Valorización de la biomasa remanente del proceso de extracción de aceite de la diatomea Phaeodactylum tricornutum.
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Date
2024
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Universidad de Concepción
Abstract
Las microalgas, organismos eucariontes unicelulares fotosintéticos, son consideradas una de las materias primas más prometedoras para su aplicación en sectores industriales como la gestión ambiental, industria energética, cosmética, farmacéutica y alimentaria. En las últimas décadas, se han estado estudiando distintas moléculas antioxidantes que podrían ser empleadas a nivel industrial, entre esas moléculas se encuentran las proteínas y péptidos de microalgas, que actúan a través de mecanismos como, la eliminación de radicales libres, eliminación enzimática de oxidantes y quelación de metales. La actividad antioxidante de los péptidos depende de su secuencia de aminoácidos, los grupos funcionales que presenten, la longitud de los péptidos y la orientación de los mismos en la proteína nativa, por lo que, para promover dicha bioactividad, se requiere aislar los péptidos desde las proteínas nativas mediante hidrólisis enzimática para obtener hidrolizados proteicos.
Phaeodactylum tricornutum es una diatomea penada marina, que se caracteriza por presentar tres morfotipos diferentes: fusiforme, trirradiado y ovalado; además es una especie industrialmente relevante, ya que acumula metabolitos como el pigmento carotenoide fucoxantina y el ácido graso poliinsaturado omega-3 ácidoeicosapentaenoico (EPA). La industrialización y comercialización de microalgas aún no es del todo rentable, siendo una de las causas el que se obtenga solo un producto desde su biomasa, lo cual ha motivado aplicar un nuevo enfoque, en el que todos los componentes de la biomasa se valoricen, lo cual puede lograrse aplicando el concepto de biorrefinería. En el caso de la extracción de ácidos grasos y fucoxantina desde la biomasa de P. tricornutum, se obtiene una biomasa residual que contiene entre un 25-30% de proteínas y que, mediante hidrólisis enzimática de dichas proteínas, se pueden obtener hidrolizados proteicos, con propiedades funcionales mejoradas respecto a la proteína cruda y de gran interés para la industria como son los antioxidantes.
Bajo este contexto, la hipótesis que se planteó en este estudio fue que los péptidos derivados de la hidrólisis de las proteínas de P. tricornutum tienen mayor actividad antioxidante que las proteínas crudas. Para lo cual: 1- Se analizó el contenido proteico de la biomasa remanente del proceso de extracción de aceite de P. tricornutum y 2- Se dilucidó si las proteínas hidrolizadas de la biomasa remanente del proceso de extracción de aceite de P. tricornutum tienen mayor actividad antioxidante que las proteínas crudas.
Para ello se estableció un cultivo de 20 L de P. tricornutum para obtener biomasa, esta se mezcló junto con otra biomasa liofilizada proveniente de otros cultivos realizados en condiciones similares. Dicha biomasa se sometió a un procedimiento de extracción de lípidos obteniéndose una biomasa sin aceite (biomasa remanente). A la biomasa con aceite y biomasa remanente se les cuantificó lípidos totales y proteínas totales. El análisis lipídico indicó que la biomasa con aceite tuvo un 11,21% de lípidos mientras que la biomasa residual tuvo un 7,65%. El contenido de proteínas de la biomasa con aceite fue del 8,86% y de la biomasa residual un 11,15%. Con el objetivo de recuperar la mayor cantidad de proteínas desde la biomasa remanente, se procedió a hidratar esta biomasa con agua destilada (pH 7), buffer fosfato (pH 7), tampón de lisis (pH 7) e hidróxido de sodio (pH 10) para posteriormente lisar independientemente con un sonicador y un ultra turrax. Los resultados indicaron que la combinación hidróxido de sodio/sonicador fue el tratamiento con el que se obtuvo la mayor extracción de proteínas (5173,57 ± 247,38 μg/mL) y con una eficiencia extractiva del 74,23%. Con el propósito de concentrar las proteínas, se ensayaron dos métodos de precipitación: 1- Sulfato de amonio y 2- Partición en tres fases (TPP), mediante el primer método se recuperó un 0,25% de las proteínas que contenía la biomasa remanente (11,15% de proteína/biomasa) mientras que, con el segundo método se recuperó un 0,19%. Considerando estos resultados se decidió no precipitar las proteínas. Para evaluar si las proteínas hidrolizadas de la biomasa residual tenían mayor actividad antioxidante que las proteínas crudas, se realizó la hidrólisis enzimática del extracto proteico con tripsina y Protamex, en diferentes tiempos de digestión enzimática (2 h, 4 h y 6 h) y luego se midió la actividad antioxidante de los extractos de proteínas sin hidrolizar e hidrolizados, mediante el ensayo de capacidad de reducción del hierro (FRAP). Los resultados revelaron que los extractos hidrolizados tuvieron mayor actividad antioxidante que el extracto sin hidrolizar (287,58 μmoles de TE/L). Además, los hidrolizados proteicos producidos por tripsina y Protamex tuvieron diferente actividad antioxidante entre los diferentes tiempos de hidrólisis. Estos resultados sugieren que a partir de la biomasa remanente del proceso de extracción de aceite desde P. tricornutum, se pueden generar hidrolizados proteicos con mayor actividad antioxidante que la proteína cruda y, por lo tanto, con mayor valor agregado en comparación al residuo sin tratar.
Microalgae, photosynthetic unicellular eukaryotic organisms, are considered one of the most promising raw materials for application in industrial sectors such as environmental management, energy, cosmetics, pharmaceutical and food industries. In recent decades, different antioxidant molecules have been studied that could be used at an industrial level. Among these molecules are proteins and peptides from microalgae, which act through mechanisms such as the elimination of free radicals, enzymatic elimination of oxidants. and metal chelation. The antioxidant activity of peptides depends on their amino acid sequence, the functional groups they present, the length of the peptides and their orientation in the native protein, therefore, to promote said bioactivity, it is necessary to isolate the peptides from native proteins, through enzymatic hydrolysis to obtain protein hydrolysates. Phaeodactylum tricornutum is a marine pennate diatom, which is characterized by presenting three different morphotypes: fusiform, triradiate and oval; It is also an industrially relevant species, since it accumulates metabolites such as the carotenoid pigment fucoxanthin and the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA). The industrialization and commercialization of microalgae is not yet completely profitable, one of the causes being that only one product is obtained from its biomass, which has motivated the application of a new approach, in which all the components of the biomass are valorized. which can be achieved by applying the biorefinery concept. In the case of the extraction of fatty acids and fucoxanthin from the biomass of P. tricornutum, a residual biomass is obtained that contains between 25-30% of proteins and, through enzymatic hydrolysis of said proteins, protein hydrolysates can be obtained. with improved functional properties compared to crude protein and of great interest to the industry such as antioxidants. Under this context, the hypothesis raised in this study was that peptides derived from the hydrolysis of P. tricornutum proteins have greater antioxidant activity than crude proteins. For which: 1- The protein content of the remaining biomass from the P. tricornutum oil extraction process was analyzed and 2- It was elucidated whether the hydrolyzed proteins of the remaining biomass from the P. tricornutum oil extraction process have greater antioxidant activity than crude proteins. For this, a 20 L culture of P. tricornutum was established to obtain biomass, this was mixed together with other freeze-dried biomass from other cultures carried out under similar conditions. Said biomass was subjected to a lipid extraction procedure, obtaining a biomass without oil (remaining biomass). Total lipids and total proteins were quantified in the biomass with oil and remaining biomass. The lipid analysis indicated that the biomass with oil had 11,21% lipids while the residual biomass had 7,65%. The protein content of the biomass with oil was 8,86% and of the residual biomass was 11,15%. With the objective of recovering the greatest amount of proteins from the remaining biomass, this biomass was hydrated with distilled water (pH 7), phosphate buffer (pH 7), lysis buffer (pH 7) and sodium hydroxide (pH 10) to subsequently lyse independently with a sonicator and an ultra-turrax. The results indicated that the sodium hydroxide/sonicator combination was the treatment with which the highest protein extraction was obtained (5173,57 ± 247,38 μg/mL) and with an extractive efficiency of 74,23%. With the purpose of concentrating the proteins, two precipitation methods were tested: 1- Ammonium sulfate and 2- Three-phase partition (TPP). Using the first method, 0,25% of the proteins contained in the remaining biomass were recovered (11,15% protein/biomass) while, with the second method, 0,19% was recovered. Considering these results, it was decided not to precipitate the proteins. To evaluate whether the hydrolyzed proteins from the residual biomass had greater antioxidant activity than the crude proteins, the enzymatic hydrolysis of the protein extract was carried out with trypsin and Protamex, at different enzymatic digestion times (2 h, 4 h and 6 h) and then measured the antioxidant activity of unhydrolyzed and hydrolyzed protein extracts, using the Ferric Reducing Antioxidant Power (FRAP). The results revealed that the hydrolyzed extracts had greater antioxidant activity than the unhydrolyzed extract (287,58 μmoles of TE/L). Furthermore, the protein hydrolysates produced by trypsin and Protamex had different antioxidant activities between the different hydrolysis times. These results suggest that from the remaining biomass of the oil extraction process from P. tricornutum, protein hydrolysates can be generated with greater antioxidant activity than crude protein and, therefore, with greater added value compared to the untreated residue.
Microalgae, photosynthetic unicellular eukaryotic organisms, are considered one of the most promising raw materials for application in industrial sectors such as environmental management, energy, cosmetics, pharmaceutical and food industries. In recent decades, different antioxidant molecules have been studied that could be used at an industrial level. Among these molecules are proteins and peptides from microalgae, which act through mechanisms such as the elimination of free radicals, enzymatic elimination of oxidants. and metal chelation. The antioxidant activity of peptides depends on their amino acid sequence, the functional groups they present, the length of the peptides and their orientation in the native protein, therefore, to promote said bioactivity, it is necessary to isolate the peptides from native proteins, through enzymatic hydrolysis to obtain protein hydrolysates. Phaeodactylum tricornutum is a marine pennate diatom, which is characterized by presenting three different morphotypes: fusiform, triradiate and oval; It is also an industrially relevant species, since it accumulates metabolites such as the carotenoid pigment fucoxanthin and the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA). The industrialization and commercialization of microalgae is not yet completely profitable, one of the causes being that only one product is obtained from its biomass, which has motivated the application of a new approach, in which all the components of the biomass are valorized. which can be achieved by applying the biorefinery concept. In the case of the extraction of fatty acids and fucoxanthin from the biomass of P. tricornutum, a residual biomass is obtained that contains between 25-30% of proteins and, through enzymatic hydrolysis of said proteins, protein hydrolysates can be obtained. with improved functional properties compared to crude protein and of great interest to the industry such as antioxidants. Under this context, the hypothesis raised in this study was that peptides derived from the hydrolysis of P. tricornutum proteins have greater antioxidant activity than crude proteins. For which: 1- The protein content of the remaining biomass from the P. tricornutum oil extraction process was analyzed and 2- It was elucidated whether the hydrolyzed proteins of the remaining biomass from the P. tricornutum oil extraction process have greater antioxidant activity than crude proteins. For this, a 20 L culture of P. tricornutum was established to obtain biomass, this was mixed together with other freeze-dried biomass from other cultures carried out under similar conditions. Said biomass was subjected to a lipid extraction procedure, obtaining a biomass without oil (remaining biomass). Total lipids and total proteins were quantified in the biomass with oil and remaining biomass. The lipid analysis indicated that the biomass with oil had 11,21% lipids while the residual biomass had 7,65%. The protein content of the biomass with oil was 8,86% and of the residual biomass was 11,15%. With the objective of recovering the greatest amount of proteins from the remaining biomass, this biomass was hydrated with distilled water (pH 7), phosphate buffer (pH 7), lysis buffer (pH 7) and sodium hydroxide (pH 10) to subsequently lyse independently with a sonicator and an ultra-turrax. The results indicated that the sodium hydroxide/sonicator combination was the treatment with which the highest protein extraction was obtained (5173,57 ± 247,38 μg/mL) and with an extractive efficiency of 74,23%. With the purpose of concentrating the proteins, two precipitation methods were tested: 1- Ammonium sulfate and 2- Three-phase partition (TPP). Using the first method, 0,25% of the proteins contained in the remaining biomass were recovered (11,15% protein/biomass) while, with the second method, 0,19% was recovered. Considering these results, it was decided not to precipitate the proteins. To evaluate whether the hydrolyzed proteins from the residual biomass had greater antioxidant activity than the crude proteins, the enzymatic hydrolysis of the protein extract was carried out with trypsin and Protamex, at different enzymatic digestion times (2 h, 4 h and 6 h) and then measured the antioxidant activity of unhydrolyzed and hydrolyzed protein extracts, using the Ferric Reducing Antioxidant Power (FRAP). The results revealed that the hydrolyzed extracts had greater antioxidant activity than the unhydrolyzed extract (287,58 μmoles of TE/L). Furthermore, the protein hydrolysates produced by trypsin and Protamex had different antioxidant activities between the different hydrolysis times. These results suggest that from the remaining biomass of the oil extraction process from P. tricornutum, protein hydrolysates can be generated with greater antioxidant activity than crude protein and, therefore, with greater added value compared to the untreated residue.
Description
Tesis presentada para optar al optar al título de Biólogo
Keywords
Biomasa, Diatomeas, Productos biológicos