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WO2025114751A1 - Method for producing sodium alginate microcapsules of homogeneous composition - Google Patents

Method for producing sodium alginate microcapsules of homogeneous composition Download PDF

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Publication number
WO2025114751A1
WO2025114751A1 PCT/IB2023/061995 IB2023061995W WO2025114751A1 WO 2025114751 A1 WO2025114751 A1 WO 2025114751A1 IB 2023061995 W IB2023061995 W IB 2023061995W WO 2025114751 A1 WO2025114751 A1 WO 2025114751A1
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Prior art keywords
microcapsules
sodium alginate
microns
extract
preparation
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Pending
Application number
PCT/IB2023/061995
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Spanish (es)
French (fr)
Inventor
Milton BARCOS ARIAS
Nardy DIEZ GARCIA
Luis GALARZA ROMERO
Rodrigo OVIEDO ANCHUNDIA
Daynet SOSA DEL CASTILLO
Jeffrey VARGAS PEREZ
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Escuela Superior Politecnica Del Litoral Espol
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Escuela Superior Politecnica Del Litoral Espol
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Priority to PCT/IB2023/061995 priority Critical patent/WO2025114751A1/en
Publication of WO2025114751A1 publication Critical patent/WO2025114751A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/645Fungi ; Processes using fungi

Definitions

  • the present invention relates to a process for obtaining microcapsules containing an enzyme extract that includes in its composition the activity of an extracellular protein expressed by the endophytic fungus Pestalotiopsis microspora.
  • the complex catalyzes the oxidation of phenolic hydroxyl groups to phenoxy radicals while oxygen (O2) is reduced to water, useful for the treatment of wastewater that presents contamination by color, chemical oxygen demand and biological oxygen demand; also, a process for the preparation of microcapsules and their use for the treatment of residual effluents is revealed.
  • Textiles have been used by civilization since around 5000 BC. This industry, which was originally domestic and family-based, changed starting in the 1500s when the first factory was established. However, spinning and weaving machinery did not appear until the Industrial Revolution of the 18th century (Neefus, J.D. "Textile Industrial Processes," in Industrial Hygiene Aspects of Plant Operations, Volume 1, 1982).
  • the production of textiles requires a large number of unit operations that use cotton, wool, synthetic fibers, or mixtures of these as raw materials, with a highly variable environmental impact from their liquid effluents.
  • the process requires a quantity of water that varies depending on the specific processes and the equipment used by the plant. For example, in dyeing with disperse dyes, between 100 and 150 liters of water are used. liters of water per kilogram of product. In dyeing with reactive dyes, between 125 and 170 liters per kilogram of product (EPA Profile of the Textile Industry, 1997).
  • microorganisms most commonly used in the bioremediation of contaminated effluents are fungi. Endophytic fungi are characterized by their ability to degrade lignin and other tree components such as cellulose. They produce extracellular enzymes that oxidize phenolic compounds. Among the characterized enzymes are manganese peroxidases, lignin peroxidases, peroxide-producing enzymes, and laccases. The activity of these m.o on dyes is based on the non-specificity of the enzyme system to depolymerize and mineralize lignin (Moeller, G., Garzón, M. Anuario Imta, 2003).
  • the second stage of the process involves bringing the wastewater into contact with the microencapsulated fungi.
  • the fungi are cultivated at temperatures between 20 and 30°C, using nitrogen, carbon, and mineral salt sources for their growth.
  • enzymes can be added to the pretreated water in encapsulated form.
  • the polymers comprising these matrices are made from biodegradable, natural, and nontoxic materials and are selected from the group consisting of alginates, maltodextrins, corn starch, kappa carrageenan salts, and iota carrageenan.
  • Patent W003/035561 also points out that it is possible to use the activity of hydrolytic enzymes, cellulolytic enzymes and ligninolytic enzymes to achieve the elimination of contaminants.

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  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Dispersion Chemistry (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention relates to a method for producing sodium alginate microcapsules of homogenous composition that contain an enzyme extract of which the composition includes the activity of an enzyme of extracellular proteins expressed by microorganisms that break down lignocellulose material, which includes an extract obtained from fungal secretion, an alginate salt preparation dissolved under agitation, and the resuspension, drying and sorting of the microcapsules.

Description

Procedimiento para la elaboración de microcápsulas de alginato de sodio de composición homogénea Procedure for the production of sodium alginate microcapsules of homogeneous composition

La presente invención se refiere a un proceso para obtener microcápsulas que contienen un extracto enzimatico que incluye en su composición la actividad de una proteína extracelular expresadas por el hongo endófito Pestalotiopsis microspora. El complejo cataliza la oxidación de grupos fenólicos hidroxilo a radicales fenoxy mientras que el oxígeno (02) se reduce a agua, útil para el tratamiento de aguas residuales que presentan contaminación por color, demanda química de oxígeno y demanda biológica de oxígeno; asimismo, se revela un procedimiento para la elaboración de las microcápsulas y su uso para el tratamiento de efluentes residuales. The present invention relates to a process for obtaining microcapsules containing an enzyme extract that includes in its composition the activity of an extracellular protein expressed by the endophytic fungus Pestalotiopsis microspora. The complex catalyzes the oxidation of phenolic hydroxyl groups to phenoxy radicals while oxygen (O2) is reduced to water, useful for the treatment of wastewater that presents contamination by color, chemical oxygen demand and biological oxygen demand; also, a process for the preparation of microcapsules and their use for the treatment of residual effluents is revealed.

Antecedentes y Descripción Background and Description

Los textiles han sido utilizados por la humanidad desde alrededor de 5000 A.C.. Esta industria que fue originalmente domestica y familiar, cambio a partir del 1500 donde se estableció la primera fabrica. Aunque no aparecerían maquinarias para hilar y tejer hasta la revolución industria en el siglo XVIII (Neefus, J.D. "Textile Industrial Processes," in Industrial Hygiene Aspects of Plantoperations, Volume 1 , 1982). Para la elaboración de textiles se requiere un gran número de operaciones unitarias que utilizan como materias primas: algodón, lana, fibras sintéticas, o mezclas de las mismas, con un impacto ambiental de sus efluentes líquidos muy variado. En los efluentes se pueden encontrar sales, almidón, peróxidos, EDTA, tensioactivos, enzimas, colorantes, metales y otros compuestos orgánicos de variadas estructuras que provienen de las distintas etapas del proceso global (Mansilla, H.D., et al., "Tratamiento de residuos líquidos de la industria de celulosa y textil" en Eliminación de Contaminantes por Fotocatálisis Heterogénea, CYTED, 2001 ) Textiles have been used by humanity since around 5000 BC. This industry, which was originally domestic and family-based, changed starting in the 1500s when the first factory was established. However, spinning and weaving machinery did not appear until the Industrial Revolution of the 18th century (Neefus, J.D. "Textile Industrial Processes," in Industrial Hygiene Aspects of Plant Operations, Volume 1, 1982). The production of textiles requires a large number of unit operations that use cotton, wool, synthetic fibers, or mixtures of these as raw materials, with a highly variable environmental impact from their liquid effluents. In the effluents, salts, starch, peroxides, EDTA, surfactants, enzymes, dyes, metals and other organic compounds of varied structures that come from the different stages of the global process can be found (Mansilla, H.D., et al., "Treatment of liquid waste from the cellulose and textile industry" in Elimination of Contaminants by Heterogeneous Photocatalysis, CYTED, 2001).

El proceso requiere de una cantidad de agua empleada que varía según sea los procesos específicos y del equipamiento utilizado por la planta. Por ejemplo, en el teñido con colorantes dispersos, se utilizan entre 100 y 150 i litros de agua por kilogramo de producto. En la tinción con colorantes reactivos, entre 125 y 170 litros por kilogramo de producto (EPA Profile of the Textilelndustry, 1997). The process requires a quantity of water that varies depending on the specific processes and the equipment used by the plant. For example, in dyeing with disperse dyes, between 100 and 150 liters of water are used. liters of water per kilogram of product. In dyeing with reactive dyes, between 125 and 170 liters per kilogram of product (EPA Profile of the Textile Industry, 1997).

La mayoría de los colorantes utilizados en la actualidad en la industria textil son de origen sintético, solubles en agua, altamente resistentes a la acción de agentes químicos y poco biodegradables. Aproximadamente el 60% de los colorantes en uso contienen en su estructura grupos azo, antraquinona o ftalocianina altamente reactivos que forman una unión del tipo éter con la fibra (Mansilla, H.D. , et al., "Tratamiento de residuos líquidos de la industria de celulosa y textil" en Eliminación de Contaminantes por Fotocatálisis Heterogénea, CYTED, 2001 ). Most of the dyes currently used in the textile industry are synthetic, water-soluble, highly resistant to chemical agents, and poorly biodegradable. Approximately 60% of the dyes in use contain highly reactive azo, anthraquinone, or phthalocyanine groups in their structure, which form ether-like bonds with the fiber (Mansilla, H.D., et al., "Treatment of liquid waste from the cellulose and textile industry" in Pollutant Removal by Heterogeneous Photocatalysis, CYTED, 2001).

Diversas técnicas fisicoquímicas son utilizadas para la remoción de colorantes sintéticos, como coagulación y floculación combinadas con filtración y flotación, precipitación-floculación con Fe(ll)/Ca(OH)2, oxidación con ozono, filtración por membrana y osmosis inversa, entre otras, las cuales producen efluentes de buena calidad (Fernández, J.A., et al. Rev. Colomb. Biotecnol. 2009), todas estas técnicas suelen ser bastantes costosas. Various physicochemical techniques are used for the removal of synthetic dyes, such as coagulation and flocculation combined with filtration and flotation, precipitation-flocculation with Fe(ll)/Ca(OH) 2 , ozone oxidation, membrane filtration and reverse osmosis, among others, which produce good quality effluents (Fernández, JA, et al. Rev. Colomb. Biotecnol. 2009), all of these techniques are usually quite expensive.

Los esfuerzos para buscar alternativas de tratamiento como la utilización de tratamientos biológicos, pueden ser más económicos ya que utilizan materia prima de bajo costo y más amigables con el ambiente (Moeller, G., Garzón, M. Anuario Imta, 2003). Efforts to seek treatment alternatives such as the use of biological treatments can be more economical since they use low-cost raw materials and are more environmentally friendly (Moeller, G., Garzón, M. Anuario Imta, 2003).

Los microorganismos (m.o) más empleados en la bioremediación de efluentes contaminados son los hongos, los hongos endófitos se caracterizan por su capacidad de degradar lignina y otros componentes de los árboles como la celulosa. Producen enzimas extracelulares que oxidan compuestos fenólicos. Dentro de las enzimas caracterizadas se encuentran manganeso peroxidasas, lignina peroxidasas, enzimas productoras de peróxido y las lacasas. La actividad de estos m.o sobre los colorantes se basa en la no especificidad del sistema enzimático para depolimerizar y mineralizar la lignina (Moeller, G., Garzón, M. Anuario Imta, 2003). The microorganisms (m.o) most commonly used in the bioremediation of contaminated effluents are fungi. Endophytic fungi are characterized by their ability to degrade lignin and other tree components such as cellulose. They produce extracellular enzymes that oxidize phenolic compounds. Among the characterized enzymes are manganese peroxidases, lignin peroxidases, peroxide-producing enzymes, and laccases. The activity of these m.o on dyes is based on the non-specificity of the enzyme system to depolymerize and mineralize lignin (Moeller, G., Garzón, M. Anuario Imta, 2003).

Dada las características de estos m.o. de secretar los complejos enzimáticos con actividad sobre los pigmentos, se realizó un protocolo de inmovilización de los extractos enzimáticos en micro encapsulados de alginato de sodio. La segunda etapa del proceso consiste en poner en contacto las aguas servidas con los microencapsulados. Los hongos se cultivan a temperaturas entre los 20 y 30°C, se utilizan fuentes de nitrógeno, carbono y sales minerales para su crecimiento. Durante el tratamiento, las enzimas pueden ser adicionados a las aguas pretratadas en forma encapsulada. Los polímeros que comprenden dichas matrices están fabricados a partir de materiales biodegradables, naturales y no tóxicos y se eligen del grupo que consiste de alginatos, maltodextrinas, almidón de maíz, sales de kappa carragenina e iota carragenina. Given the characteristics of these mo of secreting the enzymatic complexes with activity on the pigments, a protocol of immobilization of the enzymatic extracts in microencapsulated sodium alginate was carried out. The second stage of the process involves bringing the wastewater into contact with the microencapsulated fungi. The fungi are cultivated at temperatures between 20 and 30°C, using nitrogen, carbon, and mineral salt sources for their growth. During the treatment process, enzymes can be added to the pretreated water in encapsulated form. The polymers comprising these matrices are made from biodegradable, natural, and nontoxic materials and are selected from the group consisting of alginates, maltodextrins, corn starch, kappa carrageenan salts, and iota carrageenan.

La patente W003/035561también señala que es posible utilizar la actividad de enzimas hidrolíticas, enzimas celulolíticas y de enzimas ligninolíticas para conseguir la eliminación de los contaminantes. Patent W003/035561 also points out that it is possible to use the activity of hydrolytic enzymes, cellulolytic enzymes and ligninolytic enzymes to achieve the elimination of contaminants.

Claims

REIVINDICACIONES 1. Un procedimiento para la elaboración de microcápsulas de alginato de sodio de composición homogénea que contienen un extracto enzimatico que incluye en su composición la actividad de una enzima de proteínas extracelulares expresadas por microorganismos que degradan el material lignocelulósico. Dicho procedimiento COMPRENDE: un extracto obtenido de la secreción del hongo, un preparado de sal de alginato disuelto bajo agitación, y la resuspensión, secado y selección de las microcápsulas. El complejo cataliza la oxidación de grupos fenólicos hidroxilo a radicales fenoxy mientras que el oxígeno (02) se reduce a agua. 1. A process for the production of sodium alginate microcapsules of homogeneous composition containing an enzymatic extract that includes in its composition the activity of an enzyme of extracellular proteins expressed by microorganisms that degrade lignocellulosic material. Said process INCLUDES: an extract obtained from the secretion of the fungus, a preparation of alginate salt dissolved under stirring, and the resuspension, drying and selection of the microcapsules. The complex catalyzes the oxidation of phenolic hydroxyl groups to phenoxy radicals while oxygen (O2) is reduced to water. 2. Un procedimiento según reivindicación 1, caracterizado porque para obtener la composición según reivindicación 1, se inicia con el extracto obtenido de la secreción del hongo o los hongos endófitos u otros microorganismos, cuando se ha cultivado en un medio especifico que contiene un colorante textil como fuente de carbono (1). 2. A process according to claim 1, characterized in that to obtain the composition according to claim 1, it begins with the extract obtained from the secretion of the fungus or endophytic fungi or other microorganisms, when it has been cultivated in a specific medium containing a textile dye as a carbon source (1). 3. Un procedimiento según reivindicación 2, caracterizado porque se elabora un preparado de solución de una sal de alginato (alginato sódico) al 1,5%, y disuelto bajo agitación a temperatura ambiente (2), incorporando el extracto de proteínas a una solución de acetato de calcio al 2% (3) 3. A process according to claim 2, characterized in that a solution preparation of an alginate salt (sodium alginate) at 1.5% is prepared, and dissolved under stirring at room temperature (2), incorporating the protein extract into a 2% calcium acetate solution (3). 4. Un procedimiento según reivindicación 3, caracterizado porque con el preparado se resuspenden las microcápsulas formadas y aisladas (4) (5) y son secadas posteriormente durante 30 a 120 minutos (6). 4. A method according to claim 3, characterized in that the formed and isolated microcapsules (4) (5) are resuspended with the preparation and subsequently dried for 30 to 120 minutes (6). 5. Un procedimiento según reivindicación 4, caracterizado porque se selecciona la fracción de microcápsulas de tamaño entre 250 y 1000 micrones (7), descartando las microcápsulas con tamaño inferior a 250 micrones y con tamaño superior a 1000 micrones, separando y guardando las microcápsulas para posterior uso (8). 5. A method according to claim 4, characterized in that the fraction of microcapsules with a size between 250 and 1000 microns (7) is selected, discarding the microcapsules with a size smaller than 250 microns and with a size greater than 1000 microns, separating and storing the microcapsules for later use (8).
PCT/IB2023/061995 2023-11-28 2023-11-28 Method for producing sodium alginate microcapsules of homogeneous composition Pending WO2025114751A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003035561A2 (en) * 2001-09-10 2003-05-01 Universite Catholique De Louvain Sustainable process for the treatment and detoxification of liquid waste
WO2019143526A1 (en) * 2018-01-18 2019-07-25 Pioneer Hi-Bred International, Inc. Alginate encapsulation of fungal microsclerotia

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003035561A2 (en) * 2001-09-10 2003-05-01 Universite Catholique De Louvain Sustainable process for the treatment and detoxification of liquid waste
WO2019143526A1 (en) * 2018-01-18 2019-07-25 Pioneer Hi-Bred International, Inc. Alginate encapsulation of fungal microsclerotia

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DESKA MAłGORZATA, KOńCZAK BEATA: "Laccase Immobilization on Biopolymer Carriers – Preliminary Studies", JOURNAL OF ECOLOGICAL ENGINEERING, vol. 23, no. 4, pages 235 - 249, XP093327447, ISSN: 2299-8993, DOI: 10.12911/22998993/146611 *
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