Resumen de Producción de espermaceti biotecnológico mediante procesos enzimáticos en sistemas solvent-free
- español
Los productos obtenidos a partir de los mamíferos marinos han sido una fuente ancestral de riqueza para el ser humano. Entre dichos productos destaca por su valor industrial el espermaceti, una sustancia cérea extraída de la cavidad craneal del cachalote compuesta mayoritariamente por laurato, miristato, palmitato y estearato de cetilo en distintas proporciones. Durante siglos, el espermaceti ha sido utilizado como materia prima para la fabricación de velas, fármacos, cosméticos y lubricantes.
Desde la prohibición de la caza de ballenas en los años 80, se utiliza espermaceti sintético en la industria cosmética sobre todo, donde es empleado como emoliente de alta consistencia. También se usa en farmacia debido a su semejanza con el sebo humano y a sus propiedades emolientes.
El proceso de obtención de ésteres grasos como los del espermaceti a nivel industrial se basa en el uso de temperaturas elevadas y catalizadores no selectivos, que conducen a productos con impurezas que deben ser sometidos diversas etapas de post-tratamiento para que puedan cumplir con los requerimientos del sector cosmético.
Por el contrario, la síntesis biocatalítica permite eliminar las reacciones secundarias, obteniéndose productos con un elevado grado de pureza y propiedades organolépticas adecuadas a la vez que se reduce la generación de residuos contaminantes y el consumo de energía. Además, la legislación actual permite el etiquetado "natural" a los productos obtenidos por vía enzimática, lo cual resulta atractivo para productores y consumidores. Este proceso biocatalítico se basa en el uso de lipasas, enzimas lipolíticas capaces de catalizar esterificaciones cuando se hallan en medios con baja actividad del agua. Por lo tanto, la síntesis en medios en ausencia de disolventes resulta especialmente atractiva al trabajar con lipasas para la obtención de ésteres cosméticos, ya que permite evitar la utilización de disolventes peligrosos o prohibidos. No obstante, el principal inconveniente de los sistemas biocatalíticos es el elevado coste de las enzimas, que, con el fin de hacer el proceso económicamente rentable, deben ser inmovilizadas, ya que esto posibilita su recuperación y reutilización después de cada ciclo de síntesis.
En consecuencia, el objetivo de esta Tesis Doctoral es el estudio comparativo de la síntesis "solvent-free" de una mezcla de ésteres similar al espermaceti natural, empleando para ello derivados inmovilizados de lipasa comerciales o elaborados en el laboratorio. Para ello, se ha empleado el reactor de altas prestaciones modelo 5101 de Parr Instruments Co., de 100 mL de capacidad y que está equipado con un agitador de cuatro palas inclinadas. Dicho reactor permite trabajar a presión reducida y en condiciones de atmósfera inerte.
Los biocatalizadores comerciales seleccionados para este trabajo son CalB immo Plus y Novozym¿ 435, dos preparaciones de lipasa B de Candida antarctica, Lipozyme¿ RM IM, un derivado de Rhizomucor miehei y Lipozyme¿ TL IM, un preparado de Thermomyces lanuginosus. En lo que respecta a los biocatalizadores propios, éstos han sido elaborados por adsorción o enlace covalente de la lipasa B de Candida antarctica contenida en la disolución comercial Lipozyme¿ CALB L sobre las siguientes matrices poliméricas: Lewatit¿ VP OC 1064 MD PH, Amberlite¿ XAD¿ 1180, Purolite¿ Lifetech¿ ECR 1090 M, Purolite¿ Lifetech¿ ECR 8806 M and Purolite¿ Lifetech¿ ECR 8285 M. Los derivados desarrollados en el laboratorio poseen un contenido en proteína entre 71.8 y 124.5 mg por gramo de biocatalizador seco, poniendo en evidencia el elevado rendimiento de los protocolos de inmovilización seleccionados y la importancia de las propiedades del soporte (diámetro de poro, área superficial, composición) en los resultados.
La influencia de la concentración de biocatalizador fue estudiada en primer lugar para la síntesis de los ésteres cetílicos por separado, observándose que la mejora de la velocidad de reacción al incrementar dicho parámetro no era proporcional a la masa de derivado en el medio. Estos experimentos pusieron de manifiesto además que la longitud de cadena del compuesto sintetizado influye ligeramente en la velocidad de reacción. Asimismo, se evaluó el efecto de la temperatura sobre el proceso, escogiéndose 70 ºC como valor óptimo, ya que una mayor temperatura de proceso implica un mayor consumo de energía y a que cuando se trabaja a 60 ºC se requieren mayores tiempos de acondicionamiento de los reactivos. La etapa controlante de las reacciones de esterificación es la transferencia externa de materia, de forma que el proceso puede describirse con un buen grado de ajuste a través de un modelo de pseudo-primer orden.
Los ensayos de estabilidad operacional realizados apuntan a que todos los biocatalizadores comerciales y los derivados inmovilizados de Amberlite y ECR 8806 M presentan unas características adecuadas para ser utilizados en la síntesis de espermaceti a escala industrial, ya que éstos fueron recuperados y utilizados en una nueva síntesis hasta 15 veces. Durante el estudio económico de la síntesis optimizada a escala de laboratorio, se observó que dicho parámetro juega un papel fundamental en la reducción del coste del proceso biocatalítico y que la inversión económica a realizar con el mejor de los derivados propios es similar a la necesaria con el mejor de los comerciales.
Por otra parte, durante el análisis de las propiedades fisicoquímicas de los productos enzimáticos se comprobó que éstos presentan un alto grado de pureza y características similares a los ésteres comercializados actualmente y que son obtenidos por síntesis clásica. Las emulsiones cosméticas realizadas con los ingredientes biotecnológicos, presentaban una microestructura heterogénea, que junto con la longitud de cadena del éster utilizado, condicionaba la estabilidad y la resistencia a los cambios térmicos de las cremas.
Para concluir, cabe destacar que, con el propósito final de ser implantada a nivel comercial, la síntesis biocatalítica en una sola etapa de un producto similar al espermaceti natural está lista para ser llevada a una etapa intermedia de escalado, y este trabajo constituye una base sólida para la puesta a punto de la síntesis enzimática de otros muchos ésteres de utilidad en la industria cosmética.
Products obtained from marine mammals have ever been a source of wealth for the humanity. One of the most valuable for industrial purposes is the spermaceti, a waxy substance extracted from the head cavity of the sperm whale which is mainly composed by cetyl laurate, myristate, palmitate and stearate in different proportions. The spermaceti has been used for centuries as a raw material in candle-making, pharmaceutical, cosmetic and lubricants industry.
Since the banning of whales' hunting in the 80s, synthetic spermaceti is used, mainly in cosmetic industry as a hard-consistency emollient, but it is also found in pharmacy because of its similarity to human's skin sebum and its emollient properties. The conventional industrial process to obtain fatty acid esters such as the spermaceti's ones, entails high temperatures and non-selective catalysts that lead to impure products which need to undergo a host of reprocessing steps to meet the high standards of the cosmetic field.
In contrast, the decrease of side reactions through biocatalysis enables obtaining ultra-pure esters with good organoleptic properties while reducing contaminating wastes and energy consumption. In addition, according to current legislation, the cosmetic ingredients obtained by enzymatic methods can be labelled as "natural", which means another attractive feature both for producers and consumers. The biocatalytic synthesis of such esters is based on the use of lipases, lipolytic enzymes that are able to catalyse esterification reactions when placed in low water activity media. As a consequence, solvent-free synthesis is particularly interesting for the enzymatic production of such products, as the use of harmful or prohibited organic solvents is avoided.
Nevertheless, the main drawback of the biocatalytic pathway is the expensive cost of the enzymes, which must be immobilised to enable its recovery and reuse after each reaction cycle in order to make the process economically profitable.
In view of the foregoing, the aim of this Doctoral Thesis is the comparative study of the solvent-free synthesis of a mixture of esters similar to natural spermaceti by using different immobilised lipases, both from commercial origin or in-situ elaborated. For that purpose, a high performance batch reactor (Parr Instruments Co. 5101 model) has been used. The 100 mL reactor is equipped with a four-bladed impeller and allows working under low pressure and inert atmosphere conditions.
The commercial immobilised derivatives studied are CalB immo Plus and Novozym¿ 435, two Candida antarctica lipase B preparations, Lipozyme¿ RM IM, a Rhizomucor miehei biocatalyst and Lipozyme¿ TL IM, a Thermomyces lanuginosus derivative. On the other hand, several biocatalysts have been prepared by adsorption or covalent binding of the Candida antarctica lipase B contained in the commercial solution Lipozyme¿ CALB L on several polymeric matrices: Lewatit¿ VP OC 1064 MD PH, Amberlite¿ XAD¿ 1180, Purolite¿ Lifetech¿ ECR 1090 M, Purolite¿ Lifetech¿ ECR 8806 M and Purolite¿ Lifetech¿ ECR 8285 M. Protein loadings ranging between 71.8 and 124.5 mg per gram of dry biocatalyst were obtained, proving the high immobilisation yield of the process and the major influence of the carrier properties (pore diameter, surface area, chemical composition) on these results.
The influence of the concentration of biocatalysts used was first studied during the synthesis of the cetyl esters separately, pointing out that the improvement of the reaction rate observed was not proportional to the amount of enzyme in the reaction medium. In addition, it has been observed that the chain length of the products had a slight influence in the reaction rate. The reaction temperature was also evaluated, being selected 70 ºC as the optimum value for the one-step synthesis of the spermaceti analogue, as higher temperatures significantly increase energy consumption and a longer conditioning time is required when operating at 60 ºC. The esterification process is chiefly controlled by mass transfer limitations and can be described through a pseudo-first order kinetic model with quite good accuracy.
The operational stability test predicts a good long-term reusability of the commercial and Amberlite and ECR 8806 M biocatalysts for the synthesis of spermaceti at industrial scale under optimal conditions, as they were successfully recovered and recycled up to 15 times. The economic evaluation of the optimised synthesis at laboratory scale, put into evidence that this parameter has a key role in reducing the cost of the biocatalytic process and that the economic investment is similar when using the best commercial biocatalyst or the best in-lab derivative.
The analysis of the physicochemical properties of the enzymatic products proved their high purity and that they are similar to the ones currently commercialised, which are obtained through classic chemical routes. The cosmetic emulsions containing the biotechnological compounds turned out to have a heterogeneous microstructure, which along with the enzymatic ester used while formulating, it determines the cream's stability and resistance to thermal changes.
To sum up, the one-step biocatalytic synthesis of a product similar to natural spermaceti is ready to be scaled-up from the laboratory to a pilot plant with the final purpose of implementing this product at commercial level. Moreover, this work constitutes a solid basis for the development of the enzymatic synthesis of many other cosmetic esters.
- English
Products obtained from marine mammals have ever been a source of wealth for the humanity. One of the most valuable for industrial purposes is the spermaceti, a waxy substance extracted from the head cavity of the sperm whale which is mainly composed by cetyl laurate, myristate, palmitate and stearate in different proportions. The spermaceti has been used for centuries as a raw material in candle-making, pharmaceutical, cosmetic and lubricants industry.
Since the banning of whales' hunting in the 80s, synthetic spermaceti is used, mainly in cosmetic industry as a hard-consistency emollient, but it is also found in pharmacy because of its similarity to human's skin sebum and its emollient properties. The conventional industrial process to obtain fatty acid esters such as the spermaceti's ones, entails high temperatures and non-selective catalysts that lead to impure products which need to undergo a host of reprocessing steps to meet the high standards of the cosmetic field.
In contrast, the decrease of side reactions through biocatalysis enables obtaining ultra-pure esters with good organoleptic properties while reducing contaminating wastes and energy consumption. In addition, according to current legislation, the cosmetic ingredients obtained by enzymatic methods can be labelled as "natural", which means another attractive feature both for producers and consumers. The biocatalytic synthesis of such esters is based on the use of lipases, lipolytic enzymes that are able to catalyse esterification reactions when placed in low water activity media. As a consequence, solvent-free synthesis is particularly interesting for the enzymatic production of such products, as the use of harmful or prohibited organic solvents is avoided.
Nevertheless, the main drawback of the biocatalytic pathway is the expensive cost of the enzymes, which must be immobilised to enable its recovery and reuse after each reaction cycle in order to make the process economically profitable.
In view of the foregoing, the aim of this Doctoral Thesis is the comparative study of the solvent-free synthesis of a mixture of esters similar to natural spermaceti by using different immobilised lipases, both from commercial origin or in-situ elaborated. For that purpose, a high performance batch reactor (Parr Instruments Co. 5101 model) has been used. The 100 mL reactor is equipped with a four-bladed impeller and allows working under low pressure and inert atmosphere conditions.
The commercial immobilised derivatives studied are CalB immo Plus and Novozym¿ 435, two Candida antarctica lipase B preparations, Lipozyme¿ RM IM, a Rhizomucor miehei biocatalyst and Lipozyme¿ TL IM, a Thermomyces lanuginosus derivative. On the other hand, several biocatalysts have been prepared by adsorption or covalent binding of the Candida antarctica lipase B contained in the commercial solution Lipozyme¿ CALB L on several polymeric matrices: Lewatit¿ VP OC 1064 MD PH, Amberlite¿ XAD¿ 1180, Purolite¿ Lifetech¿ ECR 1090 M, Purolite¿ Lifetech¿ ECR 8806 M and Purolite¿ Lifetech¿ ECR 8285 M. Protein loadings ranging between 71.8 and 124.5 mg per gram of dry biocatalyst were obtained, proving the high immobilisation yield of the process and the major influence of the carrier properties (pore diameter, surface area, chemical composition) on these results.
The influence of the concentration of biocatalysts used was first studied during the synthesis of the cetyl esters separately, pointing out that the improvement of the reaction rate observed was not proportional to the amount of enzyme in the reaction medium. In addition, it has been observed that the chain length of the products had a slight influence in the reaction rate. The reaction temperature was also evaluated, being selected 70 ºC as the optimum value for the one-step synthesis of the spermaceti analogue, as higher temperatures significantly increase energy consumption and a longer conditioning time is required when operating at 60 ºC. The esterification process is chiefly controlled by mass transfer limitations and can be described through a pseudo-first order kinetic model with quite good accuracy.
The operational stability test predicts a good long-term reusability of the commercial and Amberlite and ECR 8806 M biocatalysts for the synthesis of spermaceti at industrial scale under optimal conditions, as they were successfully recovered and recycled up to 15 times. The economic evaluation of the optimised synthesis at laboratory scale, put into evidence that this parameter has a key role in reducing the cost of the biocatalytic process and that the economic investment is similar when using the best commercial biocatalyst or the best in-lab derivative.
The analysis of the physicochemical properties of the enzymatic products proved their high purity and that they are similar to the ones currently commercialised, which are obtained through classic chemical routes. The cosmetic emulsions containing the biotechnological compounds turned out to have a heterogeneous microstructure, which along with the enzymatic ester used while formulating, it determines the cream's stability and resistance to thermal changes.
To sum up, the one-step biocatalytic synthesis of a product similar to natural spermaceti is ready to be scaled-up from the laboratory to a pilot plant with the final purpose of implementing this product at commercial level. Moreover, this work constitutes a solid basis for the development of the enzymatic synthesis of many other cosmetic esters.
