IT201800010421A1 - BIODEGRADABLE AND BIOCOMPATIBLE COMPOSITE MICRO AND NANOSPHERE THAT GENERATE A MAGNETICALLY SENSITIVE MICROBIAL CATALYST FOR BIOTRANSFORMATION - Google Patents
BIODEGRADABLE AND BIOCOMPATIBLE COMPOSITE MICRO AND NANOSPHERE THAT GENERATE A MAGNETICALLY SENSITIVE MICROBIAL CATALYST FOR BIOTRANSFORMATION Download PDFInfo
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- IT201800010421A1 IT201800010421A1 IT102018000010421A IT201800010421A IT201800010421A1 IT 201800010421 A1 IT201800010421 A1 IT 201800010421A1 IT 102018000010421 A IT102018000010421 A IT 102018000010421A IT 201800010421 A IT201800010421 A IT 201800010421A IT 201800010421 A1 IT201800010421 A1 IT 201800010421A1
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- biocompatible
- biodegradable
- acid
- nanospheres
- microbial
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/20—After-treatment of capsule walls, e.g. hardening
- B01J13/206—Hardening; drying
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L29/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers
- C08L29/02—Homopolymers or copolymers of unsaturated alcohols
- C08L29/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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Description
Descrizione della domanda di brevetto per invenzione industriale dal titolo : micro e nanosfere composite biodegradabili e biocompatibili che generano un catalizzatoremicrobico magneticamente sensibile per la biotrasformazione. Description of the patent application for industrial invention entitled: biodegradable and biocompatible composite micro and nanospheres that generate a magnetically sensitive microbial catalyst for biotransformation.
Campo dell’invenzione Field of the invention
La presente invenzione si riferisce al campo della chimica e in particolare al campo delle nanotecnologie poiché fornisce microsfere e nanosfere composite biodegradabili e biocompatibili generanti catalizzatori microbici magneticamente sensibili per la fermentazione microbica o qualsiasi altra biotrasformazione, il loro processo di produzione, il loro uso per innescare la proliferazione di microrganismi catalitici o di biomassa microbica selezionata per i processi di bioconversione e fermentazioni microbiche o qualsiasi altra biotrasformazione. The present invention refers to the field of chemistry and in particular to the field of nanotechnologies since it provides biodegradable and biocompatible composite microspheres and nanospheres generating magnetically sensitive microbial catalysts for microbial fermentation or any other biotransformation, their production process, their use to trigger the proliferation of catalytic microorganisms or microbial biomass selected for the processes of bioconversion and microbial fermentation or any other biotransformation.
Stato dell’arte State of the art
L'immobilizzazione di celle batteriche è una tecnica comune utilizzata in molte aree delle bioscienze e delle biotecnologie, basata sull'adesione di cellule batteriche a un’area o luogo specifico (Cassidy M, Lee H, Trevors J, Applicazioni ambientali di cellule microbiche immobilizzate: una revisione, J Ind Microbiol 1996, 16, 79-101). Sono note diverse tecniche per l'immobilizzazione delle cellule batteriche, comprendenti l'adsorbimento su superfici (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418; Van Iersel M, Brouwer-Post E, Rombouts F, Abee T, Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer. Enzyme Microb Technol, 2000, 26, 602-607), intrappolamento (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418), l’incapsulamento in gel biopolimerici (Cassidy M, Lee H, Trevors J, Environmental applications of immobilized microbial cells: a review, J Ind Microbiol 1996, 16, 79-101; Galazzo JL, Bailey JE, Growing Saccharomyces cerevisiae in calcium-alginate beads induces cell alterations which accelerate glucose conversion to ethanol, Biotechnol Bioeng, 1990, 36, 417-426; Hilge-Rotmann B, Rehm H-J, Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae, Appl Microbiol Biotechnol, 1991, 34, 502-508, Eun Y-J, Utada A S, Copeland M F, Takeuchi S, Weibel D B, Encapsulating Bacteria in Agarose Microparticles Using Microfluidics for High-Throughput Cell Analysis and Isolation, ACS Chem. Biol. 2011, 6, 260–266, Polakovicˇ M, Sˇvitel J, Bucˇko M, Filip J, Nedeˇla V, Ansorge-Schumacher M B, Gemeiner P, Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications, Biotechnol Lett 2017 39:667–683), e tramite legame covalente/reticolazione con supporti inorganici (Jirku V, Whole cell immobilization as a means of enhancing ethanol tolerance, J Ind Microbiol Biotechnol, 1999, 22147-151; Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418; Zucca P, Sanjust E, Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms, Molecules, 2014, 19, 14139-14194; Zur J, Wojcieszyn ́ska D, Guzik U, Metabolic Responses of Bacterial Cells to Immobilization. Molecules 2016, 21, 958). Tuttavia, le suddette tecniche di immobilizzazione hanno ancora molti svantaggi, come la limitata diffusione del substrato e dei prodotti, specialmente nelle tecniche di intrappolamento e incapsulamento altri svantaggi come la perdita di cellule, la bassa capacità di carico della matrice, perdita di attività e usura del materiale di supporto durante l'immobilizzazione, (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418). Il legame covalente ha lo svantaggio della non specificità delle interazioni e della perdita di vitalità cellulare a causa della citotossicità di alcuni degli agenti utilizzati (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418). Le nanoparticelle metalliche, come quelle di ossido di ferro, sono utilizzate nell'immobilizzazione delle cellule batteriche, grazie alle loro proprietà come responsività a un campo magnetico, l'ampio rapporto superficie-volume, la biocompatibilità e la facile metodologia di separazione (Ranmadugala D, Ebrahiminezhad A, Manley-Harris M, Ghasemi Y, Berenjian A, immobilizzazione magnetica dei batteri mediante nanoparticelle di ossido di ferro, Biotechnol Lett (2018) 40: 237-248) e miglioramento dell’efficienza di ossigenazione nel mezzo di fermentazione (Labbeiki G, Attar H, Heydarinasab A, Sorkhabadi S, Rashidi A, Enhanced oxygen transfer rate and bioprocess yield by using magnetite nanoparticles in fermentation media of erythromycin, DARU Journal of Pharmaceutical Sciences 2014, 22, 66). L'immobilizzazione di celle batteriche su nanoparticelle magneticamente sensibili con adeguate proprietà chimiche di superficie è principalmente utilizza nelle separazioni tramite magnete di cellule batteriche bersaglio da una miscela di batteri in alimenti, campioni ambientali o biologici (Ranmadugala D, Ebrahiminezhad A, Manley-Harris M, Ghasemi Y, Berenjian A, Magnetic immobilization of bacteria using iron oxide nanoparticles, Biotechnol Lett (2018) 40:237–248, Chinese patent n. CN105203577) perché consente la separazione di cellule senza compromettere la vitalità e la riutilizzabilità delle cellule immobilizzate nel sistema, facilitando il processamento a valle (Ebrahiminezhad A, Varma V, Yang S, Berenjian A, Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation, Appl Microbiol Biotechnol, 2016, 100, 173-180). È nota nell’arte, la funzionalizzazione del rivestimento con molecole biologiche, come anticorpi, proteine e ligandi mirati per promuovere specifiche interazioni con i batteri nell'immobilizzazione delle cellule batteriche (Ebrahiminezhad A, Varma V, Yang S, Berenjian A, Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation, Appl Microbiol Biotechnol, 2016, 100, 173-180). È noto che una maggiore adesione delle cellule batteriche alle microparticelle determina un aumento della resa del prodotto e l'efficienza complessiva del processo, facilitando il recupero e la riusabilità delle cellule (Ranmadugala D, Ebrahiminezhad A, Manley-Harris M, Ghasemi Y, Berenjian A, Magnetic immobilization of bacteria using iron oxide nanoparticles, Biotechnol Lett 201) 40:237–248). I copolimeri noti come materiali per il rivestimento di particelle magnetiche sono polimeri naturali, come algar polisaccaridiagar, agarosio, alginato, carragenina, polimeri sintetici come poliacrilammide, polistirene, poliuretano, chitosano, che vengono utilizzati per aumentare l'adesione di cellule batteriche e lieviti (Safarik I, Pospiskova K, Maderova Z, Baldikova E, Horska K, Safarikova M, Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells, Yeast 2015; 32, 239–243). Immobilization of bacterial cells is a common technique used in many areas of bioscience and biotechnology, based on the adhesion of bacterial cells to a specific area or place (Cassidy M, Lee H, Trevors J, Environmental applications of immobilized microbial cells : a review, J Ind Microbiol 1996, 16, 79-101). Several techniques are known for the immobilization of bacterial cells, including adsorption on surfaces (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418; Van Iersel M, Brouwer-Post E, Rombouts F, Abee T, Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer. Enzyme Microb Technol, 2000, 26 , 602-607), entrapment (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418), encapsulation in biopolymer gels (Cassidy M, Lee H, Trevors J, Environmental applications of immobilized microbial cells: a review, J Ind Microbiol 1996, 16, 79-101; Galazzo JL, Bailey JE, Growing Saccharomyces cerevisiae in calcium-al ginate beads induces cell alterations which accelerate glucose conversion to ethanol, Biotechnol Bioeng, 1990, 36, 417-426; Hilge-Rotmann B, Rehm H-J, Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae, Appl Microbiol Biotechnol, 1991, 34, 502-508, Eun Y-J, Utada A S, Copeland M F, Takeuchi S, Weibel D B, Encapsulating Bacteria in Agarose Microparticles Using Microfluidics for High-Throughput Cell Analysis and Isolation, ACS Chem. Biol. 2011, 6, 260–266, Polakovicˇ M, Sˇvitel J, Bucˇko M, Filip J, Nedeˇla V, Ansorge-Schumacher M B, Gemeiner P, Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications, Biotechnol Lett 2017 39: 667–683), and by covalent bonding / crosslinking with inorganic supports (Jirku V, Whole cell immobilization as a means of enhancing ethanol tolerance, J Ind Microbiol Biotechnol, 1999, 22147-151; Martins SCS, Martins CM, Fiu ́Za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418; Zucca P, Sanjust E, Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms, Molecules, 2014, 19, 14139-14194; Zur J, Wojcieszyn ́ska D, Guzik U, Metabolic Responses of Bacterial Cells to Immobilization. Molecules 2016, 21, 958). However, the above immobilization techniques still have many disadvantages, such as limited spread of substrate and products, especially in trapping and encapsulation techniques other disadvantages such as cell loss, low matrix load capacity, loss of activity and wear. of support material during immobilization, (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418 ). Covalent bonding has the disadvantage of non-specificity of interactions and loss of cell viability due to the cytotoxicity of some of the agents used (Martins SCS, Martins CM, Fiu ́za LMCG, Santaella ST 2013 Immobilization of microbial cells: a promising tool for treatment of toxic pollutants in industrial wastewater, Afr J Biotechnol 2013, 12, 4412-4418). Metal nanoparticles, such as iron oxide nanoparticles, are used in the immobilization of bacterial cells, thanks to their properties such as responsiveness to a magnetic field, the large surface-to-volume ratio, biocompatibility and easy separation methodology (Ranmadugala D , Ebrahiminezhad A, Manley-Harris M, Ghasemi Y, Berenjian A, magnetic immobilization of bacteria by iron oxide nanoparticles, Biotechnol Lett (2018) 40: 237-248) and improvement of oxygenation efficiency in the fermentation medium (Labbeiki G, Attar H, Heydarinasab A, Sorkhabadi S, Rashidi A, Enhanced oxygen transfer rate and bioprocess yield by using magnetite nanoparticles in fermentation media of erythromycin, DARU Journal of Pharmaceutical Sciences 2014, 22, 66). Immobilization of bacterial cells on magnetically sensitive nanoparticles with suitable surface chemical properties is mainly used in magnet separations of target bacterial cells from a mixture of bacteria in food, environmental or biological samples (Ranmadugala D, Ebrahiminezhad A, Manley-Harris M , Ghasemi Y, Berenjian A, Magnetic immobilization of bacteria using iron oxide nanoparticles, Biotechnol Lett (2018) 40: 237–248, Chinese patent n. CN105203577) because it allows the separation of cells without compromising the viability and reusability of cells immobilized in system, facilitating downstream processing (Ebrahiminezhad A, Varma V, Yang S, Berenjian A, Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation, Appl Microbiol Biotechnol, 2016, 100, 173-180). It is known in the art, the functionalization of the coating with biological molecules, such as antibodies, proteins and ligands targeted to promote specific interactions with bacteria in the immobilization of bacterial cells (Ebrahiminezhad A, Varma V, Yang S, Berenjian A, Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation, Appl Microbiol Biotechnol, 2016, 100, 173-180). It is known that greater adhesion of bacterial cells to microparticles leads to an increase in product yield and overall process efficiency, facilitating cell recovery and reusability (Ranmadugala D, Ebrahiminezhad A, Manley-Harris M, Ghasemi Y, Berenjian A, Magnetic immobilization of bacteria using iron oxide nanoparticles, Biotechnol Lett 201) 40: 237–248). Copolymers known as magnetic particle coating materials are natural polymers, such as algar polysaccharidiagar, agarose, alginate, carrageenan, synthetic polymers such as polyacrylamide, polystyrene, polyurethane, chitosan, which are used to increase the adhesion of bacterial and yeast cells ( Safarik I, Pospiskova K, Maderova Z, Baldikova E, Horska K, Safarikova M, Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells, Yeast 2015; 32, 239–243).
Problema tecnico Technical problem
Le tecniche utilizzate per l'immobilizzazione di cellule batteriche su supporti presentano inconvenienti quali la diffusione limitata del substrato e dei prodotti, specialmente nelle tecniche di intrappolamento e incapsulamento, alle quali si aggiungono perdita di cellule, la bassa capacità di carico, la disattivazione e l’usura del materiale di supporto durante l'immobilizzazione. Quando è usata la tecnica di immobilizzazione tramite legame covalente i principali svantaggi sono la non- specificità delle interazioni e la perdita di vitalità cellulare dovuta alla citotossicità di alcuni degli agenti utilizzati. The techniques used for the immobilization of bacterial cells on supports have drawbacks such as limited diffusion of the substrate and products, especially in the techniques of entrapment and encapsulation, to which are added cell loss, low load capacity, deactivation and l wear of the support material during immobilization. When the covalent bonding immobilization technique is used, the main disadvantages are the non-specificity of the interactions and the loss of cell viability due to the cytotoxicity of some of the agents used.
L'immobilizzazione dei batteri sulla superficie delle particelle dipende da molti fattori, tra cui le caratteristiche chimicofisiche della superficie delle particelle, le caratteristiche della superficie delle cellule microbiche, come ad esempio la produzione di sostanze polimeriche extracellulari (EPS) che coprono la superficie cellulare, così come le condizioni ambientali e di coltura. I polimeri o copolimeri utilizzati come materiali di rivestimento di particelle magnetiche non sono applicabili per la sintesi su larga scala e non possono essere utili nei processi biotecnologici industriali. The immobilization of bacteria on the particle surface depends on many factors, including the chemical and physical characteristics of the particle surface, the surface characteristics of microbial cells, such as the production of extracellular polymeric substances (EPS) covering the cell surface, as well as the environmental and cultivation conditions. Polymers or copolymers used as magnetic particle coating materials are not applicable for large-scale synthesis and cannot be useful in industrial biotechnological processes.
Uno scopo della presente invenzione è di fornire microsfere e nanosfere biodegradabili e biocompatibili generanti catalizzatori microbici magneticamente sensibili per processi di bioconversione e fermentazioni microbiche o qualsiasi altra biotrasformazione e per sintesi su larga scala. An object of the present invention is to provide biodegradable and biocompatible microspheres and nanospheres generating magnetically sensitive microbial catalysts for bioconversion processes and microbial fermentations or any other biotransformation and for large scale synthesis.
Un altro scopo della presente invenzione è di fornire un metodo per migliorare la resa e il valore della bioconversione di substrati in prodotti chimici o intermedi, ad esempio diminuendo il tempo di purificazione dei prodotti dopo le fermentazioni microbiche o qualsiasi altra biotrasformazione, eliminando il bisogno di filtrazione della biomassa microbica, eliminando la necessità di centrifugazione del brodo di coltura, superando i problemi di composizione mista, superando la variabilità della composizione mista della biomassa microbica da lotto a lotto, superando i problemi legati alla presenza di inibitori nella composizione mista. Another object of the present invention is to provide a method for improving the yield and value of the bioconversion of substrates into chemicals or intermediates, for example by decreasing the purification time of products after microbial fermentations or any other biotransformation, eliminating the need for filtration of microbial biomass, eliminating the need for centrifugation of the culture broth, overcoming the problems of mixed composition, overcoming the variability of the mixed composition of microbial biomass from batch to batch, overcoming the problems related to the presence of inhibitors in the mixed composition.
Il sudetto problema tecnico viene risolto progettando una entità catalitica, ovvero un biocatalizzatore microbico magneticamente sensibile, da utilizzare per le fermentazioni biocatalitiche industriali o qualsiasi altro processo di biotrasformazione, riutilizzabile per più cicli, capace di diminuire i costi dei processi e aumentare la redditività dei processi biocatalitici. The aforementioned technical problem is solved by designing a catalytic entity, that is a magnetically sensitive microbial biocatalyst, to be used for industrial biocatalytic fermentation or any other biotransformation process, reusable for several cycles, capable of decreasing the costs of the processes and increasing the profitability of the processes. biocatalytic.
Il biocatalizzatore microbico magneticamente sensibile è un sistema complesso che manifesta emergenza, quindi presenta funzionalità che le sue parti non avevano quando da sole. Queste caratteristiche non erano in precedenza possedute dai suoi singoli componenti. Per progettare l'entità catalitica, sono stati sintetizzati specifici copolimeri, in considerazione delle caratteristiche specifiche del biocatalizzatore microbico magneticamente sensibile progettato. The magnetically sensitive microbial biocatalyst is a complex system that manifests emergence, therefore it presents features that its parts did not have when alone. These characteristics were not previously possessed by its individual components. To design the catalytic entity, specific copolymers were synthesized, taking into account the specific characteristics of the designed magnetically sensitive microbial biocatalyst.
Oggetto dell’invenzione Object of the invention
Oggetto della presente invenzione sono microsfere e nanosfere composite, biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biocompatibile e biodegradabile e/o almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile, e un nucleo interno magneticamente sensibile. Object of the present invention are composite, biodegradable and biocompatible microspheres and nanospheres comprising an outer layer consisting of at least one biocompatible and biodegradable hydrophilic polymer and / or at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible metalloid, and an inner core magnetically sensitive.
Un ulteriore oggetto della presente invenzione è un processo per la preparazione delle microsfere e delle nanosfere composite magnetiche biodegradabili e biocompatibili mediante atomizzazione (sprydrying), coacervazione, coprecipitazione e assorbimento. A further object of the present invention is a process for the preparation of biodegradable and biocompatible magnetic composite microspheres and nanospheres by atomization (sprydrying), coacervation, coprecipitation and absorption.
Un ulteriore oggetto della presente invenzione è l'uso di dette microsfere e nanosfere composite biodegradabili e biocompatibili come supporto magneticamente sensibile per la proliferazione di biomasse microbiche sulla loro superficie esterna. A further object of the present invention is the use of said biodegradable and biocompatible composite microspheres and nanospheres as magnetically sensitive support for the proliferation of microbial biomass on their external surface.
Un ulteriore oggetto della presente invenzione è l'uso di dette microsfere e nanosfere composite biodegradabili e biocompatibili come supporto magneticamente sensibile per aumentare l’attività biocatalitica del film microbico risultante dalla proliferazione di biomasse microbiche sulla loro superficie esterna. A further object of the present invention is the use of said biodegradable and biocompatible composite microspheres and nanospheres as a magnetically sensitive support to increase the biocatalytic activity of the microbial film resulting from the proliferation of microbial biomass on their external surface.
Un ulteriore oggetto della presente invenzione sono microsfere e nanosfere composite biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biodegradabile e biocompatibile e/o almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile e nucleo interno magneticamente sensibile, rivestite con un film microbico. A further object of the present invention are biodegradable and biocompatible composite microspheres and nanospheres comprising an outer layer consisting of at least one biodegradable and biocompatible hydrophilic polymer and / or at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible nonmetal and magnetically inner core sensitive, coated with a microbial film.
Un ulteriore oggetto della presente invenzione è l'uso di microsfere e nanosfere composite biodegradabili e biocompatibili rivestite con un film microbico come biocatalizzatore microbico magneticamente sensibile. A further object of the present invention is the use of biodegradable and biocompatible composite microspheres and nanospheres coated with a microbial film as a magnetically sensitive microbial biocatalyst.
Un ulteriore oggetto della presente invenzione è una formulazione comprendente almeno un polimero idrofilo biodegradabile e biocompatibile, e facoltativamente comprendente almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile, che costituisce lo strato esterno biodegradabile e biocompatibile delle microsfere e nanosfere composite magneticamente sensibili. A further object of the present invention is a formulation comprising at least one biodegradable and biocompatible hydrophilic polymer, and optionally comprising at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible nonmetal, which constitutes the biodegradable and biocompatible outer layer of the microspheres and nanospheres magnetically sensitive composite.
Un ulteriore oggetto della presente invenzione è un fornire un processo per la sintesi di un copolimero idrofilo biodegradabile e biocompatibile comprendente i seguenti stadi: A further object of the present invention is to provide a process for the synthesis of a biodegradable and biocompatible hydrophilic copolymer comprising the following steps:
a) attivazione di gruppi reattivi di almeno un polimero di partenza aggiungendo un agente attivante in un solvente adatto, a) activation of reactive groups of at least one starting polymer by adding an activating agent in a suitable solvent,
b) reazione del polimero attivato come ottenuto nello stadio a) con almeno un metalloide biocompatibile in cui il rapporto molare tra moli di metalloide e moli di unità ripetitive di polimero attivato è compreso tra 0,01 e 1, per ottenere il copolimero biodegradabile e biocompatibile come prodotto finale. b) reaction of the activated polymer as obtained in step a) with at least one biocompatible nonmetal in which the molar ratio between moles of non-metal and moles of repetitive units of activated polymer is between 0.01 and 1, to obtain the biodegradable and biocompatible copolymer as a final product.
Ulteriori caratteristiche della presente invenzione saranno evidenti nella seguente descrizione dettagliata con riferimento ai risultati sperimentali. Further features of the present invention will be evident in the following detailed description with reference to the experimental results.
Descrizione dettagliata dell’invenzione Detailed description of the invention
Oggetto della presente invenzione sono microsfere e nanosfere composite, biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biocompatibile e/o almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile, e un nucleo interno magneticamente sensibile. Object of the present invention are composite, biodegradable and biocompatible microspheres and nanospheres comprising an outer layer consisting of at least one biocompatible hydrophilic polymer and / or at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible metalloid, and a magnetically sensitive inner core .
Un ulteriore oggetto della presente invenzione sono microsfere e nanosfere composite, biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biocompatibile e/o almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile, e un nucleo interno magneticamente sensibile, rivestite con una film microbico. A further object of the present invention are composite, biodegradable and biocompatible microspheres and nanospheres comprising an outer layer consisting of at least one biocompatible hydrophilic polymer and / or at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible metalloid, and an inner core magnetically sensitive, coated with a microbial film.
Preferibilmente, il polimero idrofilo biodegradabile e biocompatibile è scelto dal gruppo costituito da: poli (alcool vinilico) (PVA), polisaccaridi, chitina, chitosano, poliammidi. Preferably, the biodegradable and biocompatible hydrophilic polymer is selected from the group consisting of: poly (vinyl alcohol) (PVA), polysaccharides, chitin, chitosan, polyamides.
Preferibilmente, i polisaccaridi sono scelti dal gruppo costituito da: cellulosa e derivati della cellulosa , maltodestrine, amidi, acido ialuronico, inulina. Preferably, the polysaccharides are selected from the group consisting of: cellulose and cellulose derivatives, maltodextrins, starches, hyaluronic acid, inulin.
Preferibilmente, le poliammidi sono scelte dal gruppo costituito da: poli-idrossietil-aspartamide, poliamminoacidi. Preferably, the polyamides are selected from the group consisting of: poly-hydroxyethyl-aspartamide, polyamino acids.
Più preferibilmente il polimero biocompatibile è PVA o poli (N-2-idrossietil)-D,L-aspartammide (PHEA). More preferably, the biocompatible polymer is PVA or poly (N-2-hydroxyethyl) -D, L-aspartamide (PHEA).
Preferibilmente il copolimero idrofilo biodegradabile e biocompatibile è un copolimero innestato (grafted). Preferably the biodegradable and biocompatible hydrophilic copolymer is a grafted copolymer.
I metalloidi biocompatibili sono in grado di legare e rilasciare ossigeno, di agire come nutrienti, di aumentare la vitalità e l'attività dei biocatalizzatori microbici di generare proprietà addizionali come la sensibilità al pH, sono capaci di generare mucoadesione, di generare auto-interazione con il biofilm microbico o la biomassa, di generare elettrofilatura. Biocompatible metalloids are able to bind and release oxygen, to act as nutrients, to increase the vitality and activity of microbial biocatalysts, to generate additional properties such as pH sensitivity, are able to generate mucoadhesion, to generate self-interaction with microbial biofilm or biomass, to generate electrospinning.
Preferibilmente il metalloide biocompatibile è scelto dal gruppo costituito da acido 3-amminofenilboronico, acido 4-amminofenilboronico, acido 3-carbossifenilboronico, acido 4-carbossifenilboronico, acido 3-idrossifenilboronico, acido 4-idrossifenilboronico, acido 3-(idrossimetil) fenilboronico, acido 4-(Idrossimetil) fenilboronico, acido 3-(idrossietil) fenilboronico, acido 4-(idrossietil) fenilboronico, acido 3-(idrossipropil) fenilboronico, 4-(idrossipropil) acido fenilboronico, acido 3-formilfenilboronico, acido 4-formilfenilboronico, acido 3-vinilfenilboronico. Preferably the biocompatible nonmetal is selected from the group consisting of 3-aminophenylboronic acid, 4-aminophenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, 3-hydroxyphenylboronic acid, 4-hydroxyphenylboronic acid, 3- (hydroxymethyl) phenylboronic acid, 4- (hydroxymethyl) phenylboronic acid - (Hydroxymethyl) phenylboronic acid, 3- (hydroxyethyl) phenylboronic acid, 4- (hydroxyethyl) phenylboronic acid, 3- (hydroxypropyl) phenylboronic acid, 4- (hydroxypropyl) phenylboronic acid, 3-formylphenylboronic acid, 4-formylphenylboronic acid, 3-acid -vinylphenylboronic.
Preferibilmente il copolimero idrofilo biodegradabile e biocompatibile può avere una struttura reticolata che racchiude chimicamente il metalloide biocompatibile. Preferably the biodegradable and biocompatible hydrophilic copolymer can have a crosslinked structure which chemically encloses the biocompatible nonmetal.
I metalloidi biocompatibili possono aumentare la dinamicità delle proprietà supramolecolari del materiale. Biocompatible metalloids can increase the dynamism of the supramolecular properties of the material.
Più preferibilmente il copolimero idrofilo biodegradabile e biocompatibile è l'acido 4-carbossifenilboronico (CPBA) legato ai gruppi ossidrile del PHEA o del PVA di partenza. More preferably, the biodegradable and biocompatible hydrophilic copolymer is 4-carboxyphenylboronic acid (CPBA) bound to the hydroxyl groups of the starting PHEA or PVA.
Preferibilmente, il nucleo interno magneticamente sensibile è costituito da ossidi di ferro o magnetite. Preferably, the magnetically sensitive inner core consists of iron oxides or magnetite.
La preparazione del copolimero idrofilo biodegradabile e biocompatibile può essere eseguita mediante una reazione di coniugazione, reazioni di condensazione e accoppiamento di ammina che consente il legame tra i sostituenti nucleofili ed elettrofili dei polimeri di partenza e almeno un acido boronico selezionato dal gruppo costituito da: Acido 3-amminofenilboronico, acido 4-amminofenilboronico, acido 3carbossifenilboronico, acido 4-carbossifenilboronico, acido 3-idrossifenilboronico, acido 4-idrossifenilboronico, acido 3-(idrossimetil) fenilboronico, acido 4-(idrossimetil) fenilboronico, acido 3-(idrossietile ) fenilboronico, acido 4-(idrossietil) fenilboronico, acido 3-(idrossipropil) fenilboronico, acido 4-(idrossipropil) fenilboronico, acido 3-formilfenilboronico, Acido 4-formilfenilboronico, acido 3-vinilfenilboronico, acido 4-vinilfenilboronico. The preparation of the biodegradable and biocompatible hydrophilic copolymer can be carried out by means of a conjugation reaction, condensation reactions and amine coupling that allows the bond between the nucleophilic and electrophilic substituents of the starting polymers and at least one boronic acid selected from the group consisting of: Acid 3-aminophenylboronic, 4-aminophenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, 3-hydroxyphenylboronic acid, 4-hydroxyphenylboronic acid, 3- (hydroxymethyl) phenylboronic acid, 4- (hydroxymethyl) phenylboronic acid, phenylboronic (3- hydroxymethyl) phenylboronic acid , 4- (hydroxyethyl) phenylboronic acid, 3- (hydroxypropyl) phenylboronic acid, 4- (hydroxypropyl) phenylboronic acid, 3-formylphenylboronic acid, 4-formylphenylboronic acid, 3-vinylphenylboronic acid, 4-vinylphenylboronic acid.
Preferibilmente le microsfere e le nanosfere composite magnetiche biodegradabili e biocompatibili hanno una dimensione compresa tra 0.1 e 1000 micron. Preferably, the biodegradable and biocompatible magnetic composite microspheres and nanospheres have a size between 0.1 and 1000 microns.
Un ulteriore oggetto della presente invenzione è una formulazione comprendente almeno un polimero idrofilo biodegradabile biocompatibile, e facoltativamente comprendente almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile, che costituisce lo strato esterno delle microsfere e nanosfere magnetiche composite biodegradabile e biocompatibili. A further object of the present invention is a formulation comprising at least one biocompatible biodegradable hydrophilic polymer, and optionally comprising at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible nonmetal, which constitutes the outer layer of the biodegradable composite magnetic microspheres and nanospheres and biocompatible.
Un ulteriore oggetto della presente invenzione è un processo per la sintesi di un copolimero idrofilo biocompatibile e biodegradabile comprendente i seguenti stadi: A further object of the present invention is a process for the synthesis of a biocompatible and biodegradable hydrophilic copolymer comprising the following steps:
a) attivazione di gruppi reattivi di almeno un polimero di partenza aggiungendo agenti attivanti in un solvente adatto, a) activation of reactive groups of at least one starting polymer by adding activating agents in a suitable solvent,
b) reazione del polimero attivato come ottenuto nello stadio a) con un metalloide biocompatibile in cui il rapporto molare tra moli di metalloide e moli di unità ripetitive di polimero attivato è compreso tra 0,01 e 1, per ottenere il copolimero idrofilo biocompatibile biodegradabile come prodotto finale. b) reaction of the activated polymer as obtained in step a) with a biocompatible nonmetal in which the molar ratio between moles of nonmetal and moles of repetitive units of activated polymer is between 0.01 and 1, to obtain the biocompatible biodegradable hydrophilic copolymer such as final product.
Preferibilmente nello stadio a) il polimero di partenza è scelto dal gruppo costituito da poli (vinil alcol) (PVA), polisaccaridi (cellulosa, derivati della cellulosa, maltodestrine, amidi, acido ialuronico, inulina), chitina, chitosano, poliammidi, poli (N-2-idrossietil)-D,L-aspartammide (PHEA). Preferably in step a) the starting polymer is selected from the group consisting of poly (vinyl alcohol) (PVA), polysaccharides (cellulose, cellulose derivatives, maltodextrins, starches, hyaluronic acid, inulin), chitin, chitosan, polyamides, poly ( N-2-hydroxyethyl) -D, L-aspartamide (PHEA).
Più preferibilmente nello stadio a) il polimero di partenza è PVA o poli (N-2-idrossietile)-D,L-aspartammide (PHEA). More preferably in step a) the starting polymer is PVA or poly (N-2-hydroxyethyl) -D, L-aspartamide (PHEA).
Preferibilmente nello stadio a) l'agente attivante è selezionato dal gruppo costituito da (4-nitrofenil)carbonato (4-NPBC), N-idrossi-succinimmide (NHS), N-3(3-dimetilamminopropil) -N-etilcarbodiimmide idrocloruro (EDC-HCl), N,N'-Dicyclohexylcarbodiimide (DCC), 4-(Dimethylamino)piridina (DMAP). Preferably in step a) the activating agent is selected from the group consisting of (4-nitrophenyl) carbonate (4-NPBC), N-hydroxy-succinimide (NHS), N-3 (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride ( EDC-HCl), N, N'-Dicyclohexylcarbodiimide (DCC), 4- (Dimethylamino) pyridine (DMAP).
Preferibilmente nello stadio a) il solvente adatto è scelto dal gruppo costituito da dimetilformammide (DMF), acqua. Preferably in step a) the suitable solvent is selected from the group consisting of dimethylformamide (DMF), water.
Più preferibilmente, nello stadio a) l'agente attivante è DCC e DMAP in soluzione DMF, o NHS ed EDC-HCl in soluzione acquosa o 4-NPBC in soluzione DMF anidra. More preferably, in step a) the activating agent is DCC and DMAP in DMF solution, or NHS and EDC-HCl in aqueous solution or 4-NPBC in anhydrous DMF solution.
Preferibilmente nello stadio a) la reazione viene condotta a temperatura costante, compresa tra 25 e 60 °C. Preferably in step a) the reaction is carried out at a constant temperature, between 25 and 60 ° C.
Preferibilmente nello stadio a) il peso molecolare dei polimeri di partenza è compreso tra 1 e 1000 kDa. Preferably in step a) the molecular weight of the starting polymers is between 1 and 1000 kDa.
Preferibilmente nello stadio b) il metalloide biocompatibile è scelto dal gruppo costituito da acido 3-amminofenilboronico, acido 4-amminofenilboronico, acido 3-carbossifenilboronico, acido 4-carbossifenilboronico, acido 3-idrossifenilboronico, acido 4-idrossifenilboronico, 3-(idrossimetil) acido fenilboronico, 4-(idrossimetil) acido fenilboronico, 3-(Acido idrossietil) fenilboronico, acido 4-(idrossietil) fenilboronico, acido 3- (idrossipropil) fenilboronico, acido 4-(idrossipropil) fenilboronico, acido 3-formilfenilboronico, acido 4-formilfenilboronico, acido 3-vinilfenilboronico. Preferably in step b) the biocompatible nonmetal is selected from the group consisting of 3-aminophenylboronic acid, 4-aminophenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, 3-hydroxyphenylboronic acid, 4-hydroxyphenylboronic acid, 3- (hydroxymethyl) acid phenylboronic, 4- (hydroxymethyl) phenylboronic acid, 3- (hydroxyethyl) phenylboronic acid, 4- (hydroxyethyl) phenylboronic acid, 3- (hydroxypropyl) phenylboronic acid, 4- (hydroxypropyl) phenylboronic acid, 3-formylphenylboronic acid, 4- formylphenylboronic, 3-vinylphenylboronic acid.
Più preferibilmente nello stadio b) il metalloide biocompatibile è acido 4-carbossifenilboronico (CPBA), acido 4-amminofenilboronico o acido 4-(idrossimetil)fenilboronico. More preferably in step b) the biocompatible nonmetal is 4-carboxyphenylboronic acid (CPBA), 4-aminophenylboronic acid or 4- (hydroxymethyl) phenylboronic acid.
Preferibilmente nello stadio b) la reazione viene condotta ad una temperatura compresa tra 15 e 60 ° C. Preferably in step b) the reaction is carried out at a temperature between 15 and 60 ° C.
Preferibilmente il peso molecolare dei copolimeri ottenuti dal processo alla fine dello stadiob) è compreso tra 1 e 1000 kDa. Preferably the molecular weight of the copolymers obtained from the process at the end of step b) is between 1 and 1000 kDa.
Più preferibilmente i copolimeri ottenuti dal processo alla fine dello stadio b) è l'acido 4-carbossifenilboronico (CPBA) legato a gruppi ossidrile del PHEA o PVA di partenza. More preferably, the copolymers obtained from the process at the end of step b) are 4-carboxyphenylboronic acid (CPBA) linked to the hydroxyl groups of the starting PHEA or PVA.
Un ulteriore oggetto della presente invenzione è un processo per la preparazione delle microsfere e delle nanosfere composite magnetiche, biodegradabili e biocompatibili, mediante atomizzazione (sprydrying) nonché coacervazione, coprecipitazione e assorbimento. A further object of the present invention is a process for the preparation of magnetic, biodegradable and biocompatible composite microspheres and nanospheres, by means of atomization (sprydrying) as well as coacervation, coprecipitation and absorption.
Preferibilmente le microsfere e le nanosfere composite magnetiche, biodegradabili e biocompatibili sono prodotte mediante atomizzazione (sprydrying) della formulazione comprendente almeno un polimero idrofilo biocompatibile biodegradabile e facoltativamente comprendente almeno un copolimero di almeno un polimero idrofilo biodegradabile e biocompatibile con almeno un metalloide biocompatibile sul nucleo interno magneticamente sensibile, per ottenere lo strato esterno delle microsfere e delle nanosfere magnetiche composite biodegradabili e biocompatibili. Preferably the magnetic, biodegradable and biocompatible composite microspheres and nanospheres are produced by atomization (sprydrying) of the formulation comprising at least one biodegradable biocompatible hydrophilic polymer and optionally comprising at least one copolymer of at least one biodegradable and biocompatible hydrophilic polymer with at least one biocompatible metalloid on the inner core magnetically sensitive, to obtain the outer layer of the biodegradable and biocompatible composite magnetic microspheres and nanospheres.
Preferibilmente, il processo per la preparazione delle microsfere e delle nanosfere magnetiche composite, biodegradabili e biocompatibili comprende disperdere in un solvente mediante agitazione almeno un polimero idrofilo biocompatibile e biodegradabile e/o almeno un copolimero di almeno un copolimero biocompatibile idrofilo biodegradabile con almeno un metalloide biocompatibile e aggiungendo alla dispersione ottenuta di ossidi di ferro o magnetite in polvere. Preferably, the process for the preparation of the biodegradable and biocompatible composite magnetic microspheres and nanospheres comprises dispersing in a solvent by stirring at least one biocompatible and biodegradable hydrophilic polymer and / or at least one copolymer of at least one biocompatible hydrophilic biocompatible copolymer with at least one biocompatible metalloid and adding to the obtained dispersion of powdered iron oxides or magnetite.
Preferibilmente la dispersione è un polimero o copolimero e maltodestrina (rapporto ponderale compreso tra 1: 1 e 1:100 a 100:1). Preferably the dispersion is a polymer or copolymer and maltodextrin (weight ratio between 1: 1 and 1: 100 to 100: 1).
Preferibilmente il solvente è acqua. Preferably the solvent is water.
Preferibilmente gli ossidi di ferro o di magnetite hanno una dimensione delle particelle compresa tra 0,01 e 1000 micron. Preferably, the iron or magnetite oxides have a particle size of between 0.01 and 1000 microns.
La resa del processo è compresa tra il 20 e il 100%. The yield of the process is between 20 and 100%.
Un ulteriore oggetto della presente invenzione è l'uso delle microsfere e nanosfere magnetiche composite, biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biocompatibile biodegradabile e/o almeno un copolimero di almeno un metalloide biocompatibile e un nucleo interno magneticamente sensibile come supporto per la proliferazione di biomasse microbiche sulla loro superficie esterna. A further object of the present invention is the use of composite, biodegradable and biocompatible magnetic microspheres and nanospheres comprising an outer layer consisting of at least one biodegradable biocompatible hydrophilic polymer and / or at least one copolymer of at least one biocompatible nonmetal and a magnetically sensitive inner core such as support for the proliferation of microbial biomass on their external surface.
Con biomasse microbiche si intende, nell’ambito della presente invenzione, microrganismi eucariotici e procariotici. By microbial biomass we mean, in the context of the present invention, eukaryotic and prokaryotic microorganisms.
Un ulteriore oggetto della presente invenzione è l'uso di microsfere e nanosfere composite magnetiche, biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biocompatibile e biodegradable e/o almeno un copolimero di almeno un polimero idrofilo biocompatibile e biodegradabile con almeno uno metalloide biocompatibile e un nucleo interno magneticamente sensibile come supporto magneticamente sensibile per aumentare l'attività biocatalitica del film microbico risultante sulla loro superficie esterna. A further object of the present invention is the use of magnetic, biodegradable and biocompatible composite microspheres and nanospheres comprising an outer layer consisting of at least one biocompatible and biodegradable hydrophilic polymer and / or at least one copolymer of at least one biocompatible and biodegradable hydrophilic polymer with at least one biocompatible nonmetal and a magnetically sensitive inner core as a magnetically sensitive support to increase the biocatalytic activity of the resulting microbial film on their outer surface.
Il film microbico è formato dalle biomasse microbiche di microrganismi eucariotici e procarioti. The microbial film is formed from the microbial biomass of eukaryotic and prokaryotic microorganisms.
Un ulteriore oggetto della presente invenzione è l'uso di microsfere e nanosfere composite magnetiche, biodegradabili e biocompatibili comprendenti uno strato esterno costituito da almeno un polimero idrofilo biodegradabile e biocompatibile e/o almeno un copolimero di almeno un polimero biocompatibile biodegradabile con almeno un metalloide biocompatibile e un nucleo interno magneticamente sensibile rivestite con un film microbico come biocatalizzatore microbico magneticamente sensibile. A further object of the present invention is the use of magnetic, biodegradable and biocompatible composite microspheres and nanospheres comprising an outer layer consisting of at least one biodegradable and biocompatible hydrophilic polymer and / or at least one copolymer of at least one biodegradable biocompatible polymer with at least one biocompatible metalloid and a magnetically sensitive inner core coated with a microbial film as a magnetically sensitive microbial biocatalyst.
In cui, nell’ambito della presente invenzione, per magneticamente sensibile si intende essere sensibile a tutti i tipi di magnetismo che vanno dai magneti permanenti agli elettromagneti. In which, in the context of the present invention, by magnetically sensitive is meant to be sensitive to all types of magnetism ranging from permanent magnets to electromagnets.
In cui nell’ambito della presente invenzione, per sensibilità magnetica del nucleo interno si intende che è superparamagnetico, paramagnetico, ferromagnetico e ferrimagnetico. In which in the context of the present invention, the magnetic sensitivity of the inner core means that it is superparamagnetic, paramagnetic, ferromagnetic and ferrimagnetic.
In cui, nell’ambito della presente invenzione, per biocatalizzatore microbico si intende migliorare la resa e il valore della conversione di substrati in prodotti chimici o intermedi, ad esempio diminuendo il tempo di purificazione dopo le fermentazioni microbiche o qualsiasi altra biotrasformazione, eliminando il bisogno di filtrazione della biomassa microbica, eliminando la necessità di centrifugazione del brodo di coltura, superando i problemi di composizione mista, superando la variabilità della composizione mista della biomassa microbica da lotto a lotto, superando i problemi legati alla presenza di inibitori nel composizione mista. In which, in the context of the present invention, by microbial biocatalyst it is intended to improve the yield and the value of the conversion of substrates into chemical or intermediate products, for example by decreasing the purification time after microbial fermentations or any other biotransformation, eliminating the need filtration of microbial biomass, eliminating the need for centrifugation of the culture broth, overcoming the problems of mixed composition, overcoming the variability of the mixed composition of microbial biomass from batch to batch, overcoming the problems related to the presence of inhibitors in the mixed composition.
Il biocatalizzatore microbico magneticamente sensibile è utilizzato nei processi di biotrasformazione microbica. The magnetically sensitive microbial biocatalyst is used in microbial biotransformation processes.
In cui, nell’ambito della presente invenzione, per processi di biotrasformazione si intende metabolismo eterotrofico. In which, in the context of the present invention, by biotransformation processes is meant heterotrophic metabolism.
In cui, nell’ambito della presente invenzione, metabolismo eterotrofo comprende la respirazione, che è un metabolismo eterotrofo che utilizza ossigeno, la fermentazione, che è un metabolismo eterotrofo in cui un composto organico piuttosto che l'ossigeno è l'accettore finale di elettroni (o idrogeno), il ciclo di Krebs che mediante piruvato è un processo ossidativo nella respirazione, il ciclo del piruvato, del gliossalato che come metabolismo eterotrofico è una modifica del ciclo di Krebs, comprendendo anche trasporto di elettroni, fosforilazione ossidativa, ipotesi di estrusione di protone, fotosintesi, autotrofia, respirazione anaerobica, ciclo dell'azoto. Wherein, within the scope of the present invention, heterotrophic metabolism includes respiration, which is heterotrophic metabolism using oxygen, fermentation, which is heterotrophic metabolism in which an organic compound rather than oxygen is the final electron acceptor (or hydrogen), the Krebs cycle which by means of pyruvate is an oxidative process in respiration, the pyruvate cycle, of glyoxalate which as heterotrophic metabolism is a modification of the Krebs cycle, also including transport of electrons, oxidative phosphorylation, hypothesis of extrusion of proton, photosynthesis, autotrophy, anaerobic respiration, nitrogen cycle.
In una forma di realizzazione preferita le microsfere e le nanosfere composite biodegradabili e biocompatibili sono utilizzate per lo screening di fenotipi di biocatalizzatori microbici candidati alla conversione di uno specifico lotto di substrato. In dettaglio, ogni fenotipo di biocatalizzatori microbici è coltivato su microsfere e nanosfere composite biodegradabili e biocompatibili e facilmente separato dal brodo di coltura con un magnete, sia magnete permanente esterno o una barra magnetica o un elettromagnete, le microsfere e le nanosfere composite biodegradabili e biocompatibili generanti un biocatalizzatore microbico magneticamente sensibile per la fermentazione microbica vengono utilizzate per valutare rapidamente quanto sia l'efficacia di un fenotipo per eseguire il bioprocesso designato, sia per prodotti bersaglio (target) esterni che internalizzati, la mancanza di centrifugazione e/o filtrazione consente di sviluppare facilmente fenotipi multipli in parallelo per valutare rapidamente quale tra i fenotipi testati è il più efficace come biocatalizzatore microbico. In a preferred embodiment the biodegradable and biocompatible composite microspheres and nanospheres are used for screening phenotypes of microbial biocatalysts candidates for the conversion of a specific substrate lot. In detail, each phenotype of microbial biocatalysts is grown on biodegradable and biocompatible composite microspheres and nanospheres and easily separated from the culture broth with a magnet, either external permanent magnet or magnetic rod or electromagnet, the biodegradable and biocompatible composite microspheres and nanospheres generating a magnetically sensitive microbial biocatalyst for microbial fermentation are used to quickly assess how effective a phenotype is to perform the designated bioprocess, for both external and internalized target products, the lack of centrifugation and / or filtration allows for Easily develop multiple phenotypes in parallel to quickly assess which of the tested phenotypes is the most effective as a microbial biocatalyst.
In una forma di realizzazione preferita il biocatalizzatore microbico magneticamente sensibile è essere utilizzato in bioprocessi multiciclici, , che sono biocatalisi o fermentazioni o qualsiasi altra biotrasformazione, può essre riutilizzato per diversi cicli biocatalitici. Una volta che il substrato nel brodo di coltura è consumato dal catalizzatore microbico e trasformato nel prodotto bersaglio (target), il catalizzatore microbico che forma un film microbico catalitico sulla superficie delle microsfere e delle nanosfere composite biodegradabili e biocompatibili può essere separato dal brodo di coltura per mezzo di un magnete. I biocatalizzatori microbici magneticamente sensibili una volta separati dal brodo di coltura vecchio ed esaurito possono essere esposti ad un nuovo e fresco brodo di coltura per innescare un nuovo ciclo di fermentazione o qualsiasi altro processo di biotrasformazione. In a preferred embodiment the magnetically sensitive microbial biocatalyst is used in multicyclic bioprocesses, which are biocatalysis or fermentation or any other biotransformation, can be reused for different biocatalytic cycles. Once the substrate in the culture broth is consumed by the microbial catalyst and transformed into the target product, the microbial catalyst that forms a catalytic microbial film on the surface of the biodegradable and biocompatible composite microspheres and nanospheres can be separated from the culture broth. by means of a magnet. Magnetically sensitive microbial biocatalysts once separated from the old and depleted culture broth can be exposed to a fresh new culture broth to trigger a new fermentation cycle or any other biotransformation process.
In una ulteriore forma di realizzazione, in processi a più stadi o più fasi, nel primo stadio uno specifico fenotipo microbico proliferante sulla superficie delle microsfere e delle nanosfere composite biodegradabili e biocompatibili trasformerà il substrato, modificando così la composizione del brodo di coltura; poi, il brodo di coltura viene chiarificato per mezzo di un magnete, separando il biocatalizzatore microbico magneticamente sensibile dai loro prodotti; un secondo fenotipo ottimizzato per la nuova composizione del brodo di coltura e che prolifera sulla superficie di nuove microsfere e nanosfere composite biodegradabili e biocompatibili, è introdotto nel bioreattore e può completare il secondo stadio della biocatalisi, trasformando i prodotti del primo stadio nel prodotto finale. Le microsfere e le nanosfere composite biodegradabili e biocompatibili possono consentire un processo biocatalitico multi-stadio, infatti ogni volta che la biomassa converte il substrato in una entità chimica, un nuovo microorganismo responsabile dell’ulteriore stadio della trasformazione può essere introdotto nel bioprocesso. Le interazioni possono essere ripetute per tante volte quanto sono necessarie per ottenere il prodotto bersaglio (target). In ogni stadio operazioni che consumano tempo e risorse come la centrifugazione e la filtrazione sono state evitate grazie alla responsività magnetica delle microsfere e nanosfere composite biodegradabili e biocompatibili. In a further embodiment, in multi-stage or multi-stage processes, in the first stage a specific microbial phenotype proliferating on the surface of the biodegradable and biocompatible composite microspheres and nanospheres will transform the substrate, thus modifying the composition of the culture broth; then, the culture broth is clarified by means of a magnet, separating the magnetically sensitive microbial biocatalyst from their products; a second phenotype optimized for the new composition of the culture broth and which proliferates on the surface of new biodegradable and biocompatible composite microspheres and nanospheres, is introduced into the bioreactor and can complete the second stage of biocatalysis, transforming the products of the first stage into the final product. The biodegradable and biocompatible composite microspheres and nanospheres can allow a multi-stage biocatalytic process, in fact every time the biomass converts the substrate into a chemical entity, a new microorganism responsible for the further stage of transformation can be introduced into the bioprocess. The interactions can be repeated for as many times as necessary to obtain the target product. At each stage, time-consuming and resource-consuming operations such as centrifugation and filtration were avoided thanks to the magnetic responsiveness of the biodegradable and biocompatible composite microspheres and nanospheres.
Esempi Examples
Esempio 1: preparazione di copolimeri contenenti acidi boronici Example 1: preparation of copolymers containing boronic acids
Gli agenti attivanti consistono in N,N'-dicicloesilcarbodiimmide (DCC) e 4-(dimetilammino)piridina (DMAP) in soluzione di dimetilformammide (DMF), il metalloide è costituito da acido 4-carbossifenilboronico (CPBA) e la reazione viene condotta a 30°C, per 24 ore. Activating agents consist of N, N'-dicyclohexylcarbodiimide (DCC) and 4- (dimethylamino) pyridine (DMAP) in dimethylformamide solution (DMF), the nonmetal consists of 4-carboxyphenylboronic acid (CPBA) and the reaction is carried out at 30 ° C, for 24 hours.
Il peso molecolare dei polimeri di partenza era di 80kDa e dei copolimeri finali di 200kDa. The molecular weight of the starting polymers was 80kDa and of the final copolymers 200kDa.
L'acido 4-carbossifenilboronico (CPBA) è legato a gruppi idrossile del PHEA o PVA di partenza. Gli schemi di reazione sono qui riportati. 4-carboxyphenylboronic acid (CPBA) is bound to hydroxyl groups of the starting PHEA or PVA. The reaction schemes are shown here.
Esempio 2: preparazione delle microsfere e nanosfere biodegradabili e biocompatibili magneticamente sensibili Example 2: preparation of the biodegradable and biocompatible magnetically sensitive microspheres and nanospheres
Il metodo di produzione utilizzato è l’atomizzazione (sprydrying): 1g di PVA (Mw di 80kDa) e maltodestrina (rapporto in peso 1:1) sono stati dispersi in 20ml di acqua distillata mediante agitazione magnetica per 24 ore e bagno ad ultrasuoni per 1 ora a 40°C. A questa dispersione, è stato aggiunto 1g di polvere di ossido di ferro (con una dimensione delle particelle di 0,05 micron). Per la produzione delle microparticelle finali viene utilizzato un Spray-Drying Buchi con i seguenti parametri: ingresso T: 120°C, aspirazione 100%, velocità della pompa di raccolta del campione 20%, ugello atomizzatore 0,7mm, gas utilizzato: aria compressa filtrata. La resa delle micro e nanoparticelle ottenuta è dell'80% circa. Le dimensioni di micro e nanoparticelle sono comprese tra 1 e 10 micron, come confermato dall'analisi al microscopio elettronico. The production method used is atomization (sprydrying): 1g of PVA (Mw of 80kDa) and maltodextrin (weight ratio 1: 1) were dispersed in 20ml of distilled water by magnetic stirring for 24 hours and an ultrasonic bath to 1 hour at 40 ° C. To this dispersion, 1g of iron oxide powder (with a particle size of 0.05 microns) was added. For the production of the final microparticles, a Buchi Spray-Drying is used with the following parameters: T inlet: 120 ° C, 100% suction, speed of the sample collection pump 20%, 0.7mm atomizing nozzle, gas used: compressed air filtered. The yield of the micro and nanoparticles obtained is about 80%. The dimensions of micro and nanoparticles are between 1 and 10 microns, as confirmed by the analysis with the electron microscope.
Esempio 3: Attrazione magnetica del biocatalizzatore microbico per fermentazioni microbiche magneticamente sensibili Example 3: Magnetic attraction of the microbial biocatalyst for magnetically sensitive microbial fermentations
Il magnete utilizzato nell'esempio è un magnete cubico permanente NdFeB 12x12x12mm; (BxH) max 45-48MGOe, 358-382kJ/m³. Il magnete può essere usato per attrarre i biocatalizzatori magnetici microbici e separare rapidamente la biomassa catalitica dal terreno di coltura evitando la centrifugazione e/o la filtrazione. The magnet used in the example is a permanent cubic NdFeB 12x12x12mm magnet; (BxH) max 45-48MGOe, 358-382kJ / m³. The magnet can be used to attract microbial magnetic biocatalysts and quickly separate the catalytic biomass from the culture medium avoiding centrifugation and / or filtration.
Esempio 4: Preparazione delle microsfere e nanosfere composite magnetiche biodegradabili e biocompatibili generanti catalizzatori microbici magneticamente sensibili per fermentazioni microbiche a qualsiasi altra trasformazione e loro applicazioni per l’incremento del valore generato dai processi di fermentazioni microbica per la trasformazione di substrati in prodotti chimici. Example 4: Preparation of biodegradable and biocompatible magnetic composite microspheres and nanospheres generating magnetically sensitive microbial catalysts for microbial fermentations to any other transformation and their applications for increasing the value generated by microbial fermentation processes for the transformation of substrates into chemical products.
Sospensioni di spore di Streptomyces coelicolor M145 sono state preparate in soluzione acquosa di glicerolo (20% v/v) secondo (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, Hopwood, DA (2000) Practical Streptomyces Genetica: John Innes Fondazione, Norwich Research Park, Colney, Norwich NR4 7UH, Regno Unito). Il titolo di sospensione delle spore è stato quantificato contando le unità formanti colonie (CFU) sul terreno di crescita dell'agar e a diluizioni in serie. Al fine di eseguire colture su mezzo di crescita liquido, spore di S.coelicolore (108/ml) sono state inoculate in matracci sterili da 50ml contenenti 10ml di terreno R5A (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, Hopwood , DA (2000) Practical Streptomyces Genetica: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, Regno Unito) con microsfere e nanosfere composite biodegradabili e biocompatibili precedentemente sterilizzate con plasma O2 (concentrazione finale di microsfere 50 mg/ml). Parallelamente colture di controllo delle spore di S.coeliocolor sono state preparate come sopra descritto ma senza l'aggiunta di microsfere composite biocompatibili e biodegradabili. Tutte le colture sono state incubate a 30°C su agitatore orbitale (200rpm). Spore suspensions of Streptomyces coelicolor M145 were prepared in aqueous glycerol solution (20% v / v) according to (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, Hopwood, DA (2000) Practical Streptomyces Genetica : John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom). The spore suspension titer was quantified by counting colony forming units (CFUs) on the agar growth medium and at serial dilutions. In order to perform cultures on liquid growth medium, S.coelicolor spores (108 / ml) were inoculated into sterile 50ml flasks containing 10ml of R5A medium (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, Hopwood, DA (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK) with biodegradable and biocompatible composite microspheres and nanospheres previously sterilized with O2 plasma (final concentration of microspheres 50 mg / ml). In parallel, control cultures of S.coeliocolor spores were prepared as described above but without the addition of biocompatible and biodegradable composite microspheres. All cultures were incubated at 30 ° C on an orbital shaker (200rpm).
Tutte le procedure sopra descritte sono state eseguite in condizioni sterili. Le produzioni di Actinorodina (ACT) e Undecilprodigiosina (RED) sono state quantificate dopo 5 giorni di incubazione come descritto da (Kieser, 2000), con piccole modificazioni. In particolare, è stata applicata la procedura basata sul trattamento con KOH (1N) per lisare le cellule miceliali e per solubilizzare l'ACT totale (cioè intra/extracellulare) (5 min, miscelazione per vortice) a 1mL di brodo di coltura di tutte le colture. Dopo il trattamento con KOH, al fine di rimuovere il materiale insolubile in acqua, è stata sfruttata la responsività magnetica del biocatalizzatore microbico magneticamente sensibile, costituito da microsfere e nanosfere composite magnetiche biodegradabili e biocompatibili, per separare il biofilm catalitico microbico dal brodo di coltura contenente il loro prodotti (ACT e RED) (Il magnete permanente NdFeB 12x12x12 mm; (BxH) max 45-48 MGOe, 358-382 kJ/m³). Pertanto, il brodo di coltura è stato raccolto e la concentrazione di ACT è stata quantificata spettrofotometricamente a 640nm usando il coefficiente di estinzione molare del composto puro (ε640 = 25,320 M<-1 >cm<-1>). Il restante pellet è stato quindi utilizzato per quantificare il ROS legato al micelio che è un composto insolubile in acqua. In particolare, il pellet è stato lavato tre volte utilizzando tampone Tris-HCl (1M, pH 7,5). Quindi, RED è stato solubilizzato usando 0,5M HCl sciolto in metanolo (60min, miscelazione per inversione) e quantificato spettrofotometricamente a 530nm usando il coefficiente di estinzione molare del composto puro (ε530 = 100,500 M<-1 >cm<-1>). Le concentrazioni di ACT e RED, la cui identità era confermata dall'assorbimento dello spettri visibile (Horinouchi, 1984) è il risultato del valore medio di tre colture distinte. La grande intensità di colore del matraccio numero 3 indica una maggiore conversione del substrato nel prodotto chimico finale. La presenza di microsfere e nanosfere composite biodegradabili e biocompatibili aumenta la produttività dei catalizzatori microbici rispetto alla coltura planctonica e aumenta la resistenza a eventuali inibitori catalitici. Inoltre, la presenza di microsfere e nanosfere composite biodegradabili e biocompatibili aumenta di circa il 20% la resa dei prodotti ACT e RED. All the procedures described above were performed under sterile conditions. The productions of Actinorodina (ACT) and Undecilprodigiosina (RED) were quantified after 5 days of incubation as described by (Kieser, 2000), with small modifications. In particular, the procedure based on the treatment with KOH (1N) was applied to lysate the mycelial cells and to solubilize the total ACT (i.e. intra / extracellular) (5 min, mixing by vortex) to 1mL of culture broth of all crops. After the treatment with KOH, in order to remove the insoluble material in water, the magnetic responsiveness of the magnetically sensitive microbial biocatalyst, consisting of biodegradable and biocompatible composite magnetic microspheres and nanospheres, was exploited to separate the microbial catalytic biofilm from the culture broth containing their products (ACT and RED) (The permanent magnet NdFeB 12x12x12 mm; (BxH) max 45-48 MGOe, 358-382 kJ / m³). Therefore, the culture broth was collected and the ACT concentration was spectrophotometrically quantified at 640nm using the molar extinction coefficient of the pure compound (ε640 = 25.320 M <-1> cm <-1>). The remaining pellet was then used to quantify the ROS bound to the mycelium which is a water insoluble compound. In particular, the pellet was washed three times using Tris-HCl buffer (1M, pH 7.5). Then, RED was solubilized using 0.5M HCl dissolved in methanol (60min, mixing by inversion) and quantified spectrophotometrically at 530nm using the molar extinction coefficient of the pure compound (ε530 = 100,500 M <-1> cm <-1>) . The concentrations of ACT and RED, whose identity was confirmed by the absorption of the visible spectra (Horinouchi, 1984) is the result of the average value of three distinct cultures. The large color intensity of flask number 3 indicates a greater conversion of the substrate into the final chemical. The presence of biodegradable and biocompatible composite microspheres and nanospheres increases the productivity of microbial catalysts compared to planktonic culture and increases resistance to any catalytic inhibitors. Furthermore, the presence of biodegradable and biocompatible composite microspheres and nanospheres increases the yield of ACT and RED products by approximately 20%.
Le microsfere e le nanosfere composite biodegradabili e biocompatibili possono essere utilizzate per valutare rapidamente l'efficacia di un fenotipo ad eseguire il bioprocesso specifico, sia per i prodotti bersaglio (target) esternalizzati che internalizzati. La mancanza di centrifugazione e/o filtrazione consente di valutare facilmente fenotipi multipli in parallelo per valutare rapidamente qual è il fenotipo più efficace tra i biocatalizzatori microbici testati. Biodegradable and biocompatible composite microspheres and nanospheres can be used to rapidly assess the effectiveness of a phenotype to perform the specific bioprocessing, for both externalized and internalized target products. The lack of centrifugation and / or filtration makes it possible to easily evaluate multiple phenotypes in parallel to quickly assess which is the most effective phenotype among the tested microbial biocatalysts.
Nei processi multistadio o biocatalisi multifasiche o fermentazione o in qualsiasi altro processo di biotrasformazione, durante il primo stadio uno specifico fenotipo microbico, proliferante sulla superficie delle microsfere e nanosfere composite biodegradabili e biocompatibili trasformerà il substrato, modificando così la composizione del brodo di coltura. Il brodo di coltura viene chiarificato per mezzo di un magnete, separante il biocatalizzatore microbico magneticamente sensibile dai loro prodotti. Un secondo fenotipo ottimizzato per la nuova composizione del brodo di coltura e proliferante sulla superficie di nuove microsfere e nanosfere composite biodegradabili e biocompatibili è introdotto nel bioreattore e può completare il secondo stadio della biocatalisi, trasformando i prodotti del primo stadio nel prodotto finale. Le microsfere e le nanosfere composite biodegradabili e biocompatibili possono consentire un processo di biocatalisi in più fasi, infatti ogni volta che la biomassa converte il substrato in un'entità chimica, un nuovo microrganismo responsabile dell'ulteriore passaggio della trasformazione può essere introdotto nei processi. L'iterazione può essere ripetuta per quante volte si rende necessario all’ottenimento del prodotto target. In ogni stadio, operazioni che consumano risorse e tempo come la centrifugazione e la filtrazione, sono state evitate grazie alla responsività magnetica delle microsfere e nanosfere composite biodegradabili e biocompatibili. In multistage or multiphase biocatalysis or fermentation processes or in any other biotransformation process, during the first stage a specific microbial phenotype, proliferating on the surface of the biodegradable and biocompatible composite microspheres and nanospheres will transform the substrate, thus modifying the composition of the culture broth. The culture broth is clarified by means of a magnet, separating the magnetically sensitive microbial biocatalyst from their products. A second phenotype optimized for the new composition of the culture broth and proliferating on the surface of new biodegradable and biocompatible composite microspheres and nanospheres is introduced into the bioreactor and can complete the second stage of biocatalysis, transforming the products of the first stage into the final product. Biodegradable and biocompatible composite microspheres and nanospheres can allow a multi-step biocatalysis process, in fact every time the biomass converts the substrate into a chemical entity, a new microorganism responsible for the further step of the transformation can be introduced into the processes. The iteration can be repeated as many times as is necessary to obtain the target product. At each stage, resource and time consuming operations such as centrifugation and filtration were avoided thanks to the magnetic responsiveness of the biodegradable and biocompatible composite microspheres and nanospheres.
Il biocatalizzatore microbico magneticamente sensibile può essere riutilizzato per diversi cicli biocatalitici. Una volta che il substrato nel brodo di coltura viene consumato dal catalizzatore microbico e trasformato nel prodotto bersaglio (target), il catalizzatore microbico che forma un film catalitico microbico sulla superficie delle microsfere e delle nanosfere composte biodegradabili e biocompatibili può essere separato dal brodo di coltura per mezzo di un magnete. I biocatalizzatori microbici sensibili magneticamente, una volta separati dal brodo di coltura esaurito, possono essere esposti a un nuovo e fresco brodo di coltura per innescare un nuovo ciclo di fermentazione o qualsiasi altra biotrasformazione. The magnetically sensitive microbial biocatalyst can be reused for several biocatalytic cycles. Once the substrate in the culture broth is consumed by the microbial catalyst and transformed into the target product, the microbial catalyst that forms a microbial catalytic film on the surface of the biodegradable and biocompatible compound microspheres and nanospheres can be separated from the culture broth by means of a magnet. The magnetically sensitive microbial biocatalysts, once separated from the spent culture broth, can be exposed to a fresh new culture broth to trigger a new fermentation cycle or any other biotransformation.
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