ES2697301B2 - NANOEMULSIONS TO PREVENT BIOLOGICAL COLONIZATION OF SURFACES - Google Patents

Description

DESCRIPTION

NANOEMULSIONS TO PREVENT BIOLOGICAL COLONIZATION OF SURFACES

Field of the Invention

The present invention relates to nanoemulsions with nanoencapsulated essential oils for the treatment and prevention of biological contamination of inorganic surfaces in general and their manufacture.

Background of the invention

Biological colonization represents one of the processes of deterioration that most affect wall and mortar surfaces. When favorable environmental conditions occur (high humidity values, direct contact with sunlight), different types of fungi, bacteria, algae and molds can colonize the walls causing chemical, physical and aesthetic damage. Although most of the degradations can only be appreciated on a microscopic scale, this problem becomes very important when the colonization takes place on surfaces of interest such as materials and structures that belong to the immovable cultural heritage (wall paintings, archaeological sites, historical buildings, etc. .).

Protecting these and other surfaces in general from the attack of microorganisms is an extremely complicated task since, today, there is no eco-compatible and reversible product capable of preventing colonization. For this reason, the only way currently available to limit biodeterioration phenomena is to resort to the periodic elimination of the biological patinae that are developing cyclically.

Today, the elimination treatment most used by restaurateurs is the application of antimicrobial products followed by the mechanical export of the treated patina. The most used antimicrobial products for this purpose are acids, pyrimidines and organometallic compounds, while mechanical removal of biological skates is usually carried out using scalpels and spatulas that cause damage to the substrates. However, using this cleaning technique to treat a mural surface of cultural interest can be very expensive, since the price of these treatments can vary from 450 to 700 euros per m2.

Despite the efficacy of the antimicrobial compounds currently used, the world of research is constantly searching for eco-compatible alternatives that respect the principles of green chemistry. In this context, several research groups are experimenting with the use of essential oils with antimicrobial properties as a biological alternative to the conventional chemicals mentioned above. Research carried out so far has produced very promising results. For example, essential oils extracted from aromatic plants such as Origanum vulgare, Rosmarinas officinalis and Lavandula angustifolia, have been shown to successfully neutralize some of the biodeteriogens that most commonly compromise the conservation of the immovable cultural heritage.

However, its use in the pure state does not prevent long-term biological colonization, mainly due to their high volatility, which means that this treatment can only be used to neutralize the biological patina that has already colonized the wall surface. This limitation can be overcome through the encapsulation of the essential oil in intelligent nanoemulsions that prevent evaporation of the active compound and allow its release to be controlled. In fact, the use of biological compounds such as zein, casein, egg, pectin, alginate and chitosan is well known to form biocompatible and stable nanocapsules that are being used in the medical and food field to prevent degradation and evaporation of essential oils and At the same time control your release.

In view of the above, and despite the progress achieved so far, there is still a need to provide effective alternative products for the treatment of contaminated surfaces and especially for the effective prevention of biological colonization, overcoming at least part of the disadvantages of The products of the state of the art.

Surprisingly, the authors of the invention have discovered that it is possible to obtain a nanoemulsion in a simple way by encapsulating essential oils in nanocapsules, formed by a central core based on a zein matrix, in which the essential oil is embedded, and protected by two layers of pectin and casein coating respectively. This new nanocapsule of nanocapsules is capable of providing better results in the treatment and prevention of biological contamination, guaranteeing high durability and stability of the product at room temperature.

Description of the figures

Figure 1: Optical image and characterization of the nanoemulsion using a Nano Zetasizer ZS system.

Figure 2: Characterization of the structure of the nanocapsules by Fourier transform infrared spectroscopy (FTIR) in transmission mode or total attenuated reflectance (ATR)

Figure 3: Transmission electron microscopy (TEM) images taken after 6 months of aging at room temperature.

Description of the invention

In a first aspect the invention relates to a process for preparing a nanoemulsion comprising nanocapsules with essential oil. This procedure, hereinafter the method of the invention, comprises the following steps:

(i) prepare a solution of zein and essential oil in water-alcohol (solution A); a solution of casein in water (solution B) and a solution of pectin in water (solution C);

(ii) add solution A on the mixture of solutions B and C, or alternatively, add solution A and solution B simultaneously on solution C, and

(iii) obtaining a nanoemulsion of nanocapsules.

The process of the invention leads to the spontaneous formation of stable nanocapsules due to the physicochemical processes that take place when the solutions A, B and C are combined in the manner defined in step (ii). The resulting nanocapsules are characterized in that they comprise:

(a) a central core comprising a zein matrix and an essential oil;

(b) a first core core coating layer comprising casein and

(c) a second outer coating layer on the first coating layer comprising pectin.

Solution A to practice the invention is prepared from zein and essential oil that are dissolved in a water-alcohol mixture, where the alcohol is preferably ethanol. The amount of alcohol in solution A may vary between 50% and 99.5% v / vt (total volume / volume), preferably between 55 and 90% v / vt, more preferably between 60 and 85% v / vt and even more preferably between 65 and 75% v / vt. Solution A is prepared by stirring at room temperature. The concentration of zein and essential oil in solution A is not particularly limited. In general, the concentration of zein is between 0.1 and 1.5% m / vt, preferably between 0.2 and 1.0% m / vt, more preferably between 0.3 and 0.8% m / vt and even more preferably between 0.4 and 0.6% m / vt (mass / total volume). In general, the concentration of essential oil is between 0.5 and 5% v / vt, preferably between 1.0 and 4.0% v / vt, more preferably between 1.7 and 3.0% v / vt and even more preferably between 2.3 and 2.7% v / vt. Solution B is prepared by dispersing a salt derived from casein in water, for example sodium caseinate. The dispersion is carried out at room temperature under stirring. Solution C is prepared by dispersing pectin in water. The dispersion is carried out at room temperature under stirring. In a preferred embodiment the pH of solution C is adjusted to a value below pH 4.6 with a base, for example a basic solution, preferably a sodium hydroxide solution.

The water used to prepare the solutions is preferably ultrapure water.

In the context of the present invention, the ambient temperature should be understood as between 20 ° C and 30 ° C, preferably around 25 ° C.

The step (ii) in which the 3 solutions are contacted can be carried out according to two different alternatives.

According to a particular embodiment of the process, solutions B and C are mixed first, and solution A is added thereto. The mixture of B and C is preferably done under magnetic stirring and at room temperature. Typically stirring is carried out for a time of 1 hour at 1000rpm. Solution A is then added onto the mixture of solutions B and C. Preferably solution A is added dropwise manually (for example, by micro pipette) or automatically (for example, by peristaltic pump) and the mixture which results, is subjected to magnetic stirring at room temperature. In general, stirring is carried out for a time equal to or greater than 30 minutes, preferably for 1 hour, at 1000 rpm.

According to another particular embodiment of the process, solutions A and B are added dropwise simultaneously (manually or, more preferably, automatically by peristaltic pumps) to solution C. The resulting mixture is subjected to magnetic stirring. at room temperature. In general, stirring is carried out for a time equal to or greater than 30 minutes, preferably for 1 hour, at 1000 rpm.

In the process of the invention the spontaneous formation of the nanocapsules occurs because the concentration of alcohol in the mixture of solutions A, B and C decreases progressively. The essential oil and zein, becoming immiscible in the final solution, form (under stirring) drops of essential oil / zein of nanometric scale. Casein, being an amphiphilic protein, creates a first protective layer that covers the drops of essential oil / zein (through hydrophobic interactions), and provides steric and electrostatic repulsion between the nanoparticles (through the hydrophilic portion). Finally, pectin, having a net charge opposite to casein, creates a complex of electrostatic nature with it. Being a surfactant, the outer pectin layer stabilizes the nanoparticles in the aqueous solution.

The inventors have observed that the formation of the nanocapsules takes place when the concentration of the alcohol in the solution mixture is equal to, or less than 15% v / vt, preferably equal to or less than 12% v / vt, more preferably equal to or less than 10% v / vt and even more preferably equal to or less than 7% v / vt. The person skilled in the art can calculate in each particular case the volume ratio in which solutions A, B and C have to be mixed to reach said alcohol concentration and achieve formation spontaneous nanoemulsion of the invention.

The nanoemulsion of the invention has between 0.05% and 2.00% m / vt (mass / total volume) of zein, preferably between 0.05 and 0.5% m / vt, and more preferably between 0.05 and 0.25% m / vt; between 0.05 and 2.00% v / vt (total volume / volume) of essential oil preferably between 0.10 and 1.00% v / vt, and more preferably between 0.20 and 0.50% v / vt ; between 0.10 and 1.50% m / vt of pectin, preferably between 0.10 and 0.50% m / vt, more preferably between 0.20 and 0.40% m / vt; and between 0.10 and 1.50% m / vt of casein, preferably between 0.10 and 0.50% m / vt, more preferably between 0.20 and 0.40% m / vt. The rest up to 100% corresponds to the aqueous phase (water-alcohol).

The essential oil that can be used in the invention can be one or a mixture of two or more essential oils with antimicrobial properties such as, for example, lavender, eucalyptus, oregano, cinnamon, clove and white thyme, rosemary, among others. Its use as an antimicrobial is very attractive because they are natural compounds that can be obtained from a wide variety of plants.

Regardless of whether the process is carried out according to one or another particular embodiment, a nanocapsule nanoemulsion with an average diameter around 300 nm to 200 nm is spontaneously obtained. In a particular embodiment the average diameter is between 275 nm and 225 nm, preferably around 250 nm. The resulting nanocapsules have high stability which is important to ensure that they maintain their antimicrobial activity for a long time after being applied to the surfaces to be treated.

In a preferred embodiment of the process of the invention, once the nanocapsule nanoemulsion is obtained in step (iii), the pH thereof is adjusted to a value close to the isoelectric point of the zein, preferably between 6.2 and 8, 2, and more preferably between 6.6 and 7.0 and a heat treatment is applied which comprises subjecting the nanoemulsion to a temperature of between 60 and 90 ° C, preferably between 65 and 85 ° C, and more preferably between 70 and 80 ° C. In this way, a nanoemulsion of even more stable nanocapsules is obtained and with a smaller average diameter between 120 nm and 190 nm, preferably between 125 nm and 180 nm, more preferably between 130 nm and 160 nm. In a particular embodiment the average diameter is equal to or less than 170 nm. In another particular embodiment it is equal to or less than 150 nm. The reduction in size that takes place is due on the one hand, because the increase in temperature facilitates the denaturation of proteins, in this case the caseinate, and causes their consequent compaction. On the other hand, it is favored that the alcohol present (for example, ethanol) evaporates, causing a subsequent decrease in the solubility of the zein and the essential oil, with the consequent compaction of the nanocapsules.

In a particular embodiment the heat treatment is done by placing the nanoemulsion resulting from step (iii) in a water bath. Generally the heat treatment is carried out for a time equal to or greater than 1 hour and under constant agitation.

The nanoemulsion obtained in this process constitutes an additional aspect of the present invention. The nanoemulsion comprises nanocapsules characterized in that they comprise:

(a) a central core comprising a zein matrix and an essential oil;

(b) a first core core coating layer comprising casein and

(c) a second outer coating layer on the first coating layer comprising pectin.

The nanoemulsion has been characterized by various techniques that have shown that the nanocapsules are spherical, uniform with a polydispersity index between 0.18 and 0.25 (preferably between 0.21 and 0.23), have an average diameter which varies as defined above, and are stable with Z potentials well below -30 d.nm, values that also remain fairly constant over time (see Example 1, Table 1).

Example 2 shows a comparative test in which a nanoemulsion was prepared following the method of Example 1, but without casein. The results shown in Table 2 of average diameter, polydispersity index and Z potential indicate that the nanocapsules of the nanoemulsion, and the nanoemulsion itself are less stable over time than in the case of the present invention. In this sense it can be seen that while the average diameter of the nanocapsules in Example 2 increases by 25% in Example 1 in two months, the average diameter of the nanocapsules of the invention increases by only 15%. This data indicates that the nanoemulsion of the invention makes the latter more compact and therefore more stable. On the other hand, the Z potential data reflects the stability of the nanoemulsions. In this sense, although the nanocapsules without casein at time 0 have a more negative value in Example 2 than in Example 1 it is found that over time the potential Z is decreasing more rapidly: specifically, in 8 weeks (56 days) the nanocapsules of Example 2 lose 7.6% of charge, while those of the invention with casein lose only 2.3%. This clearly indicates that in the long term the nanocapsules in the nanoemulsion of the invention remain surprisingly stable for longer without precipitating.

The antimicrobial essential oil is embedded in the zein matrix, and also protected by the first and second layers of oxidation and evaporation phenomena, giving it high stability, and safeguarding its long-term antimicrobial properties. On the other hand, the very nature and disposition of the nanocapsule compounds is advantageous in that it allows the intelligent release of the essential oil, that is, the release only takes place in the presence of microorganisms such as fungi, algae, bacteria and molds that , synthesize enzymes which depolymerize the nanocapsule compounds releasing the essential oil that can then act as an antimicrobial.

Therefore, in a further aspect the invention relates to the use of the nanoemulsion of the invention for the antimicrobial treatment of a contaminated inorganic substrate and / or for the prevention of biological colonization of an inorganic substrate.

The use comprises bringing the nanoemulsion into contact with the inorganic surface of said substrate. The nanoemulsion of the invention can be applied directly, or it can be previously diluted in a suitable solvent and in the appropriate proportion in each case. The contact of the surface of the substrate and the nanoemulsion is carried out by any conventional method, for example by spraying. The appropriate amount in each case will depend on the particular particular embodiment, such as the type of substrate, the surface area of the substrate to be treated, etc.

The use of nanoemusion for the antimicrobial treatment of a substrate means reducing or eliminating the biological colonization of the substrate. The use of nanoemusion for the prevention of the biological colonization of a substrate means avoiding the biological colonization of a substrate. The use according to the invention also has the advantage that it does not affect the appearance, or the chemical-physical properties of the treated substrate, which is especially advantageous in the case of substrates of cultural interest such as those of Cultural Heritage.

The essential oils, being 100% volatile, leave no deposits on the treated surface. Therefore, the nanoemulsion can be advantageous and potentially applied to any type of wall, mortar and stone surface susceptible to the phenomenon of biological colonization.

From an economic point of view, the low cost of the compounds used, and the process of obtaining allows to obtain an extremely economical product. Besides being a nanoemulsion obtained from natural, ecological and biodegradable products, it guarantees the safety of workers and respect for the environment. The procedure for obtaining the nanoemulsion is simple, and only requires simple stirring at room temperature.

The present invention manages to provide a solution to a problem of great importance in the world of the conservation of cultural heritage, of surfaces in general and in particular of wall surfaces, mortars and sculptures by biodeteriogens.

The following are examples to illustrate the invention that should not be considered as limiting the scope of the invention.

Examples

Example 1: Preparation of a nanoemulsion

A nanoemulsion was prepared with stable nanocapsules and less than 150 nm in size using essential oil extracted from the clove plant (whose fungicidal and bactericidal capacity has been proven in numerous works). To do this, first solutions A, B and C were prepared as follows:

• Solution A : 30mg of zein (type Z3625 Sigma Aldrich Corp) and 150pl of essential oil were dissolved in 5ml of 70% aqueous ethanol solution. The dispersion was subjected to magnetic stirring (1000rpm) for 150 minutes at 25 ° C;

• Solution B : 120mg of casein (type C8654 Sigma Aldrich Corp) were dissolved in 5ml of ultrapure water. The dispersion was subjected to magnetic stirring (1000 rpm) for 120 minutes at 25 ° C;

• Solution C : 48mg of pectin (type P9135, Sigma-Aldrich Corp.) were dissolved in 20ml of ultrapure water. The dispersion was subjected to magnetic stirring (1000 rpm) for 240 minutes at 25 ° C; Next, the pH of the solution was adjusted to pH 4.0 using a 10mM HCl solution;

1 ml of solution A and 1 ml of solution B were simultaneously poured into 10 ml of solution C by using a multichannel pipette (eppendorf). Then, the solution was magnetically stirred for 1 hour, at 1000 rpm and at a temperature of 25 ° C.

In this way, a 6.4% aqueous ethanol solution was obtained and composed of 0.05% m / vt of zeine, 0.25% v / vt of essential oil, 0.20% m / vt of casein and 0.20% m / vt of pectin.

Finally, the pH of the suspension was adjusted to a value equal to 6.6 by a solution of 10mM NaOH. After 60 minutes of magnetic stirring at 1000rpm and at room temperature, a heat treatment (80 ° C water bath) was applied for 60 minutes in order to induce protein denaturation and evaporation of ethanol.

Characterization of nanoemulsion and nanocapsules:

Once the synthesis process is finished, the analytical techniques have been used in order to characterize the nanoemulsion.

The analyzes carried out using the Nano Zetasizer ZS system (Malvern Instruments, Ltd., Worcestershire, UK) have allowed to verify that the nanoemulsion obtained is composed of nanocapsules, uniform (that is to say with a polydispersity index (PDI) = 0.229 ± 3 , 4), stable (potential Z: = -47.2 ± 2.6) and average particle size less than 150nm (Z size: 133.2 ± 3.4 nm) (see Figure 1).

A part of the nanoemulsion was spray dried using a Nano Spray Dryer B-90 (BUCHI, Labortechnik Ag, Flawill, Switzerland) and the powder obtained was characterized with Fourier transform infrared spectroscopy (FTIR) in ATR mode.

As shown in Figure 2, the main peaks of the zein, casein and pectin compounds have been detected in the spectrum of the pulverized sample (lower spectrum: S1). The absence of the essential oil identification peaks confirms that the active compound is located in the core of the nanocapsules.

It should also be noted that, in order to assess its stability over time, the nanoemulsion was analyzed weekly using the Nano Zetasizer equipment for a period of two months. As can be seen in the following Table 1, nanoparticles have been shown to have excellent stability over time.

Finally, thanks to the images captured through a transmission electron microscope (TEM) it has been possible to observe that the nanocapsules, after 6 months of aging, still had a perfectly spherical shape (Figure 3, where a) is the image in time zero and b) is the image at 6 months).

Comparative Example 2: Preparation of a nanoemulsion

A nanoemulsion was prepared following exactly the method described in Example 1 except that solution B did not contain casein and was only ultrapure water. The following was characterized in the same manner as in Example 1. The results are shown in the following Table 2:

Claims (5)

1. Use of a nanoemulsion comprising nanocapsules characterized in that they comprise: (a) a central core comprising a zein matrix and an essential oil; (b) a first coating layer of the central core comprising casein and (c) a second outer coating layer on the first coating layer comprising pectin. for the antimicrobial treatment of a contaminated inorganic substrate and / or for the prevention of biological colonization of an inorganic substrate. 2. Use of the nanoemulsion according to claim 1, wherein the nanocapsules have an average diameter between 300 nm and 200 nm. 3. Use of the nanoemulsion according to claim 1 or 2, wherein the nanocapsules have an average diameter between 120 nm and 190 nm. 4. Use of the nanoemulsion according to any one of claims 1 to 3 which comprises contacting the nanoemulsion with the inorganic surface of said substrate by applying it directly or after dilution in a solvent. 5. Use of the nanoemulsion according to any of claims 1 to 3 which comprises contacting the nanoemulsion with the inorganic surface of said substrate by spraying.