adhesividad de las lonchas. Este efecto en la textura provocado por una intensa degradación de proteínas podría explicar el número de puntos notablemente mas alto (P <0,05) obtenido en las muestras de alto índice de proteólisis durante el estudio proteómico. Así mismo, cinco fragmentos de proteína estuvieron sobrerrepresentadas en el grupo de alto índice de proteólisis, observándose los mayores valores de cambio relativo (RC >0,4) para MYH1, ACTS y MYH4. Según esto, y teniendo en cuenta evidencias previas de una mayor degradación de la miosina en respuesta a la proteólisis, MYH1 y MYH4 podrian ser adecuados marcadores de proteólisis y, por tanto, dada su relación, de adhesividad. En cuanto a los aminoácidos libres, el contenido total no mostró diferencias significativas entre grupos. Sin embargo, seis de los 18 aminoácidos analizados (serina, taurina, cisteína, metionina, isoleucina y leucina) mostraron diferencias significativas (P <0.05) entre los grupos con distintos niveles de proteólisis. En todos los grupos, la leucina fue el aminoácido mayoritario, siendo mayor su contenido cuanto mayor era el índice de proteólisis. Esta misma tendencia se observó para la serina, metionina e isoleucina, mientras que los valores de cisteína y taurina mostraron una tendencia opuesta.
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