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    • 专利摘要:
    Procedure for the protection of biological material and thermolabile compounds for possible industrial applications. Procedure for the protection of biological material in general and thermolabile substances, specifically applicable to microorganisms and viruses and any other biological material, as well as to any substance derived or not, that needs protection to extend its useful life or extend its field of application, through processing electrohydrodynamic or aerodynamic. This processing allows to extend the useful life of the product in a unique way since it does not involve exposure to temperature of the substance to be protected. In addition, depending on the characteristics or variables used during the coating process, the encapsulation allows maintaining the viability of the product under stress conditions such as temperature, relative humidity or ph among others, providing an effective protection during its subsequent preparation, processing, storage and even for example during the passage through the gastrointestinal tract for the case of probiotic microorganisms. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2541203A2
    申请号:ES201430034
    申请日:2014-01-15
    公开日:2015-07-16
    发明作者:José María LAGARÓN CABELLO；Rocío PÉREZ MASÍA；Amparo LÓPEZ RUBIO
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    SECTOR AND OBJECT OF THE INVENTION
    Food and pharmaceutical sectors. Materials to stabilize microorganisms,
    viruses and biological material, as well as compounds or substances of added value
    10 thermolabile and bioactive.
    The object of the present invention is a method of protecting biological material in general and thermolabile substances, specifically applicable to microorganisms and viruses and any other biological material, as well as to any substance derived or not, that needs protection to extend its useful life. or expand its scope, by electrohydrodynamic or aerodynamic processing. Specifically, micro-, submicro- and nanoparticles of different polymers are obtained by electrohydrodynamic or aerodynamic processing, also known as electrospraying and electrospraying and blowing and spraying, 20 respectively, called in English electrospinning, electrospraying and solution blow spinning and solution blow spraying, respectively. These micro-, submicro- and nanoparticles are processed directly on preparations of microorganisms (lyophilized or dehydrated preparations by any other method of preservation), of viruses, of biological material or thermolabile substances, so that 25 remain adhered by the high surface volume ratio of the morphology obtained by forming an effective protective coating or capsule that covers all the interstices of the product, being therefore more efficient than other methods or processes in the preservation of viability (count of microorganisms or number of UFC colony forming units) or stability of the product. This processing allows to extend the life of the product in a unique way since it does not entail the temperature exposure of the substance to be protected. In addition, depending on the characteristics or variables used during the coating process, encapsulation allows maintaining the viability of the product under stress conditions such as temperature, relative humidity or pH, among others, providing effective protection during
    subsequent preparation, processing, storage and even for example during the passage through the gastrointestinal tract in the case of probiotic microorganisms.
    STATE OF THE TECHNIQUE
    5 In the field of probiotics there is a growing demand for functional products based on microorganisms beneficial to health. However, it is necessary that these microorganisms are in the product in quantities greater than 107, ideally 109 colony forming units (CFU / mL) at the time of consumption for its effect to be efficient [Gerez, CL, Font de Valdez, G.,
    10 Gigante, M.L., Grosso, C.R.F. (2012). Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH. Lelters in Applied Microbiology 54 (6), pp. 552-556). Because most of these microorganisms are sensitive to environmental conditions (humidity, oxygen concentration, light ..), it is important to develop strategies that protect them from the environment to be able to
    15 keep them. Thus, numerous processes for the development of structures capable of providing the necessary conditions for the optimal metabolism of microorganisms are being studied, while protecting them from the surrounding environment. Some of the techniques used for the immobilization and protection of microorganisms are cellular adsorption on solid supports [Rezaee, A.,
    20 Godini, H., Bakhtou, H. (2008). Microbial cellulose as support material for the immobilization of denitrifying bacteria. Environmental Engineering and Management Journal, 7 (5), pp. 589-594], [4] or "oculation [5], [D. Yin, D., liu, C., li, F., Ge, X., Bai, F. (2011). Development of observed kinetic model for self-flocculating yeast, Huagong Xuebao / CIESC Journal, 62 (11), pp. 3149-3155] Both techniques require
    25 that the microorganisms are able to adhere naturally on the supports (ability to form biofilm) or aggregate to flocculate respectively. In addition, other strategies such as encapsulation have been studied [Rathore, S., Desai, P.M., Uew, CV., Chan, L.W., Heng, P.W.S. (2013). Microencapsulation of microbial cells. Journal of Food Engineering 116 (2), pp. 369-381). Among the most
    30 common are extrusion encapsulation, coacervation, spray drying and emulsification, (de Vos, P., Faas, MM, Spasojevic, M. Sikkema, J. (2010). Encapsulation for preservation of functionality and targeted delivery of bioactive food components International Dairy Journal, 20 (4) pp. 292-302] However, these technologies require a warming of the solutions or the use of agents
    organic at least in one of the production phases, which could damage or destroy encapsulated microorganisms. Therefore, it would be desirable to have new technologies that do not involve aggressive conditions (both temperature and solvents used) and give rise to small particle sizes. The technique 5 of electrohydrodynamic processing comprising processes called electrospinning and electrospraying (electro-stretching or electrospraying by high voltage) is a simple and highly versatile method for obtaining fibers and / or capsules by means of the action of an external electric field that is applied between two electrodes and to which it is subjected to the polymer solution. Some of the properties of the structures 10 obtained by electrohydrodynamic processing are their nanometric size and do not require the use of temperature, therefore the technique has already been proposed for the encapsulation of probiotics with good results [Lopez-Rubio, A., Sanchez , E., Sanz, Y., and Lagaron, JM (2009). Encapsulation of living bifidobacteria in ultrathin PVOH electrospun fibers. Biomacromolecules 10, 2823-2829]. On the other hand, the aerodynamic processing technique 15 comprising stretching and spraying by blowing (solution blow spinning and spraying) consists in the application of pressure differences to accelerate a fluid and thus obtain the different polymeric structures. This technology has been used for the encapsulation of active agents [Oliveira, J.E., Medeiros, E.S., Cardozo, L., VolI, F., Madureira, E.H., Mattoso, L.H.C., Assis, O.8.G. 20 (2013). Development of poly (Iactic acid) nanostructured membranes for the controlled delivery of progesterone to livestock animals. Materials Science and Engineering e 33 (2), pp. 844-849], as well as for probiotics [Spanish patent application P201131048]. In any case, the direct encapsulation, disperses and confines the microorganisms in an enclosed space, requires their dissolution, stability and compatibility with the media and solvents, and also requires that the materials used to form the capsules correctly conform to the metabolic needs of encapsulated microorganisms (adequate mechanical properties, solvents and permeability to nutrients, gases and metabolites) and thus maintain their viability over time. In addition, all encapsulation technologies require that microorganisms be subjected to various conditions that could affect their viability (temperature, organic solvents, high voltage or pressure difference). In this way the microorganism is always affected to a greater or lesser extent and although long-term viability can be maintained, the initial count often decreases. In this sense, the technique of
    Coating protection by electrohydrodynamic or aerodynamic processing of the present invention provides very efficient protection of microorganism preparations since micro-, submicro- and protective nanostructures that adhere to them are obtained, protecting them from external conditions, 5 and therefore maintaining their viability for longer. In addition, it avoids any type of direct processing of the material, so that the initial viability is maintained, an aspect of great industrial relevance. On the other hand, by means of this technique the microorganism is not confined in a rigid matrix but simply wrapped by an encapsulating coating so it does not interfere with the metabolism.
    EXPLANATION OF THE INVENTION
    The present invention consists of a methodology to protect microorganisms,
    virus, biological material in general and any heat-labile substance that you need
    protection to extend its useful life or extend its scope through a
    15 coating composed of micro-, submicro or polymeric nanostructures obtained by electrohydrodynamic or aerodynamic processing and which remain attached to the microorganisms generating a protective encapsulation effect. In this way, thermolabile compounds and biological materials to be encapsulated are not subjected to any type of processing, thus being protected and isolated from the environment.
    20 environment. For this purpose they are obtained by electrospinning / electrospraying, more preferably by electrospraying or electrosprayado, or drawn / sprayed by blow, more preferably by sprayed by blow, micro-, submicro or nanoparticles from synthetic or natural polymers (biopolymers), preferably soluble in water and mainly biopolymers, since they are usually
    25 more beneficial for microorganisms.
    The process of protection of biological material and thermolabile compounds of industrial interest comprises the following stages:
    30 preparation of polymeric solutions forming micro-, submicro- and nanoparticles that make up coating materials
    - coating of the biological material or thermolabile compounds by means of the materials prepared in the previous stage, the coating of the material being carried out
    biological or thermolabile compounds by electrohydrodynamic or aerodynamic processing.
    The biological material is preferably microorganisms or viruses and particularly
    5 microorganisms are selected from Lactobacillus, Bifidobacterium, Cyanobacterium, RhodobacteraJes, Saccharomyces, or any other microorganism that needs protection.
    Thermolabile compounds are enzymes, vitamins, essential elements, or any molecule or derivative compound.
    The coating materials are selected from proteins, oligosaccharides, polysaccharides, lipids, polymers and combinations thereof.
    Among the polymers, water-soluble, alcohols and mixtures thereof, such as polyethylene oxide, copolymers of ethylene and vinyl alcohol, polyvinyl alcohol, polyvinyl pyrrolidone and combinations thereof, are more particularly selected.
    Among the proteins, zein and whey protein of milk are particularly selected.
    Among the polysaccharides, dextran, maltodextrin and starch and any combination or preparation thereof such as the commercial preparation based on a resistant starch called fibersol are particularly selected.
    The solvent used for the preparation of polymer solutions is selected from water, mixtures of water alcohols and other organic solvents, preferably water or mixtures of alcohol and water.
    At the stage of preparing the polymer solutions, processing aids can be added that facilitate the formation of the micro-, submicro and nanoparticles and are selected from plasticizers, surfactants, emulsifiers, surfactants, antioxidants or any combination thereof, preferably sorbitan monolaureate (commercially known as span-20).
    Homogenization treatments can also be included by shaking, turning or ultrasonic
    In a preferred embodiment, electrohydrodynamic processing or
    5 aerodynamic is electro-sprayed or blow-sprayed in at least one stage.Optionally, electrohydrodynamic or aerodynamic processing is performed in severalstages with a homogenization and / or sieving treatment between them.
    Electrohydrodynamic or aerodynamic processing can be performed directly
    10 on the biological material or thermolabile compounds previously dehydrated by lyophilization or other non-electrohydrodynamic or aerodynamic process or without dehydration, producing dehydration and application of the coating in successive processes by electrohydrodynamic or aerodynamic processing.
    15 In case it is necessary before or after the coating stage, a sieving treatment is carried out to control the particle size, preferably by mechanical sieving.
    DETAILED DESCRIPTION OF THE INVENTION
    20 The methodology for obtaining coatings of microorganisms by electrohydrodynamic or aerodynamic processing is described in detail below. The objective of these coatings is to stabilize and maintain the viability of various microorganisms or thermolabile substances with industrial application so that they can, for example, remain high
    25 concentrations at the time of application (consumption, sensors, water treatment, etc.)
    The first stage consists in the preparation of the polymeric solutions forming the micro-submicro and nanoparticles that will comprise the
    30 coatings In this phase, other substances (such as plasticizers, surfactants, emulsifiers, surfactants, antioxidants, process aids in general or any combination thereof) that facilitate the formation of structures can be added.
    Among the surfactants, different types of compounds commonly referred to as span, different types of tween and lecithin, and more preferably that which is commercially called span-20, whose composition is sorbitan monolaureate, will preferably be selected without limitation.
    This stage also includes a homogenization treatment by agitation and / or ultrasound. Stirring can be vigorous to favor dispersion of the additives in the polymer matrix.
    10 The second stage consists of coating the microorganisms by electrohydrodynamic or aerodynamic processing. This process can be performed directly on the commercial product formulated and dehydrated by some method such as lyophilization or electrohydrodynamic or aerohydrodynamic processing or it can be obtained in situ by electrohydrodynamic or aerodynamic processing or by any
    15 another procedure without limiting sense prior to coating.
    Electrohydrodynamic processing comprising electro-stretching (electrospinning) and electrospraying is a technology based on the application of high electric fields to produce electrically charged fluids from 20 viscoelastic polymer solutions, which when dried produce micro-and nanofibers or micro-and nanocapsules, respectively. Electrospraying and electroplating equipment consists of a power supply that supplies current, a pump where the tanks with solutions are placed and one or more conductive propellants, typically needles, of a conductive material. The needles are connected to the tanks and are focused towards the collector where the dry material will be collected. In the case of aerodynamic processing, which includes blow spray and blow stretch, a pump is used where the tanks with the solutions are placed. These tanks are connected to a nozzle through which a flow of pressurized gas is also passed. The mouthpiece is focused towards
    30 the collector where the material is collected. Thus, the preparation of microorganisms is placed on the collector and the protective particles with controlled size will be stretched or sprayed directly on them forming a coating that provides stability to the product during its processing, postprocessing, storage and even during use during application.
    Within electrohydrodynamic processing, preferred use is made in the present invention of electrospray and within aerodynamic processing, preferential use is made in the present invention of blow-molding.
    5 In all cases, a gravity, mechanical or other sieve treatment can be carried out to prepare the microorganisms before and after the coating to control the particle size.
    The coating process can be carried out in one or several steps according to the
    10 protective needs of the material and with one or more materials, and may involve processes of turning and homogenization of the coating using any known industrial means to increase the efficiency of the coating process.
    Materials used for coatings obtained in the manner described
    15 above comprise proteins, oligosaccharides, polysaccharides, lipids, other water soluble polymers such as and without limitation polyvinyl alcohol, polyethylene oxide or polyvinylpyrrolidone and water alcohol mixture such as and without limitation copolymers of ethylene and vinyl alcohol and any preparation commercial thereof or mixture of the above. Preferably, the proteins will be used,
    20 oligosaccharides, polysaccharides and lipids due to their sustainable character and greater stability and compatibility with microorganisms.
    Among the proteins, animal, vegetable and microbial proteins, such as those from whey, will preferably be selected without limitation,
    25 caseins, natural polypeptides or obtained by genetic modification of microorganisms, collagen, soy protein and zein, and more preferably the zein and whey protein.
    Among the oligosaccharides, lactose, sucrose, maltose and fructooligosaccharides and more preferably fructooligosaccharides will preferably be selected and not limited to.
    Among the polysaccharides, alginate, pectins, chitosan, gums, carrageenans, starch, dextran, maltrodextrin, cellulose, glycogen, chitin and more preferably starch, dextran, maltrodextrin and any preparation thereof such as the fibersol or prepared similar.
    Among the lipids, acids are preferably selected without limitation.5 fatty
    As for solvents, water is preferred, but since the technique eliminates the solvent very efficiently, the process of the invention is compatible with any type of solvent, whether or not it is compatible with the product to be protected, 10 this being One of the advantages of this process. As for microorganisms, in the case of probiotics, both facultative aerobic bacteria belonging to the genus Lactobaci / Jus, and strict anaerobes of the genus Bifidobacterium have been tested. Also fungi of the genus Saccharomyces can be used in this application. In the case of water treatment or sensor applications,
    15 preferred families are Cyanobacterium or RhodobacteraJes.
    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 experts in the field, other objects, advantages and characteristics of the
    The invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    BRIEF DESCRIPTION OF THE FIGURES
    25 Figure 1. Scanning Electron Microscopy (SEM) image of electroplated structures of starch in aqueous solution.
    Figure 2. Sample of an optical microscopy image of the freeze-dried crystals of Lactobacillus plantarum coated by the polymeric structures (alginates, 30 pectins and modified starch) obtained by electrospraying.
    Figure 3. Coated lyophilisate counts vs. lyophilisate not coated over time stored at 37 ° C.
    Figure 4. Coated lyophilisate counts vs. lyophilisate not coated over time stored at 53% relative humidity (RH).
    Figure 5. Scanning Electron Microscopy (SEM) image of the 5-lyophilic crystals of Lactobacillus planfarum.
    Figure 6. Scanning Electron Microscopy (SEM) image of the lactobacillus plantarum lyophilic crystals coated with whey proteins by the method of the present invention.
    10 Figure 7. Scanning Electron Microscopy (SEM) image of the zein capsules covering the lyophilic crystals of Lactobacillus plantarum.
    MODE OF REALIZATION OF THE INVENTION
    15 Example 1. Obtaining the nanostructured coating from a polysaccharide
    In this example, a typical process of obtaining coatings based on a polysaccharide using the electrospray technique is described.
    In a first stage, the solution of polysaccharide (commercial brand fibersol) in distilled water is prepared. The concentration of the polysaccharide used is 40% by weight with respect to the volume of the solvent. The solution is stirred at room temperature until a homogeneous solution is obtained.
    25 Once the solution is obtained, it is used to generate the micro-and nanocapsules using the electro-spray technique using a commercial equipment of the Fluidnatek brand of Bioinicia, S.L., Paterna (Valencia). The solution is introduced into 5 mL syringes connected through Teflon tubes to a needle
    30 stainless steel diameter 0.9 mm. The needle is connected to an electrode that in turn is connected to a 0-30 KV power supply. A voltage between 15-20 KV is applied and the solution is pumped through said needle with a flow of 0.1 mUh. The counter electrode is connected to a stainless steel plate (collector) where the lyophilized probiotic is placed. The distance between the needle and the collector is about 15 cm. The process is carried out at room temperature. In this way the micro-and nanocapsules shown in Figure 1 are obtained that are adhered to the deliophile particles.
    5 Capsule sizes obtained in this way range between 50 nm and 3microns (ie capsules with sizes below 100 nm).
    Example 2. Obtaining a nanostructured coating from a mixture of 10 biopolymers (alginates, pectins and modified starch) and their application on the lyophilisate of Lactobacillus plantarum.
    The first stage consists in the preparation of the nanoparticle coating forming solution. For this, the mixture of alginate, pectin and modified starch is dissolved in a concentration of 3.1 and 10% by weight with respect to volume
    of the solvent (distilled water) respectively. The mixture is kept under stirring until the components are completely dissolved. The final solution is introduced into a 5 ml syringe and then passed through the electrohydrodynamic processing equipment.
    Secondly, a fine and homogeneously distributed deposition of the lyophilisate (approx. 5x1010 cfu / gram) is carried out by means of a sieve and distributed over the collector of the electrohydrodynamic processing equipment. The electro-spray of the solution is then carried out on the lyophilisate. The equipment consists of a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow of the solution and a stainless steel needle. The needle is connected to the syringe through a Teflon cable and placed perpendicular to the manifold. For this example, the electro-stretching conditions were as follows: solution flow rate of 0.15 ml / h; 30 14 kV of current; 10 cm distance between the needle and the collector. After a controlled time of electrospraying the material is turned over and then again electrosprayed to ensure a complete coating of all lyophilisate. The material collected under these conditions consists of a nanostructured system
    composed of spherical capsules of the mixture of alginates, pectins and starch that cover the lyophilisate crystals (as shown in Figure 2).
    Example 35 Obtaining a nanostructured coating from polyethylene oxide(PEO) and its application on lyophilisate of Lactobacillus plantarum.
    The first stage consists in the preparation of the nanoparticle coating forming solution. For this, the PEO is dissolved in a concentration of 4% by weight with respect to the volume of the solvent (distilled water). The solution is kept under stirring until the PEO is completely dissolved. The final solution is introduced into a 5 ml syringe and then passed through
    of electrohydrodynamic processing equipment.
    Secondly, the lyophilisate deposition is performed and distributed homogeneously over the electrohydrodynamic processing equipment collector. Next, the electrospraying of the solution on the lyophilisate is carried out. The equipment consists of a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the
    20 solution flow rate and a stainless steel needle. The needle is connected to the syringe through a tetlon cable and placed perpendicular to the manifold. For this example, the electrospray conditions were as follows: solution flow rate of 0.15 ml / h; 14 kV current; 10 cm distance between the needle and the collector. After a controlled time of electrosprayado the material turns and then
    25 is electrosprayed again to ensure a complete coating of all lyophilisate. The material collected under these conditions consists of a nanostructured system composed of PEO fibers that cover the freeze-dried crystals.
    Example 4. Obtaining a nanostructured coating from a mixture of polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVOH) and its application on the lyophilisate of Lactobacillus plantarum.
    The first stage consists in the preparation of the nanoparticle coating forming solution. For this, the PVP and the PVOH are dissolved in a concentration of 20% and 4% by weight with respect to the volume of the solvent (distilled water) respectively. The solution is kept under stirring until all
    5 the components are completely dissolved. The final solution is introduced into a 5 ml syringe and then passed through the electrospray equipment.
    Secondly, the lyophilisate deposition is performed and distributed
    10 homogeneously on the collector of the electrospray equipment. Next, the electrospray of the solution on the lyophil is performed. The electrospray equipment consists of a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow of the solution and a stainless steel needle. The needle is
    15 connects to the syringe through a Teflon cable and is placed perpendicular to the manifold. For this example, the processing conditions were as follows: solution flow rate of 0.15 ml / h; 14 kV current; 10 cm distance between the needle and the collector. After a controlled time of electrosprayado the material is turned and then again electrosprayar to ensure a coating
    20 full of all lyophilisate. The material collected under these conditions consists of a nanostructured system composed of PVP and PVOH capsules that cover the freeze-dried crystals.
    Example 5
    25 Encapsulation of probiotics (Lactobacillus plantarum) from a whey protein concentrate using different electrohydrodynamic processes and comparison of the initial viability of the different materials.
    30 The first stage consists in the preparation of the forming solutions of the encapsulates and of the coating. For this, the whey protein concentrate is dissolved in a concentration of 20% by weight with respect to the volume of the solvent (skim milk). In addition, a surfactant (Span-20) is added in a concentration of 20% with respect to the protein weight to improve processing. The mixture is kept under stirring until the components are completely dissolved.
    Secondly, the probiotic microorganism is added according to the processing
    5 will carry out. For uniaxial electrospray, lyophilisate is added to thesolution in a concentration of up to 50% by weight with respect to the weight ofpolymer. In the case of coaxial electrospray, a solution oflyophilisate of 50% by weight with respect to the volume of solvent (saline solution).Finally, in the case of the coating, the lyophilisate is deposited and distributed in
    10 homogeneous manner on the collector of the electrostimulation equipment. The electro-spray of the solutions is then carried out. In the case of the coating, after a controlled time of electrospraying the material is turned over and then again electrosprayed to ensure a complete coating of the entire lyophilisate. The equipment for the uniaxial process and the coating consists of a source of
    15 supply that supplies between O and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow of the solution and a stainless steel needle. The needle is connected to the syringe through a Teflon cable and placed perpendicular to the manifold. In the case of coaxial electrospray, the equipment consists of the same elements as before, but
    20 also has another digital pump to place the syringe with the lyophilisate and two concentric needles, through which the polymer solution (through the outer needle) and the lyophilisate solution (through the needle inside) will circulate.
    The electrospray conditions were as follows: dissolution flow rate of
    0.15 ml / h; 14 kV current; 7 cm distance between the needle and the collector in the case of uniaxial processing and the coating. In the case of coaxial processing were: 0.16 mUh for protein dissolution, 0.08 mLlh for lyophilisate solution, 20 kV of current and 7 cm distance between the needle and the collector.
    Finally, the viability of the microorganisms of the different materials obtained was measured by observing that the coating method provided higher counts and similar to the counts of the unprotected starting lyophil, than the lyophil encapsulated by the uniaxial and coaxial method (Table 1 ). Figure 6 shows the electron microscopy image of the coated lipophilic compared to the image of the uncoated lyophilic crystals (Figure 5).
    Table 1
    EncapsulatedEncapsulated
    coaxial uniaxialCovering
    Counts (UFC / g) 2.3x1082.1 x 1088.2x109
    Example 6Obtaining submicrometric encapsulation structures from amaltodextrin using the spinníng / spraying blow technique(stretched / spray sprayed)
    In this example, the procedure for obtaining submicron capsules using the stretch drawing / spray technique is detailed. First, a solution of the maltodextrin in water is prepared, using a polysaccharide concentration of 40% by weight with respect to the volume used, while stirring
    20 at room temperature until a homogeneous solution is obtained.
    The solution was introduced into a 5 ml syringe located in a syringe pump and connected through Teflon tubes to an internal stainless steel needle with a diameter of 0.9 mm. This needle was mounted in a coaxial configuration, the outer needle 25 through which pressurized nitrogen gas flows at high speed (230-250 mIs). The nitrogen flow pumped coaxially through the outer needle accelerates and stretches the polymer solution that flows through the inner needle and aids in the formation of encapsulation structures. The solution flow with maltodextrin was 0.5 mUh. The nitrogen gas pressure in the bottle was 20-30 bar. The structures
    30 generated and solidified were collected in a collector located at a distance of about 18-20 cm.
    Example 7
    Feasibility results of Lactobacillus plantarum after application of the nanostructured coating obtained from various polymer formulations.
    5 This example shows the feasibility results of L. plantarum obtained as soon as they are coated by electrohydrodynamic processing. First, the different polymer solutions were prepared with the corresponding proportions and additives according to each material. These formulations were passed through the electrospray equipment to obtain the nanostructured coating. TO
    10 A fixed amount (0.2 g) of lyophilisate was homogeneously deposited on the collector where the nanostructured material is collected. Finally, the electrospray of the solutions on the lyophilisate is carried out. After a controlled time of electrospraying the material is turned over and then again electrosprayed to ensure a complete coating of all lyophilisate. The team consists of
    15 a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow of the solution and a stainless steel needle. The needle is connected to the syringe through a Teflon cable and placed perpendicular to the manifold. The electrospray conditions were adjusted to the different polymers used. The material
    20 collected under these conditions consists of a nanostructured system composed of pOlimer capsules that cover the freeze-dried crystals. Immediately after collecting it, the material was seeded in the optimal medium for the growth of the bacteria and the microorganism counts were made. The feasibility results obtained are shown in Table 2.
    Table 2.
    Coating Forming Materials Alginate
    Alginate + Pectins + Fibersol + Span-20
    Alginate + Fibersol + Span-20 Starch Starch + Polyvinyl Alcohol (PVOH)
    Starch + Span-20 Dextran
    Dextran + Span-20
    Maltodextrin + PVOH Maltodextrin + Span-20
    Polyethylene Oxide (PEO)
    Pululane Pululane + PVOH
    PVOH
    Polyvinyl pyrrolidone (PVP) + PVOH
    PVP + Span-20
    Whey Protein Concentrate
    (WPC)
    W PC + PVOH
    WPC + Span-20 Zeina Initial feasibility (CFUlg)
    1.10E + 10 5.70E + 09 4.60E + 09 2.30E + 09 1.20E + 09 1.20E + 10 3.50E + 09 1.20E + 10 1.30E + 09 2.40E + 09 1.30E + 10 1.20E + 10 1.10E +10 2.40E + 10 3.40E + 10 2.30E + 10
    6.20E + 09 3.10E + 09 1.80E + 09 1.10E + 10
    Example 8
    Lactobacillus plantarum coating with process resistant starch
    electrohydrodynamic and study of the feasibility over time in conditions of temperature
    10 The first stage consists in the preparation of the nanoparticle coating forming solution. For this, the resistant starch is dissolved in a concentration of 20% by weight with respect to the volume of the solvent (distilled water).
    In addition, 2% of the Span 20 surfactant is added to facilitate the electrospray process. The solution is kept under stirring until all the components are completely dissolved. The final solution is introduced into a 5 ml syringe and then passed through the equipment.
    Secondly, the deposition of the lyophilisate is carried out and distributed homogeneously over the collector of the electro-stretching equipment by means of a sieve. The electro-spray of the solution is then carried out on the lyophilisate. After a controlled time of electrospraying the material is turned over and then again electrosprayed to ensure a complete coating of all lyophilisate. The electrospray equipment consists of a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow of the solution and a stainless steel needle. The needle is connected to the syringe through a Teflon cable and placed perpendicular to the manifold. For this example, the electrospray conditions were as follows: solution flow rate of 0.15 ml / h; 14 kV current; 10 cm distance between the needle and the collector. The material collected under these conditions consists of a nanostructured system composed of resistant starch capsules that cover the freeze-dried crystals. This material was stored together with uncoated lyophilisate at 37 oC for an accelerated study of the viability of the microorganisms. Figure 3 shows the counts of the 2 stored samples (lyophilized coated and uncoated). It is observed that the initial counts are similar in both cases. However, by increasing the storage time under these conditions, the viability of the uncoated lyophilisate
    25 decreases faster than that of protected lyophilisate.
    Example 9. Coating of Lactobacillus plantarum with zein by electrohydrodynamic processing and study of the feasibility over time under conditions of
    30 humidity
    The first stage consists in the preparation of the nanoparticle coating forming solution. For this, the zein is dissolved in a concentration of 12% by weight with respect to the volume of the solvent (a mixture of alcohol and water in a 15:85 ratio respectively). The solution is kept under stirring until the zein is completely dissolved. The final solution is introduced into a 5 ml syringe and then passed through the electrospray equipment.
    Secondly, the lyophilisate deposition is performed and distributed homogeneously over the electrohydrodynamic processing equipment collector
    Then, the electrospray of the solution on the lyophilisate is performed. After
    10 a controlled time of electrospraying the material is turned over and then re-electrosprayed to ensure a complete coating of all lyophilisate. The equipment consists of a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow of the solution and a stainless steel needle. The needle connects to the
    15 syringe through a Teflon cable and placed perpendicular to the collector. For this example, the electrospray conditions were as follows: solution flow rate of 0.30 ml / h; 12 kV current; 10 cm distance between the needle and the collector. The material collected under these conditions consists of a nanostructured system composed of zein capsules that cover the crystals of
    20 lyophilisate, as seen in Figure 7, compared to the image of the crystals of the uncoated lyophilisate (Figure 5). This material was stored together with uncoated lyophilisate at 53% relative humidity to allow an accelerated study of the viability of microorganisms. Figure 4 shows the counts of the 2 stored samples (lyophilized coated and uncoated). It is observed that
    25 at the beginning the viability of the coated lyophilisate has a greater fall, however, after a short time it stabilizes, while the unprotected lyophilisate drops exponentially.
    Example 10
    30 Coating and encapsulation of fJ-carotene with zein by electrohydrodynamic processing and comparative study of antioxidant stability under ultraviolet (UV) light
    The first stage consists in the preparation of the forming solutions of the nanoparticle coating and the encapsulates. For the coating, the zein is dissolved in a concentration of 12% by weight with respect to the volume of the solvent (a mixture of alcohol and water in a 15:85 ratio respectively). To obtain the encapsulated 3-carotene, a solution of 33% zein by weight with respect to the volume of the solvent (a mixture of alcohol and water in a 15:85 ratio respectively) is prepared and the antioxidant is added in a zein ratio: 3-carotene of 95: 5 respectively. The solutions are kept under stirring until the components are completely dissolved. The final solutions
    10 are introduced into a syringe of 5 ml to then pass through the equipment.
    The antioxidant is then deposited and distributed evenly over the equipment collector for the coating. Finally, the electrospray of the solutions on the collector is performed. In the case of the coating, after a controlled time of electrosprayado, the material is turned and then again electrosprayar to ensure a complete coating of the entire carotene. The equipment consists of a power supply that supplies between 0 and 30 kV of current, a digital pump where the syringe is placed and that allows to control the flow rate of the solution and a stainless steel needle. The needle is connected to the syringe through a Teflon cable and placed perpendicular to the manifold. For this example, the processing conditions were for both materials (coating and encapsulation) the following: solution flow rate of 0.30 ml / h; 12 kV current; 10 cm distance between the needle and the collector. The uncoated 3-carotene, the coated 3-carotene and the 3-carotene encapsulates with zein were exposed to UV light for 10 h. Table 3 shows the evolution of the absorbance at 455 nm of ¡3-carotene, where this compound has a characteristic band. The initial absorbance and absorbance are shown after 10h under UV light of the uncoated, coated and encapsulated 3-carotene. It is observed that the coating was the technology
    30 that best protected the antioxidant against UV degradation.
    Table 3
    3-carotene Encapsulated Coating
    Absorbance initial(455
    nm) 0.2550.2550.255
    Absorbance 10h (455 nm) 0.0860.1900.100
    权利要求:
    Claims (14)
    [1]
    1.-Procedure for the protection of biological material and thermolabile compounds of industrial interest comprising the following stages:
    5 preparation of polymeric solutions forming micro-, submicro- and nanoparticles that make up coating materials
    - coating of the biological material or thermolabile compounds by means of
    10 materials prepared in the previous stage, characterized in that the coating of the biological material or thermolabile compounds is carried out by electrohydrodynamic or aerodynamic processing.
    [2]
    2. Method according to claim 1, characterized in that the biological material 15 are microorganisms or viruses.
    [3]
    3. Method according to claim 2, characterized in that the microorganisms are Lactobacillus, Bifidobacterium, Cyanobacterium, Rhodobacterales, Saccharomyces, or any other microorganism that needs protection.
    Method according to claim 1, characterized in that the thermolabile compounds are enzymes, vitamins, essential elements, or any molecule or compound derived or not, that needs protection.
    Method according to any one of claims 1 to 4, characterized in that the coating materials are selected from proteins, oligosaccharides, polysaccharides. lipids, polymers and combinations thereof.
    [6]
    6. Method according to claim 5, characterized in that the materials of
    Polymers coating are selected from polyethylene oxide, copolymers of ethylene and vinyl alcohol, polyvinyl alcohol, polyvinyl pyrrolidone and combinations thereof.
    [7]
    7. Method according to claim 5, characterized in that the proteins used as coating materials are selected from animal, vegetable and microbial proteins, particularly those from whey, caseins, natural polypeptides or obtained by genetic modification of
    5 microorganisms, collagen, soy protein and zein.
    [8]
    8. Method according to claim 7, characterized in that the proteins used as coating materials are selected between the zein and the whey protein.
    Method according to claim 5, characterized in that the oligosaccharides used as coating materials are selected from lactose, sucrose, maltose and fructooligosaccharides and particularly fructooligosaccharides.
    [10]
    10. Method according to claim 5, characterized in that the polysaccharides used as coating materials are selected from alginate, pectins, chitosan, gums, carrageenans, starch, dextran, maltrodextrin, cellulose, glycogen and chitin.
    11. Method according to claim 10, characterized in that the polysaccharides used as coating materials are selected from dextran, maltodextrin and starch and any combination thereof.
    12. Method according to any one of claims 1 to 11, characterized in that the solvent used for the preparation of polymer solutions is selected from water, alcohols, mixtures of alcohols and water and other organic solvents.
    13. Method according to claim 12, characterized in that the solvent is water or mixtures of alcohol and water.
    [14]
    14. Method according to any one of claims 1 to 13, characterized in that in the stage of preparation of the polymer solutions, processing aids are added that facilitate the formation of the micro-, submicro and nanoparticles and are selected from plasticizers, surfactants, emulsifiers, surfactants, antioxidants or any combination thereof.
    15. Process according to claim 14, characterized in that the substanceprocessing aid that is added is sorbitan monolaureate.
    [16]
    16. Process according to any one of claims 1 to 15, characterized in that the step of preparing the polymer solutions includes a homogenization treatment 10 by stirring.
    [17]
    17. Method according to any one of claims 1 to 15, characterized
    because the stage of preparation of polymer solutions includes an ultrasonic homogenization treatment.
    [18]
    18. Method according to any one of claims 1 to 17, characterized in that the electrohydrodynamic or aerodynamic processing is electrosprayed or blow-sprayed in at least one stage.
    19. Method according to claim 18, characterized in that the electrohydrodynamic or aerodynamic processing is carried out in several stages with a homogenization, turning and / or sieving treatment between them.
    [20]
    20. Method according to any one of claims 1 to 19,
    25 characterized in that the coating by electrohydrodynamic or aerodynamic processing is performed directly on the biological material or thermolabile compounds previously dehydrated by lyophilization or other non-electrohydrodynamic or aerodynamic process.
    21. Method according to any one of claims 1 to 19, characterized in that the electrohydrodynamic or aerodynamic processing is performed directly on the biological material or thermolabile compounds without dehydration, the dehydration of these occurring and then the coating by electrohydrodynamic or aerodynamic processing.
    [22]
    22. Method according to any one of claims 1 to 21, characterized
    because prior to the coating stage a treatment of
    sieved to control particle size.
    [23]
    23. Method according to any one of claims 1 to 21, characterized
    because after the coating stage a treatment of
    sieved to control particle size.
    24. Method according to claims 22 or 23, characterized in that the screening treatment is mechanical.
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    同族专利:
    公开号 | 公开日
    ES2541203R2|2015-08-06|
    EP3095854A1|2016-11-23|
    EP3095854A4|2017-09-27|
    WO2015107246A1|2015-07-23|
    ES2541203B1|2016-05-13|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2395553B1|2011-06-22|2013-12-26|Consejo Superior De Investigaciones Científicas |PROCEDURE FOR OBTAINING MICRO-, SUBMICRO- AND NANOCAPSULES BASED ON PROTEINS OF MILK.|
    ES2402612B1|2011-10-24|2014-03-11|Consejo Superior De Investigaciones Científicas |MICRO- SUBMICRO AND Nanostructures BASED ON PROTEIN OF AMARANTO.|DE102016125182A1|2016-12-21|2018-06-21|Groz-Beckert Kg|Process for producing fibers and nonwovens by solution blow spinning and nonwoven fabric made therewith|
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    EP15737796.1A| EP3095854A4|2014-01-15|2015-01-15|Method for protecting biological material and thermolabile compounds for possible industrial uses|
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