WO2011067441A2 - Bacterial strain cect7625, uses thereof and xeroprotectant product produced from same - Google Patents

Bacterial strain cect7625, uses thereof and xeroprotectant product produced from same Download PDF

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Publication number
WO2011067441A2
WO2011067441A2 PCT/ES2010/000517 ES2010000517W WO2011067441A2 WO 2011067441 A2 WO2011067441 A2 WO 2011067441A2 ES 2010000517 W ES2010000517 W ES 2010000517W WO 2011067441 A2 WO2011067441 A2 WO 2011067441A2
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composition
biological material
microorganism
fructose
xeroprotective
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PCT/ES2010/000517
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Spanish (es)
French (fr)
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WO2011067441A3 (en
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Maximino Manzanera Ruiz
Jesús Juan GONZÁLEZ LÓPEZ
Juan Jesús NARVÁEZ REINALDO
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Universidad De Granada
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Publication of WO2011067441A3 publication Critical patent/WO2011067441A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07H3/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • Bacterial strain CECT7625 uses and xeroprotector product produced by it.
  • the present invention relates to a microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. Also the present invention 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, ⁇ -hydroxybutyrate and fructose.
  • the present invention also 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, a isolated organ, an isolated biological tissue or a cell, or a molecule with biological activity such as an enzyme with lipase activity.
  • the present invention relates to a method of obtaining the xeroprotective composition or a method for the conservation of said biological material.
  • amorphous crystal-forming protective molecules such as trehalose disaccharide (aD-glucopyranosyl-aD-glucopyranoside).
  • trehalose disaccharide aD-glucopyranosyl-aD-glucopyranoside.
  • the formation and use of amorphous crystals is well documented (Manzanera et al., 2002. Appl Environ Microbiol, 68: 4328-4333).
  • preservatives that form these crystals are suitable for this type of preservation and include non-reducing carbohydrates such as trehalose, hydroxyectoin, maltitol, lactitol (4-OaD-glucopyranosyl-D-glucitol), palatinit [GPS mixture (aD -glucopyranosyl-1-6-sorbitol) and GPM (aD-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.
  • the presence of water in the dry state is generally less than 0.2 g / g dry cell weight in most cryptobionts. These water levels are sufficient for these microorganisms to resist extreme dehydration, high temperatures, ionizing radiation or, in some species of tardigrades, pressures of up to 600 MPa.
  • biofilms (Subaerial biofilms), formed by bacteria of the genus Rhodococcus sp among others, are capable of producing osmoprotective compounds, that is, polymeric extracellular substances (EPS) (Gorbushina, 2007.
  • EPS polymeric extracellular substances
  • Ortega-Morales et al. (2007) (Ortega-Morales et al. 2007. Journal of Applied Microbiology, 102: 254-264), describe bacteria from biofilms in tropical intertidal zones, where bacteria belonging to the genus Microbacterium sp. how source of new protective exopolymers of cells against desiccation.
  • LeBlanc (2008) (LeBlanc, 2008. Applied and environmental microbiology, 74 (9): 2627-2636), refers to the microorganism Rhodococcus jostii RHA1, an actinomycete with favorable metabolic capacities for bioremediation of contaminated soils, capable of secreting the ectoin and trehalose osmoprotectors.
  • the present invention relates to a microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. Also the present invention 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, ⁇ -hydroxybutyrate and fructose.
  • the present invention also 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, a isolated organ, an isolated biological tissue or a cell, or a molecule with biological activity such as an enzyme with lipase activity.
  • the present invention relates to a method of obtaining the xeroprotective composition or a method for the conservation of said biological material.
  • the ability to conserve sensitive biological material for indefinite periods in an active or viable way is of importance in applications for the medical, agricultural and industrial sectors.
  • tools are offered to solve the conservation of biological material that presents difficulties for its stabilization.
  • the biological material preserved with the xeroprotective composition produced by the microorganism with access number CECT7625 is stable for long periods of time.
  • 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 activity
  • Enzymatic of a composition containing acetate, or lactate, or glutamic acid, or ⁇ -hydroxybutyrate or fructose is less than the preservation effect produced by a composition containing all components, that is, the xeroprotective composition has a synergistic effect on its ability to conserve biological material.
  • one aspect of the present invention relates 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 Type Crop Collection (CECT) on November 10, 2009 and was assigned the deposit number CECT7625. The address of said International Deposit Authority is: University of Valencia / Research building / Campus of Burjassot / 46100 Burjassot (Valencia).
  • strain CECT7625 of the present invention is: Kingdom: Bacteria I Phylum: Actinobacteria I Order: Actinomycetales I Family: Nocardiaceae I Genus: Rhodococcus.
  • the maximum temperature tolerated for the growth of this strain is 40 ° C.
  • the minimum temperature to detect growth was between 15 ° C and 20 ° C, while its optimum growth temperature was around 35 ° C. It was unable to grow at pH 12 although if at pH 9.
  • the minimum pH tolerated for the growth of this strain was found between pH 5 and pH 7, this being considered as the optimum pH for the growth of this strain.
  • the present invention also relates 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.
  • Another aspect of the present invention relates to a bacterial population comprising the microorganism deposited with access number CECT7625. The bacterial population can be formed by other strains of microorganisms of any species.
  • the bacterial population is a set of microorganism cells where there is at least one cell of said microorganism deposited with access number CECT7625, at any stage of the development state and at any stage of growth, seasonal or stationary, regardless of the morphology that present, in the form of coconut, bacillus or intermediate morphologies of the above.
  • microorganism or the bacterial population may be referred to as the "microorganism of the present invention” or the “microorganism of the invention”.
  • Another aspect of the present invention relates to the use of the microorganism of the invention for the production of an xeroprotective composition.
  • a further aspect of the present invention relates to the xeroprotective composition produced by the microorganism of the invention.
  • the term Bacterial Milking Product (POB) can be used.
  • POB Bacterial Milking Product
  • xeroprotective composition refers to a composition that prevents the adverse effects of total or partial drying of material of biological origin or material of synthetic origin.
  • 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 may be, for example, but without limited, 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 a pharmacological, immunogenic and / or physiological effect 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) agents against hypertension, diuretics, vasodilators, central nervous system stimulants, 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 synthetic, as recombinant products), nucleic acid molecules (both in polymeric form of two or more DNA or RNA nucleotides, including both double chain and single chain molecules, genetic constructs, expression vectors, antis
  • Material of synthetic origin refers to a type of material that has not been produced or synthesized directly by a living organism but has been created by humans, for example, but not limited to, a DNA sequence amplified by PCR, or a protein or enzyme modified intentionally or not.
  • a preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or a synthetic xeroprotective composition, comprising fructose, glutamic acid, ⁇ -hydroxybutyrate, acetate and lactate.
  • acetate refers to the acetate anion present in a solution, ie the acetate (C2H3O2) anion, " is a carboxylate and is a conjugate base of acetic acid.
  • lactate refers to the lactate anion present in a solution , ie the lactate anion (C3H5O3) " , is a carboxylate and is a conjugate base of lactic acid.
  • Glutamate an ionized form of glutamic acid, refers to one of the 20 essential amino acids that are part of proteins.
  • ⁇ -Hydroxybutyrate or beta-hydroxybutyrate
  • Fructose (or levulose) is a ketohexose (6 carbon atoms) with chemical formula C6Hi 2 O 6 , monosaccharide with the same empirical formula as glucose but with different structure.
  • a more preferred embodiment relates to the xeroprotective composition produced by the microorganism of the invention, or to the synthetic xeroprotective composition, which comprises a ratio of between 35 and 45 of 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 relates 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.
  • the xeroprotective composition has a ratio of (fructose) :( glutamic acid) :( acetate) of (41) :( 2,4) :( 1), respectively.
  • a more preferred embodiment relates to the xeroprotective composition produced by the microorganism of the invention, or to the synthetic xeroprotective composition, which comprises a ratio between 14 and 18 of fructose: 3 and 5 of glutamic acid: 0.6 and 1 of ⁇ -hydroxybutyrate: 0.5 and 1, 5 of acetate: 1 and 2 of lactate. That is, a ratio (fructose) :( glutamic acid) :( 3-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 relates to the xeroprotective composition where the ratio of (fructose) :( glutamic acid) :( -hydroxybutyrate) :( acetate) :( lactate) is (15 to 17) :( 3.5 to 4 , 5) :( 0.7 to 0.9) :( 0.7 to 1, 2) :( 1, 2 to 1, 6).
  • the xeroprotective composition has a ratio of (fructose) :( glutamic acid): ⁇ - hydroxybutyrate) :( acetate) :( lactate) of (16) :( 4) :( 0.8) :( 1) :( 1 , 4), respectively.
  • portion refers to the corresponding correspondence of the elements of the composition (fructose, glutamic acid, ⁇ -hydroxybutyrate, acetate and lactate) related to each other. That is, it refers to a mathematical relationship that links the elements of the composition.
  • the xeroprotective composition that has a proportion of (fructose) :( glutamic acid) :( P- 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 .
  • composition of the present invention or “composition of the invention”.
  • Another aspect of the present invention is the use of the composition of the invention for the conservation of biological material with a residual moisture content 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.
  • biological material in dry state can be used the expression "biological material in dry state". Under these conditions, the preservative or stabilizer 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.
  • the dried biological material is embedded in the amorphous crystal formed by the stabilizer.
  • 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.
  • 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, moldings or solids in 3 dimensions such as, but not limited to, blocks, pads, patches, sheets, balls, or seeds of dry biological material.
  • residual moisture refers to the amount of moisture a product contains after it has passed through some type of process capable of removing water from it.
  • Residual humidity is the mass percentage 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 10 g of every 100 g of the product correspond to water. Residual humidity can be measured by methods known in the state of the art such as, but not limited to, by the titrimetric method, the azeotropic method or the gravimetric method.
  • the term “conservation of biological material” refers to the maintenance or care of the permanence of the intrinsic characteristics of the biological material.
  • a preferred embodiment relates 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 isolated organ, an isolated biological tissue or a cell.
  • the cell can be prokaryotic or eukaryotic.
  • the cell can be a cell of a microorganism in any state of development.
  • the cell can be somatic or germinal, plant or animal.
  • Said cell can come from any organism or microorganism and can occur in any state of differentiation, such as, but not limited to, from a culture of a cellular tissue or organs, sperm, ovules or embryos.
  • 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 Bifidobacterium spp.
  • the multicellular organism can be, for example, but not limited to, a nematode.
  • 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.
  • the cell may have been treated, manipulated or mutated before preservation.
  • a cell may have become 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.
  • 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.
  • 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, under conditions of high osmolarity (high concentration of salts) that stimulate the intracellular production of trehalose or other amorphous crystal-forming stabilizers.
  • the invertebrate organism is, but is not limited to, an insect larva 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.
  • 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 Tetrapoda Superclass (with four limbs),
  • Amphibia class (amphibians) or Reptilia class (reptiles), or any of its parts (Vernon and Jackson, 1931. The biological bulletin, 60: 80-93).
  • Vernon and Jackson carried out a study on the Leopard frog (Rana pipiens), in which their skin, tongue, spleen, and liver naturally dries with a water loss of between 43-81% of the water content total.
  • An isolated organ, or an isolated biological tissue (including blood) can be preserved by the composition of the present invention.
  • Serrato et al. (2009) results of cryopreservation protocols of biological tissues can be observed (Serrato et al., 2009. Histology and histopathology, 24: 1531-1540).
  • a more preferred embodiment relates to the use of the composition of the invention, where the biological material is a molecule with biological activity.
  • molecule with biological activity refers to a biological molecule whose origin is a living organism or that has been alive, or derivatives or analogs of said molecule.
  • derivatives refers to molecules obtained by the modification of a molecule with biological activity, which have similar functionality.
  • analogs refers to molecules that have a function similar to the molecule with biological activity.
  • the molecule with biological activity is an enzyme.
  • An even more preferred embodiment of the present invention relates 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 (Triacylglycerol lipase), EC 3.1.1 .4 (Phospholipase A (2)), EC 3.1.1.5 (Lysophospholipase), EC 3.1.1 .23 (Acylglycerol lipase), EC 3.1.1.
  • Another aspect of the present invention relates to a method of obtaining the xeroprotective composition of the invention comprising: a) culturing the microorganism of the invention in a culture medium with fructose as a carbon source,
  • step (b) dehydrate the microorganisms obtained in the culture of step (a) until they have a residual humidity equal to or less than 10%
  • step (b) rehydrate the dehydrated microorganisms of step (b) in a hypotonic medium
  • step (d) select the liquid fraction of the product obtained in step (c) comprising the xeroprotective composition.
  • 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) medium in which xeroprotectors, or natural osmolytes are not present, because the bacteria would prefer to take them from outside to synthesize them.
  • LB Luria Bertani
  • a preferred embodiment of the present invention relates 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 gelled culture medium to a greater or lesser extent, that is, comprising agar to facilitate its gelation or any gelling compound.
  • 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 relates to the method, where the dehydration of the microorganisms according to step (b) is carried out.
  • 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 environment than in the cytoplasm of the cells of the microorganism of the present invention, therefore, the cell releases water, that is, it is dehydrated.
  • 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 medium than in the cytoplasm of the cell of the microorganism of the present invention, therefore, the cell recovers water, that is, it is rehydrated.
  • a further preferred embodiment relates to the method, where the hypotonic means for rehydration of the microorganisms according to step (c) is water partially or totally distilled, partially or totally deionized or partially or totally demineralized.
  • the cells and the hypotonic medium must be in contact for at least 5 minutes. Preferably they will be in contact for at least 20 minutes with stirring.
  • Obtaining the medium containing the stabilizing substances will be carried out by any method that guarantees the separation of cells from the liquid content, preferably by gentle centrifugation, followed by a filtration step. Preferably 0.4 micrometer pore diameter filters will be used.
  • step (d) Another preferred embodiment relates to the method, where, in addition, the liquid fraction of step (d) is dehydrated until the xeroprotector product has a residual humidity equal to or less than 10%.
  • the xeroprotector product of the invention can be separated from the culture medium by any method of concentration. Preferably it they will use freeze dryers that produce the stabilizer in a dry state.
  • the stabilizing molecules may be dissolved or dispersed in a proportion of between 10 and 30%. This solution or dispersion is added to the biological material that is to be preserved and will be dried.
  • Another aspect of the present invention relates to the method for the conservation of biological material comprising: a) mixing the composition of the invention with a sample of biological material, and
  • step (a) dehydrate the product obtained in step (a) to a residual humidity equal to or less than 10%.
  • 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.
  • Another preferred embodiment relates to the method for the conservation of biological material, where the biological material is a molecule with biological activity.
  • a more preferred embodiment of the present invention relates to the method, where the molecule with biological activity is an enzyme.
  • the enzyme is a lipase.
  • Another even more preferred embodiment relates to any of the methods for the conservation of biological material, where dehydration of the mixture of the xeroprotective composition and the biological material according to the step (b) is carried out by means of a hypertonic solution or by means of an air current.
  • 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 through the use of the composition of the present invention.
  • the invention includes extrusions or molds in which the material conserved by any method of the present invention is included.
  • a method of producing a molding containing active biomaterial by stabilizers may include solutions of plastic materials.
  • a molding of plastic material with encapsulated active biomaterial can be useful as components of biosensors.
  • a biosensor can include bacteria that are capable of detecting toxic substances, pathogens or toxicity in general.
  • FIG. 1 Shows the viability of the different bacterial isolates selected by exposure to chloroform after 24 hours of air drying. Error bars show the standard deviation of at least three replicas.
  • microorganisms represented in the figure are: 1 J3A, 1 J14, 2J2, 2J8A, 2J12B, 2J15B, 2J16A, 2J30, 3J18, 6J30, Acitenobacter calcoaceticus, Pseudomonas putida, in addition to strain 4J2A2.
  • FIG. 2 Shows the lipase activity (in percentage relative to 100% positive control activity) after drying of the lipase enzyme in the presence of the different components of 10% bacterial milking products (w / v).
  • the positive control is trehalose at 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 drying stabilization 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. Shows lipase activity (in percentage relative to 100% positive control activity) after drying of the lipase enzyme in presence of the different chemical compounds (fructose, glutamic acid, beta-hydroxybutyrate, acetate and 10% lactate).
  • 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, acetate and 10% lactate in the same proportion found in the POBSIA of strain 4J2A2 by drying treatment and subsequent hydration being the ratio of 16: 4: 0, 8: 1: 1, 4 respectively.
  • 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).
  • POB bacterial milking product
  • POBSIAs Air Drying
  • 4J2A2 is the value of lipase activity recorded after drying stabilization 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 polyvinylpyrrolidone
  • T is trehal.
  • strain CECT7625 with the term "4J2A2".
  • EXAMPLE 1 Isolation of microorganism 4J2A2 (CECT7625), extraction of POB (Bacterial Milking Products) and enzyme xeroprotection assay.
  • 1 g of dry soil was homogenized, from Granada (37,182 Latitude N and 3,624 Longitude O), not exposed to rain or irrigation, 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 would guarantee their contact with the chloroform.
  • the earth was deposited in glass vials to which 3 ml 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 contact of the sample with the solvent.
  • the soil samples were deposited in a sterile glass petri dish without a lid until the evaporation of the chloroform was completed. 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, 2-10 5 CFU / g of soil was detected in the untreated samples, while these were reduced to 1, 3-10 5 CFU / g of soil after 30 minutes of treatment.
  • CFU colony forming units
  • microplates were placed under a stream of sterile air in a laminar flow hood for 24 hours. The plates remained dry 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 sowing, 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 the wet data as a reference of 100%. The standard deviation to the mean allowed the calculation of the student's T to study if the differences in survival were significant.
  • the 4J2A2 microorganism was identified as a strain with levels of drying tolerance significantly higher than those of the positive control Acinetobacter calcoaceticus (PADD68).
  • Said strain 4J2A2 belongs to the genus 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 through BIOLOG metabolic studies and DNA-DNA hybridization with the nearest species.
  • FIG. 1 shows the tolerance values of the isolated strains. 1.2. Extraction of products from bacterial milking.
  • Halomononas elongata was included as a positive control, since it is a strain recognized as a halotolerant (Saber and Galinski, 1998. Biotechnol. Bioeng., 57: 306-313) and P. putida KT2440 as a strain Halosensitive (De Castro et al., 2000. Appl. Environ. Microbiol., 66: 4142-4144).
  • the strain selected as hypertolerant at 4J2A2 drying was inoculated.
  • H. elongata was included as a positive control since it had already been used by Sauer and Galinski to obtain hydroxyectoin
  • E. coli was included as an example of a halo and xerosensitive strain. Said inoculums were incubated with shaking for 48 hours. The samples were then centrifuged for 10 minutes at 10,000 rpm in a Beckman Avanti-J25 centrifuge and the supernatant fraction was removed, the bacterial sediment was resuspended in 20 ml of sterile M9 solution and deposited on filters.
  • the filters After 24 hours, the filters, subjected to desiccation by a stream of sterile air, were separated from the medium and deposited in tubes with sterile distilled water allowing to incubate 20 minutes at 30 ° C and 150 rpm. Consecutively the same centrifugation process was repeated, the bacterial precipitate was discarded and the supernatant was filtered (using a 0.22 pm filter). The filtrate of each sample was divided into two fractions, one of which was used to determine the xeroprotective activity of the supernatant (milking fraction) and the other for the identification and characterization of the compounds present in this fraction.
  • 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 air incubation (4J2A2D). Also, in FIG. 3 lipase activity (in percentage relative to 100% positive control activity) is shown after drying of the lipase enzyme in the presence of the various chemical compounds of the synthetic 4J2A2D composition (fructose, glutamic acid, beta-hydroxybutyrate, acetate and 10% lactate). The positive control is trehalose at 10%. 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 and lactate, 16: 4: 0.8: 1: 1, 4 respectively.
  • the combination of the compounds fructose, glutamic acid, beta-hydroxybutyrate, acetate and lactate has a synergistic effect on the conservation of lipase activity since the sum of the Lipase activity shown by compositions containing the compounds in isolation is less than the result obtained with said mixture.
  • Table 1 Composition of the bacterial milking product (POB) of strain 4J2A2.
  • Enzyme xeroprotection assay The objective of this test was to determine the ability of the bacterial milking product fraction to protect enzymes from drying out. For this, the enzyme lipase was used. Starting from 1 pl containing 0.00554 units of Burkholderia cepacia lipase (Sigma-Aldrich 62309-100 mg), 15 pl of the product fraction of the bacterial milking to study were added.
  • the substrate solution (SS) was prepared by mixing 10 ml of solution A (30 mg of pNPP in 10 ml of sopropanol) with 90 ml of solution B (0.1 g of gum arabic and 0.4 ml of Triton X-100 in
  • FIG. 2 shows the tolerance values of the isolated strains.
  • the objective of this test was to determine the ability to protect live cells of Escherichia coli MC4100 against desiccation by using the bacterial milking product (POB) as well as those extracted by Air Drying (POBSIAs) of various xerotolerant strains analogous to that described by Manzanera et al., (2002) (Manzanera et al., 2002. Applied and Environmental Microbiology 68:
  • E. coli pre-inoculum was used, from which a minimum medium plus glucose was inoculated as an added carbon source of 0.6 M NaCl until an initial optical density of 0.05 was reached.
  • E. coli cells were resuspended in a 10% solution of the POBs or POBSIAs extracted from the xerotolerant cells (extracted as described above) and in addition, 1.5% polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • Synthetic POB / POBSIA mixing was performed in a 34.2% solution.
  • the suspension of cells in the different solutions were subjected to vacuum drying conditions without freezing, in a freezer- Modified desiccator (Dura - Stop ⁇ P; FTS Systems, Stone Ridge, NY) at 30 ° C medium temperature and 100 mTorr (2Pa; 2x10 "5 atmospheres) for 20 seconds, with a temperature ramp of 2.5 ° C / min with 15 minutes of pause after each increase of 2 ° C, until reaching the maximum temperature of 40 ° C.
  • Dura - Stop ⁇ P FTS Systems, Stone Ridge, NY

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Abstract

The invention relates to a microorganism of the Rhodococcus sp. species (access number CECT7625). The invention also relates to the use of said microorganism or a population of same for the production of a xeroprotectant composition, said composition comprising acetate, lactate, glutamic acid, β-hydroxybutyrate and fructose. The invention further relates to the use of the xeroprotectant composition for preserving biological material with a residual moisture content equal to or less than 10%, in which the biological material is an invertebrate organism, a seed, a plantlet, a microorganism, an isolated organ, an isolated biological tissue or a cell or a molecule with biological activity such as, for example, an enzyme with lipase activity. Furthermore, the invention relates to a method for obtaining the xeroprotectant composition or a method for preserving said biological material.

Description

Cepa bacteriana CECT7625, usos y producto xeroprotector producido por la misma.  Bacterial strain CECT7625, uses and xeroprotector 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 aislado o 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. Also the present invention 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, β-hydroxybutyrate and fructose. The present invention also 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, 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.
ESTADO DE LA TÉCNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
La conservación de materiales biológicos mediante deshidratación y osmoconcentración es una tecnología conocida. Cuando la tarea de conservar biomoléculas sensibles se hizo necesaria, el simple secado mediante deshidratación fracasó, ya que se eliminaba el agua estructural, produciendo la desnaturalización posterior y la pérdida de actividad 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. Los métodos actuales de conservación requieren de gran costo en energía y generalmente necesitan de almacenaje a bajas temperaturas. En ocasiones, después de su conservación el material biológico tiene una actividad y/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. The conservation of biological materials by dehydration and osmoconcentration 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. Current conservation methods require high energy costs and generally need storage at low temperatures. Sometimes, after its conservation, 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 un nivel modesto de termotolerancia en el producto final, y se requiere aún refrigeración para reducir el deterioro 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 eventually lead to vaccination of patients with a lower than standard vaccine or, in some cases, unusable.
Durante la selección natural evolutiva, ciertas especies de microorganismos, plantas y animales adquirieron la notable y elegante capacidad de tolerar la deshidratación extrema, permaneciendo latentes en medios hostiles durante períodos muy largos de tiempo y aún capaces de adquirir una actividad vital completa una vez 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 y el procedimiento por el que sobreviven se conoce como anhidrobiosis. Todas las especies de animales y plantas que presentan esta capacidad, contienen moléculas protectoras formadoras de cristales amorfos como el disacárido trehalosa (a-D-glucopiranosil-a-D- glucopiranósido). La formación y 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 (4-O-a-D-glucopyranosyl-D- glucitol), palatinit [mezcla de GPS (a-D-glucopiranosil-1 -6-sorbitol) y GPM (a-D-glucopiranosil-1 -6-manitol)] y sus componentes individuales GPS y 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. During evolutionary natural 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 procedure by which they survive is known as anhydrobiosis. All species of animals and plants that present This capacity contains amorphous crystal-forming protective molecules such as trehalose disaccharide (aD-glucopyranosyl-aD-glucopyranoside). 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 (4-OaD-glucopyranosyl-D-glucitol), palatinit [GPS mixture (aD -glucopyranosyl-1-6-sorbitol) and GPM (aD-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 presencia de agua en el estado seco es generalmente inferior a 0,2 g/g de peso celular seco en la mayoría de los criptobiontes. Estos niveles de agua son suficientes para que estos microorganismos resistan la deshidratación extrema, 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 0.2 g / g dry cell weight in most cryptobionts. These water levels are sufficient for these microorganisms to resist extreme dehydration, high temperatures, ionizing radiation or, 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. Journal of Applied Microbiology, 102: 254-264), describen bacterias de las biopelículas de las zonas intermareales tropicales, donde se han aislado bacterias que pertenecen al género Microbacterium sp. como fuente de nuevos exopolímeros protectores de las células contra la desecación. Por otra parte, LeBlanc (2008) (LeBlanc, 2008. Applied and environmental microbiology, 74(9): 2627-2636), se refiere al microorganismo Rhodococcus jostii RHA1 , un actinomiceto con capacidades metabólicas favorables para la biorremediación de suelos contaminados, capaz de secretar los osmoprotectores ectoina y trehalosa. It is known that biofilms (Subaerial biofilms), 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. Journal of Applied Microbiology, 102: 254-264), describe bacteria from biofilms in tropical intertidal zones, where bacteria belonging to the genus Microbacterium sp. how source of new protective exopolymers of cells against desiccation. On the other hand, LeBlanc (2008) (LeBlanc, 2008. Applied and environmental microbiology, 74 (9): 2627-2636), refers to the microorganism Rhodococcus jostii RHA1, an actinomycete with favorable metabolic capacities for bioremediation of contaminated soils, capable of secreting the ectoin and trehalose osmoprotectors.
EXPLICACIÓN DE LA INVENCIÓN 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 aislado o 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. EXPLANATION OF THE INVENTION The present invention relates to a microorganism of the bacterial species Rhodococcus sp. with access number CECT7625. Also the present invention 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, β-hydroxybutyrate and fructose. The present invention also 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, 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 capacidad para conservar material biológico sensible por periodos de tiempo indefinido en forma activa o viable es de importancia en aplicaciones para los sectores médico, agrícola e industrial. En la presente invención se ofrecen herramientas para solucionar la conservación de material biológico que presenta dificultades para su estabilización. El material biológico preservado con la composición xeroprotectora producida por el microorganismo con n° de acceso CECT7625 es estable por periodos largos de tiempo. Tal como se muestra en los ejemplos de la presente invención, 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 ácido glutámico, 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 in an active or viable way is of importance in applications for the medical, agricultural and industrial sectors. In the present invention tools are offered to solve the conservation of biological material that presents difficulties for its stabilization. The biological material preserved with the xeroprotective composition produced by the microorganism with access 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 activity Enzymatic of a composition containing acetate, or lactate, or glutamic acid, or β-hydroxybutyrate or fructose, is less than the preservation effect produced by a composition containing all 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 en la Colección Española de Cultivos Tipo (CECT) el 10 de noviembre de 2009 y le correspondió el n° de depósito CECT7625. La dirección de dicha Autoridad Internacional de depósito es: Universidad de Valencia / Edificio de investigación / Campus de Burjassot / 46100 Burjassot (Valencia). Therefore, one aspect of the present invention relates 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 Type Crop Collection (CECT) on November 10, 2009 and was assigned the deposit number CECT7625. The address of said International Deposit Authority is: University of Valencia / Research building / Campus of Burjassot / 46100 Burjassot (Valencia).
En adelante, para hacer referencia a dicha cepa se puede emplear el término "4J2A2". Hereinafter, the term "4J2A2" can be used to refer to said strain.
La clasificación científica de la cepa CECT7625 de la presente invención es: Reino: Bacteria I Filo: Actinobacteria I Orden: Actinomycetales I Familia: Nocardiaceae I Género: Rhodococcus. The scientific classification of strain CECT7625 of the present invention is: Kingdom: Bacteria I Phylum: Actinobacteria I Order: Actinomycetales I Family: Nocardiaceae I Genus: Rhodococcus.
Las características de dicha cepa son: The characteristics of said strain are:
- Metabolismo caracterizado por oxidación/fermentación, Dextrina, tween 40, tween 80, N-acetil-D-glucosamina, D-arabitol, D-fructosa, ácido D- glucónico, α-D-glucosa, D-manitol, D-manosa, D-psicosa, D-ribosa, D- sorbitol, ácido α-cetovalérico, ácido L-láctico, ácido L-málico, metil piruvato, mono-metil succinato, ácido propiónico, ácido pirúvico, L-alanil- glicina, glicerol, 2'-deoxiadenosina. - La temperatura máxima tolerada para el crecimiento de esta cepa son 40°C. La temperatura mínima para detectar crecimiento se encontró entre 15°C y 20°C, mientras que su temperatura óptima de crecimiento fue de entorno a 35°C. Fue incapaz de crecer a pH 12 aunque si a pH 9. - El pH mínimo tolerado para el crecimiento de esta cepa se encontró entre pH 5 y pH 7, considerándose éste como pH óptimo para el crecimiento de esta cepa. - 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-psychosa, D-ribose, D- sorbitol, α-ketovaleric acid, L-lactic acid, L-malic acid, methyl pyruvate, mono-methyl succinate, propionic acid, pyruvic acid, L-alanylglycine, glycerol, 2'- deoxyadenosine. - The maximum temperature tolerated for the growth of this strain is 40 ° C. The minimum temperature to detect growth was between 15 ° C and 20 ° C, while its optimum growth temperature was around 35 ° C. It was unable to grow at pH 12 although if at pH 9. - The minimum pH tolerated for the growth of this strain was found between pH 5 and pH 7, this being considered as the optimum pH for the growth of this strain.
- Igualmente fue incapaz de proliferar en medio LB con una concentración de NaCI igual o superior a 2 M, quedando la concentración máxima tolerada entre 0,8 M y 2 M de NaCI. Esta cepa mostró un crecimiento óptimo a una concentración de 0,2 M de NaCI, aunque también fue capaz de proliferar en este medio en ausencia de NaCI. - Ensayos de sensibilidad a antibióticos mostraron halos de inhibición del crecimiento en los cinco antibióticos ensayados en disco: rifampicina3o (2,83 cm); estreptomicina25 (2,20 cm); tetraciclina2o (0,87 cm); cloramfenicol5o (4,12 cm); kanamicina3o (1 ,93 cm). Asimismo, la presente invención también se refiere a un microorganismo derivado del microorganismo depositado con n° 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. Otro aspecto de la presente invención se refiere a una población bacteriana que comprende el microorganismo depositado con n° 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 n° de acceso CECT7625, en cualquier fase del estado de desarrollo y en cualquier fase de crecimiento, estacional o estacionaria, independientemente de la morfología que presente, en forma de coco, bacilo o morfologías intermediarias de las anteriores. - 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 0.8 M and 2 M NaCl. This strain showed optimal growth at a concentration of 0.2 M NaCl, although it was also able to proliferate in this medium in the absence of NaCl. - Antibiotic sensitivity tests showed growth inhibition halos in the five antibiotics tested on disk: rifampicin3 (2.83 cm); Streptomycin 2 5 (2.20 cm); tetracycline 2 or (0.87 cm); chloramphenicol 5 or (4.12 cm); Kanamycin 3 or (1, 93 cm). Likewise, the present invention also relates 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. Another aspect of the present invention relates to a bacterial population comprising the microorganism deposited with access number CECT7625. The bacterial population can be formed by other strains of microorganisms of any species. The bacterial population is a set of microorganism cells where there is at least one cell of said microorganism deposited with access number CECT7625, at any stage of the development state and at any stage of growth, seasonal or stationary, regardless of the morphology that present, in the form of coconut, bacillus or intermediate morphologies of the above.
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 aspecto más de la presente invención se refiere al uso del microorganismo de la invención para la producción de una composición xeroprotectora. Un aspecto más de la presente invención se refiere a la composición xeroprotectora producida por el microorganismo de la invención. Para referirse a la composición xeroprotectora de la presente invención se puede emplear el término Producto de Ordeñado Bacteriano (POB). El término "composición xeroprotectora" tal como se entiende en 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 material biológico 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 de los elementos anteriores, o cualquier molécula orgánica que tenga un efecto farmacológico, inmunogénico y/o fisiológico de acción local y/o sistémica. El material biológico puede comprender agentes terapéuticos; agentes anti-infectivos 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, ¡nmunosupresores, relajantes musculares, psicoestimulantes, sedantes, tranquilizantes, proteínas, péptidos o fragmentos de los mismos (tanto naturales, como sintéticos, como productos recombinantes), moléculas de ácidos nucleicos (tanto en forma polimérica de dos o más nucleótidos de ADN o ARN, incluyendo tanto 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 o dUTP, de utilidad en la técnica de PCR, secuenciación, etc). El material de origen sintético se refiere a un tipo de material que no ha sido producido o sintetizado por un organismo vivo directamente sino que ha sido creado por el ser humano como por ejemplo, pero sin limitarse, una secuencia de ADN amplificada por PCR, o una proteína o enzima modificada intencionadamente o no. Una realización preferida de la presente invención se refiere a la composición xeroprotectora producida por el microorganismo de la invención, o a una composición xeroprotectora sintética, que comprende fructosa, ácido glutámico, β-hidroxibutirato, acetato y lactato. Another aspect of the present invention relates to the use of the microorganism of the invention for the production of an xeroprotective composition. A further aspect of the present invention relates to the xeroprotective composition produced by the microorganism of the invention. To refer to the xeroprotective composition of the present invention, the term Bacterial Milking Product (POB) can be used. The term "xeroprotective composition" as understood in the present invention refers to a composition that prevents the adverse effects of total or partial drying of material of biological origin or material of synthetic origin. 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 may be, for example, but without limited, 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 a pharmacological, immunogenic and / or physiological effect 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) agents against hypertension, diuretics, vasodilators, central nervous system stimulants, 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 synthetic, as recombinant products), nucleic acid molecules (both in polymeric form of two or more DNA or RNA nucleotides, including both double chain and single chain molecules, genetic constructs, expression vectors, antisense RNA, sen tido or RNAi molecules) or nucleotides (such as, but not limited to, dATP, dCTP, dGTP, dTTP 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 directly by a living organism but has been created by humans, for example, but not limited to, a DNA sequence amplified by PCR, or a protein or enzyme modified intentionally or not. A preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or a synthetic xeroprotective 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 carboxilato y es base conjugada del ácido acético. El término "lactato" se refiere al anión lactato presente en una solución, es decir el anión lactato (C3H5O3)", es un carboxilato y es base conjugada del ácido láctico. El glutamato, forma ionizada del ácido glutámico, se refiere a uno de los 20 aminoácidos esenciales que forman parte de las proteínas. El β-hidroxibutirato (o beta-hidroxibutirato) es el anión que deriva de la disolución del ácido 3-hidroxibutírico. La fructosa (o levulosa) es una cetohexosa (6 átomos de carbono) con formula química C6Hi2O6, monosacárido con la misma fórmula empírica que la glucosa pero con diferente estructura. The term "acetate" refers to the acetate anion present in a solution, ie the acetate (C2H3O2) anion, " is a carboxylate and is a conjugate base of acetic acid. The term" lactate "refers to the lactate anion present in a solution , ie the lactate anion (C3H5O3) " , is a carboxylate and is a conjugate base of lactic acid. Glutamate, an ionized form of glutamic acid, refers to one of the 20 essential amino acids that are part of proteins. Β-Hydroxybutyrate (or beta-hydroxybutyrate) is the anion that derives from the dissolution of 3-hydroxybutyric acid. Fructose (or levulose) is a ketohexose (6 carbon atoms) with chemical formula C6Hi 2 O 6 , 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, o a la composición xeroprotectora sintética, que comprende una proporción de entre 35 y 45 de fructosa: 1 ,4 y 3,4 de ácido glutámico: 0,5 y 1 ,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 relates to the xeroprotective composition produced by the microorganism of the invention, or to the synthetic xeroprotective composition, which comprises a ratio of between 35 and 45 of 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 relates 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, o a la composición xeroprotectora sintética, que comprende una proporción de entre 14 y 18 de fructosa: 3 y 5 de ácido glutámico: 0,6 y 1 de β-hidroxibutirato: 0,5 y 1 ,5 de acetato: 1 y 2 de lactato. Es decir, una proporción (fructosa):(ácido glutámico):(3-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):( -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). Preferiblemente la composición xeroprotectora tiene una proporción de (fructosa):(ácido glutámico):^- hidroxibutirato):(acetato):(lactato) de (16):(4):(0,8):(1 ):(1 ,4), respectivamente. A more preferred embodiment relates to the xeroprotective composition produced by the microorganism of the invention, or to the synthetic xeroprotective composition, which comprises a ratio between 14 and 18 of fructose: 3 and 5 of glutamic acid: 0.6 and 1 of β -hydroxybutyrate: 0.5 and 1, 5 of acetate: 1 and 2 of lactate. That is, a ratio (fructose) :( glutamic acid) :( 3-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 relates to the xeroprotective composition where the ratio of (fructose) :( glutamic acid) :( -hydroxybutyrate) :( acetate) :( lactate) is (15 to 17) :( 3.5 to 4 , 5) :( 0.7 to 0.9) :( 0.7 to 1, 2) :( 1, 2 to 1, 6). Preferably the xeroprotective composition has a ratio of (fructose) :( glutamic acid): ^ - 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 elementos de la composición. Para que sirva de ejemplo, la composición xeroprotectora que tiene una proporción de (fructosa):(ácido glutámico):(P- 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 elements of the composition (fructose, glutamic acid, β-hydroxybutyrate, acetate and lactate) related to each other. That is, it refers to a mathematical relationship that links the elements of the composition. To serve as an example, the xeroprotective composition that has a proportion of (fructose) :( glutamic acid) :( P- 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". Otro aspecto de la presente invención es el uso de la composición de la invención para la conservación de material biológico con un contenido en humedad residual igual o inferior al 10%. El contenido de humedad residual del 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 conservante o estabilizador coalesce para alcanzar un estado no-cristalino, vitreo, y sólido (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. 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. El material biológico seco en estas formas puede encontrarse en estado no particulado y puede suministrarse en formas por ejemplo, pero sin limitarse, molduras o sólidos en 3 dimensiones como por ejemplo, pero sin limitarse, bloques, pastillas, parches, hojas, bolas, o pepitas de material biológico seco. Hereinafter reference may be made to any of the above compositions as the "composition of the present invention" or "composition of the invention". Another aspect of the present invention is the use of the composition of the invention for the conservation of biological material with a residual moisture content 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, to refer to this type of biological material can be used the expression "biological material in dry state". Under these conditions, the preservative or stabilizer 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. In this way the dried biological material is embedded in the amorphous crystal formed by the stabilizer. 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. 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, moldings or solids in 3 dimensions such as, but not limited to, blocks, pads, patches, sheets, balls, or seeds of dry biological material.
El término "humedad residual" tal como se emplea en la presente invención se refiere a la cantidad de humedad que contiene un producto después de pasado por algún tipo de proceso capaz de eliminar agua del mismo. La humedad residual es el porcentaje de masa del producto que corresponde a agua respecto del total de la masa. Es decir, un valor de humedad residual de un producto igual a un 10% significa que 10 g de cada 100 g del producto corresponden a agua. La humedad residual puede ser medida mediante métodos conocidos en el estado de la técnica como por ejemplo, pero sin limitarse, mediante el método titrimétrico, el método azeotrópico o el método gravimétrico. El término "conservación de material biológico" hace referencia al mantenimiento o cuidado de la permanencia de las características intrínsecas del material biológico. 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, un órgano aislado, un tejido biológico aislado o una célula. La célula puede ser procariota o eucariota. La célula puede ser una célula de un microorganismo en cualquier estado de desarrollo. La célula puede ser somática o germinal, vegetal o animal. Dicha célula puede proceder de cualquier organismo o microorganismo y puede presentarse 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. The term "residual moisture" as used in the present invention refers to the amount of moisture a product contains after it has passed through some type of process capable of removing water from it. Residual humidity is the mass percentage 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 10 g of every 100 g of the product correspond to water. Residual humidity can be measured by methods known in the state of the art such as, but not limited to, by the titrimetric method, the azeotropic method or the gravimetric method. The term "conservation of biological material" refers to the maintenance or care of the permanence of the intrinsic characteristics of the biological material. A preferred embodiment relates 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 isolated organ, an isolated biological tissue or a cell. The cell can be prokaryotic or eukaryotic. The cell can be a cell of a microorganism in any state of development. The cell can be somatic or germinal, plant or animal. Said cell can come from any organism or microorganism and can occur in any state of differentiation, such as, but not limited to, from a culture of a cellular tissue or organs, sperm, ovules or embryos. 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 Bifidobacterium 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 replicación y división celular. Por otra parte la célula puede haber sido tratada, manipulada o mutada antes de su conservación. Por ejemplo, pero sin limitarse, una célula puede haberse hecho competente para transformaciones o transfecciones, o puede contener ácidos nucleicos recombinantes. Las células que se conservan pueden formar una población homogénea o heterogénea, por ejemplo, pero sin 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 or mutated before preservation. For example, but not limited to, a cell may have become 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, en condiciones de alta osmolaridad (alta concentración de sales) que estimulen la producción intracelular de 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, under conditions of high osmolarity (high concentration of salts) that stimulate the intracellular production of trehalose or other amorphous crystal-forming stabilizers.
El organismo invertebrado es pero sin limitarse, una larva de insecto o un crustáceo. Dichos organismos invertebrados 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, an insect larva 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 biológico es un organismo vertebrado perteneciente a la Superclase Tetrápoda (con cuatro extremidades), 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 Tetrapoda Superclass (with four limbs),
Clase Amphibia (anfibios) o Clase Reptilia (reptiles), o cualquiera de sus partes (Vernon y Jackson, 1931 . The biological bulletin, 60: 80-93). Vernon y Jackson llevaron a cabo un estudio sobre la rana Leopardo (Rana pipiens), en el que de forma natural se seca su piel, lengua, bazo, e hígado con una pérdida de agua de entre un 43-81 % del contenido de agua total. Un órgano aislado, o un tejido biológico aislado (incluida la sangre) pueden conservarse mediante la 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). Amphibia class (amphibians) or Reptilia class (reptiles), or any of its parts (Vernon and Jackson, 1931. The biological bulletin, 60: 80-93). Vernon and Jackson carried out a study on the Leopard frog (Rana pipiens), in which their skin, tongue, spleen, and liver naturally dries with a water loss of between 43-81% of the water content total. 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 refiere a moléculas que presentan una función similar a la molécula con actividad biológica. A more preferred embodiment relates 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 the modification of a molecule with biological activity, which have similar functionality. On the other hand, the term "analogs" refers to molecules that have a function similar 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 (Triacilglicerol lipasa), EC 3.1.1 .4 (Fosfolipasa A(2)), EC 3.1.1.5 (Lisofosfolipasa), EC 3.1.1 .23 (Acilglicerol lipasa), EC 3.1.1 .24 (3-oxoadipato enol-lactonasa), EC 3.1.1 .25 (1 ,4-lactonasa), EC 3.1 .1 .26 (Galactolipasa), EC 3.1 .1.32 (Fosfolipasa A(1 )), EC 3.1 .1 .33 (6-acetilglucosa deacetilasa), EC 3.1 .1 .34 (Lipoproteína lipasa). Preferiblemente la enzima lipasa tiene actividad Triacilglicerol lipasa (EC 3.1 .1 .3). Otro aspecto de la presente invención se refiere a un método de obtención de la composición xeroprotectora de la invención que comprende: a) cultivar el microorganismo de la invención en un medio de cultivo con fructosa como fuente de carbono, 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 relates 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 (Triacylglycerol lipase), EC 3.1.1 .4 (Phospholipase A (2)), EC 3.1.1.5 (Lysophospholipase), EC 3.1.1 .23 (Acylglycerol lipase), EC 3.1.1. 24 (3-oxoadipate enol-lactonase), EC 3.1.1 .25 (1, 4-lactonase), EC 3.1 .1 .26 (Galactolipase), EC 3.1 .1.32 (Phospholipase A (1)), EC 3.1 .1 .33 (6-acetylglucose deacetylase), EC 3.1 .1 .34 (Lipoprotein lipase). Preferably the enzyme lipase has Triacylglycerol lipase activity (EC 3.1 .1 .3). Another aspect of the present invention relates to a method of obtaining the xeroprotective composition of the invention comprising: a) culturing the microorganism of the invention in a culture medium with fructose as a 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 hipotónico y c) rehydrate the dehydrated microorganisms of step (b) in a hypotonic medium and
d) seleccionar la fracción líquida del producto obtenido en el paso (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) en los que hay presentes xeroprotectores, u osmolitos naturales no sirven, porque la bacteria preferiría tomarlos del exterior a sintetizarlos. 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) medium in which xeroprotectors, or natural osmolytes are not present, 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 medio de cultivo gelificado en mayor o menor grado, es decir, que comprende agar para facilitar su gelificación o cualquier compuesto gelificante. A preferred embodiment of the present invention relates 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 gelled culture medium to a greater or lesser extent, that is, comprising agar to facilitate its gelation 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 hipertónica es una solución que tiene mayor concentración de soluto en el medio externo que en el 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 relates 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 environment than 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 del microorganismo de la presente invención, por tanto, la célula recupera agua, es decir, se rehidrata. Una realización preferida más se refiere al método, donde el medio hipotónico para la 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 medium than in the cytoplasm of the cell of the microorganism of the present invention, therefore, the cell recovers water, that is, it is rehydrated. A further preferred embodiment relates to the method, where the hypotonic means for rehydration of the microorganisms according to step (c) is water partially or totally distilled, partially or totally deionized or partially or totally demineralized.
Las células y el medio hipotónico han de estar en contacto al menos 5 minutos. Preferiblemente estarán en contacto al menos 20 minutos en agitación. La obtención del medio que contiene las sustancias estabilizantes se realizará por cualquier método que garantice la separación de células del 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 must be in contact for at least 5 minutes. Preferably they will be in contact for at least 20 minutes with stirring. Obtaining the medium containing the stabilizing substances will be carried out by any method that guarantees the separation of cells from the liquid content, preferably by gentle centrifugation, followed by a filtration step. Preferably 0.4 micrometer pore diameter filters 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%. El producto xeroprotector de la invención se puede separar del medio de cultivo por cualquier método de concentración. Preferiblemente se utilizarán secadores de tipo liofilizador que produzcan el estabilizador en estado seco. Las moléculas estabilizadoras se podrán disolver o dispersar en una proporción de entre el 10 y el 30%. Esta disolución o dispersión se añade al material biológico que se desee conservar y se someterá a desecación. Another preferred embodiment relates to the method, where, in addition, the liquid fraction of step (d) is dehydrated until the xeroprotector product has a residual humidity equal to or less than 10%. The xeroprotector product of the invention can be separated from the culture medium by any method of concentration. Preferably it they will use freeze dryers that produce the stabilizer in a dry state. The stabilizing molecules may be dissolved or dispersed in a proportion of between 10 and 30%. This solution or dispersion is added to the biological material that is to be preserved and will be dried.
Otro aspecto de la presente invención se refiere al método para la conservación de material biológico que comprende: a) mezclar la composición de la invención con una muestra de material biológico, y Another aspect of the present invention relates to the method for the conservation of biological material comprising: 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%. 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. Otra realización preferida se refiere al método para la conservación de material biológico, donde el material biológico es una molécula con actividad biológica. b) dehydrate the product obtained in step (a) to a residual humidity equal to or less than 10%. 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. Another preferred embodiment relates 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 preferida de la presente invención la enzima es una lipasa. A more preferred embodiment of the present invention relates 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.
Otra realización aún más preferida se refiere a cualquiera de los métodos para la conservación de material biológico, donde la deshidratación de la mezcla de la composición xeroprotectora y del material biológico según el paso (b) se lleva a cabo por medio de una solución hipertónica o por medio de una corriente de aire. Another even more preferred embodiment relates to any of the methods for the conservation of biological material, where dehydration of the mixture of the xeroprotective composition and the biological material according to the 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 mediante el uso de la composición de la presente invención. La invención incluye extrusiones o moldes en los que se incluye el material conservado por cualquier método de la presente invención. Un método de producción de una moldura que contenga biomaterial activo mediante 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 through the use of the composition of the present invention. The invention includes extrusions or molds in which the material conserved by any method of the present invention is included. A method of producing a molding containing active biomaterial by stabilizers 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.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Las siguientes figuras y ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. DESCRIPCION DE LAS FIGURAS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from 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. DESCRIPTION OF THE FIGURES
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 ilustrativo y no limitante, las siguientes figuras: FIG. 1. Muestra la viabilidad de los distintos aislados bacterianos seleccionados mediante exposición a cloroformo tras 24 horas de secado al aire. Las barras de error muestran la desviación estándar de al menos tres réplicas. 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. Shows the viability of the different bacterial isolates selected by exposure to chloroform after 24 hours of air drying. Error bars show the standard deviation of at least three replicas.
Los microorganismos representados en la figura son: 1 J3A, 1 J14, 2J2, 2J8A, 2J12B, 2J15B, 2J16A, 2J30, 3J18, 6J30, Acitenobacter calcoaceticus, Pseudomonas putida, además de la cepa 4J2A2. The microorganisms represented in the figure are: 1 J3A, 1 J14, 2J2, 2J8A, 2J12B, 2J15B, 2J16A, 2J30, 3J18, 6J30, Acitenobacter calcoaceticus, Pseudomonas putida, in addition to strain 4J2A2.
FIG. 2. Muestra la actividad lipasa (en porcentaje relativo a un 100% de actividad del 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 lipase activity (in percentage relative to 100% positive control activity) after drying of the lipase enzyme in the presence of the different components of 10% bacterial milking products (w / v).
El control positivo es trehalosa al 10%. El control negativo (-) se corresponde con la ausencia de compuesto alguno como aditivo previo a la desecación de la enzima. 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 cepa 4J2A2 mediante choque hiper/hipoosmótico respectivamente. 4J2A2D es la actividad lipasa registrada tras la estabilización por secado y posterior reconstitución de la enzima en presencia de POBSIA (Producto de Ordeñado Bacteriano extraído por Secado mediante Incubación al Aire) extraídos de la cepa 4J2A2 mediante tratamiento de secado y posterior hidratación. FIG. 3. Muestra la actividad lipasa (en porcentaje relativo a un 100% de actividad del control positivo) tras secado de la enzima lipasa en presencia de los distintos compuestos químicos (fructosa, ácido glutámico, beta-hidroxibutirato, acetato y lactato al 10%). The positive control is trehalose at 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 drying stabilization 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. Shows lipase activity (in percentage relative to 100% positive control activity) after drying of the lipase enzyme in presence of the different chemical compounds (fructose, glutamic acid, beta-hydroxybutyrate, acetate and 10% 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, acetato y lactato al 10% en la misma proporción encontrada en el POBSIA de la cepa 4J2A2 mediante tratamiento de secado y posterior hidratación siendo la proporción de 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, acetate and 10% lactate in the same proportion found in the POBSIA of strain 4J2A2 by drying treatment 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 cepa 4J2A2 mediante choque hiper/hipoosmótico respectivamente. 4J2A2D es la actividad lipasa registrada tras la estabilización por secado y posterior reconstitución de la enzima en presencia de POBSIA. 4J2A2 is the value of lipase activity recorded after drying stabilization 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.
T es trehalosa. T is trehal.
EJEMPLOS EXAMPLES
A continuación se ilustrará la invención mediante unos ensayos ilustrativos y de 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 now be illustrated by illustrative and non-limiting tests carried out by the inventors describing the isolation of the strain of the present invention as well as the ability to Production of xeroprotective compounds. Hereinafter reference can be made to strain CECT7625 with the term "4J2A2".
EJEMPLO 1. Aislamiento del microorganismo 4J2A2 (CECT7625), extracción de POB (Productos del Ordeñado Bacteriano) y ensayo de xeroprotección de enzimas. EXAMPLE 1. Isolation of microorganism 4J2A2 (CECT7625), extraction of POB (Bacterial Milking Products) and enzyme xeroprotection assay.
1.2. Aislamiento de la cepa 4J2A2 (CECT7625). 1.2. Isolation of strain 4J2A2 (CECT7625).
Se homogeneizó 1 g de suelo seco, procedente de Granada (37.182 Latitud N y 3.624 Longitud O), no expuesto a 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 grano de tierra fino que garantizara su contacto con el cloroformo. La tierra se depositó en viales de vidrio a los que se les añadió 3 mi de cloroformo puro. Tras la adición del cloroformo se incubaron a temperatura ambiente durante 30 minutos con agitación esporádica para maximizar el contacto de la muestra con el disolvente. Para eliminar el cloroformo de las muestras una vez transcurrido el 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 mi de TSB. Con las suspensiones de suelo se realizaron diluciones seriadas y se sembraron en placas de TSA que se incubaron 48 horas a 30°C. Trascurrido este tiempo se cuantificó el número de unidades formadoras de colonias (UFC) por mililitro de cada muestra. Así, se detectaron 2- 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. Se seleccionaron aleatoriamente 36 cepas y para identificar las cepas que producían esporas se realizó un ensayo basado en la diferente sensibilidad de las células vegetativas con respecto a las esporas al tratamiento con calor y en base al método descrito por Vilchez y colaboradores (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 resuspendieron en 1 mi de solución M9 en microtubos estériles de 1 ,5 mi. Seguidamente se sembraron 10 μΙ de esta suspensión en TSA. Acto seguido se incubó el resto de la suspensión en termobloque Mixing Block MB- 02 a 72°C durante 30 minutos. Nuevamente se tomaron 10 μΙ de cada muestra y se sembraron en placa de TSA. Aquellas cepas con capacidad para tolerar el tratamiento con calor se consideraron como esporulantes, mientras que aquellas que no toleraron el tratamiento por calor se consideraron no esporulantes. Como controles positivos y negativos se utilizaron colonias de Bacillus pumilus y de Burkholderia cepacia respectivamente. La proporción de cepas esporulantes pasó de niveles inferiores al 40% para muestras no tratadas a niveles superiores al 50% tras 30 min de exposición de las muestras de suelo al cloroformo. 1 g of dry soil was homogenized, from Granada (37,182 Latitude N and 3,624 Longitude O), not exposed to rain or irrigation, 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 would guarantee their contact with the chloroform. The earth was deposited in glass vials to which 3 ml 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 contact of the sample with the solvent. To remove the chloroform from the samples after the contact time had elapsed, the soil samples were deposited in a sterile glass petri dish without a lid until the evaporation of the chloroform was completed. 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, 2-10 5 CFU / g of soil was detected in the untreated samples, while these were reduced to 1, 3-10 5 CFU / g of soil after 30 minutes of treatment. 36 strains were randomly selected and to identify the strains that produced spores, a test was conducted based on the different sensitivity of the vegetative cells with respect to the spores to heat treatment and based on the method described by Vilchez et al. (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 microtubes of 1.5 ml. Then 10 μΙ of this suspension was seeded in TSA. The rest of the suspension was then incubated in Mixing Block MB-02 thermoblock at 72 ° C for 30 minutes. Again 10 μΙ 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 went from levels below 40% for untreated samples to 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 estudio de desecación al aire. En este ensayo se incluyó una cepa de Acinetobacter calcoaceticus (PADD68) aislada del desierto de Tabernas (Almería) identificada como tolerante a la desecación en un estudio previo y que se utilizó como control positivo. Además también se incluyó en el estudio células de Pseudomonas putida KT2440 que se utilizaron como controles negativos al ser una cepa sensible a la desecación (Antheunisse et al., 1981. Antonie Leeuwnhoek, 47: 539-545; Manzanera et al., 2002. Appl. Environ. Microbiol., 68: 4328-4333). Para este ensayo se utilizaron colonias independientes de las 17 cepas identificadas como no esporulantes aisladas en el estudio anterior y procedentes de placas de TSA de 72 horas para calcular su nivel de tolerancia a la desecación tal y como se indica a continuación. 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 en 1 mi de solución M9 estéril. Partiendo de esta suspensión celular se realizaron diluciones seriadas que se sembraron en placas de TSA con objeto de identificar el número de UFC/ml de partida. Por otra parte se tomaron 100 μΙ de cada suspensión y se depositaron en gotas de 5-10 pl sobre 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 tras 2-3 horas de incubación. Transcurridas 24 horas de incubación se resuspendieron en 1 mi 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 al primer conteo. Estos ensayos se realizaron por triplicado. Los resultados se expresaron como la media de los tres ensayos en porcentaje, tomando como referencia del 100% los datos húmedos. La desviación estándar a la media permitió el cálculo de la T de student para estudiar si las diferencias en supervivencia fueron significativas. De las 17 cepas aisladas en estas condiciones se identificó el microorganismo 4J2A2 por ser una cepa con niveles de tolerancia a la desecación significativamente superiores a las del control positivo Acinetobacter calcoaceticus (PADD68). Dicha cepa 4J2A2 pertenece al género 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 16S y 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. 1.2. Extracción de productos del ordeñado bacteriano. In order to analyze the ability to tolerate desiccation of isolated non-sporulant strains, an air drying study was conducted. In this trial, a strain of Acinetobacter calcoaceticus (PADD68) isolated from the Tabernas desert (Almería) identified as tolerant to desiccation was included in a previous study and was used as a positive control. In addition, Pseudomonas putida KT2440 cells that were used as negative controls were also included in the study because they were a strain sensitive to desiccation (Antheunisse et al., 1981. Antonie Leeuwnhoek, 47: 539-545; Manzanera et al., 2002. Appl. Environ. Microbiol., 68: 4328-4333). Independent colonies of the 17 strains identified as non-sporulants were used for this test. isolated in the previous study and coming from 72-hour TSA plates to calculate their level of desiccation tolerance as indicated below. 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 and resuspended in 1 ml of sterile M9 solution. Starting from this cell suspension, serial dilutions were made that were seeded on TSA plates in order to identify the starting CFU / ml number. On the other hand, 100 μΙ of each suspension was taken and deposited in 5-10 pl drops 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 remained dry 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 sowing, 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 the wet data as a reference of 100%. The standard deviation to the mean 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 4J2A2 microorganism was identified as a strain with levels of drying tolerance significantly higher than those of the positive control Acinetobacter calcoaceticus (PADD68). Said strain 4J2A2 belongs to the genus 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 through BIOLOG metabolic studies and DNA-DNA hybridization with the nearest species. FIG. 1 shows the tolerance values of the isolated strains. 1.2. Extraction of products from bacterial milking.
Para la extracción de los productos del ordeñado bacteriano y con el fin de identificar las moléculas acumuladas con capacidad para proteger biomoléculas sensibles a la desecación se recurrió a una estrategia basada en tres 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 in order to identify the accumulated molecules with the capacity to protect biomolecules sensitive to desiccation, a strategy based on three steps was used. 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 que la producción y acúmulo de sustancias xeroprotectoras se realizó en medio con minerales, se procedió a identificar las fuentes de carbono más apropiadas para el cultivo de las cepas en medio mineral M9. Para la obtención de sustancias con capacidad xeroprotectora se cultivaron en medio mineral con la fuente de carbono apropiada (fructosa) las células de dicha cepa hasta la obtención de una biomasa suficiente. Tras este acúmulo, las células se depositaron sobre placas del mismo medio con agar para la producción de medio sólido. Entre las células y el medio se depositó un filtro estéril de 0,4 mieras para permitir el paso de nutrientes a las células y su posterior recogida y manipulación. Las placas con filtros y células se sometieron a un secado por corriente de aire estéril durante 24 horas. Como control positivo se incluyó Halomononas elongata, dado que es una cepa reconocida como halotolerante (Saber y Galinski, 1998. Biotechnol. Bioeng., 57: 306-313) y a P. putida KT2440 como cepa halosensible (De Castro et al., 2000. Appl. Environ. Microbiol., 66:4142- 4144). Given that the production and accumulation of xeroprotective substances was carried out in a medium with minerals, we proceeded to identify the most appropriate carbon sources for the cultivation of the strains in M9 mineral medium. To obtain substances with xeroprotective capacity, the cells of said strain were grown in mineral medium with the appropriate carbon source (fructose) until obtaining a sufficient biomass. After this accumulation, the cells were deposited on plates of the same medium with agar for the production of solid medium. A sterile 0.4 micron filter was deposited between the cells and the medium to allow the passage of nutrients to the cells and their subsequent collection and handling. The plates with filters and cells were 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 (Saber and Galinski, 1998. Biotechnol. Bioeng., 57: 306-313) and P. putida KT2440 as a strain Halosensitive (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 Sauer y Galinski para la obtención de hidroxiectoína, y como control negativo se incluyó E. coli como ejemplo de cepa halo y xerosensible. Dichos inóculos se incubaron en agitación durante 48 horas. A continuación, se centrifugaron las muestras durante 10 minutos a 10.000 rpm en una centrifuga Beckman Avanti-J25 y se retiró la fracción sobrenadante, resuspendiendo el sedimento bacteriano en 20 mi de solución M9 estéril y se depositaron sobre filtros. Tras 24 horas, los filtros, sometidos a desecación mediante una corriente de aire estéril, se separaron del medio y se depositaron en tubos con agua destilada estéril dejando incubar 20 minutos a 30°C y 150 rpm. Consecutivamente se repitió el mismo proceso de centrifugado, se desechó el precipitado bacteriano y se filtró el sobrenadante (utilizando un filtro de 0,22 pm). El producto filtrado de cada muestra se dividió en dos fracciones, una de las cuales se utilizó para determinar la actividad xeroprotectora del sobrenadante (fracción de ordeñado) y la otra para la identificación y caracterización de los compuestos presentes en esta fracción. Ambas fracciones se sometieron a un proceso de secado utilizando un liofilizador (Labconco Freezone 6) durante 48 horas obteniéndose un sedimento que fue resuspendido en 100 μΙ de agua milliQ estéril. Una vez liofilizado, el Producto de Ordeñado Bacteriano (POB) se solubilizó en 100 microlitros de agua. Estas soluciones de POBs se utilizaron en estudios de xeroprotección. To this end, the strain selected as hypertolerant at 4J2A2 drying was inoculated. In addition, H. elongata was included as a positive control since it had already been used by Sauer and Galinski to obtain hydroxyectoin, and as a negative control, E. coli was included as an example of a halo and xerosensitive strain. Said inoculums were incubated with shaking for 48 hours. The samples were then centrifuged for 10 minutes at 10,000 rpm in a Beckman Avanti-J25 centrifuge and the supernatant fraction was removed, the bacterial sediment was resuspended in 20 ml of sterile M9 solution and deposited on filters. After 24 hours, the filters, subjected to desiccation by a stream of sterile air, were separated from the medium and deposited in tubes with sterile distilled water allowing to incubate 20 minutes at 30 ° C and 150 rpm. Consecutively the same centrifugation process was repeated, the bacterial precipitate was discarded and the supernatant was filtered (using a 0.22 pm filter). The filtrate of each sample was divided into two fractions, one of which was used to determine the xeroprotective activity of the supernatant (milking 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 μΙ of sterile milliQ water. Once lyophilized, the Bacterial Milking Product (POB) was solubilized in 100 microliters of water. These POB solutions were used in xeroprotection studies.
En la tabla 1 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 al aire (4J2A2D). Asimismo, en la FIG. 3 se muestra la actividad lipasa (en porcentaje relativo a un 100% de actividad del control positivo) tras secado de la enzima lipasa en presencia de los distintos compuestos químicos de la composición 4J2A2D sintética (fructosa, ácido glutámico, beta-hidroxibutirato, acetato y lactato al 10%). El control positivo es trehalosa al 10%. El control negativo se corresponde con la ausencia de compuestos como aditivo previo a la desecación de la enzima. 4J2A2D sintético es la mezcla de fructosa, ácido glutámico, beta-hidroxibutirato, acetato y lactato al 10% en la misma proporción encontrada en el POBSIA de la cepa 4J2A2 mediante tratamiento de secado por incubación al aire y posterior hidratación siendo la proporción de fructosa, ácido glutámico, beta-hidroxibutirato, acetato y lactato, 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 air incubation (4J2A2D). Also, in FIG. 3 lipase activity (in percentage relative to 100% positive control activity) is shown after drying of the lipase enzyme in the presence of the various chemical compounds of the synthetic 4J2A2D composition (fructose, glutamic acid, beta-hydroxybutyrate, acetate and 10% lactate). The positive control is trehalose at 10%. 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 and lactate, 16: 4: 0.8: 1: 1, 4 respectively.
Tal como puede observarse en dicha FIG. 3, la combinación de los compuestos fructosa, ácido glutámico, beta-hidroxibutirato, acetato y lactato (en igual proporción que la mostrada en la tabla 1 (4J2A2D) presenta un efecto sinérgico en la conservación de la actividad lipasa ya que la suma de la actividad lipasa mostrada por 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, beta-hydroxybutyrate, acetate and lactate (in the same proportion as shown in Table 1 (4J2A2D) has a synergistic effect on the conservation of lipase activity since the sum of the Lipase activity shown by compositions containing the compounds in isolation is less than the result obtained with said mixture.
Tabla 1. Composición del producto de ordeñado bacteriano (POB) de la cepa 4J2A2. Table 1. Composition of the bacterial milking product (POB) of strain 4J2A2.
Figure imgf000028_0001
Figure imgf000028_0001
1.3. Ensayo de xeroprotección de enzimas. El objetivo de este ensayo fue determinar la capacidad de la fracción producto del ordeñado bacteriano para proteger enzimas frente a la desecación. Para ello se utilizó la enzima lipasa. Partiendo de 1 pl que contenía 0,00554 unidades de lipasa de Burkholderia cepacia (Sigma- Aldrich 62309-100 mg) se adicionaron 15 pl de la fracción producto del ordeñado bacteriano a estudiar. Como control positivo se adicionaron 15 μΙ de una solución al 10% de trehalosa a 1 pl (0,00554 U) de solución de lipasa y como control negativo se añadió 15 μΙ de agua a 1 μΙ (0,00554 U) de solución de lipasa. Las mezclas de 16 μΙ con lipasa se depositaron en un microtubo de 2ml de capacidad y se secaron a 50°C durante 120 minutos. Una vez secas se incubaron a 100°C durante 5 minutos. Finalmente fueron almacenadas en un desecador a temperatura ambiente durante 24 horas. Pasado el tiempo de incubación, las reacciones se resuspendieron en 50 μΙ de una solución de TrisHCI (50 mM) y se transfirieron a un microtubo junto con 950 μΙ de Tris HCI (50 mM) pH8 y 1 mi de Solución de Sustrato. La determinación de la capacidad xeroprotectora de cada fracción producto de ordeñado bacteriano (POB) se determinó por el ensayo de medición de la actividad lipasa. 1.3. Enzyme xeroprotection assay. The objective of this test was to determine the ability of the bacterial milking product fraction to protect enzymes from drying out. For this, the enzyme lipase was used. Starting from 1 pl containing 0.00554 units of Burkholderia cepacia lipase (Sigma-Aldrich 62309-100 mg), 15 pl of the product fraction of the bacterial milking to study were added. As a positive control, 15 μΙ of a 10% solution of trehalose was added to 1 pl (0.00554 U) of lipase solution and as a negative control 15 μΙ of water was added to 1 μΙ (0.00554 U) of the solution of lipase The 16 μΙ mixtures with lipase were deposited in a 2 ml capacity microtube and dried at 50 ° C for 120 minutes. Once dried, they were incubated at 100 ° C for 5 minutes. They were finally stored in a desiccator at room temperature for 24 hours. After the incubation time, the reactions were resuspended in 50 μΙ of a TrisHCI solution (50 mM) and transferred to a microtube together with 950 μΙ of Tris HCI (50 mM) pH8 and 1 ml of Substrate Solution. The xeroprotective capacity determination of each bacterial milking product fraction (POB) was determined by the lipase activity measurement test.
Para la medida de la actividad lipasa se utilizó una variación del método descrito por Gupta y colaboradores (2002) consistente en la cuantificación espectrofotométrica del p-nitrofenol liberado por la enzima lipasa de Burkholderia cepacia (Sigma-Aldrich 62309-100 mg) a partir del sustrato p- nitrofenol palmitato (pNPP) (Gupta et al., 2002. Analytical Biochemistry, 31 1 : 98-99). Para ello se utilizó 1 mi de medio libre de células (985 μΙ de Tris-HCI 0,05M junto a 15 μΙ de POB obtenido por el método del "ordeñado bacteriano") mezclado con 1 mi de solución sustrato de un cultivo en fase estacionaria. Esta mezcla de ensayo se incubó a 30°C durante 30 minutos en microtubos estériles de 2 mi. La reacción se paró mediante incubación a 100°C durante 4 minutos en termobloque y 2 minutos a -20°C. La t For the measurement of lipase activity, a variation of the method described by Gupta et al. (2002) consisting of the spectrophotometric quantification of p-nitrophenol released by the enzyme lipase of Burkholderia cepacia (Sigma-Aldrich 62309-100 mg) from the p-nitrophenol palmitate (pNPP) substrate (Gupta et al., 2002. Analytical Biochemistry, 31 1: 98-99). For this, 1 ml of cell-free medium (985 μΙ of 0.05M Tris-HCI was used together with 15 μΙ of POB obtained by the "bacterial milking" method) mixed with 1 ml of substrate solution of a stationary phase culture . This test mixture was incubated at 30 ° C for 30 minutes in sterile 2 ml microtubes. The reaction was stopped by incubation at 100 ° C for 4 minutes in thermoblock and 2 minutes at -20 ° C. The t
28 absorbancia se midió en un espectrofotómetro Hitachi U-2000 a una longitud de onda de 410 nm. La solución sustrato (SS) se preparó mezclando 10 mi de solución A (30 mg de pNPP en 10 mi de ¡sopropanol) con 90 mi de solución B (0,1 g de goma arábiga y 0,4 mi de Tritón X-100 en  28 absorbance was measured on a Hitachi U-2000 spectrophotometer at a wavelength of 410 nm. The substrate solution (SS) was prepared by mixing 10 ml of solution A (30 mg of pNPP in 10 ml of sopropanol) with 90 ml of solution B (0.1 g of gum arabic and 0.4 ml of Triton X-100 in
90 mi tampón Tris-HCI 50mM pH8). La mezcla de solución A y B se agitó suavemente hasta su total disolución. La FIG. 2 muestra los valores de tolerancia de las cepas aisladas. 90 my 50mM Tris-HCI buffer pH8). The mixture of solution A and B was gently stirred until completely dissolved. FIG. 2 shows the tolerance values of the isolated strains.
EJEMPLO 2. Ensayo de xeroprotección de microorganismos. EXAMPLE 2. Test of xeroprotection of microorganisms.
El objetivo de este ensayo fue determinar la capacidad para proteger células vivas 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) de diversas cepas xerotolerantes de forma análoga a la descrita por Manzanera et al., (2002) (Manzanera et al., 2002. Applied and Environmental Microbiology 68: The objective of this test was to determine the ability to protect live cells of Escherichia coli MC4100 against desiccation by using the bacterial milking product (POB) as well as those extracted by Air Drying (POBSIAs) of various xerotolerant strains analogous to that described by Manzanera et al., (2002) (Manzanera et al., 2002. Applied and Environmental Microbiology 68:
4328-33). Para ello se utilizó un preinoculo de E. coli, a partir del cual se inoculó medio mínimo más glucosa como fuente de carbono adicionados de 0,6 M de NaCI hasta alcanzar una densidad óptica ¡nicial de 0,05. Tras 4328-33). For this, an E. coli pre-inoculum was used, from which a minimum medium plus glucose was inoculated as an added carbon source of 0.6 M NaCl until an initial optical density of 0.05 was reached. After
12 horas de incubación a 37°C en agitación, se centrifugaron alícuotas de 12 hours incubation at 37 ° C under stirring, aliquots of centrifuges were centrifuged
1 ml del cultivo crecido. Las células de E. coli se resuspendieron en una solución al 10% de los POBs o POBSIAs extraídos de las células xerotolerantes (extraídos según se describe anteriormente) y además, 1 ,5% de polivinilpirrolidona (PVP). Igualmente se realizó la misma experiencia mediante la combinación y mezcla de sustratos comerciales identificados en POBs y 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 1 ml of the grown culture. E. coli cells were resuspended in a 10% solution of the POBs or POBSIAs extracted from the xerotolerant cells (extracted as described above) and in addition, 1.5% polyvinylpyrrolidone (PVP). The same experience was also carried out by combining and mixing commercial substrates identified in POBs and POBSIAs in equal proportions, which were called synthetic POB or synthetic POBSIA as opposed to directly extracted from cells we call natural. In the case of
POB/POBSIA sintéticos la mezcla se realizó en una solución al 34,2%. La suspensión de células en las distintas soluciones se sometieron a condiciones de desecación por vacío sin congelación, en un congelador- desecador modificado (Dura - Stop μ P; FTS Systems, Stone Ridge, NY) a 30°C de temperatura media y 100 mTorr (2Pa; 2x10"5 atmósferas) durante 20 segundos, con una rampa de temperatura de 2,5°C/min con 15 minutos de pausa después de cada incremento de 2°C, hasta llegar a la temperatura máxima de 40°C. Synthetic POB / POBSIA mixing was performed in a 34.2% solution. The suspension of cells in the different solutions were subjected to vacuum drying conditions without freezing, in a freezer- Modified desiccator (Dura - Stop μ P; FTS Systems, Stone Ridge, NY) at 30 ° C medium temperature and 100 mTorr (2Pa; 2x10 "5 atmospheres) for 20 seconds, with a temperature ramp of 2.5 ° C / min with 15 minutes of pause after each increase of 2 ° C, until reaching the maximum temperature of 40 ° C.
Las muestras se sellaron al vacío y se almacenaron a 30°C hasta su ensayo de viabilidad a tiempo 1 , 15 y 30 días. Pasado este tiempo las muestras se resuspendieron en 1 ml de LB y se realizaron ensayos de viabilidad mediante siembra en placa de LB sólido que se incubaron 24 horas a 37°C, para el conteo de UFC y comparación con las UFC antes del secado, para de esta forma poder calcular la supervivencia de las muestras. En la FIG. 4 se observa cómo las composiciones sintéticas POBs y 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 xeroprotectores y durante el primer día de conservación así como a las dos semanas. Se observa que la aportación del PVP 1 ,5% a la supervivencia es nula. The samples were sealed under vacuum and stored at 30 ° C until their viability test was carried out at 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 planting in solid LB plate that were incubated 24 hours at 37 ° C, for CFU counting and comparison with CFUs before drying, for in this way to be able to calculate the survival of the samples. 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 xeroprotective compounds and during the first day of conservation as well as at two weeks. It is observed that the contribution of PVP 1.5% to survival is nil.

Claims

REIVINDICACIONES
1 . Microorganismo de la especie bacteriana Rhodococcus sp. con número de acceso CECT7625. one . Microorganism of the bacterial species Rhodococcus sp. with access number CECT7625.
2. Población bacteriana que comprende el microorganismo según la reivindicación 1 . 2. Bacterial population comprising the microorganism according to claim 1.
3. Uso del microorganismo según la reivindicación 1 o de la población bacteriana según la reivindicación 2 para la producción de una composición xeroprotectora. 3. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 for the production of an xeroprotective composition.
4. Composición xeroprotectora producida por el microorganismo según la reivindicación 1 o por la población bacteriana según la reivindicación 2. 4. Xeroprotective composition produced by the microorganism according to claim 1 or by the bacterial population according to claim 2.
5. Composición según la reivindicación 4 que comprende fructosa, ácido glutámico, β-hidroxibutirato, acetato y lactato. 5. Composition according to claim 4 comprising fructose, glutamic acid, β-hydroxybutyrate, acetate and lactate.
6. Composición xeroprotectora sintética que comprende fructosa, ácido glutámico, β-hidroxibutirato, acetato y lactato. 6. Synthetic xeroprotective composition comprising fructose, glutamic acid, β-hydroxybutyrate, acetate and lactate.
7. Composición según cualquiera de las reivindicaciones 5 ó 6 que comprende una proporción de fructosa : ácido glutámico : acetato, de entre (35 y 45) : (1 ,4 y 3,4) : (0,5 y 1 ,5) respectivamente. 7. Composition according to any of claims 5 or 6 comprising a proportion of fructose: glutamic acid: acetate, between (35 and 45): (1, 4 and 3.4): (0.5 and 1, 5) respectively.
8. Composición según la reivindicación 7, donde la proporción de fructosa : ácido glutámico : acetato es de entre (38 y 44) : (2 y 3) : (0,7 y 1 ,3), respectivamente. 8. Composition according to claim 7, wherein the proportion of fructose: glutamic acid: acetate is between (38 and 44): (2 and 3): (0.7 and 1, 3), respectively.
9. Composición según cualquiera de las reivindicaciones 5 ó 6 que comprende una proporción de fructosa : ácido glutámico : β- hidroxibutirato : acetato : lactato, de entre (14 y 18) : (3 y 5) : (0,6 y 1 ) : (0,5 y 1 ,5) : (1 y 2), respectivamente. 9. Composition according to any of claims 5 or 6 comprising a proportion of fructose: glutamic acid: β- hydroxybutyrate: acetate: lactate, between (14 and 18): (3 and 5): (0.6 and 1): (0.5 and 1, 5): (1 and 2), respectively.
10. Composición según la reivindicación 9, donde la proporción de fructosa: ácido glutámico: β-hidroxibutirato: acetato: lactato es de entre10. Composition according to claim 9, wherein the proportion of fructose: glutamic acid: β-hydroxybutyrate: acetate: lactate is between
(15 y 17) : (3,5 y 4,5) : (0,7 y 0,9) : (0,7 y 1 ,2) : (1 ,2 y 1 ,6), respectivamente. (15 and 17): (3.5 and 4.5): (0.7 and 0.9): (0.7 and 1, 2): (1, 2 and 1, 6), respectively.
1 1 . Uso de la composición según cualquiera de las reivindicaciones 4 a 10 para la conservación de material biológico con un contenido de humedad residual igual o inferior al 10%. eleven . Use of the composition according to any of claims 4 to 10 for the conservation of biological material with a residual moisture content equal to or less than 10%.
12. Uso de la composición según la reivindicación 1 1 , donde el material biológico es un microorganismo o una célula. 12. Use of the composition according to claim 1, wherein the biological material is a microorganism or a cell.
13. Uso de la composición según la reivindicación 11 , donde el material biológico es un organismo invertebrado, una semilla, una plántula, un órgano aislado o un tejido biológico aislado. 13. Use of the composition according to claim 11, wherein the biological material is an invertebrate organism, a seed, a seedling, an isolated organ or an isolated biological tissue.
14. Uso de la composición según la reivindicación 1 1 , donde el material biológico es una molécula con actividad biológica. 14. Use of the composition according to claim 1, wherein the biological material is a molecule with biological activity.
15. Uso de la composición según la reivindicación 14, donde la molécula con actividad biológica es una enzima. 15. Use of the composition according to claim 14, wherein the molecule with biological activity is an enzyme.
16. Uso de la composición según la reivindicación 15, donde la enzima es una lipasa. 16. Use of the composition according to claim 15, wherein the enzyme is a lipase.
17. Método de obtención de la composición xeroprotectora según cualquiera de las reivindicaciones 4 a 10 que comprende: a) cultivar el microorganismo de la reivindicación 1 o la población de la reivindicación 2 en un medio mineral con fructosa como fuente de carbono, 17. Method of obtaining the xeroprotective composition according to any of claims 4 to 10 comprising: a) culturing the microorganism of claim 1 or the population of claim 2 in a mineral medium with fructose as a 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 hipotónico, y  c) rehydrate the dehydrated microorganisms of step (b) in a hypotonic medium, and
d) seleccionar la fracción líquida del producto obtenido en el paso (c) que comprende la composición xeroprotectora.  d) select the liquid fraction of the product obtained in step (c) comprising the xeroprotective composition.
18. Método según la reivindicación 17, donde el medio mineral del paso (a) es sólido. 18. Method according to claim 17, wherein the mineral medium of step (a) is solid.
19. Método según cualquiera de las reivindicaciones 17 ó 18, 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. 19. Method according to any of claims 17 or 18, 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.
20. Método según cualquiera de las reivindicaciones 17 a 19, donde el medio hipotónico para la rehidratación de los microorganismos según el paso (c) es agua parcial o totalmente destilada, desionizada o desmineralizada. 20. Method according to any one of claims 17 to 19, wherein the hypotonic means for rehydration of the microorganisms according to step (c) is water partially or totally distilled, deionized or demineralized.
21 . Método según cualquiera de las reivindicaciones 17 a 20, donde además, la fracción líquida del paso (d) se deshidrata hasta que el producto xeroprotector tenga una humedad residual igual o inferior al 10%. twenty-one . Method according to any of claims 17 to 20, 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%.
22. Método para la conservación de material biológico que comprende a) mezclar la composición xeroprotectora según cualquiera de las reivindicaciones 4 a 10 con una muestra de material biológico, y 22. Method for the conservation of biological material comprising a) mixing the xeroprotective composition according to any of claims 4 to 10 with a sample of biological material, and
b) deshidratar el producto obtenido en el apartado (a) hasta una humedad residual igual o inferior al 10%.  b) dehydrate the product obtained in section (a) to a residual humidity equal to or less than 10%.
23. Método según la reivindicación 22, donde el material biológico es un microorganismo o una célula. 23. Method according to claim 22, wherein the biological material is a microorganism or a cell.
24. Método según la reivindicación 22, donde el material biológico del paso (a) es un organismo invertebrado, una semilla, una plántula, un órgano aislado o un tejido biológico aislado. 24. Method according to claim 22, wherein the biological material of step (a) is an invertebrate organism, a seed, a seedling, an isolated organ or an isolated biological tissue.
25. Método según la reivindicación 22, donde el material biológico es una molécula con actividad biológica. 25. Method according to claim 22, wherein the biological material is a molecule with biological activity.
26. Método según la reivindicación 25, donde la molécula con actividad biológica es una enzima. 26. Method according to claim 25, wherein the molecule with biological activity is an enzyme.
27. Método según la reivindicación 26, donde la enzima es una lipasa. 27. Method according to claim 26, wherein the enzyme is a lipase.
28. Método según cualquiera de las reivindicaciones 22 a 27, donde la deshidratación de la mezcla de la composición xeroprotectora y del material biológico según el paso (b) se lleva a cabo por medio de una solución hipertónica o por medio de una corriente de aire. 28. Method according to any of claims 22 to 27, wherein the dehydration of the mixture of the xeroprotective composition and the biological material according to step (b) is carried out by means of a hypertonic solution or by means of an air stream .
PCT/ES2010/000517 2009-12-04 2010-12-03 Bacterial strain cect7625, uses thereof and xeroprotectant product produced from same WO2011067441A2 (en)

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WO2011067441A3 (en) 2011-12-01
ES2389367B2 (en) 2013-05-09

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