ES2361308B2 - BACTERIAN CEPA CECT7625, USES AND XEROPROTECTOR PRODUCT PRODUCED BY THE SAME. - Google Patents
BACTERIAN CEPA CECT7625, USES AND XEROPROTECTOR PRODUCT PRODUCED BY THE SAME. Download PDFInfo
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- ES2361308B2 ES2361308B2 ES200931117A ES200931117A ES2361308B2 ES 2361308 B2 ES2361308 B2 ES 2361308B2 ES 200931117 A ES200931117 A ES 200931117A ES 200931117 A ES200931117 A ES 200931117A ES 2361308 B2 ES2361308 B2 ES 2361308B2
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
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Abstract
Cepa bacteriana CECT7625, usos y producto xeroprotector producido por la misma.#Microorganismo de la especie bacteriana Rhodococcus sp. con número de acceso CECT7625. La presente invención también se refiere al uso de dicho microorganismo o de una población del mismo para la producción de una composición xeroprotectora, donde dicha composición comprende acetato, lactato, ácido glutámico, {be}hidroxibutirato y fructosa. Además, la presente invención se refiere al uso de la composición xeroprotectora para la conservación de material biológico con un contenido de humedad residual igual o inferior al 10%, donde el material biológico es un organismo invertebrado, una semilla, una plántula, un microorganismo, un órgano aislado, un tejido biológico aislado o una célula, o una molécula con actividad biológica como por ejemplo una enzima con actividad lipasa. Y, la presente invención también se refiere a un método de obtención de la composición xeroprotectora o a un método para la conservación de dicho material biológico.Bacterial strain CECT7625, uses and xeroprotective product produced by it. # Microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. The present invention also relates to the use of said microorganism or a population thereof for the production of an xeroprotective composition, wherein said composition comprises acetate, lactate, glutamic acid, {be} hydroxybutyrate and fructose. In addition, the present invention relates to the use of the xeroprotective composition for the conservation of biological material with a residual moisture content equal to or less than 10%, where the biological material is an invertebrate organism, a seed, a seedling, a microorganism, an isolated organ, an isolated biological tissue or a cell, or a molecule with biological activity such as an enzyme with lipase activity. And, the present invention also relates to a method of obtaining the xeroprotective composition or a method for the conservation of said biological material.
Description
Cepa bacteriana CECT7625,usosyproductoxeroprotector producidoporla misma. Bacterial strain CECT7625, uses and x-ray protective product produced by it.
La presente invención se refiere a un microorganismo de la especie bacteriana Rhodococcus sp. con número de acceso CECT7625. Asimismo la presente invención se refiere al uso de dicho microorganismo o de una población del mismo para la producción de una composición xeroprotectora, donde dicha composición comprende acetato, lactato, ácido glutámico, β-hidroxibutirato y fructosa. La presente invención también se refiere al uso de la composición xeroprotectora para la conservación de material biológico con un contenido de humedad residual igual o inferior al 10%, donde el material biológico es un organismo invertebrado, una semilla, una plántula, un microorganismo, un órgano aislado, un tejido biológico aisladoo una célula,o una molécula con actividad biológica como por ejemplouna enzima con actividad lipasa. Además, la presente invención se refiere a un método de obtención de la composición xeroprotectoraoa un métodoparala conservaciónde dicho material biológico. The present invention relates to a microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. The present invention also refers to the use of said microorganism or a population thereof for the production of an xeroprotective composition, wherein said composition comprises acetate, lactate, glutamic acid, β-hydroxybutyrate and fructose. The present invention also refers to the use of the xeroprotective composition for the conservation of biological material with a residual moisture content equal to or less than 10%, where the biological material is an invertebrate organism, a seed, a seedling, a microorganism, a isolated organ, an isolated biological tissue or a cell, or a molecule with biological activity such as an enzyme with lipase activity. In addition, the present invention relates to a method of obtaining the x-protective composition or a method for the conservation of said biological material.
Estado de la técnica anterior Prior art
La conservaciónde materiales biológicos mediante deshidrataciónyosmoconcentraciónes una tecnología conocida. Cuando la tarea de conservar biomoléculas sensibles se hizo necesaria, el simple secado mediante deshidratación fracasó,yaquese eliminabaelagua estructural, produciendola desnaturalización posteriorylapérdidadeactividad vital. La liofilización se ha convertido en el método más aceptado para la conservación a largo plazo de biomoléculas sensibles, usándose por ejemplo de forma muy extendida para la conservación de vacunas atenuadas vivas. The conservation of biological materials by dehydration and concentration is a known technology. When the task of conserving sensitive biomolecules became necessary, simple drying by dehydration failed, since the structural water was eliminated, producing subsequent denaturation and loss of vital activity. Lyophilization has become the most accepted method for the long-term preservation of sensitive biomolecules, for example widely used for the conservation of live attenuated vaccines.
Los métodos actualesde conservación requierende gran costo en energíaygeneralmente necesitande almacenaje abajas temperaturas.En ocasiones,despuésdesuconservaciónelmaterial biológicotieneunaactividady/o viabilidad que no alcanza los niveles satisfactorios. Los métodos de conservación, tales como el secado a temperatura ambiente, formulaciones en líquido, el congelado con crioprotectores o la liofilización producen reducciones significativas en la actividad/viabilidad del material conservado. Current conservation methods require a high cost in energy and generally need storage at low temperatures. Sometimes, after their preservation the biological material has an activity and / or viability that does not reach satisfactory levels. Conservation methods, such as drying at room temperature, liquid formulations, freezing with cryoprotectants, or lyophilization produce significant reductions in the activity / viability of the conserved material.
Los procesos usados actualmente son lentos e implican un elevado consumo de energía. Además, la liofilización confiere sólo unnivel modestode termotolerancia enel producto final,y se requiere aún refrigeración para reducireldeterioro durante el almacenamiento. Éste es un problema particular para vacunas vivas destinadas a usarse en climas tropicales, ya que éstas pierden actividad, con el desafortunado resultado de que los programas de vacunación realizados en el campo en países tropicales, donde el control de la “cadena de frío” es difícil, pueden conducir finalmente a la vacunación de pacientes con vacuna inferior a la estándar o, en algunos casos, inservible. The processes currently used are slow and involve high energy consumption. In addition, lyophilization confers only a modest level of thermotolerance in the final product, and cooling is still required to reduce deterioration during storage. This is a particular problem for live vaccines intended for use in tropical climates, since they lose activity, with the unfortunate result that vaccination programs carried out in the countryside in tropical countries, where the control of the “cold chain” is difficult, they can ultimately lead to the vaccination of patients with a lower than standard vaccine or, in some cases, unusable.
Durantela selección naturalevolutiva, ciertas especiesde microorganismos, plantasyanimales adquirieronla notableyelegante capacidadde tolerarla deshidratación extrema, permaneciendo latentes en medios hostiles durante períodosmuylargosde tiempoyaún capacesde adquirir una actividad vital completa unavez hidratadas nuevamente. Ejemplos incluyen la “planta de la resurrección” Selaginella lepidophyla, el camarón de mar Artemia salina, la levadura Saccharomyces cerevisiae o el tardígrado Macrobiotus hufelandi. Estos organismos se denominan criptiobióticos yel procedimientoporelque sobrevivense conoce como anhidrobiosis.Todaslas especiesdeanimalesyplantas que presentan esta capacidad, contienen moléculas protectoras formadoras de cristales amorfos como el disacárido trehalosa(α-D-glucopiranosil-α-D-glucopiranósido). During the nature-selective selection, certain species of microorganisms, plants and animals acquired the remarkable and elegant ability to tolerate extreme dehydration, remaining dormant in hostile environments for very long periods of time and still capable of acquiring a complete vital activity once hydrated again. Examples include the "resurrection plant" Selaginella lepidophyla, Artemia salina sea shrimp, Saccharomyces cerevisiae yeast or Macrobiotus hufelandi tardigrade. These organisms are called cryptiobiotics and the surviving procedure known as anhydrobiosis.All animal species and plants that have this capacity contain protective molecules that form amorphous crystals such as trehalose disaccharide (α-D-glucopyranosyl-α-D-glucopyranoside).
La formacióny uso de los cristales amorfos está bien documentada (Manzanera et al., 2002. Appl Environ Microbiol, 68: 4328-4333). Algunos de los conservantes que forman estos cristales son adecuados para este tipo de conservación e incluyen hidratos de carbono no reductores como la trehalosa, hidroxiectoina, maltitol, lactitol (4O-α-D-glucopyranosyl-D-glucitol), palatinit [mezcla de GPS(α-D-glucopiranosil-1-6-sorbitol)yGPM(α-D-glucopiranosil-1-6-manitol)]y sus componentes individuales GPSy GPM. Los glicósidos no reductores de compuestos polihidroxilados tales como neotrehalosa, laconeotrehalosa,galactosil-trehalosa, sacarosa, lactosacarosa, rafinosa, etc. Otros conservantes formadores de cristales amorfos incluyen aminoácidos tales como la hidroxiectoina. The formation and use of amorphous crystals is well documented (Manzanera et al., 2002. Appl Environ Microbiol, 68: 4328-4333). Some of the preservatives that form these crystals are suitable for this type of preservation and include non-reducing carbohydrates such as trehalose, hydroxyectoin, maltitol, lactitol (4O-α-D-glucopyranosyl-D-glucitol), palatinit [GPS mixture (α-D-glucopyranosyl-1-6-sorbitol) and GPM (α-D-glucopyranosyl-1-6-mannitol)] and its individual components GPS and GPM. Non-reducing glycosides of polyhydroxy compounds such as neotrehalosa, laconeotrehalosa, galactosyl trehalose, sucrose, lactose sucrose, raffinose, etc. Other amorphous crystal-forming preservatives include amino acids such as hydroxyectoin.
La presenciadeaguaenel estado secoes generalmente inferiora0,2g/gdepeso celular secoenlamayoríadelos criptobiontes.Estosnivelesdeaguasonsuficientesparaqueestos microorganismos resistanla deshidrataciónextrema, temperaturas elevadas, radiaciones ionizantes o también, en algunas especies de tardígrados, presiones de hasta 600 MPa. The presence of water in the dry state is generally less than 0.2g / g of dry cell weight in the majority of cryptons. These levels are sufficient for these microorganisms to resist extreme dehydration, high temperatures, ionizing radiation or also, in some species of tardigrades, pressures of up to 600 MPa.
Es conocido que las biopelículas(Subaerial biofilms), formadas por bacterias del género Rhodococcus sp entre otras, son capaces de producir compuestos osmoprotectores, es decir, sustancias extracelulares poliméricas (EPS) (Gorbushina, 2007. Environmental Microbiology, 9(7): 1613-1631). Asimismo, Ortega-Morales et al. (2007) (Ortega-Morales et al.2007. JournalofApplied Microbiology,102:254-264), describen bacteriasdelas biopelículasdelaszonas intermareales tropicales, donde se han aislado bacterias que pertenecen al género Microbacterium sp. como fuente denuevosexopolímeros protectoresdelas células contraladesecación.Porotraparte, LeBlanc(2008)(LeBlanc,2008. Applied and environmental microbiology, 74(9): 2627-2636), se refiere al microorganismo Rhodococcus jostii RHA1, un actinomiceto con capacidades metabólicasfavorables para la biorremediación de suelos contaminados, capaz de secretar los osmoprotectores ectoinaytrehalosa. It is known that biofilms (Subaerial bio fi lms), formed by bacteria of the genus Rhodococcus sp among others, are capable of producing osmoprotective compounds, that is, polymeric extracellular substances (EPS) (Gorbushina, 2007. Environmental Microbiology, 9 (7): 1613 -1631). Likewise, Ortega-Morales et al. (2007) (Ortega-Morales et al. 2007. JournalofApplied Microbiology, 102: 254-264), describe bacteria from the biofilms of tropical intertidal zones, where bacteria belonging to the genus Microbacterium sp. as a source of new protective polymers of anti-drying cells. Porotraparte, LeBlanc (2008) (LeBlanc, 2008. Applied and environmental microbiology, 74 (9): 2627-2636), refers to the microorganism Rhodococcus jostii RHA1, an actinomycete with metabolic capacities for bioremediation Contaminated soils, capable of secreting the ectoinaytrehalosa osmoprotectors.
Explicación de la invención Explanation of the invention.
La presente invención se refiere a un microorganismo de la especie bacteriana Rhodococcus sp. con número de acceso CECT7625. Asimismo la presente invención se refiere al uso de dicho microorganismo o de una población del mismo para la producción de una composición xeroprotectora, donde dicha composición comprende acetato, lactato, ácido glutámico, β-hidroxibutirato y fructosa. La presente invención también se refiere al uso de la composición xeroprotectora para la conservación de material biológico con un contenido de humedad residual igual o inferior al 10%, donde el material biológico es un organismo invertebrado, una semilla, una plántula, un microorganismo, un órgano aislado, un tejido biológico aisladoo una célula,o una molécula con actividad biológica como por ejemplo una enzima con actividad lipasa. Además, la presente invención se refiere a un método de obtención de la composición xeroprotectora o a un método para la conservación de dicho material biológico. The present invention relates to a microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. The present invention also refers to the use of said microorganism or a population thereof for the production of an xeroprotective composition, wherein said composition comprises acetate, lactate, glutamic acid, β-hydroxybutyrate and fructose. The present invention also refers to the use of the xeroprotective composition for the conservation of biological material with a residual moisture content equal to or less than 10%, where the biological material is an invertebrate organism, a seed, a seedling, a microorganism, a isolated organ, an isolated biological tissue or a cell, or a molecule with biological activity such as an enzyme with lipase activity. In addition, the present invention relates to a method of obtaining the xeroprotective composition or a method for the conservation of said biological material.
La capacidadpara conservar material biológico sensiblepor periodosdetiempo indefinidoen formaactivao viable es de importancia en aplicaciones para los sectores médico, agrícola e industrial. En la presente invención se ofrecen herramientas para solucionarla conservacióndematerial biológicoque presenta dificultades parasu estabilización.El material biológico preservado con la composición xeroprotectora producida por el microorganismo con nº de acceso CECT7625es establepor periodoslargosde tiempo.Tal comose muestraenlos ejemplosdela presenteinvención,el uso de una composición que contiene cada uno de los componentes por separado para la conservación de la actividad de una enzima lipasa, es decir,la suma del efecto en la conservación de la actividad enzimática de una composición que contiene acetato,o lactato,o ácidoglutámico,o β-hidroxibutirato o fructosa, es menor que el efecto de conservación que produce una composición que contiene todos los componentes, es decir, la composición xeroprotectora tiene un efecto sinérgico en su capacidad de conservar material biológico. The ability to conserve sensitive biological material for indefinite periods of time in an active or viable way is of importance in applications for the medical, agricultural and industrial sectors. In the present invention, tools are provided to solve the biological material preservation that presents difficulties for its stabilization.The biological material preserved with the xeroprotective composition produced by the microorganism with accession number CECT7625 is stable for long periods of time.As shown in the examples of the present invention, the use of a composition containing each of the components separately for the conservation of the activity of a lipase enzyme, that is, the sum of the effect on the conservation of the enzymatic activity of a composition containing acetate, or lactate, or glutamic acid, or β-hydroxybutyrate or Fructose is less than the conservation effect produced by a composition that contains all the components, that is, the xeroprotective composition has a synergistic effect on its ability to conserve biological material.
Por tanto, un aspecto de la presente invención se refiere a un microorganismo de la especie bacteriana Rhodococcus sp. con número de acceso CECT7625. Dicho microorganismo es tolerante a la desecación. Dicha cepa ha sido depositada enla Colección EspañoladeCultivosTipo (CECT)el10de noviembrede 2009yle correspondióelnºde depósito CECT7625.La direccióndedicha Autoridad Internacionalde depósito es: UniversidaddeValencia/Edificio deinvestigación/CampusdeBurjassot/46100 Burjassot(Valencia). Therefore, one aspect of the present invention refers to a microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. Said microorganism is tolerant to desiccation. This strain has been deposited in the Spanish Collection of Type Cultures (CECT) on November 10, 2009 and the deposit number CECT7625 corresponded. The address of the International Deposit Authority is: UniversidaddeValencia / Building de Investigation / CampusdeBurjassot / 46100 Burjassot (Valencia).
En adelante, para hacer referencia a dicha cepa se puede emplear el término “4J2A2”. Hereinafter, to refer to said strain, the term "4J2A2" can be used.
La clasificación científica de la cepa CECT7625 de la presente invención es: Reino: Bacteria/Filo: Actinobacteria/Orden: Actinomycetales/Familia: Nocardiaceae/Género: Rhodococcus. The scientific classification of strain CECT7625 of the present invention is: Kingdom: Bacteria / Phylum: Actinobacteria / Order: Actinomycetales / Family: Nocardiaceae / Genus: Rhodococcus.
Las características de dicha cepa son: The characteristics of said strain are:
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- Metabolismo caracterizado por oxidación/fermentación, Dextrina, tween 40, tween 80,N-acetil-D-glucosamina,D-arabitol,D-fructosa, ácidoD-glucónico, α-D-glucosa,D-manitol,D-manosa,D-psicosa,D-ribosa,D-sorbitol, ácido α-cetovalérico, ácidoL-láctico, ácidoL-málico, metil piruvato, mono-metil succinato, ácido propiónico, ácido pirúvico, L-alanil-glicina, glicerol, 2’-deoxiadenosina. Metabolism characterized by oxidation / fermentation, Dextrin, tween 40, tween 80, N-acetyl-D-glucosamine, D-arabitol, D-fructose, D-gluconic acid, α-D-glucose, D-mannitol, D-mannose, D -psicosa, D-ribose, D-sorbitol, α-ketovaleric acid, L-lactic acid, L-malic acid, methyl pyruvate, mono-methyl succinate, propionic acid, pyruvic acid, L-alanyl glycine, glycerol, 2'-deoxyadenosine .
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- La temperatura máxima tolerada para el crecimiento de esta cepa son 40ºC. La temperatura mínima para detectar crecimientose encontró entre15ºCy20ºC, mientrasquesu temperaturaóptimade crecimientofuede entornoa35ºC. Fue incapazde crecerapH12 aunquesiapH9. The maximum temperature tolerated for the growth of this strain is 40 ° C. The minimum temperature to detect growth was between 15ºC and 20ºC, while its optimum growth temperature was around 35ºC. He was unable to grow H12 although siapH9.
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- ElpHmínimo toleradoparael crecimientodeestacepase encontró entrepH5ypH7, considerándoseéste como pH óptimo para el crecimiento de esta cepa. The minimum tolerated for the growth of this stage was found between p5 and H7, this being considered the optimum pH for the growth of this strain.
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- Igualmente fue incapaz de proliferar en medio LB con una concentración de NaCl igual o superior a 2 M, quedandola concentración máxima tolerada entre0,8My2Mde NaCl. Esta cepa mostróun crecimiento óptimoa una concentraciónde0,2MdeNaCl, aunque tambiénfuecapazde proliferarenestemedioen ausenciadeNaCl. It was also unable to proliferate in LB medium with a concentration of NaCl equal to or greater than 2 M, with the maximum concentration tolerated between 0.8M and 2M NaCl. This strain showed optimal growth at a concentration of 0.2M NaCl, although it was also able to proliferate in the absence of NaCl.
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- Ensayos de sensibilidad a antibióticos mostraron halos de inhibición del crecimiento en los cinco antibióticos ensayados en disco: rifampicina30 (2,83 cm); estreptomicina25 (2,20 cm); tetraciclina20 (0,87 cm); cloramfenicol50 (4,12 cm); kanamicina30 (1,93 cm). Antibiotic sensitivity tests showed growth inhibition halos in the five antibiotics tested on disk: rifampin 30 (2.83 cm); streptomycin25 (2.20 cm); tetracycline20 (0.87 cm); chloramphenicol50 (4.12 cm); Kanamycin 30 (1.93 cm).
Asimismo,lapresenteinvención también se refierea un microorganismo derivado del microorganismo depositado con nº de acceso CECT7625. El microorganismo derivado puede producirse de forma intencionada, por métodos mutagénicos conocidos en el estado de la técnica como por ejemplo, el crecimiento de dicho microorganismo original en exposición con conocidos agentes capaces de forzar mutagénesis. Likewise, the present invention also refers to a microorganism derived from the microorganism deposited with accession number CECT7625. The derived microorganism can be produced intentionally, by mutagenic methods known in the state of the art such as, for example, the growth of said original microorganism on exposure with known agents capable of forcing mutagenesis.
Otro aspecto de la presente invención se refiere a una población bacteriana que comprende el microorganismo depositado con nº de acceso CECT7625. La población bacteriana puede estar formada por otras cepas de microorganismos de cualquier especie. La población bacteriana es un conjunto de células de microorganismos donde al menos hay una célula de dicho microorganismo depositado con nº de acceso CECT7625, en cualquierfase del estadode desarrolloy en cualquierfasede crecimiento, estacional o estacionaria, independientementedela morfologíaque presente, en forma de coco, bacilo o morfologías intermediarias de las anteriores. Another aspect of the present invention relates to a bacterial population comprising the microorganism deposited with accession number CECT7625. The bacterial population can be formed by other strains of microorganisms of any species. The bacterial population is a group of cells of microorganisms where at least one cell of said microorganism is deposited with accession number CECT7625, in any phase of the state of development and in any phase of growth, seasonal or stationary, regardless of the morphology that is present, in the form of coconut, bacillus or intermediary morphologies of the previous ones.
En adelante se podrá hacer referencia al microorganismo o a la población bacteriana como el “microorganismo de la presente invención” o el “microorganismo de la invención”. Hereinafter, the microorganism or the bacterial population may be referred to as the "microorganism of the present invention" or the "microorganism of the invention".
Otro aspectomásdelapresenteinvenciónse refierealusodel microorganismodelainvenciónparala producción de una composición xeroprotectora. Another aspect of this invention is the real reliance on the microorganism of the invention for the production of an x-protective composition.
Un aspecto más de la presente invención se refiere a la composición xeroprotectora producida por el microorganismodelainvención.Para referirseala composiciónxeroprotectoradela presenteinvención se puede emplearel términoProductode Ordeñado Bacteriano (POB).El término “composiciónxeroprotectora”tal comose entiendeen la presente invención, se refiere a una composición que previene los efectos adversos de la desecación total o parcial de material de origen biológico o material de origen sintético. El materialbiológico se refiere a cualquier compuesto producido directamente por un organismo vivo en cualquier estado de desarrollo, en cualquier compartimento celular, sea cual sea la naturaleza, composición o estructura del mismo, o que procede de un organismo que ya no está vivo. Dicho material biológico puede ser, por ejemplo, pero sin limitarse, una célula, ácido nucleico, proteína, enzima, polisacárido, lípido, fosfolípido, liposomas, virus, partículas virales o cualquier molécula que comprenda cualquiera delos elementos anteriores,ocualquier moléculaorgánicaquetengaun efectofarmacológico, inmunogénicoy/ofisiológico de acción local y/o sistémica. El material biológico puede comprender agentes terapéuticos; agentes antiinfectivos como antibióticos, antivirales; analgésicos o combinaciones de analgésicos; agentes antiartríticos, antiasmáticos, antiinflamatorios, antioplásticos, antipruríticos, antipsicóticos, antipiréticos, antiespasmódicos, preparaciones cardiovasculares (incluyendo bloqueantes de canales de calcio, bloqueadores beta, o antiarrítmicos), agentes contra la hipertensión, diuréticos, vasodilatadores, estimuladores del sistema nervioso central, antitusivos, preparaciones anti resfriados, descongestionantes, agentes de diagnóstico, hormonas, estimuladores del crecimiento óseo, inhibidores de la resorción de la médula ósea, inmunosupresores, relajantes musculares, psicoestimulantes, sedantes, tranquilizantes, proteínas, péptidos o fragmentos de los mismos (tanto naturales, como sintéticos, como productos recombinantes), moléculasdeácidos nucleicos(tantoenforma poliméricadedosomás nucleótidosdeADNoARN, incluyendotanto moléculas de cadena doble como de cadena sencilla, construcciones genéticas, vectores de expresión, ARN antisentido, sentido o moléculas ARNi) o nucleótidos (como por ejemplo, pero sin limitarse, el dATP, dCTP, dGTP, dTTP A further aspect of the present invention refers to the x-protective composition produced by the microorganism of the invention.To refer to the protective composition of the present invention, the term Bacterial Milked Product (POB) may be used. The term "protective-composition" as understood by the present invention refers to a composition that prevents adverse effects of the total or partial drying of material of biological origin or material of synthetic origin. The biological material refers to any compound produced directly by a living organism in any state of development, in any cellular compartment, whatever the nature, composition or structure thereof, or that comes from an organism that is no longer alive. Said biological material can be, for example, but not limited to, a cell, nucleic acid, protein, enzyme, polysaccharide, lipid, phospholipid, liposomes, viruses, viral particles or any molecule comprising any of the above elements, or any organic molecule that has an anthropharmacological, immunogenic and / ophysiological of local and / or systemic action. The biological material may comprise therapeutic agents; anti-infective agents such as antibiotics, antivirals; analgesics or combinations of analgesics; antiarthritic, anti-asthmatic, anti-inflammatory, antioplastic, antipruritic, antipsychotic, antipyretic, antispasmodic, cardiovascular preparations (including calcium channel blockers, beta blockers, or antiarrhythmic agents), anti-hypertension agents, diuretics, vasodilators, central nervous system stimulants , anti-cold preparations, decongestants, diagnostic agents, hormones, bone growth stimulators, bone marrow resorption inhibitors, immunosuppressants, muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides or fragments thereof (both natural, as synthetics, as recombinant products), nucleic acid molecules (both polymeric form of two more nucleotides of DNARNA, including both double chain and single chain molecules, genetic constructs, expression vectors, antisense RNA, sense or molecule). RNAi) or nucleotides (such as, but not limited to, dATP, dCTP, dGTP, dTTP
o dUTP, de utilidad en la técnica de PCR, secuenciación, etc). El material de origen sintético se refiere a un tipo de materialquenohasido producidoo sintetizadoporunorganismovivo directamentesinoquehasido creadoporelser humano como por ejemplo, pero sin limitarse, una secuencia de ADN amplificada por PCR, o una proteína o enzima modificada intencionadamente o no. or dUTP, useful in the technique of PCR, sequencing, etc.). Material of synthetic origin refers to a type of material that has not been produced or synthesized by a live organism directly but that has been created by human beings such as, but not limited to, a DNA sequence amplified by PCR, or a protein or enzyme modified intentionally or not.
Una realización preferida de la presente invención se refiere a la composición xeroprotectora producida por el microorganismodelainvención,oa una composiciónxeroprotectora sintética,que comprende fructosa, ácido glutámico, β-hidroxibutirato, acetatoylactato. A preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or to a synthetic airborne composition, comprising fructose, glutamic acid, β-hydroxybutyrate, acetate and lactate.
El término “acetato” se refiere al anión acetato presente en una solución, es decir el anión acetato (C2H3O2)−, es un carboxilatoy es base conjugadadel ácido acético.El término “lactato”se refiereal anión lactato presenteenuna solución, es decir el anión lactato (C3H5O3)−,esun carboxilatoy esbaseconjugadadelácido láctico.Elglutamato, formaionizadadelácido glutámico,se refiereaunodelos20aminoácidos esencialesqueformanpartedelasproteínas. El β-hidroxibutirato(o beta-hidroxibutirato) esel anión que derivadela disolución delácido3-hidroxibutírico.La fructosa(olevulosa) es una cetohexosa(6 átomosde carbono) con formula químicaC6H12O6, monosacárido conla misma fórmula empíricaquela glucosa pero con diferente estructura. The term "acetate" refers to the acetate anion present in a solution, ie the acetate anion (C2H3O2) -, is a carboxylate and is a conjugate base of acetic acid. The term "lactate" refers to a lactate anion present in a solution, ie the anion Lactate (C3H5O3) - is a carboxylate and is based on lactic acid. Glutamate, formed from glutamic acid, refers to one of the 20 essential amino acids that form part of the proteins. Β-Hydroxybutyrate (or beta-hydroxybutyrate) is the anion that derives from the solution of 3-hydroxybutyric acid. Fructose (olevulose) is a ketohexose (6 carbon atoms) with chemical formula C6H12O6, monosaccharide with the same empirical formula as glucose but with different structure.
Una realización más preferida se refiere a la composición xeroprotectora producida por el microorganismo de la invención,oala composiciónxeroprotectora sintética,que comprende una proporciónde entre35y45de fructosa: 1,4y3,4 de ácido glutámico: 0,5y1,5 de acetato. Es decir, una proporción (fructosa):(ácido glutámico):(acetato) de (35 a 45):(1,4 a 3,4):(0,5 a 1,5), respectivamente. Una realización aún más preferida se refiere a la composición xeroprotectora donde la proporción de (fructosa):(ácido glutámico):(acetato) es de (38 a 44):(2 a 3):(0,7 a 1,3), respectivamente. Preferiblemente la composición xeroprotectora tiene una proporción de (fructosa):(ácido glutámico):(acetato) de (41):(2,4):(1), respectivamente. A more preferred embodiment refers to the xeroprotective composition produced by the microorganism of the invention, or to the synthetic airborne composition, which comprises a proportion of between 35 and 45 fructose: 1.4 and 3.4 of glutamic acid: 0.5 and 1.5 of acetate. That is, a ratio (fructose) :( glutamic acid) :( acetate) of (35 to 45) :( 1.4 to 3.4) :( 0.5 to 1.5), respectively. An even more preferred embodiment refers to the xeroprotective composition where the ratio of (fructose) :( glutamic acid) :( acetate) is (38 to 44) :( 2 to 3) :( 0.7 to 1.3) respectively. Preferably the xeroprotective composition has a ratio of (fructose) :( glutamic acid) :( acetate) of (41) :( 2,4) :( 1), respectively.
Una realización más preferida se refiere a la composición xeroprotectora producida por el microorganismo de la invención,oala composiciónxeroprotectora sintética,que comprende una proporciónde entre14y18de fructosa: 3y5 de ácido glutámico: 0,6y1deβ-hidroxibutirato: 0,5y1,5 de acetato:1y2 de lactato. Es decir, una proporción (fructosa):(ácido glutámico):(β-hidroxibutirato):(acetato):(lactato) de (14 a 18):(3 a 5):(0,6 a 1):(0,5 a 1,5):(1 a 2). Una realización aún más preferida se refiere a la composición xeroprotectora donde la proporción de (fructosa):(ácido glutámico):(β-hidroxibutirato):(acetato):(lactato) es de (15 a 17):(3,5 a 4,5):(0,7 a 0,9):(0,7 a 1,2):(1,2 a 1,6).Preferiblementela composiciónxeroprotectora tiene una proporciónde (fructosa):(ácido glutámico):(β-hidroxibutirato):(acetato):(lactato) de (16):(4):(0,8):(1):(1,4), respectivamente. A more preferred embodiment refers to the xeroprotective composition produced by the microorganism of the invention, or to the synthetic airborne composition, which comprises a proportion between 14 and 18 of fructose: 3 and 5 of glutamic acid: 0.6 and 1 of beta-hydroxybutyrate: 0.5 and 1.5 of acetate: 1 and 2 of lactate That is, a ratio (fructose) :( glutamic acid) :( β-hydroxybutyrate) :( acetate) :( lactate) of (14 to 18) :( 3 to 5) :( 0.6 to 1) :( 0 , 5 to 1.5) :( 1 to 2). An even more preferred embodiment refers to the xeroprotective composition where the ratio of (fructose) :( glutamic acid) :( β-hydroxybutyrate) :( acetate) :( lactate) is (15 to 17) :( 3.5 a 4.5) :( 0.7 to 0.9) :( 0.7 to 1.2) :( 1.2 to 1.6). Preferably the airborne composition has a proportion of (fructose) :( glutamic acid) :( β-hydroxybutyrate) :( acetate) :( lactate) of (16) :( 4) :( 0.8) :( 1) :( 1.4), respectively.
El término “proporción” tal como se entiende en la presente invención se refiere a la correspondencia debida de los elementos de la composición (fructosa, ácido glutámico, β-hidroxibutirato, acetato y lactato) relacionados entre sí.Es decir, se refiere a una relación matemática que vincula los elementosdela composición.Para quesirva de ejemplo, la composición xeroprotectora que tiene una proporción de (fructosa):(ácido glutámico):(β-hidroxibutirato):(acetato):(lactato) de (16):(4):(0,8):(1):(1,4),respectivamente, puede tener por ejemplo, concentraciones de (32):(8):(1,6):(2):(2,8) mg de cada elemento respectivamente/ml. The term "proportion" as understood in the present invention refers to the corresponding correspondence of the related elements of the composition (fructose, glutamic acid, β-hydroxybutyrate, acetate and lactate). That is, it refers to a mathematical relationship that links the elements of the composition. For example, the x-protective composition that has a proportion of (fructose) :( glutamic acid) :( β-hydroxybutyrate) :( acetate) :( lactate) of (16) :( 4 ) :( 0.8) :( 1) :( 1.4), respectively, may have, for example, concentrations of (32) :( 8) :( 1.6) :( 2) :( 2.8) mg of each element respectively / ml.
En adelante se podrá hacer referencia a cualquiera de las composiciones anteriores como la “composición de la presente invención” o “composición de la invención”. Hereinafter, reference may be made to any of the above compositions as the "composition of the present invention" or "composition of the invention".
Otro aspecto de la presente invención es el uso de la composición de la invención para la conservación de material biológicoconun contenidoenhumedadresidualigualoinferioral10%.El contenidode humedad residualdel material biológico puede ser igual o inferior al 9, 8, 7, 6, 5, 4, 3, 2 ó 1% de humedad residual. La conservación de dicho material puede llevarse a cabo mediante la estabilización del mismo. En la presente invención, para referirse a este tipo de material biológico se puede emplear la expresión “material biológico en estado seco”. En estas condiciones, el conservanteo estabilizador coalesce para alcanzar un estado no-cristalino, vítreo,ysólido (por ejemplo un cristal amorfo). Las partículas de cristal orgánico que están formadas al secar el material biológico con el estabilizador están cubiertas por el estabilizador que produce una alta estabilidad al reducir drásticamente las reacciones químicas.De esta forma el material biológico seco está incrustado en el cristal amorfo formado por el estabilizador. Another aspect of the present invention is the use of the composition of the invention for the conservation of biological material with a moisture content equal to equal to or less than 10%. The residual moisture content of the biological material may be equal to or less than 9, 8, 7, 6, 5, 4, 3, 2 or 1% residual moisture. The preservation of said material can be carried out by stabilizing it. In the present invention, the term "dry material biological material" can be used to refer to this type of biological material. Under these conditions, the stabilizing preservative coalesces to reach a non-crystalline, vitreous, and solid state (for example an amorphous crystal). The organic crystal particles that are formed by drying the biological material with the stabilizer are covered by the stabilizer that produces high stability by drastically reducing chemical reactions.This way the dried biological material is embedded in the amorphous crystal formed by the stabilizer. .
El material biológico seco en presencia del estabilizador que forma el cristal amorfo es resistente a plásticos en estado líquido, mientras que el material que no está seco en presencia de estos estabilizadores no es resistente a plásticos en estado líquido. The dry biological material in the presence of the stabilizer that forms the amorphous crystal is resistant to plastics in the liquid state, while the material that is not dry in the presence of these stabilizers is not resistant to plastics in the liquid state.
El material biológico seco en estas formas puede encontrarseen estado no particuladoypuede suministrarse en formas por ejemplo,pero sin limitarse, molduraso sólidos en3dimensiones como por ejemplo, pero sin limitarse, bloques, pastillas, parches,hojas, bolas,o pepitasde material biológico seco. The dry biological material in these forms can be in a non-particulate state and can be supplied in forms for example, but not limited to, solid molding in dimensions such as, for example, but not limited to, blocks, pads, patches, sheets, balls, or pips of dry biological material.
El término “humedad residual” tal como se emplea en la presente invención se refiere a la cantidad de humedad que contieneunproductodespuésdepasadoporalgúntipodeprocesocapazdeeliminaraguadelmismo.Lahumedad residual es el porcentaje de masa del producto que corresponde a agua respecto del total de la masa. Es decir, un valor de humedadresidualdeun productoigualaun10% significaque10gdecada100gdel producto correspondenaagua. La humedad residualpuede ser medida mediante métodos conocidosenel estadodela técnica comopor ejemplo,pero sin limitarse, mediante el método titrimétrico, el método azeotrópico o el método gravimétrico. The term "residual moisture" as used in the present invention refers to the amount of moisture that a product contains after the process is processed for the same type of process of the same. Residual moisture is the percentage of mass of the product that corresponds to water with respect to the total mass. That is, a residual moisture value of a product equal to 10% means that 10g of each product corresponds to water. Residual humidity can be measured by methods known in the state of the art as an example, but not limited, by the titrimetric method, the azeotropic method or the gravimetric method.
El término “conservaciónde material biológico”hace referenciaal mantenimientoo cuidadodela permanenciade las características intrínsecas del material biológico. The term "conservation of biological material" refers to the maintenance or care of the permanence of the intrinsic characteristics of the biological material.
Una realización preferida se refiere al uso de la composición de la invención para la conservación de material biológico en estado seco, donde el material biológico es un organismo invertebrado, una semilla, una plántula, un microorganismo,unórganoaislado,untejidobiológicoaisladoounacélula.Lacélulapuedeserprocariotaoeucariota. La célulapuedeseruna céluladeun microorganismoen cualquier estadode desarrollo.Lacélulapuedeser somáticao germinal,vegetaloanimal.Dichacélulapuedeprocederdecualquierorganismoomicroorganismoypuedepresentarse en cualquier estado de diferenciación, como por ejemplo,pero sin limitarse, procedente de un cultivo de un tejido celular o de órganos, esperma, óvulos o embriones. La célula puede ser una célula madre totipotente, multipotente o unipotente. El microorganismo puede ser unicelular o multicelular. El organismo unicelular se selecciona de la lista que comprende, pero sin limitarse, E. coli, S. typhimurium, P. putida., Salmonella spp, Rhizobium spp, Pseudomonas spp, Rhodococcus spp, Lactobacillus spp. o Bifidobacterium spp. El organismo pluricelular puede ser por ejemplo, pero sin limitarse, un nematodo. A preferred embodiment refers to the use of the composition of the invention for the conservation of biological material in the dry state, where the biological material is an invertebrate organism, a seed, a seedling, a microorganism, an organoisolated, an isolated biological tissue or a cellulose. The cell can be a cell of a microorganism in any state of development. The cell can be somatic or germinal, animal-like. The cell can come from any organism or organism and can be presented in any state of differentiation, such as, but not limited to, from a culture of a cell tissue or organs, sperm, embryo. The cell can be a totipotent, multipotent or unipotent stem cell. The microorganism can be unicellular or multicellular. The unicellular organism is selected from the list comprising, but not limited to, E. coli, S. typhimurium, P. putida., Salmonella spp, Rhizobium spp, Pseudomonas spp, Rhodococcus spp, Lactobacillus spp. or Bi fi dobacterium spp. The multicellular organism can be, for example, but not limited to, a nematode.
La célula conservada por la composición de la presente invención es una célula viable es decir, es capaz de realizar las funciones normales de la célula incluyendo la replicaciónydivisión celular. Por otra parte la célula puede haber sidotratada,manipuladaomutadaantesdesu conservación.Porejemplo,perosinlimitarse,unacélulapuedehaberse hecho competente para transformaciones o transfecciones, o puede contener ácidos nucleicos recombinantes. Las célulasquese conservanpuedenformarunapoblaciónhomogéneao heterogénea,porejemplo,perosin limitarse,una librería de células en la que cada una contiene una variación de algún ácido nucleico. Preferentemente, las células son células no anhidrobióticas (células sensibles a desecación) como por ejemplo, pero sin limitarse, células procedentes de microorganismos procariotas no anhidrobiontes que generalmente no sean esporulantes. The cell conserved by the composition of the present invention is a viable cell that is, it is capable of performing the normal functions of the cell including cell replication and division. On the other hand, the cell may have been treated, manipulated, mutated before its conservation. For example, but not limited, a cell may have been made competent for transformations or transfections, or it may contain recombinant nucleic acids. The cells that are conserved can form a homogeneous or heterogeneous population, for example, but not limited to, a library of cells in which each one contains a variation of some nucleic acid. Preferably, the cells are non-anhydrobotic cells (desiccation sensitive cells) such as, but not limited to, cells from non-anhydrobial prokaryotic microorganisms that are generally not sporulant.
La conservación del microorganismo puede mejorarse mediante cultivo bajo condiciones que aumenten la concentración intracelular de trehalosa o de otros estabilizadores formadores de cristales amorfos. Por ejemplo, pero sin limitarse,encondicionesdealta osmolaridad(alta concentracióndesales)que estimulenla producción intracelularde trehalosa o de otros estabilizantes formadores de cristales amorfos. The conservation of the microorganism can be improved by culture under conditions that increase the intracellular concentration of trehalose or other amorphous crystal-forming stabilizers. For example, but not limited to, high osmolarity conditions (high concentration of desalins) that stimulate the intracellular production of trehalose or other amorphous crystal-forming stabilizers.
El organismoinvertebrado es pero sin limitarse, una larvadeinsecto o un crustáceo. Dichos organismosinvertebrados pueden ser preservados en condiciones de desecación, permitiendo la actividad vital del mismo, de modo que, cuando se rehidratan, dichos organismos presentan la capacidad de movimiento. La plántula es una planta en sus primeros estadios de desarrollo, desde que germina hasta que se desarrollan las primeras hojas verdaderas. The invertebrate organism is, but is not limited to, a larvadeinsect or a crustacean. Said invertebrate organisms can be preserved under drying conditions, allowing the vital activity thereof, so that, when rehydrated, said organisms have the ability to move. The seedling is a plant in its early stages of development, from germinating until the first true leaves develop.
La composición de la invención puede usarse para la conservación de material biológico en estado seco, donde el material biológicoesunorganismovertebrado pertenecienteala SuperclaseTetrapoda(con cuatroextremidades), Clase Amphibia(anfibios)o Clase Reptilia (reptiles),o cualquierade sus partes(VernonyJackson, 1931. The biologicalbulletin,60:80-93).VernonyJacksonllevaronacaboun estudiosobrela ranaLeopardo(Rana pipiens), enel quedeformanaturalsesecasupiel,lengua,bazo,ehígadoconunapérdidadeaguadeentreun43-81%del contenido de agua total. The composition of the invention can be used for the conservation of biological material in the dry state, where the biological material is a vertebrate organism belonging to the Superclass Tetrapoda (with four extremities), Amphibia Class (amphibians) or Reptilia Class (reptiles), or any part thereof (VernonyJackson, 1931. The biologicalbulletin, 60: 80-93). Vernony Jackson has just finished a study on the frog Leopard (Rana pipiens), in the form of a natural one in his own leg, tongue, spleen, liver with a loss of water between 43-81% of the total water content.
Unórgano aislado,o un tejido biológico aislado (incluidala sangre) pueden conservarse mediantela composición de la presente invención. En Serrato et al. (2009) pueden observarse resultados de protocolos de crioperservación de tejidos biológicos (Serrato et al., 2009. Histology and histopathology, 24: 1531-1540). An isolated organ, or an isolated biological tissue (including blood) can be preserved by the composition of the present invention. In Serrato et al. (2009) results of cryopreservation protocols of biological tissues can be observed (Serrato et al., 2009. Histology and histopathology, 24: 1531-1540).
Una realización más preferida se refiere al uso de la composición de la invención, donde el material biológico es una molécula con actividad biológica. El término “molécula con actividad biológica” tal como se entiende en la presente invención se refiere a una molécula biológica cuyo origen sea un organismo vivo o que haya estado vivo, o derivados o análogos de dicha molécula. El término “derivados” se refiere a moléculas obtenidas por la modificación de una molécula con actividad biológica, que presentan una funcionalidad similar. Por otra parte, el término “análogos” se refierea moléculas quepresentan una función similarala molécula con actividadbiológica. A more preferred embodiment refers to the use of the composition of the invention, where the biological material is a molecule with biological activity. The term "molecule with biological activity" as understood in the present invention refers to a biological molecule whose origin is a living organism or that has been alive, or derivatives or analogs of said molecule. The term "derivatives" refers to molecules obtained by modifying a molecule with biological activity, which have similar functionality. On the other hand, the term "analogs" refers to molecules that have a similar function to the molecule with biological activity.
Según otra realización aún más preferida de la composición de la presente invención la molécula con actividad biológica es una enzima. Una realización todavía más preferida de la presente invención se refiere al uso de la composición de la invención, donde la enzima es una enzima con actividad lipasa. La enzima con actividad lipasa se selecciona de la lista de enzimas con números EC(Enzyme Commission numbers)que comprende las hidrolasas de éster carboxílico(EC 3.1.1)EC 3.1.1.1 (Carboxilesterasa),EC 3.1.1.2 (Arilesterasa),EC 3.1.1.3(Triacilglicerollipasa),EC 3.1.1.4(FosfolipasaA(2)),EC 3.1.1.5 (Lisofosfolipasa),EC 3.1.1.23 (Acilglicerol lipasa),EC 3.1.1.24(3oxoadipato enol-lactonasa),EC 3.1.1.25 (1,4-lactonasa),EC 3.1.1.26 (Galactolipasa),EC 3.1.1.32(FosfolipasaA(1)), EC3.1.1.33(6-acetilglucosa deacetilasa),EC 3.1.1.34 (Lipoproteína lipasa). Preferiblementelaenzima lipasa tiene actividadTriacilglicerollipasa (EC 3.1.1.3). According to another even more preferred embodiment of the composition of the present invention the molecule with biological activity is an enzyme. An even more preferred embodiment of the present invention refers to the use of the composition of the invention, where the enzyme is an enzyme with lipase activity. The enzyme with lipase activity is selected from the list of enzymes with EC numbers (Enzyme Commission numbers) comprising carboxylic ester hydrolases (EC 3.1.1) EC 3.1.1.1 (Carboxylesterase), EC 3.1.1.2 (Arilesterase), EC 3.1.1.3 (Triacylglycerollipase), EC 3.1.1.4 (PhospholipaseA (2)), EC 3.1.1.5 (Lysophospholipase), EC 3.1.1.23 (Acylglycerol lipase), EC 3.1.1.24 (3oxoadipate enol-lactonase), EC 3.1.1.25 (1,4-lactonase), EC 3.1.1.26 (Galactolipase), EC 3.1.1.32 (PhospholipaseA (1)), EC3.1.1.33 (6-acetylglucose deacetylase), EC 3.1.1.34 (Lipoprotein lipase). Preferably the enzyme lipase has Triacylglycerollipase activity (EC 3.1.1.3).
Otro aspectodelapresenteinvenciónse refiereaun métodode obtencióndela composiciónxeroprotectoradela invención que comprende: Another aspect of the present invention refers to a method of obtaining the protective composition of the invention comprising:
a) cultivarel microorganismodelainvención en un mediode cultivo con fructosa comofuentede carbono, a) cultivate the microorganism of the invention in a culture medium with fructose as carbon source,
b) deshidratar los microorganismos obtenidos en el cultivo del paso (a) hasta que tengan una humedad residual igual o inferior al 10%, b) dehydrate the microorganisms obtained in the culture of step (a) until they have a residual humidity equal to or less than 10%,
c) rehidratar los microorganismos deshidratados del paso (b) en un medio hipotónicoy c) rehydrate the dehydrated microorganisms of step (b) in a hypotonic environment
d) seleccionar la fracción líquida del producto obtenido en elpaso (c) que comprende la composición xeroprotectora. d) select the liquid fraction of the product obtained in step (c) comprising the xeroprotective composition.
El medio de cultivo es cualquier medio de cultivo conocido en el estado de la técnica para el crecimiento de un microorganismo de la presente invención, por ejemplo pero sin limitarse, el medio mineral M9. Medios ricos como el medio Luria Bertani(LB)enlosquehay presentesxeroprotectores,u osmolitos naturalesno sirven, porquela bacteria preferiría tomarlos del exterior a sintentizarlos. The culture medium is any culture medium known in the state of the art for the growth of a microorganism of the present invention, for example but not limited to, the M9 mineral medium. Rich media such as Luria Bertani (LB) media in which there are airborne protectors, or natural osmolytes, do not work, because the bacteria would prefer to take them from outside to synthesize them.
Una realización preferida de la presente invención se refiere al método de obtención de la composición xeroprotectora, donde el medio de cultivo del paso (a) es sólido. El término “sólido” tal como se entiende en la presente invención se refiere a un mediode cultivo gelificado en mayoro menor grado,es decir, que comprendeagarpara facilitar su gelificación o cualquier compuesto gelificante. A preferred embodiment of the present invention refers to the method of obtaining the xeroprotective composition, where the culture medium of step (a) is solid. The term "solid" as understood in the present invention refers to a medium of geli fi cated culture to a lesser degree, that is, comprising grasping to facilitate its gelling or any gelling compound.
La deshidratación de los microorganismos del paso (b) se lleva a cabo por medio de cualquier técnica conocida en el estado de la técnica. Otra realización preferida de la presente invención se refiere al método, donde la deshidratación de los microorganismos según el paso (b) se lleva a cabo por medio de una solución hipertónica o por medio de una corriente de aire. Preferiblemente la solución hipertónica o la corriente de aire tienen condiciones de esterilidad. La solución hipertónicaes una soluciónque tiene mayor concentraciónde solutoenel medioexternoqueenel citoplasma de la células del microorganismo de la presente invención, por tanto, la célula libera agua, es decir, se deshidrata. Dehydration of the microorganisms in step (b) is carried out by means of any technique known in the state of the art. Another preferred embodiment of the present invention refers to the method, where the dehydration of the microorganisms according to step (b) is carried out by means of a hypertonic solution or by means of an air current. Preferably the hypertonic solution or the air stream have sterility conditions. The hypertonic solution is a solution that has a higher concentration of solute in the external medium in the cytoplasm of the cells of the microorganism of the present invention, therefore, the cell releases water, that is, it is dehydrated.
La deshidratación de los microorganismos, descrita en el paso (b), se lleva a cabo en un medio hipotónico. El medio hipotónico o solución hipotónica es una solución que tiene menor concentración de soluto en el medio externo que en el citoplasma de la célula delmicroorganismo de la presente invención, por tanto, la célula recupera agua, es decir,se rehidrata.Una realización preferidamásse refiereal método, dondeelmedio hipotónicoparala rehidratación de los microorganismos según el paso (c) es agua parcial o totalmente destilada, parcial o totalmente desionizada o parcial o totalmente desmineralizada. The dehydration of the microorganisms, described in step (b), is carried out in a hypotonic medium. The hypotonic medium or hypotonic solution is a solution that has a lower concentration of solute in the external environment than in the cytoplasm of the microorganism cell of the present invention, therefore, the cell recovers water, that is, it is rehydrated. method, where the hypotonic medium for rehydration of microorganisms according to step (c) is water partially or completely distilled, partially or totally deionized or partially or totally demineralized.
Las célulasyel medio hipotónicohande estaren contactoal menos5minutos. Preferiblemente estaránen contacto al menos 20 minutos en agitación. La obtención del medio que contiene las sustancias estabilizantes se realizará por cualquier métodoquegaranticela separaciónde célulasdel contenido líquido, preferiblemente mediante centrifugado suave, seguido de un paso de filtración. Preferiblemente se utilizarán filtros de 0,4 micrómetros de diámetro de poro. The cells and the hypotonic medium will be in contact at least 5 minutes. Preferably they will be in contact for at least 20 minutes while stirring. Obtaining the medium containing the stabilizing substances will be carried out by any method that ensures the separation of cells from the liquid content, preferably by gentle centrifugation, followed by a filtration step. Preferably, fi lters of 0.4 micrometer in pore diameter will be used.
Otra realización preferida se refiere al método, donde además, la fracción líquida del paso (d) se deshidrata hasta que el producto xeroprotector tenga una humedad residual igual o inferior al 10%. Another preferred embodiment refers to the method, where in addition, the liquid fraction of step (d) is dehydrated until the xeroprotective product has a residual humidity equal to or less than 10%.
El producto xeroprotector de la invención se puede separar del medio de cultivo por cualquier método de concentración. Preferiblemente se utilizarán secadoresde tipo liofilizador que produzcanel estabilizador en estadoseco.Las moléculas estabilizadoras se podrán disolvero dispersar en una proporciónde entreel10yel 30%. Esta disolucióno dispersión se añadeal material biológico que se desee conservary se someteráa desecación. The xeroprotector product of the invention can be separated from the culture medium by any method of concentration. Preferably, lyophilizer-type dryers that produce the stabilizer in the dry state will be used. The stabilizing molecules may be dissolved or dispersed in a proportion between 10 and 30%. This dispersion solution is added to the biological material that is desired to be preserved and desiccated.
Otro aspecto de la presente invención se refiere al método para la conservación de material biológico que comprende: Another aspect of the present invention relates to the method for the conservation of biological material comprising:
a) mezclarla composicióndelainvención con una muestrade material biológico,y a) mixing the composition of the invention with a sample of biological material, and
b) deshidratar el producto obtenido en el paso (a) hasta una humedad residual igual o inferior al 10%. b) dehydrate the product obtained in step (a) to a residual humidity equal to or less than 10%.
Una realización preferida se refiere al método para la conservación de material biológico, donde el material biológico del paso (a) es un organismo invertebrado, una semilla, una plántula, un microorganismo, un órgano aislado, un tejido biológico aislado o una célula. A preferred embodiment refers to the method for the conservation of biological material, where the biological material of step (a) is an invertebrate organism, a seed, a seedling, a microorganism, an isolated organ, an isolated biological tissue or a cell.
Otra realización preferida se refiere al método para la conservación de material biológico, donde el material biológico es una molécula con actividad biológica. Another preferred embodiment refers to the method for the conservation of biological material, where the biological material is a molecule with biological activity.
Una realización más preferida de la presente invención se refiere al método, donde la molécula con actividad biológica es una enzima.Según una realización más preferidadela presenteinvenciónla enzima es una lipasa. A more preferred embodiment of the present invention refers to the method, where the molecule with biological activity is an enzyme. According to a more preferred embodiment of the present invention, the enzyme is a lipase.
Otrarealizaciónaúnmás preferidase refiereacualquieradelos métodosparala conservacióndematerial biológico, dondela deshidratacióndela mezcladela composiciónxeroprotectoraydel materialbiológicosegúnel paso(b)se lleva a cabo por medio de una solución hipertónica o por medio de una corriente de aire. Another embodiment is still more preferred than any of the methods for biological material preservation, where dehydration of the mixture of the protective composition and the biological material in step (b) is carried out by means of a hypertonic solution or by means of an air current.
Cualquier método para la conservación de material biológico de la presente invención permite el empleo de dicho material en procesos de manufacturación en los cuales el material biológico sería demasiado inestable si no estuviera conservado medianteelusodela composicióndela presenteinvención.Lainvención incluyeextrusioneso moldesen losquese incluyeel material conservadopor cualquier métododela presenteinvención.Un métodode producciónde una moldura que contengabiomaterial activomediante estabilizadores puede incluir soluciones de materiales plásticos. Por ejemplo una moldura de material plástico con biomaterial activo encapsulado puede ser útil como componentes de biosensores. Por ejemplo un biosensor puede incluir bacterias que sean capaces de detectar sustancias tóxicas, patógenos o toxicidad en general. Any method for the conservation of biological material of the present invention allows the use of said material in manufacturing processes in which the biological material would be too unstable if it were not conserved by using the composition of the present invention. The invention includes extrusion or molds in which the material conserved by any method of the present invention is included. A method of producing a molding containing biomaterial stabilizer material may include solutions of plastic materials. For example, a molding of plastic material with encapsulated active biomaterial can be useful as components of biosensors. For example, a biosensor can include bacteria that are capable of detecting toxic substances, pathogens or toxicity in general.
Alo largodela descripciónylas reivindicacionesla palabra “comprende”y susvariantes no pretendenexcluir otras características técnicas, aditivos, componenteso pasos.Para losexpertos enla materia, otros objetos,ventajas ycaracterísticas de la invención se desprenderán en parte de la descripciónyen parte de la práctica de la invención. Las siguientes figurasyejemplosse proporcionana modode ilustración,y nosepretendeque sean limitativosdela presente invención. Throughout the description and the claims the word "comprises" and its variants is not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and features of the invention will be derived in part from the description in part of the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
Descripción de las figuras Description of the fi gures
Con la intención de complementar la descripción que se ha llevado a cabo, así como de ayudar a un mejor entendimiento de las características de la invención, de acuerdo con algunos ejemplos realizados, se muestran aquí, con carácter ilustrativoy no limitante, las siguientes figuras: With the intention of complementing the description that has been carried out, as well as helping to better understand the characteristics of the invention, according to some examples made, the following figures are shown here, for illustrative and non-limiting purposes:
Fig. 1. Muestrala viabilidaddelosdistintos aislados bacterianos seleccionados medianteexposiciónacloroformo tras 24 horas de secado al aire. Fig. 1. Show the viability of the different bacterial isolates selected by chloroform exposure after 24 hours of air drying.
Las barrasde error muestranla desviación estándardeal menos tres réplicas. Error bars show the standard deviation of at least three replicas.
Los microorganismos representados en la figura son: The microorganisms represented in the figure are:
1J3A, 1J14, 2J2, 2J8A, 2J12B, 2J15B, 2J16A, 2J30, 3J18, 6J30, Acitenobacter calcoaceticus,Pseudomonas putida, además de la cepa 4J2A2. 1J3A, 1J14, 2J2, 2J8A, 2J12B, 2J15B, 2J16A, 2J30, 3J18, 6J30, Acitenobacter calcoaceticus, Pseudomonas putida, in addition to strain 4J2A2.
Fig. 2. Muestrala actividadlipasa(enporcentajerelativoaun100%de actividaddel control positivo)tras secado de la enzima lipasa en presencia de los distintos componentes de productos del ordeñado bacteriano al 10% (p/v). Fig. 2. Shows the activity lipase (in relative percentage at 100% positive control activity) after drying of the lipase enzyme in the presence of the different components of the 10% bacterial milking products (w / v).
El controlpositivoes trehalosaal 10%.El controlnegativo(-)secorresponde conla ausenciade compuesto alguno como aditivo previo a la desecación de la enzima. 4J2A2 es el valor de actividad lipasa registrada tras la estabilización por secadoyposterior reconstitucióndela enzima en presenciade POBextraídosdela cepa 4J2A2 mediante choque hiper/hipoosmótico respectivamente. 4J2A2D es la actividad lipasa registrada tras la estabilizaciónpor secadoy posterior reconstitucióndela enzima en presenciade POBSIA (Productode Ordeñado Bacteriano extraído por Secado mediante Incubación al Aire) extraídos de la cepa 4J2A2 mediante tratamiento de secado y posterior hidratación. The trehalose control device is 10%. The negative control (-) corresponds to the absence of any compound as an additive prior to the drying of the enzyme. 4J2A2 is the value of lipase activity recorded after stabilization by drying and subsequent reconstitution of the enzyme in the presence of POB extracted from strain 4J2A2 by hyper / hypoosmotic shock respectively. 4J2A2D is the lipase activity recorded after stabilization by drying and subsequent reconstitution of the enzyme in the presence of POBSIA (Bacterial Milking Product extracted by Drying by Air Incubation) extracted from strain 4J2A2 by drying and subsequent hydration treatment.
Fig. 3. Muestrala actividadlipasa(enporcentajerelativoaun100%de actividaddel control positivo)tras secado de la enzima lipasa en presencia de los distintos compuestos químicos (fructosa, ácido glutámico, beta-hidroxibutirato, acetatoylactatoal 10%). Fig. 3. Show the activity of lipase (in relative percentage at 100% positive control activity) after drying of the lipase enzyme in the presence of the different chemical compounds (fructose, glutamic acid, beta-hydroxybutyrate, 10% acetate and lactate).
El control positivo (C+) es trehalosa al 10%. El control negativo (C-) se corresponde con la ausencia de compuesto alguno como aditivo previo a la desecación de la enzima. 4J2A2D sintético es la mezcla de fructosa, ácido glutámico, beta-hidroxibutirato, acetatoy lactato al 10% en la misma proporción encontrada en el POBSIA de la cepa 4J2A2 mediante tratamientode secadoyposterior hidratación siendola proporciónde 16:4:0,8:1:1,4 respectivamente. The positive control (C +) is trehalose at 10%. The negative control (C-) corresponds to the absence of any compound as an additive prior to the drying of the enzyme. Synthetic 4J2A2D is the mixture of fructose, glutamic acid, beta-hydroxybutyrate, 10% acetate and lactate in the same proportion found in the POBSIA of strain 4J2A2 by treatment of drying and subsequent hydration being the ratio of 16: 4: 0.8: 1: 1, 4 respectively.
Fig. 4. Muestra la evolución de la supervivencia de Escherichia coli MC4100 frente a desecación mediante el empleo del producto de ordeñado bacteriano (POB) así como los extraídos por Secado mediante Incubación al Aire (POBSIAs). Fig. 4. It shows the evolution of the survival of Escherichia coli MC4100 against desiccation through the use of the bacterial milking product (POB) as well as those extracted by Air Drying (POBSIAs).
4J2A2 es el valor de actividad lipasa registrada tras la estabilización por secado y posterior reconstitución de la enzima en presencia de POB extraídos de la cepa4J2A2 mediante choque hiper/hipoosmótico respectivamente. 4J2A2D es la actividad lipasa registrada tras la estabilización por secadoyposterior reconstitución de la enzima en presencia de POBSIA. 4J2A2 is the value of lipase activity recorded after stabilization by drying and subsequent reconstitution of the enzyme in the presence of POB extracted from strain 4J2A2 by hyper / hypoosmotic shock respectively. 4J2A2D is the lipase activity recorded after stabilization by drying and subsequent reconstitution of the enzyme in the presence of POBSIA.
PVP indica que además se ha adicionado 1,5% de polivinilpirrolidona (PVP). PVP indicates that 1.5% polyvinylpyrrolidone (PVP) has also been added.
SIN es sintético. SIN is synthetic.
Tes trehalosa. Trehalose tees.
Ejemplos Examples
Acontinuación se ilustrará la invención mediante unos ensayos ilustrativosyde carácter no limitante, realizados por los inventores que describen el aislamiento de la cepa de la presente invención así como la capacidad de producción de compuestos xeroprotectores. En adelante se puede hacer referencia a la cepa CECT7625 con el término “4J2A2”. The invention will be illustrated below by means of illustrative and non-limiting tests carried out by the inventors describing the isolation of the strain of the present invention as well as the production capacity of xeroprotective compounds. Hereinafter reference can be made to strain CECT7625 with the term "4J2A2".
Ejemplo1 Example 1
Aislamiento del microorganismo 4J2A2 (CECT7625), extracción de POB (Productos del Ordeñado Bacteriano) y ensayo de xeroprotección de enzimas Isolation of the 4J2A2 microorganism (CECT7625), extraction of POB (Bacterial Milking Products) and enzyme xeroprotection assay
Se homogeneizó1gde suelo seco, procedentede Granada (37.182 LatitudNy3.624 LongitudO),noexpuestoa lluvia, ni riego, por un periodo superior a tres meses. El suelo se tomó del área circundante a raíces de adelfa(Nerium oleander). Las muestras fueron homogeneizadas para obtener un granode tierra fino quegarantizara su contacto con el cloroformo.La tierrase depositóen vialesde vidrioalosqueseles añadió3mlde cloroformo puro.Trasla adición del cloroformo se incubaron a temperatura ambiente durante 30 minutos con agitación esporádica para maximizar el contactodela muestra conel disolvente.Para eliminarel cloroformodelas muestras unavez transcurridoel tiempo de contacto, las muestras de suelo fueron depositadas en placa petri de vidrio estéril sin tapadera hasta completar la evaporación del cloroformo. Una vez secas las muestras de suelo se resuspendieron en 10 ml de TSB. Con las suspensiones de suelo se realizaron diluciones seriadasy se sembraron en placas de TSA que se incubaron 48 horas a 30ºC.Trascurrido este tiempo se cuantificó el número de unidades formadoras de colonias (UFC) por mililitro de cada muestra. Así, se detectaron2·105 UFC/g de suelo en las muestras sin tratar, mientras que estas se redujeron a 1,3·105 UFC/g de suelo tras 30 minutos de tratamiento. 1g of dry soil, from Granada (37,182 Latitude N and 3,624 Longitude O), not exposed to rain, or irrigation, was homogenized for a period exceeding three months. The soil was taken from the area surrounding oleander roots (Nerium oleander). The samples were homogenized to obtain a fine earth grain that guaranteed their contact with the chloroform. The earth was deposited in glass vials to which 3ml of pure chloroform was added. After the addition of the chloroform they were incubated at room temperature for 30 minutes with sporadic agitation to maximize the contact of the sample with the solvent. Remove the chloroform samples once the contact time has elapsed, the soil samples were deposited in sterile glass petri dish without a lid until the evaporation of the chloroform is complete. Once the soil samples were dried, they were resuspended in 10 ml of TSB. Serial dilutions were made with the soil suspensions and seeded in TSA plates that were incubated 48 hours at 30 ° C. After this time the number of colony forming units (CFU) per milliliter of each sample was quantified. Thus, 2 · 105 CFU / g of soil was detected in the untreated samples, while these were reduced to 1.3 · 105 CFU / g of soil after 30 minutes of treatment.
Se seleccionaron aleatoriamente 36 cepasypara identificar las cepas que producían esporas se realizó un ensayo basadoenla diferente sensibilidaddelas célulasvegetativas con respectoalas esporasal tratamiento con calory en basealmétodo descritoporVilchezycolaboradores(Vilchez et al., 2008. Extremophiles, 12: 297-299). De esta forma se tomaron colonias independientes procedentes de placas de TSA de al menos 24 horas de crecimiento. Estas colonias se resuspendieronen1mlde soluciónM9en microtubos estérilesde1,5ml.Seguidamentese sembraron10 µlde esta suspensión en TSA. Acto seguido se incubó el resto de la suspensión en termobloque Mixing Block MB-102 a 72ºC durante 30 minutos. Nuevamente se tomaron 10 µlde cadamuestrayse sembraron en placade TSA. Aquellas cepas con capacidad para tolerar el tratamiento con calor se consideraron como esporulantes, mientras que aquellas que no toleraronel tratamiento por calor se consideraron no esporulantes. Como controles positivosy negativos se utilizaron colonias de Bacillus pumilus ydeBurkholderia cepacia respectivamente. La proporción de cepas esporulantes pasó deniveles inferioresal 40% para muestras no tratadasaniveles superioresal 50% tras30 mindeexposicióndelas muestras de suelo al cloroformo. 36 strains were randomly selected and to identify the spore-producing strains, an assay was conducted based on the different sensitivity of the vegetative cells with respect to the sporesal treatment with calory on the basis of the method described by Vilchezy and collaborators (Vilchez et al., 2008. Extremophiles, 12: 297-299). In this way independent colonies from TSA plates of at least 24 hours of growth were taken. These colonies were resuspended in 1 ml of M9 solution in sterile 1.5 ml microtubes. 10 µl of this suspension was then seeded in TSA. The rest of the suspension was then incubated in Mixing Block MB-102 thermoblock at 72 ° C for 30 minutes. Again 10 µl of each sample was taken and seeded on TSA plate. Those strains capable of tolerating heat treatment were considered sporulants, while those that did not tolerate heat treatment were considered non-sporulants. As positive and negative controls, colonies of Bacillus pumilus and Burkholderia cepacia were used respectively. The proportion of sporulant strains passed levels lower than 40% for untreated samples levels above 50% after 30 min of exposure of soil samples to chloroform.
Con el fin de analizar la capacidad de tolerar la desecación de las cepas aisladas no esporulantes se realizó un estudiode desecaciónalaire.Enesteensayoseincluyóunacepade Acinetobacter calcoaceticus (PADD68) aislada del desiertodeTabernas(Almería) identificada como toleranteala desecaciónenun estudioprevioyquese utilizó como control positivo. Además también se incluyó en el estudio células de Pseudomonas putida KT2440 que se utilizaron como controles negativosal ser unacepa sensibleala desecación (Antheunisse et al., 1981. Antonie Leeuwnhoek, 47: 539-545; Manzanera et al.,2002.Appl.Environ. Microbiol.,68:4328-4333).Paraeste ensayose utilizaron colonias independientes de las 17 cepas identificadas como no esporulantes aisladas en el estudio anteriory procedentes de placasde TSAde72 horas para calcular sunivelde toleranciaala desecación taly como se indicaa continuación. In order to analyze the ability to tolerate the desiccation of isolated non-sporulant strains, a study of desiccation was conducted in the air. In this case, an Acinetobacter calcoaceticus (PADD68) strain isolated from the Tabernas desert (Almería) identified as tolerant to desiccation was used in a positive study and used as a positive control. In addition, Pseudomonas putida KT2440 cells that were used as negative controls were also included in the study, being a sensitive strain at drying (Antheunisse et al., 1981. Antonie Leeuwnhoek, 47: 539-545; Manzanera et al., 2002.Appl.Environ. Microbiol., 68: 4328-4333) .For this trial they used independent colonies of the 17 strains identified as non-sporulants isolated in the previous study and from 72-hour TSA plates to calculate the level of tolerance to desiccation as indicated below.
Para calcular la tolerancia a la desecación se partió de colonias aisladas procedentes de placas de TSA de 48 horas de crecimiento. Utilizando un asa estéril se tomó una única colonia que se resuspendió en1 ml de solución M9 estéril.Partiendode esta suspensión celular se realizaron diluciones seriadas que se sembraron en placasde TSA con objeto de identificar el número de UFC/ml de partida. Por otra parte se tomaron 100 µlde cada suspensiónyse depositaron en gotas de5-10 µlsobre microplacas de petri estériles sin medio. Las microplacas se situaron bajo una corriente de aire estéril en una campana de flujo laminar durante 24 horas. Las placas quedaron secas tras2-3horas de incubación. Transcurridas 24 horas de incubación se resuspendieron en 1 ml de solución M9 estéril. Partiendo de esta solución se realizaron diluciones seriadas que se sembraron en placas de TSA. Del recuento de UFC/ml de esta segunda siembra se calcularon las proporciones de supervivencia en referencia alprimer conteo. Estos ensayos se realizaron por triplicado. Los resultados se expresaron como la media de los tres ensayos en porcentaje, tomando como referenciadel 100%los datos húmedos.La desviación estándarala media permitióel cálculodelaTde student para estudiar si las diferencias en supervivencia fueron significativas. De las 17 cepas aisladas en estas condiciones se identificó el microorganismo 4J2A2por ser una cepa con niveles de tolerancia a la desecación significativamente superioresalas del control positivo Acinetobacter calcoaceticus (PADD68). Dicha cepa 4J2A2 pertenece algénero Rhodococcus sp. Estos resultados supusieron un 17% de aislados tolerantes a la desecación frente a un 0,5% de los aislados de las mismas muestras de suelo sin tratar con cloroformo. Esta cepa se caracterizó mediante secuenciación del ADNr 16Sy comparación de la secuencia con los presentes en la bases de datos, así como mediante estudios metabólicos BIOLOG e hibridación del ADN-ADN con la especie más cercana. La Fig. 1 muestra los valores de tolerancia de las cepas aisladas. To calculate the tolerance to desiccation, we started from isolated colonies from TSA plates with 48 hours of growth. Using a sterile handle, a single colony was taken that was resuspended in 1 ml of sterile M9 solution. Serial dilutions were made from this cell suspension that were seeded on TSA plates in order to identify the starting CFU / ml number. On the other hand, 100 µl of each suspension was taken and deposited in drops of 5-10 µl on sterile petri dishes without medium. The microplates were placed under a stream of sterile air in a laminar flow hood for 24 hours. The plates were dried after 2-3 hours of incubation. After 24 hours of incubation, they were resuspended in 1 ml of sterile M9 solution. Starting from this solution, serial dilutions were made and seeded on TSA plates. From the CFU / ml count of this second planting, the survival rates were calculated in reference to the first count. These tests were performed in triplicate. The results were expressed as the average of the three trials in percentage, using 100% wet data as reference. The standard deviation of the average allowed the calculation of the student's T to study if the differences in survival were significant. Of the 17 strains isolated under these conditions, the microorganism 4J2A2 was identified as a strain with significantly higher levels of drying tolerance than those of the positive control Acinetobacter calcoaceticus (PADD68). Said strain 4J2A2 belongs to Rhodococcus sp. These results accounted for 17% of isolates tolerant to desiccation versus 0.5% of the isolates of the same soil samples untreated with chloroform. This strain was characterized by sequencing the 16S rDNA and comparing the sequence with those present in the databases, as well as by BIOLOG metabolic studies and DNA-DNA hybridization with the nearest species. Fig. 1 shows the tolerance values of the isolated strains.
Paralaextraccióndelosproductosdel ordeñado bacterianoyconelfinde identificarlas moléculas acumuladas con capacidadparaproteger biomoléculas sensiblesala desecaciónse recurrióaunaestrategia basadaentres pasos.En un primer paso se realizó una extracción de las moléculas acumuladas mediante una variación de la técnica conocida como “ordeñado bacteriano” generada por los inventores. En un segundo paso se realizó un ensayo de xeroprotección con las sustancias obtenidas para identificar la capacidad de las mismas para proteger enzimas frente a la desecación. For the extraction of the products of the bacterial milking and with the purpose of identifying the accumulated molecules with the capacity to protect sensitive biomolecules on drying, a strategy was used based on three steps. In a first step an extraction of the accumulated molecules was carried out by means of a variation of the technique known as “bacterial milking” generated by the inventors. In a second step, an xeroprotection test was carried out with the substances obtained to identify their capacity to protect enzymes against desiccation.
Dado quela producciónyacúmulode sustanciasxeroprotectoras se realizó en medio con minerales, se procedióa identificarlas fuentesde carbonomás apropiadasparael cultivodelas cepasen medio mineralM9.Paralaobtenciónde sustancias con capacidadxeroprotectora se cultivaron enmedio mineral conla fuentede carbono apropiada (fructosa) las célulasde dicha cepa hastala obtenciónde una biomasa suficiente.Tras este acúmulo,lascélulasse depositaron sobreplacasdelmismomedioconagarparala produccióndemedio sólido.Entrelas célulasyelmediosedepositó un filtro estérilde0,4micraspara permitirelpasode nutrientesalas célulasy su posterior recogidaymanipulación. Las placas con filtrosycélulas se sometierona un secado por corrientede aire estéril durante24 horas. Como control positivo se incluyó Halomononas elongata, dado que es una cepa reconocida como halotolerante (SaberyGalinski, 1998. Biotechnol. Bioeng., 57: 306-313)y a P. putidaKT2440 como cepa halosensible (De Castro et al., 2000. Appl. Environ. Microbiol., 66:4142-4144). Given that the production and accumulation of airborne substances was carried out in the middle with minerals, the most appropriate carbon sources were identified for the cultivation of the strains in mineral medium M9. For the collection of substances with an air-protective capacity the cells of said strain were grown with the appropriate source of carbon (fructose) the cells of said strain until obtaining their biomass. , the cells were deposited on the plates of the average medium to the solid medium production. Between the cells and the medium a sterile filter was placed 0.4 microns to allow the passage of nutrients to the cells and their subsequent collection manipulation. The plates with fi lters and cells are subjected to sterile air drying for 24 hours. Halomononas elongata was included as a positive control, since it is a strain recognized as a halotolerant (SaberyGalinski, 1998. Biotechnol. Bioeng., 57: 306-313) and P. putidaKT2440 as a halosensitive strain (De Castro et al., 2000. Appl. Environ. Microbiol., 66: 4142-4144).
Con este fin se inocularon la cepa seleccionada como hipertolerantes a desecación 4J2A2. Además se incluyó a H. elongata como control positivo dado que ya había sido empleada por Sauery Galinski para la obtención de hidroxiectoína,ycomo control negativo se incluyó E. coli como ejemplode cepa haloyxerosensible. Dichos inóculos se incubaron en agitación durante 48 horas. A continuación, se centrifugaron las muestras durante 10 minutos a To this end, the strain selected as hypertolerants at 4J2A2 drying was inoculated. In addition, H. elongata was included as a positive control since it had already been used by Sauery Galinski to obtain hydroxyectoin, and as a negative control, E. coli was included as an example of a haloxerosensitive strain. Said inoculums were incubated with shaking for 48 hours. The samples were then centrifuged for 10 minutes at
10.000 rpm enuna centrifuga BeckmanAvanti-J25y se retiróla fracción sobrenadante, resuspendiendoel sedimento bacteriano en 20 ml de solución M9 estéril y se depositaron sobre filtros. Tras 24 horas, los filtros, sometidos a desecación mediante una corrientede aire estéril, se separaron del medioyse depositaron en tubos con agua destilada estéril dejando incubar20 minutosa 30ºCy150 rpm. Consecutivamente se repitióel mismo procesode centrifugado, se desechóel precipitado bacterianoyse filtróel sobrenadante(utilizandoun filtrode0,22 µm). El producto filtrado de cada muestra se dividió en dos fracciones, una de las cuales se utilizó para determinar la actividad xeroprotectora del sobrenadante (fracciónde ordeñado)yla otraparala identificaciónycaracterizacióndelos compuestos presentesen esta fracción. Ambas fraccionesse sometieronaun procesode secado utilizandoun liofilizador(LabconcoFreezone 6)durante 48 horas obteniéndose un sedimento que fue resuspendido en 100 µl de agua milliQ estéril. Una vez liofilizado, el Producto de Ordeñado Bacteriano (POB) se solubilizó en 100 microlitros de agua. Estas solucionesde POBs se utilizaron en estudios de xeroprotección. 10,000 rpm in a BeckmanAvanti-J25 centrifuge and the supernatant fraction was removed, the bacterial sediment was resuspended in 20 ml of sterile M9 solution and deposited on fi lters. After 24 hours, the filters, subjected to desiccation by means of a sterile air stream, were separated from the medium and deposited in tubes with sterile distilled water, allowing incubation for 20 minutes at 30 ° C and 150 rpm. The same centrifugation process was subsequently repeated, the bacterial precipitate was discarded and the supernatant filtered (using a 0.22 µm filter). The filtered product of each sample was divided into two fractions, one of which was used to determine the x-protective activity of the supernatant (milked fraction) and the other for the identification and characterization of the compounds present in this fraction. Both fractions were subjected to a drying process using a lyophilizer (Labconco Freezone 6) for 48 hours, obtaining a sediment that was resuspended in 100 µl of sterile milliQ water. Once lyophilized, the Bacterial Milking Product (POB) was solubilized in 100 microliters of water. These POBs solutions were used in xeroprotection studies.
En la tabla1 se pueden observar las composiciones de los productos de ordeñado bacteriano de la cepa 4J2A2 tras su extracción mediante choque hiper/hipoosmótico (4J2A2), o tras su extracción por secado mediante incubación alaire (4J2A2D). Asimismo,enlaFig.3 se muestralaactividadlipasa(en porcentaje relativoaun100%deactividad del control positivo) tras secado de la enzima lipasa en presencia de los distintos compuestos químicos dela composición 4J2A2D sintética (fructosa, ácido glutámico, beta-hidroxibutirato, acetatoylactato al 10%). El control positivo es trehalosaal 10%.Elcontrol negativo se corresponde conla ausenciade compuestos como aditivo previoa la desecación de la enzima. 4J2A2D sintético es la mezcla de fructosa, ácido glutámico, beta-hidroxibutirato, acetato ylactato al 10% en la misma proporción encontrada en el POBSIA de la cepa 4J2A2 mediante tratamiento de secado por incubaciónal aireyposterior hidratación siendola proporciónde fructosa, ácido glutámico, beta-hidroxibutirato, acetatoylactato,de 16:4:0,8:1:1,4 respectivamente. Table 1 shows the compositions of the bacterial milking products of strain 4J2A2 after extraction by hyper / hypoosmotic shock (4J2A2), or after extraction by drying by means of alaire incubation (4J2A2D). Likewise, Fig. 3 shows the activity of lipase (in relative percentage of 100% of the positive control activity) after drying of the lipase enzyme in the presence of the different chemical compounds of the synthetic composition 4J2A2D (fructose, glutamic acid, beta-hydroxybutyrate, 10% acetate and lactate). The positive control is 10% trehalose. The negative control corresponds to the absence of compounds as an additive prior to the drying of the enzyme. Synthetic 4J2A2D is the mixture of fructose, glutamic acid, beta-hydroxybutyrate, acetate and 10% lactate in the same proportion found in the POBSIA of strain 4J2A2 by air-drying treatment and subsequent hydration being the proportion of fructose, glutamic acid, beta-hydroxybutyrate , acetate: 16: 4: 0.8: 1: 1.4, respectively.
Tal como puede observarse en dicha Fig. 3, la combinación de los compuestos fructosa,ácido glutámico, betahidroxibutirato, acetato y lactato (en igual proporción que la mostrada en la tabla 1 (4J2A2D) presenta un efecto sinérgicoenla conservacióndela actividad lipasayaquela sumadela actividad lipasa mostradapor composiciones que contienen los compuestos de forma aislada es menor que el resultado obtenido con dicha mezcla. As can be seen in said Fig. 3, the combination of the compounds fructose, glutamic acid, betahydroxybutyrate, acetate and lactate (in the same proportion as shown in Table 1 (4J2A2D) has a synergistic effect in the conservation of lipasayaquela activity added to the lipase activity shown by Compositions containing the compounds in isolation is less than the result obtained with said mixture.
TABLA1 TABLE 1
Composición del producto de ordeñado bacteriano (POB) de la cepa 4J2A2 Composition of the bacterial milking product (POB) of strain 4J2A2
El objetivo de este ensayo fue determinar la capacidad de la fracción producto del ordeñado bacteriano para proteger enzimas frenteala desecación.Para ello se utilizóla enzima lipasa.Partiendode1 µl que contenía 0,00554 unidades de lipasa de Burkholderia cepacia (Sigma-Aldrich 62309-100 mg) se adicionaron 15 µlde la fracción producto del ordeñado bacteriano a estudiar. Como control positivo se adicionaron 15 µl de una solución al 10% de trehalosa a1 µl(0,00554U)de soluciónde lipasaycomo controlnegativose añadió15 µlde aguaa1µl(0,00554 U) de solución de lipasa. Las mezclas de 16 µlcon lipasa se depositaron en un microtubo de2 ml de capacidady se secaron a 50ºC durante 120 minutos. Unavez secas se incubarona 100ºC durante5minutos. Finalmente fueron almacenadasenun desecadoratemperatura ambiente durante24 horas.Pasadoel tiempode incubación,las reacciones se resuspendieron en 50 µlde una solucióndeTrisHCl(50mM)yse transfirierona un microtubo junto con 950 µl deTrisHCl(50mM)pH8y1mlde Soluciónde Sustrato.La determinacióndela capacidadxeroprotectoradecada fracción producto de ordeñado bacteriano (POB) se determinó por el ensayo de medición de la actividad lipasa. The objective of this test was to determine the ability of the product fraction of the bacterial milking to protect enzymes against desiccation. To this end, the enzyme lipase was used, leaving 1 µl containing 0.00554 units of lipase from Burkholderia cepacia (Sigma-Aldrich 62309-100 mg ) 15 µl of the product fraction of the bacterial milking to study were added. As a positive control, 15 µl of a 10% solution of trehalose was added to 1 µl (0.00554U) of lipase solution and as negative control was added 15 µl of water to 1 µl (0.00554 U) of lipase solution. The 16 µl mixtures with lipase were deposited in a 2 ml capacity microtube and dried at 50 ° C for 120 minutes. Once dry, it is incubated 100 ° C for 5 minutes. Finally, they were stored in a dried room temperature for 24 hours.After incubation time, the reactions were resuspended in 50 µl of a solution of TrisHCl (50mM) and a microtube was transferred together with 950 µl of TrisHCl (50mM) pH8 and 1ml of Substrate Solution. ) was determined by the lipase activity measurement test.
Parala medidadelaactividadlipasase utilizóunavariacióndel método descritoporGuptaycolaboradores(2002) consistente en la cuantificación espectrofotométrica del p-nitrofenol liberado por la enzima lipasa de Burkholderia cepacia (Sigma-Aldrich 62309-100mg)a partirdel sustrato p-nitrofenol palmitato (pNPP) (Gupta et al., 2002. Analytical Biochemistry,311:98-99).Paraelloseutilizó1mlde mediolibrede células(985 µldeTris-HCl 0,05M juntoa 15 µldePOB obtenidoporel métododel “ordeñado bacteriano”) mezclado con1mlde solución sustratodeun cultivo enfase estacionaria.Esta mezclade ensayoseincubóa30ºC durante30 minutosen microtubos estérilesde2 ml.La reacciónseparó mediante incubacióna 100ºC durante4minutosen termobloquey2minutosa-20ºC.La absorbancia se midió en un espectrofotómetro HitachiU-2000a una longitudde ondade 410 nm.La solución sustrato (SS)se preparó mezclando10mlde soluciónA(30mgdepNPPen10mlde isopropanol) con90mlde soluciónB(0,1gde gomaarábigay0,4mldeTritónX-100en90mltampónTris-HCl50mMpH8).Lamezclade soluciónAyBseagitó suavemente hasta su totaldisolución.La Fig.2muestra losvaloresde toleranciade las cepas aisladas. For the measurement of the activity, lipase was used an variation of the method described by Gupta and collaborators (2002) consisting of the spectrophotometric quantification of the p-nitrophenol released by the enzyme lipase from Burkholderia cepacia (Sigma-Aldrich 62309-100mg) from the substrate p-nitrophenol palmitate (Gppta p. 2002). Analytical Biochemistry, 311: 98-99) For 1 ml of half-free cells (985 µl of Tris-HCl 0.05M together with 15 µl of POP obtained by the "bacterial milking" method) mixed with 1 ml of substrate solution of a stationary culture culture, this mixture was incubated at 30 ° C for 30 minutes in 30 minutes The reaction was separated by incubation at 100 ° C for 4 minutes in thermoblock and 2 minute-20 ° C. The absorbance was measured in a HitachiU-2000 spectrophotometer at a wavelength of 410 nm. The substrate solution (SS) was prepared by mixing 10ml of solution A (30mg of PNP in 10ml of isopropanol) with 90ml of rubber B and 0.4 ml of rubber B nX-100en90mltampónTris-HCl50mMpH8) .Lamezclade soluciónAyBseagitó gently until totaldisolución.La Fig.2muestra losvaloresde toleranciade isolates.
Ejemplo2 Example 2
Ensayo de xeroprotección de microorganismos Xeroprotection test of microorganisms
El objetivo de este ensayo fue determinar la capacidad para proteger células vivas de Escherichia coli MC4100 frentea desecación medianteelempleodelproductode ordeñado bacteriano(POB)asícomolosextraídosporSecado mediante IncubaciónalAire(POBSIAs)dediversascepasxerotolerantesdeformaanálogaala descritapor Manzanera et al., (2002) (Manzanera et al., 2002. Applied andEnvironmental Microbiology 68: 4328-33).Para ello se utilizó un preinóculo de E. coli, a partir del cual se inoculó medio mínimo más glucosa como fuente de carbono adicionados de 0,6Mde NaCl hasta alcanzar una densidad óptica inicialde 0,05.Tras12 horasde incubacióna 37ºC en agitación, se centrifugaron alícuotas de1 ml del cultivo crecido. Las células de E. coli se resuspendieron en una solución al 10%de los POBso POBSIAsextraídosde las célulasxerotolerantes(extraídos según se describe anteriormente)y además,1,5%depolivinilpirrolidona(PVP). Igualmentese realizólamismaexperiencia mediantela combinacióny mezclade sustratos comerciales identificados en POBsy POBSIAs en iguales proporciones, a los que se llamaron POB sintético o POBSIA sintético en contraposición al directamente extraído de células que llamamos natural. En el caso de los POB/POBSIA sintéticos la mezcla se realizó en una solución al 34,2%. La suspensión de células en las distintas solucionesse sometierona condicionesde desecaciónporvacíosin congelación,enun congelador-desecador modificado (Dura -Stop µ P;FTS Systems, Stone Ridge,NY)a 30ºCde temperatura mediay100mTorr(2Pa; 2x10−5 atmósferas) durante 20 segundos, con una rampa de temperatura de 2,5ºC/min con 15 minutos de pausa después de cada incremento de 2ºC, hasta llegar a la temperatura máxima de 40ºC. The objective of this test was to determine the ability to protect live cells of Escherichia coli MC4100 against desiccation through the use of bacterial milked product (POB) as well as extracted by Drying by Incubation to Air (POBSIAs) of various types of allergen-deforming and analogous described by Manzanera et al., 2002, Applied (2002). andEnvironmental Microbiology 68: 4328-33) .To that end, a pre-circle of E. coli was used, from which a minimum medium plus glucose was inoculated as an added carbon source of 0.6M NaCl until an initial optical density of 0.05 was reached. After 12 hours of incubation at 37 ° C under stirring, 1 ml aliquots of the grown culture were centrifuged. The E. coli cells were resuspended in a 10% solution of the POOBs or POBSIA extracted from the xerotolerant cells (extracted as described above) and in addition, 1.5% depolivinylpyrrolidone (PVP). The same experience was also carried out through the combination and mixture of commercial substrates identified in POBs and POBSIAs in equal proportions, which were called synthetic POB or synthetic POBSIA as opposed to the directly extracted from cells we call natural. In the case of synthetic POB / POBSIA, the mixing was performed in a 34.2% solution. The suspension of cells in the different solutions is subjected to freeze-drying conditions in a modified freezer-desiccator (Dura -Stop µP; FTS Systems, Stone Ridge, NY) at 30 ° C medium temperature and 100mTorr (2Pa; 2x10-5 atmospheres) for 20 seconds, with a temperature ramp of 2.5ºC / min with 15 minutes of pause after each increase of 2ºC, until reaching the maximum temperature of 40ºC.
Las muestras se sellaronalvacíoy se almacenarona 30ºC hasta su ensayode viabilidada tiempo1,15y30 días. Pasado este tiempo las muestras se resuspendieron en1 ml de LBy se realizaron ensayos de viabilidad mediante siembra en placadeLB sólido que se incubaron24 horasa 37ºC, parael conteode UFCycomparación con las UFC antes del secado, para de esta forma poder calcular la supervivencia de las muestras. The samples were sealed under vacuum and stored at 30 ° C until their feasibility test time 1,15 and 30 days. After this time the samples were resuspended in 1 ml of LB and viability tests were carried out by solid plate seeding that was incubated 24 hours at 37 ° C, for the CFU count and comparison with the CFUs before drying, in order to calculate the survival of the samples.
En la Fig.4 se observa cómo las composiciones sintéticas POBsy POBSIAs de la cepa 4J2A2 produjeron un aumento de la supervivencia de los microorganismos respecto de la supervivencia experimentada por dichos microorganismos mediante una solución de trehalosa, cuando las soluciones estaban al 34,2% de dichos compuestos xeroprotectoresyduranteel primerdíade conservaciónasí comoalasdos semanas.Seobservaquela aportacióndelPVP 1,5% a la supervivencia es nula. In Fig. 4 it is observed how the synthetic compositions POBs and POBSIAs of strain 4J2A2 produced an increase in the survival of the microorganisms with respect to the survival experienced by said microorganisms by means of a trehalose solution, when the solutions were at 34.2% of said airborne compounds during the first day of conservation as well as for two weeks. Note that the contribution of PVP 1.5% to survival is nil.
Claims (28)
- 1. one.
- Microorganismo de la especie bacteriana Rhodococcus sp. con número de acceso CECT7625. Microorganism of the bacterial species Rhodococcus sp. with access number CECT7625.
- 2. 2.
- Población bacteriana que comprende el microorganismo según la reivindicación 1. Bacterial population comprising the microorganism according to claim 1.
- 3. 3.
- Uso delmicroorganismo segúnla reivindicación1odela población bacteriana segúnla reivindicación2parala producción de una composición xeroprotectora. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 for the production of an xeroprotective composition.
- 4. Four.
- Composición xeroprotectora producida por el microorganismo según la reivindicación 1 o por la población bacteriana según la reivindicación 2. Xeroprotective composition produced by the microorganism according to claim 1 or by the bacterial population according to claim 2.
- 6. 6.
- Composiciónsegúnlareivindicación5que comprende una proporciónde fructosa:ácido glutámico:acetato,de entre (35y45):(1,4y3,4):(0,5y1,5) respectivamente. Composition according to claim 5 comprising a proportion of fructose: glutamic acid: acetate, between (35 and 45) :( 1.4 and 3.4) :( 0.5 and 1.5) respectively.
- 7. 7.
- Composición según la reivindicación 6, donde la proporción de fructosa:ácido glutámico:acetato es de entre (38 y44):(2y3):(0,7y1,3), respectivamente. Composition according to claim 6, wherein the proportion of fructose: glutamic acid: acetate is between (38 and 44) :( 2y3) :( 0,7y1,3), respectively.
- 8. 8.
- Composición según la reivindicación5que comprende una proporción defructosa:ácido glutámico:β-hidroxibutirato:acetato:lactato,de entre (14y18):(3y5):(0,6y1):(0,5y1,5):(1y2), respectivamente. Composition according to claim 5 which comprises a ratio of fructose: glutamic acid: β-hydroxybutyrate: acetate: lactate, between (14y18) :( 3y5) :( 0.6y1) :( 0.5y1.5) :( 1y2), respectively.
- 9. 9.
- Composición segúnla reivindicación8, dondela proporciónde fructosa:ácidoglutámico:β-hidroxibutirato:acetato:lactatoesde entre(15y17):(3,5y4,5):(0,7y0,9):(0,7y1,2):(1,2y1,6), respectivamente. Composition according to claim 8, wherein the proportion of fructose: glutamic acid: β-hydroxybutyrate: acetate: lactate between (15 and 17) :( 3.5 and 4.5) :( 0.7 and 0.9) :( 0.7 and 1.2) :( 1.2 and 1 , 6), respectively.
- 11. eleven.
- Uso de la composición según la reivindicación 10, donde el material biológico es un microorganismo o una célula. Use of the composition according to claim 10, wherein the biological material is a microorganism or a cell.
- 12. 12.
- Uso de la composición según la reivindicación 10, donde el material biológico es un organismo invertebrado, una semilla, una plántula, un órgano aislado o un tejido biológico aislado. Use of the composition according to claim 10, wherein the biological material is an invertebrate organism, a seed, a seedling, an isolated organ or an isolated biological tissue.
- 13. 13.
- Uso de la composición según la reivindicación 10, donde el material biológico es una molécula con actividad biológica. Use of the composition according to claim 10, wherein the biological material is a molecule with biological activity.
- 14. 14.
- Uso de la composición según la reivindicación 13, donde la molécula con actividad biológica es una enzima. Use of the composition according to claim 13, wherein the molecule with biological activity is an enzyme.
- 15. fifteen.
- Uso de la composición según la reivindicación 14, donde la enzima es una lipasa. Use of the composition according to claim 14, wherein the enzyme is a lipase.
- 18. 18.
- Método según cualquiera de las reivindicaciones 16ó 17, donde la deshidrataciónde los microorganismos según el paso (b) se lleva a cabo por medio de una solución hipertónica o por medio de una corriente de aire. Method according to any of claims 16 or 17, wherein the dehydration of the microorganisms according to step (b) is carried out by means of a hypertonic solution or by means of an air current.
- 19. 19.
- Método según cualquiera de las reivindicaciones 16 a 18, donde el medio hipotónico para la rehidratación de los microorganismos según elpaso (c) es agua parcial o totalmente destilada, desionizada o desmineralizada. Method according to any of claims 16 to 18, wherein the hypotonic means for rehydration of the microorganisms according to step (c) is water partially or totally distilled, deionized or demineralized.
- 20. twenty.
- Método según cualquiera de las reivindicaciones 16 a 19, donde además, la fracción líquida del paso (d) se deshidrata hasta que el producto xeroprotector tenga una humedad residual igual o inferior al 10%. Method according to any of claims 16 to 19, wherein, in addition, the liquid fraction of step (d) is dehydrated until the xeroprotective product has a residual humidity equal to or less than 10%.
- 21. twenty-one.
- Método para la conservación de material biológico que comprende: a) mezclarla composiciónxeroprotectorasegún cualquieradelasreivindicaciones4 a9 con una muestra Method for the preservation of biological material comprising: a) mixing the airborne composition according to any of claims 4 to 9 with a sample
- 22. 22
- Método según la reivindicación 21, donde el material biológico es un microorganismo o una célula. Method according to claim 21, wherein the biological material is a microorganism or a cell.
- 23. 2. 3.
- Método según la reivindicación 21, donde el material biológico del paso (a) es un organismo invertebrado, una semilla, una plántula, un órgano aislado o un tejido biológico aislado. Method according to claim 21, wherein the biological material of step (a) is an invertebrate organism, a seed, a seedling, an isolated organ or an isolated biological tissue.
- 24. 24.
- Método según la reivindicación 21, donde el material biológico es una molécula con actividad biológica. Method according to claim 21, wherein the biological material is a molecule with biological activity.
- 25. 25.
- Método según la reivindicación 24, donde la molécula con actividad biológica es una enzima. Method according to claim 24, wherein the molecule with biological activity is an enzyme.
- 26. 26.
- Método según la reivindicación 25, donde la enzima es una lipasa. Method according to claim 25, wherein the enzyme is a lipase.
- Categoría Category
- Documentos citados Reivindicaciones afectadas Documents cited Claims Affected
- A TO
- LEBLANC J.C., et al. Global response to desiccation stress in the soil actinomycete Rhodococcus jostii RHA1. 2008. Applied and Environmental Microbiology. Vol. 74, No. 9, páginas 2627-2636. 1-27 LEBLANC J.C., et al. Global response to desiccation stress in the soil actinomycete Rhodococcus jostii RHA1. 2008. Applied and Environmental Microbiology. Vol. 74, No. 9, pages 2627-2636. 1-27
- A TO
- SHUKLA M., et al. Multiple-stress tolerance of ionizing radiation-resistant bacterial isolates obtained from various habitats: correlation between stresses. 2007. Current Microbiology. Vol. 54, páginas 142-148. 1-27 SHUKLA M., et al. Multiple-stress tolerance of ionizing radiation-resistant bacterial isolates obtained from various habitats: correlation between stresses. 2007. Current Microbiology. Vol. 54, pages 142-148. 1-27
- A TO
- ALVAREZ H.M., et al. Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress.2004. FEMS Microbiology Ecology. Vol. 50, páginas 75-86. 1-27 ALVAREZ H.M., et al. Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress. 2004. FEMS Microbiology Ecology. Vol. 50, pages 75-86. 1-27
- A TO
- WANG Y., et al. Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by Rhodococcus sp. M5. 1995. Gene. Vol. 164, páginas 117-122. 1-27 WANG Y., et al. Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl / chlorobiphenyl degradation by Rhodococcus sp. M5 1995. Gene. Vol. 164, pages 117-122. 1-27
- Categoría de los documentos citados X: de particular relevancia Y: de particular relevancia combinado con otro/s de la misma categoría A: refleja el estado de la técnica O: referido a divulgación no escrita P: publicado entre la fecha de prioridad y la de presentación de la solicitud E: documento anterior, pero publicado después de la fecha de presentación de la solicitud Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application
- El presente informe ha sido realizado • para todas las reivindicaciones □ para las reivindicaciones nº: This report has been prepared • for all claims □ for claims no:
- Fecha de realización del informe 09.05.2011 Date of realization of the report 09.05.2011
- Examinador I. Rueda Molins Página 1/4 Examiner I. Rueda Molins Page 1/4
- Novedad (Art. 6.1 LP 11/1986) Novelty (Art. 6.1 LP 11/1986)
- Reivindicaciones Reivindicaciones 1-27 SI NO Claims Claims 1-27 IF NOT
- Actividad inventiva (Art. 8.1 LP11/1986) Inventive activity (Art. 8.1 LP11 / 1986)
- Reivindicaciones Reivindicaciones 1-27 SI NO Claims Claims 1-27 IF NOT
- Documento Document
- Número Publicación o Identificación Fecha Publicación Publication or Identification Number publication date
- D01 D01
- LEBLANC J.C., et al. Global response to desiccation stress in the soil actinomycete Rhodococcus jostii RHA1. Applied and Environmental Microbiology. Vol. 74, No. 9, páginas 2627-2636. 2008 LEBLANC J.C., et al. Global response to desiccation stress in the soil actinomycete Rhodococcus jostii RHA1. Applied and Environmental Microbiology. Vol. 74, No. 9, pages 2627-2636. 2008
- D02 D02
- SHUKLA M., et al. Multiple-stress tolerance of ionizing radiationresistant bacterial isolates obtained from various habitats: correlation between stresses. Current Microbiology. Vol. 54, páginas 142-148. 2007 SHUKLA M., et al. Multiple-stress tolerance of ionizing radiationresistant bacterial isolates obtained from various habitats: correlation between stresses. Current Microbiology Vol. 54, pages 142-148. 2007
- D03 D03
- ALVAREZ H.M., et al. Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress.2004. FEMS Microbiology Ecology. Vol. 50, páginas 75-86. 2004 ALVAREZ H.M., et al. Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress. 2004. FEMS Microbiology Ecology. Vol. 50, pages 75-86. 2004
- D04 D04
- WANG Y., et al. Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by Rhodococcus sp. M5. Gene. Vol. 164, páginas 117-122. 1995 WANG Y., et al. Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl / chlorobiphenyl degradation by Rhodococcus sp. M5 Gene. Vol. 164, pages 117-122. nineteen ninety five
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200931117A ES2361308B2 (en) | 2009-12-04 | 2009-12-04 | BACTERIAN CEPA CECT7625, USES AND XEROPROTECTOR PRODUCT PRODUCED BY THE SAME. |
| ES201100031A ES2389367B2 (en) | 2009-12-04 | 2009-12-04 | SYNTHETIC COMPOSITION WITH XEROPROTECTOR EFFECT. |
| PCT/ES2010/000517 WO2011067441A2 (en) | 2009-12-04 | 2010-12-03 | Bacterial strain cect7625, uses thereof and xeroprotectant product produced from same |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200931117A ES2361308B2 (en) | 2009-12-04 | 2009-12-04 | BACTERIAN CEPA CECT7625, USES AND XEROPROTECTOR PRODUCT PRODUCED BY THE SAME. |
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| ES2361308A1 ES2361308A1 (en) | 2011-06-16 |
| ES2361308B2 true ES2361308B2 (en) | 2011-11-15 |
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| ES201100031A Active ES2389367B2 (en) | 2009-12-04 | 2009-12-04 | SYNTHETIC COMPOSITION WITH XEROPROTECTOR EFFECT. |
| ES200931117A Active ES2361308B2 (en) | 2009-12-04 | 2009-12-04 | BACTERIAN CEPA CECT7625, USES AND XEROPROTECTOR PRODUCT PRODUCED BY THE SAME. |
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| WO (1) | WO2011067441A2 (en) |
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| US20040064846A1 (en) * | 2001-02-28 | 2004-04-01 | Henry Daniell | Genetic engineering of drought tolerance via a plastid genome |
| FR2819414A1 (en) * | 2001-01-15 | 2002-07-19 | Cognis France Sa | COSMETIC AND / OR PHARMACEUTICAL PREPARATIONS COMPRISING EXTRACTS FROM PLANTS RESOURCING |
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| Publication number | Publication date |
|---|---|
| ES2389367B2 (en) | 2013-05-09 |
| ES2389367A1 (en) | 2012-10-25 |
| ES2361308A1 (en) | 2011-06-16 |
| WO2011067441A2 (en) | 2011-06-09 |
| WO2011067441A3 (en) | 2011-12-01 |
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